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curing type acetoxycolor tranlucentdensity approximately 1.04 g / ccskin formation time approximately 5 min at 30ccuring time approximately 24 h. for 2 mm thickness at 30cmovement accommodation approximately 0.61 n / mmelasticity approximately 260%
curing type acetoxy density approximately 1.04 g / cc skin formation time approximately 5 min at 30c curing time approximately 24 h. for 2 mm thickness at 30c movement accommodation approximately 15% tensile strength step 1 : ear impression is made using irreversible hydrocolloid alginate and custom made wax rim . wet gauze pieces are layered on top of alginate for the interlocking of the plaster to be poured [ figure 1 ] . making of ear impression step 2 : undercut areas in the impression are identified and blocked out with wax . block out impression partly with wax and cast poured step 3 : working model is retrieved from the impression carefully and base trimmed [ figure 3 ] . working model with dental stone prepared step 4 : custom tray using soft tray sheets prepared . a complete ear cast is made by making alginate impression for a person related to the patient [ figure 4 ] . custom tray using soft tray sheets prepared for complete ear cast step 5 : skin tone of the patient is matched to the oil colors . colors mixed with silicone step 6 : silicone material is dispensed to the under cuts of the custom tray and filled completely [ figure 6 ] . material dispensed with syringe into soft sheet custom tray step 7 : separating medium is applied on working cast and under cuts are filled with silicone material . it is pressed against the soft sheet custom tray carrying the silicone material [ figure 7 ] . silicone filled tray pressed against working cast step 8 : prosthesis is tried on the model and trimmed before insertion on patient [ figure 8 ] . over a period of time various materials such as wood , leather , polyurethane and polyvinyl chloride have been used to produce esthetic properties to prosthesis , but silicone has proved to be the material of choice because of its lifelike effects and flexibility . methyl methacrylate resin is been used as a maxillo - facial material , but its use is limited due to its rigidity . the flexibility and staining of silicone this industrial silicone has the property of elasticity and has the potential to incorporate stains and colors to mimic natural skin . the advantage of this industrial silicone material is the low cost factor , soft nature , flexibility , easy to fabricate and having good esthetic property and it will be an ideal material for the post - graduate student to train himself and acquire skills during his pre - clinical training program , which includes maxilla facial prosthodontics . since the cost is not prohibitive and as this material is quite similar to the current silicones , it can be recommended . | this article describes an industrial elastic silicone as a material for the laboratory fabrication of ear prosthesis .
it has been tested for toxicity in lab animals by the sgs india pvt .
ltd and approved as a material to pass the parameter of abnormal toxicity .
this material therefore can be safely recommended for laboratory exercise to fabricate facial prosthesis .
the high cost of the maxillo facial silicone materials prohibits their use for facial prosthesis in pre - clinical training of post - graduate students in maxillofacial prosthodontics . for this reason ,
pre - clinical laboratory exercise in facial prosthesis is inadequate .
a few institutions use polymethyl methacrylate resins which are rigid and do not have elastic characteristics of silicone , which is used for facial defects .
this cost - effective industrial silicone material which mimics the elastic and color characteristics of the conventional silicones can be recommended for preclinical exercises . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
since the late 1990s , several institutions and cooperative research groups have reported success in the treatment of locally advanced muscle - invasive bladder cancer with the use of combined modality therapy ( cmt ) , including transurethral resection of the bladder tumor ( tur - bt ) , radiotherapy ( rt ) and chemotherapy ( 13 ) . however , it is well recognized that the bladder continually changes its volume and position on a daily basis and that treating a bladder by rt traditionally requires at least a 1.52 cm isotropic setup margin ( 4,5 ) . such a large margin and treatment field may result in late bowel toxicity . while discussing tolerance doses of the bladder , marks et al . ( 6 ) suggested that it might be useful to classify the bladder complications into symptoms of global injury include urinary frequency , urgency , decrease in bladder capacity and cystitis . when the entirety of the bladder is irradiated to a modest dose ( < 4050 gy ) , global injury is infrequent and the development of a bladder injury is most dependent on the maximum bladder dose ( focal injury ) . total doses of 6575 gy delivered to very small portions of the bladder ( less than 20% ) are associated with an 510% complication rate . when the whole - bladder dose approaches 5060 gy , the risk of global bladder dysfunction starts to increase . in this setting , significant bladder complications may be seen even when the maximum bladder dose is low . based on the findings in their report , a partial bladder boost treatment technique with image guidance was selected to minimize the dose to the whole bladder at our institution . the real - time tumor - tracking radiotherapy ( rtrt ) system that we have used clinically since 1999 has the advantage that it enables us to correct the target location and also to observe the location of the target ( through the fiducial markers ) even during the beam delivery . to minimize the influence of bladder mobility and subsequent radiation - induced toxicity , we conducted a prospective phase ii study on the safety and efficacy of definitive rt for locally advanced bladder cancer with an image - guided local - boost rt using fiducial markers and an rtrt system for positioning during radiation delivery . this study was set up as a single - center phase ii trial following fleming 's single - stage design . the primary endpoint was overall survival and the secondary endpoints were treatment - related toxicity and local control . we set the minimum required 5-year overall survival at 30% and the minimum required level of efficacy at 60% in 5-year overall survival . the required sample size was calculated to be 20 with 80% power and a 2.5% one - sided significance level . all enrolled patients met each of the following criteria : ( i ) histologically confirmed urothelial cancer , ( ii ) t2t4n0m0 with or without hydronephrosis , ( iii ) who performance status 02 , ( iv ) clinically inoperable or refused surgery and ( v ) provision of written informed consent . patients with tumors that covered all or most of the bladder wall and patients with small cell carcinoma were excluded . this study was approved by the ethics committee of the hokkaido university graduate school of medicine . transurethral tumor resection was followed by 40 gy irradiation in 1620 fractions to the whole bladder and the regional lymph node area , or the whole bladder with an anisotropic margin of 1.52 cm . to minimize the duration between the implantation of fiducial markers and the end of rt and to avoid the dropout of the markers , implantation was performed after the delivery of 40 gy . until january 2002 , one or two markers were transurethrally implanted in the bladder wall near the primary tumor bed ( i.e. tur - bt scar ) ; thereafter , four to six markers were implanted around the primary tumor bed . the interruption of rt to implant the marker was scheduled to be < 12 days in principle . a localized boost for the primary tumor ( 25 gy/10 fractions ) was given using the rtrt system . a percutaneous full - thickness tumor biopsy was performed at the time of marker implantation . patients with adequate renal function ( creatinine clearance 45 ml / min ) received concurrent chemoradiotherapy ( ccrt ) with nedaplatin ( 70 mg / m intravenously , day 1 , day 22 and day 50 ) ; the other patients received radiotherapy alone ( rt - alone ) . the ccrt patients received an initial 40 gy at 2 gy / fraction , and the rt - alone patients received an initial 40 gy at 2.5 gy / fraction . nedaplatin ( cis - diammineglycolatoplatinum ; aqupla , shionogi & co. , osaka , japan ) is a second - generation platinum complex with reduced nephrotoxicity and gastrointestinal toxicity ( 7 ) . it was reported that nedaplatin also has a radiosensitizing effect ( 8) , and that its anticancer efficacy was comparable with that of cisplatin ( cddp ) and higher than that of carboplatin ( cbdca ) against bladder cancer ( 9 ) . for the present patient population , follow - up cystoscopy and urine cytology were performed every 3 months after the completion of the treatment . toxicity was evaluated using the national cancer institute common terminology criteria for adverse events ( nci - ctcae ) version 4.0 . five - year local control and survival rates were estimated by the kaplan meier method . all statistical analyses were performed using jmp 9.0.3 ( sas institute , cary , nc , usa ) . until 2004 at our institution , to ensure a constant bladder volume , an intravesical instillation of 100 ml sterile normal saline was performed , followed by computed tomography ( ct ) scanning of the small pelvis for rt planning with the patient in the supine position on a flat carbon table . the pre - scanning procedure was changed in 2005 , because the intravesical instillation was suspected of causing retrograde pyelonephritis in a patient ; thereafter , patients were instructed to void 30 min before ct scanning to allow the bladder to fill with urine . either a pinnacle ( hitachi medical , tokyo ) or a xio ( cms / elekta , st . louis , mo , usa ) was used as the three - dimensional radiation treatment planning system ( 3drtp ) . the clinical target volume ( ctv ) was defined as the volume within the contours of the bladder tumor and bladder wall surrounded by fiducial markers . the planning target volume ( ptv ) was obtained by expanding the ctv by a margin of 510 mm in all three dimensions . a 10-mm margin was used for patients in whom only one marker had been implanted . if multiple markers were implanted and we were certain that these markers were outside the tumor based on cystoscopy , a 5-mm margin was applied . the coordinates of the fiducial markers and the ctv were determined on the 3drtp using ct images . the radiation dose ( 25 gy/10 fractions ) was prescribed to the isocenter placed at the centroid of the ptv ( fig . 1 ) . at the time we started this study , dose volume histogram ( dvh ) was not routinely used and no specific dose constraint has been used for the bladder and organs at risk ( oars ) in this study .
figure 1.dose distribution of the boost plan for a patient with right posterolateral bladder tumor . clinical target volume ( ctv ) ( red mesh ) , planning target volume ( ptv ) ( cyan contour ) , bladder ( yellow - green contour ) and rectum ( purple contour ) . clinical target volume ( ctv ) ( red mesh ) , planning target volume ( ptv ) ( cyan contour ) , bladder ( yellow - green contour ) and rectum ( purple contour ) . the rtrt system consists of a 6 or 10 mv linear accelerator , two diagnostic x - ray fluoroscopic systems in the linear accelerator room , image processing units and an image display unit ( originally mitsubishi ; changed to varian medical japan co. , tokyo ) , as we have reported previously ( 10 ) . the actual position of the markers can be visualized on the fluoroscopic image during irradiation . the planned marker position is transferred from 3drtp and superimposed on the display unit of the rtrt system . for positional registration , one of the markers or the gravity center of the three markers was used as the fiducial point . for patients who were implanted with only one or two markers , one of the markers was used ; for patients who were implanted with more than three markers , the gravity center was used . after the manual setup using skin marks , the coordinates of the fiducial points were measured at the start of treatment and during irradiation . a fluoroscopic image from an rtrt system is digitized with a pixel size of 0.09 0.09 mm and image processors compared the digitized image with the template image of a round - shaped metallic marker to detect the location of the marker ( 11 ) . details of the calculation of the parallel and rotational setup errors have been reported ( 12 ) . in short , the position of the patient can be corrected by adjusting the patient 's table position by using a remote control bar on the treatment console . when the displacement of the fiducial point exceeds the threshold , the operator can correct the patient 's table position using the remote control unit . the threshold used in this study was 2.0 mm in each direction anterior posterior , cranial caudal and left right and if the displacement exceeded 2.0 mm in any direction , the table position was corrected accordingly . the table position can be changed in the lateral , vertical and longitudinal directions within 0.1 mm of the specifications . we calculated the rotational setup errors around each axis but intentionally did not correct them in this study . all enrolled patients met each of the following criteria : ( i ) histologically confirmed urothelial cancer , ( ii ) t2t4n0m0 with or without hydronephrosis , ( iii ) who performance status 02 , ( iv ) clinically inoperable or refused surgery and ( v ) provision of written informed consent . patients with tumors that covered all or most of the bladder wall and patients with small cell carcinoma were excluded . this study was approved by the ethics committee of the hokkaido university graduate school of medicine . transurethral tumor resection was followed by 40 gy irradiation in 1620 fractions to the whole bladder and the regional lymph node area , or the whole bladder with an anisotropic margin of 1.52 cm . to minimize the duration between the implantation of fiducial markers and the end of rt and to avoid the dropout of the markers , implantation was performed after the delivery of 40 gy . until january 2002 , one or two markers were transurethrally implanted in the bladder wall near the primary tumor bed ( i.e. tur - bt scar ) ; thereafter , four to six markers were implanted around the primary tumor bed . the interruption of rt to implant the marker was scheduled to be < 12 days in principle . a localized boost for the primary tumor ( 25 gy/10 fractions ) was given using the rtrt system . a percutaneous full - thickness tumor biopsy was performed at the time of marker implantation . patients with adequate renal function ( creatinine clearance 45 ml / min ) received concurrent chemoradiotherapy ( ccrt ) with nedaplatin ( 70 mg / m intravenously , day 1 , day 22 and day 50 ) ; the other patients received radiotherapy alone ( rt - alone ) . the ccrt patients received an initial 40 gy at 2 gy / fraction , and the rt - alone patients received an initial 40 gy at 2.5 gy / fraction . nedaplatin ( cis - diammineglycolatoplatinum ; aqupla , shionogi & co. , osaka , japan ) is a second - generation platinum complex with reduced nephrotoxicity and gastrointestinal toxicity ( 7 ) . it was reported that nedaplatin also has a radiosensitizing effect ( 8) , and that its anticancer efficacy was comparable with that of cisplatin ( cddp ) and higher than that of carboplatin ( cbdca ) against bladder cancer ( 9 ) . for the present patient population , follow - up cystoscopy and urine cytology were performed every 3 months after the completion of the treatment . toxicity was evaluated using the national cancer institute common terminology criteria for adverse events ( nci - ctcae ) version 4.0 . five - year local control and survival rates were estimated by the kaplan meier method . all statistical analyses were performed using jmp 9.0.3 ( sas institute , cary , nc , usa ) . until 2004 at our institution , to ensure a constant bladder volume , an intravesical instillation of 100 ml sterile normal saline was performed , followed by computed tomography ( ct ) scanning of the small pelvis for rt planning with the patient in the supine position on a flat carbon table . the pre - scanning procedure was changed in 2005 , because the intravesical instillation was suspected of causing retrograde pyelonephritis in a patient ; thereafter , patients were instructed to void 30 min before ct scanning to allow the bladder to fill with urine . either a pinnacle ( hitachi medical , tokyo ) or a xio ( cms / elekta , st . louis , mo , usa ) was used as the three - dimensional radiation treatment planning system ( 3drtp ) . the clinical target volume ( ctv ) was defined as the volume within the contours of the bladder tumor and bladder wall surrounded by fiducial markers . the planning target volume ( ptv ) was obtained by expanding the ctv by a margin of 510 mm in all three dimensions . a 10-mm margin was used for patients in whom only one marker had been implanted . if multiple markers were implanted and we were certain that these markers were outside the tumor based on cystoscopy , a 5-mm margin was applied . the coordinates of the fiducial markers and the ctv were determined on the 3drtp using ct images . the radiation dose ( 25 gy/10 fractions ) was prescribed to the isocenter placed at the centroid of the ptv ( fig . 1 ) . at the time we started this study , dose volume histogram ( dvh ) was not routinely used and no specific dose constraint has been used for the bladder and organs at risk ( oars ) in this study .
figure 1.dose distribution of the boost plan for a patient with right posterolateral bladder tumor . clinical target volume ( ctv ) ( red mesh ) , planning target volume ( ptv ) ( cyan contour ) , bladder ( yellow - green contour ) and rectum ( purple contour ) . clinical target volume ( ctv ) ( red mesh ) , planning target volume ( ptv ) ( cyan contour ) , bladder ( yellow - green contour ) and rectum ( purple contour ) . the rtrt system consists of a 6 or 10 mv linear accelerator , two diagnostic x - ray fluoroscopic systems in the linear accelerator room , image processing units and an image display unit ( originally mitsubishi ; changed to varian medical japan co. , tokyo ) , as we have reported previously ( 10 ) . the actual position of the markers can be visualized on the fluoroscopic image during irradiation . the planned marker position is transferred from 3drtp and superimposed on the display unit of the rtrt system . for positional registration , one of the markers or the gravity center of the three markers was used as the fiducial point . for patients who were implanted with only one or two markers , one of the markers was used ; for patients who were implanted with more than three markers , the gravity center was used . after the manual setup using skin marks , the coordinates of the fiducial points were measured at the start of treatment and during irradiation . a fluoroscopic image from an rtrt system is digitized with a pixel size of 0.09 0.09 mm and image processors compared the digitized image with the template image of a round - shaped metallic marker to detect the location of the marker ( 11 ) . details of the calculation of the parallel and rotational setup errors have been reported ( 12 ) . in short , the position of the patient can be corrected by adjusting the patient 's table position by using a remote control bar on the treatment console . when the displacement of the fiducial point exceeds the threshold , the operator can correct the patient 's table position using the remote control unit . the threshold used in this study was 2.0 mm in each direction anterior posterior , cranial caudal and left right and if the displacement exceeded 2.0 mm in any direction , the table position was corrected accordingly . the table position can be changed in the lateral , vertical and longitudinal directions within 0.1 mm of the specifications . we calculated the rotational setup errors around each axis but intentionally did not correct them in this study . between 1999 and 2008 , 20 patients with invasive bladder cancer were enrolled , and 14 of these patients were treated by ccrt . the median follow - up period was 55.5 months ( range : 9126 months ) , and all survivors were followed for more than 2 years . the number of patients for each tumor location when the bladder wall was divided into two portions ( left versus right , cranial versus caudal and anterior versus posterior ) were 10 versus 10 , 4 versus 16 and 6 versus 14 , respectively . ten patients received intravesical instillation of sterile normal saline as a preparation and the remaining 10 patients were instructed to void 3060 min before ct scanning and treatment . table 1.characteristics of the 20 bladder cancer patientsmedian age ( range ) ( years)77 ( 5885)gender ( % ) male15 ( 75% ) female5 ( 25%)tumor stage ( % ) t211 ( 55% ) t37 ( 35% ) t42 ( 10%)pathological grade ( % ) 25 ( 25% ) 315 ( 75%)concurrent chemoradiotherapy ( % ) 14 ( 70%)median no . of implanted markers ( range)4 ( 16)no . of patients with less than three markers ( % ) 8 ( 40% ) characteristics of the 20 bladder cancer patients nedaplatin was administered to the 14 ccrt patients , with good compliance ; 13 patients completed the planned dose . the median overall treatment time ( ott ) was 65 days ( range : 5185 days ) . all patients completed the planned rt except for one patient who wanted , for religious reasons , to avoid the risk of requiring a blood transfusion in the event of side effects . for this patient we could not calculate the precise cumulative dose of bladder and other oars ( rectum and intestinal cavity that was defined as the volume of abdominal cavity bordered anteriorly and laterally to the abdominal / pelvic wall and inferiorly to the rectum / bladder including all visible bowel loops ) because initial plan for whole bladder ( 40 gy ) and boost plan ( 25 gy ) were created based on different treatment planning ct series . therefore , assuming that the entire bladder and oars received 40 gy in the initial plan for whole bladder , we assessed the standard dvh parameters of bladder and oars of the last nine patients whose data were available for analysis ( 1315 ) although not always the entire oars were actually received 40 gy . median v65 gy ( % ) of bladder was 3.5% ( range , 0.0112.7% ) , median v60 gy ( % ) of rectum was 1.3% ( range , 0.0021.4% ) and median v45 gy ( cm ) of intestinal cavity was 172.9 cm ( range , 127.2419.1 cm ) . two patients experienced acute treatment - related toxicities during treatment ; the toxicity was a grade 3 urinary tract infection in both cases . one of these patients developed retrograde pyelonephritis just after the intravesical instillation of sterile normal saline when the ct scanning was performed ; treatment had to be interrupted in this case . of the 14 patients treated by ccrt , only one patient had to skip chemotherapy due to side effects ( grade 3 thrombocytopenia on day 50 ) . no other acute toxicity greater than grade 3 or higher was observed , nor was any significant adverse event thought to be caused by the implantation of the fiducial markers . with respect to late treatment - related toxicities of these patients , one required endoscopic hemostasis ( grade 3 ) and the other patients were improved by conservative treatment ( grade 2 ) . one patient who had a tumor relapse in the bladder and a pelvic lymph node after the initial protocol treatment underwent additional irradiation to the metastatic pelvic lymph node for palliation . she developed a vesicovaginal fistula at 1.5 years after the initial treatment ; this was attributed to tumor progression . she also developed an adhesive intestinal obstruction at 1.9 years and required a laparotomy ( grade 3 ) . no other urethral or bowel toxicity of grade 2 or higher was observed ( table 2 ) . we could not determine the relationship between the tumor location and the gastrointestinal complication in this study ; in the patient who had grade 3 gastrointestinal toxicity ( adhesive intestinal obstruction ) after irradiation to the metastatic pelvic lymph nodes in addition to the initial protocol study , the tumor was located in the lower posterior part of the bladder . table 2.number of patients with acute and late complicationsnumber of patientsgrade ( nci - ctcae ver.4.0)12345acute complications urinary frequency71 diarrhea1 urinary tract infection2 thrombocytopenia1late complications cystitis noninfective ( hemorrhagic cystitis)41 adhesive intestinal obstruction1 number of patients with acute and late complications among the 20 patients , 14 had achieved complete response ( cr ) at the time of the implantation of fiducial markers , based on re - evaluation with a percutaneous full - thickness bladder biopsy of the primary tumor site . the local - control rate ( fig . eight patients locally relapsed in the bladder ; of these , five had relapse in the boost field , two had relapse in the marginal area of ctv ( i.e. inside the markers ) and one had relapse outside the boost field ( extensive superficial relapse ) . although the number of patients was less , when dividing patients into two groups according to the preparation method , no significant difference was observed in the rate of local tumor control in this study ( 50% for the intravesical instillation group and 79% for the patients who voided 3060 min before treatment , p = 0.22 , wilcoxon test ) . all patients who had local recurrence were managed with conservative treatment , such as tur - bt and/or intravesical drug therapy , and no patient received a salvage cystectomy . in other words , figure 3.overall survival , cause - specific survival and relapse - free survival for all patients . overall survival , cause - specific survival and relapse - free survival for all patients . between 1999 and 2008 , 20 patients with invasive bladder cancer were enrolled , and 14 of these patients were treated by ccrt . the median follow - up period was 55.5 months ( range : 9126 months ) , and all survivors were followed for more than 2 years . the number of patients for each tumor location when the bladder wall was divided into two portions ( left versus right , cranial versus caudal and anterior versus posterior ) were 10 versus 10 , 4 versus 16 and 6 versus 14 , respectively . ten patients received intravesical instillation of sterile normal saline as a preparation and the remaining 10 patients were instructed to void 3060 min before ct scanning and treatment . table 1.characteristics of the 20 bladder cancer patientsmedian age ( range ) ( years)77 ( 5885)gender ( % ) male15 ( 75% ) female5 ( 25%)tumor stage ( % ) t211 ( 55% ) t37 ( 35% ) t42 ( 10%)pathological grade ( % ) 25 ( 25% ) 315 ( 75%)concurrent chemoradiotherapy ( % ) 14 ( 70%)median no . of implanted markers ( range)4 ( 16)no . of patients with less than three markers ( % ) 8 ( 40% ) characteristics of the 20 bladder cancer patients nedaplatin was administered to the 14 ccrt patients , with good compliance ; 13 patients completed the planned dose . the median overall treatment time ( ott ) was 65 days ( range : 5185 days ) . all patients completed the planned rt except for one patient who wanted , for religious reasons , to avoid the risk of requiring a blood transfusion in the event of side effects . for this patient we could not calculate the precise cumulative dose of bladder and other oars ( rectum and intestinal cavity that was defined as the volume of abdominal cavity bordered anteriorly and laterally to the abdominal / pelvic wall and inferiorly to the rectum / bladder including all visible bowel loops ) because initial plan for whole bladder ( 40 gy ) and boost plan ( 25 gy ) were created based on different treatment planning ct series . therefore , assuming that the entire bladder and oars received 40 gy in the initial plan for whole bladder , we assessed the standard dvh parameters of bladder and oars of the last nine patients whose data were available for analysis ( 1315 ) although not always the entire oars were actually received 40 gy . median v65 gy ( % ) of bladder was 3.5% ( range , 0.0112.7% ) , median v60 gy ( % ) of rectum was 1.3% ( range , 0.0021.4% ) and median v45 gy ( cm ) of intestinal cavity was 172.9 cm ( range , 127.2419.1 cm ) . two patients experienced acute treatment - related toxicities during treatment ; the toxicity was a grade 3 urinary tract infection in both cases . one of these patients developed retrograde pyelonephritis just after the intravesical instillation of sterile normal saline when the ct scanning was performed ; treatment had to be interrupted in this case . of the 14 patients treated by ccrt , only one patient had to skip chemotherapy due to side effects ( grade 3 thrombocytopenia on day 50 ) . no other acute toxicity greater than grade 3 or higher was observed , nor was any significant adverse event thought to be caused by the implantation of the fiducial markers . with respect to late treatment - related toxicities of these patients , one required endoscopic hemostasis ( grade 3 ) and the other patients were improved by conservative treatment ( grade 2 ) . one patient who had a tumor relapse in the bladder and a pelvic lymph node after the initial protocol treatment underwent additional irradiation to the metastatic pelvic lymph node for palliation . she developed a vesicovaginal fistula at 1.5 years after the initial treatment ; this was attributed to tumor progression . she also developed an adhesive intestinal obstruction at 1.9 years and required a laparotomy ( grade 3 ) . no other urethral or bowel toxicity of grade 2 or higher was observed ( table 2 ) . we could not determine the relationship between the tumor location and the gastrointestinal complication in this study ; in the patient who had grade 3 gastrointestinal toxicity ( adhesive intestinal obstruction ) after irradiation to the metastatic pelvic lymph nodes in addition to the initial protocol study , the tumor was located in the lower posterior part of the bladder . table 2.number of patients with acute and late complicationsnumber of patientsgrade ( nci - ctcae ver.4.0)12345acute complications urinary frequency71 diarrhea1 urinary tract infection2 thrombocytopenia1late complications cystitis noninfective ( hemorrhagic cystitis)41 adhesive intestinal obstruction1 number of patients with acute and late complications among the 20 patients , 14 had achieved complete response ( cr ) at the time of the implantation of fiducial markers , based on re - evaluation with a percutaneous full - thickness bladder biopsy of the primary tumor site . the local - control rate ( fig . eight patients locally relapsed in the bladder ; of these , five had relapse in the boost field , two had relapse in the marginal area of ctv ( i.e. inside the markers ) and one had relapse outside the boost field ( extensive superficial relapse ) . although the number of patients was less , when dividing patients into two groups according to the preparation method , no significant difference was observed in the rate of local tumor control in this study ( 50% for the intravesical instillation group and 79% for the patients who voided 3060 min before treatment , p = 0.22 , wilcoxon test ) . all patients who had local recurrence were managed with conservative treatment , such as tur - bt and/or intravesical drug therapy , and no patient received a salvage cystectomy . in other words , figure 3.overall survival , cause - specific survival and relapse - free survival for all patients . . overall survival , cause - specific survival and relapse - free survival for all patients . up to the early 1990s , rt for patients with t2-t4n0m0 bladder cancer achieved a 5-year overall survival of 30% . one report has suggested that severe and chronic adverse effects affecting the bladder and bowel are not uncommon for this cancer , including a treatment - related death rate of 9.5% ( 16 ) . meanwhile , the 5-year overall survival rate after radical cystectomy for these patients was 50% . since the late 1990s , several groups have reported on bladder preservation therapy through a combined modality approach for muscle - invasive bladder cancer ( 13 ) . in all of these studies , to the best of our knowledge , patients with a cr after induction treatment were selected as candidates for bladder preservation , whereas in all the other patients , cystectomy was performed whenever feasible ( i.e. selective bladder preservation ) . shipley et al . ( 17 ) and efstathiou et al . ( 18 ) reported that , in two series of patients at massachusetts general hospital , cmt preserved the native bladder of more than 70% of patients while offering a long - term survival rate similar to that of a contemporary cystectomy series ; the details of treatment - related toxicities were not reported . a recent study in italy on 121 patients reported 5-year overall , local - control and bladder - intact survival rates of 67.7 , 56.3 and 51.2% , respectively ( 19 ) . in this study , 15% ( 18/121 ) of patients had grade 2 , 3% ( 4/121 ) grade 3 and 1% ( 1/121 ) grade 4 bladder chronic adverse effects ; moreover , 13% ( 16/121 ) had grade 2 and 2% ( 2/121 ) had grade 4 bowel chronic adverse effects . reviewed the results of bladder - sparing cmt and reported 5060% 5-year overall survival with 10% ( 18/186 ) grade 2 , 3% ( 5/186 ) grade 3 and 2% ( 3/186 ) grade 4 bladder chronic adverse effects ; moreover , 5% ( 20/415 ) of patients had grade 2 and 1.5% ( 6/415 ) grade 4 bowel chronic adverse effects ( 20,21 ) . efstathiou et al . ( 22 ) reported , based on the data of radiation therapy oncology groups ( rtog ) 8903 , 9506 , 9706 and 9906 , that the rate of late grade 3 or more genitourinary toxicity was 5.7% and that of grade 3 or more gastrointestinal toxicity was 1.9% . ( 23 ) also reported the results of a urodynamic and quality - of - life study on long - term survivors ; the majority of patients retained good bladder function , but one - fifth of them had persistent bowel symptoms such as difficulty with bowel control or abdominal cramping or pain . these studies suggest that recent improvements in the rt technology are reducing bladder and bowel chronic adverse effects but that there is still room for improvement . the large margins and treatment fields used in the past often resulted in chronic adverse effects , but the small margins based on recent static computed tomography in treatment planning may cause geographical error . the present study was based on a feasibility study conducted between 1998 and 1999 that showed that fiducial markers and two sets of fluoroscopy were useful to reduce setup errors compared with a skin - based setting for bladder and prostate cancer ( 10 ) . we have also found that a method using multiple gold markers with frequent correction of the table position during rt is useful to reduce the error due to intrafractional organ motion ( 24 ) . we inserted more than three markers around the tumor bed to be covered in the image - guided boost in principle , since the bladder wall is so flexible . at our institute , as well as at other institutes , a radical cystectomy is generally regarded as the first treatment of choice for muscle - invasive bladder cancer . patients who were referred for rt were not suited for surgical treatment due to their poor medical condition or they refused radical cystectomy . therefore , we did not perform radical cystectomy for the patients in this study , even in patients with residual tumor after induction treatment . for these reasons , it was difficult to compare our results with those of selective bladder preservation in both survival and local control . we set the minimum required 5-year overall survival at 30% based on the results of rt without any patient selection . the minimum required level of efficacy was set at 60% in 5-year overall survival because we believed that the efficacy of the image - guided definitive rt combined with maximal tur - bt and chemotherapy were comparable with radical cystectomy . moreover , we excluded patients who were unsuited for local - boost rt ( i.e. patients with tumors that covered all or most of the bladder wall ) . currently , several types of image - guidance technology using not only implanted fiducial markers but also on - board kilovoltage cone - beam computed tomography ( cbct ) and ultrasonography are being used . offline and online adaptive rt using pre - planned treatment plans and cbct has received much attention in light of its ability to reduce setup error and the margins required , thereby reducing treatment volumes and the volume of irradiated small bowel in external beam rt for bladder cancer ( 2530 ) . as for the motion detected by cbct , foroudi et al . ( 31 ) reported about the intrafractional bladder motion estimated from pretreatment and posttreatment using cbct recently . in the article they found that the margins required to cover the intrafractional bladder changes from pretreatment to posttreatment in the superior , inferior , right , left , anterior and posterior were 1.25 cm ( range , 1.191.50 cm ) , 0.67 cm ( range , 0.581.12 cm ) , 0.74 cm ( range , 0.590.94 cm ) , 0.73 cm ( range , 0.511.00 cm ) , 1.20 cm ( range , 0.851.32 cm ) and 0.86 cm ( range , 0.730.99 cm ) , respectively . they concluded that care is required while using image - guided radiation therapy protocols that reduce ctv to ptv margins based only on daily pretreatment soft tissue position . the advantage of our method using fiducial markers and the rtrt system is that it enables us to observe the location of the target through the fiducial markers even during the beam delivery . in our series , the incidence of the sessions that the table position adjustments were required was 19.7% for correcting the intrafractional errors . the maximum offset in the superior , inferior , right , left , anterior and posterior direction were 1.47 , 0.53 , 0.38 , 1.33 , 0.89 and 0.50 cm , respectively . based on the foroudi 's findings and ours , we consider that the intrafractional motion of the bladder may not be negligible . the relative positions of the markers in the bladder are not always constant , but the tumor exists in the area surrounded by the markers . therefore , we believe that the multiple markers implanted around the tumor are still a useful surrogate of the tumor position . in the case that only one or two markers are implanted as in the early phase of this study , a larger margin is considered to be required . a shortcoming of our method is that we are not seeing the bladder volume or the anatomical relationship between the marker and the tumor during irradiation . regarding the doses to the bowel extrapolating from the report of tuomikoski et al . ( 27 ) , which concluded that adaptive rt was beneficial to decrease irradiation of the small bowel in bladder cancer treatment , we could apply a smaller margin in boost irradiation than that used in their study without fiducial markers and therefore reduce the doses to the bowel . however , we should investigate more quantitatively the benefit of reducing bowel volume with our technique compared with other methods without fiducial markers and an rtrt system . we suspect that the combination of two sets of fluoroscopy with in - room ct or cone - beam ct is the best solution for bladder cancer at present . the interruption of rt for marker implantation might deteriorate the effect of rt ; however , our method achieved the minimum required level of 5-year overall survival and was comparable with other reports in terms of selective bladder preservation . in general , clinical data on the influence of ott on the outcome of rt for bladder cancer is scarce and not conclusive . in a recent analysis , majewski et al . ( 32 ) concluded that the ott did not significantly influence the treatment outcome . ( 33 ) also reported that there is no evidence to support short otts or large fraction sizes in bladder cancer . considering these reports , the interruption in this study might have had relatively less influence on the clinical outcome . our results showed that the incidence of grade 3 toxicity was similar to or slightly higher than that in other non - igrt series , despite the less number of patients . the possible reasons are ( i ) the overall dose irradiated to the bladder wall adjacent to the tumor could not be reduced and ( ii ) the patient who experienced grade 3 gastrointestinal toxicity had received additional irradiation after the protocol treatment for pelvic lymph node recurrence . the major limitation of our study is the less number of patients enrolled over a long period . because the current standard treatment for locally advanced muscle - invasive bladder cancer ( i.e. radical cystectomy and bladder - sparing cmt ) is suboptimal , we were able to treat only patients who were clinically inoperable or refused surgery . japan has a lower incidence of bladder cancer than those reported in north america , southern europe and western europe . therefore , we believe that the less number of patients in this series was not related to the treatment itself . the clinical outcome of the present method was at least comparable with the results of the recent cmt series with respect to local control and survival , despite the inclusion of patients who were considered inoperable and those who had hydronephrosis in this series . although the less number of patients and the long recruitment period limit the value of this study , our data suggest that both the implantation of fiducial markers and image - guided local - boost rt using an rtrt system can be safely accomplished , and the clinical outcome is encouraging . a larger prospective multi - institutional study is warranted for more precise evaluations of the technological efficacy and patients ' quality of life . this work was supported by the funding program for world - leading innovative r&d on science and technology ( first program), initiated by the council for science and technology policy ( cstp ) , japan . funding to pay the open access publication charges for this article was also provided by the | objectivethe real - time tumor - tracking radiotherapy system with fiducial markers has the advantage that it can be used to verify the localization of the markers during radiation delivery in real - time .
we conducted a prospective phase ii study of image - guided local - boost radiotherapy for locally advanced bladder cancer using a real - time tumor - tracking radiotherapy system for positioning , and here we report the results regarding the safety and efficacy of the technique.methodstwenty patients with a t2-t4n0m0 urothelial carcinoma of the bladder who were clinically inoperable or refused surgery were enrolled .
transurethral tumor resection and 40 gy irradiation to the whole bladder was followed by the transurethral endoscopic implantation of gold markers in the bladder wall around the primary tumor .
a boost of 25 gy in 10 fractions was made to the primary tumor while maintaining the displacement from the planned position at less than 2 mm during radiation delivery using a real - time tumor - tracking radiotherapy system .
the toxicity , local control and survival were evaluated.resultsamong the 20 patients , 14 were treated with concurrent chemoradiotherapy .
the median follow - up period was 55.5 months .
urethral and bowel late toxicity ( grade 3 ) were each observed in one patient .
the local - control rate , overall survival and cause - specific survival with the native bladder after 5 years were 64 , 61 and 65%.conclusionsimage - guided local - boost radiotherapy using a real - time tumor - tracking radiotherapy system can be safely accomplished , and the clinical outcome is encouraging .
a larger prospective multi - institutional study is warranted for more precise evaluations of the technological efficacy and patients ' quality of life . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
a 54-year - old insulin dependent diabetic female presented to a district hospital , where she was found to be in diabetic ketoacidotic coma ( dka ) , with peri - orbital cellulitis and neurological impairment . on arrival at the referral hospital , the ent surgeons elicited a two - day history of right sided facial and eye swelling , associated with progressively decreasing vision of the right eye . the patient was hiv negative and had no other medical or surgical history of note besides diabetes mellitus . on physical examination , she was found to be well orientated with a glascow coma scale of 15/15 , blood pressure of 102/52 mm / hg , a pulse rate of 112 and a respiratory rate of 15 . she was noted to have an extensive right sided periorbital cellulitis with marked proptosis of the right eye . opthalmological examination of the right eye , revealed opthalmoplegia ( frozen eye ) with mild chemosis , a clear cornea and lens , with the presence of a pale optic disc . the patient was assessed as having irreversible blindness of the right eye secondary to optic atrophy . on neurological examination , she was found to have multiple cranial nerve palsies , involving cranial nerves 4 , 5 , 6 , 7 , 8 and 9 . routine laboratory investigations revealed a glucose level of 12.5 mmol / l with 1 + ketones present on urine dipstick testing . the c reactive protein was raised to 38 , whilst the full blood count , urea and electrolytes , and liver function tests were all within normal ranges . an urgent computerized tomography ( ct ) scan of the paranasal sinuses showed partial opacification of the right maxillary , bilateral ethmoid and right sphenoid sinuses , with a possible filling defect of the right cavernous sinus . in view of these findings the patient was booked for an emergency endoscopic antrostomy for debridement of the necrotic tissue , as well as an external fronto - ethmoidectomy ( efe ) . intra - operative findings revealed extensive right sided intra - nasal necrotic tissue requiring autoethmoidectomy . although the mucosa of the superior turbinate had a dusky appearance , it remained intact . the normal anatomy of the sphenoid sinus was distorted and there was difficulty accessing the ostium . antrostomy of the right maxillary sinus revealed a large defect in the wall caused by the disease process . efe revealed that the frontal sinus was clear with no evidence of pus or mucosal damage . in light of these intra operative findings , the patient was commenced empirically on intravenous amphotericin b. the patient was clinically stable post operatively . however , on consultation with the neurologist , a ct scan of the brain was ordered to exclude a possible infarct in the temporal lobe . ct scan findings showed a right medial hypodensity with possible early cerebritis . a magnetic resonance imaging ( mri ) of the brain was therefore requested and showed extensive sinus disease involving the orbit with spread to the extra ocular muscles . a non enhancing hypo - density of the right temporal lobe , representing ischaemia was seen . intra - operative specimens from the right nasal cavity , middle turbinate and maxillary sinus were submitted to the laboratory for histology and microbiological investigation . microscopy of these specimens revealed broad , aseptate hyphae suggestive of zygomycetes sp . and thin , septate hyphae with dichotomous branching suggestive of aspergillus sp histology confirmed invasive co - infection with rhizopus sp . and aspergillus sp . while in hospital intra - operative findings revealed extensive bony and tissue necrosis of the septum , lamina papyracae and maxillary sinus . all necrotic and non - viable tissue was debrided and biopsies were taken from the septum and inferior turbinate . microbiology and histology of intra operative specimens were negative for both rhizopus sp . and aspergillus sp . intra operative findings included crusts involving the right nasal cavity and maxillary sinus with an anterior septal perforation . once again , specimens received at the laboratory did not reveal any fungi on microscopy and culture . the patient remained clinically stable and the ct prior to discharge revealed residual disease in right nasal cavity and sphenoid . a naso - endoscopy was performed which revealed right sided crusting due to possible inadequate douching . the patient completed 4 weeks of amphotericin b and was thereafter discharged on oral posaconazole 200 mg three times daily . on follow up 6 weeks later , the irreversible blindness in the right eye as well as cranial nerve palsies were still present . acute invasive fungal sinusitis is an aggressive disease seen particularly in immunocompromised individuals with neutropenia , hiv and diabetes mellitus . mortality rates remains very high , especially if the diagnosis and appropriate management is not timely instituted . etiological agents responsible for invasive fungal sinusitis include aspergillus spp . ; that are more commonly seen in patients with neutropenia ; and zygomycetes ( rhizopus spp . , mucor spp . , absidia spp . ) that are more commonly seen in diabetic patients , particularly in the setting of dka as was seen in this patient . invasive fungal infections of the oro - rhinocerebral area , due to mixed mucormycosis and aspergillosis infections have rarely been reported , , , , . the disease is acquired by inhalation of fungal spores into the nasal cavity with subsequent invasion of the sinus mucosa . fever , headache and nasal congestion , accompanied by facial pain and swelling are the earliest symptoms reported . disease thereafter rapidly progresses with extensive involvement of the cavernous sinus , orbit or intracranial compartment . subsequently , peri - orbital cellulitis , restriction of ocular movements , ptosis , proptosis and visual deterioration due to occlusion of the central retinal artery may follow , which is highlighted in this case report , . diagnosis is currently dependent on microscopy and culture as well as histological examination of specimens , . however , microscopic identification of fungal species is highly dependent on the quantity and quality of hyphae present . identification of mixed fungal infections is even more challenging , as illustrated in this case , wherein , the mixed infection resulted in only the rhizopus sp . this was attributed to the rapid growth of rhizopus , which hindered the growth of aspergillus sp . this illustrates the important role that initial direct microscopy plays in making the diagnosis of mixed fungal infections . the introduction of molecular techniques that use broad - range pcr amplification and reverse line blot hybridization ( pcr / rlb ) also allow for the detection and differentiation of mixed fungal infections . although these molecular methods have the advantage of providing a rapid diagnosis ; they are not freely available in low resource countries . in the diagnosis of invasive sinusitis , ct is sensitive for the detection of changes in the ostium ; however mri remains superior for the evaluation of intracranial and intraorbital extension . these investigations were an integral part of the management in this patient . a combination of early surgical intervention , appropriate antifungal therapy and the correction of underlying causes such as metabolic acidosis improves prognosis . in addition , serial endoscopic debridements have been shown to improve cure rates , as was illustrated in this patient . a disadvantage is that it can only be administered parenterally and is also potentially nephrotoxic . posaconozole is the only triazole with activity against both rhizopus sp . and aspergillus sp . . its availability in oral formulations has facilitated the outpatient management of patients with severe fungal disease . this case highlights the importance of close liaison between clinicians and the laboratory in the management of rare and complicated infections . | we report on an unusual case of oro - rhinocerebral disease caused by mucormycosis and aspergillus co - infection in a 54-year - old insulin dependent diabetic patient .
although she was successfully treated with parenteral amphotericin b followed by oral posaconazole , she was left with irreversible blindness of the right eye and multiple cranial nerve palsies . |
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the ebola outbreak
in 2014 raised new questions about routes of
transmission of the disease . experiments
with non - human primates have shown that inhalation exposure to ebola
virus can lead to fatal infection . patients with ebola virus
disease expel large volumes of diarrhea , which may contain
up to 10 genome copies of virus per milliliter , and toilets , sewer systems , and wastewater treatment
plants are known to produce bioaerosols . the combination of these factors indicates that the potential exists
for aerosolization of ebola virus from wastewater systems . sanitation
facilities that are common in developed countries , such as pressure - assisted
flush toilets and aeration basins , may provide opportunities for aerosolization
of the virus that do not exist in western africa , where the vast majority
of cases of ebola virus disease have occurred . given that ebola
is a high - consequence pathogen , it is critical
to consider all possible exposure routes . what we define as the secondary aerosolization exposure route ( i.e. , aerosolization from sources other
than the infected host ) has been established as a concern for other
diseases . for example , a combination of epidemiological , experimental ,
and modeling approaches suggests that aerosolization from toilets
and sewer pipes contributed to an outbreak of severe acute respiratory
syndrome in an apartment complex in hong kong in 2003 . an important factor in determining the potential
for a pathogen to spread via aerosolization from wastewater is partitioning
among the aqueous phase , biosolids , which are not as easily aerosolized ,
and material surfaces . if a pathogen partitions preferentially to
biosolids or surfaces , then the potential for aerosolization is reduced . the goal of this research was to assess the partitioning of viruses
in fluids and materials characteristic of modern wastewater systems . we measured the partitioning of an unenveloped virus ( ms2 ) and an
enveloped virus ( phi6 ) among the liquid phase , solids , and porcelain ,
polyvinyl chloride ( pvc ) , polypropylene , and concrete surfaces , using
both synthetic sludge and real , anaerobically digested sludge as model
fluids . results from this work will provide information not only about
the potential for aerosolization of viruses but also about their fate
in wastewater systems . because surface chemistry
affects partitioning , we considered both
unenveloped and lipid - enveloped viruses : ms2 and phi6 , respectively . ms2 ( atcc 15597-b1 ) is an icosahedral , single - stranded rna bacteriophage
27 nm in diameter and is widely used as a surrogate for enteric
viruses in environmental studies . phi6 ( kindly provided by p. turner
of yale university , new haven , ct ) is an icosahedral , double - stranded
rna bacteriophage 85 nm in diameter and has been proposed
as a surrogate for ebola virus , although
the structures of the two viruses differ ( i.e. , roughly spherical
vs filamentous ) . table s1 of the supporting information compares the structure
of ebola virus and the two surrogates . we propagated the bacteriophages
using host bacteria , escherichia coli and pseudomonas syringae , and standard culture methods . concentrations
in stock suspensions were 1010 plaque - forming
units per milliliter ( pfu ml ) . we tested
four bowl - shaped materials commonly used in wastewater
systems : porcelain , pvc , polypropylene , and concrete . the supporting information provides further details
about the containers used in this study . as a surrogate for
diarrhea , we tested both synthetic sludge and
real sludge collected from a wastewater treatment plant . we considered
both types of sludge to balance a well - defined composition of the
fluid against real - world conditions . for synthetic sludge we used a strain of bacillus and e. coli isolated from anaerobically digested
sludge as model gram - positive and gram - negative microorganisms of
enteric relevance , rather than yeast . for real sludge , we collected
anaerobically digested sludge from a wastewater treatment plant whose
flow is dominated by domestic sources ( > 99% ) . we stored the sludge
at 4 c and used it within a few weeks of collection to ensure
a robust population of microorganisms . we did not sterilize the sludge out of concern that doing so would
alter its properties and affect partitioning results . we seeded both
types of sludge with ms2 or phi6 to achieve a final bacteriophage
concentration of 10 pfu ml . we conducted experiments in triplicate for each combination of
bacteriophage , material , and type of sludge ( 2 4 2 =
16 combinations ) , as illustrated in figure s1 . for each replicate , we filled a container with 10 ml of sludge
seeded with bacteriophage and 40 ml of autoclaved , ultrapure water
to achieve a dilution similar to what might occur with diarrhea in
a toilet . after 510 min , we transferred the fluid , including
suspended solids , to a sterile , 50 ml tube and centrifuged it at 1700
rcf for 3 min . we poured excess fluid off the pelleted solids , briefly
vortexed them , and denoted these as the solid fraction . following established methods for recovery of viruses from surfaces , we collected the surface fraction using three sterile
cotton swabs ( puritan 22029488 ) in series , each premoistened with
sterile lb broth . we swabbed systematically and with constant pressure
over the entire exposed surface area and subsequently eluted virus
from the three swabs into 1 ml of lb broth by compressing and swirling
the swab in a sample tube . the swabbed surface area was 115
cm ( 10% ) for all materials . to prepare samples
for analysis by quantitative polymerase chain
reaction ( qpcr ) , we first converted rna to cdna . immediately upon
separating the fractions , we extracted rna from 140 l subsamples
of each fraction using a qiagen qiaamp viral rna kit . for calibration
standards , we also extracted viral rna from serially diluted stock
suspensions of each bacteriophage , whose concentration ( pfu ml ) was determined by a plaque assay , and carried it
through the entire preparation and analysis process . we omitted carrier rna from the extraction processes to increase
rna extraction efficiency , and we subjected phi6 rna to a postextraction
heat shock treatment ( 110 c , 5 min ) . we immediately synthesized cdna using a bio - rad iscript cdna synthesis
kit with 5 l of template rna , 4 l of iscript 5x , 1 l
of reverse transcriptase , and 10 l of nuclease - free water ,
using the kit s recommended thermocycler protocol . we used
qpcr to quantify virus concentration in each fraction and
in the calibration standards in terms of pfu equivalents per milliliter . we analyzed fractions from synthetic sludge in triplicate ( experimental
replicates ) by intercalating dye - based qpcr using bio - rad sybr green
mastermix with 5 l of sybr green , 400 nm forward and reverse
primers , 2.4 l of nuclease - free water , and
1 l of cdna template . we analyzed fractions from real sludge
in triplicate ( experimental replicates ) by probe - based qpcr using
bio - rad iq supermix with 5 l of iq supermix , 400 nm forward
and reverse primers , 300 nm probe , 1.72
l of nuclease - free water , and 1 l of cdna template . table s3 shows the primers and probes , and table s4 lists the qpcr conditions . for
quality control , we employed serial dilutions of genomically
sequenced calibration standards , triplicate reactions , negative template
controls , and melt curve analysis . the calibration curve covered a
minimum of 6 orders of magnitude to equate qpcr amplification of genomic
copies to the concentration determined by a plaque assay in standards . we prepared serial dilutions of cdna and analyzed all
standards and samples in triplicate ( technical replicates ) on the
qpcr machine ( bio - rad cfx96 real - time system and c1000 thermal cycler ) . additional controls are
described in the supporting information . of the 144 individual experimental replicates , we discarded four
that failed to amplify . we reviewed preliminary qpcr results using
the machine s software to verify the calibration curve and
melt curve , including quantification cycle results . melt curve analysis
showed a single , well - defined peak at the expected melt temperature
for the genome . we spiked
ms2 , an unenveloped bacteriophage , and phi6 , an enveloped
bacteriophage , into diluted synthetic and real sludge in porcelain ,
concrete , pvc , and polypropylene containers and measured partitioning
among the liquid , solids , and the interior surface of the container . figures 1 and 2 show the concentrations of ms2 and phi6 , respectively , in
terms of pfu equivalents in three experimental replicates of each
fraction derived from diluted synthetic sludge . the three fractions
were 46 ml of liquid , 4 ml of wetted solids , and 1 ml of surface extract . ms2 and phi6 partitioned similarly in synthetic sludge ; concentrations
were highest in the liquid and solid phases for all materials tested ,
with the exception of phi6 in porcelain , where phi6 concentrations
were comparable in all three fractions . mean concentrations of ms2
in each fraction were within 1 order of magnitude of each other , while
mean concentrations of phi6 varied by up to 2 orders of magnitude . background concentrations of genomic material were detectable in both
types of sludge , particularly for phi6 , but were at least 4 orders
of magnitude lower than in the seeded sludge . concentration of ms2
bacteriophage , determined by qpcr and reported
in pfu equivalents per milliliter , in synthetic sludge diluted 5-fold
in water in each of three phases : 46 ml of liquid , 4 ml of wetted
solids , and 1 ml of material surface extract for porcelain , concrete ,
pvc , and polypropylene . concentration of phi6 bacteriophage , determined
by qpcr and reported
in pfu equivalents per milliliter , in synthetic sludge diluted 5-fold
in water in each of three phases : 46 ml of liquid , 4 ml of wetted
solids , and 1 ml of material surface extract for porcelain , concrete ,
pvc , and polypropylene . figures 3 and 4 show the concentrations
of ms2 and phi6 , respectively ,
in terms of pfu equivalents in three experimental replicates of each
fraction derived from diluted , real , anaerobically digested sludge . the variability was much larger in real sludge than in synthetic sludge ,
likely due to heterogeneities in the real sludge . the average concentration
of ms2 in the solid fraction was 2 orders of magnitude lower
than in the other fractions for all materials except concrete . average
concentrations of ms2 were highest in the liquid fraction for concrete ,
porcelain , and pvc ( figure 3 ) . in polypropylene , the average concentrations of ms2 in the liquid
and surface extract from concrete were of a similar order of magnitude ;
however , the surface samples exhibited much larger variability . average
concentrations of phi6 in real , anaerobically digested sludge were
highest in the liquid fraction for all materials . average concentrations
were 12 orders of magnitude lower in the surface extract
and were 24 orders of magnitude lower in the wetted solids
( figure 4 ) . concentration
of ms2 bacteriophage , determined by qpcr and reported
in pfu equivalents per milliliter , in anaerobically digested sludge
diluted 5-fold in water in each of three phases : 46 ml of liquid ,
4 ml of wetted solids , and 1 ml of material surface extract for porcelain ,
concrete , pvc , and polypropylene . concentration of phi6 bacteriophage , determined
by qpcr and reported
in pfu equivalents per milliliter , in anaerobically digested sludge
diluted 5-fold in water in each of three phases : 46 ml of liquid ,
4 ml of wetted solids , and 1 ml of material surface extract for porcelain ,
concrete , pvc , and polypropylene . each point represents an experimental
replicate , and there are three nearly overlapping points for solids
in concrete . a mass balance indicated
that the sum of virions recovered ( pfus )
from all three fractions was roughly within 1 order of magnitude of
the number of virions initially spiked into the samples ( table s5 ) , so the amount of virus recovered was
close to the amount spiked into the samples . one exception was phi6
in real sludge , for which the total amount of virus recovered was
23 orders of magnitude higher , on average , than the number
of virions spiked into the samples for all four material types . a
negative control consisting of sludge that was not spiked with virus
failed to amplify , it appears that phi6 replicated rapidly in host bacteria present in
the sludge during the experiment , and thus , the partitioning results
represent both spiked and new virus . figure s2 shows the partitioning of
ms2 and phi6 among the three fractions for synthetic and real sludge
and all four materials . in all cases , at least 94% of the virions
partitioned into the liquid fraction . in synthetic sludge , up to 4%
of virions partitioned to the solids , while in real sludge , no more
than 0.8% of virions partitioned to the solids . in synthetic sludge ,
partitioning to the material surface was low , at most 1% except for
phi6 in porcelain , where 6% of virions partitioned to the surface .
in real sludge , partitioning to the material surface was also low ,
although both ms2 and phi6 partitioned more to the surface of concrete
and polypropylene than to the surface of porcelain and pvc this study demonstrates that partitioning of viruses in wastewater
among the liquid , biosolids , and material surface does not mitigate
the potential for aerosolization of virus , as most of the virus remains
in the liquid phase . airborne viruses have been detected at wastewater
treatment facilities at concentrations as high as 3 10 genome copies m for adenovirus . previous studies have shown that virus removal efficiency in wastewater
treatment plants ranges widely from 0 to 4 logs . in contrast
to our results , some of these studies have shown that viruses adsorb
well to solids . our results are consistent with the general finding
that the extent of removal of bacteria is greater than the extent
of removal of virus in conventional treatment plants . with respect
to concerns about transmission of ebola virus , one
limitation of this study is that the surrogate viruses may not be
representative of ebola virus . its filamentous shape may make it more
or less prone to adsorb to solids and surfaces . another limitation
is that the time allowed for partitioning was only 510 min ,
which could be representative of the time excreta remains in a toilet
before flushing but is much shorter than the residence time in sewers
and at wastewater treatment plants . we have identified five
potential sources of uncertainty in quantification
of virus concentrations . although our qpcr calibration curves had
high r values , the differing matrices
of the three fractions may have produced varying extraction and amplification
efficiencies . it is possible that genomic material from inactivated
virus and/or exogenous genomic material was counted as pfu equivalents . inhibitors are a challenge for any pcr analysis and if severe will
result in false negatives or underestimates of gene copy levels . in
this study , we expect that any effects of inhibitors would have been
minimal , as we were operating at the mid to upper end of the calibration
curve and did not have difficulty detecting virus in any of the samples .
also , the mass balances did not suggest a major loss of virus . because
of requirements of the experimental approach , the surface fraction
also included any virions present in the residual liquid that remained
in the bowls after pouring out the contents ; allowing the bowls to
dry out completely prior to swabbing previous studies have shown that the swabbing method recovers
758% of viruses from dry environmental surfaces , depending
on the type of swab , virus , surface material , and virus assay ( culture
vs molecular ) . thus , there is the possibility
for both overestimation and underestimation of the amount of virus
that partitioned to porcelain , concrete , pvc , and polypropylene in
this study . the mass balance ( table s5 )
suggests that large amounts of virus were not lost during the experiment ,
but even if the actual numbers of virions on surfaces were 10 times
higher , the majority of virions would still be associated with the
liquid phase . | to gain insight into the potential
for aerosolization of viruses
in wastewater systems , we investigated the partitioning of ms2 and
phi6 bacteriophages in synthetic sludge and anaerobically digested
sludge from a wastewater treatment plant .
we evaluated partitioning
among the liquid , solids , and material surfaces of porcelain , concrete ,
polyvinyl chloride ( pvc ) , and polypropylene . in all cases , at least
94% of the virions partitioned into the liquid fraction . in real sludge ,
no more than 0.8% of virions partitioned to the solids and no more
than 6% to the material surface .
both ms2 and phi6 partitioned more
to the surface of concrete and polypropylene than to the surface of
porcelain or pvc .
partitioning of viruses in wastewater among the
liquid , biosolids , and material surface does not appear to mitigate
the potential for aerosolization of virus , as most of the virus remains
in the liquid phase . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
nonpharmacological interventions to support children and adults with asthma include self - management education and support , information giving , behavioral change techniques , and efforts to enhance communication between the person with asthma and health care professionals . these diverse interventions have been provided by an equally diverse range of individuals from physicians to nurses , multidisciplinary teams , pharmacists , and lay educators . this review set out to determine the type of interventions offered by various professionals and what type of outcomes they achieve . in the past decade , the quality and quantity of asthma educational and behavioral interventions have increased , probably as a result of high asthma prevalence , advances in the understanding of clinical management and management by patients , and increasing interest in theories of education and behavior change.13 educational and behavioral programs evaluated in clinical trials have been delivered by a variety of health professionals and lay people . rigorously evaluated interventions have aimed at achieving a variety of outcomes and have used a variety of program components . the financial and material costs of delivering interventions for asthma , although not always discussed in reports of findings , can be expected to differ , at least , according to whose time must be covered to implement the program . theoretically , different types of program providers may differ in their ability to produce desired outcomes . for example , it may be that clinicians could be expected to be more able to address correct use of medical regimens and lay persons more able to advise regarding day - to - day barriers to managing asthma effectively . this review of interventions aimed at ( a ) describing the outcomes of clinical trials of asthma educational and behavioral interventions undertaken by different types of providers in the past decade and ( b ) exploring differences in program components employed by them . articles appearing in the english language in the cochrane collaboration data base , medline , pubmed , and google scholar were searched . search terms included asthma self - management , asthma behavior , asthma randomized controlled trials , asthma outcomes , asthma education , and asthma patient education . inclusion criteria were publication in a peer - reviewed journal between 1990 and 2009 ; randomized clinical trial ( rct ) to assess an educational or behavioral intervention for asthma ; and evidence of statistical assessment of asthma - related outcomes on at least one variable including asthma symptoms , pulmonary function , medicine use , psychosocial factors , days absent from work or school , days of restricted activity due to asthma , self - management , self - efficacy , quality of life , emergency department use , hospital in - patient stays , and office visits . success in achieving outcomes was accepted as statistical difference between interventions and control in a patient sample of at least 100 subjects . virtually no study provided sample size calculations , and as asthma outcomes related to health care use generally require larger samples , 100 was considered a generous cut point . the initial search was broad , accepting any article related to evaluation of social and behavioral interventions to ensure a comprehensive view of available work , and generated 1650 articles . preliminary application of study criteria identified 249 potential studies for inclusion that met one or more criteria . further review of these investigations by two independent reviewers yielded 50 rcts that fully met all inclusion criteria . no further review of methodological quality of the studies was conducted beyond that it appeared in a peer review journal and comprised an rct . the 50 eligible articles were again closely examined by two individuals and data extracted using a standard protocol regarding target population , sample size , program provider , program content , intervention components , processes , and outcomes . comparison among provider type was computation of differences between percent of successful program to number attempted . the majority of the 50 rct evaluated programs were conducted by teams of providers ( n = 20 ) and the least by pharmacists ( n = 4 ) . just above 28% were offered to adults with asthma , just under 65% were for children , and 7% included both . table 1 illustrates that among the most frequently studied outcomes ( health care use , symptoms , self - management / self - efficacy , and quality of life ) , health care use was the outcome most frequently reported . in the majority of studies , no delineation was made as to which were primary versus secondary goals of the research . a number of studies described more than one outcome resulting from the program , and not all reported about the same or included all the major outcomes . considering the number of interventions undertaken by type of provider and the number reporting success achieving health care use reductions , physicians had a 83% success rate ( ie , the percent of times positive health care reduction outcomes were reported given the number of studies by that type of provider ) , nurses reported success in 73% of their undertakings , pharmacists reported no success , multidisciplinary teams reported 50% success , and lay people 35% success . for reports of symptom reduction , reported success for physicians was 33% , nurses 36% , pharmacists 50% , multidisciplinary teams 51% , and lay people 11% . multidisciplinary teams reported achieving quality of life outcomes in 50% of the studied programs and lay persons self - management and/or self - efficacy outcomes in 33% of programs . auxiliary outcomes of a more mediating or psychosocial type beyond the most frequently reported major outcomes were described in some studies . the most frequently reported outcome of a mediating or psychosocial type was use of medicines and delivery devices . table 4 presents the components and processes of the interventions by provider type and outcome . program approaches varied from providing highly specific asthma information along with specialist consultations , for example , levy et al,14 to enhancing patient clinician interactions including emphasis on communication , for example , cabana et al,4 to paying indirect attention to asthma in literacy education , for example , robinson et al.32 table 5 presents program focus , content , and processes by outcome . patient communication , self - management skills , control of the environment , and medicine and device use were all employed in programs that reduced health care use . action plans , peak expiratory flow ( pef ) monitoring , control of the environment , and clinician patient communication skills were employed in interventions that reduced asthma symptoms . clinician communication and patient asthma self - management education were included in interventions improving quality of life and self - management outcomes . two areas of focus , interactions between patients and clinicians and patient education for self - management , were evident in all interventions reporting major outcomes , that is , those related to health care use , symptoms , self - management / self - efficacy , or quality of life . physician - directed programs emphasized one - on - one counseling , self - monitoring , and use of diaries / action plans . nurses used individual , group , and telephone learning sessions and employed activities to elicit patient participation such as role plays and problem - solving exercises . teams used a range of these activities and , in addition , case managers . lay people - led programs involved individual , group , and home visit sessions and use of peer educators findings from this review of asthma interventions demonstrate that several types of providers have led programs assessed through rcts using various program components and reporting varying results . no rationale was provided in research reports for why given program planners sought to emphasize certain outcomes and not others or included certain program components and not others or deployed certain program providers and not others . there is a degree of consistency in outcomes achieved across the interventions as measured by frequency of reports of reaching a category of major outcome . almost half of the interventions achieved reductions in health care use and about one - third reduced frequencies of asthma symptoms . so many have demonstrated symptom or health care use improvements that these may have become the unofficial bench mark of success . patient communication and patient self - management may be the most promising to include in efforts to change health care use and reduce asthma symptoms as these elements have been included in all programs to date reporting such outcomes . a number of studies have described only outcomes related to self - efficacy , medicine use , school / work absenteeism , feelings about asthma , etc . some , in fact , may be the mediating factors producing what we have termed major outcomes . the frequency with which these auxiliary results have been sought and achieved has been less than attempts to achieve change in symptoms , health care use , self - management / self - efficacy , and quality of life . these more distal outcomes have likely been assumed by program planners to be associated with major outcomes . however , in other words , support for these being the sole outcome sought and achieved in interventions , until they are proven to be the route to clinical changes , is questionable . important considerations regarding the type of program leader and interventions themselves could not be addressed in this exploration . for example , the relative costs of delivering a program and the cost of training different types of individuals to lead programs differ . physician time is usually expensive whether providing an intervention solo or as part of a team . peer leaders may be the least expensive in implementation but not in training and needed backup support . a program with many components may be the most powerful or as this study suggests one or two very effective elements may produce the best results . for example , many trials involving teams have been conducted , while only four concern pharmacists . exclusion of studies of fewer than 100 subjects may have worked against some studies where sample size recalculation would indicate smaller numbers could ascertain differences . studies of teams of providers did not describe fully the relative roles of team members or assess which provider had the most influence on success . no multifactorial research designs were used in the studies included here to uncover which element or combination of elements in the intervention produced the outcome . thus , our findings may be subject to publication bias . in one study , for example , griffiths et al24 not all patients had asthma and the whole may not reflect subgroup differences . a few studies focused on specific ethnic / racial groups ( eg , african - american , chinese , south asian ) , but no comparison between approaches for differing ethnicities was available . as components of interventions may have differing effects on subgroups of the population , comparative effective studies appear needed . further , investigations in this review comprise those targeted at children , at adults , and sometimes both . the relative advantages of approaches identified here for younger and older patients were not clear in the available data and deserve attention in future studies . how , by necessity , we have looked at the extant studies that also reflect weaknesses in the field more generally . for example , measures used to assess asthma outcomes are not standard and/or are not applied in a standard way . the rationale and/or theory underlying the components of an intervention were not described in study reports inhibiting theoretical conclusions regarding why an intervention may or may not have worked . descriptions of the organizational context for program delivery , or success in institutionalizing an effective intervention , were not presented , so characteristics of sustainability or longevity of programs can not be assessed . nonetheless , the findings from this review are instructive concerning the current situation regarding the type of providers and components of interventions apparently associated with specific asthma outcomes . a number of recommendations are evident in the results of this review . one , as noted , is the need for standard asthma outcome measures and uniform application of them . new efforts by the us national heart , lung , and blood institute and a joint committee of the european respiratory association and american thoracic society to identify and assess the validity and reliability of asthma outcome measures should help in this regard.60 another is to consider health care use and symptom reduction as the gold standards of intervention success . if programs do not , at minimum , achieve these results , their added value and a strong rationale for their association with clinical or quality of life improvements would appear to be needed . also needed as part of standard practice in program planning is a clearer rationale for selection of a ) intended outcomes b ) program provider selected to pursue the outcomes , and c ) the program components included to achieve it . specific intervention studies are needed that evaluate the comparative effectiveness of programs as provided by one type of health professional versus another . the only such study identified in this review was one by partridge et al,47 where lay providers were compared to nurse program providers . the relative advantage of different providers appears to have important implications for both the type of outcomes achieved and the frequency of achieving them , as well as , cost of program implementation . an implication of these findings is that those with a specific professional background may benefit from adopting the techniques successfully used by other professionals . multifactorial studies are needed to compare program components for their relative effectiveness in producing outcomes . needed personnel , supervision , as well as , intensity and duration evident in the interventions studied varied greatly . research is needed to examine the costs of program delivery against the savings generated by outcomes . cost pressures in most health care systems make acquisition of this information necessary to ensure adoption and institutionalization of interventions that can assist patients to reduce the burden of asthma on them , their families , and their communities . in the past decade , multidisciplinary teams have been the most frequent providers of asthma educational and behavioral interventions . two elements , self - management skills and physician - patient communication , have been the program components most deployed by providers successfully reaching these outcomes . apparent emerging gold standards for asthma interventions are outcomes related to reductions in symptoms and/or health care use . outcomes produced by different program components and different providers vary with some having more success with clinically related results and some with more potentially mediating psychosocial - related results . comparative effectiveness studies are needed to assess outcomes associated with different program providers and program components . | objectives : randomized clinical trial ( rct ) data reviewed for outcomes and processes associated with asthma educational and behavioral interventions provided by different types of health professionals.methods:cochrane collaboration , medline , pubmed , google scholar search from 1998 to 2009 identified 1650 articles regarding asthma educational and behavioral interventions resulting in 249 potential studies and following assessment produced a final sample of 50 rcts.results:approaches , intended outcomes , and program providers vary greatly . no rationale provided in study reports for the selection of specific outcomes , program providers , or program components .
health care utilization and symptom control have been the most common outcomes assessed .
specific providers favor particular teaching approaches .
multidisciplinary teams have been the most frequent providers of asthma interventions .
physician - led interventions were most successful for outcomes related to the use of health care .
multidisciplinary teams were best in achieving symptom reduction and quality of life .
lay persons were best in achieving self - management / self - efficacy outcomes .
components most frequently employed in successful programs are skills to improve patient
clinician communication and education to enhance patient self - management .
fifty percent of interventions achieved reduction in the use of health care and one - third in symptom control . a combination approach including self - management and patient clinician communication involving multidisciplinary team members may have the greatest effect on most outcomes.conclusions:the extent to which and how different providers achieve asthma outcomes through educational and behavioral interventions is emerging from recent studies .
health care use and symptom control are evolving as the gold standard for intervention outcomes .
development of self - management and clinician patient communication skills are program components associated with success across outcomes and providers . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
chemical contents of goat milk are different from cow milk , in which the total fat ( 3.8% ) , protein ( 3.4% ) , mineral and vitamin ( vitamin a , b , b12 and d ) is higher than cow milk(1 , 2 , 3 , 4 ) . besides that , goat milk is easier to digest in human digestion systems than cow milk ( 2 , 5 ) . goat milk is the milk proteins that are the main source of bioactive peptides ( 6 , 7 ) . a bioactive peptide from milk protein was activated and released through the proteolysis enzymatic process during food digestion and processing on gastrointestinal ( 8 , 9 ) . a protein on milk could divide into two different groups , soluble protein ( whey protein ) and insoluble protein ( casein ) at isoelectrical point ( pi ) and in milk caseins are soluble as micelles ( 10 ) . whey protein is contained of -lactoalbumin and -lactoglobulin , while casein are contains of s1- , s2- , - , and -casein ( 11 , 12 ) . milk protein and whey from goat milk is a precursor of bioactive components that contribute to antimicrobial activity with a wide spectrum of bacterial pathogen types of food materials ( 13 , 14 , 15 ) . as many studies was made in order to analyze the ability of bioactive peptide from milk protein on inhibition or destruction of pathogen ( 16 , 17 , 18 , 19 ) . previous study , reported that fresh milk and yoghurt from goat ethawah crossbreed had specific protein with molecular weight 36kda , in which this specific protein did not find in fresh cow milk ( 20 ) . this protein was identified by matrix - assisted laser desorption / ionization time - of - flight ( maldi - tof ) analysis ( 21 ) as protein -s2 casein ( csn1s2 ) contents eight bioactive peptides that each peptide has specific biological function in difference mechanism as anti - oxidant ( 22 ) anti - osteoporosis , and anti - inflamation ( 23 , 24 ) . we predict that some peptides of goat csn1s2 protein may able to inhibit the pathogen bacteria growth as anti - microbial agent . research related with antimicrobial activity for alpha casein from goat milk is relatively less than other casein components . herein , we reported the effect of antimicrobial activity from csn1s2 protein as a member of casein protein from ethawah breed goat milk and yoghurt against pathogen bacteria in some gram negative and positive . gram positive bacteria ( listeria monocytogenes , staphylococcus aureus and bacillus cereus ) and gram negative bacteria ( escherichia coli , salmonella typhi and shigella flexneri ) was obtained from the medical faculty , brawijaya university type culture collection . all bacteria were maintained on luria bertani broth agar plates ( sigma - aldrich , usa ) . a csn1s2 protein with molecular weight 36kda was isolated from ethawah goat milk and yoghurt using sodium dodecyl sulphate- polyacrylamide gel electrophoresis ( sds - page ) according to the previous study ( 20 ) . diffusion agar analysis for antimicrobial activity was carried out with some modification ( 25 ) . the isolated pathogenic bacteria were grown in luria berthani ( lb ) broth at 37c for 24 hours and adjusted approximately to a density of 1.510 cfu / ml and then each pathogen bacteria were then transferred to luria berthani agar with cotton swab . well was made on agar for seeded 200 l of sample csn1s2 protein and yoghurt was added to each well . the plates were then incubated aerobically at 37c for 24 h and 48 h. the diameters of the clear zones ( in the top agar layer ) were measured to the nearest mm . the determination of effect of the antimicrobial peptide csn1s2 protein from goat milk and yoghurt ethawah on microbial cells were performed with minor modification ( 25 ) . luria berthani ( lb ) broth ( containing 10 g tryptone , 5 g yeast extract , 10 g sodium chloride per liter , ph 7.2 at 37 c ) was carried out in serial dilution test tube preparation . serial dilutions of the two csn1s2 protein samples were made in luria berthani ( lb ) broth medium containing 1 ml to give a final concentration of 1.25 , 2.5 and 5 mg / ml . briefly , tubes were seeded 20 l of the test organisms with approximately 110 cfu / ml using early log - phase culture , 1.510 cfu / ml using middle log - phase culture , and 310 cfu / ml using end log - phase culture . negative controls tubes contained csn1s2 protein milk or yoghurt . the determination microbial count was employed in triplicate samples plated onto lb agar and incubated at 37 c for 24 h. data analysis results were expressed as means . the data was variance analyzed ( anova ) statistically with spss 16.0 software ( spss inc . , gram positive bacteria ( listeria monocytogenes , staphylococcus aureus and bacillus cereus ) and gram negative bacteria ( escherichia coli , salmonella typhi and shigella flexneri ) was obtained from the medical faculty , brawijaya university type culture collection . all bacteria were maintained on luria bertani broth agar plates ( sigma - aldrich , usa ) . a csn1s2 protein with molecular weight 36kda was isolated from ethawah goat milk and yoghurt using sodium dodecyl sulphate- polyacrylamide gel electrophoresis ( sds - page ) according to the previous study ( 20 ) . diffusion agar analysis for antimicrobial activity was carried out with some modification ( 25 ) . the isolated pathogenic bacteria were grown in luria berthani ( lb ) broth at 37c for 24 hours and adjusted approximately to a density of 1.510 cfu / ml and then each pathogen bacteria were then transferred to luria berthani agar with cotton swab . well was made on agar for seeded 200 l of sample csn1s2 protein and yoghurt was added to each well . the plates were then incubated aerobically at 37c for 24 h and 48 h. the diameters of the clear zones ( in the top agar layer ) were measured to the nearest mm . the determination of effect of the antimicrobial peptide csn1s2 protein from goat milk and yoghurt ethawah on microbial cells were performed with minor modification ( 25 ) . luria berthani ( lb ) broth ( containing 10 g tryptone , 5 g yeast extract , 10 g sodium chloride per liter , ph 7.2 at 37 c ) was carried out in serial dilution test tube preparation . serial dilutions of the two csn1s2 protein samples were made in luria berthani ( lb ) broth medium containing 1 ml to give a final concentration of 1.25 , 2.5 and 5 mg / ml . briefly , tubes were seeded 20 l of the test organisms with approximately 110 cfu / ml using early log - phase culture , 1.510 cfu / ml using middle log - phase culture , and 310 cfu / ml using end log - phase culture . negative controls tubes contained csn1s2 protein milk or yoghurt . the determination microbial count was employed in triplicate samples plated onto lb agar and incubated at 37 c for 24 h. data analysis results were expressed as means . the data was variance analyzed ( anova ) statistically with spss 16.0 software ( spss inc . , effect of antimicrobial peptides on the inhibition of bacterial growth base of clear zone analysis qualitative antimicrobial activity analysis was done using the agar diffusion methods of measuring the diameter of the clear zone ( figure 1 ) . treatment uses csn1s2 from milk , both in 24 and 48 hours incubation , was showed highest inhibition at concentration 5 mg / ml in almost all bacteria comparable to the other concentrations . b. cereus was obtained the highest inhibition with clear zone diameter 13.01 + 1.25 mm and 11.96 + 0.81 mm in 24 and 48 hours incubation , respectively . the lowest inhibition at concentration 5 mg / ml in 24 and 48 hours incubation was obtained in two different bacteria , s. typhi ( 2.91 + 0.35 mm ) and l. monocytogenes ( 0.51 + 0.16 mm ) , respectively . ( b ) inhibition zone diameter of goat milk csn1s2 against pathogen bacteria ( p<0.05 ) . a , b , c , d the mean value is indicate a significant difference ( p<0.05 ) . although the highest inhibition was obtain by different bacteria compare to the csn1s2 from milk , but in term of concentration , similar result was obtained by treatment using csn1s2 from yoghurt . incubation for 24 hours at concentration 5 mg / ml was resulted the highest inhibition in s. flexneri with clear zone diameter 9.73 + 0.22 mm . there were no significant different between s. flexneri and e. coli in 48 hours incubation , both were resulted the highest inhibition . in contrast with result from treatment milk csn1s2 , although the lowest inhibition was obtained also from s. typhi and l. monocytogenes , but with different incubation time , 48 and 24 hours respectively . effect of antimicrobial peptides on the microbial cell pathogen numbers , during exponential growth phase : early , middle , and late . antimicrobial activity of csn1s2 from goat ethawah breed milk and yoghurt in all concentrations to gram positive and negative bacteria at three different growth phases ( early , middle , and late exponential ) were shown various results ( figure 2 , table 1 ) . in all phases , for treatment using milk , most of the bacteria were showed significant decreasing , especially at concentration 5 mg / ml compared to control and other treatments , except for s. typhi . although s. typhi was decreased significantly at concentration 1.25 mg / ml , but the number was almost did not . values are presented as mean amount of microbial cells , indicated a significant different ( p < 0.05 ) compared with control group all bacterial species . antimicrobial activity of csn1s2 goat milk and goat yoghurt against microbial cells of listeria monocytogenes , staphylococcus aureus , bacillus cereus , escherichia coli , salmonella typhi and shigella flexneri at ( a ) early exponential growth phase , ( b ) . a , b , c , d the mean value is indicate a significant difference ( p<0.05 ) . antimicrobial activity of csn1s2 goat milk and goat yoghurt against microbial pathogen cells at early , middle and end exponential growth phase change significantly in other following concentrations , especially in the early and late phase . the highest decreasing for gram positive and negative bacteria at early , middle and late phase were obtained from b. cereus ( 3.4e+03 ) and s. flexneri ( 4.57e+03 ) ; l. monocytogenes ( 2.55e+03 ) and s. flexneri ( 3.35e+03 ) ; and b. cereus ( 4.78e+03 ) and s. flexneri ( 3.13e+03 ) , respectively . similar with milk , in all phases , the highest decreasing for treatment using yoghurt were also resulted at concentration 5 mg / ml . although , almost all bacteria were decreased significantly at concentration 5 mg / ml compare to control and other treatments , but except for e. coli in the late phase . there was no significant difference on e. coli between concentration 2.5 and 5 mg / ml treatment . the highest decreasing for gram positive and negative bacteria at early , middle and late phase for treatment using yoghurt were obtained from b. cereus ( 2.02e+03 ) and s. flexneri ( 5.35e+03 ) ; l. monocytogenes ( 3.32e+03 ) and s. typhi ( 3.88e+03 ) ; and l. monocytogenes ( 3.82e+03 ) and s. typhi ( 5.73e+03 ) , respectively . there are very weak for diameter < 5 mm ; weak for diameter > 5 mm ; intermediate for diameter 5 - 10 mm ; strong for diameter > 10 - 20 mm ; and very strong or the most sensitive for diameter > 20 - 30 mm ( 26 ) . based on those criteria , the strong inhibition level was obtained in treatment of csn1s2 from milk at concentration 5 mg / ml on b. cereus and s. flexneri . vice versa , in treatment of csn1s2 from yoghurt at same concentration was resulted only a intermediate inhibition level . the antimicrobial activity of csn1s2 from milk and yoghurt in this study could be categorized as bacteriostatic , because at least in part , the growth inhibition was observed in all pathogen bacteria . bacteriostatic activity can be shown by the slow growth activity when the treatment was applied . in contrast , bactericidal activity can be shown by no growth activity after treatment ( 4 , 27 , 28 ) . effects of milk protein to the bacteria in middle exponential phase having antimicrobial activity with high sensitivity when compared to the right when approaching or on the stationary phase ( 29 , 30 , 31 ) . milk protein effect toward bacteria on middle exponential phase has a high sensitivity of antimicrobial activity when compared to early or exact stationary phase ( 31 ) . based on those two analyses , antimicrobial activity of csn1s2 from milk and yoghurt was showing a higher activity on gram positive bacteria comparable to gram negative bacteria . the outer membrane of gram negative bacteria was consist of lipopolysaccharides , in which this molecule when binding to the peptide could decrease the peptide affinity and in turn will decrease the antimicrobial activity toward this bacteria ( 32 , 33 ) . basically , our study was showing a higher potential inhibition by csn1s2 from milk than yoghurt in all pathogenic bacteria . this result was almost similar to previous studies , ( 34 , 35 ) it reported that whey protein from goat and cow milk was showed antimicrobial activity on several pathogen bacteria . at least in part , from our study we could conclude that antimicrobial activity of csn1s2 from milk and yoghurt was based on dose dependent manner , higher concentration will resulted higher antimicrobial activity . in our study , the optimum concentration was obtained at 5 mg / ml . compare to csn1s2 from yoghurt , milk had a higher antimicrobial activity . and overall , our result was showed the higher inhibition on gram positive bacteria than gram negative bacteria . | background : goat milk is reported to have antimicrobial activity of several pathogen bacteria that contained on food materials .
the research related with antimicrobial activity of alpha - s2 casein from goat milk is relatively less than other casein components .
herein , we reported the antimicrobial activity of caprine alpha - s2 casein ( csn1s2 ) protein from ethawah breed goat milk and yoghurt in gram positive ( listeria monocytogenes , staphylococcus aureus and bacillus cereus ) and negative pathogen bacteria ( escherichia coli , salmonella typhi and shigella flexneri ) . those bacteria were known as pathogens that caused gastrointestinal infection.methods:serial dilution and agar diffusion analysis with three different concentrations of caprine csn1s2 , 1.25 mg / ml , 2.5 mg / ml , and 5 mg / ml were used to test the inhibition effect of protein on the viability of bacteria cells .
the inhibitory activity of caprine csn1s2 was based on dose dependent manner .
agar diffusion analysis was showed the larger diameter of clear zone at b. cereus and s. flexneri.results:the serial dilution analysis was shown the inhibition of almost in all groups of bacteria with concentration 5 mg / ml higher by csn1s2 protein of goat fresh milk than yogurt .
the inhibitory activity caprine csn1s2 protein of fresh milk was shown a vary inhibition clear zone with optimal concentration 5 mg / ml , however csn1s2 protein of goat yogurt intermediate effectively was only in gram negative bacteria .
the weakness bacteria against inhibition activity caprine csn1s2 protein was b. cereus ( gram positive ) and s. flexneri ( gram negative ) .
meanwhile the strongest bacteria against inhibition activity caprine csn1s2 protein was s. typhi ( gram negative ) , may cause in this bacteria has lipopolysaccharide prevent to interact with that protein as proper.conclusion:this study result concluded that the caprine csn1s2 protein has inhibition activity in opposition to pathogenic bacteria by optimal concentration 5 mg / ml in all bacteria and indicated caprine csn1s2 protein as anti - microbial agent . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
levobupivacaine is a frequently used local anesthetic ( la ) due to its longer sensory block , lower cardiac toxicity , and shorter motor block properties . the addition of opioids to la spinal anesthesia increases anesthesia quality and ensures effective analgesia during intraoperative and early postoperative periods . for this reason , however , these agents may cause dose - dependent side effects on fetal heart rate in newborns such as bradycardia , as well as various side effects in the mother such as maternal hypotension , pruritus , nausea , vomiting , and respiratory depression . the doses of fentanyl and sufentanil that would provide effective analgesia and minimum side effects were reported to be 10 to 25 g and 2.5 to 5 g , respectively . furthermore , the equivalent dose range between fentanyl and sufentanil during intrathecal administration was identified as 3.4 to 4.5:1 . although there are studies in the literature comparing the effectiveness of fentanyl and sufentanil added as adjuvants to 0.5% levobupivacaine during spinal anesthesia , there is no consensus regarding which agents would be more effective . in our prospective , randomized , double - blind , controlled study , our aim was to compare the effect of adding low - dose fentanyl ( 10 g ) or sufentanil ( 2.5 g ) to 0.5% levobupivacaine ( 2.2 0.2 ml ) on anesthesia quality , block characteristics , side effects , duration of postoperative analgesia , and surgeon satisfaction score . ka08/48 ) and receipt of written informed consents from patients , 93 pregnant women older than age 18 years without fetal distress / anomaly ( ie , gestational pregnancy age 36 weeks , height 155 cm , weight 110 kg , and fetal weight 2500 g ) and with american society of anesthesiologists physical status of i to ii who were undergoing elective caesarean section were enrolled in the study . patients with allergies to any local anesthetic , with a history of hypersensitivity and anaphylactic reactions , who were taken to emergency surgery , and who had pre - eclampsia were excluded . following monitoring with noninvasive blood pressure , electrocardiogram , and pulse oxymeter in all patients , ringer s lactate solution ( 15 ml / kg ) was administered for 10 to 15 minutes . spinal anesthesia was performed in the sitting position with midline approach at the l3l4 intervertebral space by an anesthesiologist blinded to the drug injected . the drug syringes were prepared before injection by another anesthesiologist who was not involved in the study . intraoperative and postoperative assessments were performed by an anesthesiologist blinded to patient allocations and study drugs . the study drugs included 2.2 ml ( 11 mg ) levobupivacaine ( chirocaine ; abbott laboratories , abbott park , illinois ) administered to patients with height < 163 cm , whereas 2.4 ml ( 12 mg ) levobupivacaine was administered to patients with height 163 cm . in addition , 2.2 0.2 ml 0.5% levobupivacaine plus 10 g fentanyl ( fentanyl citrate ampule 50 g / ml ; johnson & johnson , new brunswick , new jersey ) was administered to group f and 2.2 0.2 ml levobupivacaine plus 2.5 g sufentanil ( sufenta ; johnson & johnson , new brunswick , new jersey ) was administered to group s at rate of 3 ml/30 sec and completed to a volume of 3 ml with the addition of saline in all groups . following intrathecal administration , patients were placed in the supine position and their heads were slightly elevated . patients were then directed to a left lateral position with a 15 to 20 angle to prevent aortocaval compression , and oxygen ( at 24 l / min ) was provided with a facemask . sensory block was evaluated every 2 minutes with a pinprick test ; motor block was evaluated with the bromage scale ( 0 = no motor loss , 1 = inability to flex the hip , 2 = inability to flex the knee , and 3 = inability to flex the ankle ) . if sensory block did not achieve the t5 level within 20 minutes , general anesthesia was administered . onset time of sensory block at the l1-t10 level , the highest level of sensory block , duration of sensory block , time for 2-segment regression of sensory block through the t10 level , and the onset and duration of motor block were recorded . systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) , heart rate ( hr ) , and oxygen saturation were evaluated every 3 minutes during the first 15 minutes , and every 5 minutes afterward . if sbp values decreased > 20% with respect to the baseline values , or decreased to < 100 mm hg , fluid loading and ephedrine ( 5 mg ) were administered . a decrease in heart rate to < 55 beats / min was considered bradycardia and atropine ( 0.5 mg ) was administered . a decrease in the respiratory rate to < 10/min , and a decrease in oxygen saturation to < 90% were considered indications of respiratory depression . apgar scores were evaluated at first and fifth minutes by a pediatrician with no information regarding the groups . for blood gas measurements intraoperative pain evaluation was performed with the visual analog scale ( vas ) ( 0 = no pain and 10 = worst pain possible ) while performing surgical incision , uterine incision , and skin closure . in cases where vas > 3 , iv fentanyl ( 50 g ) was administered . sedation levels were monitored , and propofol ( up to 0.5 mg / kg ) was administered if patients had still discomfort . when patients required > 50 g fentanyl and/or 0.5 mg / kg propofol , the block was considered unsuccessful and general anesthesia was administered . intraoperative and postoperative nausea , vomiting , pruritus , respiratory depression , and other side effects were recorded at the first , second , sixth , and 12th hours . metoclopramide ( 10 mg iv ) was administered for nausea , and diphenhydramine ( 25 mg ) was administered for severe pruritus . surgeon satisfaction score was evaluated according to the sufficiency of muscle relaxation and the provision of adequate surgical conditions ( 0 = poor , 1 = fair , 2 = good , and 3 = excellent ) . the time of first analgesic requirement was recorded for the patients , and pethidine hydrochloride ( 50 mg i m ) was administered as the first analgesic choice when vas score was > 3 and additional analgesic requirements were satisfied with diclofenac sodium ( 75 mg i m ) . the total amount of diclofenac sodium required during the first 24 hours was also recorded . data analysis was performed using spss ( version 17.0 , ibm - spss inc , armonk , new york ) . the mean duration of sensory block was assumed to be 140 34.4 minutes based on a previous study , in which a group of patients received fentanyl . a power analysis indicated that 25 patients per group were required to detect an increase in duration of sensory block by 20% . we allowed for 6 more patients in each group to compensate for drop - outs during the study period . categorical measurements were recorded as numbers and percentages , continuous measurements as mean ( sd ) , and also the median ( minimum maximum ) where necessary . in the comparison of categorical variables , the anova test was used for distributions in the comparison of continuous measurements between the groups , whereas the kruskal - wallis test was used for parameters without normal distribution . the error was set at 0.05 , type ii error was set at 0.20 , and a p value < 0.05 was considered significant for all comparisons . no significant intergroup differences were identified with regard to individual characteristics , the duration of surgery , and total fluid use ( table i ) . all groups had a significant sbp and dbp decrease with respect to the baseline values ( p = 0.001 and p = 0.029 , respectively ) during the third minute , with the highest decrease in group s , and the lowest decrease in group c ( figure 2 ) . the number of patients requiring ephedrine , and the administered ephedrine doses were similar between all groups ( table i ) . in group s , 4 patients were treated with atropine due to bradycardia ( p < 0.05 ) , and hr values were significantly higher than the basal values at the sixth , ninth , and 12th minutes ( p = 0.007 , p = 0.007 , and p = 0.004 , respectively ) . hr values did not differ in comparison to baseline values in group f and group c. oxygen saturation values were similar between groups and above 90% at all times . the onset time of sensory block , the onset time of the block reached the t10 level the highest sensory block level and the onset time of motor block were significantly longer in group c ( p < 0.001 ) , whereas no significant differences were identified between group s and group f. the highest block level ( t4 ) and the duration of motor block was similar in all 3 groups , and no significant difference was observed between groups with regard to the time of 2-segment regression of the sensory block . the time of regression of the sensory block to the t10 level and the duration of sensory block was significantly longer for group s ( p = 0.003 and p < 0.001 , respectively ) ( table ii ) . vas values for surgical incision , uterus incision , and skin closure , as well as intravenous fentanyl demand and sedation requirement were significantly higher in group c ( p < 0.05 for each comparisons ) ( table iii ) . the time to first analgesic requirement was significantly longer and the analgesic demand during the first 24 hours was significantly lower in group s ( p < 0.001 ) ( table ii ) . apgar scores and umbilical venous blood gas values ( ph , oxygen partial pressure , carbon dioxide partial pressure , and base excess ) were similar between groups and within normal ranges ( table iv ) . during the intraoperative period , all groups were similar with regard to the frequency of complications . postoperatively , the frequency of pruritus was significantly higher in group s for the first 2 hours ( p < 0.001 ) . one patient in group s had pruritus and eruption on the legs , whereas 1 patient in group c developed lumbar pain . however , no further complications were observed in the patient follow - ups ( table v ) . surgeon satisfaction score was statistically lower in group c in comparison to the other groups ( p = 0.003 ) ( figure 3 ) . spinal anesthesia is preferred over epidural anesthesia due to its rapid onset , the higher level of muscle relaxation , and the lower dose requirement of la during caesarean section . it ensures reliable and good quality of block for both the mother and the newborn . in our study , the duration of sensory block and first analgesic requirement were longer and analgesic requirement during the 24-hour period was lower in group s , and sufficient intraoperative anesthesia could not be ensured . surgeon satisfaction score was insufficient in all groups , being the lowest in the control group . in caesarean section surgeries performed under spinal anesthesia , it has been reported that the administration of la alone has a short duration of effect , that it is insufficient for preventing visceral pain and nausea especially during uterus manipulation and peritoneum closure , and that it leads to postoperative analgesic requirement at an earlier stage . the most frequently used agent among intrathecal opioids is fentanyl , which was demonstrated to be effective for 180 to 240 minutes when administered at doses of 10 to 25 g . sufentanil is also a lipophilic opioid ; it has been reported that its onset of effect is more rapid , and that its duration of effect is 25% longer than fentanyl . due to the rapid onset of its effects , as well as the effective postoperative analgesia that it ensures , sufentanil is an alternative to fentanyl and other opioids , and its use is becoming increasingly more common in caesarean section surgeries . in our study , we also aimed to determine the effectiveness of sufentanil . the selection of different combinations and suitable doses when using opioids with las requires a careful consideration of factors such as the formation of sensory and motor block , the quality and duration of postoperative analgesia , and the side effects that might be observed in the mother and the newborn . in previous studies , the fact that different results were reported in normal individuals despite the use of similar dosages is associated with the differing distribution of the drugs during pregnancy . for caesarean section surgeries , the minimum intrathecal dose was reported as 10.58 mg levobupivacaine , 6.25 g fentanyl , and 1.5 to 2.5 g sufentanil . we have also used a low - dose la and adjuvant opioid in our study . in vercauteren et al an intrathecally administered 2 ml 0.125% levobupivacaine plus 1.5 g sufentanil plus 2.5 g epinephrine combination was used for delivery analgesia , and reported onset times for sensory block ( 4.4 minutes ) were longer than the times obtained for the sufentanil and fentanyl groups in our study . this can be explained with the higher sufentanil concentrations and doses in our study . with a combination of 8 mg levobupivacaine plus 2.5 g sufentanil , gautier et al reported that the mean time to the highest level of sensory block was 17 minutes . in our study , this time was determined as 8.54 minutes with the levobupivacaine plus sufentanil combination , 7.52 minutes with the levobupivacaine plus fentanyl combination , and 12.7 minutes with the levobupivacaine only group . the shorter times in our study might be associated with the higher levobupivacaine doses . in studies where intrathecal levobupivacaine was used alone , motor block onset times were reported as 10.0 minutes by glaser et al and as 15 minutes by burke et al . in our study , the addition of opioids to la decreased onset times of motor block for levobupivacaine ( group s 3.75 minutes , group f 3.0 minutes , and group c 10.0 minutes ) . in a study comparing the effects of fentanyl addition ( 10 , 15 , or 25 g ) to intrathecal levobupivacaine ( 5 , 7.5 , or 10 mg ) on sensory and motor blocks , the time to maximum motor block was reported as being shorter in the 10 mg levobupivacaine plus fentanyl group . similarly in our study , motor and sensory block levels increased more rapidly in comparison to the control group ; this effect was probably associated with the lipophilic character of sufentanil and fentanyl . in studies comparing sufentanil with fentanyl , longer spinal anesthesia durations have been reported with sufentanil . in the study by dahlgren et al , where 2.5 or 5 g sufentanil and10 g fentanyl was added to bupivacaine , and in the study by meininger et al , where 2.5 or 5 g sufentanil and10 g fentanyl was added to 60 mg 2% mepivacaine , effective spinal analgesia durations were found to be higher in sufentanil groups in comparison to fentanyl and placebo groups . in our study , the duration of sensory block was , in agreement with the literature , significantly longer in the sufentanil group . the fact that this duration was shorter in the fentanyl group compared with the sufentanil group ( 179 minutes vs 211 minutes ) is also consistent with other studies in which intrathecal 10 g fentanyl was added to treatment . in our study , the durations of motor block were found to be shorter than the duration of the sensory block . continuation of postoperative analgesia despite an early end to the motor block significantly extended the time to the first analgesic requirement in the sufentanil group . for spinal anesthesia performed during caesarean sections , gautier et al reported the time to first analgesic requirement as 136 minutes for a 8 mg levobupivacaine plus 2.5 g sufentanil combination . this duration was longer in our study , and was identified as 219 minutes for the sufentanil group . this difference might be due to the higher dose of levobupivacaine used in our study . in a study where fentanyl and sufentanil were added to bupivacaine , dahlgren et al reported intraoperative intravenous fentanyl requirement for 1 patient in the 5 g sufentanil group . in their study where 20 g fentanyl , 2.5 g sufentanil , or saline was added to 1.5 to 2.4 ml 0.5% bupivacaine , lee et al reported that intraoperative intravenous fentanyl administration was only required by 22 of 24 patients in the control group . intraoperative pain and discomfort caused by separation of the rectus muscle and stretching of the peritoneum also necessitated the use of additional analgesia and sedation in our study . during uterus incision and skin closure , 22% of control patients required intravenous fentanyl use . despite higher vas values in the sufentanil group that were not statistically significant , 19% of patients required intravenous fentanyl . intraoperative analgesia was best ensured in the group with fentanyl added to levobupivacaine , with none of the patients requiring any iv fentanyl for intraoperative analgesia . in our study , the requirement for fentanyl and sedation affected anesthesia quality . when fentanyl and propofol use were considered , it was determined that the quality of anesthesia was lowest in the control group , and was highest in the fentanyl group . this is in agreement with the characteristics of the blocks determined in our study , with inadequate block levels in the control group leading to an increase in both fentanyl and propofol use . this also suggests that the intraoperative administration of intravenous fentanyl may have contributed to the sensory block duration and effective postoperative analgesia in the sufentanil group . methods such as fluid loading , having the patient assume a left lateral position , and using vasoconstrictor agents have been recommended for its prevention . significant levels of hypotension compared with the basal values was observed in all groups at the third minute following the block ; such cases were corrected with rapid administration of fluid loading and ephedrine . in their study comparing addition of 5 g sufentanil and 10 and 20 g fentanyl to10 mg levobupivacaine and10 mg bupivacaine during spinal anesthesia , bremeric et al reported that the hemodynamic data of both groups were similar , with no differences regarding side effects . gunusen et al demonstrated that the addition of 10 g fentanyl to 10 mg levobupivacaine increased the incidence of hypotension , whereas lower doses of levobupivacaine led to insufficient anesthesia and analgesia . in our study , no intergroup differences were observed with regard to intraoperative side effects . pruritus was significantly more frequent in the sufentanil group during the postoperative period , whereas pruritus did not develop in any control group patients . incidence of pruritus among pregnant women with various opioids ( especially lipophilic opioids ) administered intrathecally was reported at between 30% and 95% , although the pruritus was generally transient and mild . in their study performed with varying intrathecal fentanyl doses ( 545 g ) , palmer et al described that pruritus was not dose - dependent , and that it was observed in all patients . in certain studies , intrathecal sufentanil increased the incidence of pruritus in a dose - dependent fashion . in their study adding placebo , 1.5 , 2.5 , or 5 g sufentanil to 0.5% hyperbaric bupivacaine during elective caesarean section surgery , demiraran et al reported that the lowest incidence of pruritus was observed in the group with 1.5 g sufentanil added to their la . similarly in our study , the frequency of pruritus increased in the group with 2.5 g sufentanil combined with their treatment . however , the cases of pruritus were transient , and only 1 patient required treatment . best postoperative analgesia in our study was provided by the addition of sufentanil to levobupivacaine , which affected the use of additional analgesics within the postoperative first 24 hours . it has been reported in the literature that intrathecal doses of 2.5 to 5 g sufentanil reduced the postoperative requirement for analgesia , especially during the first 6 hours . dahlgren et al determined in their study a significantly lower use of additional analgesia in the sufentanil group during the first 24 hours ( especially during the first 6 hours ) . although the additional use of analgesics during the postoperative first 24 hours in our study was significantly lower in the sufentanil group , the use of additional analgesics was highest in the control group . the addition of intrathecal fentanyl or other opioids to la administration during caesarean sections did not affect the apgar scores and newborn blood gas values . it was emphasized that the depth and duration of hypotension could have a considerable influence on hypotension s effect on newborns . no differences were observed between the apgar scores and the gas values of the umbilical blood , and fetal acidosis was not seen in any of the groups . this might be associated with the transient nature of the changes in blood pressure , and also its rapid correction . surgeon satisfaction score in our study was significantly lower in the control group . however , muscle relaxation at surgery site was generally moderate in the other groups , and considered excellent by only 5 patients . sufficient muscle relaxation was not achieved in some patients ; this adversely affected the quality of anesthesia and surgeon satisfaction . a study published in recent years reported a 50% effective dose of 6.2 mg for levobupivacaine , and a 95% effective dose of 12.9 mg . the study described that the addition of 2.5 g sufentanil to the 95% effective dose did not affect hemodynamics , that sufficient block and effective anesthesia and analgesia were achieved , and that lower doses resulted were not effective . this could have let us evaluate the compatibility of patients and surgeons satisfaction scores about the anesthetic technique . our study demonstrated that the addition of sufentanil and fentanyl to intrathecal levobupivacaine during caesarean section surgery is more effective than the administration of levobupivacaine alone . the addition of sufentanil to levobupivacaine allowed rapid onset time for sensory and motor block levels . it also extended the duration of postoperative analgesia , and led to a decrease in total analgesic requirement . however , physicians should keep in mind that it may be insufficient in terms of intraoperative anesthesia quality and sufentanil might invoke perioperative pruritus . the authors have indicated that they have no conflicts of interest regarding the content of this article . | backgroundthe addition of opioids to local anesthetics contributes to the quality of spinal anesthesia and postoperative analgesia.objectivein our prospective , randomized , double - blind , controlled study , our aim was to compare the effect of low - dose sufentanil plus levobupivacaine or a fentanyl plus levobupivacaine mixture on anesthesia quality , block characteristics , newborn and mother well - being , surgeon satisfaction , and duration of postoperative analgesia.methodsninety-three patients were randomized into 3 groups ( n = 31 ) .
patients in group c received 0.5% levobupivacaine ( 2.2 0.2 ml ) , group s received 2.5 g sufentanil plus 0.5% levobupivacaine ( 2.2 0.2 ml ) , and group f received 10 g fentanyl plus 0.5% levobupivacaine ( 2.2 0.2 ml ) intrathecally completed to a volume of 3 ml with the addition of saline in all groups .
patients demographics , sensory and motor block characteristics , hemodynamics , apgar scores , umbilical blood gas values , maternal side effects , surgeon satisfaction score , time to first analgesia requirement , and additional analgesic use within 24 hours were recorded.resultsin group s and group f , target levels of sensory and motor block were achieved more rapidly ( p < 0.001 ) .
the hemodynamic values were lower ( p < 0.05 ) , and the duration of sensory blockade and the time of first analgesic requirement were longer ( p < 0.001 ) in group s. additional analgesic requirement during first 24-hour period was lowest in group s , and highest in group c ( p < 0.001 ) .
apgar scores and umbilical blood gas samples were similar between groups .
postoperative pruritus was more frequent in group s ( p < 0.001 ) and surgeon satisfaction score was significantly lower in group c ( p = 0.003).conclusionswe suggest that the addition of sufentanil and fentanyl to intrathecal levobupivacaine during caesarean section surgery is more effective than the administration of levobupivacaine alone .
the addition of sufentanil to levobupivacaine allowed rapid onset time for sensory and motor block levels .
it also extended the duration of postoperative analgesia and led to a decrease in total analgesic requirement .
clinicaltrials.gov
identifier : nct01858090 . |
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pyomyoma ( suppurative leiomyoma ) is a rare , but life - threatening , condition resulting from infarction and infection of uterine leiomyoma ( 1,2 ) . incidence of pyomyoma has decreased due to the development of antibiotics . since 1945 , only 50 pyomyoma cases have been documented in the literature , with a mortality rate of 6% ( 3/50 ) ( 35 ) . the most likely cause of mortality was delayed and difficult diagnosis . although the triad of pyomyoma are sepsis , leiomyoma , and no other source of infection ( 5 ) , it may present with silent or non - specific symptoms , which results in delayed diagnosis and treatment . visualization of intratumoral gas formation may be suggestive of pyomyoma , but has not been consistently reported in all cases . furthermore , large abdominal complex masses are likely to be first suspected as pelvic malignancies if found incidentally in perimenopausal women . the present study experienced a rare case of large uterine pyomyoma in a perimenopausal woman who presented with anemia gravis , severe inflammatory reaction and cachexia . a total of 50 reported cases of pyomyoma in the literature since 1945 were also studied . a 53-year - old multigravida woman with 7 months of amenorrhea was referred to the department of obstetrics and gynecology ( wakayama medical university , wakayama , japan ) due to gradual abdominal distension starting 2 years previously . the patient exhibited muscle weakness and walking difficulty , but no fever , abdominal pain or metrorrhagia . her abdomen was swollen to the size of a beach ball , with a 126 cm abdominal circumference and body weight of 84.1 kg . an intrauterine device ( iud ) had been inserted following her third birth , and no history of any lower abdominal or pelvic discomfort , leiomyoma , pelvic surgery , or other predisposing factors were known . the present study was unable to determine the uterine cervix with pelvic examination due to its deviation , and was unable to acquire cytopathological findings of the cervix and endometrium . transvaginal and transabdominal ultrasound examinations revealed a large abdominal mass with heterogeneous echogenicity . computed tomography ( ct ) and magnetic resonance imaging ( mri ) revealed a large unilocular mass with an irregular surface and thickened wall , occupying the abdominal cavity without gas formation ( fig . 1 ) . contrast ct revealed an expanded branch of the left internal iliac artery , which was suspected to be the uterine artery , surrounding the mass ( fig . hemoglobin levels were 5.7 g / dl , white blood cell count was 57,300/l and c - reactive protein was elevated to 20.24 mg / dl . cancer antigen ( ca)125 was also elevated to 200 u / ml . blood and vaginal cultures were negative . possible diagnoses of the large abdominal mass included gynecologic tumor types ( benign or malignant ovarian tumor , uterine sarcoma or pyometra ) and gastrointestinal stromal tumor or mucocele of the appendix . gastroscopy revealed no specific findings and colonoscopy revealed difficulty of insertion above the sigmoid colon due to pressure from the mass . due to the finding of the uterine artery by contrast ct , the origin of the mass was suspected to be the uterus . following antibiotic medication and blood transfusion , the patient underwent a total abdominal hysterectomy and bilateral salpingo - oophorectomy . intraoperative findings demonstrated a solid tumor arising from the back of the uterine body with normal bilateral adnexa . prior to removal , 12.4 liters of purulent , malodorous fluid was drained from the tumor ( fig . the surrounding myometrium appeared normal and measured 3 cm in thickness , and the cut surface of the mass was purple - yellowish ( fig . 2c ) . the pathological diagnosis was leiomyoma with marked necrosis and chronic inflammation , with no evidence of malignancy ( fig . the post - operative course was uneventful and the patient was discharged from the hospital on post - operative day 14 . at 4 months follow - up , the patient 's weight had increased to 62 kg due to a good appetite . pyomyoma occurs in both post - and pre - menopausal women , however , the risk of suppurative myoma is increased by pregnancy ( 1,2 ) . for post - menopausal patients , systemic vascular changes necrosis of the leiomyoma caused by vascular flow insufficiency in the uterus following menopause is also a possible cause . a history of uterine leiomyoma , pregnancy , abortion , menopause , uterine artery embolization ( uae ) , iud , vascular insufficiency ( diabetes , hypertension and atherosclerosis ) and systemic disease or infection may be predisposing factors for pyomyoma ; however , definitive diagnoses remain difficult . although rapid clinical diagnosis for pyomyoma is often difficult due to its low incidence and the requirement to rule out the possibility of malignancy , mortality has decreased due to the improvement in surgical treatments , including myomectomy and hysterectomy , and broad - spectrum antibiotics . a medline search since 1945 revealed only 50 reported cases of pyomyoma : 27 were non - pregnant woman ( mean age , 51.8 years ; range , 3669-years - old ; table i ) ( 329 ) and 23 cases were associated with pregnancy or abortion ( mean age , 33.6 years ; range , 2844-years - old ) . the mean pyomyoma size in non - pregnancy and pregnancy - associated cases were 16.2 cm ( 338 cm ) and 13.5 cm ( 558 cm ) , respectively . non - pregnancy pyomyoma tended to be larger compared with pregnancy - associated pyomyoma , and the present case was the largest among all reported cases of non - pregnancy - associated pyomyoma . severe anemia gravis and inflammatory reaction were described in numerous pyomyoma cases , including the present study . it has been shown that 2/50 cases had a history of iud usage ( 7,30 ) , 5/50 cases had uae ( 812 ) , 6/50 cases had vascular insufficiency ( 4,1317 ) and 8/50 cases demonstrated gas production ( 6,810,12,3133 ) . although pyomyoma arises spontaneously , post - partum , post - instrumentation or post - surgery have been reported in the literature , only two cases include a iud , and only one case has been previously reported in a non - pregnant woman ( 7 ) . infection by staphylococcus species was reported in 8 cases , streptococcus species in 7 cases , escherichia coli in 6 cases , enterococcus faecalis in 5 cases , clostridium species in 3 cases , proteus species in 2 cases and candida species in 2 cases . in the present case , streptococcus agalactiae was cultured from the pus and tumor , however , not from the blood and vagina , suggesting the infection existed focally within the tumor . therefore , conservative management with pre - operative broad - spectrum antibiotics and appropriate surgery was performed successfully . for surgical treatment , hysterectomy was performed in 32 cases , myomectomy in 10 cases and drainage in 6 cases . diagnosis of large pyomyoma is difficult since surgery is required for a definitive diagnosis . a malignant tumor , in particular ovarian cancer , was initially suspected due to the findings of a large abdominal mass with signs of necrosis in a cachexic perimenopausal woman with an elevated ca125 level . in a previous report , imaging analysis was shown to identify only non - specific results ( 6 ) . although intratumoral gas formation is markedly suggestive of pyomyoma on ultrasound and ct imaging , gas formation is not consistently observed , as with the present case . in the present case , contrast ct contributed to the diagnosis of pyomyoma due to the identification of the uterine artery location . ca125 has been observed to be increased in other gynecologic and non - gynecologic malignancies , as well as in a variety of benign disorders , including leiomyoma . among the previously reported cases of pyomyoma , 5 cases reported ca125 levels ( 6,7,14,15,18 ) , with a mean level of 56.3 u / ml ( 29.98109.7 u / ml ) . ca125 was measured only in non - pregnancy - associated cases , possibly due to suspicion of gynecological malignancy based on the age of the patient and size of tumor . furthermore , in pregnancy - associated cases , diagnosis of myoma was likely during pre - natal examination prior to the onset of the symptom , which may have contributed to the identification of the tumor origin . in the present case , to the best of our knowledge , this is the third iud - associated case of pyomyoma , with the largest tumor size and highest ca125 level compared with previously reported non - pregnancy cases . a large pyomyoma is difficult to distinguish from a gynecological malignant tumor , particularly in perimenopausal , cachectic women with non - specific clinical presentation and without a history of leiomyoma . a history of iud usage and contrast ct may be helpful in the diagnosis of pyomyoma . gynecologists and general surgeons must be aware of the possibility of pyomyoma when presented with a large abdominal tumor , and in certain cases , surgery in combination with broad spectrum antibiotics may result in a good outcome . | pyomyoma is a rare complication , which withoug antibiotics or surgical intervention , may cause sepsis and mortality . the present study reported a case of large uterine pyomyoma in a perimenopausal female .
a 53-year - old multigravida woman was referred to the department of obstetrics and gynecology ( wakayama medical university , wakayama , japan ) due to progressive abdominal distension . the patient presented with anemia gravis , severe inflammatory reaction and cachexia .
computed tomography revealed a large unilocular mass , 50 cm in size , with an irregular surface and thickened wall , occupying the entire abdomen .
following antibiotic medication , the patient underwent a total abdominal hysterectomy and bilateral salpingo - oophorectomy .
intraoperative findings demonstrated a solid tumor arising from the back of the uterine body .
a total of 12 liters of purulent , malodorous fluid was drained from the tumor .
the resected mass was 50 cm in size and 13.5 kg in weight .
cultures of the pus revealed the presence of streptococcus agalactiae .
pathological findings revealed suppurative leiomyoma with no malignancy .
large pyomyoma is difficult to distinguish from a gynecological malignant tumor types , particularly in perimenopausal women with non - specific clinical presentation .
although pyomyoma is a benign tumor , care must be taken to discriminate these from large abdominal tumors . |
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worldwide , hepatocellular carcinoma ( hcc ) is the fifth most common malignancy and the third most common cause of cancer - related death . liver cancer is a major health problem and its incidence is increasing in china , although several trials have attempted to control its progression . furthermore , conventional treatments such as chemotherapy and radiotherapy can increase the risk of potential complications . hence , herbal medicines have attracted considerable interest as a novel anticarcinogenic agent because of their high efficacy , low toxicity and costs . radix astragali is a key herb in many traditional formulations in china , which was recorded more than 2000 years ago in sheng nong 's materia medica . in recent years , astragalus injection ( ai ) , as an extract from radix astragali , has been extensively used for clinical adjunctive therapy to ameliorate the side effects of antineoplastic drugs . several clinical studies showed that the active constituents of ai , including polysaccharides , astragalosides and total flavonoids could ameliorate the side effects of chemotherapeutic drugs . recently , there has been growing evidence that the astragalus extract may be a potential anti - tumorigenic agent . for instance , astragalus polysaccharides can prevent hepatocarcinogenesis in rats . on the other hand , the total saponins of radix astragali ( ast ) also exhibited anti - carcinogenic effects in ht-29 human colon cancer cells . however , the precise anti - carcinogenic effect and mechanism of ai remains unclear . in the present study , we aimed to examine the effect of ai on the growth of h22 transplanted tumor in mice , and to further elaborate its growth - inhibitory mechanism . our key findings demonstrate that ai exerts inhibitory effects on hcc cells in vivo by inducing cell apoptosis . these results suggest that ai is an attractive candidate drug to prevent the tumor proliferation . astragalus injection was obtained from diao pharmaceutical co. , ltd ( chengdu , china ) . cyclophosphamide ( ctx ) , ( jiangsu hengrui medicine co. , ltd , batch number - 08122621 ) was dissolved in distilled water . kunming male mice , five - week old , weighing 18~22 g , were obtained from the experimental animal center at sichuan university . the mice were housed in plastic cages with hardwood chip bedding in an air - conditioned room at 232c and 555% humidity , and with a 12 h light / dark cycle on basal diet ( animal center ) . the animal handlings and experimental procedures were approved by the animal ethics committee of sichuan university ( permit number : 2003 - 149 ) . five - week old male kunming mice were inoculated with h22 cell suspension ( 110/ml ) , 0.1 ml/10 g , through subcutaneous injection at right side axilla . twenty - four hour after the tumor cell inoculation , the mice were randomized equally into 5 groups of 15 each . the minimum effective dose of astragalus in treating hepatocellular carcinoma was 4 g / kg . according to the method of prasad et al . the negative control group mice were injected with an equal volume of normal saline ( ns ) while mice in other three groups were given ai ( i.p . , 12 , 8 , 4 g / kg ) once every alternate day from day 1 ( six times totally ) . from the fourth day , tumor volume ( v ) was measured on alternate days , and calculated according to the formula : v ( cm ) = ab/2 , where a is largest superficial diameter and b is smallest superficial diameter . all mice were sacrificed 13 days after inoculation with h22 cells , and the transplanted tumors were excised and weighed . to evaluate the anticancer activity of astragalus , tumor inhibitory rates were calculated with the following formula : tumor inhibitory rate ( % ) = 1-(tumor weight of treated group/ tumor weight of control group ) 100 . apoptotic rate was assessed by flow cytometry ( fcm)after staining with propidium iodide ( pi ) and annexin vfitc ( sigma , st . briefly , tumor tissues were immediately removed and the rbcs were lysed using an ammonium chloride lysis buffer and filtered through a double layer of stainless - steel mesh using a syringe plunger to obtain single cell suspension . 1 10 cells per sample were incubated in 1 binding buffer containing annexin v - fitc and pi . data were analyzed with flow cytometry software ( beckman coulter ) and the gating included all cells . sections were cleared of paraffin , and endogenous peroxidases were blocked by incubation with 3% h2o2 and washed . sections of the tissues were then incubated with rabbit serum for 10 min at ambient temperature . subsequently , the sections were incubated overnight with a goat polyclonal anti - bax antibody ( boster , china , 1:50 ) and anti - bcl-2 antibody ( boster , china , 1:50 ) at 4c , followed by the addition of biotinylated rabbit anti - goat igg secondary antibody ( jinshan , bj , china ) . to verify the binding specificity for bax and bcl-2 , some sections were also incubated with primary antibody or the secondary antibody . in these situations , no positive staining was found in the sections , indicating that the immunoreactions were positive in all the experiments . immunohistochemistry staining was processed in accordance with the manufacturer 's instructions and visualized by use of diaminobenzidine ( dab ) staining . samples were observed in four randomly selected optical fields under microscopy ( 200 ) and the average optical density was analyzed with image - pro plus ( media cybernetics , usa ) by an observer blind to the treatment groups . the data were expressed as meanstandard deviation ( sd ) . the statistical package for social sciences version 13.0 ( spss inc , chicago , il ) was used for standard statistical analysis including one - way anova and student 's t - test . a value of p < 0.05 was considered as statistically significant . astragalus injection was obtained from diao pharmaceutical co. , ltd ( chengdu , china ) . cyclophosphamide ( ctx ) , ( jiangsu hengrui medicine co. , ltd , batch number - 08122621 ) was dissolved in distilled water . kunming male mice , five - week old , weighing 18~22 g , were obtained from the experimental animal center at sichuan university . the mice were housed in plastic cages with hardwood chip bedding in an air - conditioned room at 232c and 555% humidity , and with a 12 h light / dark cycle on basal diet ( animal center ) . the animal handlings and experimental procedures were approved by the animal ethics committee of sichuan university ( permit number : 2003 - 149 ) . five - week old male kunming mice were inoculated with h22 cell suspension ( 110/ml ) , 0.1 ml/10 g , through subcutaneous injection at right side axilla . twenty - four hour after the tumor cell inoculation , the mice were randomized equally into 5 groups of 15 each . the minimum effective dose of astragalus in treating hepatocellular carcinoma was 4 g / kg . according to the method of prasad et al . the negative control group mice were injected with an equal volume of normal saline ( ns ) while mice in other three groups were given ai ( i.p . , 12 , 8 , 4 g / kg ) once every alternate day from day 1 ( six times totally ) . from the fourth day , tumor volume ( v ) was measured on alternate days , and calculated according to the formula : v ( cm ) = ab/2 , where a is largest superficial diameter and b is smallest superficial diameter . all mice were sacrificed 13 days after inoculation with h22 cells , and the transplanted tumors were excised and weighed . to evaluate the anticancer activity of astragalus , tumor inhibitory rates were calculated with the following formula : tumor inhibitory rate ( % ) = 1-(tumor weight of treated group/ tumor weight of control group ) 100 . apoptotic rate was assessed by flow cytometry ( fcm)after staining with propidium iodide ( pi ) and annexin vfitc ( sigma , st . briefly , tumor tissues were immediately removed and the rbcs were lysed using an ammonium chloride lysis buffer and filtered through a double layer of stainless - steel mesh using a syringe plunger to obtain single cell suspension . 1 10 cells per sample were incubated in 1 binding buffer containing annexin v - fitc and pi . data were analyzed with flow cytometry software ( beckman coulter ) and the gating included all cells . sections were cleared of paraffin , and endogenous peroxidases were blocked by incubation with 3% h2o2 and washed . sections of the tissues were then incubated with rabbit serum for 10 min at ambient temperature . subsequently , the sections were incubated overnight with a goat polyclonal anti - bax antibody ( boster , china , 1:50 ) and anti - bcl-2 antibody ( boster , china , 1:50 ) at 4c , followed by the addition of biotinylated rabbit anti - goat igg secondary antibody ( jinshan , bj , china ) . to verify the binding specificity for bax and bcl-2 , some sections were also incubated with primary antibody or the secondary antibody . in these situations , no positive staining was found in the sections , indicating that the immunoreactions were positive in all the experiments . immunohistochemistry staining was processed in accordance with the manufacturer 's instructions and visualized by use of diaminobenzidine ( dab ) staining . samples were observed in four randomly selected optical fields under microscopy ( 200 ) and the average optical density was analyzed with image - pro plus ( media cybernetics , usa ) by an observer blind to the treatment groups . the data were expressed as meanstandard deviation ( sd ) . the statistical package for social sciences version 13.0 ( spss inc , chicago , il ) was used for standard statistical analysis including one - way anova and student 's t - test . a value of p < 0.05 was considered as statistically significant . astragalus injection was obtained from diao pharmaceutical co. , ltd ( chengdu , china ) . cyclophosphamide ( ctx ) , ( jiangsu hengrui medicine co. , ltd , batch number - 08122621 ) was dissolved in distilled water . kunming male mice , five - week old , weighing 18~22 g , were obtained from the experimental animal center at sichuan university . the mice were housed in plastic cages with hardwood chip bedding in an air - conditioned room at 232c and 555% humidity , and with a 12 h light / dark cycle on basal diet ( animal center ) . the animal handlings and experimental procedures were approved by the animal ethics committee of sichuan university ( permit number : 2003 - 149 ) . five - week old male kunming mice were inoculated with h22 cell suspension ( 110/ml ) , 0.1 ml/10 g , through subcutaneous injection at right side axilla . twenty - four hour after the tumor cell inoculation , the mice were randomized equally into 5 groups of 15 each . the minimum effective dose of astragalus in treating hepatocellular carcinoma was 4 g / kg . according to the method of prasad et al . the negative control group mice were injected with an equal volume of normal saline ( ns ) while mice in other three groups were given ai ( i.p . , 12 , 8 , 4 g / kg ) once every alternate day from day 1 ( six times totally ) . from the fourth day , tumor volume ( v ) was measured on alternate days , and calculated according to the formula : v ( cm ) = ab/2 , where a is largest superficial diameter and b is smallest superficial diameter . all mice were sacrificed 13 days after inoculation with h22 cells , and the transplanted tumors were excised and weighed . to evaluate the anticancer activity of astragalus , tumor inhibitory rates were calculated with the following formula : tumor inhibitory rate ( % ) = 1-(tumor weight of treated group/ tumor weight of control group ) 100 . apoptotic rate was assessed by flow cytometry ( fcm)after staining with propidium iodide ( pi ) and annexin vfitc ( sigma , st . briefly , tumor tissues were immediately removed and the rbcs were lysed using an ammonium chloride lysis buffer and filtered through a double layer of stainless - steel mesh using a syringe plunger to obtain single cell suspension . 1 10 cells per sample were incubated in 1 binding buffer containing annexin v - fitc and pi . data were analyzed with flow cytometry software ( beckman coulter ) and the gating included all cells . sections were cleared of paraffin , and endogenous peroxidases were blocked by incubation with 3% h2o2 and washed . sections of the tissues were then incubated with rabbit serum for 10 min at ambient temperature . subsequently , the sections were incubated overnight with a goat polyclonal anti - bax antibody ( boster , china , 1:50 ) and anti - bcl-2 antibody ( boster , china , 1:50 ) at 4c , followed by the addition of biotinylated rabbit anti - goat igg secondary antibody ( jinshan , bj , china ) . to verify the binding specificity for bax and bcl-2 , some sections were also incubated with primary antibody or the secondary antibody . in these situations , no positive staining was found in the sections , indicating that the immunoreactions were positive in all the experiments . immunohistochemistry staining was processed in accordance with the manufacturer 's instructions and visualized by use of diaminobenzidine ( dab ) staining . samples were observed in four randomly selected optical fields under microscopy ( 200 ) and the average optical density was analyzed with image - pro plus ( media cybernetics , usa ) by an observer blind to the treatment groups . the data were expressed as meanstandard deviation ( sd ) . the statistical package for social sciences version 13.0 ( spss inc , chicago , il ) was used for standard statistical analysis including one - way anova and student 's t - test . a value of p < 0.05 was considered as statistically significant . to determine whether astragalus inhibits tumor growth in vivo , h22 cells were injected into kunming mice . growth curve of hepatocarcinoma h22 showed that astragalus inhibited tumor growth significantly in a dose- and time - dependent manner from 4 to 12 g / kg [ figure 1 ] . treated with astragalus , the tumor inhibition rates ranged from 17.28 to 52.36% [ table 1 ] . effects of different dosages of astragalus injection and cyclophosphamide ( ctx ) on tumor growth in h22 bearing mice . * p < 0.05 vs control inhibitory effect of astragalus injection on h22 bearing mice after astragalus treatment for 10 days , a significantly high rate of apoptosis was found , compared with saline controls in hepatocarcinoma h22 bearing mice [ figure 2 ] . apoptosis rates of h22 transplanted tumor cells , values are shown as mean sd ( n=5 ) . * p < 0.05 vs control . ctx = cyclophosphamide ( 60 mg / kg ) ; aih = astragalus injection ( 12g / kg ) ; aim = astragalus injection ( 8 g / kg ) ; ail = astragalus injection ( 4 g / kg ) . to test the effect of ai on the regulation of apoptosis gene , figure 3 shows that the protein levels of bcl-2 and bax protein changed in astragalus treated h22 bearing mice.ai treatment decreased the expression of anti - apoptotic protein bcl-2 compared with the control ( p < 0.05 ) . the expression of the pro - apoptotic protein bax was increased significantly by ai treatment in a dose - dependent manner as compared with control ( p < 0.05 ) . a marked enhancement of the bax / bcl-2 ratio was observed after ai treatment [ figure 3 ] . expression of bax and bcl-2 proteins in transplanted tumor cells of h22 bearing mice ( a ) representative immunostaining for bax proteins in h22 bearing mice ( 200 . * p < 0.05 vs control . ctx = cyclophosphamide ( 60 mg / kg ) ; aih = astragalus injection ( 12 g / kg ) ; aim = astragalus injection ( 8 g / kg ) ; ail = astragalus injection ( 4 g / kg ) . to determine whether astragalus inhibits tumor growth in vivo , h22 cells were injected into kunming mice . growth curve of hepatocarcinoma h22 showed that astragalus inhibited tumor growth significantly in a dose- and time - dependent manner from 4 to 12 g / kg [ figure 1 ] . treated with astragalus , the tumor inhibition rates ranged from 17.28 to 52.36% [ table 1 ] . effects of different dosages of astragalus injection and cyclophosphamide ( ctx ) on tumor growth in h22 bearing mice . * p < 0.05 vs control inhibitory effect of astragalus injection on h22 bearing mice after astragalus treatment for 10 days , a significantly high rate of apoptosis was found , compared with saline controls in hepatocarcinoma h22 bearing mice [ figure 2 ] . apoptosis rates of h22 transplanted tumor cells , values are shown as mean sd ( n=5 ) . * p < 0.05 vs control . ctx = cyclophosphamide ( 60 mg / kg ) ; aih = astragalus injection ( 12g / kg ) ; aim = astragalus injection ( 8 g / kg ) ; ail = astragalus injection ( 4 g / kg ) . to test the effect of ai on the regulation of apoptosis gene , figure 3 shows that the protein levels of bcl-2 and bax protein changed in astragalus treated h22 bearing mice.ai treatment decreased the expression of anti - apoptotic protein bcl-2 compared with the control ( p < 0.05 ) . the expression of the pro - apoptotic protein bax was increased significantly by ai treatment in a dose - dependent manner as compared with control ( p < 0.05 ) . a marked enhancement of the bax / bcl-2 ratio was observed after ai treatment [ figure 3 ] . expression of bax and bcl-2 proteins in transplanted tumor cells of h22 bearing mice ( a ) representative immunostaining for bax proteins in h22 bearing mice ( 200 . * p < 0.05 vs control . ctx = cyclophosphamide ( 60 mg / kg ) ; aih = astragalus injection ( 12 g / kg ) ; aim = astragalus injection ( 8 g / kg ) ; ail = astragalus injection ( 4 g / kg ) . to determine whether astragalus inhibits tumor growth in vivo , h22 cells were injected into kunming mice . growth curve of hepatocarcinoma h22 showed that astragalus inhibited tumor growth significantly in a dose- and time - dependent manner from 4 to 12 g / kg [ figure 1 ] . treated with astragalus , the tumor inhibition rates ranged from 17.28 to 52.36% [ table 1 ] . effects of different dosages of astragalus injection and cyclophosphamide ( ctx ) on tumor growth in h22 bearing mice . * after astragalus treatment for 10 days , a significantly high rate of apoptosis was found , compared with saline controls in hepatocarcinoma h22 bearing mice [ figure 2 ] . apoptosis rates of h22 transplanted tumor cells , values are shown as mean sd ( n=5 ) . * p < 0.05 vs control . ctx = cyclophosphamide ( 60 mg / kg ) ; aih = astragalus injection ( 12g / kg ) ; aim = astragalus injection ( 8 g / kg ) ; ail = astragalus injection ( 4 g / kg ) . to test the effect of ai on the regulation of apoptosis gene , immunohistochemistry staining was performed for bcl-2 and bax protein . figure 3 shows that the protein levels of bcl-2 and bax protein changed in astragalus treated h22 bearing mice.ai treatment decreased the expression of anti - apoptotic protein bcl-2 compared with the control ( p < 0.05 ) . the expression of the pro - apoptotic protein bax was increased significantly by ai treatment in a dose - dependent manner as compared with control ( p < 0.05 ) . a marked enhancement of the bax / bcl-2 ratio was observed after ai treatment [ figure 3 ] . expression of bax and bcl-2 proteins in transplanted tumor cells of h22 bearing mice ( a ) representative immunostaining for bax proteins in h22 bearing mice ( 200 . values are shown as meansd ( n=10 ) . * p < 0.05 vs control . ctx = cyclophosphamide ( 60 mg / kg ) ; aih = astragalus injection ( 12 g / kg ) ; aim = astragalus injection ( 8 g / kg ) ; ail = astragalus injection ( 4 g / kg ) . apoptosis , the program of cellular suicide , is critical in tumorigenesis and tumor progression . thus , targeting apoptosis is considered as an effective therapeutic strategy for treatment of cancer . astragalus , a traditional chinese medicine , has so far been used as a modulating agent for all kinds of immunological diseases and in combination with cytotoxic drugs to ameliorate their side effects . recently , it has been demonstrated that the anti - tumor mechanism of astragalus may be an induction of the apoptosis of cancer cell.[1214 ] however , there has been little scientific advancement with regard to the effect of astragalus on cancer . among the effective components of astragalus mongholicus , total astragalus saponins ( ast ) possess anti - tumor properties on human colon cancer cells and human hepatocellular hepg2 cell line by inducing apoptosis in vitro . our study demonstrated that both the weight and volume of tumor were inhibited by astragalus in a dose- and time - dependent manner from 4 to 12 g / kg . the apoptosis rate also exhibited a dose- and time - dependence . to obtain further information regarding apoptotic modulation , we have examined the immunoreactivity of bax and bcl-2 proteins in h22 transplanted tumors . bcl-2 and bax function as tumor anti - apoptotic and pro - apoptotic factors and are the two main members of the bcl-2 protein family , which might interact with each other to form homodimers and heterodimers in regulating apoptosis . therefore , alteration in the levels of bax and bcl-2 protein influences apoptosis and the ratio between them is the key to determine the cell survival or death . in our study , we showed the astragalus injection induced apoptosis in h22 transplanted tumors and it was accompanied by up - regulation of bax and the down - regulation of bcl-2 . these results indicate that imbalance of bcl-2 family protein expression plays a critical role in this anti - tumor treatment . in the present study , the antitumor effect and the preliminary growth - inhibitory mechanism of astragalus injection on h22 bearing mice were investigated . from the results , it is concluded that astragalus injection can inhibit tumor growth by inducing apoptosis via up - regulating bax expression and the ratio of bax / bcl-2 and down - regulating bcl-2 expression . | objective : the objective of the present study is to investigate the anti - proliferation activity of astragalus on human hepatocellular carcinoma ( hcc ) cells and its mechanism.materials and methods : hepatic cancer h22 bearing mice were used to study the anti - hepatocarcinoma activity of astragalus in vivo .
the growth curve and inhibitory rate of tumor growth were measured .
cell apoptosis of each group was measured by flow cytometry ( fcm ) .
protein expression of bax and bcl-2 were analyzed by immunohistochemistry ( ihc ) .
the statistical package for social sciences version 13.0 ( spss inc , chicago , il ) was used for standard statistical analysis including one - way anova and student 's t - test .
a value of p<0.05 was considered to be statistically significant.results:astragalus significantly inhibited the growth of h22 carcinoma , with an inhibitory rate of 17.28 - 52.36% .
fcm and immunohistochemical assay show that the cell apoptosis rate and protein expression of bax and bax / bcl-2 ratio of h22 transplanted tumor in astragalus treated group were significantly higher than the control group ( p<0.05 ) .
the protein expression of bcl-2 was significantly lower than control ( p<0.05).conclusion : the results of the present study suggest that astragalus has significant anti - tumor effect in vivo in inducing apoptosis of h22 tumor cells by promoting protein expression of bax , decreasing protein expression of bcl-2 gene , and markedly increasing the bax / bcl-2 ratio . |
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acromegaly is a rare disease , and in the large majority of cases , is caused by a growth hormone - producing pituitary microadenoma or macroadenoma . the resulting excess growth hormone secretion affects multiple organ systems , and biochemical disease control is essential in order to reduce the mortality to the normal level of the background population.1 the severity and complexity of the disease warrant that all patients be offered lifelong follow - up at highly specialized centers.2 denmark is privileged in having several national medical registries and databases through which routine collection of relevant health - related data is secured for administrative ( eg , reimbursement ) and quality surveillance purposes . these administrative registries enable us to rapidly assemble a cohort for epidemiologic studies and have the additional advantage of reducing the risk of certain types of bias , eg , recall bias or non - responder - related selection bias in the setting of questionnaire - based surveys.3,4 finally , the registries have been shown to be characterized by a high completeness of registrations.4,5 as the quality of data collected for administrative purposes may not automatically translate into equally useful and valid data in a subsequent research setting , validation studies become a prerequisite before considering such use of registry data.5 so far , no studies have provided data regarding the quality of hospital discharge diagnoses of acromegaly . we therefore aimed to validate the international classification of diseases ( icd ) codes for acromegaly as used in the danish national registry of patients ( dnrp ) . we wished to evaluate whether incident cases of acromegaly during a specific period of time could be correctly identified through the dnrp , and to estimate the positive predictive value ( ppv ) of icd codes of relevance to a diagnosis of acromegaly , thereby evaluating the overall usefulness of the dnrp as a source for identifying patients with incident acromegaly . in addition , we aimed to examine the accuracy of the first registration with respect to the time of diagnosis and to give estimates of the acromegaly incidence rate as well as of age and sex distribution . the study area covers the central region of denmark with an average of 1.3 million inhabitants ( 23% of the danish population ) during the period january 1 , 1991 to december 31 , 2009 . the geographic area is formally defined by the danish region codes 65 , 70 , and 76 ; hence including former counties of ringkbing , aarhus , and viborg , respectively , in combination with hospital code 6006 ( horsens hospital ) . denmark has 5.6 million inhabitants , and the national health service provides universal tax - financed health care . all persons living permanently in denmark are assigned a unique 10-digit personal identification number ( the cpr number ) of which six digits denote the date of birth and the sex is mirrored by the last digit . the cpr number is used for all purposes requiring exact person identification and remains unchanged throughout life.3 since 1977 , the dnrp has tracked danish hospital admissions through linkage with the cpr number , recording dates of admission and discharge and up to 20 discharge diagnoses . diagnoses are coded by hospital physicians using the danish versions of the icd : the 8th revision ( icd-8 ) was used from 1977 to 1993 , while the 10th revision has been used since 1994 . since 1995 , the dnrp also includes information on outpatient contacts and emergency room visits.6 we identified patients registered with one or more icd codes of presumed relevance for a diagnosis of acromegaly . in addition to explicit acromegaly codes , we identified patients registered with other codes indicating a pituitary disorder , eg , various pituitary adenoma codes ( table 1 ) . data on the institutional origin of each registration and the number of relevant dnrp registrations for individual patients were also recorded and analyzed . all available information in the medical records , including clinical symptoms and findings , written radiology reports , and results from laboratory tests was taken into consideration . a diagnosis of acromegaly was made when laboratory testing ( elevated insulin - like growth factor 1 and nadir growth hormone serum concentrations ) or imaging ( visible pituitary adenoma ) and treatment were in agreement with acromegaly . all cases with an uncertain diagnosis based on the available information were discussed with a specialist in endocrinology and acromegaly until a consensus was reached . computerized journals were reviewed in all accessible cases , supplemented with a review of paper journals in patients within the study population . the data validity was expressed as the ppv of individual icd-8 or icd-10 discharge codes , and 95% confidence intervals ( cis ) were calculated . the ppv was defined as the proportion of patients having confirmed acromegaly when registered with a diagnosis of acromegaly in the dnrp . normally distributed and non - gaussian distributed data are presented as the mean and standard deviation or a median with 25% and 75% percentiles , respectively . the incidence rate was calculated assuming a poisson distribution and given as number of new cases per million per year . the study area covers the central region of denmark with an average of 1.3 million inhabitants ( 23% of the danish population ) during the period january 1 , 1991 to december 31 , 2009 . the geographic area is formally defined by the danish region codes 65 , 70 , and 76 ; hence including former counties of ringkbing , aarhus , and viborg , respectively , in combination with hospital code 6006 ( horsens hospital ) . denmark has 5.6 million inhabitants , and the national health service provides universal tax - financed health care . all persons living permanently in denmark are assigned a unique 10-digit personal identification number ( the cpr number ) of which six digits denote the date of birth and the sex is mirrored by the last digit . the cpr number is used for all purposes requiring exact person identification and remains unchanged throughout life.3 since 1977 , the dnrp has tracked danish hospital admissions through linkage with the cpr number , recording dates of admission and discharge and up to 20 discharge diagnoses . diagnoses are coded by hospital physicians using the danish versions of the icd : the 8th revision ( icd-8 ) was used from 1977 to 1993 , while the 10th revision has been used since 1994 . since 1995 , the dnrp also includes information on outpatient contacts and emergency room visits.6 we identified patients registered with one or more icd codes of presumed relevance for a diagnosis of acromegaly . in addition to explicit acromegaly codes , we identified patients registered with other codes indicating a pituitary disorder , eg , various pituitary adenoma codes ( table 1 ) . data on the institutional origin of each registration and the number of relevant dnrp registrations for individual patients were also recorded and analyzed . all available information in the medical records , including clinical symptoms and findings , written radiology reports , and results from laboratory tests was taken into consideration . a diagnosis of acromegaly was made when laboratory testing ( elevated insulin - like growth factor 1 and nadir growth hormone serum concentrations ) or imaging ( visible pituitary adenoma ) and treatment were in agreement with acromegaly . all cases with an uncertain diagnosis based on the available information were discussed with a specialist in endocrinology and acromegaly until a consensus was reached . computerized journals were reviewed in all accessible cases , supplemented with a review of paper journals in patients within the study population . denmark has 5.6 million inhabitants , and the national health service provides universal tax - financed health care . all persons living permanently in denmark are assigned a unique 10-digit personal identification number ( the cpr number ) of which six digits denote the date of birth and the sex is mirrored by the last digit . the cpr number is used for all purposes requiring exact person identification and remains unchanged throughout life.3 since 1977 , the dnrp has tracked danish hospital admissions through linkage with the cpr number , recording dates of admission and discharge and up to 20 discharge diagnoses . diagnoses are coded by hospital physicians using the danish versions of the icd : the 8th revision ( icd-8 ) was used from 1977 to 1993 , while the 10th revision has been used since 1994 . since 1995 , the dnrp also includes information on outpatient contacts and emergency room visits.6 we identified patients registered with one or more icd codes of presumed relevance for a diagnosis of acromegaly . in addition to explicit acromegaly codes , we identified patients registered with other codes indicating a pituitary disorder , eg , various pituitary adenoma codes ( table 1 ) . data on the institutional origin of each registration and the number of relevant dnrp registrations for individual patients were also recorded and analyzed . all available information in the medical records , including clinical symptoms and findings , written radiology reports , and results from laboratory tests was taken into consideration . a diagnosis of acromegaly was made when laboratory testing ( elevated insulin - like growth factor 1 and nadir growth hormone serum concentrations ) or imaging ( visible pituitary adenoma ) and treatment were in agreement with acromegaly . all cases with an uncertain diagnosis based on the available information were discussed with a specialist in endocrinology and acromegaly until a consensus was reached . computerized journals were reviewed in all accessible cases , supplemented with a review of paper journals in patients within the study population . the data validity was expressed as the ppv of individual icd-8 or icd-10 discharge codes , and 95% confidence intervals ( cis ) were calculated . the ppv was defined as the proportion of patients having confirmed acromegaly when registered with a diagnosis of acromegaly in the dnrp . normally distributed and non - gaussian distributed data are presented as the mean and standard deviation or a median with 25% and 75% percentiles , respectively . the incidence rate was calculated assuming a poisson distribution and given as number of new cases per million per year . our dnrp search identified 287 patients in the study area who had been registered with icd codes e22.0 , 25300 or 25301 , for whom records were accessible in 275 ( 96% ) cases ( table 2 ) . overall , review of hospital records allowed the diagnosis of acromegaly to be confirmed in 149 cases , yielding a ppv of 54.2 ( ci 48.360.0 ) . in 110 cases , the patients were diagnosed within the central region of denmark and within the chosen time frame with a ppv of 40.0 ( 95% ci 34.445.9 ) . the remaining 39 cases with confirmed acromegaly had either been diagnosed outside the study period ( n=20 ) , in another geographic region ( n=16 ) , were non - residents ( n=2 ) , or not evaluable due to missing data ( n=1 ) . the incidence rate of acromegaly within the study population was 4.5 ( 95% ci 3.65.5 ) per million per year and constant through the study period . the mean age at diagnosis was 49.6 ( 95% ci 46.853.4 ) years and a sex distribution with 48% female . the median time difference between the first dnrp registration and the diagnostic blood sample was one day ( 25- , 75-percentile : 120 ) in 103 cases . among the group of patients registered with the remaining relevant icd codes related to various pituitary diseases , all available records were reviewed except those registered only with codes e22.1 hyperprolactinaemia or d35.2 benign neoplasm of other and unspecified endocrine glands , pituitary gland , among which 20% were randomly selected for review . in all , 372 cases were reviewed and no cases of acromegaly had been misclassified as other diseases . one case was recorded as e22.9 hyperfunction of pituitary gland , unspecified and only after the end of the study period was diagnosed with and recorded as e22.0 acromegaly ( table 1 ) . when applying the same dnrp search criteria but restricting the search to the highly specialized department of endocrinology at aarhus university hospital ( code 7003.07x ) , all 110 confirmed cases of acromegaly within the study population were identified , whereas eleven other cases of confirmed acromegaly , all of whom were diagnosed in another geographic region , were lost . thus , the ppv increased to 66.7% ( 95% ci 60.072.7 ) and 53.1% ( 95% ci 46.459.8 ) for all patients with acromegaly and for the study population , respectively ( table 2 ) , at the cost of a decline in the number of identified cases with confirmed acromegaly from 100% to 92.6% ( 95% ci 87.395.8 ) in the group of all patients , but no loss of cases in the study population . the pattern of hospital registrations varied between different subgroups of patients identified by the original dnrp search . in three groups , ie , acromegaly cases within the study population , acromegaly outside the study population , and false positive cases , the median total number of per - patient registrations was 22.7 , 11.3 , and 3.3 , respectively . moreover , when applying a restriction of at least one , two , or three registrations to our dnrp search criteria , the ppv increased with a concurrent loss of cases with confirmed acromegaly ( table 3 ) . our dnrp search identified 287 patients in the study area who had been registered with icd codes e22.0 , 25300 or 25301 , for whom records were accessible in 275 ( 96% ) cases ( table 2 ) . overall , review of hospital records allowed the diagnosis of acromegaly to be confirmed in 149 cases , yielding a ppv of 54.2 ( ci 48.360.0 ) . in 110 cases , the patients were diagnosed within the central region of denmark and within the chosen time frame with a ppv of 40.0 ( 95% ci 34.445.9 ) . the remaining 39 cases with confirmed acromegaly had either been diagnosed outside the study period ( n=20 ) , in another geographic region ( n=16 ) , were non - residents ( n=2 ) , or not evaluable due to missing data ( n=1 ) . the incidence rate of acromegaly within the study population was 4.5 ( 95% ci 3.65.5 ) per million per year and constant through the study period . the mean age at diagnosis was 49.6 ( 95% ci 46.853.4 ) years and a sex distribution with 48% female . the median time difference between the first dnrp registration and the diagnostic blood sample was one day ( 25- , 75-percentile : 120 ) in 103 cases . among the group of patients registered with the remaining relevant icd codes related to various pituitary diseases , all available records were reviewed except those registered only with codes e22.1 hyperprolactinaemia or d35.2 benign neoplasm of other and unspecified endocrine glands , pituitary gland , among which 20% were randomly selected for review . in all , 372 cases were reviewed and no cases of acromegaly had been misclassified as other diseases . one case was recorded as e22.9 hyperfunction of pituitary gland , unspecified and only after the end of the study period was diagnosed with and recorded as e22.0 acromegaly ( table 1 ) . when applying the same dnrp search criteria but restricting the search to the highly specialized department of endocrinology at aarhus university hospital ( code 7003.07x ) , all 110 confirmed cases of acromegaly within the study population were identified , whereas eleven other cases of confirmed acromegaly , all of whom were diagnosed in another geographic region , were lost . thus , the ppv increased to 66.7% ( 95% ci 60.072.7 ) and 53.1% ( 95% ci 46.459.8 ) for all patients with acromegaly and for the study population , respectively ( table 2 ) , at the cost of a decline in the number of identified cases with confirmed acromegaly from 100% to 92.6% ( 95% ci 87.395.8 ) in the group of all patients , but no loss of cases in the study population . the pattern of hospital registrations varied between different subgroups of patients identified by the original dnrp search . in three groups , ie , acromegaly cases within the study population , acromegaly outside the study population , and false positive cases , the median total number of per - patient registrations was 22.7 , 11.3 , and 3.3 , respectively . moreover , when applying a restriction of at least one , two , or three registrations to our dnrp search criteria , the ppv increased with a concurrent loss of cases with confirmed acromegaly ( table 3 ) . we evaluated the data quality of the dnrp in relation to the diagnosis of acromegaly through a population - based cohort study including 1.3 million inhabitants in the central region of denmark during the period 19912009 . by reviewing 647 discharge records suggesting a diagnosis of acromegaly ( n=275 ) or closely related pituitary diseases ( n=372 ) , as shown in table 1 , we were able to estimate the ppv values of the icd-8 and icd-10 codes used to identify new cases of acromegaly in a national hospital discharge registry . we identified 149 cases of acromegaly , including 110 cases diagnosed between 1991 and 2009 ( ie , during the relevant study period ) within the central region of denmark . since a thorough review of cases with only icd codes indicating other or non - specific pituitary disease identified no additional cases of acromegaly , and since previous studies indicate a high degree of completeness of the dnrp , we believe that only very few cases of acromegaly , if any , may have been missed by our dnrp search . moreover , although cases of acromegaly misclassified as non - related pituitary disease can not be completely ruled out , repeated misclassification seems unlikely when taking into account that patients with acromegaly are offered lifelong follow - up at a highly specialized department . admittedly , cases of acromegaly with cosecretion of prolactin diagnosed as a prolactinoma , rare cases of a somatotropinoma making its first appearance as a pituitary apoplexy with adenoma destruction , or cases of acromegaly where the diagnosis simply was not considered , can not be ruled out.7,8 likewise , some limitations must be taken into account regarding the diagnostic verification process that we applied to suspected cases of acromegaly . firstly , the reviewer could not be blinded because all records were named according to the icd codes . secondly , in some cases the icd code could not be confirmed during the manual review process . instead , the electronic journal was reviewed and used to confirm or reject the diagnosis of acromegaly . however , in most cases , the relevant icd code was identified and the particular hospital file was examined . when comparing the characteristics of our cohort of acromegaly with respect to the mean age at diagnosis ( 49.6 years ) , the sex distribution ( 48% female ) , and the incidence rate ( 4.5/million / year ) , our cohort seems comparable with other cohorts in the literature.2 however , a more precise estimate of the true incidence rate should be calculated on the basis of a larger population , ie , a national cohort . we found the overall ppv for acromegaly in the dnrp to be only 54% , and the ppv further decreased to 40% for truly positive ( confirmed ) cases within the study population as defined by geography and time period criteria . to improve the ppv , we refined the search criteria by restriction to a selected highly specialized department of endocrinology . without losing any acromegaly cases from the study population , the ppv of icd codes for acromegaly in the dnrp increased to 53% in the study population . finally , when analyzing and further including the number of registrations into the search strategy , we found that patients with acromegaly both inside and outside the study population had higher numbers of registrations than patients in the false positive group ( table 3 ) . when applying a restriction of at least one , two , or three registrations to the search , the ppv gradually increased from 53% to 75% in the study population , with a less pronounced decline in the percentage of identified cases with confirmed acromegaly from 100% to 95% . cases of acromegaly with only a few registrations were characterized by recent diagnosis , older age , and the coexistence of other medical conditions . the findings are not surprising , given that acromegaly is a lifelong condition requiring continuity of care at a highly specialized endocrine department . our study indicates that no or only very few patients with acromegaly fail to be registered by the regional highly specialized medical department of endocrinology , and that these patients typically are registered several times due to repeated hospital contacts . according to our study , the effect of applying both aspects of this knowledge to a search for new cases of acromegaly in a well defined geographic area and for a well defined time period will be a relative increase in ppv of the search result by almost 90% ( from 40% to 75% ) while preserving the identification of most cases of confirmed acromegaly . in this study covering a recent 19-year period and including approximately one fourth of the danish population , the dnrp appears to be a useful tool for identifying new cases of acromegaly , especially when restricting the search criteria to the regional highly specialized medical department of endocrinology . the initial ppv was well below 50% , but could be markedly increased by adding several registrations as a supplementary search criterion . we speculate that this approach in searching the dnrp may be successfully applied to other rare diseases in which continuity of care is provided by highly specialized departments . | backgroundthe incidence of acromegaly is uncertain , since population - based studies are few . in the absence of a specific acromegaly registry
, the danish national registry of patients ( dnrp ) becomes a potential source of data for studying the epidemiology of acromegaly , by linking all hospital discharge diagnoses to the personal identification numbers of individual danish inhabitants .
the validity of the dnrp with respect to acromegaly , however , remains to be tested .
the aim of this study was to validate the international classification of diseases ( icd ) codes for acromegaly ( icd-8 : 25300 , 25301 .
icd-10 : e22.0 ) as used in the dnrp , and to assess the influence of various registration patterns on the accuracy of registry data.methodswe identified patients registered with icd codes for the diagnosis of acromegaly or other pituitary disorders during the period 19912009 .
data on the institutional origin of each registration and the number of relevant dnrp registrations were recorded , and systematic patient chart reviews were performed to confirm the diagnosis.resultsin total , 110 cases of acromegaly were confirmed , compared with 275 registered cases , yielding a positive predictive value ( ppv ) of 40% . when restricting the search to the regional highly specialized department of endocrinology , the ppv increased to 53% with no loss of cases with confirmed acromegaly . with a requirement of at least one , two , or three dnrp registrations ,
the ppv increased , but with a concurrent loss of confirmed cases.conclusionthe dnrp seems to be a useful source for identifying new cases of acromegaly , especially when restricting the search to a relevant regional highly specialized department .
the ppv of dnrp data used for this purpose can be increased by including only cases with several registrations .
a similar approach may be successfully applied to other rare diseases in which continuity of care is provided by highly specialized departments . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the ubiquity of computers in our lives is undisputed . for every modern - age college student it is virtually a must to own or have access to a computer for him or her to succeed in course work . almost every assignment in today 's classroom requires some computer work , be it researching literature , performing calculations , or writing a report . in addition , many young people take advantage of a variety of online communication resources , such as e - mail and instant messengers , or social websites such as myspace , facebook , and twitter , and thus are familiar with many types of computer software . given this image , one might think that the undergraduates of today are naturally ready to dive into the world of , presently highly computerized , scientific research . although many students are far more adaptable to computers and their applications than older generations , they seem daunted by more sophisticated uses , such as data manipulation and analysis for the purpose of scientific research ( goodall , 2008 ) . it is widely believed that this is at least partially due to the insufficient preparation of the students in terms of basic mathematical skills , and especially logic and statistics , which are indispensable in life sciences research ( rao , 2008 ) . this article describes an innovative undergraduate - level course , a four - credit - hour elective open to all life science majors , titled computer literacy for life sciences , which aims at partially remedying this situation . although it is not easy to fill in all the gaps resulting from many years of underpreparation in the area of quantitative skills development , this course is intended to provide the students with some basic proficiency needed for scientific research in the digital era . the overall purpose of computer literacy for life sciences is for students to develop a hands - on ( and brains - on ! ) working experience in using standard computer software tools as well as computer techniques and methodologies used in life sciences research . researchers should be familiar with the computer 's operating system and its main and most useful applications . they must know how to effectively manipulate documents and data files , including those downloaded from libraries and data repositories available on the world wide web ( www ) . scientists must be able to efficiently manage and query the data resulting from wet lab experiments as well as those derived from computer simulations . such data are then subject to statistical analysis , or other means of data mining , to answer the underlying scientific questions . finally , researchers should be able to present their discoveries in the form of reports or scientific articles and audiovisual presentations . to fulfill all of the above - mentioned objectives , the students in the computer literacy for life sciences course are mentored throughout the process of scientific pursuit , which is implemented in the form of several group virtual research projects to be completed with the use of state - of - the - art computer techniques and applications . each virtual project includes the following components : background literature research , data manipulation , formulation of scientific questions and verifiable hypotheses , statistical data analysis , and presentation of scientific discoveries . several aspects of this course , such as the idea of semester - long virtual research projects , modules , and skills - checks as well as the grading scheme , were based on a somewhat similar course developed at san jose state university ( macdonald et al . , 2010 ) . many of the experiences derived from a course on information literacy in biology education described by porter ( 2005 ) also were very useful in the preparation of computer literacy for life sciences . the first installment of computer literacy for life sciences was taught at emory university in fall semester 2009 . the enrollment in the course was 12 highly motivated students from two majors : biology , and neuroscience and behavioral biology ( nbb ) . the course was taught once a week in a 3-h - long session consisting of a short ( up to 30 min ) lecture on the main concepts of the particular session followed by a 2.5-h hands - on computer laboratory under the supervision of , and with help from , the instructor . the course was taught in a modular manner , with each module representing a major stage of a research project , as outlined below . the students were divided into several groups , each working on a specific virtual research project , selected from a list of projects provided by the instructor . in each module , the students were to acquire a set of skills that would allow them to complete the specific phase of their project and traverse to the next step , until a successful conclusion of their pursuit . each of the stages was concluded by a short group presentation to the class , as well as an in - class practical competency exam ( described below ) . the presentations were designed to give each group an opportunity to update the class on their progress , as well as to discuss any problems or difficulties they might have encountered . to avoid a situation in which only some group members are actually performing all the work and thus learning the material , each member of the group was expected to partake in the presentations . the students were expected to complete readings , cooperate in the group project , prepare presentations for each of the course modules , participate in class discussions , and successfully pass the competency exams . table 1 presents the grade computation scheme used , and a discussion of the specific subareas of assessment follows . as mentioned , the module group presentations were implemented to allow each team to provide an update on their progress as well as to have the opportunity to discuss any problems or difficulties they might have encountered , so that the whole class could brainstorm , learn , and benefit from the experiences of others . the presentations were graded based on a rubric that included the following categories : organization , content knowledge , supporting material , mechanics , and delivery . in the module 1 group presentations , only the instructor provided the scores , but all other presentations also were scored by the other classmates . the purpose for this was twofold : 1 ) the grading students provided more feedback and thus increased the perceived fairness of the grades ( all the grades , both from the instructor and the students , were included in the final score ) ; but more importantly , 2 ) they were forced to think about the aspects of the presentations that were included in the rubric and inadvertently reflect on their own performance in those categories . competency exams . similarly to the group presentations concluding each of the first three modules , there were three competency exams administered during the semester , to be completed by each student individually . the competency exams were based on skills - check handouts provided for each module by the instructor , before the exam . each handout contained a list of tasks that a student was expected to be able to complete after attaining the learning objectives of a given module . only the tasks included in a skills - check were to be included on an actual exam , although some variations ( e.g. , different input files , different variables ) were to be expected . thus , if the students mastered the material on a given skills - check , they should have no trouble with the exam to follow . the skills - checks were practiced by the students in class during special review sessions as well as in their own time . final presentation . toward the end of the term , all groups had a chance to orally present their discoveries in the form of a final team presentation . this presentation , in contrast to the three prior presentations , was to provide an overview of the complete project , similarly to what might be presented at a scientific conference . the students were expected to discuss their entire experience , without simply repeating all of the material from the previous presentations , and concentrate on their hypotheses and how they attempted to verify them . chronologically , the presentations were scheduled before the final article , so that the students could obtain valuable feedback to be included in the final product . final article . at the conclusion of the semester , each group had to summarize their projects and discoveries in the form of a short journal - like coauthored article . the students were given proper instruction and resources for how to prepare such a publication beforehand . the articles were to contain a discussion of the underlying big - picture problem , the state of current research in the field , a presentation of the group 's ideas and hypotheses , a description of all the methods used throughout the project , and a summary of the main discoveries resulting from the entire virtual research project . the last category included in the grading scheme was a somewhat subjective method to reward the students for their added effort , realized in the form of numerous extra - credit assignments , and overall interest in the subject matter . based on the above - described scheme , a standard percentage grading scale rounded to the nearest whole number ( by rounding up from 0.5 ) was used to calculate the final grades with a+ the course was divided into four separate modules , each having a different set of overarching learning objectives . the modules and their corresponding main learning goals are presented in table 2 , and a detailed description of their contents follows . modules and their corresponding main learning goals module 1 : operating systems and web - based project repositories ; background literature research . in the beginning of the course , the students had a chance to familiarize themselves with the two operating systems widely used in the scientific community , windows ( microsoft , redmond , wa ) and unix / linux ( developer linus torvalds and thousands of collaborators ) , with the emphasis on effective file management . both operating systems provide a collection of tools that can be applied to scientific pursuits and they also often complement each other . therefore , it is important to have some degree of mastery over both and to be able to make them interact with each other . also at this stage , the students were asked to select a virtual research project from the list provided by the instructor to be pursued by them and their teammates for the remainder of the course . there were 14 project proposals from the following three areas : basic biology , biomedicine , and neuroscience ( due to the obligation imposed by the nbb major , which required a neuroscience component in the course ) . to be on the list , a topic had to be supported by a relatively large number of works in the literature and have underlying data available . the data sets , and the corresponding principal publications in addition to the instructor 's own neuroscience - related projects , were obtained from www resources , such as the university of california irvine machine learning repository ( http://archive.ics.uci.edu/ml ) . table 3 lists the 14 proposed virtual research project themes . proposed virtual research project topics not surprisingly , because most of the 12 students enrolled in the course were premed students , the most popular topics were among the biomedicine - related topics . actually , based on the students ' selections , four groups were formed , each dealing with one of the biomedicine topics listed in table 3 . after the selections had been made , the students were asked to perform background literature research on their topics . the underlying background information about a scientific inquiry is usually acquired by using computer tools to research the literature or the www . the internet is also abundant with existing and publicly available data sets derived from experiments performed by others in the scientific community that not only can provide invaluable insight into the inquiry of interest but also may be used for further analysis complementing previous studies . topics such as browsing and searching online scientific databases as well as retrieving and storing the appropriate information were covered in this part of the course . the students learned how to use this kind of information to form their own scientific questions and delineate plans for answering them . at this stage , in addition to the microsoft windows operating system , which was used in virtually all other tasks in the course , the students were also introduced to the unix / linux - type operating systems . using the popular tectia ssh secure shell application , they learned how to connect to a linux - based server provided by emory university , transfer files between their local windows - based workstation and the server , and effectively manipulate those files on the remote server . these skills were not only important for completion of the final part of this module ( described in the next paragraph ) but also are , in general , fundamental if one plans to perform computational simulations , often done on unix / linux supercomputers , as the basis for life sciences research . using their freshly acquired knowledge about the two operating systems and their interconnectivity , the students learned how to create a simple website to serve as a repository of their project 's records and documentation . they learned the basics of hypertext markup language ( html)a principal programming language used in webpage development as well as some aspects of website design provided by the popular web development application , macromedia ( currently adobe systems , mountain view , ca ) dreamweaver . the main purpose of this task was trifold : the students were not only expected to 1 ) create a resource to store all the materials pertaining to their projects ( accessible to them from wherever they were ) , and 2 ) learn the basics of website development , but also to 3 ) have a smooth introduction to teamwork and begin cooperating with their group - mates . it is also important to point out that the students , as opposed to using the dreamweaver as the only method of webpage development , were expected to use html for a reason . most of the students in the class had never used a programming language before and learning html was a method for easing them into the concept of coding by allowing them to program something that would be simple , yet palpable and exciting . this experience also was expected to make the introduction of the other programming language used in the course , sql ( described below ) , easier . research studies can be performed in the laboratory setting as wet experiments , or they can take the form of computer simulations , but regardless of the method , the results are rather large amounts of computerized data . such data can be effectively stored in database management systems that allow for easy access and retrieval . in this section of the course , the students learned the basics of relational database management systems ( rdbms ) along with how to efficiently query such systems to obtain the desired portions of the data . specifically , microsoft access , with its structured query language ( sql ; the most commonly used data querying language ) capabilities was utilized for this purpose . the students also learned how to upload data retrieved from the www in the previous module into the rdbms and then effectively query the system to extract the relevant parts of the data . finally , the students became skilled at exporting such subsets of data to other types of software for the purpose of data analysis . most of the material presented in this module was acquired by the students in the form of tutorials and in - class and/or take - home assignments ( referred to as the tutorial / assignment model ) . for example , some of the tutorials included in this module were as follows : creating a new , blank database in microsoft access 2007 ; uploading data into access from external sources ; or sorting , searching , and filtering data in access . in the sql portion of the module , some of the tutorials were as follows : simple queries using sql ; data extraction using sql ; and data summarization using sql . as mentioned , the tutorials list the specific steps needed to complete a given task ( e.g. , upload data to access ) . initially , the students were given the tutorials and asked to complete the steps individually , under the supervision and with help of the instructor , as needed . however , the students insisted , and it turned out to be more efficient , on completing the tutorials as a group , to make sure that everybody is on the same page and all questions are answered . in addition to tutorials , the students had to complete assignments that were either take - home or to be performed in - class . the purpose of the assignments was for the students to extrapolate from the knowledge and skills they acquired by completing the tutorials to new , yet similar , circumstances and problems . for example , based on several previously completed tutorials , the students were asked to upload a new data set into access , create a series of cooperating sql queries to extract specific information about the data , and export that information from access to a text file . the assignments were to be performed either individually , in pairs , or in groups , depending on the particular material . an example of such an assignment can be found at www.biology.emory.edu/research/prinz/tomasz/cl_for_ls . after attaining the learning objectives of this module , the students should be able to import the data of interest into access from an external source , manipulate them to extract particular subsets of the data , and export those pieces of data into external data files , to be used for further analyses . calculations and manipulations needed for data analysis can be performed using various statistical programs and spreadsheets . in this part of the course , the students experimented with two different approaches , using two different programs , to statistical data analysis . the first approach was based on excel ( microsoft ) to extract and graph basic statistics from data samples . the students learned how to get to know the data and how to summarize their essential characteristics , all by using the standard and relatively simple functions provided by excel . the second approach used a more sophisticated statistical data analysis software package , spss ( spss , chicago , il ) , to investigate interactions between variables in the data . the students learned how to examine interdependencies between variables , by using various measures and methods such as correlations and regression analysis . in this module , , the students learned several fairly basic statistical methods used for data analysis . in excel , the students were introduced to performing simple descriptive statistical analysis ( e.g. , in terms of means , medians , modes , standard deviations ) , as well as creating basic plots to graphically describe the data . in spss , the students applied more sophisticated statistical techniques including : box - and - whisker analysis , correlations , t tests , anova , and regression analysis . the purpose here was by no means to turn the students into statistical analysis experts , but to make them capable of choosing and performing appropriate statistical analyses and tests ( out of this quite limited arsenal of tests ) using one of the two introduced software packages , and to be able to understand the meaning of the results . therefore , a special emphasis was placed on stating testable hypotheses and the interpretation of a hypothesis testing result . in several assignments that accompanied the tutorials , students were asked to brainstorm and propose several hypotheses pertaining to their virtual research projects , and test those hypotheses using excel or spss . module 4 : presentation of scientific discoveries . finally , in the last part of the course , the students had a chance to summarize their inquiries and discoveries in the form of a short ( 45 pages ) coauthored journal - like article and a final class presentation . the articles , as well as the presentations , were to be organized into sections corresponding to the modules of the course . each section contained a description of the methods used and a discussion of how they were applied to a particular problem . the articles and the presentations concluded with a summary of the main discoveries resulting from the entire virtual research project . the students were expected to complete readings , cooperate in the group project , prepare presentations for each of the course modules , participate in class discussions , and successfully pass the competency exams . table 1 presents the grade computation scheme used , and a discussion of the specific subareas of assessment follows . as mentioned , the module group presentations were implemented to allow each team to provide an update on their progress as well as to have the opportunity to discuss any problems or difficulties they might have encountered , so that the whole class could brainstorm , learn , and benefit from the experiences of others . the presentations were graded based on a rubric that included the following categories : organization , content knowledge , supporting material , mechanics , and delivery . in the module 1 group presentations , only the instructor provided the scores , but all other presentations also were scored by the other classmates . the purpose for this was twofold : 1 ) the grading students provided more feedback and thus increased the perceived fairness of the grades ( all the grades , both from the instructor and the students , were included in the final score ) ; but more importantly , 2 ) they were forced to think about the aspects of the presentations that were included in the rubric and inadvertently reflect on their own performance in those categories . similarly to the group presentations concluding each of the first three modules , there were three competency exams administered during the semester , to be completed by each student individually . the competency exams were based on skills - check handouts provided for each module by the instructor , before the exam . each handout contained a list of tasks that a student was expected to be able to complete after attaining the learning objectives of a given module . only the tasks included in a skills - check were to be included on an actual exam , although some variations ( e.g. , different input files , different variables ) were to be expected thus , if the students mastered the material on a given skills - check , they should have no trouble with the exam to follow . the skills - checks were practiced by the students in class during special review sessions as well as in their own time . final presentation . toward the end of the term , all groups had a chance to orally present their discoveries in the form of a final team presentation . this presentation , in contrast to the three prior presentations , was to provide an overview of the complete project , similarly to what might be presented at a scientific conference . the students were expected to discuss their entire experience , without simply repeating all of the material from the previous presentations , and concentrate on their hypotheses and how they attempted to verify them . chronologically , the presentations were scheduled before the final article , so that the students could obtain valuable feedback to be included in the final product . final article . at the conclusion of the semester , each group had to summarize their projects and discoveries in the form of a short journal - like coauthored article . the students were given proper instruction and resources for how to prepare such a publication beforehand . the articles were to contain a discussion of the underlying big - picture problem , the state of current research in the field , a presentation of the group 's ideas and hypotheses , a description of all the methods used throughout the project , and a summary of the main discoveries resulting from the entire virtual research project . the last category included in the grading scheme was a somewhat subjective method to reward the students for their added effort , realized in the form of numerous extra - credit assignments , and overall interest in the subject matter . based on the above - described scheme , a standard percentage grading scale rounded to the nearest whole number ( by rounding up from 0.5 ) was used to calculate the final grades with a+ the course was divided into four separate modules , each having a different set of overarching learning objectives . the modules and their corresponding main learning goals modules and their corresponding main learning goals module 1 : operating systems and web - based project repositories ; background literature research . in the beginning of the course , the students had a chance to familiarize themselves with the two operating systems widely used in the scientific community , windows ( microsoft , redmond , wa ) and unix / linux ( developer linus torvalds and thousands of collaborators ) , with the emphasis on effective file management . both operating systems provide a collection of tools that can be applied to scientific pursuits and they also often complement each other . therefore , it is important to have some degree of mastery over both and to be able to make them interact with each other . also at this stage , the students were asked to select a virtual research project from the list provided by the instructor to be pursued by them and their teammates for the remainder of the course . there were 14 project proposals from the following three areas : basic biology , biomedicine , and neuroscience ( due to the obligation imposed by the nbb major , which required a neuroscience component in the course ) . to be on the list , a topic had to be supported by a relatively large number of works in the literature and have underlying data available . the data sets , and the corresponding principal publications in addition to the instructor 's own neuroscience - related projects , were obtained from www resources , such as the university of california irvine machine learning repository ( http://archive.ics.uci.edu/ml ) . table 3 lists the 14 proposed virtual research project themes . proposed virtual research project topics not surprisingly , because most of the 12 students enrolled in the course were premed students , the most popular topics were among the biomedicine - related topics . actually , based on the students ' selections , four groups were formed , each dealing with one of the biomedicine topics listed in table 3 . after the selections had been made , the students were asked to perform background literature research on their topics . the underlying background information about a scientific inquiry is usually acquired by using computer tools to research the literature or the www . the internet is also abundant with existing and publicly available data sets derived from experiments performed by others in the scientific community that not only can provide invaluable insight into the inquiry of interest but also may be used for further analysis complementing previous studies . topics such as browsing and searching online scientific databases as well as retrieving and storing the appropriate information were covered in this part of the course . the students learned how to use this kind of information to form their own scientific questions and delineate plans for answering them . at this stage , in addition to the microsoft windows operating system , which was used in virtually all other tasks in the course , the students were also introduced to the unix / linux - type operating systems . using the popular tectia ssh secure shell application , they learned how to connect to a linux - based server provided by emory university , transfer files between their local windows - based workstation and the server , and effectively manipulate those files on the remote server . these skills were not only important for completion of the final part of this module ( described in the next paragraph ) but also are , in general , fundamental if one plans to perform computational simulations , often done on unix / linux supercomputers , as the basis for life sciences research . using their freshly acquired knowledge about the two operating systems and their interconnectivity , the students learned how to create a simple website to serve as a repository of their project 's records and documentation . they learned the basics of hypertext markup language ( html)a principal programming language used in webpage development as well as some aspects of website design provided by the popular web development application , macromedia ( currently adobe systems , mountain view , ca ) dreamweaver . the main purpose of this task was trifold : the students were not only expected to 1 ) create a resource to store all the materials pertaining to their projects ( accessible to them from wherever they were ) , and 2 ) learn the basics of website development , but also to 3 ) have a smooth introduction to teamwork and begin cooperating with their group - mates . it is also important to point out that the students , as opposed to using the dreamweaver as the only method of webpage development , were expected to use html for a reason . most of the students in the class had never used a programming language before and learning html was a method for easing them into the concept of coding by allowing them to program something that would be simple , yet palpable and exciting . this experience also was expected to make the introduction of the other programming language used in the course , sql ( described below ) , easier . research studies can be performed in the laboratory setting as wet experiments , or they can take the form of computer simulations , but regardless of the method , the results are rather large amounts of computerized data . such data can be effectively stored in database management systems that allow for easy access and retrieval . in this section of the course , the students learned the basics of relational database management systems ( rdbms ) along with how to efficiently query such systems to obtain the desired portions of the data . specifically , microsoft access , with its structured query language ( sql ; the most commonly used data querying language ) capabilities was utilized for this purpose . the students also learned how to upload data retrieved from the www in the previous module into the rdbms and then effectively query the system to extract the relevant parts of the data . finally , the students became skilled at exporting such subsets of data to other types of software for the purpose of data analysis . most of the material presented in this module was acquired by the students in the form of tutorials and in - class and/or take - home assignments ( referred to as the tutorial / assignment model ) . for example , some of the tutorials included in this module were as follows : creating a new , blank database in microsoft access 2007 ; uploading data into access from external sources ; or sorting , searching , and filtering data in access . in the sql portion of the module , some of the tutorials were as follows : simple queries using sql ; data extraction using sql ; and data summarization using sql . as mentioned , the tutorials list the specific steps needed to complete a given task ( e.g. , upload data to access ) . initially , the students were given the tutorials and asked to complete the steps individually , under the supervision and with help of the instructor , as needed . however , the students insisted , and it turned out to be more efficient , on completing the tutorials as a group , to make sure that everybody is on the same page and all questions are answered . in addition to tutorials , the students had to complete assignments that were either take - home or to be performed in - class . the purpose of the assignments was for the students to extrapolate from the knowledge and skills they acquired by completing the tutorials to new , yet similar , circumstances and problems . for example , based on several previously completed tutorials , the students were asked to upload a new data set into access , create a series of cooperating sql queries to extract specific information about the data , and export that information from access to a text file . the assignments were to be performed either individually , in pairs , or in groups , depending on the particular material . an example of such an assignment can be found at www.biology.emory.edu/research/prinz/tomasz/cl_for_ls . after attaining the learning objectives of this module , the students should be able to import the data of interest into access from an external source , manipulate them to extract particular subsets of the data , and export those pieces of data into external data files , to be used for further analyses . calculations and manipulations needed for data analysis can be performed using various statistical programs and spreadsheets . in this part of the course , the students experimented with two different approaches , using two different programs , to statistical data analysis . the first approach was based on excel ( microsoft ) to extract and graph basic statistics from data samples . the students learned how to get to know the data and how to summarize their essential characteristics , all by using the standard and relatively simple functions provided by excel . the second approach used a more sophisticated statistical data analysis software package , spss ( spss , chicago , il ) , to investigate interactions between variables in the data . the students learned how to examine interdependencies between variables , by using various measures and methods such as correlations and regression analysis . in this module , in excel , the students were introduced to performing simple descriptive statistical analysis ( e.g. , in terms of means , medians , modes , standard deviations ) , as well as creating basic plots to graphically describe the data . in spss , the students applied more sophisticated statistical techniques including : box - and - whisker analysis , correlations , t tests , anova , and regression analysis . the purpose here was by no means to turn the students into statistical analysis experts , but to make them capable of choosing and performing appropriate statistical analyses and tests ( out of this quite limited arsenal of tests ) using one of the two introduced software packages , and to be able to understand the meaning of the results . therefore , a special emphasis was placed on stating testable hypotheses and the interpretation of a hypothesis testing result . in several assignments that accompanied the tutorials , students were asked to brainstorm and propose several hypotheses pertaining to their virtual research projects , and test those hypotheses using excel or spss . module 4 : presentation of scientific discoveries . finally , in the last part of the course , the students had a chance to summarize their inquiries and discoveries in the form of a short ( 45 pages ) coauthored journal - like article and a final class presentation . the articles , as well as the presentations , were to be organized into sections corresponding to the modules of the course . each section contained a description of the methods used and a discussion of how they were applied to a particular problem . the articles and the presentations concluded with a summary of the main discoveries resulting from the entire virtual research project . as mentioned , in the first module , students had the opportunity to familiarize themselves with windows and unix / linux operating systems , perform a background literature research on their group virtual topics , and create a simple website to serve as a repository of their project 's records and documentation . the module proved to be a success , as not only did the students develop interesting and comprehensive websites for their projects ( two examples shown in figure 1 ) but also the stage was set for further group cooperation on the virtual research projects . virtual research project repository websites developed by the students in two selected groups . in the second module , the students were presented with the actual data sets underlying their projects and , utilizing the skills they had learned based on tutorials and assignments , were asked to explore the data and extract the important subsets from the data sets . many of the data sets behind the virtual projects contained superfluous attributes , such as names and ids , hardly useful for classification . the students were expected to identify such fields and remove them from consideration , which all groups successfully performed by the means of the sql language . in addition , because some of the fields in the data sets contained missing entries , the students , after researching various methods for dealing with missing data ( which was one of their take - home assignments ) , implemented some of those approaches using sql . the third module , concerned with the actual analysis of the data pertaining to the group projects , allowed the students to utilize their newly acquired knowledge of several statistical techniques ( e.g. , graphing methods , correlations , t tests , analysis of variance [ anova ] , and regression analysis ) to test and interpret their hypotheses . based on several tutorials and assignments , the students learned how to perform statistical analyses and tests in either excel or spss , and which of those methods would work best for their particular data set . because each of the projects was quite different in terms of the characteristics of the underlying data , the outcomes of analyses ranged from a description of a set of quite clear and concise classification rules ( analysis of the cell nuclei from digitized images of a fine needle aspirate [ fna ] of a breast mass to determine malignancy of cancer and diagnosing heart abnormalities based on cardiac single proton emission computed tomography [ spect ] images ) , through comparisons of different regression methods ( breast cancer surgery survival rates analysis ) , to the creation of new attributes based on the existing ones ( e.g. , body mass index , based on other physical attributes of patients ) for the purpose of creation of the best possible classifier ( diagnosing heart arrhythmia based on clinical patient data ) . finally , each group delivered a presentation and returned a comprehensive and interesting article in which they provided an overview of their project , as well as the results of their studies . as an illustration of the entire virtual research experience , consider the group working on the heart arrhythmia project . after building their online project repository , the students in that group obtained a data set containing 452 patient records in terms of their age , sex , height , and weight , as well as different characteristics of the patients ' electrocardiogram ( ecg ) . the data set also included the information about the patients ' diagnoses : either normal or one of 15 types of arrhythmia ( gvenir et al . , 1997 ) . the students uploaded the data set into access and used sql to explore the data and to perform preprocessing in terms of missing entries or other inconsistencies , to prepare the data set for analysis . importantly , in addition to the relatively simple data manipulation procedures the students carried out in this step , they already started forming hypotheses as to the importance of specific attributes in terms of the overall diagnosis . for example , based on various combinations of the sql queries that the members of this group implemented , they hypothesized that weight and height by themselves may be quite misleading predictors of arrhythmia , but if combined into the body mass index , the diagnosis accuracy could be improved . in the next step , the students in the arrhythmia group used excel and spss to further analyze their data . they looked more closely at the distributions of values in all the attributes and , based on those analyses , hypothesized that three additional fields ( qrs - duration , t - interval , and p - interval , all representing specific characteristics of the ecg ) also may be useful for diagnostic purposes . to test their hypotheses , the students performed one - way anova tests and built a multinomial logistic regression model and indeed confirmed the significance of the selected attributes . it is important to point out that the analyses performed by the students in this group , as well as the other three groups , were based on their own open - ended hypotheses and ideas and were not merely a recreation of other published studies . although the students had a rather limited selection of data analysis methods at their disposal ( due to the time constraints of the course ) and did not actively seek other bioinformatics or data mining approaches beyond what was covered in the class , they learned how to choose and apply correct statistical tests to evaluate their own hypotheses . the computer literacy for life sciences course has proved to be a very successful endeavor , as indicated by the students ' positive feedback on unofficial surveys and official evaluations , as well as their high performance on assignments and tests . in their anonymous comments , students expressed their appreciation for the tutorial / assignment approach and claimed it made a tough class easy and helped them navigate the unfamiliar material . they also appreciated the freedom to apply knowledge and experiment with data . most students enjoyed the group work and hands - on experience . the most difficult aspect of the course was the timing and length of the sessions . one 3-h - long class - meeting late in the afternoon was very taxing for both the students and the instructor . in the second installment of the course , which was taught again in spring semester 2010 , finally , to answer the underlying question of this article , are the digital - era undergraduates computer literate enough to face today 's research ? , based on my observations in the classroom , i would have to draw the conclusion that no , this is not quite the case , because in spite of their familiarity with computers , the students of today seem to be quite helpless in the face of more advanced computer applications , especially when they require some background knowledge of mathematics , statistics , or computer science ( as opposed to just computer use ) . however , because students are so familiar with and accustomed to computers , with a little help and guidance , as provided by the computer literacy for life sciences course , for example , they can achieve proper research - centered computer literacy quite easily . | computer literacy plays a critical role in today 's life sciences research . without the ability to use computers to efficiently manipulate and analyze large amounts of data resulting from biological experiments and simulations , many of the pressing questions in the life sciences could not be answered .
today 's undergraduates , despite the ubiquity of computers in their lives , seem to be largely unfamiliar with how computers are being used to pursue and answer such questions .
this article describes an innovative undergraduate - level course , titled computer literacy for life sciences , that aims to teach students the basics of a computerized scientific research pursuit .
the purpose of the course is for students to develop a hands - on working experience in using standard computer software tools as well as computer techniques and methodologies used in life sciences research .
this paper provides a detailed description of the didactical tools and assessment methods used in and outside of the classroom as well as a discussion of the lessons learned during the first installment of the course taught at emory university in fall semester 2009 . |
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whole slide digital imaging ( wsdi ) offers an alternative to glass slides for diagnostic interpretation . prior work has mainly focused on wsdi for frozen section interpretation , with fewer studies on routine pathologic diagnosis and secondary consults.[19 ] wsdi presents an attractive alternative to shipping glass slides for expert consultation in diagnostically challenging cases , eliminating turnaround time and potential damage or loss of diagnostically critical glass slides . our institution has provided digital slide consultations for 2 affiliated hospital , zhejiang university , china , since 2010 and has utilized digital pathology embedded within the department for research and tumor boards since 2008 . one high volume and diagnostically challenging area in pathology that could benefit from rapid expert consultation is the evaluation and grading of dysplasia in esophageal and gastric biopsies . we are unaware of prior studies addressing the use of wsdi for the evaluation of dysplasia in upper gi tract biopsies . thus , we undertook the present study to examine the accuracy and efficiency of wsdi in diagnosing gi dysplasia . both inter- and intraobserver variability in the glass slide diagnosis of esophageal dysplasia , even among expert pathologists , has been reported,[1013 ] and our intention in this study was not to repeat these prior studies . rather , given our prior experience with digital image diagnosis and our expertise in gastrointestinal pathology , we sought to establish whether diagnostic variability between glass slides and wsdi was similar or different than those reported for glass slide to glass slide evaluation . one hundred of cases of upper gi biopsies with diagnosis of negative , indefinite , low grade or high grade dysplasia were identified from the ucla pathology database . two of the authors ( dg and sd ) performed case selection and subsequent statistical analysis , but were not involved in interpreting digital images for this study . forty - two cases ( 37 esophageal and 5 gastric ) were selected for inclusion , as were a control set of 10 cases with classic diagnostic features ( tubular adenoma - 3 cases , sessile serrated adenoma - 2 cases , hyperplastic polyp , rectal adenocarcinoma , barrett 's esophagus without dysplasia , esophageal squamous mucosa with no dysplasia , and normal colonic mucosa ) . criteria for inclusion included cases in which the two case screeners agreed on the diagnosis and slides that were free of artifacts ( folded tissue , uneven sections , bubbles , thick sectioning , unusually dark or light h and e stains ) that can result in poor quality digital images . the final study set included 13 cases negative for dysplasia , 9 cases indefinite for dysplasia , 11 cases of low grade dysplasia , and 9 cases of high grade dysplasia [ figure 1 ] . degrees of dysplasia : negative ( a ) indefinite ( b ) low grade ( c ) high grade ( d ) ( hematoxylin - eosin stain , original magnification : 10 ) glass slides were anonymized to ensure pathologists were blinded to the diagnosis and scanned at 20x with an aperio xt scanner ( aperio technologies , vista , ca ) by translational pathology core laboratory ( ucla ) . the initial screeners ( dg and sd ) reviewed all digital images to ensure they were in focus and of high quality . four specialty trained gastro - intestinal pathologists ( bn , sh , dd , gc ) with varying experience as an attending pathologist ( 6 months , 3 years , 9 years , and 13 years of experience ) and computer skills reviewed the wsdi , then the glass slides after a minimum period of 34 weeks . digital images were reviewed on standard dell monitors with 1280 1024 pixel resolution and our computers were equipped with ati radeon hd2400xt video cards . final diagnosis , time required , problems encountered and whether or not the pathologist felt the need for 40x magnification were recorded for all cases . the time required for the diagnosis was recorded by each pathologist from the time the digital slide opened in the aperio viewer to diagnosis or from the time the slide was placed on the microscope stage to diagnosis . since the intention of this study was to examine variability in diagnosis between wsdi and glass slides , only intraobserver variability was calculated . three different sets of intraobserver variability statistics were calculated for each pathologist : wsdi to glass diagnosis for the set of control slides , wsdi to glass diagnosis for dysplasia and glass to glass diagnosis for dysplasia . for dysplasia cases , intraobserver variability was calculated using two different criteria , as used by previous authors : one approach used three clinically relevant categories ( negative , indefinite / low grade , high grade ) and the second approach used all four diagnostic categories ( negative , indefinite , low grade , and high grade ) . a major diagnostic discrepancy was defined as a diagnosis of negative for dysplasia in one media versus a finding of any other diagnosis ( indefinite , low grade , or high grade dysplasia ) in the other . a minor discrepancy was defined as a difference in the grade of dysplasia assigned . for the control slides , a minor diagnostic discrepancy was defined as a difference in dysplasia grade and a major diagnostic discrepancy was defined as any diagnosis other than that 's pathologist 's glass slide diagnosis or the diagnosis of the two authors who selected the cases . kappa coefficient can be thought of as the chance - corrected proportional agreement , and possible values range from + 1 ( perfect agreement ) to 0 ( no agreement above that expected by chance ) to 1 ( complete disagreement ) . all pathologists showed perfect correlation ( 1.0 ) between wsdi and glass diagnosis for the set of 10 control slides . all diagnoses also correlated perfectly with the diagnoses assigned by the two authors selecting the cases . as expected , in the set of dysplasia slides , all pathologists had major and minor discrepant diagnoses on wsdi to glass slide review and for glass to glass slide review . intraobserver kappa values for both wsdi to glass and glass to glass review were improved when calculated on the three clinically relevant categories [ table 1 ] . intraobserver k values for three clinically relevant categories and for four categories overall , there was more variation in kappa values for wsdi to glass interpretation . table 1 shows the kappa values for each pathologist as calculated by the three clinically relevant categories or by all four categories . for wsdi to glass review , the kappa statistics were 0.36 ( fair ) , 0.42 ( moderate ) , 0.71 ( good ) , and 0.78 ( good ) for the three clinically relevant categories and 0.28 ( fair ) , 0.37 ( fair ) , 0.63 ( good ) , and 0.75 ( good ) for all four categories . for glass to glass review , the kappa statistics were 0.58 ( moderate ) , 0.70 ( good ) , 0.70 ( good ) , and 0.75 ( good ) for the three clinically relevant categories and 0.50 ( moderate ) , 0.56 ( moderate ) , 0.64 ( good ) and 0.68 ( good ) for all categories . two pathologists had lower kappa values for wsdi in both sets of kappa calculations , while one pathologist had higher kappa value for wsdi [ table 1 ] . table 2 shows the percentage of discrepant diagnoses by pathologist for both wsdi - glass and glass glass review . with wsdi , total discrepant diagnoses ( major plus minor ) for each pathologist ranged from 17% to 55% . major discrepancies ranged from 7% to 29% and minor discrepancies from 10% to 33% [ table 2 ] . for glass glass review , total discrepant diagnoses ( major plus minor ) for each pathologist ranged from 21% to 36% ; major discrepancies ranged from 7% to 14% and minor discrepancies from 12% to 24% [ table 2 ] . no correlation was noted between a pathologist 's experience and percentage of discrepant diagnoses on wsdi interpretation . review time and discrepancies for each pathologist the specific diagnoses on glass and wsdi slides in the discrepant cases are listed in table 3 . discrepant dysplasia diagnoses between wsdi and glass slides occurred in 57 of 168 ( 34% ) total possible incidences ( 4 pathologists 42 slides = 168 incidences ) . in 14 of these 57 discrepancies ( 25% ) , this involved only one pathologist with a discrepancy between their glass and wsdi diagnosis . in 16 of the 57 discrepancies ( 28% ) , two pathologists were discrepant between their glass and wsdi diagnosis , and in 27 of the 57 incidences ( 47% ) , three of four pathologists had discrepancies between their glass and wsdi diagnosis . discrepant dysplasia diagnoses from glass to glass review occurred in 48 of 168 ( 29% ) total possible incidences . in 14 of these 48 incidences ( 29% ) , this involved only one pathologist with a discrepancy between the two passes on glass slide . in 9 of the 48 incidents ( 19% ) , two pathologists were discrepant , in 4 of the 48 incidents ( 8% ) three of four pathologists had discrepancies , and in 1 of the 47 incidents ( 2% ) all four pathologists were discrepant between their first and second review of the glass slides . specific diagnoses in the discrepant cases ( wsdi vs glass ) pathologists generally downgraded the degree of dysplasia on wsdi compared to the glass slides [ tables 4 and 5 ] . dysplasia downgrading occurred in 53 of 60 wsdi cases ( 88% ) and in 24 of 48 second review glass cases ( 50% ) . for wsdi , this included 26 of 29 cases ( 90% ) with major discrepancies [ table 3 ] . seven cases were upgraded on wsdi by three of the four pathologists ; three cases indefinite on wsdi were called negative on glass , two cases low grade on wsdi were diagnosed as indefinite on glass , and two cases high grade on wsdi were interpreted as low grade on glass . scanning at 40 was requested in 10 cases ; in 4 of these cases , the pathologist 's diagnosis was discrepant between wsdi and glass images . dysplasia downgrading or upgrading ( wsdi vs glass ) dysplasia downgrading or upgrading ( glass pass#1 vs glass pass#2 ) wsdi review required more time than glass slide review ( range 50% to 400% longer ) , depending on the case and the pathologist [ table 2 ] . the time required for glass slide review was similar for the first and second glass slide review . other comments for wsdi interpretation included three cases for which the pathologist mentioned that the location of the biopsy would have helped and one case was far too blue / dark to accurately evaluate nuclear atypia . prior studies in frozen section correlation and second opinion diagnoses have indicated that digital slide review can enhance efficient use of expert pathologists time , increase the experience of reviewers for frozen sections and result in more rapid second opinion diagnoses.[14 ] the number of hospitals and reference labs with scanners continues to increase , and it is likely this will increasingly become part of routine pathology practice . wsdi has been utilized by the ucla department of pathology since 2008 , including substantial experience with the use of wsdi for tumor boards and research studies . since 2010 , prior internal studies involving digital interpretation of routine gi cases show our pathologists have perfect concordance ( kappa 1.0 ) between wsdi and glass slides for routine cases such as gastric adenocarcinoma , barrett 's esophagus without dysplasia , chronic gastritis with or without h. pylori and candida esophagitis . however , we wished to further establish whether there might be difficulties in digital interpretation of more diagnostically challenging cases such as gi dysplasia , especially as questions related to gi dysplasia represent a large part of our current glass slide consultation practice . indeed , for wsdi interpretation to become fully integrated into pathologists routine practice , wsdi interpretations need to be comparable to their glass slide interpretations for all cases , both routine and challenging . our results show a greater range of intraobserver variability for wsdi to glass versus glass to glass interpretation among a group of four specialty trained gi pathologists . as reported by others , our kappa values were higher when clinically relevant categories ( negative , indefinite / low grade , high grade ) were considered . kappa values for three clinically relevant categories ( negative , indefinite / low grade , high grade dysplasia ) on wsdi were fair for one pathologist ( 0.36 ) , moderate for one pathologist ( 0.42 ) and good for two pathologists ( 0.71 and 0.78 ) ; by contrast , for glass - to - glass kappa values were moderate for one pathologist ( 0.58 ) and good for three pathologists ( 0.70 , 0.70 , and 0.75 ) . our kappa values for intraobserver variation of dysplasia for glass - to - glass interpretation are similar to that previously reported by montgomery and colleagues . in that study , kappa values ranged from 0.64 to 0.68 for the clinically relevant categories and 0.420.76 for the distinct histologic categories , compared to our values of 0.580.75 and 0.500.68 , respectively . one pathologist in our group showed a large jump in kappa scores between media , from a fair score for wsdi - glass ( 0.42 ) to the highest kappa score ( 0.75 - good ) for glass - glass review . interestingly , this pathologist reported a lack of diagnostic confidence in several wsdi cases ( 10 of 42 total cases , or 24% ) , and indicated the need for glass slide review in these cases to render an accurate diagnosis . this suggests that pathologists will have different degrees of comfort in adapting this new technology to their practice , especially for more subtle and problematic diagnoses ( like dysplasia grading ) as compared to diagnosing cancer versus normal tissue . another pathologist had the lowest kappa score on both wsdi and glass , which suggests that intrinsic factors that account for intraobserver variability may not be significantly influenced by the medium ( digital versus glass ) used for interpretation . these findings suggest that pathologists who decide to use wsdi for interpretation of gi dysplasia cases may benefit from regular , on - going , re - review of paired digital and glass images for continuing education , at least in the early stages of adoption . the most unexpected and significant result of our study is the finding that pathologists overwhelmingly downgraded dysplasia grade in wsdi versus glass slides . we did not expect this result at the beginning of the study , and thus did not design our study to address this finding specifically . possible causes include scanning magnification , color balance / dynamic range of the wsdi relative to glass slides , quality / resolution of the viewing monitors , and pathologist experience with dysplasia diagnosis using digital microscopy . while the scanning magnification used in this study ( 20 ) could play a role , pathologists only indicated the further need for 40 magnification in 10 out of a total of 168 cases . moreover , only 4 discrepant diagnoses were found out of the 10 cases where 40 magnification was requested . thus , while scanning magnification may have contributed to some discrepant diagnoses , it did not account for all discrepancies . in our study , pathologists used their standard computer monitor for imaging viewing . it is possible that interpretation using a high resolution monitor ( such as barco coronis fusion wide - screen diagnostic color display system ) or a larger monitor would have led to different results . one pathologist did mention that one wsdi was too blue and dark to interpret nuclear detail properly ; it is possible that subtle variations in color balance or dynamic range in the digital images led to misinterpretation of nuclear features which are critical for the evaluation of dysplasia . finally , it is possible that , with experience , there would be fewer cases of downgrading on digital images . wsdi took two to four times longer than glass slide interpretation for all pathologists . with multiple slides and cases pathologists may become faster in wsdi interpretation with experience , and we plan additional studies to address this question . however , for the present , wsdi does present additional technical challenges relative to glass slides . these include the speed with which images can be loaded into the viewer and the quality of the display monitor . furthermore , most digital slide viewers continue to rely on standard computer accessories ( mouse and keyboard ) that limit efficiency and ease of viewing . differences in hardware ( scanners ) and software ( viewers ) may impact the pathologist 's interpretation of the image reviewed or their diagnostic confidence . scanning at 40 takes significantly longer than 20 scans , and varies depending on the size of tissue being scanned and the scanner itself . we find 40 scans require from 50% to 500% more scanning time in our laboratory and result in files several fold larger than those of 20x scans . in a routine high throughput clinical practice , these incremental differences could significantly impact scanning time and server storage space . as a profession , we believe several of the concerns raised by this study ( time required , discrepancies , confidence in diagnosis ) are related to our limited experience with digital dysplasia diagnosis and speculate these problems will dissipate with increased usage of the digital platform . moreover , the rapid improvements witnessed in the hardware and software used for digital slide scanning and interpretation also would be expected to mitigate some of these concerns . in conclusion , our results indicate that pathologists can interpret upper gi dysplasia similarly on wsdi and glass slides , and thus wsdi potentially can be used for clinical diagnosis . however , some pathologists may encounter more challenges than others in using wsdi for this purpose . pathologists who adopt wsdi for the evaluation of gi dysplasia cases presumably should benefit from regular , on - going , re - review of paired digital and glass images for continuing education , at least in the early stages of adoption . furthermore , until additional studies are available , we believe our data support a judicious approach to digital dysplasia interpretation and indicate that , in some cases , pathologists may prefer to review the glass slides in order to render a diagnosis with confidence . | background : whole slide digital imaging ( wsdi ) offers an alternative to glass slides for diagnostic interpretation . while prior work has concentrated on the use of whole slide digital imaging for routine diagnostic cases , this study focuses on diagnostic interpretation of digital images for a highly challenging area , upper gastro - intestinal ( gi ) dysplasia . the aim of this study is to study the accuracy and efficiency of wsdi in the diagnosis of upper gi tract dysplasia.materials and methods : forty - two hematoxylin and eosin ( h and e)-stained slides representing negative , indefinite , low grade and high grade dysplasia were selected and scanned at 20x ( aperio xt ) .
four attending gi pathologists reviewed the wsdi , then glass slides , with at least 34 weeks between each media ; glass slides were re - reviewed 1618 months later.results:intraobserver variability for three clinically relevant categories ( negative , indefinite / low grade , high grade ) was wider for wsdi to glass ( kappa range 0.360.78 ) than glass to glass ( kappa range 0.580.75 ) . in comparison to glass slide review ,
wsdi review required more time and was associated with an unexpected trend toward downgrading dysplasia.conclusions:our results suggest : ( 1 ) upper gi dysplasia can be diagnosed using wsdi with similar intraobserver reproducibility as for glass slides ; however , this is not true for all pathologists ; ( 2 ) pathologists may have a tendency to downgrade dysplasia in digital images ; and ( 3 ) pathologists who use wsdi for interpretation of gi dysplasia cases may benefit from regular , on - going , re - review of paired digital and glass images to ensure the most accurate utilization of digital technology , at least in the early stages of implementation . |
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postmortem examinations were conducted on 1,620 red deer and 796 roe deer shot by hunters during hunting seasons in 20052008 ( technical appendix ) . sex , age , body condition , and macroscopic aspects of hooves , mucosae , and internal organs were recorded . were detected by using a competition elisa kit ( id - vet , montpellier , france ) . results were expressed as percentage negativity compared with kit negative control serum , and cutoff values were established . serum samples from 80 red deer and 40 roe deer were also analyzed by using a virus neutralization ( vn ) test for btv-1 and btv-8 . spleen samples obtained in 2006 and 2007 were used for detection of btv rna segment 5 and cellular -actin transcripts by reverse transcription quantitative pcr ( rt - qpcr ) according to a modified procedure of toussaint et al . ( 2 ) . to assess performance of the elisa , we performed receiver operating characteristic analysis . to estimate effects of potential factors ( sex and age , year , month and area of sampling ) on risk for seropositivity , we used a multivariate logistic regression model . a total of 237 pairs of elisa and rt - qpcr results from red deer were used for receiver operating characteristic analysis , which yielded an area under the curve of 0.811 and a cutoff value for the elisa that maximized sensitivity ( 86% ) and specificity ( 98% ) . serologic status was defined as positive ( < 66% ) , doubtful ( > 66%<75% ) , or negative ( > 75% ) and was comparable to that found for domestic ruminants ( 3 ) . for btv-8 , concordance between elisa and vn results was 95% for red deer and 82% for roe deer . neutralizing antibodies to btv-1 were not detected . from 2006 on , no gross lesions compatible with bluetongue disease were found . in 2005 , for hunting seasons in 2006 , 2007 , and 2008 , seroprevalences were 1.51% ( 95% confidence interval [ ci ] 0.89%2.07% ) , 52.33% ( 95% ci 49.91%54.78% ) , and 33.95% ( 95% ci 31.64%36.26% ) for red deer and 2.56% ( 95% ci 1.43%3.60% ) , 2.75% ( 95% ci 1.62%3.90% ) , and 1.67% ( 95% ci 0.75%2.51% ) for roe deer and showed a significant difference between species ( p<0.0001 by cochran - mantel test ) . unimodal negatively skewed distributions of percentage negativity in 2005 and 2006 likely reflect seronegative populations . conversely , bimodal profiles in 2007 and 2008 are compatible with ongoing infections in the red deer population . spatial evolution of humoral responses in red deer is shown in figure 2 . in 2006 , seropositive animals were detected in only 5 districts , of which 4 were the most eastern districts among the 20 sampled ; deer in most districts were infected in 2007 ( 22/25 ) and 2008 ( 17/21 ) . frequency distribution of results of a competitive elisa for detecting antibodies against bluetongue virus in serum samples from roe deer ( white columns ) and red deer ( black columns ) during the hunting seasons of a ) 2005 , b ) 2006 , c ) 2007 , and d ) 2008 , belgium . hunting was conducted in 30 ( area 12,851 km ) of 37 ( area 16,844 km ) forest districts known to contain wild cervids . the study population of wild cervids in southern belgium ( 4930n5048n ) is estimated to be 11,000 red deer ( cervus elaphus ) and 33,000 roe deer ( capreolus capreolus ) . serum samples with a percentage negativity value ( relative to the negative control serum ) < 66 were considered positive . distribution of red deer samples obtained in belgium ( wallonia ) in a ) 2005 , b ) 2006 , c ) 2007 , and d ) 2008 , and location of forest districts . white circles indicate districts where only seronegative animals were detected , and black circles indicate districts where seropositive animals were detected . multiple logistic regression analysis showed that risk for seropositivity was significantly affected by age ( 84.53 , p<0.0001 ) , year ( 282.75 , p<0.0001 ) , and location of sampling ( 63.10 , p<0.0001 ) , but not by sex ( 0.19 , p>0.90 ) or month of sampling ( 2.45 , p>0.45 ) . seroprevalence was lower for juveniles than for subadults ( odds ratio 2.11 , 95% ci 1.473.04 ) and adults ( odds ratio 3.79 , 95% ci 2.854.62 ) . the decrease in 2008 was significant only for juveniles , and the seropositivity gradient decreased toward southern part of the study region ( table ) . for roe deer , adults , > 2 y of age ; subadults , 12 y of age ; juveniles , < 1 y of age . fourteen forest districts were distributed among 3 nonadjacent areas , 5 in the eastern area , 4 in the central area , and 5 in the southern area . the 343 spleen samples ( 230 red deer and 113 roe deer ) tested by rt - qpcr in 2006 and the 193 samples ( roe deer ) tested in 2007 were negative for btv rna . conversely , 14% ( 33/237 ) of red deer -actin positive samples ( 237/331 ) were positive for btv rna ( technical appendix ) . these 33 animals did not have gross lesions suggestive of bluetongue disease . of 32 serum samples available , 26 were seropositive , 1 was doubtful , and 5 were seronegative , which suggested that these animals had been infected recently . two pan - btv rna positive spleen fragments , sampled at the end of hunting season in 2008 , were reassessed by using a btv-8 genotype our study provides evidence that btv-8 infects wild cervid populations in belgium . for red deer , a few infections occurred in 2006 in the eastern part of belgium , i.e. , the area in which the ovine and bovine cases had been detected ( 5 ) . in 2007 , the infection spread west and southwest , and its seroprevalence increased . in 2008 , distribution remained stable but overall seroprevalence decreased , mostly among juveniles . a large number of serum samples showed percentage negativity values between positive and negative values , which is indicative of ongoing virus transmission by vector midges during the hunting season . more animals were infected in eastern and central than in southern wallonia , a finding that resembles the spatial distribution of the virus in livestock and might be correlated with lower density of cattle populations and cooler temperatures in hilly southern districts ( 6 ) . the proportion of seropositive animals increased with age , probably resulting more from prolonged exposure of adults to the vector , than to any resistant status of juveniles . decreasing overall seroprevalence in 2008 might be caused by mandatory vaccination of domestic ruminants and spontaneously acquired herd immunity within the red deer population , which reduced virus prevalence among insect vector populations in 2008 . because seropositive subadults and adults sampled in 2008 could have been infected in 2007 , seroprevalence among juveniles should more accurately reflect the level of exposure to infected insect vectors . if this explanation was true , the decrease in seroprevalence among juveniles in 2008 would confirm reduction of the virus insect reservoir . although red deer and roe deer samples were collected at the same locations and during the same hunting events , seroprevalence was lower among roe deer . because 5 of 12 elisa - positive and none of the elisa - negative roe deer serum samples were positive by btv-8 vn test and a similar between - species pattern was found by rt - qpcr , poor sensitivity of the elisa in roe deer as the cause of between - species difference can be ruled out . host - related and vector - related factors might account for this difference . because red deer live in large groups and move more , they might be more exposed to insects / pathogens than are roe deer , which live in small groups in winter and have smaller home - range sizes and are seasonally territorial ( 7 ) . midge feeding patterns reported variations in host attractiveness , which could correlate with exhaled carbon dioxide and acetone ( 8) , specific phenolic compounds emitted from urine ( 9 ) or hair fragrance ( 10 ) . btv might be maintained in an as yet unknown reservoir host population with a long or relapsing viremia and in which clinical signs are absent . because no excess illness or death occurred in 20072008 , btv-8 infection of wild cervids is probably benign enough to go unnoticed . some spleen samples from dead red deer found during winter also showed positive results by rt - qpcr even if btv was not the cause of death ( a. linden , unpub . data ) . coupled with the high seroprevalence we report , the possibility that red deer are btv reservoirs warrants further investigation . | to investigate bluetongue virus serotype 8 infection in belgium , we conducted a virologic and serologic survey on 2,416 free - ranging cervids during 20052008 .
infection emerged in 2006 and spread over the study area in red deer , but not in roe deer . |
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we consecutively screened entire glass slides of 105 radical nephrectomy specimens diagnosed with rcc from the archives of the pathology department , that were collected between january 2000 and december 2007 . from among these tissue sufficiency was defined as the presence of renal parenchyma that contained more than 50 glomeruli located at least 5-mm from the tumor tissue to avoid tumor effects . electronic medical charts were reviewed to exclude cases with other pre - existing or concurrent neoplasms ( except for the presence of hypertension or diabetes ) . one paraffin tissue block was retrieved for each case fulfilling the definition of tissue sufficiency described above . sections were cut 4-m thick and stained with hematoxylin and eosin and periodic acid - schiff methods . the histologic review was performed without knowledge of clinical information ( by m.j.k . and b.j.l . ) . non - neoplastic lesions in glomerular , tubular , interstitial , and vascular compartments were evaluated quantitatively and semi - quantitatively . the percentage of globally and segmentally sclerosed glomeruli and the presence of glomerular hyalinosis were recorded . the extent of global glomerulosclerosis was graded as follows : grade 0 , no global glomerulosclerosis ; grade 1 , global glomerulosclerosis < 10% ; grade 2 , global glomerulosclerosis from 11% to 20% ; and grade 3 , global glomerulosclerosis > 20% . tubular atrophy , interstitial fibrosis , interstitial inflammation , and arteriolar hyalinosis were semi - quantitatively graded from 0 to 3 according to the banff classification.8,9 the presence of nephrocalcinosis was also recorded . first , arterial intima fibrous thickening was graded from 0 to 3 as follows : grade 0 , no sub - intimal fibrosis ; grade 1 , mild sub - intimal sclerosis without significant occlusion of the arterial lumen ( < 10% ) ; grade 2 , moderate sub - intimal sclerosis with arterial lumen occlusion < 50% ; and grade 3 , severe occlusion of > 50% arterial lumen associated with thinning of the muscle layer and replacement by fibrous tissue.5 secondly , intimal thickness and outer diameter ( od ) were measured in arteries with an od between 150 and 300 m , and the ratio was expressed as a percentage of od , which is referred to as r.10,11 the od was measured along the shortest axis from one outer media to the other , excluding the adventitia . diabetic nephropathy was diagnosed and classified according to tervaert et al.12 : class i , glomerulomegaly and isolated glomerular basement membrane thickening ; class ii , mesangial expansion ; class iii , nodular glomerulosclerosis ; and class iv , advanced diabetic glomerulosclerosis . gender and age at the time of surgery and the presence of hypertension or diabetes were recorded . serum creatinine levels were obtained preoperatively , and one and five years after surgery in 69 , 59 , and 45 cases , respectively . estimated glomerular filtration rate ( egfr ) was calculated using the abbreviated modification of diet in renal disease equation : 175plasma creatinineage ( 0.742 if female).13 chronic kidney disease was defined as egfr<60 ml / min/1.73 m. data was analysed using sas ver a linear mixed model was used to assess correlations among histologic findings , clinical features , and egfr . post - hoc analysis was used to assess the impact of global glomerulosclerosis on egfr . we performed t - tests and chi - square tests to compare features between genders . we consecutively screened entire glass slides of 105 radical nephrectomy specimens diagnosed with rcc from the archives of the pathology department , that were collected between january 2000 and december 2007 . from among these tissue sufficiency was defined as the presence of renal parenchyma that contained more than 50 glomeruli located at least 5-mm from the tumor tissue to avoid tumor effects . electronic medical charts were reviewed to exclude cases with other pre - existing or concurrent neoplasms ( except for the presence of hypertension or diabetes ) . one paraffin tissue block was retrieved for each case fulfilling the definition of tissue sufficiency described above . sections were cut 4-m thick and stained with hematoxylin and eosin and periodic acid - schiff methods . the histologic review was performed without knowledge of clinical information ( by m.j.k . and b.j.l . ) . non - neoplastic lesions in glomerular , tubular , interstitial , and vascular compartments were evaluated quantitatively and semi - quantitatively . the percentage of globally and segmentally sclerosed glomeruli and the presence of glomerular hyalinosis were recorded . the extent of global glomerulosclerosis was graded as follows : grade 0 , no global glomerulosclerosis ; grade 1 , global glomerulosclerosis < 10% ; grade 2 , global glomerulosclerosis from 11% to 20% ; and grade 3 , global glomerulosclerosis > 20% . tubular atrophy , interstitial fibrosis , interstitial inflammation , and arteriolar hyalinosis were semi - quantitatively graded from 0 to 3 according to the banff classification.8,9 the presence of nephrocalcinosis was also recorded . first , arterial intima fibrous thickening was graded from 0 to 3 as follows : grade 0 , no sub - intimal fibrosis ; grade 1 , mild sub - intimal sclerosis without significant occlusion of the arterial lumen ( < 10% ) ; grade 2 , moderate sub - intimal sclerosis with arterial lumen occlusion < 50% ; and grade 3 , severe occlusion of > 50% arterial lumen associated with thinning of the muscle layer and replacement by fibrous tissue.5 secondly , intimal thickness and outer diameter ( od ) were measured in arteries with an od between 150 and 300 m , and the ratio was expressed as a percentage of od , which is referred to as r.10,11 the od was measured along the shortest axis from one outer media to the other , excluding the adventitia . diabetic nephropathy was diagnosed and classified according to tervaert et al.12 : class i , glomerulomegaly and isolated glomerular basement membrane thickening ; class ii , mesangial expansion ; class iii , nodular glomerulosclerosis ; and class iv , advanced diabetic glomerulosclerosis . gender and age at the time of surgery and the presence of hypertension or diabetes were recorded . serum creatinine levels were obtained preoperatively , and one and five years after surgery in 69 , 59 , and 45 cases , respectively . estimated glomerular filtration rate ( egfr ) was calculated using the abbreviated modification of diet in renal disease equation : 175plasma creatinineage ( 0.742 if female).13 chronic kidney disease was defined as egfr<60 ml / min/1.73 m. data was analysed using sas ver . 9.2 ( sas institute inc . , cary , nc , usa ) . a linear mixed model was used to assess correlations among histologic findings , clinical features , and egfr . post - hoc analysis was used to assess the impact of global glomerulosclerosis on egfr . we performed t - tests and chi - square tests to compare features between genders . the study group included 57 males ( 67.1% ) and 28 females ( 32.9% ) . stages of rcc at diagnosis for t1 , t2 , and t3 were 56 cases ( 65.9% ) , 15 cases ( 17.6% ) , and 14 cases ( 16.5% ) , respectively . six patients ( 7.1% ) had both hypertension and diabetes at the time of operation . various non - neoplastic lesions were observed in the renal parenchyma ( table 1 ) . the most common findings were tubular atrophy , arteriosclerosis , and glomerulosclerosis , which were present in 94.1% , 90.6% , and 88.2% , respectively . focal segmental glomerular sclerosis was observed in 10 cases ( 11.8% ) and associated with hyalinosis in three cases . enlarged glomeruli greater than 250 m in bowman 's diameter were frequently found in the cortex of four cases ( 4.7% ) . tubular atrophy and interstitial fibrosis involving more than 25% of the cortical area ( grades 2 and 3 ) were present in 14.1% and 10.6% of cases , respectively . interstitial inflammation occupying more than 25% of the cortical interstitium was observed in 9.4% of patients . the ratio of arterial intimal thickness to the arterial wall , revealed as r , in 150- to 300-m arteries was 12:43 . chronic pyelonephritis was observed in four cases , revealing focal destructive tubular atrophy , interstitial inflammation , and interstitial fibrosis . diabetic nephropathy was diagnosed in two cases , while advanced nodular glomerulosclerosis ( class iii ) and an early lesion ( class i ) were each noted in one case . among the original 85 cases , 69 , 59 , and 45 had preoperative data and 1- and 5-year follow - up data , respectively . chronic kidney disease was present in 12 cases preoperatively and 21 cases five years after surgery . twenty cases were lost to follow - up after surgery . of the remaining cases , we could find a record of death in three cases with stage t1b at the time of surgery . two cases died of cancer with distant metastasis at seven and 10 years after nephrectomy . the linear mixed model was applied to examine histologic factors that might predict egfr at surgery and one and five years after radical nephrectomy . among these tumor size and other histologic parameters , including the grades of tubular atrophy , interstitial inflammation , interstitial fibrosis , arteriosclerosis , and small arterial intimal thickness ( r ) were not correlated with egfr ( table 2 ) . they were all males with an average age of 61.7 years ( median , 65 years ) . , they also had moderate to severe degrees of arteriosclerosis . excluding two cases without available egfr values , four of the seven cases had chronic renal disease defined by egfr<60 ml / min/1.73 m at the time of surgery ( cases 1 , 3 , 6 , and 9 ) . in the remaining three cases , two cases ( cases 2 and 5 ) progressed to chronic renal disease one year after surgery ( table 3 ) . when histologic features were compared between genders , males showed significantly more severe renal histologic injuries than females . preoperative and postoperative egfr values were significantly lower in males than in females ( table 4 ) . consequently , in the analytic model including both clinical and histologic parameters , age and gender were significantly correlated with egfr ( p=.0038 and p<.001 , respectively ) , but glomerulosclerosis was not ( p=.0964 ) . the study group included 57 males ( 67.1% ) and 28 females ( 32.9% ) . stages of rcc at diagnosis for t1 , t2 , and t3 were 56 cases ( 65.9% ) , 15 cases ( 17.6% ) , and 14 cases ( 16.5% ) , respectively . six patients ( 7.1% ) had both hypertension and diabetes at the time of operation . various non - neoplastic lesions were observed in the renal parenchyma ( table 1 ) . the most common findings were tubular atrophy , arteriosclerosis , and glomerulosclerosis , which were present in 94.1% , 90.6% , and 88.2% , respectively . focal segmental glomerular sclerosis was observed in 10 cases ( 11.8% ) and associated with hyalinosis in three cases . enlarged glomeruli greater than 250 m in bowman 's diameter were frequently found in the cortex of four cases ( 4.7% ) . tubular atrophy and interstitial fibrosis involving more than 25% of the cortical area ( grades 2 and 3 ) were present in 14.1% and 10.6% of cases , respectively . interstitial inflammation occupying more than 25% of the cortical interstitium was observed in 9.4% of patients . the ratio of arterial intimal thickness to the arterial wall , revealed as r , in 150- to 300-m arteries was 12:43 . chronic pyelonephritis was observed in four cases , revealing focal destructive tubular atrophy , interstitial inflammation , and interstitial fibrosis . diabetic nephropathy was diagnosed in two cases , while advanced nodular glomerulosclerosis ( class iii ) and an early lesion ( class i ) were each noted in one case . among the original 85 cases , 69 , 59 , and 45 had preoperative data and 1- and 5-year follow - up data , respectively . chronic kidney disease was present in 12 cases preoperatively and 21 cases five years after surgery . twenty cases were lost to follow - up after surgery . of the remaining cases , we could find a record of death in three cases with stage t1b at the time of surgery . two cases died of cancer with distant metastasis at seven and 10 years after nephrectomy . the linear mixed model was applied to examine histologic factors that might predict egfr at surgery and one and five years after radical nephrectomy . among these tumor size and other histologic parameters , including the grades of tubular atrophy , interstitial inflammation , interstitial fibrosis , arteriosclerosis , and small arterial intimal thickness ( r ) were not correlated with egfr ( table 2 ) . they were all males with an average age of 61.7 years ( median , 65 years ) . , they also had moderate to severe degrees of arteriosclerosis . excluding two cases without available egfr values , four of the seven cases had chronic renal disease defined by egfr<60 ml / min/1.73 m at the time of surgery ( cases 1 , 3 , 6 , and 9 ) . in the remaining three cases , two cases ( cases 2 and 5 ) progressed to chronic renal disease one year after surgery ( table 3 ) . when histologic features were compared between genders , males showed significantly more severe renal histologic injuries than females . preoperative and postoperative egfr values were significantly lower in males than in females ( table 4 ) . consequently , in the analytic model including both clinical and histologic parameters , age and gender were significantly correlated with egfr ( p=.0038 and p<.001 , respectively ) , but glomerulosclerosis was not ( p=.0964 ) . recently published renal cancer checklists recommend that abnormal histologic findings in non - neoplastic kidney be reported.14 in practice , however , concurrent non - neoplastic lesions / diseases are often neglected or omitted in pathologic reports . in a review of our original surgical pathology reports , only seven cases ( 8.2% ) had records of non - neoplastic findings . the major reason for omission is that pathologists probably consider the renal parenchyma relatively well preserved or with minimal alterations in most cases . however , rcc patients are older and have a high prevalence of hypertension and diabetes , which may negatively impact postoperative renal function.15,16 although partial nephrectomy is advocated , especially in patients with hypertension or diabetes,17 radical or total nephrectomy is still a major surgical procedure for rcc . nonetheless , patient survival rate after surgery has progressively increased as tumors are being detected early , and also as new therapeutic target drugs are used for advanced cases.18 obesity and hyperlipidemia are also frequently found in older patients and may jeopardize renal function as they live longer . in that regard , nephrectomy tissue provides a good opportunity to examine the non - neoplastic renal parenchyma to obtain information on the histologic factors influencing postoperative renal function . it was present in 17.4% preoperatively and its proportion increased to 46.7% five years after surgery . among the histologic parameters assessed , only global glomerulosclerosis correlated with egfr at one and five years postsurgery . six of the seven patients with > 20% glomerulosclerosis had egfr values < 60 ml / min/1.73 m at one year after surgery . two - thirds of them had other clinical risk factors that could affect renal function . however , the presence of hypertension and diabetes did not correlate with egfr ( p=.705 and p=.515 , respectively ) . although glomerulosclerosis was correlated with egfr , its significance disappeared when age and gender were considered . preoperative and postoperative egfr values were significantly higher in females , likely due to the higher percentages of glomerulosclerosis , interstitial inflammation , and fibrosis in males ( table 4 ) . estrogen has been shown to protect renal endothelial barrier function in vitro and in vivo.19 estrogen exerts beneficial effects on the kidney by decreasing renin levels , angiotensin - converting enzyme activity , and aldosterone production.20 age also correlated with declining egfr . according to the formula ( age/2)-10,21 nine cases presented glomerulosclerosis over the age limit . to our surprise , all patients with > 20% glomerulosclerosis were male and also had glomerulosclerosis percentages over the age limit ; these overlaps might obscure the effects of glomerulosclerosis in the study results . interstitial fibrosis was moderate to severe in 10.6% and one - third of cases were associated with > 20% glomerulosclerosis . cases with moderate to severe arterial intimal thickening were more frequently observed and associated with hypertension , but did not correlate with renal histology either . we hypothesized that sampling error might be a major factor explaining for discrepancy with other studies.5 although we set a point of 5-mm from the tumor margin to exclude tumoral effects , it is not clear whether 5-mm is a safe limit , regardless of tumor size or associated conditions . in a study of hepatocellular carcinoma , parenchyma within 10-mm of tumor tissue still showed significant histologic changes related to the tumor.22 there has been no consensus on the size of specimen sufficient for the evaluation . we selected cases that contained more than 50 glomeruli , but tissue composition was variable . since we graded vascular wall thickening in the mostly affected arteries rather than according to average wall thickness , the grade may not represent the fibrotic areas affected by the vessel . diabetic nephropathy is the most common cause of end - stage renal disease in korea.23 although diabetes is reported to be frequent in rcc patients , only seven patients were diagnosed with diabetes before surgery , and just two of them showed morphologic features of diabetic nephropathy : one exhibited nodular glomerulosclerosis ( class iii ) and the other only showed glomerulomegaly ( class i ) . in addition , glomerulomegaly was present in two cases without a history of diabetes at the time of surgery . we suspected these two cases as having early diabetic nephropathy , as they were diagnosed with diabetes during follow - up . nephromegaly , including glomeruli and tubules , is an early feature of diabetic nephropathy and may appear even before the clinical recognition of glucose intolerance . however , clinicopathologic correlation is necessary because glomerulomegaly may develop in obesity24 as a secondary adaptation or a congenitally low number of nephrons.25 nephrocalcinosis was not rare in our study , but calcified lesions were not extensive and had no specific causes . as we measured the thickness of vessels with ods of 150 to 300 m , we might have missed histologic changes in smaller resistance vessels . however , arteriolar hyalinosis was rare , and glomerulosclerosis was well correlated with egfr . finally , it is not clear whether age and gender factors are more important than histologic factors . non - neoplastic lesions and the percentages of glomerulosclerosis should be described in surgical pathology reports to provide additional information on post - operative renal functional decline . | backgroundpre - existing non - neoplastic renal diseases or lesions may influence patient renal function after tumor removal .
however , its description is often neglected or omitted in pathologic reports . to determine the incidence and clinical significance of non - neoplastic lesions , we retrospectively examined renal tissues obtained during 85 radical nephrectomies for renal cell carcinoma.methodsone paraffin - embedded tissue block from each case containing a sufficient amount of non - tumorous renal parenchyma was cut and processed with hematoxylin and eosin and periodic acid - schiff methods .
non - neoplastic lesions of each histological compartment were semi - quantitatively and quantitatively evaluated.resultsamong the various histologic lesions found , tubular atrophy , arterial intimal thickening , and glomerulosclerosis were the most common ( 94.1% , 91.8% , and 88.2% , respectively ) .
glomerulosclerosis correlated with estimated glomerular filtration rate at the time of surgery , as well as at 1- and 5-years post - surgery ( p=.0071 ) , but tubulointerstitial fibrosis or arterial fibrous intimal thickening did not .
post - hoc analysis revealed that glomerulosclerosis of more than 20% predicted post - operative renal function .
however , its significance disappeared when gender and age were considered.conclusionsin conclusion , non - neoplastic lesions , especially with regard to glomerulosclerosis percentage , should be described in pathology reports to provide additional information on renal function decline . |
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oxaliplatin is a third - generation platinum - based chemotherapeutic drug and is mainly used for the treatment of colorectal cancer . however , oxaliplatin produces numerous side effects ( e.g. , acute neurotoxicity and chronic peripheral neuropathy ) that the regulation has extremely difficult . although cold dysesthesia is a characteristic symptom in oxaliplatin - induced peripheral neuropathy , its underlying mechanisms are not completely understood . oxalate , a metabolite of oxaliplatin , has been reported to be involved in cold dysesthesia.3 , 4 our previous report has also been shown that transient receptor potential melastatin 8 ( trpm8 ) , characterized as a cold - sensing cation channel5 , 6 , 7 , 8 is involved in oxaliplatin - induced cold dysesthesia.9 , 10 in oxaliplatin - treated patients , several drugs ( e.g. , calcium gluconate / magnesium sulfate , glutathione , carbamazepine , gabapentin , amifostine , acetyl - l - carnitine and -lipoic acid ) are used for the management of the peripheral neuropathy.3 , 11 although the above drugs are effective against mild neuropathy,3 , 11 the regulation is still difficult . shakuyakukanzoto ( skt : sho yo gn co tng ) is a traditional herbal formulation consisting of two herbal medicines : paeoniae radix ( sho yo ) and glycyrrhizae radix ( gn co ) . skt is effective in treating muscle pain , muscle spasms , joint pain , and numbness in patients.12 , 13 , 14 , 15 in rodents ( rats and mice ) , skt inhibits chemotherapy - induced mechanical allodynia . however , it is unknown whether skt is effective in oxaliplatin - induced cold dysesthesia . thus , in this study , we investigated the effects of skt on oxaliplatin - induced cold allodynia in mice . male c57bl/6ncr mice ( 6 week old at the start of the experiment ; japan slc ltd , hamamatsu , japan ) were used . five animals were housed per cage under controlled temperature ( 2123 c ) , humidity ( 4565% ) , and light ( 7:00 am to 7:00 pm ) conditions . oxaliplatin was purchased from sigma - aldrich ( st . louis , mo , usa ) and was dissolved in 5% glucose . oxaliplatin was injected intraperitoneally ; the dose ( 3 mg / kg ) was selected from our previously published report and was in accordance with the recommended clinical dose of 85 mg / m body surface area , which corresponds to a dose of 2.4 mg / kg in a person of 170 cm body height and 60 kg weight . dried extracts of skt ( lot . 2020068010 ) were obtained from tsumura and co. ltd ( tokyo , japan ) and were dissolved in 5% gum arabic . skt or the vehicle was administered orally once daily on day 1 and 2 of oxaliplatin injection . 3-[7-(trifluoromethyl)-5-[2-(trifluoromethyl)phenyl]-1h - benzimidazol-2-yl]-1-oxa-2-azaspiro[4.5]dec-2-ene ( tc - i 2014 ) ( tocris bioscience , bristol , uk ) was dissolved in 20% 2-hdroxypropyl--cyclodextrin and was administered orally on day 3 of oxaliplatin injection . mice were placed individually in a plastic cage ( 110 180 150 mm ) with a wire mesh bottom . after an acclimation of at least 60 min , acetone ( 10 l ) was applied to the plantar skin . the escape response was observed immediately after acetone application in not only oxaliplatin - treated mice but also healthy mice . thus , this response was not evaluated . the reaction following it was scored as follows : 0 , no response ; 1 , lifting of the hind paw ; 2 , flinching or licking of the hind paw . the stimulus was applied 2 times to each hind paw at intervals of more than 20 s , and the average ( total 4 times / mouse ) served as a response score . the animals were anesthetized with pentobarbital ( 80 mg / kg , intraperitoneal ) and transcardially perfused with phosphate - buffered saline ( pbs ) . the dorsal root ganglia ( l4l5 levels ) was removed , immediately frozen in liquid nitrogen , and stored at 80 c until assay . inc . , waltham , ma , usa ) and treated with dnase i ( takara bio inc . , shiga , japan ) . rt - pcr assay of trpm8 and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) was performed as described . the sequences of primers were as follows : trpm8 , 5-ggctggagatgagattgtgag-3 ( sense ) and 5-gctgaagtgggtggagaaga-3 ( antisense ) ; glyceraldehyde-3-phosphate dehydrogenase , 5-ccaaggtcatccatgacaac-3 ( sense ) and 5-ttactccttggaggccacgt-3 ( antisense ) . the expression levels of mrna were analyzed with nih image software ( national institute of health , bethesda , maryland , usa ) . the mrna expression level of trpm8 in each sample was normalized to that of gapdh and was then further normalized to its expression in the vehicle control . statistical significance was analyzed using two - way repeated measures analysis of variance ( anova ) and post hock holm idk multiple comparisons test or one - way anova and post - hoc dunnett 's test . a single intraperitoneal injection of oxaliplatin ( 3 mg / kg ) induces cold dysesthesia , which peaks on day 3 . thus , in the present study , we therefore evaluated the cold dysesthesia until day 3 after oxaliplatin injection . g / kg ) did not affect the initial development of cold dysesthesia after oxaliplatin administration , but skt did not significantly and dose - dependently inhibit the exacerbation of the dysesthesia from day 2 after oxaliplatin injection , in comparison with the vehicle ( fig . 1 ) . two - way repeated measures anova revealed a significant difference with prophylactic skt ( f3,16 = 4.292 , p < 0.05 ) and a significant interaction between oxaliplatin treatment and time ( f9,48 = 2.344 , p < 0.05 ) . our previous report has shown that the mrna expression of trpm8 in the dorsal root ganglia was observed to peak on day 3 after oxaliplatin injection , and may be involved in oxaliplatin - induced cold dysesthesia.9 , 10 thus , this study examined whether trpm8 was involved in oxaliplatin - induced cold dysesthesia using trpm8 antagonist tc - i 2014 . oral administration of trpm8 antagonist tc - i 2014 ( 10 and 30 mg / kg ) inhibited the cold dysesthesia in a dose - dependent manner ( fig . 2 ) . two - way repeated measures anova revealed a significant difference with tc - i 2014 ( f2,15 = 8.404 , p < 0.01 ) . it has been demonstrated that trpm8 is involved in oxaliplatin - induced cold dysesthesia based on the present results and our previous reports.9 , 10 in addition , single administration of skt does not inhibit oxaliplatin - induced cold dysesthesia , suggesting that skt does not have the antagonistic activity of trpm8 . we therefore examined the effect of repetitive administration of skt ( 0.11.0 g / kg ) on the expression of trpm8 mrna in the dorsal root ganglia of mice treated with oxaliplatin . repetitive administration of skt ( 1.0 g / kg ) significantly inhibited the expression of trpm8 mrna , compared with mice administered the vehicle ( fig . 3 ) . although the administration of skt at higher concentrations ( 0.3 g / kg ) had a tendency to decrease the mrna expression , skt at lower concentrations ( 0.1 g / kg ) did not have that effect ( fig . prophylactic repetitive administration of skt prevented the development of cold dysesthesia in mice treated with oxaliplatin . in our preliminary experiments , therefore , skt would need to be administered repetitively to produce an anti - dysesthesia activity , suggesting that the regulation of gene expression related in cold dysesthesia is involved in skt 's mechanism of action . a single intraperitoneal injection of oxaliplatin increases the mrna expression of trpm8 in the dorsal root ganglia of mice and peaks at day3 after the injection.9 , 10 trp ankyrin 1 ( trpa1 ) also is known as a cold - sensing trp channel . trpm8 and trpa1 are activated at temperature below 25 c and 17 c , respectively . in this study , we used acetone for the cold stimulus . in our preliminary experiment , the application of acetone to the plantar skin decreased the temperature from 28.67 0.10 c to 24.46 0.24 c ( andoh , mizoguchi and kuraishi : unpublished observation ) . in addition to our previous report , trpm8 antagonist tc - i 2014 was observed to inhibit oxaliplatin - induced cold dysesthesia in mice . taken together , however , we do not deny the involvement of trpa1 in cold dysesthesia induced by oxaliplatin , as other recent studies have shown that trpa1 is involved in oxaliplatin - induced cold dysesthesia.21 , 22 trpm8 mrna is expressed in the dorsal root ganglia , but not the brain , spinal cord , heart , skin and muscle . repetitive administration of skt inhibited the increase of trpm8 mrna in the dorsal root ganglia of mice treated with oxaliplatin . the inhibitory mechanisms of skt on trpm8 mrna expression are still unclear . camp response element - binding , c - myc , myocyte enhancer factor 2 , etc . are the reported transcription factors for the regulation of trpm8 mrna expression . there are no reports on the regulation of the above transcription factors by skt . have reported that the component shakuyaku , but not kanzo , plays an important role in the regulation of mechanical allodynia . oxaliplatin increases intracellular ca ions in primary cultures of dorsal root ganglion neurons , resulting in a greater chance that creb may be phosphorylated and the activate transcription . in contrast , paeoniflorin inhibits the phosphorylation of creb through the inhibition of an increase in intracellular ca ions . in addition , paeoniflorin activates the a1 adenosine receptor , which inhibits the activity of adenylyl cyclase . thus , the decrease in the concentration of camp induced by paeoniflorin may also be involved in the inhibition of creb phosphorylation . however , there is no evidence of the regulation of other transcription factors by paeoniflorin and other components of shakuyaku . these issues will be the focus of our future study , as skt may inhibit the mrna expression of trpm8 through the regulation of the activation of transcription factors including creb . thus , skt may be useful for the prevention of oxaliplatin - induced peripheral neuropathy . the underlying mechanism of the anti - cold dysesthesia action of skt may involve the regulation of the expression of trpm8 . | oxaliplatin - induced peripheral neuropathy characterized especially as cold dysesthesia is a major dose - limiting side effect of the drug and is very difficult to control . in the present study
, we examined whether the traditional herbal formulation shakuyakukanzoto ( skt : sho yo gn co tng ) could relieve oxaliplatin - induced cold dysesthesia in mice .
the inhibitory mechanisms were also investigated .
repetitive administration of skt ( 0.11.0 g / kg ) starting from the day after oxaliplatin injection inhibited cold dysesthesia in a dose - dependent manner .
our previous report has shown that the mrna expression of transient receptor potential melastatin 8 ( trpm8 ) , characterized as a cold - sensing cation channel , is increased in the dorsal root ganglia of mice treated with oxaliplatin .
in addition , trpm8 antagonist tc - i 2014 ( 10 and 30 mg / kg ) also attenuated cold dysesthesia in oxaliplatin - treated mice . taken together , it is suggested that trpm8 is involved in the cold dysesthesia induced by oxaliplatin .
repetitive administration of skt inhibited the mrna expression of trpm8 induced by oxaliplatin in the dorsal root ganglia .
these results suggested that prophylactic repetitive administration of skt is effective in preventing the exacerbation of oxaliplatin - induced cold dysesthesia by inhibiting the mrna expression of trpm8 in the dorsal root ganglia . |
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the differential diagnoses includes infections , autoimmune orchitis , testicular tumors , recurrent testicular torsion or trauma , chemotherapy , radiation treatment and , in rare cases , alcohol abuse ( see table 1 ) 1 . neonates will present with microphallus , absent testicles , and underdeveloped scrotum . in the peripubertal / pubertal period , a child fail to progress in puberty as evidenced by lack of testicular enlargement , poor pubertal height gain , and low testosterone levels in conjunction with elevated gonadotropins . for some individuals , particularly in the case of klinefelter syndrome which affects 1:600 men , there is usually some degree of testicular enlargement and testosterone production . , we report a case of an adolescent with evidence late gonadal pubertal staging who acquired primary gonadal failure shortly after he presented with severe diabetic ketoacidosis ( dka ) at the onset of his diagnosis of diabetes . the clinical scenario presented is consistent with hypoperfusion damage to the testes secondary to shock . a 14yearold caucasian boy with no significant past medical history presented to the emergency department when his mother was unable to wake him up . the family reported a 3 day history of flulike symptoms with polyuria and polydipsia but no vomiting . on exam , exam by the consulting endocrine team at presentation documented normal body mass index , no acanthosis , and sexual maturity staging of tanner 4 for pubic hair with testicular volume of 15 ml bilaterally . the patient was diagnosed with severe dka based on labs which showed ph 6.89 , bicarbonate 4.5 mmol / l , blood glucose 1493 mg / dl , and ketonuria . he had acute renal failure with initial blood urea nitrogen ( bun ) of 59 and serum creatinine 3.4 mg / dl . the patient was transferred to the pediatric intensive care unit ( picu ) for further management . additional labs drawn at presentation are shown in table 2 . while in the picu , in spite of biochemical resolution of dka , the patient had persistent hypotension and developed multisystem organ failure . he had a normal serum cortisol level in response to a 1 mcg cosyntropin stimulation test performed on day 3 of admission ( baseline 15 mcg / dl , t = 60 min 25 mcg / dl ; 420 mcg / dl ) , and blood cultures drawn at presentation were negative . management included mechanical ventilation , both conventional ( for 10 days ) and highfrequency oscillatory ( for 5 days ) , pressor therapy , and additional supportive care . after 25 days in the picu , the patient improved and was discharged on multiple daily injection insulin therapy . at his first diabetes outpatient clinic visit 3 months after his initial diagnosis , the patient had no complaints of poor energy . on physical exam , testicular volumes were 2 ml bilaterally with adult penile size and pubic hair tanner stage 4 . laboratory studies to assess this change in testicular volume showed 46 , xy karyotype , testosterone level of 205 ng / dl ( genital stage tanner 3 tanner 4 , drawn at 7 am ) , elevated gonadotropins ( luteinizing hormone ( lh ) 15.02 miu / ml ( 0.294.77 ) ; folliclestimulating hormone ( fsh ) 17.85 miu / ml ( 0.407.40 ) ) , and low inhibin b ( < 10 pg / ml ) . based on these finding , a diagnosis of primary gonadal failure was made , and topical testosterone replacement therapy was started . we present an unusual case of a young man who developed primary gonadal failure coincident with his presentation of new onset diabetes . differential diagnoses for hypergonadotropic hypogonadism are presented in table 1 . while individuals with klinefelter syndrome ( 47 , xxy ) are at increased risk for type 1 diabetes , the normal male karyotype rules out this diagnosis in our patient 2 . in addition , the unique finding of testicular volume regression from initial presentation is more consistent with an anatomical insult to the gonads . viral orchitis is usually accompanied by testicular pain , which was not elicited at presentation 3 , 4 . however , this diagnosis can not be completely ruled out in our patient given his altered mental status during the illness , which did not allow for localized pain assessment . , lh and fsh will both rise indicating leydig cell failure with subsequent low testosterone levels , and sertoli cell failure which will be reflected in low inhibin b levels and impaired spermatogenesis 5 . gonadal failure is rare in aps ( 5% ) , and it is unlikely that it would copresent with only type 1 diabetes , the only other autoimmune diagnosis in our patient 6 . the syndrome is characterized by development of pituitary hypofunction , which may result in gonadal failure . however , gonadotropins will be low , indicating central hypogonadism , rather than elevated , as described in our patient . the clinical scenario presented is most consistent with a direct vascular insult to the testes which would explain the abrupt regression in testicular size from the initial presentation . interruption of this arterial supply via spermatic cord torsion or disruption of blood flow secondary to shock could lead to testicular damage and subsequent primary gonadal failure . the finding of testicular regression following an episode of severe hypotension is what prompted the biochemical evaluation for our patient . this case highlights the importance of performing comprehensive physical examinations including genital exam in both inpatient and outpatient settings . without initial complete physical exam on presentation and followup exam , testicular dysfunction would not have been identified in a timely manner , and this patient would likely have developed signs and symptoms of androgen deficiency . | key clinical messagethe stalling or regression of pubertal development may be the first sign of hypergonadotropic hypogonadism in adolescent males .
we report here a case of pediatric hypergonadotropic hypogonadism that likely developed secondary to ischemic injury during severe diabetic ketoacidosis ( dka ) .
this case highlights the importance of performing genital exams during all evaluations of pediatric patients . |
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thyroiditis , which literally means thyroid inflammation , is a term that includes different conditions with some sort of controversy in nomenclature even among thyroid disorders expert endocrinologists . thyroiditis disorders can be classified in several ways , based on different etiology whether known or suspected different pathology , or different clinical presentation . they include painful conditions as subacute thyroiditis ( sat ) and suppurative thyroiditis and painless conditions in which the primary manifestations are thyroid dysfunction or enlargement ( goiter ) in the absence of clinically evident manifestations of acute inflammation . . sat , also called subacute granulomatous or de quervain thyroiditis , is an uncommon condition , yet is considered the most common cause of painful thyroiditis . the disease is thought to have a viral origin , with possible pathogens including mumps virus , hepatitis b and c viruses , cytomegalovirus , enterovirus , and coxsackie viruses a and b . however , the exact etiology of sat is unknown . studying the natural history and clinical outcome of the disease in different ethnic backgrounds has a potential significance since genetic factors that might affect individuals ' susceptibility to possible viral pathogens were reported to play a role in the pathogenesis of the disease . clinically , the condition is associated with severe pain that is usually localized to the anterior aspect of the neck and may radiate up to the jaw or ear . in addition , tenderness of the thyroid gland upon palpation and small diffuse goiter are frequently present . common initial clinical features and laboratory investigation results include low - grade fever , fatigue , mild thyrotoxic manifestations , suppressed thyroid stimulating hormone ( tsh ) , poor or no thyroid uptake , and elevated erythrocyte sedimentation rate ( esr ) . within few weeks and after the depletion of the preformed thyroid hormone , the latter might last up to several months to be followed by an euthyroid phase . clinical picture and laboratory results are the bases for the diagnosis , while histopathology and cytological diagnosis are rarely required . the uncommon incidence of the disease is reported in several parts of the world . in a reasonably comprehensive study that was published in 2003 from olmsted county in minnesota , usa , the incidence rate for the disease was reported to be 3.6 cases per 100,000/year in the most recent years of the study , with the incidence rate for women exceeding that for men . the peak incidence was in the fifth decade of life for both sexes . from italy , a study was recently published that included patients retrospectively evaluated at an endocrine and metabolic unit in a single institution during 2 years . the authors have demonstrated that 0.3% of those patients were diagnosed as sat , also with a differential gender preference toward female ( female to male ratio of ~6 : 1 ) . there are few available studies that focus on the demographic distribution and clinical outcome of sat in the kingdom of saudi arabia . about a decade ago , qari and maimani have published their work studying the disease outcome in the western province of the kingdom , an area that is located along the red sea shore and as such represents a different environment and lifestyle nature than those prevalent in the central region of the country . the study has followed up 23 patients with sat for 2-year duration and commented on the clinical presentation and outcome of the disease in this area of the country . the current study was designed with the objectives to report the clinical presentation , laboratory investigations , therapeutic approach , and clinical outcome of sat in the region of the capital city : riyadh ( central province of the kingdom of saudi arabia ) and to compare these data with the results previously published from the western province of the country and to those reported from different parts of the world . a long follow - up for the patients ' clinical outcome was an important priority in the design of this study to monitor potential long term thyroid dysfunction . to fulfill these objectives , we designed a retrospective review study in king khalid university hospital ( kkuh ) of king saud university ( ksu ) located in riyadh , where we retrieved relevant medical information from the medical record department of kkuh looking at medical records of all patients who underwent a thyroid uptake scan during an eight - year duration . initially , we have analyzed the data of all thyroiditis cases retrieved in our search ; then we focused on the confirmed cases of sat . this is a retrospective review study that was performed in the obesity research center , kkuh , ksu , riyadh . after obtaining the institutional ethical committee approval , we retrieved relevant medical information from the medical records department of the hospital looking at all patients who underwent a thyroid uptake scan between january 1 , 2004 , and december 31 , 2011 . the aim was that to study various forms of thyroiditis and specifically sat in the region of riyadh . based on the retrieved medical information , subjects were included if they manifest low normal , reduced , or absent 99 m technetium pertechnetate ( tc-99 m ) thyroid uptake with no detected visible nodules on thyroid scan . we have chosen the thyroid uptake as an initial means for medical record retrieval because it is the investigation tool most frequently requested for suspected cases of thyroiditis in our center . for those patients , the diagnosis of different types of thyroiditis was recorded from their medical files , and based on the classification published by pearce and colleagues , as follows . hashimoto 's thyroiditis was diagnosed by the presence of high persistent titer of antithyroid antibodies as antimicrosomal ( ama ) or antithyroglobulin ( ata ) antibodies and the characteristic pathological findings of lymphocytic infiltration , germinal centers , or fibrosis , when available . painless postpartum thyroiditis was diagnosed in women of childbearing age , who showed low thyroid uptake , high titer of antithyroid autoantibodies , and characteristic lymphocytic infiltration when pathological analysis was recorded , while thyroid function might show variable findings . painless sporadic thyroiditis diagnostic criteria are very similar to the painless postpartum thyroiditis and can manifest in male as well . drug - induced thyroiditis , as the name implies , occurs secondary to certain drugs as amiodarone , lithium , interferon , or interleukin-2 . painful thyroiditis includes sat , suppurative thyroiditis , radiation - induced thyroiditis , and trauma- or palpation - induced thyroiditis . sat diagnostic criteria include absent or low thyroid uptake in the thyroid uptake scan and the presence of transient thyrotoxicosis at time of presentation in the absence of both excessive iodine intake history and history of medication that could explain the transient thyrotoxicosis ( e.g. , lithium , amiodarone , and various cytokines ) . hypothyroidism or even euthyroidism might be detected depending on the natural history of the disease and the time of presentation . in addition to abnormal thyroid uptake and the thyroid function test ( tft ) results , painful sat diagnosis , as the name implies , was based on fulfilling two other criteria , namely , neck pain and/or tenderness and elevated esr . while the esr is elevated , together with other manifestation of acute infection in suppurative thyroiditis , both the thyroid uptake and the tft can be normal . information regarding the subjects demographic criteria ; age at presentation , sex , and socioeconomic state ; family and previous medical history ; clinical picture at presentation and results of laboratory investigations including esr , tft , and antithyroid antibody measurements were all recorded . the reported tft including tsh , ft4 , and ft3 were performed in kkuh using commercially available kits by roche elecsys modular analytics cobas e411 utilizing electrochemiluminescence immunoassay ( roche diagnostics , mannheim , germany ) . specific thyroid - related investigations in addition to the thyroid uptake scan , as thyroid ultrasonography and the relatively infrequently requested cytological examination of fine needle aspirated - thyroid gland specimen , were noted if available . subsequent clinics ' visits were carefully reviewed to assess the thyroid - related medications and the clinical outcome of the patients included in this study . the mean sd follow - up period for sat patients was 7.9 4.7 years . reviewing thyroid uptake scan data in kkuh , demonstrated that it was requested for 550 subjects in the current study 's specified duration . among those subjects , low normal , reduced , or absent uptake were reported in 67 subjects ( 12.2% ) , and , among the latter , the diagnosis of thyroiditis was recorded in the medical files in 25 patients ( 37.3% ) . thyroiditis patients comprise both genders ( 20 female and 5 male patients ; i.e. , 80% of thyroiditis patients are female ) with a male to female ratio of 1 : 4 and with their age at presentation ranging from 3 to 78 years old , ( average sd 35.2 11.2 years ) . the number of thyroiditis patients in different age intervals is illustrated in figure 1 , and it shows that 75% of patients in the female group were middle age at time of presentation ( age range of 2047 years old ) . similarly , 60% of patients in the male group were middle age at time of presentation ( age range of 2444 years old ) . in both genders 72% of patients were middle age at time of presentation . when we classified thyroiditis patients based on the presence or absence of neck pain , 8 subjects fulfill the criteria of thyroiditis with thyroid pain and tenderness , while the remaining 17 subjects fulfill the criteria of painless thyroiditis . those will be analyzed in more detail below while one subject ( a child ) was diagnosed with acute bacterial ( suppurative , infectious ) thyroiditis . the latter was a 3-year - old girl who presented with severe pain and tenderness in the neck region accompanied by fever and thyroid swelling . thyroid investigations demonstrated low thyroid uptake , and both ultrasound and fine needle aspiration and cytological examination have confirmed the presence of a thyroid abscess due to bacterial infection . other reported categories of painful thyroiditis were not represented in our retrieved medical records , such as traumatic , radiation , or the more rare cases of painful hashimoto 's thyroiditis . among those with painless thyroiditis , two subjects were diagnosed as hashimoto 's thyroiditis based on the clinical findings , the laboratory results especially those of high and persistent titer of ama ; one subject was diagnosed as postpartum thyroiditis ; one subject had lithium - induced thyroiditis , and 13 subjects fulfilled the criteria of painless sporadic thyroiditis . both hashimoto 's thyroiditis cases were female in the thirties of their age , one of them suffered as well from other allergic and inflammatory disorders ( adult still 's disease , allergic rhinitis , and chronic hepatitis ) . the laboratory investigations of both cases have demonstrated elevated esr , anemia , lymphocytosis , and monocytosis . their tft results differ , one hashimoto 's thyroiditis patient had overt thyrotoxicosis upon presentation and was actually treated with antithyroid drug ( atd ) and corticosteroid , while the other subject had hypothyroidism upon presentation and was started on thyroxine replacement therapy . both subjects manifested low thyroid uptake and the characteristic ultrasonography and cytological findings of hashimoto 's thyroiditis . among the painless thyroiditis , one subject a female 28-year - old at time of presentation presented with goiter . upon investigation , she had low thyroid uptake and was diagnosed as painless postpartum thyroiditis . this was 30 years old at time of presentation , psychiatric female patient who developed lithium - induced thyroiditis manifested by low thyroid uptake , transient thyrotoxicosis , and a transient ama autoantibody . the condition was spontaneously resolved , and the ama titer turned negative in 2 years . the remaining painless thyroiditis subjects were categorized as painless sporadic thyroiditis , who manifested the characteristic clinical , laboratory , thyroid uptake , imaging , and cytological criteria . among the thyroiditis subjects in general , the clinical presentation varied , with different combinations as demonstrated in table 1 . the most frequently reported symptoms and signs were palpitation , goiter , and weight change , while those least frequently reported were ear and face pain and shortness of breath . table 2 demonstrates the results of laboratory investigations and of the thyroid uptake and scan of the thyroiditis cases as a whole . in addition to the thyroid uptake scan , results of other investigation as thyroid ultrasonography and fine needle aspiration ( fna ) were available for some of the patients . thyroid ultrasonography was requested for 10 patients out of the 25 patients diagnosed as thyroiditis ( 40% ) . of these , small hypoechoic or anechoic nodules less than 1 cm and irregular thyroid surface were mainly described . furthermore , fna and cytological examination was done for 6 patients ( 24% ) showing colloid nodule ( 2 patients ) , a colloid nodule with cystic changes ( 1 patient ) , acute inflammatory cells and abscess ( 1 patient ) , and lymphocytic infiltration with criteria of chronic inflammation ( 2 patients ) . further reviewing the records of the whole group of thyroiditis patients to document the treatment approach revealed that in the initial thyrotoxic phase , -blockers were prescribed for 8 of the 25 patients ( 32% ) . only one patient ( 4% ) was prescribed gradually tapering dose of corticosteroids . in addition , during the course of the disease , 6 patients were prescribed thyroxine ; one of them during the hypothyroid phase of the disease , while in the remaining 5 patients , thyroxine was prescribed for permanent hypothyroidism . on the other hand , atd ( carbimazole ) careful revision for the details of the subsequent patients ' clinics visits for a follow - up period of 7 4 years ( average sd ) to record the clinical outcome has revealed that euthyroid state and resolution of thyroiditis was reported in 20 patients ( 80% ) , while permanent hypothyroidism occurred in 5 patients ( 20% ) . we further analyzed the painful sat group records separately to be compared to the results from the western region of the kingdom . there is obvious similarity of both gender and age distribution of the sat patients ( figure 2 ) to those of the thyroiditis group as a whole ( figure 1 ) ; sat is more represented in females ( 6 females and 1 male patients ; the subjects ' age at presentation ranges from 16 to 54 years old , ( average sd 34.7 8.6 years ) . the number of sat patients in different age intervals is illustrated in figure 2 , and it shows that 71.4% of sat patients in both genders were middle age at time of presentation . the symptoms and signs of sat at time of presentation varied , with palpitation and sore throat present in 62.5% of subjects , weight loss in 50% , goiter , anxiety , fatigability and heat intolerance in 37.5% , excessive sweating and hoarseness of voice in 25% ; and the least reported clinical presentations were tremors , fever , mood change , insomnia , loss of hair , and ear and face pain ( present in only 12.5% of the sat subjects ) . table 3 demonstrates the results of laboratory investigations and of the thyroid uptake and scan . it shows similar pattern as that of the whole thyroiditis group ( table 2 ) but with greater elevation of ft4 , ft3 , and esr average values and greater reduction of the thyroid uptake average value in the sat group relative to the whole thyroiditis group . regarding the treatment modality , it was reported that -blockers were prescribed to 57% and nsaid to 29% of sat cases in the initial thyrotoxic phase . it is also noted that atd were transiently prescribed for 3 out of the 7 patients of painful sat in the initial thyrotoxic phase . follow - up period of 7.9 4.7 years ( average sd ) demonstrated that 6 out of the 7 sat patients ( 85.7% ) had recovered , whether spontaneously ( 4 patients ) or after transient treatment with thyroxine ( 2 patients ) , while 1 patient had developed permanent hypothyroidism for which she has been prescribed thyroxine replacement therapy . the current work 's experimental approach was to retrieve all the medical records in a specified period of time that contain results of the thyroid uptake and scan , which is frequently requested to confirm the clinical and laboratory diagnoses of thyrotoxicosis and to start the treatment accordingly . therefore , using it in combination with the clinical findings , for example , thyroid pain and tenderness , and laboratory results , for example , esr and tft , dominate the major diagnostic and therapeutic considerations in our medical institute as well as elsewhere in the world . other rare conditions include traumatic , radiation , and the unusual painful hashimoto 's thyroiditis . on the other hand , painless or silent thyroiditis is sometimes called lymphocytic thyroiditis based on the pathological findings of this condition , and it includes various subcategories as previously mentioned . in the present study , we initially analyzed all thyroid disorders for which thyroid uptake and scan demonstrated low normal , reduced , or absent uptake . among 25 individuals with this abnormal uptake retrieved in a period of 8 years , 17 subjects ( 68% ) were presented as painless thyroiditis and 8 subjects ( 32% ) were presented as painful thyroiditis . different pathologic conditions of retrieved painless thyroiditis encompass the following : 13 subjects ( 52% ) with painless sporadic thyroiditis , 2 subjects ( 8% ) with hashimoto 's thyroiditis , 1 subject ( 4% ) with painless postpartum thyroiditis , and 1 subject ( 4% ) with painless drug- ( lithium ) induced thyroiditis . no riedel ( fibrous ) thyroiditis was retrieved among the included medical records of the current study . the single case of lithium - induced thyroiditis transiently developed autoantibodies to the thyroid gland ( ama ) . this is similar to other reports in the literature , where damage to the thyroid gland due to destructive thyroiditis results in releasing of thyroid follicles contents and triggering the immune system to react . regarding painful thyroiditis , 7 subjects ( 28% ) were diagnosed as sat and 1 subject ( 4% ) with infectious ( suppurative ) thyroiditis . no radiation , traumatic , or painful hashimoto 's thyroiditis were diagnosed among the included records of this work . we further analyzed the painful sat in more detail , as this condition was previously studied in a different region of the kingdom of saudi arabia , and our aim was to compare both studies ' outcomes in an effort to detect geographical or environmental differences , if any . . sat is a self - limiting inflammatory disorder of the thyroid gland , with a possible viral cause . thyroid disorders , sat is considered uncommon , with the incidence rate of 3 cases per 100,000 per year as reported in a study performed in olmsted county , minnesota , usa . in the denmark , a prospective population - based study that was recently published focusing on patients with overt hyperthyroidism in 2 cities with slightly different iodine deficiency status has confirmed the previously reported low incidence of sat . among the denmark 's cohorts of patients with various nosological types of hyperthyroidism , there is only one published study in the kingdom of saudi arabia that reported on sat cases in the western region of the kingdom , and none was reported in other districts including the central province where the capital city , riyadh , is located . in the western region , 23 patients with sat were retrieved in a duration of 2 years and were followed up for 2 years . indeed , no wide scale community - based studies have been conducted anywhere in the kingdom in order to estimate the standardized incidence rate of sat in the kingdom of saudi arabia . in the present study , the approach adopted allowed us to analyze the clinical presentation and outcome of confirmed cases of sat that have been diagnosed in kkuh and to compare these data to other investigators ' published reports . we retrieved 7 cases of confirmed sat diagnosis in 8-year duration by revising the medical records of 550 patients who underwent a thyroid uptake scan . consistent with several published reports [ 57 , 1013 ] , our findings demonstrate a clear gender preference , where women are more affected than men with similar gender ratio as reported in the literature . similarly , an age preference was noted in the present study with the age group more affected by sat being the middle age adults for both females and males . this is also in line with data reported by other groups [ 5 , 6 , 9 , 12 , 13 ] . in the present study , the patients ' leading reported symptoms and signs were those related to thyrotoxicosis ; this is consistent with other reports for sat . one patient presented as a case of fever of unknown origin , an unusual presentation that was previously reported in the literature [ 14 , 15 ] . it is noted that laboratory measurement of some of parameters assessed by other hospitals or suggested in the literature , for instance circulating thyroglobulin and crp levels [ 16 , 17 ] , respectively , are not requested in our center . future studies might be required to assess the potential advantages , if any , and to estimate the cost - effectiveness of requesting such parameters for suspected thyroiditis patients . in line with other reports on sat , fna cytology is not routinely requested for the disease diagnosis , and it is used only in cases where the diagnosis of painful / tender neck mass is ambiguous . as mentioned earlier , a report on painful sat cases in the western province of the kingdom was previously published . the kingdom 's western province is located on the red sea coast and is therefore environmentally different than the central one , where there are no seas or permanent rivers . both studies were not designed to be epidemiological ones , and hence no incidence rate per population per year can be obtained from either study . the western province study reported similar age distribution and gender preference trend but different value for the female to male ratio than in the current study ( female to male ratio 1.9 : 1 in the western region and 6 : 1 in the central region ) . the laboratory investigation results in general follow the same pattern in both regions of the kingdom . on the other hand , long term clinical outcome differs in both regions . in the present study , permanent hypothyroidism was reported in 5 patients of the whole thyroiditis group ( 20% ) , and in 1 patient of the painful sat group . the long follow - up duration for the patients ' clinical outcome in our work relative to the study performed in the western province is considered a strong aspect of the present study and might explain the difference observed in the percent of patients who developed permanent hypothyroidism along the years as compared to the western province study . furthermore , the different therapeutic approach in the initial phase of the painful sat between both regions might be a partial explanation for different clinical outcome . the study from western province reported that anti - inflammatory drugs were frequently prescribed as follows : 35% of patients were treated with corticosteroids alone , 30% with nsaid only , and 35% with both nsaid and corticosteroids . in addition , almost all their patients were prescribed a -blocker as a symptomatic therapy , and 13% of their patients were treated initially with atd . on the other hand , the present study demonstrates that in the initial thyrotoxic phase of painful sat , -blockers were prescribed for 57% of cases and nsaid for 29% of patients . it is also noted that atd were transiently prescribed for 3 out of the 7 patients of painful sat in the initial thyrotoxic phase . in addition , among the painful sat subjects , thyroxine was prescribed transiently to 2 patients and permanently to a third patient who developed permanent hypothyroidism . considering the different location and lifestyle of the central and the western regions of the country , it is intriguing to hypothesize that the clinical outcome differences between the current study and that of qari and maimani are possibly partially due to the difference in iodine deficiency between these two regions . in fact , studies on iodine status from different provinces in the kingdom are scarce and more thorough analysis is needed . however , the small number of available studies has shown a clear geographical difference in iodine state in the kingdom , as measured by the urinary iodine concentration and the percentage of subjects with a low urinary iodine concentration . clinically , the provincial difference in the iodine status was associated with a corresponding difference in the prevalence and severity of goiter . in different regions of the world , carl and coworkers have recently conducted an epidemiological study of subtypes of hyperthyroidism in 2 cities with different iodine deficiency status in denmark . they have reported a significantly higher standardized incidence rate per 100,000 person - years in the city with more iodine deficiency . based on the unpredicted nature of sat and the difference in the clinical outcomes between the western and central provinces of the kingdom , a nation - wide well - designed epidemiological study would be expected to provide population standardized statistical analysis of thyroid disorders including sat in different regions of the kingdom and equally important to help in establishing a practical guidelines for management of this disease and other thyroid disorders . the present study clearly demonstrates that prescribing corticosteroid for sat patients is not a common practice in our center even in the early phase of the disease where corticosteroids might be considered as effective symptomatic therapy . in fact , prescribing -blockers is more commonly resorted to aiming at controlling the initial thyrotoxic manifestations of the disease . the management approaches reported in the literature for sat indicate that some patients require no treatment , while many others frequently require nsaid as symptomatic analgesic and anti - inflammatory agents . if the symptoms persist despite the use of nsaid or in cases with severe symptoms , corticosteroids are usually prescribed only after excluding acute suppurative thyroiditis . it is quite interesting to study the correlation between the therapeutic approach and the clinical outcomes for patients with sat . fatourechi and colleagues have reported that among their cohort , prescribing corticosteroids did not protect against the occurrence of transient hypothyroidism in the early phase of the disease . as a matter of fact , they reported that long term hypothyroidism was significantly more common in the group who received corticosteroid therapy . in our group , none of the sat patients received corticosteroids and thus we can not comment on the association between corticosteroid and a particular clinical outcome . one limitation is imposed by the approach adopted . since sat could be diagnosed clinically without performing a thyroid uptake scan , using the latter as the method for retrieving medical records for patients to be included in the study might have underestimated the number of sat cases diagnosed in our region during the 8 years specified . on the other hand , focusing on confirmed cases and reporting on the clinical presentation and outcome for a long follow - up period are clearly strength points in the current work . in conclusion , our retrospective review study shows that sat is an uncommon disease in the central province of the kingdom of saudi arabia . the disease follows the differential age and gender preferences as reported in the western province of the kingdom and in different parts of the world . symptomatic corticosteroid therapy is not a common practice in treating the thyrotoxic initial phase of the disease in the central region as compared to the western region of the kingdom . on the other hand , permanent hypothyroidism is not uncommon among the sat patients followed up for several years in the central as compared to the western region of the country . results obtained from this study highlight the need for a nation - wide well - designed epidemiological study for all thyroid disorders including sat that would be expected to provide population - standardized statistical information and help in establishing practical guidelines for the management of thyroid disorders in various regions of the country . | few studies have been reported from the kingdom of saudi arabia ( sa ) to describe the clinical presentation and long term outcomes of subacute thyroiditis ( sat ) .
our aim was to review the demographic , anthropometric , clinical presentation , laboratory results , treatment , and disease outcome in riyadh region and to compare those with results from different regions of the kingdom and different parts of the world .
we reviewed the medical files of patients who underwent thyroid uptake scan during an 8-year period in king khalid university hospital .
only 25 patients had confirmed diagnosis of thyroiditis .
age and gender distribution were similar to other studies .
most patients presented with palpitation , goiter , and weight change .
elevated thyroid hormones , suppressed thyroid - stimulating hormone , and elevated esr were reported . among those ,
7 cases of sat were recorded .
-blockers were prescribed to 57% and nonsteroidal anti - inflammatory drugs to 29% of sat .
long follow - up demonstrated that 85.7% of sat cases recovered , while 14.3% developed permanent hypothyroidism . in conclusion , sat is uncommon in the central region of sa .
compared to the western region , corticosteroid is not commonly prescribed , and permanent hypothyroidism is not uncommon . a nation - wide epidemiological study to explain these interprovincial differences
is warranted . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the lfm system is appended to an epi - fluorescence microscope ( zeiss , axiovert200 ) equipped with a led excitation light source ( = 488 nm , 300 mw , coolled)and a standard gfp filter set ( zeiss ) . in all c. elegans imaging experiments , weused a 40 0.95na dry objective ( zeiss apochromat ) , while zebrafish imagingwas performed with a 20x 0.5na dry objective ( zeiss plan - neofluar ) . themicrolens array is mounted inside a 5-axis kinematic mount ( thorlabs ) to allowfine adjustment of the array orthogonal to the optics axis , which we found crucialfor high - quality results . the array is further imaged onto a 5.5 megapixel ( 2560 x2160 pixels ) scmos camera ( andor zyla ) utilizing a 1:1 relay macro lensobjective ( nikon af - s 105 mm 2.8 g vr if - ed micro ) ( fig details on opticaldesign choices and their effect on resolution are discussed in supplementary note 1 . to record neuronal activity from c. elegans , we loaded adult worms ( one- tofour - egg - stage ) expressing nls - gcamp5k under the punc31-promoter ( strainszim294 and zim617 ) into a microfluidic channel which was connected to areservoir containing s - basal buffer with 5 mm tetramisole , an acetylcholinereceptor specific agonist that mildly paralyzes the animal 's muscles to reducemotion . the worm was placed off the native focal plane and towards the objective using a piezo stepper motor ( pi-721 , physik instrumente gmbh ) , suchthat the entire worm was ideally contained in the region spanning -30 m to 0m . by doing so we exploited the highest resolution of lfdm while avoidingartifacts near the focal plane . when recording from the head region only , theworm 's head ganglia were placed at the center of the fov and excitation waslimited to this area by the use of an iris in the excitation pathway . for the experiments involving chemosensory stimulation , we followed the procedure described in ref . have been imaged 5 - 8 dpf ( days post fertilization ) using stable lines huc : gcamp5 g and huc : gal4/uas : gcamp5 g , respectively . the fish wereimmobilized by embedding them in 2% agarose with their mouth and tailcleared of agarose to allow for odor stimulation and tail movement . odorstimulation was performed by manually supplying water containing highly concentrated rotten fish extract into the imaging chamber during recordings . the volume reconstruction itself can be formulated as a tomographic inverseproblem , where multiple different perspectives of an 3d volume are observed , and linear reconstruction methods implemented via deconvolution after recording the raw light - field images , the digital images were cropped toregions of interest and resampled to contain 11 11 or 15 15 angular lightfieldsamples under each lenslet . two calibration images , one showing themicrolenses with collimated rear - illumination and one showing a uniformfluorescent slide , were used for digital image rectification , in which camerapixels are assigned to individual microlenses . reconstruction of each frame of animage sequence took between 2 and 30 minutes , depending on the size of theimage , number of iterations of the deconvolution algorithm , reconstruction method and workstation used . computational resources are further discussed in supplementary note 2 . to extract a fluorescence time series for individual neurons from the 4d data , different strategies were employed for c. elegans and zebrafish . for c. elegans , we first applied rigid - body motion correction to each individual z plane movie.we then computed a median - intensity projection through time for each motioncorrectedz plane movie and used a maxima - finding algorithm to identify areas of peaked intensity in each projection . a circular region of interest ( roi ) wascreated surrounding each intensity peak , and overlapping roi areas within z planes were eliminated . rois in adjacent z planes within an x - y distance of 7pixels were considered to be a component of the same neuron , up to a maximumof 5 planes , and for each neuron at each time point , the brightest 100 pixels ofthe aggregate of all pixels within the neuron 's component rois were averaged toproduce a single fluorescence value and de - trended with an exponential decay function to account for photobleaching . for zebrafish , the data was first detrendedbased on the overall intensity of each frame . then , to reduce time seriesdata , first in - active voxels were discarded based on their time - domain variance.splitting the volume into smaller sub - volumes further reduced data size . we followed the strategy proposed in reference to extract cellular signals from theca - imaging data . each sub - volume data underwent pca / ica for automated spatial filter extraction where ideally each spatial filter corresponds to thelocation of a neuron . after automatically rejecting spatial filters based on sizeand dispersion , we applied the spatial filters to the 4d data to extract their fluorescence intensity . time - points during which the fish seemed to contract where discarded and replaced with nearest neighbor fluorescence intensities.these contractions typically lasted between 200 ms and 1 sec only and weretemporally very sparse . fish which moved substantially during image acquisitionwere discarded from analysis . to extract f / f0 , we calculated f / f0 = 100 * ( f(t) f0)/f0 , with f0 being the mean fluorescence intensity of each corrected trace . the lfm system is appended to an epi - fluorescence microscope ( zeiss , axiovert200 ) equipped with a led excitation light source ( = 488 nm , 300 mw , coolled)and a standard gfp filter set ( zeiss ) . in all c. elegans imaging experiments , weused a 40 0.95na dry objective ( zeiss apochromat ) , while zebrafish imagingwas performed with a 20x 0.5na dry objective ( zeiss plan - neofluar ) . themicrolens array is mounted inside a 5-axis kinematic mount ( thorlabs ) to allowfine adjustment of the array orthogonal to the optics axis , which we found crucialfor high - quality results . the array is further imaged onto a 5.5 megapixel ( 2560 x2160 pixels ) scmos camera ( andor zyla ) utilizing a 1:1 relay macro lensobjective ( nikon af - s 105 mm 2.8 g vr if - ed micro ) ( fig details on opticaldesign choices and their effect on resolution are discussed in supplementary note 1 . to record neuronal activity from c. elegans , we loaded adult worms ( one- tofour - egg - stage ) expressing nls - gcamp5k under the punc31-promoter ( strainszim294 and zim617 ) into a microfluidic channel which was connected to areservoir containing s - basal buffer with 5 mm tetramisole , an acetylcholinereceptor specific agonist that mildly paralyzes the animal 's muscles to reducemotion . the worm was placed off the native focal plane and towards the objective using a piezo stepper motor ( pi-721 , physik instrumente gmbh ) , suchthat the entire worm was ideally contained in the region spanning -30 m to 0m . by doing so we exploited the highest resolution of lfdm while avoidingartifacts near the focal plane . when recording from the head region only , theworm 's head ganglia were placed at the center of the fov and excitation waslimited to this area by the use of an iris in the excitation pathway . for the experiments involving chemosensory stimulation , we followed the procedure described in ref . for zebrafish experiments , mitf/ larvae with pan - neuronal gcamp5 expression have been imaged 5 - 8 dpf ( days post fertilization ) using stable lines huc : gcamp5 g and huc : gal4/uas : gcamp5 g , respectively . the fish wereimmobilized by embedding them in 2% agarose with their mouth and tailcleared of agarose to allow for odor stimulation and tail movement . odorstimulation was performed by manually supplying water containing highly concentrated rotten fish extract into the imaging chamber during recordings . the volume reconstruction itself can be formulated as a tomographic inverseproblem , where multiple different perspectives of an 3d volume are observed , and linear reconstruction methods implemented via deconvolution after recording the raw light - field images , the digital images were cropped toregions of interest and resampled to contain 11 11 or 15 15 angular lightfieldsamples under each lenslet . two calibration images , one showing themicrolenses with collimated rear - illumination and one showing a uniformfluorescent slide , were used for digital image rectification , in which camerapixels are assigned to individual microlenses . reconstruction of each frame of animage sequence took between 2 and 30 minutes , depending on the size of theimage , number of iterations of the deconvolution algorithm , reconstruction method and workstation used . to extract a fluorescence time series for individual neurons from the 4d data , different strategies were employed for c. elegans and zebrafish . for c. elegans , we first applied rigid - body motion correction to each individual z plane movie.we then computed a median - intensity projection through time for each motioncorrectedz plane movie and used a maxima - finding algorithm to identify areas of peaked intensity in each projection . a circular region of interest ( roi ) wascreated surrounding each intensity peak , and overlapping roi areas within z planes were eliminated . rois in adjacent z planes within an x - y distance of 7pixels were considered to be a component of the same neuron , up to a maximumof 5 planes , and for each neuron at each time point , the brightest 100 pixels ofthe aggregate of all pixels within the neuron 's component rois were averaged toproduce a single fluorescence value and de - trended with an exponential decay function to account for photobleaching . for zebrafish , the data was first detrendedbased on the overall intensity of each frame . then , to reduce time seriesdata , first in - active voxels were discarded based on their time - domain variance.splitting the volume into smaller sub - volumes further reduced data size . we followed the strategy proposed in reference to extract cellular signals from theca - imaging data . each sub - volume data underwent pca / ica for automated spatial filter extraction where ideally each spatial filter corresponds to thelocation of a neuron . after automatically rejecting spatial filters based on sizeand dispersion , we applied the spatial filters to the 4d data to extract their fluorescence intensity . time - points during which the fish seemed to contract where discarded and replaced with nearest neighbor fluorescence intensities.these contractions typically lasted between 200 ms and 1 sec only and weretemporally very sparse . fish which moved substantially during image acquisitionwere discarded from analysis . to extract f / f0 , we calculated f / f0 = 100 * ( f(t) f0)/f0 , with f0 being the mean fluorescence intensity of each corrected trace . | high - speed large - scale 3d imaging of neuronal activity poses a major challenge in neuroscience . here , we demonstrate intrinsically simultaneous functional imaging of neuronal activity at single neuron resolution for an entire caenorhabditis elegans as well as for the whole - brain of larval zebrafish .
our technique captures dynamics of spiking neurons in volumes of ~700 m x 700 m x 200 m at 20 hz and its simplicity makes it an attractive tool for high - speed volumetric calcium imaging . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
it is attracting considerable attention in north america , europe and elsewhere as an important framework to develop better and more cost - effective health systems . the goal of the international journal of integrated care is to encourage scholarly inquiry in this new field . but , have we defined integrated care ' well enough to help the community of academicians , scientists , policy analysts and practitioners to understand this concept , and guide the search for solid evidence and lessons ? like a rorschach test , integrated care has many meanings ; it is often used by different people to mean different things . it is most frequently equated with managed care in the us , shared care in the uk , transmural care in the netherlands , and other widely recognised formulations such as comprehensive care and disease management [ 13 ] . however , it is unclear as to whether all of these appellations aptly fall under the rubric of integrated care . this lack of conceptual clarity stands as a major barrier to promoting integrated care in both theory and practice . terminology plays a critical communications role in terms of the way we think about , shape , deliver , manage , regulate , finance , and evaluate health care . phrases like integrated care can only be understood if we examine their context and logic . in the following discussion paper , we will explore what integrated care could mean and should mean , and will also discuss several important implications . we take a bottom - up , patient - centred perspective , which is largely informed by our long experience in primary care , public health , and various forms of integrated care . our aim is to stimulate fruitful dialogue and debate in the spirit of laying the groundwork for an evidence - based integrated care . it is impossible to address the meaning of integrated care without first examining the roots and core notions of integration . integration stems from the latin verb integer , that is , to complete. the adjective integrated means organic part of a whole, or reunited parts of a whole. it is mostly used to express the bringing together or merging of elements or components that were formerly separate . understanding of a situation. in other words , what is connoted is a desire to understand the relationship of elements that constitute the entirety . integration is at the heart of systems theory and , therefore , central to organisational design and performance . all organisations ( and systems ) are , to some extent , hierarchical structures that are comprised of separate , but interconnected components ; these components are supposed to play complementary roles in order to accomplish their joint tasks . however , the division , decentralisation , and specialisation found in the architecture of more complex organisations usually interfere with efficiency and quality goals . therefore , the fulfilment of system aims necessitates co - operation and collaboration among and between the various parts of the organisation or system . in this sense , integration is the glue that bonds the entity together , thus enabling it to achieve common goals and optimal results . these ideas are , of course , applicable to the health care enterprise whether we are referring to its institutions and providers , or the health , social service and related systems in which they operate . health systems and health care institutions are among the most complex and interdependent entities known to society . historically , many factors have worked to divide various types of health care institutions and services on the one hand , and administrators , physicians , nurses and allied professionals on the other differing rules , inter - sectoral boundaries ( as between health care , mental health care , and social care ) , funding streams , and institutional and professional cultures , to name the most obvious . without integration at various levels , patients get lost , needed services fail to be delivered , or are delayed , quality and patient satisfaction decline , and the potential for cost - effectiveness diminishes [ 1214 ] . managers are not the only actors with an interest in health care integration . in the 1970s and 1980s , this reflected concern that the then rapidly emerging trend of specialisation would end up disintegrating professional practice and fragmenting patient care . the rise of general or family practice at the end of the twentieth century grew out of the idea that medicine needs more integration , not less . mcwhinney sees the family physician not only acting across specialty and clinical boundaries , but also bridging the very difficult gap between medical and social problems . referring to systems thinking , he stresses that the integration of knowledge and working methods in general medical practice is necessitated by the bio - psychosocial nature of illness , as well as the complexity of the health care system in which medical care is delivered.1 consequently , general practitioners in europe and family practitioners in the united states have developed guiding principles , which , in part , advance comprehensiveness and forms of integration as key professional goals . for example , wonca europe ( 2002 ) has called for general practitioners to take a comprehensive , person - centred approach , including exercising responsibility for the co - ordination of care . institute of medicine ( 1996 ) . finally , we would be remiss if the economic imperative of health care integration was not mentioned . policy - makers and payers in both the public and private sectors place great hope in its ability to save money , or at the very least , to ensure that health care resources are used more wisely . perhaps grne and garcia - barbero do the best job of summing up what health care integration generally means : the bringing together of inputs , delivery , management and organisation of services as a means [ of ] improving access , quality , user satisfaction and efficiency . nonetheless , various authors emphasise different aspects . brown & mccool , for example , suggest that integration allows for greater efficiency and effectiveness , less duplication and waste , more flexible service provision , and better co - ordination and continuity . the who study group , on the other hand , sees virtue in its ability to encourage more holistic and personalised approaches to multidimensional health needs . the foregoing discussion should make it clear that there are different meanings and interpretations of integration in general , and integration in the health care field in particular . ours is a pragmatic , rather than an ideological or idealistic view of health care integration . it need not mean that all parts of the health system are merged , or that merger is complete . point out , it may be helpful to think of discontinuities in health care as inevitable . even in the most carefully designed systems , health professionals must find innovative ways to get around the almost certain flaws and gaps . therefore , for us , integration may be seen as a step in the process of health systems and health care delivery becoming more complete and comprehensive . surely , the lack of integration in health care touches both consumers and providers , although not equally . the largely systems- or organisation - driven logic presented in the previous discussion finds solid support for integration in efficiency terms . however , looking at health care integration from the unique vantage of patients and specific patient populations sheds badly needed light on the subject.2 professionals seldom look at the world of health care through the patient lens . traditionally , caregivers demand that their patients be compliant , that is , follow their instructions . care - seekers , therefore , usually do not expect that their concerns will come first , or even be seriously addressed . our humanistic or patient - centric view argues strongly that populations with physical , developmental or cognitive disabilities often with related chronic conditions or complex illnesses endow the concept of health care integration with a unique logic and meaning . vulnerable individuals , such as the diverse group described above , have complicated and ongoing needs ( which frequently are part - medical , part - physical , part - psychological , and part - social ) , experience difficulties in everyday living , require a mix of services delivered sequentially or simultaneously by multiple providers , and receive both cure and care in home , community and institutional settings . these mostly long term , incurable , unpredictable and costly conditions present major challenges to both patients and family carers : making sure that needed services are delivered , preventing and managing flare - ups and crises , transitioning from one type or level of care to another , maintaining health and functioning , and coping with individual and family stress . the provision of health care , social services and related supports ( e.g. housing ) at the right time and place to such individuals is equally daunting . problems typically include difficulties with obtaining needs assessments , putting together comprehensive service packages , co - ordinating multiple providers and services , ensuring continuity , monitoring health and functional status , responding to crises , supporting family carers , and , finally , performing all of these essential activities within existing funding and resource constraints . what accounts for the poor fit between the needs of these multi - problem patients and the existing infrastructure of cure and care ? according to hardy et al . and first , required services are the responsibility of many sectors , jurisdictions , institutions , and providers . second , the various components of care and cure work in parallel with separate funding streams and budgets , and frequently conflicting regulations . third , health and social care differ distinctively in terms of language , culture , professional roles and responsibilities , and clinical or service approaches . our analysis suggests more of a patient - oriented definition of integration.3 we , therefore , propose the following : integration is a coherent set of methods and models on the funding , administrative , organisational , service delivery and clinical levels designed to create connectivity , alignment and collaboration within and between the cure and care sectors . the goal of these methods and models is to enhance quality of care and quality of life , consumer satisfaction and system efficiency for patients with complex , long term problems cutting across multiple services , providers and settings.4 the result of such multi - pronged efforts to promote integration for the benefit of these special patient groups is called integrated care.5 the authors perspective , already discussed at length , is that the overriding rationale is to solve the many problems arising from the complex presentation of chronic and disabling diseases and conditions . behind this approach is the desire , above all , to enhance quality and provide a better level of service that is , one that is more sensitive to the personal circumstances and wishes of the individual patient . other aims are geared to improving the efficiency , cost - effectiveness and organisational aspects of the health system in which curing , caring and patient management takes place . conceptually , this is good as far as it goes . to obtain real results for real people , however a continuum of strategies from the macro to the micro are available to foster integrated care . the application of these strategies is not only crucial to achieving more humanistic patient outcomes , but also better results in both efficiency and cost - effectiveness terms . the list of methods and tools found in box 1 , though not exhaustive , are most useful in addressing the barriers and bottlenecks which often occur at various levels in five important , interlocking domains :
funding : more often than not , form follows financing . this means that the division , structure and flow of funds for health and social care and related services can affect virtually all aspects of integrated care.administrative : the manner in which government regulatory and administrative functions are structured and devolved can help eliminate program complexities , streamline eligibility and access , and better manage system resources.organisational : networking , both vertically and horizontally and through formal or informal means , is a major method to improve how organisations work together . joint working relationships within and between agencies in the heath and social care sectors can optimise resources , facilitate overall efficiency , and enhance the capacity for seamless care, that is , the smooth and uninterrupted provision of necessary care .service delivery : the mode of service delivery and management how staff are trained , perform their responsibilities and tasks , work together , and relate to patients and family carers and their needs have a major impact on a number of critical variables in integrated care . such variables include service access , availability and flexibility , continuity and co - ordination of care , consumer satisfaction , and quality and cost outcomes ; and , clinical : shared understanding of patient needs , common professional language and criteria , the use of specific , agreed - upon practices and standards throughout the lifecycle of a particular disease or condition , and the maintenance of ongoing patient - provider communication and feedback are essential quality ingredients in integrated care.suggested throughout this paper , there are varying degrees of completeness , comprehensiveness and formality in integrated care . however , as leutz observes , the level , type , and combination of strategies used , would depend on the characteristics of the patient population and the specific challenges they face in obtaining appropriate , quality care . therefore , patients with particularly severe and unstable conditions requiring intensive , ongoing medical and social attention from a host of providers for relatively long duration would demand a complex solution necessitating more , and more structured forms of integration from several of the operational domains described above . the corollary would also be true : less complex patient groups would require narrower span and less thoroughgoing interventions . this means that the division , structure and flow of funds for health and social care and related services can affect virtually all aspects of integrated care . administrative : the manner in which government regulatory and administrative functions are structured and devolved can help eliminate program complexities , streamline eligibility and access , and better manage system resources . organisational : networking , both vertically and horizontally and through formal or informal means , is a major method to improve how organisations work together . joint working relationships within and between agencies in the heath and social care sectors can optimise resources , facilitate overall efficiency , and enhance the capacity for seamless care, that is , the smooth and uninterrupted provision of necessary care . service delivery : the mode of service delivery and management how staff are trained , perform their responsibilities and tasks , work together , and relate to patients and family carers and their needs have a major impact on a number of critical variables in integrated care . such variables include service access , availability and flexibility , continuity and co - ordination of care , consumer satisfaction , and quality and cost outcomes ; and , clinical : shared understanding of patient needs , common professional language and criteria , the use of specific , agreed - upon practices and standards throughout the lifecycle of a particular disease or condition , and the maintenance of ongoing patient - provider communication and feedback are essential quality ingredients in integrated care . pooling of funds ( at various levels ) prepaid capitation ( at various levels ) consolidation / decentralisation of responsibilities / functions inter - sectoral planning needs assessment / allocation chain inter - agency planning and/or budgeting service affiliation or contracting jointly managed programs or services strategic alliances or care networks consolidation , common ownership or merger around - the - clock ( on - call ) coverage integrated information systems standard diagnostic criteria ( e.g. dsm iv ) uniform , comprehensive assessment procedures common decision support tools ( i.e. practice guidelines and protocols % ) regular patient / family contact and ongoing support source : adapted from . integrated care , though increasingly recognised as a critical adjunct to health care delivery and patient management , is a modern - day tower of babel . 6 as is often the case with nascent fields , especially those with a strongly multidimensional character , the defining concepts and boundaries lack specificity and clarity . thus , the definitions , which are commonly used , tend to be vague and confusing . this makes it difficult to develop the knowledge base so essential to refine and move the field ahead . the concept of integration is a defining variable in the meaning of integrated care . in this paper , we identified two different but overlapping ways of looking at this important notion . in the first , integration reflects a largely hierarchical or top - down process driven by more generalised organisational exigencies for perfection or optimisation . view , in which the characteristics and needs of specific patient groups , and their fit ( or lack thereof ) with existing systems of care and cure more or less determine the what , how , and where of integration . it endows the term integrated care with a logic and meaning of greatest relevance to multi - problem patients with serious chronic and disabling conditions . in this paper , we presented our own definitions of integration and integrated care , which reflect this understanding . we accept that there are alternative interpretations , and look forward to wider discussion of these ideas . it would , however , be in the best interest of the burgeoning field of integrated care for such discussion to ultimately arrive at a consensus on the field 's boundaries , basic concepts , terminology and typology sooner rather than later.7 there are several implications that arise from our views . in closing , we would like to sketch some of the more salient points :
patient benefit . earlier , we broadly identified a target population who we believe would distinctly benefit from integrated care . however , what is needed are more detailed insights with respect to the successful impact of integrated care on specific patient groups ( e.g. the frail elderly ) , including the level , type , and combination of strategies involved in successful initiatives.barriers . major contextual , institutional and professional factors were briefly described in this paper , and many are addressed in greater detail in hardy el al . . we must better understand these key variables and how they affect experience and performance in differing countries , patient populations , organisational environments , and program designs.costs . the total costs of integrated care including outlays for staff and support systems , services , and start - up must be carefully defined , tracked and calculated before we can make pronouncements on the strategy 's cost-effectiveness.patient and family involvement . successful integrated care ( i.e. models that are effective in meeting patient needs ) demands the ongoing involvement of patients and family carers in programme planning , implementation and oversight . this will ensure that user needs and expectations are reflected where it counts , and that consumer satisfaction issues can be realistically addressed.research and evaluation . we must not only systematically examine the interventions themselves , but also a wide range of outcomes including health , psychosocial , and economic measures . equally as important , studies should focus on the experience of patients served by such approaches . this makes it incumbent on researchers and evaluators to employ an array of quantitative and qualitative methods and techniques to answer the many efficiency and effectiveness questions that various stakeholders are likely to pose . finally , a shared research agenda national and/or cross - national in scope would be helpful in theory- , model- , and evidence - building . earlier , we broadly identified a target population who we believe would distinctly benefit from integrated care . however , what is needed are more detailed insights with respect to the successful impact of integrated care on specific patient groups ( e.g. the frail elderly ) , including the level , type , and combination of strategies involved in successful initiatives . barriers . major contextual , institutional and professional factors were briefly described in this paper , and many are addressed in greater detail in hardy el al . . we must better understand these key variables and how they affect experience and performance in differing countries , patient populations , organisational environments , and program designs . the total costs of integrated care including outlays for staff and support systems , services , and start - up must be carefully defined , tracked and calculated before we can make pronouncements on the strategy 's cost - effectiveness . successful integrated care ( i.e. models that are effective in meeting patient needs ) demands the ongoing involvement of patients and family carers in programme planning , implementation and oversight . this will ensure that user needs and expectations are reflected where it counts , and that consumer satisfaction issues can be realistically addressed . we must not only systematically examine the interventions themselves , but also a wide range of outcomes including health , psychosocial , and economic measures . equally as important , studies should focus on the experience of patients served by such approaches . this makes it incumbent on researchers and evaluators to employ an array of quantitative and qualitative methods and techniques to answer the many efficiency and effectiveness questions that various stakeholders are likely to pose . finally , a shared research agenda national and/or cross - national in scope would be helpful in theory- , model- , and evidence - building . | abstractintegrated care is a burgeoning field .
as is often the case in new areas of inquiry and action , conceptual clarification is demanded . without such attention
, it would be difficult to advance theory and practice in this increasingly important professional arena . in the following discussion paper ,
the authors explore the intellectual territory of integrated care , and underscore the need for a patient - centric imperative and meaning .
they also examine the practical applications and implications arising from their views .
the intention is to stimulate fruitful dialogue and debate about what
integrated care could and should be . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
as there is no documented data on the prevalence of sffis in diabetes in sri lanka , we investigated the prevalence of fungal foot infections among diabetic patients in a tertiary care setting . ethical clearance was obtained from the ethical committee of university of sri jayewardenepura , sri lanka ( human biology/2012/02 ) and from the ethical committee of colombo south teaching hospital , sri lanka ( 228 ) . a descriptive cross sectional study conducted over a period of one year . three hundred and eighty five type 2 diabetic patients , who were suffering from diabetes for more than 5 years and attending the diabetic clinic at a tertiary care hospital , were included in the study after obtaining their informed written consent . pregnant mothers , debilitated patients who suffered from diabetes for less than five years were excluded . in addition to demographic data , patients were asked questions on knowledge and practices about the fungal foot infections , its risk and preventive measures . sampling was done after obtaining informed written consent of the patients who volunteered for the research . clinical examinations of patients toe nails or other infected sites in the foot were performed by a clinical microbiologist . the affected areas were cleaned with 70% alcohol and sterile scissors , nail - cutters were used to cut the infected part of the toe nail . swabs were collected from the infected toe - webs and soles of the patients and were inoculated on sabourand 's dextrose agar ( sda ) ( oxoid , usa ) plates directly . nail clippings were analyzed by direct microscopy by using 10% koh ( potassium hydroxide ) . subsequently , nail clippings and swabs of each patient were inoculated on sda ( oxoid , usa ) plates with and without antimicrobials ( chloramphenicol ( 50 g / ml ) and cycloheximide ( 100 g / ml ) and incubated at 25c- 30c aerobically for 3 weeks . yeast species were identified by means of gram 's stain , germ tube test and carbohydrate assimilation test . yeast colonies were inoculated to 0.5 ml of horse serum and incubated at 37c for 2 1/2 hours and looked for the production of germ tube . carbohydrate assimilation test commercially ( difco laboratories ) available yeast nitrogen base ( ynb ; carbon free ) agar dissolved in distilled water was autoclaved in 15 ml volumes within universal containers . heavy suspensions of the yeasts to be indentified were prepared using one loopful of the yeast isolate in 5 ml of sterile distilled water and 5 drops from each suspension were added to a separate ynb agar container at 45c . each seeded agar container was poured into a 90 mm sterile petri dish and allowed to set at room temperature . few crystals of the carbohydrates glucose , maltose , sucrose , lactose , galactose , cellibiose , raffinose , inositol , xylose , trehalaose and melibiose were carefully placed on the surface of the seeded ynb agar ( difco laboratories ) plate according to a template and incubated at room temperature ( 30c ) up to 48 hours . enhanced growth of the yeast at the points where the sugars were placed was detected visually and indicated assimilation of the particular sugar and when the sugar was not utilized there was no visible growth of the yeast . statistical analysis - laboratory investigations and data from the questionnaires were entered into microsoft office excel . statistical analysis , more specifically odd ratio and chi - square tests were performed using the software package , spss version 15 . ethical clearance was obtained from the ethical committee of university of sri jayewardenepura , sri lanka ( human biology/2012/02 ) and from the ethical committee of colombo south teaching hospital , sri lanka ( 228 ) . three hundred and eighty five type 2 diabetic patients , who were suffering from diabetes for more than 5 years and attending the diabetic clinic at a tertiary care hospital , were included in the study after obtaining their informed written consent . pregnant mothers , debilitated patients who suffered from diabetes for less than five years were excluded . in addition to demographic data , patients were asked questions on knowledge and practices about the fungal foot infections , its risk and preventive measures . sampling was done after obtaining informed written consent of the patients who volunteered for the research . clinical examinations of patients toe nails or other infected sites in the foot were performed by a clinical microbiologist . the affected areas were cleaned with 70% alcohol and sterile scissors , nail - cutters were used to cut the infected part of the toe nail . swabs were collected from the infected toe - webs and soles of the patients and were inoculated on sabourand 's dextrose agar ( sda ) ( oxoid , usa ) plates directly . nail clippings were analyzed by direct microscopy by using 10% koh ( potassium hydroxide ) . subsequently , nail clippings and swabs of each patient were inoculated on sda ( oxoid , usa ) plates with and without antimicrobials ( chloramphenicol ( 50 g / ml ) and cycloheximide ( 100 g / ml ) and incubated at 25c- 30c aerobically for 3 weeks . yeast species were identified by means of gram 's stain , germ tube test and carbohydrate assimilation test . for the germ tube test , yeast colonies were inoculated to 0.5 ml of horse serum and incubated at 37c for 2 1/2 hours and looked for the production of germ tube . carbohydrate assimilation test commercially ( difco laboratories ) available yeast nitrogen base ( ynb ; carbon free ) agar dissolved in distilled water was autoclaved in 15 ml volumes within universal containers . heavy suspensions of the yeasts to be indentified were prepared using one loopful of the yeast isolate in 5 ml of sterile distilled water and 5 drops from each suspension were added to a separate ynb agar container at 45c . each seeded agar container was poured into a 90 mm sterile petri dish and allowed to set at room temperature . few crystals of the carbohydrates glucose , maltose , sucrose , lactose , galactose , cellibiose , raffinose , inositol , xylose , trehalaose and melibiose were carefully placed on the surface of the seeded ynb agar ( difco laboratories ) plate according to a template and incubated at room temperature ( 30c ) up to 48 hours . enhanced growth of the yeast at the points where the sugars were placed was detected visually and indicated assimilation of the particular sugar and when the sugar was not utilized there was no visible growth of the yeast . statistical analysis - laboratory investigations and data from the questionnaires were entered into microsoft office excel . statistical analysis , more specifically odd ratio and chi - square tests were performed using the software package , spss version 15 . three hundred and eighty five , type 2 diabetic patients who attended the diabetic clinic at a tertiary care hospital were included in the study . amongst them , 255 were women with a mean age of 56.2 years ( standard deviation ( sd 11.27 ) and 130 were men with a mean age of 57.8 years ( sd 11.76 ) . all the patients were clinically examined by a clinical microbiologist for superficial fungal foot infections ( sffis ) . among the 385 diabetic patients , 295 ( 77% ) 203 ( 69% ) were women with a mean age of 57.5 years ( sd 10.88 ) and 92 ( 31% ) were men , with a mean age of 59.9 years ( sd 11.57 ) . among the 295 patients who were clinically suspected , 255 ( 86.4% ) the tables 1 and 2 shows the proportion of the sffis by fungal culture and koh results . proportion of sffis by culture among gender results of direct microscopy ( koh ) and culture among the 236 samples , which were positive for direct microscopy technique , 227 ( 96.2% ) were culture positive . the false negativity of direct microscopy was 3.8% , as 9 samples which were positive for direct microscopy showed negative culture results . proportion of sffi with the duration of diabetes among the clinically suspected , 50 patients with positive culture results were less than 40 years of age . two hundred and five patients with positive culture results were more than 40 years of age . since the p value was 0.011 ( < 0.05 ) , occurrence of sffi according to culture table 4 explains the proportion of sffis with the fasting blood sugar ( fbs ) and post prandial blood sugar levels ( ppbs ) . since the p value was 0.015 , occurrence of sffi was found to be statistically significant according to culture with less controlled fbs levels . since the p value was 0.003 , occurrence of sffi was found to be statistically significant with less controlled ppbs levels . proportion of sffi with fbs and ppbs levels among the study population 142 ( 37% ) of patients assumed they had fungal infections , but 295 ( 77% ) were clinically suspected and 255 ( 66% ) were mycologically confirmed [ figure 1 ] . it is evident that the actual frequency of fungal infections was underestimated by the patients . in the questionnaire all the patients said that they examined their feet every day for infections and injuries . also , all of them are cutting their nails regularly and 265 ( 69% ) of the patients dry their feet after a bath . among all the patients 150 ( 39% ) herbal medication was used by 13 ( 3.4% ) of patients for foot infections . among the study population , 150 ( 39% ) patients all of them had used anti fungal ointments as a treatment for the infection . out of these patients 110 ( 73% ) showed positive culture results indicating a recurrence of sffi . according to the nail characteristics , 283 ( 73% ) had hard or brittle nails , 274 ( 71% ) had discolored nails , and 98 ( 26% ) had dry rough feet . according to regular activities , 206 ( 53% ) of patients undertook activities associated with water and 126 ( 33% ) attended on agricultural activities . the relationship between the patient assessment on sffis , clinical finding and mycological results out of the 482 organisms which were isolated from the collected specimens , 349 ( 72% ) were non dermatophyte fungal species , 78 ( 16% ) were yeast species and 55 ( 12% ) were dermatophyte fungal species . aspergillus niger [ figure 2a and b ] was predominant in nail infections followed by fusarium.spp [ figure 3a and b ] , candida albicans and aspergillus flavus . in skin mycosis candida albicans was predominant followed by aspergillus terreus , trichoderma.spp [ figure 4a and b ] and trichophyton tonsurans [ figure 5a and b ] . table 5 shows the different fungal species isolated from the specimens , while table 6 shows some of the fungal species isolated from the lesions . ( a and b ) aspergillus niger ( a and b ) fusarium.spp ( a and b ) trichoderma.spp ( a and b ) t.tonsurans spectrum of fungal pathogens isolated from the lesions fungal species isolated from the lesions out of the 482 organisms which were isolated from the collected specimens , 349 ( 72% ) were non dermatophyte fungal species , 78 ( 16% ) were yeast species and 55 ( 12% ) were dermatophyte fungal species . aspergillus niger [ figure 2a and b ] was predominant in nail infections followed by fusarium.spp [ figure 3a and b ] , candida albicans and aspergillus flavus . in skin mycosis candida albicans was predominant followed by aspergillus terreus , trichoderma.spp [ figure 4a and b ] and trichophyton tonsurans [ figure 5a and b ] . table 5 shows the different fungal species isolated from the specimens , while table 6 shows some of the fungal species isolated from the lesions . ( a and b ) aspergillus niger ( a and b ) fusarium.spp ( a and b ) trichoderma.spp ( a and b ) t.tonsurans spectrum of fungal pathogens isolated from the lesions fungal species isolated from the lesions diabetic patients represent a unique group of individuals who are more prone to develop infections than others . a broader range of etiological agents from primary pathogens to opportunistic fungal species is one of the characteristic of fungal infections combined with diabetes . the growing diabetic population in asia and the high frequency of fungal foot infections in patients with diabetes present a considerable health problem . the clinical presentations of fungal infections are unpredictable and poor , often leading to delayed diagnosis . our study assessed the prevalence of lower limb fungal foot infections in patients with type 2 diabetes at a tertiary care hospital in sri lanka . in our study , among the 385 diabetic patients , 66% were mycologically confirmed to have superficial fungal foot infections ( sffi ) . non dermatophyte fungal species were the most common pathogen causing sffi , followed by yeast and dermatophyte fungal species . nail infections was the commonest type of sffi among the study population which is in agreement with the previous study done by yehia et al . among the patients who were clinically suspected to have sffi , 14% showed negative culture results , even though they had been clinically suspected to have sffi . these patients were using anti fungal ointments at the time of collection and therefore the growth of the fungal species might have been inhibited . aspergillus niger was the most common causative pathogen which caused nail infections followed by fusarium.spp and candida albicans . in a previous study , aspergillus niger was found to be the commonest cause of superficial fungal infections , among the cleaners . although , these data can not be directly compared due to the two different populations this may have some association with the region . our results were also in accordance with previous investigations of eckhard et al . , nair et al . , where they have also found asperigillus niger as the commonest cause of fungal foot infections in diabetes . , have reported that dermatophyte fungal species such as trichophyton.spp , microsporum.spp and epidermophyton.spp were more common in toe nail infections in diabetic foot . this conflicting data may be because of the climatic factors in different geographical areas in the world and differences in habits , cultures among different nations and also may be due to the emerging pathogens . finding of aspergillus niger as the commonest pathogen with a high proportion ( 43% ) causing superficial fungal infection is a significant finding not only locally but globally , as this organism is known as a saprophytic organism found in soil . in south east asia the hot , humid climate may have contributed to the changing etiologic pattern in sffis . further it is a known fact that many individuals walk bare foot and do not pay enough attention to foot care which is especially important in diabetic foot . this is an alarming finding especially in the diabetics and may well be an emerging pathogen globally . on the other hand misuse of antifungal ointments without prescription this study is restricted to a tertiary care hospital in colombo which contains more than > 1000 beds and is one of the main teaching hospitals in sri lanka . there is no documented data available on the prevalence of superficial fungal infections in diabetics in sri lanka . hence , our findings can not be directly compared or applied to the whole sri lankan population . a study done for a period of 5 years ( 1974 - 1980 ) had found dermtophytes ( trichophyton rubrum ) as the most prevalent causative agent for superficial fungal infections in the country . in another study , which looked at the pattern of the superficial fungal infections in two time periods ( 1974 - 78 and 1990 - 94 ) have found increased tenia pedis infection in 1990 - 94 period compared to 1974 - 78 , which the author thinks is a result of increased habit of wearing shoes . this result is in contrast to the findings where we found aspergillus niger as a commonest causative agent . further our findings of aspergillus niger as the true pathogen is consolidated as 60% of the clinical specimens gave both direct smear and culture positivity for this organism . , fatar et al . , showed that candida albicans was the most common fungal pathogen isolated from skin lesions in the lower limb , followed by non albicans species . among the non albicans species , candida parapsilosis was the commonest causative agent followed by candida tropicalis and candida lusitaniae . . risk factors for candida parapsilosis infection include previous traumatic dystrophy of the foot and exposure to soil during activities such as gardening . some yeast species like candida albicans , c. parapsilosis and c. guilliermondii constitute the physiologic flora of the feet . they can shift to an invading mycelia fungal form due to the nourishment given by the hyperglycemic state in diabetic patients to cause candida infections in lower limbs . in our study , cryptococcosis is a life threatening opportunistic fungal infection . as diabetic patients are immune compromised , they are at high risk of developing the infection when they are exposed to soil contaminated with bird droppings during activities such as gardening and walking bare foot . fungal culture results were found to be highly sensitive over the koh results . according to the chi - square test , previous studies have shown that koh false negativity was 5 - 15% . in our study , as 3.8% showed positive results in direct microscopy and negative results in culture , koh false negativity was less than 15% . these results could be due to lack of viable fungal filaments in the sample due to the use of anti fungal ointments . technical errors such as use of less concentrated koh and not adequate incubated time can also give false negative koh result . even though majority of the study population who were clinically suspected to have sffi were females , the percentage of culture positive was more in specimens collected from males . occurrence of sffi was found to be statistically significant with male gender ( chi - square test , p value was 0.044 ) . fata et al . , reported that gender related factors such as differences in life style , professional activities , and sports activities can affect the skin and nail structure of the foot . therefore , the occurrence of sffi might differ among males and females . the statistical analysis of the study using odds ratio with 95% confidence interval showed that , higher number ( 88% ) of patients were reported to have suffered from diabetes for more than ten years . in concordance with chi - square , also , the presence of sffi was found to be statistically significant with the advancing age of the diabetic patient ( p value was 0.011 ) . the increase in the prevalence of fungal foot infections with advancing age has been reported in previous investigations . koklu et al . , reported that the diabetic patients more than 50 years are more susceptible to sffi because the increased thickness of the nail plate and decreased growth rate of the nail . accordingly prolong diabetes and older age can lead to sffi because these patients provide a favorable environment for the growth of fungi , therefore recognition and early intervention is advisable because of the progressive nature of the fungal infections . majority of the patients with sffi , had a raised fasting blood sugar ( fbs ) level from the standard value of < 100 mg / dl and had high post prandial blood sugar ( ppbs ) level ( standard value < 140 mg / dl ) . occurrence of sffi was found to be statistically significant with less controlled fbs and ppbs levels respectively ( p values were 0.015 and 0.003 ) . previous study of delamaire et al . , have shown that the elevated sugar levels in blood , decreases the granulocyte function , leading to tissue invasiveness and enhanced growth of superficial fungi in diabetic foot . good metabolic management and maintenance of an optimally balanced concentration of blood glucose will help to delay and reduce the incidence of sffi in lower limbs . fungal re - infection was found in 73% indicating incomplete eradication of the fungal infection . patients tend to stop treatment as soon as the swelling , itching and redness subside before the infection is fully eradicated resulting in possible re - infection . the data gathered from the questionnaire indicated gaps in knowledge with regard to the self - diagnosis of superficial mycosis which carry a possible risk of inappropriate treatment . these findings suggested the inadequate knowledge of the patients . also , may be due to less prominent symptoms associated especially with nail infections . hence examining feet for fungal infections among diabetic patients is important because it might lead to development of severe secondary bacterial infection in diabetic foot . therefore , education of diabetic patients regarding appropriate foot hygiene and the need of daily self - inspection of the feet in order to detect and manage the infections in lower limbs is important . reporting any changes such as hard , brittle nails , discoloration of the nail and skin , whitish scales of the toe webs , to medical doctors at the diabetic clinic , cutting the toe nails short , drying the feet after a bath and avoid walking bare foot , using appropriate footwear and avoiding foot trauma should be encouraged in order to prevent sffi in diabetes . aspergillus niger was the predominant pathogen causing fungal foot infections in diabetic patients followed by candida albicans . fungal foot infections were seen significantly with the increasing age , gender , duration of the diabetes and with less controlled glycaemic level . regular examination of the lower limbs and appropriate treatment is recommended in - order to minimize the possible complications associated with diabetic foot . | background : superficial fungal foot infection ( sffi ) in diabetic patients increases the risk of developing diabetic foot syndrome . sixteen percent of urban population is suffering from diabetes in sri lanka . as the diabetes patients are more prone to get fungal foot infections , early intervention is advisable owing to the progressive nature of the infection .
there is no data on the prevalence of sffis in diabetic patients in sri lanka.objective:to determine the etiological agents causing sffi in patients with type 2 diabetes.materials and methods : three hundred eighty five diabetic patients were included .
nail clippings and swabs were collected from the infected sites using the standard protocol .
laboratory identification was done and pathogens were identified to the species level by morpho physiological methods.results:clinically 295 patients showed sffi , of which 255 ( 86% ) were mycologically confirmed for infection . out of 236 direct microscopy ( koh ) positives , 227 ( 96% ) were culture positive . two hundred and fifty one patients ( 98% ) with sffi had diabetes for more than 10 years .
of the patients with sffis 92% had > 100 mg / dl fbs and 81% had > 140 mg / dl ppbs levels and 80% had both elevated fbs and ppbs .
non - dermatophyte fungal species were the commonest pathogens followed by yeast and dermatophytes.conclusion:aspergillus niger was the commonest pathogen followed by candida albicans .
sffis were seen significantly with the increasing age , gender , duration of diabetes and with less controlled glycaemic level . |
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with the advent of laparoscopy , repair of ventral hernias has undergone a revolution . with continuous improvement in technology and instrumentation , on the other hand , laparoscopic repair of large ventral hernias can be technically challenging . various factors influence the complexity of the procedure , an important one of which is the handling of mesh intraperitoneally . various techniques of laying the mesh have been used . we present the double rolling and center hitch ( drach ) technique to lay a large intraperitoneal onlay mesh . the drach technique was used in 18 patients with large ventral hernias between may 2010 and september 2011 [ table 1 ] . these included large primary paraumbilical , epigastric , and incisional hernias . all are made on one side of abdomen , just within the anterior axillary line . adhesiolysis is done and the hernia contents are reduced . in eight cases , we encountered swiss cheese defects and in one there was gross divarication of recti . the defects are measured in centimeters and the size of mesh to be placed is determined . a minimum of 5 cm margin of the mesh is kept on all sides of the defect . in 10 patients , we used 15 20 cm mesh and in five patients , 20 25 cm mesh is used . in one patient , we had to use an additional 10 15 cm mesh to cover all defects with the required margin , and a very large mesh ( 42 28 cm ) had to be used in one patient [ table 2 ] . we prefer proceed mesh ( ethicon , sonnerville , nj , usa ) , which was used in all the cases . size and time relationship the center of the defect is marked on the skin and so is the outline of the mesh , keeping the pneumoperitoneum to 10 mm of hg . four corner stitches are taken with 2 - 0 polypropylene and both ends are cut long . the mesh is tightly rolled from either side along its long axis to meet in the mid - line keeping two corner sutures inside the roll [ figure 1 ] . the anti - adhesion coating is kept on the inside , which after deployment would be adjacent to the abdominal viscera . having done this , a center hitch is taken with 2 - 0 polypropylene on the outer ( parietal ) side [ figure 2 ] . in three cases , the needle was inserted along with the mesh roll and retrieved out through the center of the defect . in the rest the mesh is drenched in saline and both rolled halves squeezed together to negotiate it through the 12-mm port [ figure 3 ] . first , the right half of mesh is unrolled by a pushing maneuver , keeping the center hitch in place [ figure 4 ] . four transfascial corner sutures ( tfs ) were placed in 14 patients , while in two patients , both with infraumbilical hernias and 20 25 cm mesh , the inferior corners were tacked to the coopers ligament after raising a peritoneal flap . the free edge between the tfs is fixed using protacks(ethicon , usa ) in two concentric rings or crowns . the outer crown is placed 1 cm from the margin of mesh , at an interval of approximately 2 cm . the inner crown is placed at least 2 - 3 cm inside the outer crown . the interval between the tacks of inner crown is variable and depends on the number of remaining tacks in tacking device . after securing the right half of the mesh the inner crown of tacks is placed after the outer crown is completed . all along the center hitch the tfs were delivered out with a suture passer needle . the total operative time was noted starting from creation of carboperitoneum till closure of last port incision . this was marked from the beginning of insertion of mesh and till firing of last tack . it includes the time taken for mesh insertion , unfolding the mesh , centering it over the defect , securing it with corner transfascial sutures , and fixing it with tacks . the relationship of mesh size and the mdt ( size - to - time ratio ) was calculated . all are made on one side of abdomen , just within the anterior axillary line . adhesiolysis is done and the hernia contents are reduced . in eight cases , we encountered swiss cheese defects and in one there was gross divarication of recti . the defects are measured in centimeters and the size of mesh to be placed is determined . a minimum of 5 cm margin of the mesh is kept on all sides of the defect . in 10 patients , we used 15 20 cm mesh and in five patients , 20 25 cm mesh is used . in one patient , we had to use an additional 10 15 cm mesh to cover all defects with the required margin , and a very large mesh ( 42 28 cm ) had to be used in one patient [ table 2 ] . we prefer proceed mesh ( ethicon , sonnerville , nj , usa ) , which was used in all the cases . size and time relationship the center of the defect is marked on the skin and so is the outline of the mesh , keeping the pneumoperitoneum to 10 mm of hg . four corner stitches are taken with 2 - 0 polypropylene and both ends are cut long . the mesh is tightly rolled from either side along its long axis to meet in the mid - line keeping two corner sutures inside the roll [ figure 1 ] . the anti - adhesion coating is kept on the inside , which after deployment would be adjacent to the abdominal viscera . having done this , a center hitch is taken with 2 - 0 polypropylene on the outer ( parietal ) side [ figure 2 ] . in three cases , the needle was inserted along with the mesh roll and retrieved out through the center of the defect . in the rest , the suture was retrieved with a suture passer needle through the center . the mesh is drenched in saline and both rolled halves squeezed together to negotiate it through the 12-mm port [ figure 3 ] . first , the right half of mesh is unrolled by a pushing maneuver , keeping the center hitch in place [ figure 4 ] . four transfascial corner sutures ( tfs ) were placed in 14 patients , while in two patients , both with infraumbilical hernias and 20 25 cm mesh , the inferior corners were tacked to the coopers ligament after raising a peritoneal flap . the free edge between the tfs is fixed using protacks(ethicon , usa ) in two concentric rings or crowns . the outer crown is placed 1 cm from the margin of mesh , at an interval of approximately 2 cm . the inner crown is placed at least 2 - 3 cm inside the outer crown . the interval between the tacks of inner crown is variable and depends on the number of remaining tacks in tacking device . after securing the right half of the mesh , the left half is unrolled with a pulling maneuver . for this , we prefer curved bladed dissectors with their jaws open [ figure 4 ] . the inner crown of tacks is placed after the outer crown is completed . all along the center hitch the tfs were delivered out with a suture passer needle . the total operative time was noted starting from creation of carboperitoneum till closure of last port incision . this was marked from the beginning of insertion of mesh and till firing of last tack . it includes the time taken for mesh insertion , unfolding the mesh , centering it over the defect , securing it with corner transfascial sutures , and fixing it with tacks . the relationship of mesh size and the mdt ( size - to - time ratio ) was calculated . mesh deployment time : the average mdt of the 18 procedures was 28.6 min with the drach technique which was less than for the usual technique . size - to - time ratio : the mean size - to - time ratio was 12.2 which was less than for the usual techniquein all cases , the mesh was adequately centered with a minimum margin of 5 cm from the defect .
size - to - time ratio : the mean size - to - time ratio was 12.2 which was less than for the usual technique in all cases , the mesh was adequately centered with a minimum margin of 5 cm from the defect . with tight double rolling of mesh , it was easier to negotiate it through a 12-mm port . in cases where mesh larger than 20 25 cm was used , a 15-mm port was utilized for mesh passage . once inside , the alignment of mesh in craniocaudal axis and centering over the defect was greatly simplified and much quicker by suspending the mesh with the center stitch retrieved percutaneously . as the mesh was tightly suspended throughout the fixation , the handling of mesh was considerably reduced . this center hitch also helped in avoiding the mesh to get folded on itself . hesselink and associates concluded that hernias over 4 cm diameter should be repaired using a prosthesis . showed that use of mesh significantly reduces the number of recurrences after incisional hernia repair . over the years jenkins et al . demonstrated an increase in operating time with every square centimeter increase in size of mesh , and with less number of years of post - graduate training . introduction of a large mesh mandates proper rolling to insert it through a 12- or 15-mm laparoscopic port . himpens et al . suggested that the mesh should be inserted in a sterile bag after removal of the 10-mm port . we use the 12-mm excel port ( ethicon , usa ) for insertion after tight doublerolling [ figure 4 ] . negotiating a mesh larger than 15 20 cm through a 12-mm port carries a higher risk of mesh damage , even after tight rolling . special care is taken with meshes having adhesion barriers to prevent its damage during insertion . in the drach technique , the adhesive coat is kept on the inside and the mesh is drenched in saline for easier passage . all the defects are covered properly with adequate margin of at least 3 cm to reduce recurrence . measuring the span of the defects from inside more accurately determines the size of mesh to be placed and avoids over- or under - sizing . , the center hitch is taken through a point on the skin which is marked under laparoscopic guidance . it suspends the mesh to the anterior abdominal wall centering over the defect / cluster of defects . injuries at the time of mesh placement are well known . handling a large mesh in a compromised domain predisposes to bowel and vascular injuries . technical challenge of handling a large mesh after an already exhaustive adhesiolysis ( especially in incisional hernias ) adds to the fatigue and frustration of the surgeon . the reduction in the technical difficulty may be of advantage to the surgeons especially in the early phase of their learning curve . | background : intraperitoneal onlay mesh repair is an established modality to treat large ventral hernias . various techniques of laying the mesh are utilized .
we present the double rolling and center hitch technique to lay a large intraperitoneal onlay mesh.objective:the aim of the study is to devise and adopt a method to reduce the difficulty in manoeuvring a large mesh inside the peritoneal cavity . it should also help in correct placement of mesh and decrease the operative time.materials and methods : the drach technique was used in eighteen patients with large ventral hernias between may 2010 and september 2011 .
the mesh size used was 15x20 cm and more ( considered to be large mesh).results : all the procedures were completed successfully .
mesh handling was significantly easier with the drach technique .
the average mesh deployment time ( mdt ) was 15mins . in all cases
the mesh was adequately centred with a margin of 3 - 5 cm from the defect.conclusion:the drach technique can be employed to lay large intraperitoneal meshes in order to reduce the handling difficulties associated with large meshes , and to aid in better placement of meshes so as to centered over the defect . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
cell contacts with the extracellular matrix ( ecm ) provide both cohesive and functional properties in a variety of tissues , such as epithelia , nerves , muscle , and stroma , through specific interactions with cell membrane receptors [ 1 , 2 ] . all ecms are made up of collagen fibrils and/or networks , proteoglycans as well as specialized glycoproteins such as fibronectin and laminins that are archetypal of interstitial ecm and basement membrane ( bm ) , respectively [ 3 , 4 ] . cells from multiple origins interact with ecm molecules using a variety of receptors , most of them being members of the integrin superfamily . over 24 distinct integrin heterodimers have been characterized to date , describing the association between 18 and 8 subunits [ 57 ] . the fact that integrin - mediated connections between the ecm and the cytoplasm regulate key cell functions such as adhesion , migration , proliferation , apoptosis , and differentiation is well recognized [ 811 ] . epithelia express a wide variety of typical integrin receptors such as the 11 , 21 , 31 , and 64 integrins that serve as collagen and/or laminin receptors [ 1215 ] . although less well documented in epithelia , the rgd - dependent integrins are another group of receptors that appears to be involved in epithelial cell homeostasis [ 1517 ] . rgd - dependent integrins include 51- , 81- , and v - containing integrins and are named as such because they specifically recognize the rgd motif , a sequence of three amino acids ( arg - gly - asp ) found in many ecm molecules such as fibronectin and osteopontin [ 5 , 12 , 14 , 15 ] . collectively , these interactions are termed the rgd - dependent adhesion system ( figure 1 ) . interestingly , rgd - dependent cell interactions represent a key role in hierarchical assembly and maturation of adhesion structures including focal complexes ( fxs ) , focal adhesions ( fas ) , and fibrillar adhesions ( fbs ) [ 1 , 2 , 18 ] . therefore , rgd adhesion can by divided into three distinct components , the extracellular component ( e.g. , fibronectin ) , the membrane receptor ( e.g. , the 51 integrin ) , and the intracellular molecule ( e.g. , vinculin ) . moreover , each component acts in concert with the others to organize and regulate rgd adhesion dynamics . in this paper , we will focus on the importance of the rgd - dependent adhesion system for human intestinal crypt cell homeostasis ( section 2 ) . we chose to elaborate on recent findings from our laboratory related to each of the rgd adhesion components , the 81 integrin ( receptor , section 3 ) , integrin - linked kinase ( ilk ) ( intracellular molecule , section 4 ) , and type vi collagen ( ecm , section 5 ) . the small intestinal epithelium is a useful model to investigate the relationship between cell state and interaction with the ecm because of the well - defined architecture of its renewing unit , the crypt - villus axis . indeed , proliferative cells , differentiating cells , and mature functional cells are topologically restricted to distinct compartments : the lower two - thirds of the crypt , upper third of the crypt , and villus , respectively . gene expression in intestinal crypt cells must therefore be tightly regulated to efficiently control stemness , proliferation , migration , and differentiation in order to ensure the right equilibrium for the production of functional cells destined to renew the villus epithelium [ 19 , 20 ] . there is strong evidence that cell - matrix interactions are involved in the regulation of these cell functions in the crypt [ 12 , 21 , 22 ] . for instance , differential spatial expression of laminins in the epithelial bm and their epithelial integrin receptors were observed along the crypt axis while in vitro studies have revealed functional relationships between laminin - binding integrins and specific intestinal cell functions such as proliferation , migration , and differentiation [ 2330 ] . a schematic illustration of the human crypt - villus axis and the spatial expression of laminins , laminin receptors of the integrin family , and the two classic rgd components fibronectin and the 51 integrin ( as depicted by dark areas ) is shown in figure 2 . moreover , another example is the transient expression of the tenascin and osteopontin receptor 91 integrin in the lower third of the crypt of the immature small intestine as well as in proliferative epithelial crypt cells and its reexpression in colon adenocarcinoma cells . the rgd archetype fibronectin is another ecm component that was found strongly expressed in the epithelial bm of the crypts in both human and small laboratory animals [ 3336 ] . synthesis and deposition of fibronectin by proliferating intestinal epithelial cells was confirmed in vitro [ 26 , 34 ] . furthermore , expression of the fibronectin receptors , 51 and v - containing integrins , was found to be associated with intestinal cell proliferation [ 21 , 29 , 37 ] . taken together , these observations suggest that fibronectin may significantly contribute to the rgd system regulating intestinal crypt cell functions . to investigate this hypothesis , we used a strategy combining expression studies in the intact human intestine and functional studies using hiec cells , a human intestinal epithelial crypt cell model well - characterized for the expression of typical features of intestinal crypt cells [ 3841 ] . as summarized in the next sections , this experimental approach has led to the identification and characterization of new components of the rgd - dependent adhesion system that emphasize the importance of this adhesion system in human intestinal crypt homeostasis . initially characterized in the chicken nervous system [ 42 , 43 ] , integrin 81 represents an important rgd - dependent receptor . ligand binding to integrin 81 was shown to be important for rhoa gtpase activation and subsequent actin stress fiber assembly in vascular smooth muscle cells [ 4547 ] . integrin 81 was also recently found to play an important role in microfilament organization which was central to rgd - dependent intestinal epithelial crypt cell adhesion . 8 subunit knockdown experiments , carried out in hiec cells , showed that this integrin is important for proper vinculin recruitment to adhesion structures ( figure 3 ) . intestinal epithelial crypt cells in which 8 was knocked down exhibited lower numbers of vinculin - positive fas compared to controls , while paxillin localization was not affected . it is well known that rhoa / rock signalling enhances actin stress fiber assembly and increases cell adhesion [ 4951 ] . rhoa activity was shown to promote scaffolding protein recruitment , including vinculin , to the developing adhesion structures [ 51 , 52 ] . thus , the increased rhoa activity displayed by 8 knockdown cells leads to the absence or reduced levels of vinculin observed within these cells [ 48 , 53 ] . based on the scheme of adhesion structures hierarchical assembly , vinculin recruitment occurs at later stages of fx formation , while paxillin is recruited at early stages . thus , observations made in intestinal epithelial crypt cells suggest that integrin 81 is essential , at this particular stage of fx maturation into fa , via its role in rhoa activation ( figure 3(a ) ) . a similar function could also be predicted for the collagen - binding integrin 21 , considering the expression of this receptor in undifferentiated intestinal epithelium cells and its participation in rhoa activation [ 44 , 55 , 56 ] . interestingly , ectopic expression of the enterocytic differentiation associated factor gata-4 in intestinal epithelial crypt cells caused a depletion of 8 subunit expression [ 39 , 48 ] . in these same cells , reduced levels of 81 were associated with a decrease in cell growth , marked by cyclin d1 inhibition and accumulation of cells in the g1 phase . similarly , decreased rhoa activity was observed in differentiated and nonproliferative ht29 cells compared to their undifferentiated and proliferative counterparts . together with the role of integrin 81 in rgd - dependent adhesion , these findings support the concept that cell - ecm interactions are crucial to maintaining a proliferative state in epithelial cells , which is anchorage and cell position dependent , reflecting its exclusive localization in the lower crypt of the intact intestine . due to the role of integrin 81 in rgd - dependent cell adhesion and rhoa gtpase activity , this receptor was shown to exert a critical influence on intestinal epithelial crypt cell motility . alteration of rhoa activity was found to modulate migration in different systems [ 49 , 57 ] . we recently reported that loss of rgd-81 interactions in intestinal epithelial cells caused increased cell migration . from a physiological perspective , proliferating intestinal epithelial cells must be restricted to the lower two - thirds of the crypt to avoid premature terminal differentiation and loss of proliferative capacity [ 38 , 40 ] . therefore , without necessarily affecting the expression of differentiation master regulator genes , rgd - dependent adhesion plays a major role in regulating cell migration , which in turn is crucial for wound healing , cell differentiation , and tissue integrity [ 24 , 58 , 59 ] . indeed , integrin receptors , such as 51 and v integrins , play a central role in controlling anoikis or apoptosis by loss of attachment [ 10 , 11 , 60 ] . specifically , engagement of 1 integrins was found to be essential to intestinal epithelial cell survival through fak signalling [ 60 , 61 ] . as mentioned above , integrin 81 is involved in efficient vinculin recruitment to developing adhesion structures [ 48 , 53 ] . the presence of vinculin in cell - ecm adhesion structures affects cell survival signal transduction . as previously described , hiec cells share a number of features with intestinal epithelial stem cells , including a proliferative and undifferentiated state as well as the expression of several putative stem cell markers . interestingly , silencing of vinculin expression in f9 embryonic teratocarcinoma cells , another cell model closely related to stem cells , has shown increased resistance to anoikis , while ectopic reexpression of vinculin restored sensitivity to anchorage - dependent survival . a similar phenomenon was observed in nonadherent 8 knockdown intestinal epithelial crypt cells . in both studies , the absence of vinculin combined with the presence of paxillin in primitive adhesion structures prior to loss of adhesion could explain such a phenomenon ( figure 3 ) . at the molecular level , it has been shown that paxillin exhibits partially overlapping binding sites for fak and vinculin . thus , the vinculin tail domain appears to compete with fak for paxillin binding . in the presence of vinculin , fak activation would depend on ecm - integrin binding . however , absence of vinculin leads to a conformational change in adhesion structures which results in constitutive activation of fak when bound to paxillin where fak activity no longer relies on ecm - integrin interactions [ 53 , 63 ] . additionally , nonadherent 81-depleted intestinal epithelial cells showed increased activity of the pi3 a summary of integrin 81 contribution to anoikis regulation in epithelial intestinal crypt cells is presented in figure 3 . considering the proliferative and highly adaptive capacities of crypt cells , such as stem and transit amplifying cells , rgd - dependent 81 interactions with ecm are suggested to act as a security switch that keeps the detachment of undifferentiated epithelial cells in check . it is worth noting that none of the five human colorectal cancer cell lines tested were found to express the integrin 8 subunit and that ectopic expression of this rgd - dependent receptor restored sensitivity of malignant cells to anoikis . the mechanism by which colon cancer cells repress 8 expression to bypass this checkpoint remains unknown . however , in normal cells , this security step mediated by 81 occupancy is potentially important to support homeostasis in the human intestinal crypt . new evidence from the literature has shown that colon cancer may originate from defective crypt stem cells . therefore , in light of the expression of 81 in the region associated with intestinal stem cells , combined with its role in sensitizing epithelial cells to anoikis , it could be speculated that 8 integrin silencing represents a key step in cancer initiation , in order to escape apoptosis upon modification of the malignant stem cell niche or location . in this context , 81 could promote defective progenitor cell elimination , and consequently prevent the onset of ecto - cryptal proliferative structures . such specific involvement of rgd - dependent integrins is not without precedent since altered expression of other heterodimers has been reported in colon cancer . for instance , integrin v3 expression has been found to be specifically decreased in anoikis - resistant caco-2 cells . the intestinal epithelial cell mediates rgd interactions through expression of specific integrin receptors , as observed with 81 , as well as through the production and deposition of rgd ligands , such as fibronectin . the efficient deposition of fibronectin into the bm relies upon its recognition by rgd - dependent integrins , which mediate its unfolding to expose specific fibronectin structural domains , which in turn mediates the formation of insoluble fibronectin fibrils . fibronectin deposition is characterized by the formation of specialized cell - matrix contact structures containing integrins , cytosolic proteins , and actin referred to as fibrillar adhesion ( fb ) points . the integrin - linked kinase ( ilk ) is a constituent of integrin containing adhesion sites where it mediates multiple cellular processes . ilk is a pseudokinase and scaffolding protein ubiquitously expressed in mammalian cells forming a trimeric complex with pinch and parvins named the ipp complex [ 6870 ] . ilk interacts with the cytoplasmic domain of integrin 1 and 3 subunits to create a physical link between integrins and the actin cytoskeleton [ 68 , 71 ] . interestingly , it has been suggested that ilk regulates fibronectin expression / deposition [ 7274 ] and other studies have placed ipp complex members within fa points [ 75 , 76 ] . in vivo , fibronectin expression is restricted to the bm underlying epithelial crypt cells and hiec cells produce copious amount of fibronectin and generate numerous well - defined adhesion structures . the expression and roles of ilk we first focused on the localization of ilk - related components in the small intestine . as previously observed for fibronectin and integrin 51 in the intact intestine [ 21 , 29 , 33 ] , ilk , pinch-1 -parvin , and -parvin were found to be predominantly expressed by the proliferative epithelial cells of the crypts . in hiec cells , ilk , pinch-1 , -parvin , and -parvin were all closely associated with fa points ( figure 4(a ) ) . a sirna strategy was used to knock down ilk expression in hiec cells in order to further investigate the role of ilk in intestinal crypt cells . interestingly , ilk knockdown in hiec was accompanied by severe disruption of the ipp complex including the loss of pinch-1 and parvins as well as major alterations in fibronectin synthesis and functional matrix deposition ( figure 4(b ) ) . overexpression of ilk was previously shown to increase fibronectin deposition in rat intestinal cells while ilk knockdown decreases fibronectin expression in mice and human colon cancer cells . indeed , the fibronectin gene promoter contains response elements that have been shown to be potentially regulated by ilk - mediated signalling [ 68 , 77 ] . however , in hiec cells , although a reduction of fibronectin was observed at the transcript level , ilk knockdown had no net effect on fibronectin protein amounts found in the culture medium suggesting that it was mainly the ability to process and deposit soluble fibronectin that was altered by the loss of the ipp complex . the exact mechanism by which ilk knockdown impairs fibronectin deposition remains to be elucidated . expression levels of the fibronectin integrin receptors were not altered in hiec ilk knockdown cells suggesting that the required receptors for fibrillogenesis remain available for binding . however , because of the important scaffolding role of ilk and ipp complexes , the decrease of fibronectin deposition in these ilk - deficient cells may reflect a reduction in the cytoskeletal tension necessary for fibrillogenesis [ 78 , 79 ] . alternatively , alteration in signalization pathways may also be involved . indeed , key signalling molecules such as src , pi3 k , and erk have been shown to modulate fibronectin deposition in various cell models [ 8082 ] and ilk and the ipp complexes can regulate these signalling molecules [ 8385 ] . in addition to an alteration in fibronectin deposition , ilk knockdown severely affected basic intestinal crypt cell functions such as cell spreading , migration restitution abilities as well as cell proliferation . alterations in these functions in ilk - knockdown hiec cells were not surprising since these functions can be stimulated by fibronectin in intestinal epithelial cells [ 8689 ] . interestingly , exogenously deposited fibronectin was found to fully rescue the ilk - knockdown hiec phenotype with regard to cell proliferation , spreading and migration . taken together , as summarized in figure 4 , these data reveal that ilk and , by extension , the ipp complexes , perform crucial roles in the control of human intestinal crypt cell homeostasis , especially as key mediator of fibronectin deposition in the bm , which in turn regulates cell proliferation , migration , and restitution . type vi collagen is a ubiquitously expressed ecm component . in interstitial ecm , collagen vi acts as an anchoring meshwork bridging collagen fibers to the surrounding matrix [ 91 , 92 ] . collagen vi has also been shown to directly interact with the bm - specific type iv collagen supporting a key role for this collagen in connecting bm to ecm [ 94 , 95 ] . however , we recently identified collagen vi as a bona fide component of the basal lamina in the intestinal bm and found that it is synthesized in significant amounts by crypt epithelial cells . to investigate the function of type vi collagen in the intestinal epithelial crypt cell , we used a similar knockdown strategy with hiec cells as described in the previous sections for integrin the 8 subunit and ilk . surprisingly , abolition of collagen vi expression resulted in a striking increase in cell size and spreading accompanied by a significant increase in the number of stress fibers and tensin recruitment at the fb points . the observations that removal of collagen vi emphasized features normally associated with fibronectin suggested that collagen vi regulates fibronectin assembly in epithelial cells . interactions between collagen vi and fibronectin have been previously reported [ 93 , 96 ] . further investigation in collagen - vi - depleted hiec cells revealed that fibronectin was increased at both protein and transcript levels and was subjected to extracellular rearrangement into long , parallel fibrils . importantly , exogenous collagen vi , but not collagen i or iv , was able to fully rescue the knockdown phenotype indicating that the effect is specific for type vi collagen . considering that exposure of fibronectin - binding sites is critical for both cell binding and fibrillogenesis [ 67 , 97 ] , one may hypothesize that , under normal conditions , collagen vi acts by limiting cellular accessibility to fibronectin through competition for integrin receptors ( figure 5(a ) ) or by a direct interaction with fibronectin in the ecm ( figure 5(b ) ) . consistent with this possibility , collagen vi has been reported to be recognized by the rgd - binding 51 and v integrins [ 98100 ] . furthermore , hiec binding to collagen vi is integrin 1 dependent and it was the fb complexes , specifically enriched in tensin and 51 integrin [ 6 , 54 , 67 ] , that were enhanced in collagen - vi - depleted hiec . to further investigate the mechanism underlying the generation of fb complexes in collagen - vi - depleted intestinal crypt cells , the regulation of actomyosin forces was analyzed . actin contractility depends on the phosphorylation of the myosin light chain ( mlc ) , which is mainly mediated by the kinases mlc ( mlck ) and rho ( rock ) acting on mlc and myosin phosphatase , respectively [ 101104 ] . interestingly , mlck - dependent activation of mlc phosphorylation was observed in poor collagen vi / rich fibronectin ecm environments consistent with the observed generation of higher numbers of tensin - enriched fb complexes and extensive fibronectin fibrillar deposition ( figure 5(c ) ) . as summarized in figure 5 , these data identified collagen vi as a major regulator of fibronectin synthesis and fibrillogenesis and suggest that collagen vi influences intestinal epithelial crypt cell behaviour by restraining cell - fibronectin interactions and their downstream events . described as a predominant epithelial bm component in the intestinal crypt more than 3 decades ago [ 3336 ] , fibronectin has been confirmed to play an important role in the rgd system regulating crypt epithelial cell functions . the recent findings summarized herein further emphasize the crucial importance of this rgd - adhesion system and its regulatory mechanisms . indeed , intestinal epithelial cells can regulate rgd interactions through expression of specific integrin receptors , as exemplified by 81 , which exerts major regulatory influences on key cell functions such as cell proliferation , migration , and survival [ 48 , 53 ] . regulation of rgd interactions can also be accomplished by regulating production and deposition of their ligands , such as fibronectin , as illustrated by the finding that ilk / ipp complexes are key mediators of fibronectin deposition into the bm , which in turn regulates cell proliferation , migration , and restitution . finally , regulation of rgd - dependent cell interactions can also be achieved by interaction with other ecm molecules as shown with type vi collagen , a basement membrane component that regulates epithelial cell - fibronectin interactions . taken together , these studies define new molecular elements and shed new light on the relative complexity of specific cell - matrix interactions in a well - defined environment such as the intestinal crypt and the critical impact these interactions have on cell function . | interactions between the extracellular matrix ( ecm ) and integrin receptors trigger structural and functional bonds between the cell microenvironment and the cytoskeleton .
such connections are essential for adhesion structure integrity and are key players in regulating transduction of specific intracellular signals , which in turn regulate the organization of the cell microenvironment and , consequently , cell function .
the rgd peptide - dependent integrins represent a key subgroup of ecm receptors involved in the maintenance of epithelial homeostasis . here
we review recent findings on rgd - dependent ecm - integrin interactions and their roles in human intestinal epithelial crypt cells . |
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rosai - dorfman disease ( rdd ) , also known as sinus histiocytosis with massive lymphadenopathy most often presents as a painless , massive bilateral enlargement of the cervical lymph nodes associated with fever , leucocytosis , elevated erythrocyte sedimentation rate ( esr ) , and polyclonal hypergammaglobulenemia . it is characterized by marked lymphoplasmacytic and hystiocytic infiltration with emperipolesis ( lymphophagocytosis ) in the lymph nodes . although extranodal rdd ( involving the orbit , upper respiratory tract , skin , subcutaneous tissue , skeletal system , central nervous system , etc . ) is found in 40% of the patients with rdd , involvement of the thyroid has been reported rarely . so far , six cases of rdd involving the thyroid were reported in literature , all occurred in females with a mean age of 56.3 years . all the cases were initially diagnosed as thyroid malignancies with lymph nodal metastasis , both clinicoradiologically and cytologically.[24 ] the cytological features of rdd involving the thyroid are virtually diagnostic and can obviate the need for a biopsy in most cases . a 15-year - old female presented with multiple , painless enlargement of the cervical and submandibular lymph nodes along with thyromegaly and fever since three months . she was initially diagnosed outside as having tuberculosis and was under antituberculous treatment since two months , to which there was no response . routine laboratory investigations revealed hemoglobin of 10 gm% , erythrocyte sedimentation rate of 90 mm at the end of first hour , total leukocyte count of 14,600 cells / mm and a differential count of neutrophils-44% , lymphocytes-52% , monocytes-2% , eosinophils-2% . fine needle aspiration cytology ( fnac ) was performed from the thyroid as well as the lymph nodes . smears were stained with giemsa as well as hematoxylin and eosin and found to be richly cellular [ figure 1 ] . smears from both the lesions , that is , thyroid and lymph nodes showed a similar cytomorphological picture . microscopic examination revealed the presence of histiocytes with single to multiple nuclei , fine nuclear chromatin and inconspicuous - to - prominent nucleoli ; no nuclear grooving or atypia was noted . the histiocytes had abundant pale cytoplasm containing numerous intact lymphocytes ( emperipolesis ) [ figure 1 lower inset ] , plasma cells and neutrophils [ figure 1 upper inset ] . based on this characteristic cytomorphology , a diagnosis of rosai - dorfman disease , nodal and extranodal , involving the thyroid , was made , ruling out malignancy . the patient was treated with steroids with a good response and thus an unnecessary total thyroidectomy was prevented . cellular smears with clusters of histiocytes over a lymphocytic background ( h and e , 100 ) . upper inset shows histiocytes with emperipolesis of neutrophils ( h and e , 400 ) and lower inset shows emperipolesis of lymphocytes ( giemsa , 1000 ) rosai - dorfman disease is a rare histiocytic disorder of unknown etiology involving the lymph nodes . extranodal rdd involving the thyroid is still rare . so far , in the literature six cases of rdd involving the thyroid have been reported . although most cases of nodal rdd occur during the first or second decade of life , with a male predominance , all the previously reported six cases of rdd involving the thyroid have occurred in elderly females with a mean age of 56.3 years . the clinical presentation of rdd as described in the initial publications consists of painless , bilateral cervical lymphadenopathy associated with fever , leucocytosis , elevated esr , and hypergammaglobulenemia . a wide range of extranodal involvement described recently includes involvement of the eyes , respiratory tract , including nose , skin , subcutaneous tissue , bone , skeletal muscle , central nervous system , intestinal tract , salivary glands , genitourinary tract , thyroid , breast , liver , kidney , heart , uterine cervix and so on . our case was a 15-year - old female , who presented with multiple cervical and submandibular lymphadenopathies , diffuse thyromegaly , fever , leucocytosis and elevated esr . however , the possibility of rdd involving thyroid was not considered until fnac was performed . the cytological features of rdd usually reveal numerous large histiocytes with abundant pale cytoplasm and ingested lymphocytes ( emperipolesis ) . the histiocytes show positive immunostaining for s100 protein , cd14 , cd11c , cd33 and cd68 antigens in the cytological smears . differential diagnosis of rdd involving the thyroid include undifferentiated carcinoma , langerhans cell histiocytosis , thyroiditis and lymphoma . in undifferentiated thyroid carcinoma , the neoplastic cells show highly pleomorphic and hyperchromatic nuclei and neoplastic spindle cells . in the langerhans cell histiocytosis , de quervain thyroiditis should be differentiated from rdd on the basis of the presence of multinucleate giant cells , degenerating thyrocytes , inflammatory cells , macrophages , colloid and dirty background and absence of emperipolesis and lymphadenopathy . in hodgkin lymphoma , the smears show lymphocytes , plasma cells , histiocytes , eosinophils and the characteristic reed sternberg ( r - s ) cells . in rdd , rdd involving the thyroid , although rare , can be diagnosed by the characteristic cytological features , obviating the need for a biopsy . ours is the first case of rdd involving the thyroid to be diagnosed by fnac , to the best of our knowledge . | rosai - dorfman disease ( rdd ) , also known as sinus histiocytosis with massive lymphadenopathy is a rare disease involving the lymph nodes .
extranodal rdd involving the thyroid is extremely rare .
so far , six cases of rdd involving thyroid have been reported in the literature ; all have occurred in females with a mean age of 56.3 years .
clinically , radiologically and cytologically , all the cases were initially diagnosed as thyroid malignancies with lymph nodal metastasis .
the final diagnosis was made histologically only after total thyroidectomy .
we herein , present a seventh case of rdd involving the thyroid in a 15-year - old female , diagnosed first on fine needle aspiration cytology ( fnac ) .
we conclude that fnac is a useful diagnostic procedure for rdd involving thyroid ; it can avoid an unnecessary thyroidectomy . |
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plants coordinate many biochemical and physiological processes with the earth 's 24 hour rotation , presumably improving their fitness by anticipating the predictable cycles of light and temperature in the environment . two recent papers have examined biological rhythms in plants using genome - wide transcript - analysis techniques . these approaches have uncovered candidate regulatory genes and have both confirmed and extended the range of physiological processes known to be under rhythmic control . the analysis of biological rhythms is complicated by the fact that many rhythmic processes are directly influenced by light ( figure 1 ) . in the natural day / night cycle , light regulation can lead to rhythmic changes that are referred to as diurnal responses . additionally , many physiological changes anticipate the coming dawn or dusk and continue to show an oscillation of roughly 24 hours even in constant conditions ; these are termed circadian rhythms . the many overt morphological rhythms include leaf movements , the opening of stomatal pores and the photoperiodic induction of flowering . in addition , biological rhythms have been found to regulate the expression of genes encoding enzymes and regulatory and structural proteins . daily cycling of these gene products appears to coordinate physiological processes in the plant with the ambient environment . together , these developmental and enzymatic rhythms are thought to optimize photon capture and avoid concurrent stresses , thus providing energy for both nitrogen and carbon uptake . ( a ) simplified regulatory circuit for the circadian system . light ( represented by a bulb ) regulates some genes directly , leading to diurnal rhythms under cycles of light and dark . light also resets the circadian oscillator , which in turn regulates the expression of clock - controlled genes . ( b ) circadian - clock - controlled genes exhibit peak expression levels at several phases with respect to the light / dark cycle ( represented by hatched and white bars above the graph ) . examples of clock - regulated gene sets are given , with peak expression before subjective dawn ( for example , the phenylpropanoid synthesis pathway ; red ) and early in the subjective day ( for example , photosynthesis - related genes ; green ) . there are two main technologies for array - based global gene - expression analysis ; both are based on nucleic - acid hybridization but one relies on cdnas ( usually attached to glass ) while the other uses synthetic oligonucleotides . in two recent papers , each of these technologies was used to undertake the global analysis of steady - state mrna levels in arabidopsis thaliana plants maintained under diurnal and/or circadian growth conditions . this is the first time global transcript analysis has been used to uncover the networks of rhythmic genes in any organism . schaffer et al . analyzed gene expression using cdna arrays generated by the arabidopsis functional genomics consortium ( a consortium , funded by the us national science foundation , that includes stanford , wisconsin and michigan state universities ) . the arabidopsis cdna array , which represents approximately 7,800 unique genes or around 30% of expressed sequences , was used to identify diurnal expression changes in samples harvested under a 12 hour light/12 hour dark cycle , and circadian regulation in plants transferred to constant light or darkness . the arabidopsis cdna array results were quite reproducible , with fewer than 2% of transcripts measured as having a greater than two - fold difference in abundance in independent , replicate rna samples . approximately 11% of genes showed differential expression at one or more of four phases tested during the light / dark cycle ( data available at ) . about 25% of genes that showed diurnal regulation were also circadian - regulated , as revealed by differential transcript abundance after 12 hours versus 24 hours under constant conditions . in total , around 2% of the genes represented on the array were scored as circadian - regulated . the same arabidopsis cdna array has been used to analyze transcripts obtained under a wide range of physiological conditions , including auxin treatment , viral challenge and acclimation to light . schaffer et al . could therefore perform cluster analysis using available published and unpublished data , in order to identify 33 genes regulated principally by the circadian clock . some of these genes will probably prove to be informative markers for future circadian studies ; they might also represent a functionally significant group . as the publicly accessible expression databases grow , such cluster analysis should prove its utility in unraveling gene regulatory networks in plants . used an oligonucleotide - based array ( genechip , pioneered by affymetrix ; santa clara , ca ) that contains information on approximately 8,200 genes . the rna samples analyzed were isolated from plants grown in a light / dark cycle but harvested following transfer to continuous light , so this experiment specifically tested circadian regulation . the steady - state mrna levels were measured in replicate hybridizations of 12 samples harvested every 4 hours , covering two circadian cycles in constant light . a dedicated biomathematical software tool was developed to detect rhythmic patterns in these data . here , around 6% of genes interestingly , all phases of expression were well represented , suggesting that the clock regulates gene expression throughout the day / night cycle , rather than clock - controlled genes being expressed only at a limited number of preferred phases . several authors have noted that genes with functions in photosynthesis are often maximally expressed in the early morning , presumably optimizing photosynthetic function during the daylight hours . propose several striking new examples in which rhythmic regulation appears orchestrated such that genes that act coordinately are expressed simultaneously . some of these gene sets function in concert with the daily environmental cycle , in so - called external coordination . for example , 23 genes involved in the synthesis of photoprotective phenylpropanoids are maximally expressed before dawn , including chalcone synthase . other gene sets might be expressed in a fixed temporal relationship to key metabolic processes ( internal coordination ) . nine genes involved in the energetically demanding process of nitrogen assimilation were expressed early in the day , for example , when cellular energy levels are increased by light harvesting . the detection of such functional clustering is highly valuable , shedding new light on the potential importance of biological rhythms for vegetative plant growth . in such large data sets , however , this analysis currently requires either encyclopedic knowledge of plant biochemistry or an extensive literature review . for many metabolic processes , diurnal regulation might allow sufficient coordination with the environment , so circadian anticipation of dawn / dusk might add little advantage . is it significant that genes conferring metabolic function were the largest identifiable class of diurnal- and circadian - controlled genes ? harmer et al . suggest that this finding reflects the better understanding of enzymatic genes than those of other functions . for instance , about 25% of circadian - regulated genes encode proteins of unknown function and another 25% are uncharacterized genes containing limited sequence homology to known genes . many of these latter genes have sequence homology to kinases , phosphatases and transcription factors . some of these presumably underlie the observed circadian ' gating ' of signal transduction , which fine tunes biological responses over the day / night cycle . patterns of coordinate gene expression might reflect regulation by common transcription factor(s ) binding to similar promoter element(s ) . indeed , some transcripts encoding transcription factors were found to oscillate in phase with transcripts of their known target genes . harmer et al . identified a conserved sequence upstream of 31 genes that were expressed at the end of the subjective day . they confirmed in transgenic plants that this ' evening element ' was required for robust circadian transcription from one such promoter . these results are profound , because bioinformatic approaches alone accurately predicted a gene - regulatory sequence . correlations between coordinate gene expression and promoter sequences should be increasingly useful in uncovering regulatory mechanisms for organisms with sequenced genomes . the simultaneous application of the two microarray methods allows some tentative technical comparisons that might highlight the importance of using both array technologies as well as of the choice of genes to analyze . the overall number of circadian - controlled genes observed in constant light was quite different : about 6% of genes for the oligonucleotide array ; about 0.5% for the cdna array , which identified fewer cycling genes in the light than in darkness . setting aside differences in the numbers of rna samples tested from circadian conditions and in the analytical methods , the identity of the genes assayed might also have contributed . genes on the oligonucleotide - based chip were a relatively random sample created using genome sequence from the arabidopsis genome initiative . genes on the cdna array are not a random sample , as these genes were isolated as part of the arabidopsis expressed sequence tag collection , which contains virtually all highly expressed arabidopsis genes and lacks many rare transcripts . global expression profiling of the entire genome will be required to test the suggestion that a smaller percentage of abundant transcripts cycle , but this will be possible now that the complete arabidopsis genome sequence is publicly available . global transcript analysis can potentially return four results : identifying new markers for research , revealing cis - acting elements and trans - acting factors that cluster with a pattern of regulation , identifying novel patterns of regulation , and finding clusters of co - regulated genes with a functional connection that suggests the physiological importance of their regulation . the current challenge is to follow up some of these data with higher - resolution methods , before microarrays designed to sample the entire transcriptome of arabidopsis uncover the complete complement of clock - controlled genes . | oligonucleotide and cdna microarrays have been used to analyse the mrna levels of 8,000 genes in arabidopsis thaliana throughout the day / night cycle .
genes involved in signal transduction and in various metabolic pathways were found to be coordinately regulated by circadian rhythms and/or by light . |
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ozonized olive oil ( ozo ) is a powerful natural remedy for a variety of health concerns , especially those related to skin health . ozo is a registered drug and has been used effectively for healing wounds and pressure ulcers in mice . clinically , ozo has been used to treat bedsores , intractable fistula and wounds after surgical operation . the olive oil ( oo ) is obtained from the fruit of the olive tree . oleic acid ( 6585% ) was detected as the main fatty acid along with 10% linoleic acid , 9% palmitic acid , and 3% stearic acid . ozo is produced by the reaction of ozone with the olive oil almost exclusively with the carbon - carbon double bonds present in the unsaturated fatty acids . this reaction produces several oxygenated compounds such as hydroperoxides , ozonides , aldehydes , peroxides , diperoxides , and polyperoxides . triolein triozonide ( ozonized triolein ) has been identified as the major oxygenated triglyceride in ozo . ozo may be slowly decomposed due to the inherent structure of the triolein triozonide molecule . this decomposition can be delayed by avoiding moisture and storing the oil at low temperature . in a few clinical cases in japan , ozo has been used to treat bedsores and intractable fistula or wounds after surgical operation [ 3 , 8 ] ; however , the detailed action mechanism of ozo is not clear . also , ozo can control the key events in the healing phases and triggering and modulating the inflammatory stage . the aim of this work was to evaluate the potential protective effect of ozo in 2,4-dinitrobenzene sulphuric acid ( dnbs ) induced colitis in rats as well as elucidate the role of some important constituents of antioxidant defense system such as superoxide dismutase ( sod ) , glutathione peroxidase ( gsh - px ) , and catalase ( cat ) in these possible effects . male albino rats of 200 20 g body weight were used in this study . animals were housed in a well - controlled environment and had free access to food and water throughout the study period . one week before starting the experimental procedure , animals were weighed and gently manipulated in the laboratory environment for 20 min every day to minimize the effects of stress per se on the parameters to be measured . animal welfare and experimental procedures were carried out in accordance with the guide for the care and use of laboratory animals . the protocol for induction of colitis was reviewed and approved by the research committee of dubai pharmacy college . all reagents used for the determinations of sod , cat , gshpx , and tbars were purchased from sigma chemicals ( st . potassium iodide , sodium thiosulfate , starch , ethanol , potassium hydroxide , and phenolphthalein were purchased from merck ( germany ) and 2,4-dinitrobenzene sulphonic acid ( dnbs ) from icn biomedicals . ozonized oil was kindly donated from ozone clinic , rashid hospital , dubai , uae . ozonation of olive oil was conducted by the procedure described by sakazaki et al . . the ozo is formed from pure virgin olive oil and it was subjected to high concentrations of ozone gas for a limited period till it would be transformed to ozo . the ozo was stored at 810c and allowed to melt at room temperature before use . they are as follows : ( i ) peroxide index ( pi ) indicates the quantity of peroxide available in ozo ; ( ii ) acid value is the number of milligrams of potassium hydroxide required to neutralize the free acids in 1.0 g of the substance ; ( iii ) viscosity was measured by dv-11 + proviscometer at room temperature . the noninflamed groups were as follows : nontreated ( negative control ) , oo- and ozo - treated groups at doses of 6 mg / kg each , while the inflamed groups were the nontreated , oo - treated at dose of 6 mg / kg , and ozo - treated at doses of 3 and 6 mg / kg . starting the day before colitis induction , the oils treatments were given orally , by gavage , once daily at the same time . the animals ' body weights were recorded daily during the experimental period . at day 6 after colitis , the animals were sacrificed by ether overdose . the animals were deprived of food for 24 hours before the colitis induction but had free access to water and the rats were lightly anaesthetized by inhalation of ethyl ether . 2,4-dinitrobenzene sulphonic acid ( 15 mg per rat ) dissolved in 0.25 ml of 50% ethanol was instilled into the distal colon of each animal using a rubber catheter , so that the tip was about 8 cm proximal to the anus . rats were killed at the sixth day after colitis induction and the distal colon was removed , opened longitudinally , and washed with phosphate - buffered saline ( pbs ) . whole - wall samples from distal - colon , taken from an area immediately adjacent to the gross macroscopic damage , were cut out and fixed in 10% formalin solution . sections of 5 m thickness of colon were cut , serially mounted on glasses , and processed for routine haematoxylin - eosin ( h&e ) staining . specimens of colonic tissue were also removed from the area of gross injury , snap frozen in liquid nitrogen , and stored at 80c until subsequent assays . the macroscopic features were based on the following features : presence of adhesions between the colon and other intra - abdominal organs , consistency of colonic faecal material , thickening of the colonic wall , presence and extension of hyperaemia . microscopic criteria for damage and inflammation were assessed by light microscope on h&e stained histological sections . histological parameters included the degree of mucosal architecture changes , cellular infiltration , external muscle thickening , presence of crypt abscess , and goblet cell depletion . colonic tissues were weighted and homogenized in 10% w / v of a solution of kcl 100 mm with edta 0.3 mm for both gsh - px and sod , while , for the assessment of both cat and tbars , tissues were homogenized in 50 mm phosphate buffer ( ph 7 ) using a tissue homogenizer ultra - turrax t25 polytron at 4c then centrifuged at 6000 g at 4c . 100 l supernatant was then added to the enzyme immunoassay 96-well plate in duplicate and assayed according to the manufacturers ' protocols for gsh - px , sod , and cat assay kits . thiobarbituric acid reactive substances ( tbars ) levels are considered as lipid peroxides index ( lp ) as per ohkawa et al . . ten percent ( w / v ) tissue homogenate was mixed with sodium dodecyl sulfate , acetate buffer ( ph 3.5 ) , and aqueous solution of thiobarbituric acid . after heating at 95c for 60 min , the red pigment produced was extracted with n - butanol - pyridine mixture and estimated by the absorbance at 532 nm and the results were expressed as nmol of tba per gram of proteins . protein concentrations were determined by the method of lowry et al . using bovine serum albumin as standard . statistical analysis was performed using one - way or two - way anova , as appropriate , with bonferroni 's correction for multiple comparisons . a p value < 0.05 was considered significant . calculations were performed using graphpad prism ( version 5.0 ; graphpad software inc . , san diego , ca , usa ) . figure 1 is representing the gain in body weight for the animals during the experimental period . intrarectal administration of dnbs significantly reduced the body weight gain with respect to noninflamed controls ( p < 0.01 ) . treatment with ozo at the doses of 3 and 6 mg / kg significantly reduced the impairment in body weight gain induced by colitis dose dependently as shown in ( figure 1 ) , whereas oo has no significant effect on body weight gain compared to its respected controls . table 2 summarizes the effects of both oo and ozo on the colonic macro- and microscopic damage scores for the entire groups . the macro- and microscopic damage scores of the inflamed nontreated rats increased by six- and tenfold , respectively , over the negative control animals . whereas there were no significant effects on both macro- and microscopic damage scores for the noninflamed animals treated with oo and ozo verses the negative control group . pretreatment with oo did not show any change in both macro- and microscopic damage score in the inflamed animals ( p > 0.05 ) . on the other hand , ozo pretreatment at both doses caused significant reduction of both macro- and microscopic damage scores in a dose dependant manner in the inflamed groups as shown in table 2 . the histological study supports the findings of the damage scores ; it shows that oo and ozo administration to the noninflamed animals do not have any destructive effects on the intestinal mucosa ( figures 2(a ) and 2(b ) ) . furthermore , dnbs at dose of 15 mg / rat caused extensive mucosal destruction with well - developed granuloma extending throughout the mucosa and submucosa , often involving the muscularis propria , which invariably appeared thickened ( figure 2(c ) ) . pretreating the inflamed animals with the oo did not show any improvement in the histological appearance over the inflamed nontreated group ( figure 2(d ) ) . nevertheless , ozo treatment at both doses showed significant improvement in the histological appearance of the mucosa and submucosa lining to be nearly normal as shown in figures 2(e ) and 2(f ) . the results of histological study confirmed clearly the protective effect of ozo against colitis induced by dnbs . table 3 also shows that dnbs induced a remarkable and significant decrease of gsh - px , cat , and sod activities in rats ' colonic tissues , p < 0.05 . the lipid peroxides index represented by tbars content in the colonic tissue increased significantly ( p < 0.05 ) from basal concentration of 0.056 0.10 nmol / g of protein to 0.67 0.30 nmol / g of protein in the inflamed nontreated animals . on the other hand , pretreating the inflamed animals with oo did not raise the levels of the three antioxidant enzymes and similarly did not reduce the levels of tbars in their colonic tissues in respect to the inflamed control ( table 3 ) . on the contrary , administration of ozo to the inflamed rats at both doses increased significantly the sod and gsh - px levels dose dependently , while only ozo at the highest dose was significantly able to increase the level of cat ( p < 0.05 ) as shown in table 3 . administration of oo per se to noninflamed rats had no significant effects on all of the three enzymes and tbars . conversely , administration of ozonized oil caused slight increase in the tbars content in the tissue of noninflamed animals , but this increment was not statistically significant . the pathogeneses of inflammatory bowel diseases ( ibd ) , including crohn 's disease and ulcerative colitis , are associated with elevated levels of reactive oxygen species ( ros ) such as peroxide anion , hydrogen peroxide ( h2o2 ) , and hypochlorous acid [ 1416 ] , and this could be attributed to the massive infiltration of polymorphonuclear and mononuclear leukocytes . in fact , free radical production is a key mechanism for the development of colonic inflammation in experimental colitis models as well [ 1719 ] . hawkins et al . reported that the injury caused by dnbs closely resembles human ulcerative colitis . this model resembles histological features of the human ulcerative colitis in many features like transmural inflammation with granuloma and diffuse ulceration and inflammation and plasma - lymphoid and eosinophil infiltrates , as well as crypt distortion . to regulate overall ros levels , the intestinal mucosa possesses a complex of antioxidant defense system , of which the sod , cat , and gsh - px are the major players , as sod is an important radical superoxide scavenger while cat and gsh - px are involved in the elimination of hydrogen peroxide and lipid hydroperoxides [ 22 , 23 ] . the efficacy of the antioxidant system is impaired during inflammation status , partially as a result of autooxidation . in a previous study , loguercio et al . showed increased level of lipid peroxidation in colonic mucosa treated with tnbs . on the other hand , ozonized oil is reported to be effective for wounds ' healing and pressure ulcers in mice and ethanol - induced ulcers in rats . in addition to ozonides , during the procedure of oil ozonization , many oxygenated compounds are generated like peroxides and aldehydes . these oxygenated compounds may be responsible for many biological activities of ozonized vegetable oils , such as antimicrobial and antifungal activities . our findings showed that dnbs increases lipid peroxidations with respect to the nontreated group , but no significant changes were found with respect to ozo - inflamed treated animals . this could be explained based on the fact of presence of many oxygenated compounds other than ozonides that have been generated during the ozonization process of the oil such as hydroperoxides and aldehydes ; this might contribute to the increased tbars content in rat colonic mucosa . the results also revealed a decrease of sod , cat , and gsh - px activities in colonic mucosa of the inflamed control animals , while the levels of both sod and gsh - px were increased significantly in rats treated with ozo in a dose dependent manner . cat activity was significantly increased by the treatment with the highest dose of ozo only , and this finding appears to be due to the fact that cat has a lower affinity for that ros comparing to sod and gsh - px . this result comes in concordances with many previous studies that reported that gsh - px plays a much greater role in the removal of h2o2 than cat [ 25 , 29 , 30 ] . the results of this study suggest that the ozo has a protective effect in the rat colonic mucosal damage induced by dnbs and this might be mediated at least partially by its enhancement effect on antioxidant enzymes such as sod and gsh - px which comprise the endogenous scavengers of ros . | the aim of the study was to evaluate the potential protective effect of ozonized olive oil ( ozo ) in 2,4-dinitrobenzene sulphuric acid ( dnbs ) induced colitis in rats and to elucidate the role of some antioxidant defense system ( superoxide dismutase
sod ,
glutathione peroxidase gsh - px , and catalase cat ) in these effects .
the physicochemical parameters including viscosity , peroxide , and acid values of olive oil and ozo were evaluated .
the animals were divided into several groups and the colitis was induced in the rats by intracolonic instillation of dnbs at dose of 15 mg / rat .
olive oil ( oo ) at dose of 6 mg / kg and ozo at doses of 3 and 6 mg / kg was administered orally for 7 days , starting the day before induction of colitis .
our results showed that macroscopic and microscopic damage scores were significantly reduced in a dose response manner in rats pretreated with ozo only .
in contrast , cat , gsh - px , and sod activities were significantly increased in the distal colon of inflamed animals pretreated with ozo with respect to control group dose dependently .
results demonstrate that ozo pretreatment exerts protective effects in dnbs induced colitis in rats and provide evidence that the protective effects of ozo are mediated by stimulation of some antioxidant enzymes . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
stressful life events trigger neurotransmitter changes in the brain via activation of the corticotropin - releasing factor ( crf ) pathway that terminates not only within the hypothalamus and other parts of the central endocrine system , but also on the locus ceruleus and raphe nuclei . this provides a biological link between stressful stimuli and the changes in the endocrine , immune , and neuro - transmitter systems that are involved in the psychopathology of depression ( figure 1 . ) . investigations of the role of the hypothalamic - pituitaryadrenal ( hpa ) axis in the psychopathology of depression commenced over 40 years ago , when it was reported that depressed patients have a higher circulating plasma cortisol concentration than those that are not depressed . at this time , the dexamethasone depression test ( dst ) was developed to provide a functional assessment of hpa axis activity . it was discovered that this synthetic glucocorticoid would normally suppress the secretion of cortisol by activating hypothalamic and pituitary glucocorticoid receptors , thereby suppressing the secretion of crf and adrenocorticotropic hormone ( acth ) which , in turn , reduced the activation of the adrenal cortex and the release of cortisol . the mechanism whereby these changes occurred was explained in terms of a negative feedback loop whereby the raised plasma glucocorticoid concentration controls the further release of the steroid . however , it soon became apparent that in patients with major depression the negative feedback loop ceased to function due to the desensitization of the central glucocorticoid receptors . nevertheless , it is now apparent that the dst lacks both specificity and sensitivity for depression , even though it may still offer reliability in the assessment of the severity of depression . hypercortisolism and a negative dst are now known to occur in patients with alzheimer 's disease and alcoholism , for example . furthermore , it has been estimated that only 60% of patients with major depression demonstrate a negative dst . nevertheless , these findings do serve to emphasize the importance of the hpa axis in psychiatric disorders . it is frequently assumed that the synthetic glucocorticoids such as dexamethasone act on glucocorticoid receptors in an identical manner to the natural glucocorticoids such as cortisol dexamethasone acts primarily on the glucocorticoid receptors in the anterior pituitary , does not readily enter the brain , and therefore differs substantially from natural glucocorticoids that activate both mineralocorticoid and glucocorticoid receptors . there is also evidence that , while dexamethasone may reduce the release of crf , it does not suppress the release of arginine vasopressin ( avp ) . there is evidence that avp , not crf , is the main activator of the hpa axis due to chronic stress and major depression . the increased action of avp is further exacerbated by the action of il-1 ; chronically administered il-1 has been shown to cause a shift in the role of crf to avp in the activation of the anterior pituitary . in addition , it has been shown that there is an age - related increase in the colocalization of avp in crf neurons in patients with major depression and dementia . thus , it seems reasonable to conclude that the hypersecretion of cortisol in patients with depression or dementia may at least be partly a consequence of an increased activation of the hpa axis by avp . additional evidence for the change in the functional activity of the pituitary gland is provided by the finding that the adrenals and the pituitary are enlarged in those with depression , these changes being associated with a hypersecretion of crf . furthermore , the density of the crf receptors in the frontal cortex are reduced , presumably as a consequence of the hypersecretion of crf . the hypersecretion of crf would appear to be a state , rather than a trait , marker of depression . if hypercortisolemia is a common feature of major depression and some types of dementia , it would be anticipated that immunosuppression would be a common feature of these conditions . however , it is apparent that both immunosuppression ( for example , of natural killer cell [ nkc ] activity ) and immune activation ( for example , macrophage activation ) are common features of depression . one possible explanation is that an increased vulnerability to environmental stress , which is a common feature of both depression and dementia , elicits a bidirectional , homeostatic interaction between the endocrine and immune systems . thus , crf has been associated with humoral activation that results in an increased release of proinflammatory cytokines . by activating the hpa axis , proinflammatory cytokines not only further release crf , but also lead to glucocorticoid resistance , thereby impairing the regulatory feedback mechanism . conversely , the increase in the concentration of plasma cortisol , together with the increased sympathetic activity that is a normal feature of the stress response , suppresses nkc and t - cell replication . there is evidence that activation of the -adrenoceptors on the nkc membrane , and which results in the decrease in activity of the nkcs , occurs independently of the activation of the hpa axis . clearly the interaction between the immune system and the hpa axis is both complex and interdependent . in the past 20 years , attention has focused on changes in the hypothalamic - pituitary - adrenal axis , together with the biogenic amine neurotransmitters noradrenaline , serotonin , and , to a lesser extent , dopamine . more recently , however , it has become apparent that both major depression and chronic stress result in more persistent structural changes in the brain as a consequence of the decrease in the synthesis of neurotrophic factors , such as bdnf and the antiapoptotic factor bcl-2 . these changes are attributed to the chronic increase in brain glucocorticoids that arise due to the desensitization of central glucocorticoid type 2 receptors that occur as a consequence of the reduction in the inhibitory feedback mechanism . such effects contribute to the failure in brain repair mechanisms which is indicated by a reduction in dendritic branching and a decrease in neurogenesis , particularly in the hippocampus and , to some extent , in the frontal cortex . such changes , together with an activation of the proinflammatory cytokines by chronic stress and depression , also enhance apoptosis through their indirect excitotoxic and metabolic actions . thus stress - induced hypercortisolemia and proinflammatory cytokines share a final common pathway that leads to impaired neuronal plasticity and deficits in central neurotransmission . the possible link between hypercortisolemia and depression is further provided by the changes induced by antidepressants and glucocorticoid receptor antagonists such as mifepristone . thus , preliminary clinical evidence has shown that the sensitization of the central glucocorticoid receptors by such treatments , that results in the re - establishment of the feedback inhibition of cortisol release , are correlated with the attenuation of the symptoms of depression . there is overwhelming evidence that inflammatory changes are an important causative factor in the pathology of alzheimer 's disease and related dementias . the increase in amyloid ( ab ) is not only a major pathological feature of such dementias , but is also responsible for stimulating inflammatory responses in the brain . these changes include an increased expression of cell adhesion molecules and proinflammatory cytokines , and the activation of microglia in the brain parenchyma . in vitro studies have also demonstrated that ab induces il - lb and ifng from vascular cells , thereby inducing a cascade of inflammatory changes . in addition , the infiltration of macrophages together with cd4 + and cd8 + t - cells , from the periphery have been detected in ab deposits in cerebral vessels in patients with cerebral amyloid angiopathy . the combination of ab and proinflammatory cytokines is linked to the increase in apoptosis in the brains of patients with dementia . for example , there is evidence that lymphocytes show a significant increase in dna fragmentation in alzheimer patients when compared with aged , but normal , controls . this change has been linked to an increase in the intracellular concentration of calcium ions , a prerequisite for apoptosis that has not been recorded in lymphocytes from aged control subjects . furthermore , apoptotic cell death is preceded by the expression of apoptosis - associated molecules such as p53 , fas ( cd95/apo-1 ) and il-1b converting enzyme . whereas the normal brain is partly immunologically privileged , in patients with inflammatory diseases such as multiple sclerosis , stroke , alzheimer 's disease , and possibly major depression , fas is widely expressed in the brain . this apoptotic protein is expressed on cd4 + and cd8 + t - cells and on nkcs . such observations provide a further link between the inflammatory changes in the brain and increased apoptosis that preludes dementia . despite these convincing observations regarding the inflammatory changes in patients with alzheimer 's disease , it is somewhat surprising to find that il-6 , a major proinflammatory cytokine that is elevated in the plasma and cerebrospinal fluid ( csf ) of patients with major depression , has been reported to be unchanged or even decreased in the blood of alzheimer 's patients . some of these differences may be accounted for by the methods used to assay il-6 . thus the concentration of il-6 in the serum and csf is often at the limit of detection , while in invitro studies , in which stimulated lymphocytes are isolated by gradient centrifugation , the cells are stressed which may alter their phenotype . it has also been argued that the decrease in proinflammatory cytokines in alzheimer 's disease is a consequence of the hypercortisolemia although this would not explain why cytokines such as il-6 remain elevated in depressed patients where hypercortisolemia also commonly occurs . the cognitive changes and dysphoria that are common symptoms in the early stages of alzheimer 's disease have been correlated with the increase in proinflammatory cytokines such as ifn. despite the equivocal evidence regarding the rise in plasma il-6 concentration in alzheimer patients , there are reports that the il-6 concentration correlates with the severity of dementia . from the numerous studies of the changes in the immune system of patients with dementias , it would appear that the inflammatory changes can trigger an increased synthesis and accumulation of ab . the accumulation of ab then initiates a further cascade of inflammatory changes in the brain involving proinflammatory cytokines and neurotoxic free radicals such as nitric oxide ( no ) ; this involves the activation of the nfk pathway and the complement system . neuronal cox 2 expression is also increased in alzheimer 's disease , and the resulting increase in pge2 contributes to the subsequent deterioration in the clinical state of the patient . in addition , the rise in il- may also indirectly contribute to the cognitive deficit by inhibiting cholinergic function ; a deficit in acetylcholine is generally accepted as the primary neurotransmitter that is causally involved in the cognitive and memory deficits in the dementias . the question arises as to whether the increase in ab is a reflection of the rise in proinflammatory cytokines , an important consideration if major depression predisposes to dementia . in support of this connection , there is evidence that severe head trauma in young persons can result in a large number of amyloid plaques shortly after the traumatic event . the accumulation of ab was shown to occur secondarily to the stress induced activation of the microglia that precipitate the release of il-1 ; the ab formed then stimulated the further evidence in support of the hypothesis linking the outcome of chronic depression with dementia comes from studies on the progression of an hiv infection to aids . it is well known that severe life stress , and bereavement of a partner with aids , is associated with a rapid progression of hiv to aids and a consequent increase in mortality for example , it has been reported that changes in immune function , such as a reduction in nk cells , correlates with the incidence of depression and the progressive deterioration in the clinical status of the patients with hiv/ aids although not all investigators have found such an association . nevertheless , such studies do provide possible support for the hypothesis that impaired immune function associated with the symptoms of depression may act not only in the progression of an aids infection but also to the onset of aids dementia in those patients who do not die as a consequence of secondary infections or cancer . evidence implicating a role for the proinflammatory cytokines in the etiology of depression has been provided by studies on the changes in il-1 , il-6 , and tnf in depressed patients and also by the effects of ifn on psychiatrically normal individuals being treated for hepatitis or a malignancy . such studies have implicated these cytokines as causative factors in the symptoms of major depression . these symptoms include depressed mood , anxiety , cognitive impairment , lack of motivation , loss of libido , sleep disturbance , and deficits in short - term memory . these changes appear to be a consequence of the neurotransmitter and endocrine changes induced by the cytokines , rather than the pathological condition for which the treatment has been administered . it is perhaps not surprising therefore to find that the symptoms of depression frequently occur in patients recovering from a chronic infection , those with multiple sclerosis , allergies , and rheumatoid arthritis . in all these situations , proinflammatory cytokines are known to be overexpressed the initial studies linking depression with an abnormality of the immune system , impaired mitogen - stimulated lymphocyte proliferation , and reduced nk cell activity in untreated depressed patients , showed changes that largely returned to normal once the patient recovered from the depressive episode . recent research into the immune changes occurring in depression has concentrated on cytokines , soluble cytokine receptors , and plasma acute - phase proteins . for example , positive acute - phase proteins have been shown to increase while the negative acute - phase proteins decreased in depression , changes that are known to be a consequence of the action of il-6 on liver function . in addition , complement proteins ( c3,c4 ) and immunoglobulin m are increased in depressed patients . such changes are evidence of immune activation involving both the inflammatory cytokines and b - cells that are activated by the proinflammatory cytokines . further evidence of immune activation in depressed patients is provided by the studies showing that the plasma concentration of il-1 , il-6 , ifng , soluble il-6 and il-2 receptors , and the il-1 receptor antagonist , are raised . in addition to the increases in proinflammatory cytokines , there is also evidence of an increased number of t - helper , t - memory , activated t - cells and b - cells that act as a source of the plasma cytokines . from these changes , it would appear that in depression there is an imbalance between the inflammatory and the anti - inflammatory arms of the immune system , the cytokines from the t1 pathway ( such as ifng ) becoming predominant over those of the anti - inflammatory t2 ( for example , il-4 ) pathway . a recent study has shown that the t3 cytokine , transforming growth factor b1 ( tgf1 ) whose function is to re - establish the balance between the t1 and t2 pathways , is increased in depressed patients following effective antidepressant treatment . though tgf1 is reported as a regulatory cytokine that keeps the balance between th1 and th2 cytokines , precisely how the increases in the proinflammatory cytokines are attenuated by tgf1 in depressed patients is unclear . localized inflammatory responses in the brain parenchyma have been associated with the pathogenesis of a number of neurological disorders including alzheimer 's disease and parkinson 's disease . at these lesion sites , activated microglia release such inflammatory mediators as tnf and pge2 . in - vitro evidence shows that pge2 secretion from lymphocytes of depressed patients is increased , as is the pge2 content of the saliva , serum , and csf of such patients . of the proinflammatory cytokines , il-6 appears to play a key role in the synthesis of this prostaglandin both in vitro and in vivo . conversely , different types of antidepressants have been shown to inhibit the secretion of proinflammatory cytokines and to reduce the synthesis of pge2 . this raises the interesting possibility that the reduction in proinflammatory cytokines and inflammatory mediators such as pge2 in the brain may be associated with the therapeutic actions of antidepressants . as it appears that the proinflammatory cytokines increase the inducible form of cyclo - oxygenase ( cox2 ) in the brain , it would be expected that cox2 inhibitors would not only attenuate the central inflammatory changes but also exert an anti - depressant effect . thus , rofecoxib , when administered to a large group of patients suffering from osteoarthritis , was found to reduce the symptoms of those who were suffering from comorbid depression ; 15% of the patients had depression at the start of the study , which decreased to 3% at the end of the period of treatment . other clinical studies have suggested that the cox2 inhibitor celecoxib has positive effects on cognitive function in depressed patients . it should be noted that celecoxib has also been shown to have beneficial effects as an add - on component to clozapine in the treatment of schizophrenia in patients who are only partially responding to the antipsychotic medication . there are several mechanisms that are postulated to be involved in the etiology of depression . it is commonly assumed that a decrease in both the noradrenergic and serotonergic functions are causally related to the changes in the mood , motivation , and cognitive changes associated with the disorder , there is now experimental evidence to show that the inhibition of cox2 is associated with a rise in the synthesis of serotonin in the cortex of the rat brain . in addition , pge2 has been shown to reduce the release of noradrenaline from central noradrenergic neurons , an effect that would be blocked by the cox2 inhibitors . thus inhibition of cox2 activity in the brain contributes not only to the reduction in inflammatory changes but also to an enhancement of biogenic amine function . pge2 is probably one of the most potent inflammatory mediators in terms of the initiation and propagation of inflammation within the brain . both clinical and experimental studies have shown that there is an increase in the tissue concentrations of pge2 in depression and in an animal model of depression . in the brain , the microglia act as macrophages . on activation , they release proinflammatory cytokines , pge2 , and neurotoxic metabolites of the kynurenine pathway . recent experimental evidence has shown that lipopoly saccharide ( lps ) , an activator of macrophage activity and a cause of brain inflammation , induces mitochondrial pge2 synthase and cox2 activity in activated microglia , thereby increasing the synthesis of pge2 at sites of inflammation in the brain . this provides a possible mechanism to explain the inflammatory changes in patients with depression or dementia ; changes that contribute to neurodegeneration . nitric oxide ( no ) can also act as an inflammatory mediator that contributes to neurodegeneration , and is raised in the plasma of depressed patients . no is produced by both the constitutive and inducible forms of no synthase ( nos ) that are associated with neurons and microglia . recent evidence suggests that proinflammatory cytokines activate inducible nos , thereby increasing no ; apoptosis results from the nitrosylation of deoxyribonucleic acid ( dna ) . the increase in peripheral and central macrophage activity associated with the inflammatory changes initiate , via the activated microglia , increases in pge2 and no that further potentiate the inflammatory changes ( figure 2.).thus in both depression and dementia , pge2 , no , and neurotoxic metabolites from the kynurenine pathway appear to play an important role central inflammatory processes that contribute to neurodegeneration . the depletion of tryptophan from the diet results in a reduction in serotonin in the brain that correlates with the onset of a depressed mood state . tryptophan is metabolized by two main pathways , by tryptophan hydroxylase leading to the synthesis of serotonin in the brain and by indoleamine 2,3-dioxygenase ( ido ) and tryptophan 2,3-dioxygease ( tdo ) resulting in the formation of kynurenine . it has been hypothesized that in depression the metabolism of tryptophan by ido and tdo is increased , thereby reducing the availability of the amino acid to synthesize serotonin . tdo is located in the liver , while ido is found in the lungs , placenta , blood and brain . as hypercortisolemia frequently occurs in both depression and dementia , it would be anticipated that tdo is overactive in patients with these disorders . by contrast , ido activity is increased by proinflammatory cytokines such as il-6 and ifng , and inhibited by anti - inflammatory cytokines such as il-4 . thus the activities of both tdo and ido are likely to be increased in depression and dementia as a consequence of the rise in circulating cortisol and the proinflammatory cytokines . there are two main stages in the metabolism of tryptophan following the actions of the dioxygenases . following the conversion of tryptophan to kynurenine by ido or tdo , kynurenine is metabolized by kynurenine hydroxylase to the neurotoxic metabolites 3-hydroxykynurenine , 3-hydroxy - anthranilic acid , and quinolinic acid . , kynurenine may be metabolized by kynurenine aminotransferase to the neuroprotective end product kynurenic acid . the mechanisms whereby quinolinic and kynurenic acids act as neurotoxic and neuroprotective agents respectively is related to their activation or inhibition of the n - methyl - d - aspartate ( nmda ) receptor , quinolinic acid and 3-hydroxyanthranilic acids being agonists of the nmda receptor while kynurenic acid is an antagonist . it has also been hypothesized that the imbalance between those nmda receptor antagonist and agonist are involved in the pathophysiology of chronic or treatment - resistant depression . in the brain , the metabolism of tryptophan by the enzymes of the kynurenine pathway occurs in both astrocytes and microglia the former producing mainly kynurenic acid while the latter produces the neurotoxic end products 3-hydroxy - kynurenine , 3-hydroxy anthranilic acid , and quinolinic acid . astrocytes have been shown to metabolize quinolinic acid and thereby reduce the neurotoxic impact that may arise following microglia activation . from the foregoing evidence , it can be hypothesized that inflammatory changes in both depression and dementia involve the activation of microglia and an increase in the inflammatory challenge to the brain . such changes also occur in patients with hepatitis who have been treated with the proinflammatory cytokine ifn and who developed depressive symptoms as a side effect of the treatment . in these patients , it has been shown that the plasma kynurenic acid concentration was reduced , thereby suggesting that the neurodegenerative metabolites were increased . more recently we have shown that similar changes occur in the blood of patients with major depression . the results of this study also showed that therapeutically effective antidepressant treatment increased the neuroprotective kynurenic acid in the blood in those patients suffering from an acute episode of depression , but not in those with chronic depression . this suggests that the progress to dementia may increase as the depression becomes more chronic . in patients with major depression , shrinkage of the hippocampus , a decrease in the number of astrocytes and a neuronal loss from the prefrontal cortex , and the striatum such findings support the view that neurodegenerative changes occur in several discrete regions of the brain in patients suffering from chronic depression . furthermore , as the astrocytes are a major source of kynurenic acid , apoptosis of these cells would result in a reduction in the neuroprotective effect of kynurenic acid . there is evidence that in the astrocytes the kynurenine pathway is limited due to the absence of kynurenine hydroxylase . as a consequence , astrocytes only produce a very low concentration of the neurotoxin quinolinic acid and a relatively high concentration of the neuroprotective agent kynurenic acid . furthermore , in astrocytes ido is preferentially induced by ifng , a cytokine that also induces the catabolism of quinolinic acid . however , it is also apparent that the increase in the synthesis of kynurenine by the astrocytes can indirectly contribute to the formation of quinolinic acid by the microglia . this situation would be compounded by the increased activation of the microglia by the proinflammatory cytokines with the consequent rise in the concentration of the inflammatory mediators pge2 and no . figure 3 . summarizes the pathways involved in the metabolism of tryptophan by the kynurenine pathway and the relationship with inflammatory cytokines in depression . the inhibition of neuronal repair mechanisms resulting from the reduction in neurotrophic factors that follow the rise in blood and tissue cortisol , apoptosis of astrocytes which are the sources of several neurotrophic factors , and the possible disruption of the phospholipase d pathway that has antiapoptotic properties and is involved in neurite formation and repair , further contribute to the neuronal loss . another association between depression and dementia is through this ido initiated kynurenine pathway related neurotoxicity . an immunohistochemical study has proven that the immunoreactivity of ido and quinolinic acid are high in the hippocampus of alzheimer 's disease patients .
so far , emphasis has been placed on the role of inflammatory mediators and neurotoxins produced by the kynurenine pathway on the possible causes of the neurodegenerative changes in the brain that eventually develops into dementia . recently , experimental evidence has shown that transgenic mice that overexpress human tau protein ( a prominent feature of different types of dementia ) show depressive - like behavior in the forced swim test . this test is widely used to predict antidepressant activity , and is based on the observation that when rodents are placed in a container of warm water from which they can not escape , they soon adopt an immobile posture . this is assumed to reflect a state of learned helplessness that reflects a depressive - like state . this behavioral state was reversed by the administration of the selective serotonin reuptake inhibitor antidepressant fluvoxamine . in - vivo microdialysis studies showed that the release of serotonin from the prefrontal cortex was reduced in the transgenic mice , an effect that was reversed by the fluvoxamine treatment . the results of this study suggest that transgenic mice overex - pressing human tau protein show symptoms of depressivelike behavior that are associated with a reduction in serotonergic function . as the behavioral and neurotransmitter changes are reversed by a selective serotonin reuptake inhibitor ( ssri ) antidepressant , it would appear that serotonin may provide a link between the pathological effects of tau protein and the subsequent depressive - like state . it would be incautious to extrapolate from this subchronic study in a transgenic mouse to the complex clinical situation in which multiple pathological changes contribute to the onset of dementia . nevertheless , the experimental studies do provide evidence in support of the hypothesis that the long - term outcome of chronic depression is often dementia . further evidence for this hypothesis comes from the study by steffens et al who demonstrated a link between late - onset depression and the rise in plasma apolipoprotein e4 which is widely considered to be a risk factor for late - onset alzheimer 's disease . the progress of major depression to dementia could result from the chronic inflammatory changes that are linked to the activation of the microglia . the activation of inducible cox2 and nos by the proinflammatory cytokines further increases the inflammatory challenge to the brain . as there is evidence that the kynurenine pathway is also activated by proinflammatory cytokines , it seems likely that the concentrations of the neurotoxins 3-hydroxy kynurenine , 3-hydroxyanthranillic acids , and quinolinic acid will also increase as a result of the activation of the microglia . the increased apoptosis of the astrocytes , with a reduction in the availability of the neuroprotective agent kynurenic acid , further adds to the impact of the neurodegenerative changes . hypercortisolemia , a common feature of both dementia and major depression , and apoptosis of astrocytes decreases the synthesis of neurotrophic factors thereby reducing neuronal repair . this process may be further enhanced by the disruption of the phospholipase d pathway that normally plays an important role in neurite formation and neuronal repair . this hypothesis may assist in explaining the degenerative changes in the hippocampus and other brain regions that are the features of chronic major depression . it may also explain why chronic depression is frequently a prelude to dementia in the elderly patient . | epidemiological studies show that there is a correlation between chronic depression and the likelihood of demential in later life .
there is evidence that inflammatory changes in the brain are pathological features of both depression and dementia .
this suggests that an increase in inflammation - induced apoptosis , together with a reductin in the synthesis of neurotrophic factors caused by a rise in brain glucocorticoids , may play a role in the pathology of these disorders .
a reduction in the neuroprotective components of the kynurenine pathway , such as kynurenic acid , and an increase in the neurodegenerative components , 3-hydroxykynurenine and quinolinic acid , contribute to the pathological changes .
such changes are postulated t cause neuronal damage , and thereby predispose chronically depressed patients to demential . |
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although more prevalent causes of loss of vision in elderly patients such as age - related macular degeneration , glaucoma , cataract , and diabetic retinopathy have been widely discussed in the medical literature , clinicians should keep in mind other less common etiologies when evaluating a patient with this complaint , especially those in which prompt treatment can potentially improve the overall clinical outcomes . one such scenario is discussed through our case presentation of an elderly lady who presented with acute onset of loss of vision to her ophthalmologist and was diagnosed with a conjunctival lymphoma . an 80-year - old woman with a history of coronary artery disease , atrial fibrillation , hypertension , diabetes mellitus , basal cell carcinoma removed via mohs procedure on the nose , diabetic retinopathy , and macular degeneration presented with a history of a recent increase in the number of falls she had been sustaining due to a recent , sudden deterioration of vision bilaterally . she was initially seen by the ophthalmologist 2 months ago for loss of vision in her right eye that was thought to be related to her previously diagnosed diabetic retinopathy . she underwent focal laser photocoagulation for diabetic retinopathy in her right eye , with no significant improvement in vision . upon further ophthalmic evaluation , a fleshy , salmon colored conjunctival tumor was identified in her left eye [ figure 1 ] . she was referred to the ocular oncology service and subsequently the conjunctival tumor was biopsied . histopathologic examination of the specimen revealed sheets of large neoplastic lymphoid cells with moderate nuclear pleomorphism , vesicular chromatin , and large nucleoli underlying the normal conjunctival epithelium [ figure 2 ] . immunohistochemical studies showed the large lymphoid cells stained positive ( diffuse , strong ) for cd20 and mum-1 , less intensely positive for pax-5 , weakly positive for bcl-6 , and negative for cd10 . histopathological findings were consistent with diffuse large b - cell lymphoma ( dlbcl ) with an immunophenotypic profile consistent with nongerminal center origin . proliferation index estimated by ki-67 labeling was found to be 80% and were positive for c - myc rearrangement . initial clinical presentation clinical composite photograph of the patient 's left eye reveals an extensive fleshy salmon solid tumor of the conjunctiva extending into the orbit inferonasally histopathology from biopsied conjunctival lesion photomicrograph reveals sheets of large neoplastic lymphoid cells with moderate nuclear pleomorphism , vesicular chromatin , and large nucleoli underlying normal epithelium . ( h&e , 30x ) immunohistochemical stains of conjunctival lesion photomicrographs of multiple immunohistochemical assays showed that the large lymphoid cells stained strongly positive for cd20 and mum-1 ; it was also positive for pax-5 and weakly positive for bcl-6 . the small background lymphocytes stained positive with cd3 the patient 's laboratory workup including complete blood count with a differential , comprehensive metabolic panel , and coagulation profile , was within normal limits . she underwent orbital computed tomography ( ct ) and magnetic resonance imaging ( mri ) scans that failed to reveal any discrete masses although the mri revealed a mild asymmetric thickening and enhancement of the sclera in the anterior left globe . additionally , both positron emission tomography - ct ( pet - ct ) and an mri of her brain failed to reveal any other systemic central nervous system ( cns ) involvement . cerebrospinal fluid ( csf ) analysis after an uncomplicated lumbar puncture ( lp ) did not reveal any malignant cells . since the patient had a biopsied conjunctival tumor on the left side but had unexplained loss of vision on the right side as well , she was presumed to have cns involvement and was treated with high dose systemic methotrexate in addition to rituximab . she received three cycles of methotrexate at 1.5 g / m ( 50% dose reduction due to age and low creatinine clearance ) and rituximab at a dose of 375 mg / m . the first cycle was complicated by severe diarrhea related to clostridium difficile infection and dehydration and the third cycle was complicated by acute renal insufficiency , fluid overload , insufficient methotrexate clearance , and prolonged hospital stay . interval mri revealed interval improvement in nonspecific scleral thickening and enhancement along the anterior aspect of the left globe . based on the documented improvement , the patient was to be continued on the same chemotherapy regimen ; however , she declined further treatment . the patient was evaluated by radiation oncology but she opted to decline further treatment at this time . she has not followed up by physicians at this hospital after that and has been lost to follow - up . non - hodgkin 's lymphoma ( nhl ) is a group of malignant neoplasms that involve excessive proliferation of b- or t - lymphocytes . nhl cases account for 4.3% of all new cancer cases in the us according to the surveillance , epidemiology , and end results ( seer ) database . ocular lymphoma ( ol ) accounts for 5 - 10% of cases of extranodal lymphomas . the seer database also suggests that there has been a rapid increase in the number of cases of ols since 1975 , with an annual increase of around 6% . this could be attributed to the availability of better imaging modalities as well as better diagnostic techniques including flow cytometry , immunohistochemistry , and molecular analysis among others . due to a significant overlap between the modes of presentation of ol with other benign diseases of the eye , it is important to recognize the clinical - pathological aspects of these tumors . early recognition is also important because many patients with orbital lymphomas will progress to develop systemic lymphomas and hence , need close surveillance . according to the 2008 world health organization ( who ) classification , there are about 70 different subtypes of lymphoma . these have been classified based on their clinical features , morphological characteristics , genetics , and immunophenotypic features . a complete discussion of all lymphoma subtypes is outside the scope of this paper but it is essential to summarize the most common subtypes of lymphoma that affect the ophthalmic structures . nhl subtypes involving the adnexal structures : ocular adnexal lymphoma ( oal ) , a rare form of nhl , comprises about 5 - 10% of all cases of ols . oal most commonly affects the orbit ( up to 40% ) followed by the conjunctiva ( 35 - 40% ) and the eyelids ( up to 10% ) . of the various who subtypes of nhl , the three most common ones to affect the adnexal structures include extranodal marginal zone lymphoma ( enmzl ) , follicular lymphoma , and diffuse large b - cell lymphoma ( dlcbl ) . enmzl of mucosa - associated lymphatic tissue ( malt ) is the most common among these , encompassing close to 50% of cases in a recent danish study . through their studies on cadaveric eyes , knop et al . have demonstrated that the conjunctiva , lacrimal gland , and the lacrimal drainage system are also a part of malt ; hence , the most common lymphoma type in these structures is the enmzl . this lymphoma has been associated with chlamydia psittaci infection in some studies with good response to antibiotic therapy . follicular lymphoma and dlbcl are the second and third most common types of nhl to affect the adnexal structures . many other nhl subtypes can manifest in adnexal structures including mantle cell lymphoma , small lymphocytic lymphoma , lymphoplasmacytic lymphoma , and anaplastic large t - cell lymphoma . interestingly , the lacrimal gland has been shown to have an equal incidence of enmzl and dlbcl . nhl subtype involving the retina / vitreous structure is generally of the dlbcl type and is classified and managed as primary cns lymphoma . oal has been described as a masquerader of other benign diseases affecting the eyes ; hence , it needs to be carefully considered when evolving the differential diagnosis of common ophthalmological clinical syndromes . the most common age for the onset of oal is between the 5 and 7 decades . systemic symptoms such as fever , fatigue , weight loss , and night sweats are rarely associated with oal . they most commonly present with symptoms from the mass including reduced visual acuity , double vision , ptosis , proptosis , dry eye , and eyelid swelling . pain is a rare clinical feature reported in only 36% of the patients . on examination , oal patients were seen to have a conjunctival mass / salmon patch similar to our patient . rarely do these patients have an erosion of the orbital wall , most commonly associated with the dlbcl type . the national comprehensive cancer network ( nccn ) guidelines suggest that standard diagnostic tests for nhl should include a detailed history for assessment of presenting clinical features , physical examination looking for systemic involvement , performance status assessment , laboratory workup including complete blood count with a differential , comprehensive metabolic panel , serum ldh level , and tests for viral infections such as human immunodeficiency virus ( hiv ) , hepatitis - b if rituximab - based regimens are planned and hepatitis c in some cases . a unilateral bone marrow biopsy with aspirate prior to starting therapy may be performed but the biopsy may not be needed in cases where the pet scan is negative unless finding another lymphoma subtype will change the management of that patient . imaging studies of the orbit such as a ct scan / mri scan are needed and finally confirmation of the diagnosis is done with biopsy , molecular analysis , and flow cytometry . patients thought to have limited stage disease of the ocular structures should be further evaluated by slit lamp biomicroscopy , gonioscopy , and dilated fundus examination . considering the fact that cns features could be the presenting symptoms of ol ( e.g. , in our patient ) , the standard evaluation of any presumed cns lymphoma includes an ophthalmic evaluation preferably by an experienced ophthalmologist . studies including csf analysis and mri scans commonly miss cns involvement . sensitivity of csf studies can be improved by serial lps to analyze several specimens . mri for suspected cns disease needs to be timed prior to the lp to prevent picking up of false positive signals in the leptomeningeal region after an lp . the treatment of oal depends on several factors associated with an oal that include several features of the tumor such as subtype of the lymphoma , cns involvement , adverse biological factors such as c - myc rearrangements with or without bcl-6 mutations and prognostic factors pertaining to the patient such as performance status and organ function . while guidelines for the treatment of lymphoma are subtype - specific , initial treatment of lymphoma in the eye ranges from surgical excision , radiation therapy for local disease control to chemotherapy for systemic disease or a combination of these modalities . enmzl type of oal needs consideration for chlamydia psittaci treatment because studies have shown regression of this lymphoma type with antibiotic use . therefore , the treatment of oal involves management of these patients in a multidisciplinary center with the availability of expert hematologists and experienced ophthalmologists to diagnose and assess response to treatment especially in atypical presentations such as in our patient . according to the 2008 world health organization ( who ) classification , there are about 70 different subtypes of lymphoma . these have been classified based on their clinical features , morphological characteristics , genetics , and immunophenotypic features . a complete discussion of all lymphoma subtypes is outside the scope of this paper but it is essential to summarize the most common subtypes of lymphoma that affect the ophthalmic structures . nhl subtypes involving the adnexal structures : ocular adnexal lymphoma ( oal ) , a rare form of nhl , comprises about 5 - 10% of all cases of ols . oal most commonly affects the orbit ( up to 40% ) followed by the conjunctiva ( 35 - 40% ) and the eyelids ( up to 10% ) . of the various who subtypes of nhl , the three most common ones to affect the adnexal structures include extranodal marginal zone lymphoma ( enmzl ) , follicular lymphoma , and diffuse large b - cell lymphoma ( dlcbl ) . enmzl of mucosa - associated lymphatic tissue ( malt ) is the most common among these , encompassing close to 50% of cases in a recent danish study . through their studies on cadaveric eyes , knop et al . have demonstrated that the conjunctiva , lacrimal gland , and the lacrimal drainage system are also a part of malt ; hence , the most common lymphoma type in these structures is the enmzl . this lymphoma has been associated with chlamydia psittaci infection in some studies with good response to antibiotic therapy . follicular lymphoma and dlbcl are the second and third most common types of nhl to affect the adnexal structures . many other nhl subtypes can manifest in adnexal structures including mantle cell lymphoma , small lymphocytic lymphoma , lymphoplasmacytic lymphoma , and anaplastic large t - cell lymphoma . interestingly , the lacrimal gland has been shown to have an equal incidence of enmzl and dlbcl . nhl subtype involving the retina / vitreous structure is generally of the dlbcl type and is classified and managed as primary cns lymphoma . oal has been described as a masquerader of other benign diseases affecting the eyes ; hence , it needs to be carefully considered when evolving the differential diagnosis of common ophthalmological clinical syndromes . the most common age for the onset of oal is between the 5 and 7 decades . systemic symptoms such as fever , fatigue , weight loss , and night sweats are rarely associated with oal . they most commonly present with symptoms from the mass including reduced visual acuity , double vision , ptosis , proptosis , dry eye , and eyelid swelling . pain is a rare clinical feature reported in only 36% of the patients . on examination , oal patients were seen to have a conjunctival mass / salmon patch similar to our patient . rarely do these patients have an erosion of the orbital wall , most commonly associated with the dlbcl type . the national comprehensive cancer network ( nccn ) guidelines suggest that standard diagnostic tests for nhl should include a detailed history for assessment of presenting clinical features , physical examination looking for systemic involvement , performance status assessment , laboratory workup including complete blood count with a differential , comprehensive metabolic panel , serum ldh level , and tests for viral infections such as human immunodeficiency virus ( hiv ) , hepatitis - b if rituximab - based regimens are planned and hepatitis c in some cases . a unilateral bone marrow biopsy with aspirate prior to starting therapy may be performed but the biopsy may not be needed in cases where the pet scan is negative unless finding another lymphoma subtype will change the management of that patient . imaging studies of the orbit such as a ct scan / mri scan are needed and finally confirmation of the diagnosis is done with biopsy , molecular analysis , and flow cytometry . patients thought to have limited stage disease of the ocular structures should be further evaluated by slit lamp biomicroscopy , gonioscopy , and dilated fundus examination . considering the fact that cns features could be the presenting symptoms of ol ( e.g. , in our patient ) , the standard evaluation of any presumed cns lymphoma includes an ophthalmic evaluation preferably by an experienced ophthalmologist . mri for suspected cns disease needs to be timed prior to the lp to prevent picking up of false positive signals in the leptomeningeal region after an lp . the treatment of oal depends on several factors associated with an oal that include several features of the tumor such as subtype of the lymphoma , cns involvement , adverse biological factors such as c - myc rearrangements with or without bcl-6 mutations and prognostic factors pertaining to the patient such as performance status and organ function . while guidelines for the treatment of lymphoma are subtype - specific , initial treatment of lymphoma in the eye ranges from surgical excision , radiation therapy for local disease control to chemotherapy for systemic disease or a combination of these modalities . enmzl type of oal needs consideration for chlamydia psittaci treatment because studies have shown regression of this lymphoma type with antibiotic use . therefore , the treatment of oal involves management of these patients in a multidisciplinary center with the availability of expert hematologists and experienced ophthalmologists to diagnose and assess response to treatment especially in atypical presentations such as in our patient . because of the significant rate of disseminated disease among patients with lymphomas in the orbit that carries a worse prognosis , early diagnosis is essential to promote better overall survival of these patients . it is important to recognize that our patient had an underlying visual deficit caused by her diabetic retinopathy and macular degeneration . although she was found to have a salmon - patch conjunctival tumor in the left eye , there was no suitable explanation for why she had experienced a sudden loss of vision in her right eye ( since there was no intraocular or epibulbar involvement detected clinically or on imaging studies ) other than hypothetical cns involvement by her lymphoma . thus , this differential diagnosis should be considered when evaluating patients presenting with vision complains in order to provide a timely referral to the ophthalmologist and hematologist for further assessment , possible biopsy , and early therapeutic intervention . moreover , when managing such patients , it is important to have the resources of a specialized multidisciplinary approach found in centers with expertise in managing both ocular and hematologic malignancies . | context : elderly patients with visual loss often have age - related macular degeneration , diabetic retinopathy , glaucoma , and cataract as common causes of visual loss .
other less common etiologies should be considered , especially in those presenting with systemic associations.case report : the patient discussed in our review is an 80-year - old female , with a history of diabetic retinopathy and macular degeneration who presented with a sudden deterioration of vision .
while this was initially attributed to diabetic retinopathy , she was eventually noted to have a salmon patch lesion in her conjunctiva , diagnosed on biopsy to be a diffuse large b - cell lymphoma.conclusion:because of the significant rate of disseminated disease among patients with lymphomas in the orbit that carries a worse prognosis , early diagnosis is essential to promote better overall survival of these patients .
we describe here a patient diagnosed with conjunctival lymphoma associated with pronounced visual loss and review the literature on this subject . |
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retinal ganglion cells ( rgcs ) can be stimulated by electrical pulses so that action potentials are generated even when the responses of photoreceptors to visual stimuli are lost due to photoreceptor degeneration ( 1 ) . a retinal implant using prosthetic electrical stimulation has been developed to provide alternative partial vision to blind people who lost their vision due to a retinal degenerative disease , such as retinitis pigmentosa ( rp ) or age - related macular degeneration ( amd ) ( 2 ) . according to the results from clinical tests of retinal implants , patients with implants can perceive phosphene with simple spatial patterns , such as large objects and letters ( 3 ) . spatial resolution may be improved to some degree by increasing the number of stimulation electrodes , which enables visual guidance of fine hand movements ( 4 ) . still , the performance of retinal implants should be improved , as a recent clinical test in multiple human subjects reported inconsistent and variable results of phosphene perception when identical stimulation parameters were applied to different subjects ( 5 ) . the observation of neural activities can provide valuable insights into the success of prosthetic electrical stimulation . we have recently shown that temporal patterns of visual input could be successfully decoded from population activities of rgcs evoked by a temporally patterned stimulation pulse train , in both normal ( 6 ) and photoreceptor - degenerated retinas ( 7 ) . cottaris and elfar ( 8) demonstrated that the spatial origin , duration , and amplitude of each stimulation pulse could be identified with multisite cortical local field potential . prior to these studies , rgc neural activities had been investigated to provide preliminary information on the requirements for electrical stimulation , such as the threshold current level , the latency of evoked neural activities , and the effect of stimulation waveforms ( 9 ) . it is essential to examine both temporal and spatial characteristics of rgc responses in order to develop the details of the stimulation methods ; i.e. , the stimulation strategy, for a faithful delivery of spatiotemporal visual information to the brain . this is obvious in that the spatial and temporal aspects of external visual inputs should be properly encoded in the neural activities evoked by stimulation so that they can be transferred to and interpreted in the brain . previous studies were mainly focused on either the temporal ( 110 ) or spatial ( 11 ) aspects of rgc activity patterns , and the encoding of spatiotemporal temporal visual information by evoked rgc activities has not been analyzed in detail . in this study , our specific aim was to investigate whether spatiotemporal visual information can be decoded from the rgc network activity evoked by patterned electrical stimulation . based on an experimental setup of an in vitro model of retinal implants ( 11 ) , we tested an amplitude - modulation scheme of biphasic current pulse trains . here , we assumed that spatiotemporal visual information would be translated to the neural activities by multiple amplitude - modulated electrical pulse trains , just as in the case of cochlear implants , in which the pulse amplitude is modulated by the amplitude of sound pressure . along with a thorough characterization of spatial spreading of stimulation current and temporal information encoding , we demonstrated that the multipixel spatiotemporal visual information can be accurately decoded from the population activities of rgcs that are stimulated by amplitude - modulated pulse trains . the rd1 ( c3h / hej strain ) mice ( age : postnatal day 56 ) were used in this study . the methods for retinal tissue preparation and for the recording of rgc activities were previously reported ( 1 ) and are briefly described here . retinal patches from rd1 mice were prepared following the method of stett et al . ( retinas were isolated , cut into patches of 3 3 mm , and mounted on a planar microelectrode array ( mea ; multichannel systems gmbh , reutlingen , germany ) so that the ganglion cell layer faced the mea . the mea contained 64 tin electrodes ( circular shape ; diameter : 30 m , interelectrode spacing : 200 m , impedance : < 50 kat 1 khz ) on a glass substrate in an 8 8 square - type grid layout . the waveforms from the electrodes were recorded with a sampling rate of 25 khz / channel ( amplification gain : 1,200 , bandwidth : 103,000 hz ) and stored on a hard drive using data acquisition software ( mc_rack ; multichannel systems gmbh ) . the data recorded by mc_rack were converted to text file by mc_datatool ( multichannel systems gmbh ) . the recorded waveforms were futher analyzed to generate single unit spike trains by spike detection and spike sorting based on principal component analysis ( 12 ) . amplitude - modulated current pulse trains were generated with a stimulus generator ( stg 1004 ; multichannel systems gmbh ) and applied to the retina via 2 or 4 channels of the mea at its vertices . the stimuli consisted of symmetric , charge - balanced biphasic pulses ( anodic first , with no temporal separation between the anodic and cathodic phases ) . for the characterization of the modulation behavior of rgc responses by pulse amplitude , trains of 20 identical pulses were applied at 1-second interpulse intervals from one of the stimulating electrodes while the pulse amplitude was increased from 260 a ( 2 , 5 , 10 , 20 , 30 , 40 , 50 , and 60 a ) . the pulse rate and pulse duration were fixed at 1 hz and 500 s , respectively . then , the amplitude - modulated pulse trains were applied to investigate whether the rgc responses can be made to encode spatiotemporal visual information . the procedure for the pulse amplitude modulations based on natural scenes is described below ( fig . black - and - white movies of natural scenes were recorded by a camcorder ( xacti hd1010 ; sanyo , osaka , japan ) for 2 minutes at 60 frames / s with a 1,280 720 resolution . two or 4 pixels of the 16 9 resolution scene were randomly selected , and the amplitude of each pulse was modulated according to the time - series of the pixel intensity within the range of 120 a ( 1 a resolution ) . the pulse trains were simultaneously applied to the retina via 2 or 4 channels of the mea to present the electrical stimulation with spatial pattern , as shown in fig . 1 . the pulse repetition rate varied from 210 hz ( 2 , 4 , 6 , 8 , and 10 hz ) . the pulse duration per phase varied from 200400 s ( 200 , 300 , and 400 s ) . pulse trains of which amplitudes were modulated according to the brightness of 2 ( or 4 ) pixels were delivered to rgcs via the stimulation electrode on the mea . the visual information was reconstructed ( i.e. , decoded ) from the evoked rgc activities . then , the goodness - of - fit between the original and decoded pulse amplitude time - series was calculated to evaluate the effectiveness of the stimulation . spike train decoding was used to estimate how accurately the spatiotemporal visual information was transferred to rgc neural activities by multichannel electrical stimulation . because each pulse train was applied to each stimulating electrode independently , it was possible to select rgcs whose evoked activities were effectively modulated by a specific pulse train ( as in fig . thus , 2 and 4 groups of rgcs were used to decode visual spatiotemporal information with 2 and 4 pixels , respectively . the raw waveforms from each electrode were transformed into single unit spike trains , and then , the rgc spike trains were transformed to a firing rate time - series by counting the numbers of spikes in 50 ms bins . rhythmic bursting patterns of ( a ) spontaneous activity and ( b ) electrically evoked response ( pulse amplitude : 20 a , pulse rate : 1 hz ) . as the arrows indicate , both spontaneous and electrically evoked activities show rhythms of bursts at similar frequencies . the response strength of the rgcs was measured by counting the number of poststimulus spikes within approximately 100 ms of the stimulus onset , which corresponds to the first peak in the psth shown in fig . 2c . rgc = retinal ganglion cell , psth = post - stimulus time histogram , isih = interspike interval histogram . the input to the decoder consisted of firing rates of multiple rgcs within a number of time bins . that is , the firing rates of multiple rgcs within a short temporal interval after the stimulus were used to decode the pulse amplitude time - series at every time bin , corresponding to the intensity time - series of multiple pixels . the input - output relationship can be described as follows : ( 1)s^(i)=p=1nj=0m-1{(rpi - jfpj ) } here , (i ) is the estimated value of the pulse amplitude of one electrode at the i time bin , and fp(j ) is the j coefficient of the linear filter corresponding to the p rgc . rp(i ) is the firing rate of the p unit at the i time bin of 50 ms duration . the decoder can be regarded as a temporal filter because it calculates the output from the past and present values of the input . when linear mapping is used , the decoding algorithm becomes an optimal linear finite impulse response ( fir ) filter ( 13 ) . in this case , ( . ) is merely a unity function , and the coefficients fp(j ) were obtained by the least squares method , as illustrated by warland et al . more generally , nonlinear regression algorithms , such as multilayer perceptron or support vector machine ( svm ) , can be used for the input - output mapping of the decoding algorithm . the specific algorithm of the svm that we used is described in hoegaerts et al . the recordings were divided into 2 groups of equal size ( i.e. 12 minutes recordings ) . the first one minute of the recording was used for the training of the decoding algorithms and the remaining one minute was used to test the trained decoding algorithms . to quantify the accuracy of the information decoded from rgc activity , similarities between the original and decoded pulse amplitude time - series were computed by correlation coefficient ( cc ) . all experimental methods and animal care procedures were approved by the institutional animal care and use committee of chungbuk national university ( approval number : cbnura-042 - 0902 - 1 ) . the rd1 ( c3h / hej strain ) mice ( age : postnatal day 56 ) were used in this study . the methods for retinal tissue preparation and for the recording of rgc activities were previously reported ( 1 ) and are briefly described here . retinal patches from rd1 mice were prepared following the method of stett et al . ( retinas were isolated , cut into patches of 3 3 mm , and mounted on a planar microelectrode array ( mea ; multichannel systems gmbh , reutlingen , germany ) so that the ganglion cell layer faced the mea . the mea contained 64 tin electrodes ( circular shape ; diameter : 30 m , interelectrode spacing : 200 m , impedance : < 50 kat 1 khz ) on a glass substrate in an 8 8 square - type grid layout . the waveforms from the electrodes were recorded with a sampling rate of 25 khz / channel ( amplification gain : 1,200 , bandwidth : 103,000 hz ) and stored on a hard drive using data acquisition software ( mc_rack ; multichannel systems gmbh ) . the data recorded by mc_rack were converted to text file by mc_datatool ( multichannel systems gmbh ) . the recorded waveforms were futher analyzed to generate single unit spike trains by spike detection and spike sorting based on principal component analysis ( 12 ) . amplitude - modulated current pulse trains were generated with a stimulus generator ( stg 1004 ; multichannel systems gmbh ) and applied to the retina via 2 or 4 channels of the mea at its vertices . the stimuli consisted of symmetric , charge - balanced biphasic pulses ( anodic first , with no temporal separation between the anodic and cathodic phases ) . for the characterization of the modulation behavior of rgc responses by pulse amplitude , trains of 20 identical pulses were applied at 1-second interpulse intervals from one of the stimulating electrodes while the pulse amplitude was increased from 260 a ( 2 , 5 , 10 , 20 , 30 , 40 , 50 , and 60 a ) . the pulse rate and pulse duration were fixed at 1 hz and 500 s , respectively . then , the amplitude - modulated pulse trains were applied to investigate whether the rgc responses can be made to encode spatiotemporal visual information . the procedure for the pulse amplitude modulations based on natural scenes is described below ( fig . black - and - white movies of natural scenes were recorded by a camcorder ( xacti hd1010 ; sanyo , osaka , japan ) for 2 minutes at 60 frames / s with a 1,280 720 resolution . two or 4 pixels of the 16 9 resolution scene were randomly selected , and the amplitude of each pulse was modulated according to the time - series of the pixel intensity within the range of 120 a ( 1 a resolution ) . the pulse trains were simultaneously applied to the retina via 2 or 4 channels of the mea to present the electrical stimulation with spatial pattern , as shown in fig . 1 . the pulse repetition rate varied from 210 hz ( 2 , 4 , 6 , 8 , and 10 hz ) . the pulse duration per phase varied from 200400 s ( 200 , 300 , and 400 s ) . encoding and decoding of visual information . pulse trains of which amplitudes were modulated according to the brightness of 2 ( or 4 ) pixels were delivered to rgcs via the stimulation electrode on the mea . the visual information was reconstructed ( i.e. , decoded ) from the evoked rgc activities . then , the goodness - of - fit between the original and decoded pulse amplitude time - series was calculated to evaluate the effectiveness of the stimulation . spike train decoding was used to estimate how accurately the spatiotemporal visual information was transferred to rgc neural activities by multichannel electrical stimulation . because each pulse train was applied to each stimulating electrode independently , it was possible to select rgcs whose evoked activities were effectively modulated by a specific pulse train ( as in fig . thus , 2 and 4 groups of rgcs were used to decode visual spatiotemporal information with 2 and 4 pixels , respectively . the overall procedure for spike train decoding the raw waveforms from each electrode were transformed into single unit spike trains , and then , the rgc spike trains were transformed to a firing rate time - series by counting the numbers of spikes in 50 ms bins . temporal patterns of the neural activities of rgcs . rhythmic bursting patterns of ( a ) spontaneous activity and ( b ) electrically evoked response ( pulse amplitude : 20 a , pulse rate : 1 hz ) . as the arrows indicate , both spontaneous and electrically evoked activities show rhythms of bursts at similar frequencies . the response strength of the rgcs was measured by counting the number of poststimulus spikes within approximately 100 ms of the stimulus onset , which corresponds to the first peak in the psth shown in fig . 2c . rgc = retinal ganglion cell , psth = post - stimulus time histogram , isih = interspike interval histogram . the input to the decoder consisted of firing rates of multiple rgcs within a number of time bins . that is , the firing rates of multiple rgcs within a short temporal interval after the stimulus were used to decode the pulse amplitude time - series at every time bin , corresponding to the intensity time - series of multiple pixels . the input - output relationship can be described as follows : ( 1)s^(i)=p=1nj=0m-1{(rpi - jfpj ) } here , (i ) is the estimated value of the pulse amplitude of one electrode at the i time bin , and fp(j ) is the j coefficient of the linear filter corresponding to the p rgc . rp(i ) is the firing rate of the p unit at the i time bin of 50 ms duration . the decoder can be regarded as a temporal filter because it calculates the output from the past and present values of the input . when linear mapping is used , the decoding algorithm becomes an optimal linear finite impulse response ( fir ) filter ( 13 ) . in this case , ( . ) is merely a unity function , and the coefficients fp(j ) were obtained by the least squares method , as illustrated by warland et al . more generally , nonlinear regression algorithms , such as multilayer perceptron or support vector machine ( svm ) , can be used for the input - output mapping of the decoding algorithm . the specific algorithm of the svm that we used is described in hoegaerts et al . the recordings were divided into 2 groups of equal size ( i.e. 12 minutes recordings ) . the first one minute of the recording was used for the training of the decoding algorithms and the remaining one minute was used to test the trained decoding algorithms . to quantify the accuracy of the information decoded from rgc activity , similarities between the original and decoded pulse amplitude time - series were computed by correlation coefficient ( cc ) . all experimental methods and animal care procedures were approved by the institutional animal care and use committee of chungbuk national university ( approval number : cbnura-042 - 0902 - 1 ) . in total , 1,185 single units of rgcs showing apparent spiking activities were identified from 27 retinal patches obtained from 27 mice . the average number of rgcs per patch was 43.89 5.66 . among these , 894 rgcs ( 33.11 7.51 rgcs / patch , 75.44% ) showed consistent and evident modulation of evoked responses according to pulse amplitude , as shown in fig . 2a and b show typical waveforms of spontaneous and electrically evoked activities of an rgc . the oscillatory bursts in the spontaneous activity produced the distinct shape of the interspike interval histogram ( isih , fig . the first peak at 10 ms resulted from an interspike interval within each burst of spikes . rhythmic burst firing patterns were also observable in electrically evoked rgc activities , as indicated by the arrows with dotted lines in fig . the rgc responses were in the form of repetitive oscillatory bursts with strong phase - locking across different trials ( fig . the phase - locked bursts of spikes were maintained up to 500700 ms ( fig . 2b and c ) . in spite of the significant alteration in firing patterns of electrically evoked activities , the strength of the evoked activities could be effectively modulated by the amplitude of the stimulation pulses ( fig . 2e ) , just as in the case of a normal retina ( 6 ) . the number of poststimulus rgc spikes within 100 ms increased essentially monotonically when the pulse amplitude was increased to 20 a and were saturated thereafter . short latency responses elicited by direct activation of rgcs were also observed after artifact removal at 3 ms poststimulus . , only a single short latency spike is evoked in response to a single current pulse , so that the decoding accuracy in our study is not depedent on the short latency response . thus , we did not included the short latency responses , which are severly contaminated by stimulation artifact and not essential for the decoding in this study ( 1617 ) . the spatial patterns of evoked rgc activities were characterized by the relationship between the response strength and the distance between stimulation and recording electrodes ( fig . the electrical stimulation was applied at one of the 2 stimulating electrodes in fig . 3a . as a typical example , for a 20 a amplitude biphasic pulse , the rgcs within 8001,000 m of the stimulation electrode fired 3 spikes ( fig . 3a and b ) . we defined the rgcs , the firing rates of which could be effectively modulated by pulse amplitude , as well - modulated rgcs ( fig . we tried to find the spatial range of effective stimulation from the distribution of the well - modulated rgcs ( fig . the rgcs were clustered into 2 groups ( group 1 and 2 ) according to the strength of response to the electrical stimulation . that is , if the stimulation at the stimulating electrode 1 ( s1 ) elicited more spikes at a specific rgc than the stimulation at the stimulating electrode ( s2 ) , then the rgc was assigned to group 1 . fig . it clearly indicates that the rgc activities were evoked and modulated more effectively by the stimulation site in closer proximity . 3d shows the ratio of well - modulated rgcs as a function of the distance from the stimulation electrode , which implies that the range of rgcs for effective encoding of information is almost linearly decreased as a function of the distance . stimulation pulse trains were applied independently to one of the 2 stimulation electrodes ( pulse amplitude : 20 a ) . ( b ) response strength as a function of the distance between the stimulation and recording electrodes . ( c ) spatial profile of the well - modulated rgcs . the number of well - modulated rgcs displayed at the location of the recording electrodes . ( d ) the percentage of the electrodes with well - modulated rgcs as a function of the distance between the stimulation and recording electrodes . the percentages were calculated by the ratio between the number of all the electrodes and the number of electrodes where the well - modulated rgcs were observed . when 2 independent amplitude - modulated pulse trains were applied simultaneously at the 2 stimulating electrodes , both of the pulse amplitude time - series obtained from 2 pixels of natural scenes could be successfully decoded from the rgc spike trains ( fig . 4 ) . a nonlinear decoder using a svm resulted in more accurate decoding , although both linear and nonlinear methods yielded satisfactory decoding . the decoding accuracy was strongly dependent on the details of stimulation , especially on the pulse amplitude range . 4a and b show the comparison of the original and decoded pulse amplitude time - series when the range of the pulse amplitude was 120 a and 110 a , respectively ( pulse rate : 8 hz , pulse duration : 300 s ) . we tested several stimulation parameters and found that the decoding accuracy was the highest for the pulse amplitude range of 120 a and the pulse duration of 300 s ( fig . when the amplitude range was changed to 110 a while fixing the pulse rate and duration , the decoding accuracy significantly deteriorated ( fig . 4c , cc was 0.5912 0.24 and 0.3827 0.23 at 120 and 110 a , respectively ; 17 retinal patches , p < 0.001 ) . 4d , when the pulse rate was changed to 2 hz , the decoding accuracy was significantly lower than at 8 hz ( cc was 0.6733 0.18 and 0.4177 0.23 at 8 and 2 hz , respectively ; 6 retinal patches , p < 0.001 ) . the pulse amplitude time - series applied to 4 stimulating electrodes could also be successfully decoded from a group of rgcs ( fig . more accurate decoding was possible by increasing the number of rgcs for the decoder input , but the increase of decoding accuracy was saturated at 10 cells ( data not shown ) . although the distance between adjacent stimulating electrodes was as short as 1,216 m , 4 independent time - series were faithfully reconstructed ( fig . the decoding accuracy was calculated for several different stimulation parameters , and it was the highest for the pulse amplitude range of 120 a and the pulse duration of 400 s ( fig . when the amplitude range was changed to 110 a while fixing the pulse rate and duration , the decoding accuracy significantly decreased ( fig . 5b , cc was 0.6679 0.10 and 0.4788 0.19 at 120 and 110 a , respectively ; 7 retinal patches , p < 0.001 ) . the decoding accuracies of 2 channel and 4 channel stimulations were not compared directly since different retinal patches were used for the 2 channel and the 4 channel stimulation experiment . examples of original and decoded pulse amplitude time - series , obtained from 2 pixels of a natural scene . ( a ) pulse amplitude range : 120 a , pulse duration : 300 s , pulse rate : 8 hz . ( b ) pulse amplitude range : 110 a , pulse duration : 300 s , pulse rate : 8 hz . the location of stimulation electrode of the mea is described in each panel ( open circle : stimulation electrode , closed circle : ground electrode ) . comparison of the decoding accuracies of ( c ) 2 different pulse amplitude ranges ( obtained from 17 retinal patches , pulse rate : 8 hz , svm ) and ( d ) 2 different pulse rates ( obtained from 6 retinal patches , pulse amplitude range : 120 a , svm ) . the decoding accuracy significantly changed in both cases ( t - test , p < 0.001 ) . examples of original and decoded pulse amplitude time - series , obtained from 4 pixels of a natural scene . ( a ) pulse amplitude range : 120 a , pulse duration : 400 s , pulse rate : 8 hz . pulse amplitude time - series applied to 4 stimulating electrodes could also be successfully decoded from a group of rgcs , just as it was with 2-channel stimulation . four - pixel decoding accuracy was significantly different at 2 different pulse amplitude ranges ( t - test , p < 0.001 , obtained from 7 retinal patches , pulse rate : 8 hz , svm ) . in this study , we demonstrated that spatiotemporal visual information can be decoded from the rgc activities of a photoreceptor - degenerated retina evoked by simultaneous multichannel stimulation based on pulse amplitude modulation . in our recent studies on temporal information encoding by electrically stimulated rgcs , the pulse amplitude time - series could be successfully reconstructed from evoked rgc activities ( 67 ) . this is supported by recent reports on human clinical trials of retinal implants , such as the report of greenwald et al . ( 18 ) , where it was shown that the brightness of the phosphene elicited by electrical stimulation of the retina followed the current amplitude . thus , we inferred that pulse amplitude modulation might be an appropriate approach for delivering temporal visual information to the retina . cottaris and elfar ( 8) showed that specific features of electrical stimulation pulses , including spatial location of the stimulation site , could be identified from local field potential in v1 . hence , it is natural to expect that spatial information of visual input might be effectively encoded by prosthetic electrical stimulation as well . our results have explicitly shown for the first time , to our knowledge , that multipixel spatiotemporal information can be decoded from the activities of the rgc network when evoked by a multichannel amplitude - modulated stimulation pulse train . these results support the plausibility of a pulse generation strategy based on amplitude modulation for successful encoding of spatial information in neural activities . the range of amplitude modulation was crucial , as it could be predicted from the characteristics of the rgc - evoked firing rate as a function of pulse amplitudes ( fig . it is obvious that an excessively wide pulse amplitude range is not beneficial for high decoding accuracy , due to the firing rate modulation characteristics shown in fig . this resulted in strong dependence of the decoding accuracy on the range of amplitude modulation . when the pulse amplitude was modulated at 110 a , which is half of the optimum ( 120 a ) , the decoding accuracy considerably deteriorated , due to the insufficient changes in the firing rate that caused ineffective encoding of the intensity variation . considering the high level of spontaneous rgc firing in photoreceptor - degenerated retinas ( 19 ) , it is also feasible that the level of electrically evoked activities was not differentiated from that of the spontaneous activities . the current spread to adjacent sites is another reason to limit the pulse amplitude range , and the spatial profile of the evoked rgc activities ( fig . other parameters , such as pulse duration or pulse rate , also significantly affected the decoding accuracy . the optimization of the stimulation strategy should be performed in a multidimensional parameter space in real clinical applications of retinal implants , as demonstrated conceptually by eckmiller et al . the decoding - based strategy of our study can be utilized for this purpose as well . for successful perception of a geometrical pattern , multiple localized phosphenes should be generated , and their brightness should be controlled individually . this is expected to be achieved by selective activation of groups of rgcs in close proximity of each stimulating electrode . we tried to investigate whether such a spatially selective activation of groups of rgcs is possible by observing the evoked rgc activities as a function of the distance from the stimulation electrode . it is known that the spatial resolution that is achievable by long - latency rgc responses ( i.e. , by indirect activation ) is limited in space by spatial extent of the ganglion cell dendritic field and is better than that of short - latency response ( i.e. , by direct activation ) ( 21 ) . however , this is for a case when the size of the stimulation electrode is sufficiently small . the spatial resolution deteriorates for a larger electrode due to the current spread . considering that the electrode size is considerably large and that the current amplitude is substantially above the threshold in our study , as well as in clinical trials conducted thus far , the spatial resolution is expected to be worse than the optimum determined by the ganglion cell dendritic field . the current spreading was indeed quite substantial , and the evoked activities were quite strong for the rgcs within 8001,000 m from the stimulating electrodes , as shown in fig . 3 ) . less than 20% of the well - modulated rgcs were outside of 800 m . considering that 1 mm corresponds to a visual angle of 3.4 in human eyes ( 22 ) , the spatial resolution achievable by our strategy is expected to be 23. it is accepted that a spatial resolution of 10 is required for perceiving large objects and movement direction ( 23 ) and thus , a spatial resolution of 23 is surely enough to be helpful in improving the quality of life of the blind . the decoding accuracy was strongly dependent on the pulse rate , as shown in fig . 4 , where the pulse rate of 8 hz was significantly more effective than a rate of 2 hz . a higher pulse rate might be beneficial because it prevents abnormal rhythmic rgc responses due to the short inter - pulse intervals . however , the pulse rate should be determined carefully because excessively high pulse rate may suppress rather than activate the rgc activities ( 10 ) . it is reported that short - width pulses can selectively evoke short - latency responses of rgcs and generate precise temporal spiking patterns by evoking only one spike with each pulse ( 10 ) . a similar strategy has recently been implemented using optogenetic stimulation as well ( 24 ) . however , this requires minute electrode size and short inter - electrode distance , which are not compatible with current retinal implant devices undergoing clinical trials , which are rather bulky . for example , the stimulating electrode size is as large 200 m ( 25 ) . the size and inter - electrode spacing of these current devices are comparable to those in our study . thus , our in vitro model can provide a basis for the evaluation and optimization of a stimulation strategy for these current devices . in conclusion , we showed that multipixel spatiotemporal visual information could be decoded accurately from rgc network activity evoked by patterned electrical stimulation when the detailed parameters of the simulation pulse trains were carefully determined . our results suggest that useful visual function may be restored by amplitude modulation - based retinal stimulation . | retinal implants have been developed as a promising way to restore partial vision for the blind .
the observation and analysis of neural activities can offer valuable insights for successful prosthetic electrical stimulation .
retinal ganglion cell ( rgc ) activities have been investigated to provide knowledge on the requirements for electrical stimulation , such as threshold current and the effect of stimulation waveforms . to develop a detailed stimulation strategy for faithful delivery of spatiotemporal visual information to the brain , it is essential to examine both the temporal and spatial characteristics of rgc responses , whereas previous studies were mainly focused on one or the other . in this study , we investigate whether the spatiotemporal visual information can be decoded from the rgc network activity evoked by patterned electrical stimulation . along with a thorough characterization of spatial spreading of stimulation current and temporal information encoding
, we demonstrated that multipixel spatiotemporal visual information can be accurately decoded from the population activities of rgcs stimulated by amplitude - modulated pulse trains .
we also found that the details of stimulation , such as pulse amplitude range and pulse rate , were crucial for accurate decoding .
overall , the results suggest that useful visual function may be restored by amplitude modulation - based retinal stimulation . |
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the boundaries are comprised by the fascia propria of the upper 2/3rd of rectum anteriorly , presacral fascia posteriorly and the endopelvic fascia with the ureters and iliac vessels on both lateral aspects . the roof is formed by the peritoneal reflection while the floor by the waldeyer s fascia . it constitutes an area for development of multiple embryologic structures between the hindgut and the proctodeum , neural elements and bone and connective tissue . the incidence has been reported in literature to be between 1 in 40,000 to 1 in 63,000 hospital admissions . due to obscure anatomic location , difficult surgical approach and etiological heterogeneity , tumors arising here pose a diagnostic and therapeutic challenge . we present our experience with 10 cases of presacral tumors with posterior approach being the major surgical approach . all patients of presacral tumors presenting in the department of general surgery , sawai man singh medical college in a single surgical unit during a time period of 14 months ( may 2013july 2014 ) were included in the study . the clinical presentation , diagnoses , surgical intervention done , post - operative outcome , and histopathological findings were reviewed . follow up was performed on outpatient visits . all patients were routine outpatient admissions . all the patients were females in the age group of 1850 years ( mean 28.4 years ) . the most common presenting symptom was lump lower abdomen ( 5/10 ) followed by swelling in lower back and perineal region ( fig . one patient presented with features of adrenergic overactivity manifested as body ache , headache and palpitations . one had edema of the left lower limb and second had neuropathic pain in left lower limb . duration of symptoms ranged from 1 to 36 months ( median of 3 months ) . three out of the 10 patients had been operated previously for similar findings and a recurrence was suspected . patient no.1 in table 1 had been operated previously for lump abdomen and histopathological examination ( hpe hereafter ) report was in favor of schwannoma ; patient no . 4 had been previously operated twice and hpe examination was in favor of leiomyosarcoma ; patient no . the swelling was palpable per rectally in 6/10 patients , and it was possible to reach the upper limit of swelling in all of these . diagnostic evaluation of the patients was done by clinical examination , ultrasonography of the abdomen and pelvis followed by ct / mri abdomen and pelvis whereever required ( fig . ultrsonography of the abdomen revealed the presence of hepatic , pulmonary and omental metastatic deposits along with ascites in patient no . she was diagnosed to have diffuse peritoneal leiomyomatosis with metastases and was referred for palliative treatment . in patient 2 , the cect abdomen and pelvis was inconclusive about the involvement of major pelvic blood vessels which was revealed on subsequent laparotomy . the surgical approach , histopathological diagnosis , icu requirement , postoperative complication , length of hospital stay is summarized in table 2 . out of the 9 patients operated , 6 were by posterior approach and rest by anterior approach . in one case , i.e. patient no . 1 , the previous surgery was done by anterior approach while the redo surgery was done by posterior approach . complete en bloc excision of the mass was achieved in all cases except one ; patient no . 2 , the mass was found to be encasing the ureters and major pelvic vasculature and hence only a biopsy was taken . we used the transverse incision in all cases of posterior approach , the location and length of the incision depending upon the size and extent of the tumor . finger dissection was done after the skin incision with minimal use of electrosurgical devices and space was created between the tumor , presacral fascia and surrounding structures . there was no need of sacrectomy in any of the cases while coccygectomy was done in 1 case . there were no major complications like pelvic hematoma , nerve injury , bowel or visceral injury . , there was no evidence of bowel or bladder dysfunction in any of the patients operated by posterior approach ; neither was there any residual limb weakness or sensation loss . preascral tumors are a rare variety of space occupying lesions whose true incidence is not known because most of the series have been published from tertiary care referral centers . despite this , various authors report an incidence between 1.4 and 6.3 cases per year [ 26 ] . the incidence of benign tumors has been reported to be more in females while malignant tumors have a slight male predominance [ 2,3,4,79 ] . several classification systems have been proposed but most commonly the retrorectal masses are classified based on their origin into congenital ( 5565% ) , neurogenic ( 1012% ) , osseous ( 511% ) , inflammatory ( 5% ) and miscellaneous ( 1216% ) . these may arise from ectoderm ( dermoids and epidermoids ) , sequestration of hindgut remnants ( tail gut cysts , rectal duplication cysts ) , neural tube defects ( anterior sacral meningocele ) , and notochord ( chordomas ) . clinical presentation is nonspecific depending on the size and location of the mass and tissue of origin . a careful digital rectal examination ( dre hereafter ) . symptoms most often arise due to compression of nearby neurological structures causing radicular pain . symptoms arising due to presacral masses depend on the size , location and origin of the tumor and also on whether the tumor is benign or malignant . in a review of five largest case series , 43% of benign tumors were free from symptoms while only 7% of malignant tumors were symptomless . a careful dre is the initial examination of choice . in a series of 120 cases from mayo clinic , if the superior limit of the tumor is palpable on dre , a posterior surgical approach may be feasible . plain radiographs may sometimes be useful and demonstrate sacral destruction ( chordomas ) , calcifications within the mass ( teratomas ) or pathognomic signs like the scimitar sign ( anterior sacral meningocele ) . computed tomography of the pelvis is widely used and is capable of differentiating solid from cystic lesions , vascular , adjacent organ involvement and bony destruction . contrast enhanced mri pelvis is the gold standard investigation of choice and the most sensitive and specific investigation also . limitations of mri are inability to distinguish between fibrosis and recurrent pelvic carcinoma in patients managed with surgery or radiation therapy . other investigations like ct / mr myelography may be done when there is involvement of cns . in cases of major vessel involvement endorectal ultrasound and lower gi endoscopy can help in evaluating the involvement of layers of rectum and superior extent of the lesion as well as the consistency of the tumor . a preoperative biopsy should only be performed if it is likely to change the management and surgical approach . currently , there is no indication for biopsy of a cystic lesion . preoperative percutaneous biopsy is , however , indicated for solid or heterogeneous tumors to facilitate decision making for neoadjuvant therapy and surgical planning . biopsy , if indicated , should never be done transperitoneally , transretroperitoneally , transrectally or transvaginally . the anterior or trans abdominal approach is recommended for very large tumors , when there is suspicion of malignancy and when the lower limit of the tumor is above the s3 vertebra . advantages of an anterior approach include better visualization of pelvic structures , bleeding control and easy mobilization of rectum . the posterior approach , first described by paul kraske ( 1885 ) , may be inter - sphincteric , trans - sphincteric parasacrococcygeal , trans - sacral , trans - sacrococcygeal , trans - anorectal , trans - vaginal . it is indicated in low lying tumors with upper limit below s3 , when there is evidence of sacral involvement , in small tumors and when malignancy is not suspected . coccygectomy indicated for better exposure as well as for complete removal of a potential communication route and consequent recurrence of cystic lesions or teratomas . advantages include a more direct approach , more commodious to adopt , operating field of the tumor is clearer , adverse influences of bowel coming in between are avoided , quicker post - operative recovery . potential drawbacks include absence of control over pelvic vessels , chances of injury to pelvic nerves and reduced working space . a combined abdomino - sacral approach is recommended for larger lesions that extend both above and below s3 , large masses , and suspected malignancy . a perineal endoscopic approach has also been described to access the presacral space and perform excision biopsies . complications of surgery include bleeding , post - operative urinary and fecal incontinence , nerve root injury leading to weakness and chances of abscess or fistula formation . rates of complications are higher in patients undergoing some form of sacral resection . even , if sacrectomy is necessary , care must be taken to preserve at least one side of s2 to reduce postoperative complications arising out of nerve damage . adequate surgical resection is the only treatment for presacral masses as most of the tumors eventually enlarge in size ; there is always a risk of malignancy and infection ; and they have the potential of causing dystocia in women of child bearing age . posterior surgical approach is an attractive option as most common variety of presacral masses is low lying developmental cysts . the prognosis of benign tumors is excellent after complete excision while that of malignant tumors is poor due to high rates of recurrence . written informed consent was obtained from the patients for publication of this case series and accompanying images . a copy of the written consent is available for review by the editor - in - chief of this journal on request . dr . dhananjay saxena study design , data acquisition , data analysis , writing , revision . | highlightspresacral tumors are rare variety of space occupying lesions which due to their location , etiological heterogeneity and difficult surgical approach , present a challenge to treating physician.surgical excision remains the treatment of choice for presacral masses.posterior surgical approach , if applied judicially , in carefully selected cases , remains an attractive option as it provides better surgical exposure , is more direct approach , commodious to adopt , easy to learn with quicker post - operative recovery . |
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from september 2010 through march 2012 , rodents were trapped in rural residences from 2 county - level divisions ( xintai and daiyue ) in taian . after each rodent was numbered and species identified , the spleen was dissected for dna extraction . during 20102011 , scrub typhus patients from xintai and daiyue were recruited from 7 hospitals in taian . before the patients received antimicrobial drugs , whole blood samples were collected and anticoagulated with edta - na2 . dna was extracted from each specimen by using a tianamp genomic dna kit ( tiangen , beijing , china ) . considering the higher sensitivity and specificity , seminested pcr was used to amplify the coding sequences spanning variable domain i vd iii of the 56-kda tsa with previously described primers a ( 5-tttcgaacgtgtctttaagc-3 ) , b ( 5-acagatgcactattaggcaa-3 ) , and e ( 5-gttggaggaatgattactgg-3 ) ( 10 ) , which yielded fragments of 733 nt . to avoid contamination , pcr preparation was performed in an area separate from dna extraction , and negative controls were included in each step . nucleotide sequences of o. tsutsugamushi determined in this study were deposited in genbank under accession nos . theses sequences were aligned with 38 reference sequences of the o. tsutsugamushi 56k - da tsa gene retrieved from genbank by using mega software with the clustalw algorithm ( 11 ) . the phylogenetic tree was constructed by using mega software with the minimum - evolution method and the kimura 2-parameter model , with bootstrapping for 1,000 replications . a percentage the species of the 385 captured rodents were 244 rattus norvegicus , 139 mus musculus , and 2 r. rattus . o. tsutsugamushi dna was detected by pcr in 10 ( 2.6% ) rodents captured in october , november , december , and march ; 6 ( 4.3% ) rodents were m. musculus and 4 ( 1.6% ) were r. norvegicus . rates of o. tsutsugamushi positivity in domestic rodents captured in spring , summer , autumn , and winter were 5.3% , 0 , 3.1% , and 1.2% , respectively ( table ) . no significant difference was observed in the rate of o. tsutsugamushi positivity determined by pcr among the 3 species of rodents ( p = 0.278 ) or among seasons ( p = 0.274 ) . among the 10 sequences obtained from rodents , we identified 3 genogroups ( kawasaki - related , fuji - related , and novel genogroup ) involving 7 genotypes with > 1 nt difference . m. musculus and r. norvegicus rodents in taian could serve as reservoirs of 5 and 3 genotypes , respectively . genotype fjs was closely related to the fuji genotype ( 96.5% identity ) , which was originally isolated from leptotrombidium fuji mites in japan ( 12 ) . genotypes sdm1 and sdm2 clustered in an independent clade , which had 70.4%77.9% identity with reference strains ( figure ) . phylogenetic relationships of orientia tsutsugamushi detected in domestic rodents and human patients with scrub typhus in rural areas of taian , shandong province , china , september 2010 through march 2012 . relationships were determined on the basis of the partial 56-kda type - specific antigen gene of o. tsutsugamushi by the minimum - evolution method with the kimura 2-parameter distance model . whole blood or eschar specimens were obtained from 19 human patients , among whom 6 ( 31.6% ) were infected with o. tsutsugamushi according to seminested pcr . two genotypes ( kws1 and kws2 ) , which were identified in domestic rodents and belonged to kawasaki - related genogroups , were also found in the patients ( figure ) . our study demonstrated the prevalence of 7 genotypes of o. tsutsugamushi in the new epidemic area in taian , northern china . given that kws1 and kws2 identified in domestic rodents could cause human infection , prevalence of o. tsutsugamushi infection in domestic rodents might be a public health concern . natural infection with the kws2 genotype of o. tsutsugamushi in rodents was found during march in taian . periodic molecular surveillance of o. tsutsugamushi in local areas might help predict changes in the epidemic features of scrub typhus . physicians should not arbitrarily exclude a diagnosis of scrub typhus for patients with fever of unknown origin during nonepidemic seasons . the kawasaki and fuji strains of o. tsutsugamushi were considered to be of low virulence ( 12,13 ) . cross - reactivities of the kawasaki strain with 118 anti - gilliam monoclonal antibodies and of the fuji strain with 5d-3 anti - kato monoclonal antibodies were observed ( 12 ) . the 56-kda tsa is responsible for adhesion to and invasion of the host cells , and its variable domains might serve as epitopes ( 5,14 ) . although the identity of the kws14 and fjs genotypes and their prototypes was > 96% , changes in virulence and antigenicity might occur because of minor variation . among the scrub typhus patients from xintai and daiyue , those that are antigenically distinguished from the commonly used antigens should be included in the antigen panel of serologic assays in the local area , which would prompt the diagnosis and prevention of scrub typhus for local residents and travelers . identity of sdm1 and sdm2 with the reference strains was < 77.9% , and they formed a distinct cluster , indicating that they might represent novel genotypes . infection of scrub typhus patients with sdm1 , sdm2 , and fuji - related genotypes has not been reported . investigation of host ranges , invasiveness , and virulence of newly defined genotypes of o. tsutsugamushi in local areas will help determine their pathogenic potential for humans . expansion of genetic diversity of o. tsutsugamushi was expected to occur in maintenance hosts ( 15 ) . we are currently screening o. tsutsugamushi in chigger mites collected from the captured rodents to evaluate the role of domestic rodents in the transmission of scrub typhus to humans . assiduous surveillance of genetic variations of o. tsutsugamushi in hosts and identification of their pathogenic potentials is essential for the improvement of diagnostic capacity , vaccine development , and assessment of epidemiologic role . | we screened orientia tsutsugamushi from 385 domestic rodents and 19 humans with scrub typhus in rural taian district , shandong province , a new scrub typhus epidemic area in northern china .
sequence analysis identified 7 genotypes in the rodents , of which 2 were also identified in the humans . |
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its etiology involves a combination of genetic factors , unfavorable growth pattern , and oral habits . habitual nonnutritive sucking , altered tongue function and posture , and persistent infantile swallowing , for example , can contribute to vertical malocclusion in the growth period.1,2 several studies have suggested a relationship among nasopharyngeal airway obstruction , mouth breathing , and skeletal and dental malocclusion . indeed , adenoid hypertrophy and chronic rhinitis may result in mouth breathing and encourage persistent infantile swallowing , which can contribute to anterior open bite.3,4,5,6 as such habits can cause relapse following orthodontic treatment,7,8 contributing factors should be uncovered when diagnosing patients with anterior open bite . considering the etiological complexity , a multidisciplinary approach involving several health professionals , such as orthodontists , otorhinolaryngologists , orofacial myofunctional therapists , and maxillofacial surgeons , in adult patients , may be necessary . here , we report the multidisciplinary correction of anterior open bite relapse secondary to upper airway obstruction . a 27-year - old man presented an anterior open bite relapse ( figures 1,2,3,4 ) . he reported that orthodontic treatment was begun when he was 13 years old and lasted 5 years . although initially satisfied with the outcome , he noticed relapse of anterior open bite one year after the brackets were removed . intraoral examination showed bilateral angle class i molar relationship , slight deviation of the upper midline , proclination of the maxillary lateral incisors , crossbite of the maxillary left canine and second premolar , negative crown inclination of the maxillary posterior teeth , and gingival recession at the mandibular canines . facial analysis showed increased lower facial height with good lip competence and large nasolabial angle . slight nasal deviation to the right was also apparent when the patient was viewed anteriorly . anterior low tongue posture was noted at rest and during swallowing , and the patient reported chronic difficulty in nose breathing . a cone - beam computed tomography ( cbct ) of the head was performed to uncover upper airway problems responsible for the breathing difficulty and relapse . the scan showed extremely thin mandibular symphysis , which can markedly affect mandibular incisor inclination during orthodontic treatment ( figure 5 ) . in addition , nasal septum deviation , right turbinate hypertrophy , and left maxillary sinus congestion were visible ( figure 6 ) . all these features were thought to contribute to the breathing problem , encourage the improper tongue posture , and thereby cause the relapse . pretreatment cephalometric analysis confirmed the class i hyperdivergent skeletal pattern associated with dental anterior open bite and moderate maxillary incisor proclination ( table 1 ) . the treatment objectives were to ( 1 ) maintain the bilateral angle class i molar relationship ; ( 2 ) correct the anterior open bite ; ( 3 ) balance maxillary gingival exposure , ensuring a pleasant smile ; ( 4 ) minimize mandibular gingival recession during the treatment ; ( 5 ) achieve positive crown inclination of the maxillary posterior teeth , creating a wider smile ; and ( 6 ) ensure stable results overall . the presence of dental ( not skeletal ) open bite , lip competence , and good facial and smile esthetics excluded combined orthodontic treatment and orthognathic surgery as well as first premolar extraction . although use of tongue spurs or a tongue crib during orthodontic treatment can achieve rapid closure of open bite , this approach was rejected because the tongue posture was judged to be secondary to the breathing problem . to meet all the treatment objectives , we offered the patient a multidisciplinary approach involving orthodontic treatment to correct the malocclusion , otorhinolaryngological intervention to resolve the airway obstruction , and periodontal treatment to correct any gingival recession arising in critical mandibular areas during the treatment . damon self ligating brackets with insignia ( ormco co. , orange , ca , usa ) were bonded to all the teeth ( figures 7 , 8) . in the digital setup , we programmed complete correction of the open bite and , because of the susceptibility to relapse , overcorrection of the overbite , placing the maxillary incisal margin 3 mm below the edge of the mandibular incisors . we also programmed positive crown torque of the maxillary posterior teeth and ensured that all the mandibular teeth would be maintained in the middle of the medullary bone to prevent bone dehiscence and fenestrations , which can cause substantial gingival recession . the brackets were threaded with the following customized archwire sequence : 0.014 inches ( in ) damon copper nickel titanium ( ni - ti ) , 0.014 0.025 in damon copper ni - ti , 0.018 0.025 in damon copper ni - ti , 0.019 0.025 in stainless steel , and 0.019 0.025 in titanium molybdenum alloy ( tma ) . leveling and alignment were performed using thermal ni - ti archwires and completed rapidly because of the previous orthodontic treatment and relapse mainly involved the anterior open bite . when the 0.018 0.025 in damon copper ni - ti archwires were fitted , the patient was also supplied with intermaxillary elastics to facilitate bite closure during the remainder of the active phase . bilateral triangular elastics ( 3/16 in , 6 oz . ) were provided for daytime use , and anterior box elastics ( 5/16 in , 6 oz . ) were added for nocturnal wear . after 10 months of orthodontic treatment , we referred the patient to an otorhinolaryngologist for bilateral turbinectomy and maxillary sinus cleaning . the surgery was performed by endonasal endoscopy and prompted immediate improvement in the patient 's breathing dynamics . the cbct scan taken 5 months postoperatively showed complete resolution of upper airway obstruction ( figure 9 ) , and at the subsequent orthodontic appointments , the patient reported that he was finally able to breathe through his nose and his sleep quality had improved . at these appointments , we reminded the patient to continue to maintain correct tongue posture at rest and during swallowing . the class i molar and canine relationship was maintained , anterior occlusion was improved , and ideal overbite and overjet was achieved . the treatment brought about mutually protected functional occlusion , with adequate canine disclusion and protrusive guidance . the temporomandibular joints showed no incoordination on opening and closing , and no temporomandibular joint or muscle problems developed during the retention and postretention periods . the lips remained competent , and the smile was balanced and pleasant . unfortunately , because of the mandibular anatomy and tooth movement , the existing gingival recession became more pronounced . the periodontist decided to improve the periodontal condition of the right mandibular central incisor alone and delay definitive mucogingival surgery for some years to stabilize the gingival conditions . superimposition of the pretreatment and post - treatment cephalometric tracings highlighted the amount of extrusion of the maxillary incisors needed to resolve the open bite and confirmed that the mandibular plane angle remained stable ( figures 10,11,12,13,14 , table 1 ) . the patient was instructed to wear a tongue elevator ( figure 15 ) for retention at night . this device featured an anterior opening that served as a tactile reminder and a chute that prevented positioning of the tongue between the dental arches . the well - aligned dentition and good overbite were maintained , facial balance was harmonious , and the smile was pleasing with good , stable gingival lines ( figure 16 ) . the success of the treatment is mainly ascribable to the thorough diagnosis and multidisciplinary approach , resolving not only the relapse but also the underlying issues . although the previous orthodontic treatment lasted 5 years , it did not guarantee stability of results . we believe that it failed predominantly because it was not aimed at tackling the airway obstruction and consequent tongue malpositioning , which were the root causes of the malocclusion . there are many options for correcting the tendency to place the tongue too forward ( tongue habit management ) . a tongue crib or tongue spurs can be used during orthodontic treatment to modify tongue posture , maintain stability of treated open bite , and prevent relapse , by establishing a new engram.9,10,11 however , many orthodontists underestimate the importance of upper airway obstruction in oral habits . anomalies of the nasal septum , turbinates , adenoids , or tonsils and chronic sinusitis can markedly alter respiratory dynamics , compromising dental arch development.4,12,13 therefore , when poor breathing is suspected , specific investigative tools , such as tomography with a wide field of view , should be used to examine the upper airway . our patient presented with anterior low tongue posture and reported chronic difficulty in nose breathing , so we examined the airway by digital volumetric tomography . the approach selected was orthodontic closure of the open bite using intermaxillary elastics and midcourse surgical turbinectomy , sinus cleaning , and correction of nasal septum deviation . the surgery had an immediate effect on the patient 's respiratory difficulty , enabling him to breathe normally and position the tongue correctly . however , as open bite presents a high risk of relapse,14,15 we prescribed nocturnal use of a tongue elevator for retention . the good results obtained by this multidisciplinary treatment are still preserved over 3 years later . dental malocclusion often needs to be treated by a multidisciplinary team to arrive at an accurate diagnosis and offer appropriate solutions . in particular , adults presenting with open bite , especially relapse cases , should be investigated for oral habits and/or airway issues before defining and selecting the best treatment option . | a 27-year - old man presented an anterior open bite relapse .
he had low tongue posture positioned anteriorly at rest and during swallowing and reported chronic difficulty in nose breathing .
head cone - beam computed tomography revealed nasal septum deviation , right turbinate hypertrophy , and left maxillary sinus congestion , which were thought to contribute to the breathing problem , encourage the improper tongue posture , and thereby cause the relapse .
multidisciplinary treatment involving an otorhinolaryngologist , an orthodontist , and a periodontist resolved the upper airway obstruction and corrected the malocclusion .
the follow - up examination after 3 years 5 months demonstrated stable results . |
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the mother of a 3-year - old girl brought her child to our traumatology orthopedic department because of the way the child 's wrist had set due to forced bending . the child was reported to have suffered , approximately 18 months before , a repetitive wrist injury by means of frequent pulling and bending of the hand by the father who may have physically abused the child for about 2 months . the mother did not indicate a specific major trauma which could lead to a conclusion that the child may have sustained the injury when the girl was 18 months old . on examination , there was diffuse swelling around the dorsum of the wrist , and the flexum approximately 20 degrees and the ulnar deviation approximately 20 degrees . she had normal sensation of the hand and good radial and ulnar pulses with capillary refill . on the basis of a clinical examination a computed tomography ( ct ) ( fig . 1 ) and a magnetic resonance imaging ( mri ) ( fig . 2 ) of the wrist were consistent with the diagnosis of the perilunate dislocation , provided by the mother and the wrist mri examination performed in our department . the surgical treatment was applied at the age of 3 years and 4 months , the reconstruction of failured ligaments and other dislocated carpal bones were planned according to mayfield classification.1 the medial dorsal approach reaching from the base of the distal radial bone to the base of the metacarpal was used between the second and the third compartments , the skin flaps were reflected to expose the extensor retinaculum , which was divided longitudinally and radiocarpal capsulotomy was made . after reduction of the dislocation , the plasty of the dorsal scapholunate ligament was performed using the capsular flap prepared from the extensor retinaculum . stabilization was achieved by two kirchner wires ( k - wires ) being introduced from radial and ulnar side ( fig . the k - wires were removed after 4 weeks and the wrist was immobilized in a cast for additional 2 weeks . two months after the surgery , the patient started to undergo the physiotherapy for 3 months post - op which included gradual strengthening , movement , and proprioceptive exercises . a medical check - up was performed after rehabilitation , 24 months after the surgery . the child did not report any pain during daily life activities or while playing sports . the patient could flex up to 40 degrees and extend up to 45 degrees ( mobility of the healthy wrist was flex up to 60 degrees , extend up to 50 degrees ) . 4 ) view showed a normal carpal alignment without instability and without the sign of the lunar avascular necrosis . at a lateral view , no disi and visi ( dorsal and volar intercalated segment instability ) was found , and the scapholunaris angle was 40 degrees while the capitolunaris angle was 25 degrees ( the normal is scapholunate angle in this age is 3060 degrees and the capitolunate angle more 30 degrees ) . because of the extremely rare type of the trauma and its being chronic in nature , the administered treatment and the result could be considered as good . the child and the rarity of occurrence of the described fractures in children and adults2
3 does not allow for a comparison between a mechanism of sudden fractures sustained which are healed as soon as the injury is diagnosed and a fracture resulting from repeating actions leading to sprains and permanent injuries that can be qualified as old chronic trauma . the authors did not refer to any similarly described case because of the apparent lack of any previous reports of this kind . however , medical literature amply describes acute perilunate dislocations2
4
5
6
7 which allows for a comparison of the type of symptoms , diagnosis , injury mechanism , treatment , effects , and further prognosis . acute dislocations , according to medical literature , are usually caused by injuries , often as a result of a traffic accident after a high - impact trauma,2 a fall from a great height or an injury associated with a given profession.4 in our case , there is a suspicion of a deliberate and continuous abuse of a 3-year - old girl which caused a chronic injury of a wrist . to the best of our knowledge the diagnosis of our case did not differ from other acute injuries described in the literature where x - ray , ct , and mri2
4
5
7 were usually performed . few complications have been described , although in some cases , they could be related to an insufficient reduction.8 as well as the resultant bone instability , volar lunate dislocation may also cause pressure and resultant dysfunction to the median nerve as it enters the carpal tunnel , therefore , emergency reduction of the perilunate dislocation is needed to reduce the pressure on the median nerve to try and prevent progression of nerve damage . there are also reports of the ulnar nerve being affected as a result of volar dislocation of the lunate.2
5
secondary dislocation or instability to closed reduction and stabilization with k - wires has led many authors to advocate for primary repair through open reduction and ligament repair.6 the perilunate luxation can rarely be treated using closed reduction.2
3
4
5
6
7
8 but in a retrospective study , primary ligamentous repair and internal fixation with k - wires managed to maintain the anatomy of the scapholunate joint and produce better results than closed methods.7
8 the procedure in this case was performed by means of open reposition using dorsal approach and the wrist was stabilized by two k - wires . next , an immobilization in a plaster brachial rail for a period of 6 weeks was recommended . after 4 weeks k - wires were removed , although in the cases described in the literature such wires were removed after 6 weeks,4 and the wrist was immobilized for an additional 64 or 8 weeks.5 some authors , however , suggest a short period of 3 weeks of immobilization in a cast with removal of the wire after 2 weeks which allows for an early mobility of the wrist.5 that can provide benefits to patients by speeding up the rate of functional recovery , minimizing stiffness , swelling , and pain.5 the authors prescribed rehabilitation , 4 months after the surgery the right wrist showed no compulsory settings in palmar flexion , ulnar deviation was in the range of 5 to 10 degrees , active palmar flexion was 25 degrees , and the dorsal one was 15 degrees . physiotherapy used in our case was introduced later than in the cases described in the literature where it was used 3 months post - op.4 because of the extremely rare type of the injury to a child as well as its late diagnosis , it is difficult to assess whether the wrist will return to its full efficiency even though there has been initially good treatment outcome . the authors claim it highly unlikely . according to the literature,2 early diagnosis and treatment are essential to maintain the range of motion and to prevent a long - term dysfunction . multiple follow - up visits after surgery are critical as perilunate dislocations may have late complications such as instabilities , degenerative joint disease , and avascular necrosis.2 none of the reported cases in the literature2
4
5
7
8 required reoperation . in the case described by the authors of the article , no secondary surgical procedures have been made . some authors point out that a delay in diagnosis results in the necessity of resection of the lunate.5 despite the fact that the diagnosis in our case was delayed because of the constant and repeated damage to the bone , the bone was not damaged in such a way as to prevent a return to the full wrist fitness . this article explores a radiological and surgical investigation of the perilunate wrist dislocation caused by chronic trauma in a 3-year - old child . | a 3-year - old girl was reported to have perilunate dislocation .
the child had suffered , approximately 18 months before , a repetitive wrist injury by means of frequent pulling and bending of the hand by the father who may have physically abused the child for approximately 2 months . at the age of 3 years and 4 months
, we performed the reduction of the perilunate dislocation and the plasty of the dorsal scapholunate ligament using the capsular flap prepared from the extensor retinaculum .
the wrist was immobilized for 6 weeks .
two months after surgery , patient started the physiotherapy for 3 months .
the patient could flex up the wrist to 40 degrees and extend up to 45 degrees ( mobility of the healthy wrist was 60 , 50 degrees ) during the examination 24 months ' post - op .
only the surgical treatment of the perilunate dislocation can lead to obtain the good surgical result . |
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deterioration of diabetic retinopathy during pregnancy is well described in women with pregestational diabetes mellitus [ 13 ] . this progression is influenced by multiple factors including the pregnancy itself , glycemic control before and during pregnancy , and the presence of preexisting retinopathy [ 4 , 5 ] . maternal medical complications including pregnancy - induced hypertension , diabetic nephropathy , and preeclampsia are also associated with progression of retinopathy [ 6 , 7 ] . unfortunately , study sample size has frequently limited the evaluation of additional risk factors and many studies predate the era of modern diabetes care in pregnancy which includes tight glycemic control and blood pressure management [ 1 , 2 , 8 , 9 ] . additionally , there is no data on screening rates within populations or on factors associated with receiving adequate retinal examinations during pregnancy . the atlantic diabetes in pregnancy ( atlantic dip ) initiative was established in 2005 and represents five antenatal centers along the irish atlantic seaboard , covering a population of 500,000 mixed urban and rural dwellers . the group offers women specialist - led , evidence - based care before , during , and after pregnancy and has significantly improved local outcomes in women with diabetes in pregnancy . the aim of this study was to review the frequency of retinal examination during pregnancy in the atlantic dip cohort and examine maternal factors associated with receiving the optimal number of examinations . additionally , we documented the progression of diabetic retinopathy during pregnancy and factors associated with this progression . this study was designed as an observational study of retinopathy status during pregnancy in women with pregestational diabetes . research ethics committee approval was obtained and women were recruited between september 2006 and december 2012 . women with singleton pregnancies who provided informed consent were included . as per previous studies , women were classified as having pregestational diabetes on the following basis : ( 1 ) an established diagnosis of type one or type two diabetes mellitus prior to conception or ( 2 ) glycosylated hemoglobin ( hba1c ) greater than 6.5% in the first trimester . data were collected from study entry until 12 weeks postpartum using an optimized digital database , namely , diamond ( hicom ) . prior to pregnancy , during annual review appointments , all women were advised regarding the need to plan pregnancy and were offered the opportunity to attend a dedicated , prepregnancy service . during pregnancy , education was provided on self - directed glucose monitoring and each woman was advised on glycemic targets . insulin was introduced ( in the setting of type two diabetes mellitus ) or adjusted if home glucose readings were outside the following ranges on more than three consecutive days : fasting glucose 5.0 mmol / l or a 2-hour postprandial reading of 7.0 mmol / l . retinal screening should occur at least twice during pregnancy in separate trimesters and if established retinopathy is present , then retinal examination should take place more frequently . for the purposes of this study , we accepted at least two retinal evaluations in separate trimesters as adequate . during the study period , retinal examination took place in the locality of each antenatal center and results were forwarded to the respective center . at each visit the pupils were then dilated with tropicamide 1% and ophthalmological examinations were performed using a two - field photography system . these images were reviewed by an accredited retinal grader . if photo screening was not performed or if the images were abnormal , an experienced ophthalmologist performed an eye examination . progression was defined as at least one stage of deterioration of diabetic retinopathy and/or development of diabetic macular edema on at least one eye . grading standards as outlined by the national screening committee in the united kingdom were followed ( table 1 ) . data were analyzed using statistical package for the social sciences ( spss ) version 20.0 ( ibm ) . hypothesis testing was performed on the data of equal variance and normal distribution using an unpaired student 's t - test . binary logistic regression was utilized to assess the association of multiple covariates with receipt of adequate retinal evaluations and the progression of retinopathy . data are expressed as means standard deviation ( sd ) or the mean , adjusted odds ratios ( aors ) , and 95% confidence intervals ( ci ) . statistical significance was accepted when the 95% ci did not contain one ( regression analyses / ratios ) or zero ( multiple group comparisons / means ) . the significance level ( ) we identified 307 women with pregestational diabetes who delivered after 22 gestational weeks from the atlantic dip database . this cohort comprised 208 ( 67.8% ) with type one diabetes and 99 ( 32.2% ) with type two diabetes . the majority of women were of caucasian ethnicity ( n = 278 , 90.6% ) . 217 ( 70.7% ) of 307 women had retinal evaluation at least once during pregnancy and 185 ( 60.3% ) had an adequate number of examinations . of the 32 women who had screening on one occasion only , 29 ( 90.6% ) had no retinopathy and 3 ( 9.4% ) had background retinopathy . table 2 outlines the characteristics of those who received an adequate number of examinations versus those who did not . those who received an adequate number of retinal evaluations were older ( 32.9 5.3 versus 31.5 5.4 years , p = 0.02 ) and a higher proportion were of caucasian ethnicity ( 94.1% versus 85.2% , p = 0.01 ) . there was no difference in gravida , parity , first or third trimester hba1c , or smoking status between the two groups . among women with type one diabetes , 64.4% received an adequate number of examinations , compared with 51.5% of women with type two diabetes . a higher proportion of women who received appropriate screening had attended prepregnancy care ( 58.4% versus 17.2% , p < 0.001 ) and taken folic acid preconceptually ( 69.7% versus 54.1% , p = 0.001 ) . a higher proportion of women received adequate examinations in the years 2009 to 2012 compared with the years 2006 to 2008 ( 74.1% versus 25.9% ) . a logistic regression model was completed to further examine maternal factors associated with receiving an adequate frequency of ophthalmological evaluation in pregnancy . the results are outlined in table 3 and they identified attendance at prepregnancy care as the only maternal factor significantly associated with receiving appropriate screening ( odds ratio 6.23 ; ci 3.3911.46 ( p < 0.001 ) ) . on evaluation of those patients who received two or more retinal evaluations ( . a higher proportion of women with type one diabetes progressed compared with type two diabetes ( 31.3% versus 11.7% , p = 0.001 ) . those women who demonstrated progression had , on average , a longer duration of diabetes ( 14.43 8.42 versus 9.79 8.36 years , although there was a higher frequency of preeclampsia in the group that experienced progression , this was not significant ( 14.6% versus 12.4% , p = 0.80 ) . however , those who did progress had a significantly higher systolic blood pressure at their initial antenatal visit ( 128.6 18.0 versus 122.1 13.0 mmhg , p = 0.03 ) . additionally , first trimester hba1c was higher ( 7.67 1.62 versus 7.03 1.39% , p = 0.01 ) and the drop in hba1c between first and third trimesters was greater ( 1.38 1.33 versus 0.74 0.90% , p = 0.004 ) among those women who had disease progression . there was no significant difference in rates of excessive gestational weight gain , smoking status , or body mass index between the two groups . logistic regression analysis revealed that increasing systolic blood pressure at booking ( or 1.03 , ci 1.011.06 , p = 0.02 ) and greater drop in hba1c between first and third trimesters of pregnancy ( or 2.05 , ci 1.093.87 , p = 0.03 ) significantly increased the odds of retinopathy progression . duration of diabetes , diabetes type , and first trimester hba1c were not associated with increased odds of progression and these results are outlined in table 5 . baseline retinal findings are outlined in table 4 . of those women who developed progression ( n = 48 ) , 32 ( 66.7% ) had no retinopathy at baseline . a total of 26 ( 54.2% ) women progressed from no retinopathy to level 1 ( background ) retinopathy only . a further 7 ( 14.6% ) progressed from level 1 to level 2 ( preproliferative ) retinopathy and 6 women ( 12.5% ) progressed to level 3 ( proliferative ) retinopathy and required laser therapy . two women in the latter group had also received prepregnancy laser therapy . finally , 6 ( 12.5% ) women developed mild maculopathy and three ( 6.3% ) experienced a worsening of preexisting maculopathy and required laser therapy . among the group with maculopathy development ( in an unselected population of women with pregestational diabetes , we demonstrate that 60.3% had an adequate number of ophthalmological examinations during pregnancy . attendance at prepregnancy care was strongly associated with receiving adequate retinal evaluation in the subsequent pregnancy . despite intensive glycemic control and antihypertensive therapy as required , progression of retinopathy was observed in 14% of the total group and in 26% of those who had more than one retinal examination during pregnancy . the american diabetes association advises an eye examination in the first trimester with close follow - up throughout pregnancy . the national institute for health and clinical excellence ( nice ) in the united kingdom recommends retinal assessment following the first antenatal clinic appointment and again at 28 weeks if the first assessment is normal . if any diabetic retinopathy is present , an additional retinal assessment should be performed at 1620 weeks . for the purposes of this study , we accepted a minimum of two retinal evaluations in separate trimesters in accordance with local guidelines . unfortunately , despite the existence of these guidelines , there was no retinal evaluation in 35% of cases and just one evaluation in a further 9% . while many studies in the area of diabetic retinopathy in pregnancy include selected patients with complete ophthalmological evaluations only [ 1 , 15 ] , a study of women with type two diabetes reported that only 73% had the available ophthalmological examinations . in relation to this current study , there is not an automatic recall system for retinal evaluation in our antenatal centers and each woman must be referred individually . it is the opinion of the authors that an automatic , standardized system of follow - up as demonstrated by hampshire et al . the majority of women who had just one eye examination were retinopathy - free and the lack of follow - up in later pregnancy may reflect a perceived minimal risk on behalf of the patient and health care provider with more focus being placed on those patients with established retinopathy . another interesting observation presented herein is the association between attendance at a prepregnancy care program and adequate retinal assessment in the subsequent pregnancy . it is reasonable to assume that the educational component of the program informs women of recommended intervals for ophthalmology review during pregnancy and these women are more likely to ensure they receive and attend appointments . the increased attendance at prepregnancy care undoubtedly explains the higher proportion of women taking prepregnancy folic acid in the group who received adequate eye assessments during pregnancy . unfortunately , we do not have information regarding the exact timing of prepregnancy care and levels of metabolic control and blood pressure at the time of attendance . although there was no significant difference in progression of retinopathy between patients attending prepregnancy care and those who did not , improved metabolic control just before pregnancy may have influenced retinopathy progression during the subsequent pregnancy . finally , the higher rates of adequate screening in the latter four years of the study reflect improvements in clinical care delivery as the atlantic dip program became established . this study highlights the ongoing risk of retinopathy progression during pregnancy particularly among women with type one diabetes . rasmussen et al . evaluated 80 patients with type two diabetes and observed progression in 14% . these studies did not find an association between glucose control and progression of retinopathy but this may be due to very tight prepregnancy glycemic control or a type - two error due to a lesser number of included subjects . however , our observations reinforce other published works that noted both significant and nonsignificant trends towards progression of retinopathy in the setting of a greater drop in hba1c during the pregnancy [ 5 , 15 , 16 ] . while the third trimester hba1c was similar between groups that did and did not progress in our study , the first trimester value was on average 0.5% higher in the group that developed retinopathy progression . the importance of prepregnancy glycemic optimization should be highlighted as it is associated with a tendency toward less progression of retinopathy compared with waiting until pregnancy is confirmed in type one diabetes [ 2 , 15 ] . in the setting of an unplanned pregnancy with poor glycemic control , the authors believe that glycemic control should be prioritized and appropriately optimized as the long - term consequences of poor glycemic control during the pregnancy appear to outweigh those of retinopathy progression [ 10 , 15 , 17 , 18 ] . this issue has also received attention in studies involving a more general diabetes population . for example , although early worsening of diabetic retinopathy was noted in a higher proportion of those assigned to intensive treatment in the diabetes control and complications trial ( dcct ) , the long - term benefits of intensive insulin treatment greatly outweighed the risks of this early worsening . the association between retinopathy progression and systolic blood pressure at booking is not unexpected as hypertensive disorders of pregnancy and indeed higher systolic blood pressure are factors known to negatively influence retinopathy [ 7 , 20 , 21 ] . in relation to the complication severity , two - thirds of women who experienced retinopathy progression developed background retinopathy only and no women with a normal retinal examination during trimester one developed sight - threatening disease or required laser therapy . these findings are reassuring , particularly as hellstedt et al . demonstrated a regression of mild retinopathy postpartum in a cohort of women with type one diabetes . a limitation of the study is that we do not have postpartum evaluations to determine the longer - term progression of retinopathy . however , arun and taylor studied women with type one diabetes for 5 years after delivery and concluded that pregnancy is not associated with postpartum worsening of retinopathy . additionally , in the pittsburgh edc pregnancy study , it was observed that the overall prevalence of retinopathy in women with prior pregnancy was similar to that of matched nulliparous women . overall , this was a robust , nested cohort analysis performed retrospectively with data managed prospectively within the atlantic dip database . although the observational study design has inherent limitations including the potential influence of unmeasured covariates such as additional medications , we have used robust statistical methods to evaluate rates of retinopathy progression and employed regression analysis to demonstrate factors associated with disease progression and adequate retinal evaluation during pregnancy . the management of the patients included in this study is a reflection of real - life clinical practice and involves patients with both type one and type two diabetes with varying durations of disease . in summary , with the establishment of a structured antenatal care program , more women are receiving an adequate number of retinal examinations during pregnancy . a significant proportion of women continue to experience deterioration in retinopathy during pregnancy and this validates the need for close follow - up . finally , the importance of prepregnancy care to fully inform women of the need for more frequent retinal assessments during pregnancy and allow preconceptual optimization of glycemic control and blood pressure should be emphasized . these results will assist the health care professional design and provid high quality antenatal care for women with pregestational diabetes mellitus . | the aim of this observational study was to evaluate screening and progression of diabetic retinopathy during pregnancy in women with pregestational diabetes attending five antenatal centres along the irish atlantic seaboard .
an adequate frequency of screening was defined as at least two retinal evaluations in separate trimesters .
progression was defined as at least one stage of deterioration of diabetic retinopathy and/or development of diabetic macular edema on at least one eye .
women with pregestational diabetes who delivered after 22 gestational weeks ( n = 307 ) were included . in total , 185 ( 60.3% ) had an adequate number of retinal examinations .
attendance at prepregnancy care was associated with receiving adequate screening ( odds ratio 6.23 ; ci 3.3911.46 ( p < 0.001 ) ) . among those who received adequate evaluations ( n = 185 ) , 48 ( 25.9% ) had retinopathy progression .
increasing booking systolic blood pressure ( or 1.03 , ci 1.011.06 , p = 0.02 ) and greater drop in hba1c between first and third trimesters of pregnancy ( or 2.05 , ci 1.093.87 , p = 0.03 ) significantly increased the odds of progression .
a significant proportion of women continue to demonstrate retinopathy progression during pregnancy .
this study highlights the role of prepregnancy care and the importance of close monitoring during pregnancy and identifies those patients at the highest risk for retinopathy progression . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
brucei bloodstream form
parasites strain 427 , variant mitat1.2 ( also known as variant 221 ) , that
express t7 polymerase and tetracycline repressor protein under g418 selection
were cultured in hmi-9 medium
( 33 ) up to a density of 2
10 cells / ml at 37 c with 5% co2 . cloning and sequencing of tbuap the tbuap open
reading frame identified in the t. brucei genome data base was
amplified by pcr from genomic dna with pfu polymerase using forward and
reverse primers containing bamhi sites ( underlined ) of
5-cgcggatccaatgagtgacagggacgtgtg-3 and
5-cgcggatccttacatgttcgatgattcgg-3 , respectively . the
products of six separate pcrs were cloned into pcr - bluntii - topo , and a
representative clone from each pcr was sequenced . the primer
5-cgcagcggttcttcgaggagaattcctac-3 was also used to obtain
complete sequence coverage of the orf . reverse transcription - pcr rna was extracted using the rneasy
extraction kits with on - column dnase digestion ( rnase - free dnase ; qiagen ) . rna
samples ( 50 ng ) were treated with ominiscript reverse transcriptase ( qiagen )
to generate cdna . the cdnas were then amplified by pcr using taq polymerase
and tbuap orf primers ( forward ,
5-aatgagtgacagggacgtgtg-3 ; reverse ,
5-ttacatgttcgatgattcgg-3 ) and dpms ( dol - p - man
synthetase ) primers ( forward , 5-aatggatgcggaccttcagcacccac-3 ;
reverse , 5-tagaaccgtgagcgcggtgccatac-3 ) to show equal rna
addition . a dna probe was made using the
tbuap orf and the random primer labeling kit ( ge healthcare ) . the
probe was then detected using the cdp - star detection kit ( ge
healthcare ) . tbuap protein expression and purification the tbuap
orf was cloned into the bamhi site of the expression vector pet15b ( novagen )
to create pet15b - tbuap , which incorporated a his6 tag when
expressed . cells
were harvested and washed in 50 mm tris - hcl , ph 8.0 , 0.3
m nacl , 1 mg / ml lysozyme , and roche complete protease inhibitor
mixture tablets ( roche applied science ) and then lysed in a french press . the
lysate was cleared by centrifugation ( 40,000 g , 60 min , 4
c ) , passed through a 0.2-m filter , and loaded onto a precharged
ni hitrap chelating hp column ( ge healthcare ) . tbuap - his6 was eluted with 50 mm tris - hcl ( ph 8.0 ) , 0.3
m nacl with 0.1 - 0.2 m imidazole . the protein was then
dialyzed overnight using a slide - a - lyser dialysis cassette ( pierce ) with
10 kda molecular mass cut - off at 4 c in 25 mm
na2hpo4-nah2po4 buffer , ph 8.0 . the sample was then filtered as above before being loaded onto a hitrap
q hp - sepharose column ( amersham biosciences ) , preequilibrated with 25
mm na2hpo4-nah2po4 , ph
8.0 . the column was washed with 25 mm
na2hpo4-nah2po4 , ph 8.0 , followed
by a gradient to 25 mm
na2hpo4-nah2po4 , ph 8.0 , 0.5
m nacl over 30 min . fractions ( 3 ml ) were collected and checked by
sds - page . tbuap - his6-containing fractions were pooled and
concentrated , and the buffer was exchanged to 50 mm tris - hcl , ph
7.5 , 10 mm mgcl2 , and 20% glycerol using a vivaspin
concentrator ( vivascience ) 10 kda molecular mass cut off at 4 c . the
protein was then stored at -80% . to obtain a prescission protease cleavable his6-tagged tbuap
protein , the tbuap open reading frame was amplified by pcr from the
aforementioned pet15b - tbuap plasmid using the forward primer ,
5-ctccatgggcagcagccatcatcatcatcatcacagcagcggcctggaagttctgttccaggggcccggatccatgagtgacagggacgtgtgcattcag-3 ,
containing an ncoi restriction site ( underlined ) , the coding sequences for
mgsshhhhhhssg ( italic type ) , and a prescission protease cleavage site of
levlfqgp ( boldface type ) , followed by a bamhi restriction site ( underlined )
and a tbuap gene - specific sequence ( uppercase ) and the reverse
primer ,
5-gctcgagatctggatccttacatgttcgatgattcggagaccacc-3 ,
containing restriction sites for bglii and bamhi ( italic type ) and a
tbuap gene - specific sequence ( uppercase ) . the pcr product was cloned
into the pgem - t easy pcr cloning vector ( promega ) and subsequently digested
with ncoi and bglii and inserted between the ncoi and bamhi sites of the
pet15b protein expression vector ( novagen ) . the resulting construct ,
pet15b - his6-pp - tbuap , that encodes the full tbuap coding
sequence preceded by the sequence mgsshhhhhhssglevlfqgpgs ( where prescission
protease cleaves between the q and the g ) was expressed in e. coli
and purified on an ni hitrap chelating hp column , as
described above . the sample was then digested with 2 mg of
gst - prescission protease ( a kind gift of bill hunter ; university of dundee ) in
50 mm tris , ph 8.0 , 100 mm nacl , 10 mm edta ,
and 1 mm dithiothreitol at room temperature for 4 - 16 h at 4 c . the sample was dialyzed for 2 h using a slide - a - lyser dialysis cassette
( 10 kda molecular mass cut - off ) at 4 c in 2 liters of 50 mm
tris - hcl , ph 8.0 , and 50 mm nacl to remove the edta , and then the
sample was passed though a 0.2-m syringe filter . the sample was passed
through a gstrap hp column ( ge healthcare ) connected to an
ni hitrap chelating hp column . the flow - through was then
dialyzed overnight using a slide - a - lyser dialysis cassette at 4 c in
1 liter of 25 mm
na2hpo4-nah2po4 , ph 8.0 , with two
changes of buffer . the sample was then passed though a 0.2-m syringe
filter and further purified using anion exchange chromatography on a
hitrap q hp - sepharose column ( ge healthcare ) . the hplc
assay used 0.05 g of tbuap - his6 incubated in 100 l of the
hplc assay buffer ( 50 mm tris - hcl , ph 7.5 , 250 m
utp , 10 mm mgcl2 , 1 mm dithiothreitol , 20%
glycerol , 250 m glcnac-1-p ) for 10 min , terminated by boiling
for 5 min . the hplc assay buffer was
altered to study substrate specificity , metal ion dependence , and ph
dependence . for substrate specificity , glcnac-1-p was changed to glucose
1-phosphate , galactose 1-phosphate , or galnac-1-p , all at 250
m . for metal ion dependence , mgcl2 was replaced with
cacl2 , cucl2 , zncl2 , or mncl2 . for
ph dependence , the tris - hcl buffer was replaced with a dual buffer of 50
mm tris , 50 mm sodium acetate with the ph adjusted with
hcl . the tbuap colorimetric assay was performed with 0.05 g of
tbuap - his6 in a 96-well plate format ( nunc ) in 90 l of
50 mm tris - hcl , ph 7.5 , 250 m utp , 250
m glcnac-1-p , 10 mm mgcl2 , 1
mm dithiothreitol , 20% glycerol , 0.04 units / ml pyrophosphatase
( sigma ) . the reaction was left for 10 min and terminated by the addition of
100 l of the color reagent ( 0.2% ammonium molybdate , 0.5% triton x-100 ,
0.7 n hcl , 0.03% malachite green ) . absorbance at 655 nm was
measured after 5 min using a spectramax 340 pc ( molecular devices ) . construction of a tbuap conditional null mutant the gene
replacement cassettes were generated by pcr amplification of 500 bp of utr
immediately flanking the 5- and 3-ends of the tbuap orf
with taq polymerase using the forward and reverse primers
5-aaggaaaaaagcggccgcagatgcgtgcacaacaaaaa-3 and
5-gtttaaacttacggaccgtcaagctttatctataacacacggagcc-3
and 5-gacggtccgtaagtttaaacggatccgtggacgttgcagcgcccgg-3
and 5-aaggaaaaaagcggccgccaccacagttcaccatccag-3 , respectively . the two
pcr products were then used in a separate pcr to produce a construct
containing the 5-utr linked to the 3-utr by a short hindiii ,
pmei , and bamhi cloning site ( italic type ) . the resulting pcr product was then
ligated into pgem-5zf(+ ) vector ( promega ) using the noti site ( uppercase ) . antibiotic resistance markers were cloned into the hindiii / bamhi restriction
sites between the two utrs to produce two constructs , one containing the
pac ( puromycin acetyltransferase ) drug resistance gene and one
containing the hyg ( hygromycin phosphotransferase ) drug resistance
gene . to generate the tetracycline - inducible ectopic copy of the
tbuap orf , the nde1 site in the orf was silenced using the primers
5-aagcttgggatagcatacgtgcagattggaa-3 and
5-attccaatctgcacgtatgctatcccaagct-3. the primers
5-catatgatgagtgacagggacgtgtg-3 and
5-ttaattaattacatgttcgatgattcgg-3 were then used to
pcr - amplify the orf , which was cloned into the vector plew100 using the ndei
and paci sites ( italic type )
( 33 ) . these constructs were purified using the qiagen maxiprep kit , digested with
noti to linearize , precipitated , washed twice with 70% ethanol , and
redissolved in sterile water . the linearized dna was electroporated into
t. brucei bloodstream cells ( strain 427 , variant 221 ) that were
stably transformed to express t7 rna polymerase and the tetracycline repressor
protein under g418 selection . cell culture , transformation , and selection were
carried out as previously described
( 33 ) . mouse infection studies the tbuap conditional null
mutant cells were subcultured and grown without selection drugs ( hygromycin ,
puromycin , phleomycin , and g418 ) for 24 h with and without 1 g / ml
tetracycline . the parasites were then introduced into groups of five mice
( dosed with and without doxycycline , respectively ) by intraperitoneal
injection of 3 10 parasites in 0.2 ml of hmi-9 medium . the
plus doxycycline group of animals were dosed with doxycycline in the drinking
water ( 0.2 mg / ml in a 5% sucrose solution ) for 1 week prior to infection and
until the experiment was terminated . infections were assessed by tail
bleeding , diluting the blood 1:200 in hmi-9 medium and counting on a neubauer
hemocytometer . tbuap localization two balb / c adult mice were used to raise
polyclonal antibodies against his6-tagged tbuap protein with
freund 's complete adjuvant . each mouse received two further immunizations with
freund 's incomplete adjuvant over 2 months . antibodies were then
affinity - purified on cnbr - sepharose - immobilized tbuap that had had its
his6 tag removed with prescission protease . wild type and tbuap conditional null mutant bloodstream form
t. brucei cells were grown in hmi-9 medium ( with or without 1
g / ml tetracycline for the conditional null mutant ) to a density of 1
10 cells / ml over 48 h , harvested by centrifugation , and
resuspended in trypanosome dilution buffer ( 0.1 m
na2hpo4 , 0.01 m nah2po4 , 0.025
m kcl , 0.4 m nacl , 5 mm mgso4 , 0.1
m glucose adjusted to ph 7.45 with hcl ) to a density of 4
10 cells / ml . aliquots ( 15 l ) were added to 13-mm coverslips
( vwr ) , left at room temperature for 15 min , fixed in 1 ml of 4%
paraformaldehyde in phosphate - buffered saline ( pbs ) for 1 h followed by three
5 min washes in 2 ml of pbs . cells were permeabilized with 0.05% triton x-100
in pbs containing 0.5 mg / ml bovine serum albumin for 10 min at room
temperature . samples were then blocked in 2 ml of pbs , 0.5% bovine serum
albumin , for 1 h at room temperature . the coverslips were incubated with mouse
anti - tbuap ( 1:5,000 dilution ) and rabbit anti - glyceraldehyde-3-phosphate
dehydrogenase ( gapdh ) antiserum ( 1:10,000 ; a kind gift of paul michels ,
catholic university of louvain ) in pbs , 0.5% bovine serum albumin . samples
were then washed , as above , in pbs , 0.5% bovine serum albumin and incubated
with 50 l of alexa 594-conjugated anti - mouse igg and alexa 488-conjugated
anti - rabbit igg ( containing 4,6-diamidino-2-phenylindole in the case of
the wild type cells ) for 1 h. coverslips were mounted on glass slides ( vwr ) ,
sealed with hydromount containing 2.5% 1,4-diazabicyclo[2.2.2]octane and left
to dry in the dark for 30 min . microscopy was performed on a zeiss axiovert
200 m fluorescence microscope for wild type cells and on a zeiss lsm 510 meta
confocal microscope for the tbuap conditional null mutant cells . recombinant tbuap - his6
was incubated with utp and different sugar-1-phosphate substrates , as
indicated , and the products were analyzed by hplc . a sugar nucleotide product
( udp - glcnac ) was observed using glcnac-1-p ( a ) but not without
glcnac-1-p ( b ) or with galnac-1-p , glc 1-phosphate , or gal
1-phosphate ( c - e , respectively ) . recombinant tbuap - his6
was incubated with utp and different sugar-1-phosphate substrates , as
indicated , and the products were analyzed by hplc . a sugar nucleotide product
( udp - glcnac ) was observed using glcnac-1-p ( a ) but not without
glcnac-1-p ( b ) or with galnac-1-p , glc 1-phosphate , or gal
1-phosphate ( c - e , respectively ) . briefly , cells were
pelleted by centrifugation , washed in ice - cold pbs , and lysed in 70% ethanol
in the presence of 10 pmol of gdp - glucose internal standard . sugar nucleotides
were extracted using envicarb columns
( 35 ) and analyzed using
multiple reaction - monitoring liquid chromatography - tandem mass spectrometry
( 19 ) . brucei cells washed
with trypanosome dilution buffer and hypotonically lysed in 300 l of water
containing 0.1 mm 1-chloro-3-tosylamido-7-amino-2-heptone ( tlck )
and 1 g / ml leupeptin . cell ghosts were harvested by centrifugation ( 13,000
g for 10 min ) , and the pellet was resuspended in sds - sample
buffer containing 8 m urea . the lysed extracts were then subjected
to electrophoresis under reducing conditions , with 1.5 10
or 5 10 cell equivalents / lane , on a nupage 4 - 12%
bistris gradient ( invitrogen ) using mops sds running buffer . proteins were
then transferred to a nitrocellulose membrane under normal western blotting
conditions . membranes were stained with ponceau s solution to demonstrate
equal loading , blocked with 0.25% bovine serum albumin , 0.05% igepal detergent
( sigma ) , 0.15 m nacl in 50 mm tris - hcl , ph 7.4 , and then
incubated with 0.33 g / ml biotinylated tomato lectin ( vector laboratories ) ,
with or without 3 mg / ml chitin hydrolysate ( vector laboratories ) , and then
with 1:10,000 diluted extraavidin - horseradish peroxidase conjugate ( sigma ) . all membranes were then developed by chemiluminescent detection ( ecl - plus ; ge
healthcare ) . to probe for p67 , t. brucei was lysed with sds - sample buffer and
loaded onto a 10% sds - polyacrylamide gel before being transferred to
nitrocellulose membrane . the membrane was then probed with mab139 ( a kind gift
from jay bangs , madison ) at a dilution of 1:2,000 as the primary antibody and
then with 1:10,000 diluted anti - mouse igg conjugated with horseradish
peroxidase , followed by ecl reagent as described above . purification and endoglycosidase digestion of soluble form variant
surface glycoprotein ( svsg)the vsg coat of trypanosomes can be
conveniently released in a soluble form through osmotic cell lysis at 37
c . this causes cleavage of the dimyristoylglycerol component of the gpi
membrane anchors by the action of an endogenous gpi - specific phospholipase c
( 30 ) . t. brucei
cultures ( 100 ml ) were washed in trypanosome dilution buffer and resuspended
in 300 l of lysis buffer ( 10 mm
nah2po4-na2hpo4 , ph 8.0 , 0.1
mm tlck , 1 g / ml leupeptin , and 1 g / ml aprotinin ) and
incubated at 37 c for 10 min . this was then cooled on ice for 2 min and
centrifuged for 5 min at 16,000 g , and the supernatant was
applied to 200 l of de52 ( whatman ) preequilibrated in 10 mm
nah2po4-na2hpo4 , ph 8.0 , buffer
and eluted with 4 200 l of fresh lysis buffer . the eluates were
pooled and concentrated to 100 l using a ym-10 spin concentrator
( microcon ) . the majority of the buffer salts were removed by diafiltration
with three additions of 0.5 ml of water . for each enzyme digestion , svsg was dissolved at 0.2 g/l in 0.5%
sds , 0.1 m dithiothreitol and boiled for 10 min . for
endoglycosidase h ( endo h ) digests , 5 l of the svsg was added to 20 l
of 50 mm sodium citrate , ph 5.5 , 10 mm
phenylmethylsulfonyl fluoride , and 0.025 units of endo h. for
n - glycosidase f ( pngase f ) digests , 5 l of svsg was added to 50
mm sodium phosphate , ph 7.5 , 0.1% triton x-100 , 10 mm
phenylmethylsulfonyl fluoride , and 0.025 units of pngase f. the digests were
then left overnight at 37 c . electrospray mass spectrometry of svsg intact svsg was
diluted to 0.05 g/l in 50% methanol , 1% formic acid and loaded into
micromass type - f nanotips . the svsg was analyzed by positive ion electrospray
tandem mass spectrometry using an applied biosystems q - starxl instrument , and
the masses were calculated using the bayesian protein reconstruction algorithm
( abi analyst software ) . identification , cloning , and expression of tbuap a blastp
search of the t. brucei predicted protein data base with homo
sapiens and saccharomyces cerevisiae uap amino acid sequences ,
accession numbers np_003106 and np _ 010180 , revealed a putative tbuap
gene ( gene number tb11.02.0120 ) . the tbuap open reading frame was
amplified by pcr using pfu polymerase from genomic dna prepared from
t. brucei strain 427 . two different consensus sequences emerged
( accession numbers am909685 and am909686 ) , suggesting that there are two
slightly different tbuap alleles , with eight base pair differences
and one amino acid difference between them ( ser or gly at position 507 ) . southern blot analysis using a tbuap orf probe confirmed that tbuap
is present as a single copy per haploid genome ( supplemental fig . the tbuap amino
acid sequence contains the expected pyrophosphorylase
lx2gxgtx6pk motif , and 13 of
15 residues shown to be involved in substrate binding in the human uap crystal
structures ( 36 ) are conserved
in the tbuap sequence ( supplemental fig . the tbuap orf was cloned into a pet15b expression vector , with a
his6 tag at the n terminus and a modified pet15b vector with a
prescission protease site between the protein and the his6 tag . both were expressed in e. coli and purified as described under
experimental procedures analysis
of the recombinant tbuap - his6 protein by analytical
ultracentrifugation ( at 1 mg / ml ) indicated that the majority ( > 80% ) of
tbuap is monomeric at this concentration ( data not shown ) . enzymatic activity of tbuap the activity and substrate
specificity of tbuap was assessed by incubating recombinant
tbuap - his6 with utp and glcnac-1-p , galnac-1-p , glc 1-phosphate , or
gal 1-phosphate and analyzing the products by hplc . using glcnac-1-p as the
substrate , a single uv - absorbing peak that co - eluted with authentic udp - glcnac
was observed ( fig . no sugar nucleotide product was observed in the
absence of tbuap - his6 ( fig . 1b ) or when galnac-1-p , glc 1-phosphate , or gal
1-phosphate was used as a substrate ( fig .
1 , c - e ) . these data show that glcnac-1-p is the preferred
substrate of tbuap under these conditions . to test the metal ion dependence of the tbuap - his6 activity , the
enzyme was preincubated with 5 mm edta and then incubated with utp ,
glcnac-1-p , and 10 mm mgcl2 , mncl2 ,
cacl2 , cucl2 , zncl2 , or no divalent cation . the products of triplicate experiments were analyzed by hplc , and relative
yields of udp - glcnac were determined . in the presence of edta alone , no
detectable udp - glcnac was formed , suggesting that tbuap is divalent metal
ion - dependent . similar levels of activity were restored with mg
and mn , whereas ca , cu , and
zn failed to restore detectable activity ( supplemental fig . the ph dependence of the activity was studied over the ph range 5.0 - 9.5 ,
using the same hplc - based assay . the enzyme displayed a broad ph optimum
between ph 6.0 and 9.0 ( supplemental fig . 6 ) . based on the aforementioned
experiments , the enzyme was assayed at ph 7.5 in the presence of 10
mm mgcl2 . in order to measure the apparent km values for the two
substrates of tbuap , a discontinuous , colorimetric , the assay relies on pyrophosphatase to convert the ppi
component of the utp + glcnac-1-p udp - glcnac + ppi reaction
to inorganic phosphate , which is subsequently measured using malachite green . the enzyme was assayed with a fixed concentration of utp and varying
concentrations of glcnac-1-p and with a fixed concentration of glcnac-1-p and
varying concentrations of utp ( supplemental figs . 7 and 8) . the apparent
km values for utp and glcnac-1-p are 26 6 and 39
13 m , respectively , with a vmax of
0.4 0.1 nmol / min and a specific activity of 24 6
mol / min / mg . these values are compared with those of other recombinant
eukaryotic uaps
( 36 - 38 )
in table 1 . table 1comparison of recombinant eukaryotic uap propertiesspeciesmolecular massglcnac-1-p kmutp kmspecific activityalternative substratesreference / sourcekda m m molminmg
1 t . brucei ( tbuap )
60.1
39 13
26 6
26 6
none
this study
g. intestinalis ( giuap )
49.6
300 120
38 17
9.2 1.9 , 55.4 11.1
glc 1-phosphate , galnac-1-p
ref . 38
s. cerevisiae ( scuap )
53.5
14
21
17
glc 1-phosphate
ref . 37
h. sapiens ( agx1 )
57.0
5.3
53
69
galnac-1-p
ref . 36
h. sapiens ( agx2 )
58.8
6.0
49
68
galnac-1-p
ref . 36
comparison of recombinant eukaryotic uap properties construction of a tbuap conditional null mutant in order to
test the hypothesis that udp - glcnac biosynthesis is essential for bloodstream
form t. brucei and that there is no alternate route to this
metabolite , a conditional null mutant of the final step of udp - glcnac
biosynthesis was created by the replacement of both endogenous copies of the
tbuap gene and the introduction of an ectopic inducible copy of
tbuap under tetracycline control . a genetically modified strain 427
t. brucei cell line was used to generate the conditional null mutant . this cell line is engineered to constitutively express t7 polymerase and
tetracycline repressor protein under the control of a t7 promoter . this cell line will be
referred to as wild type from hereon . the first endogenous allele of tbuap was replaced
with the pac gene , creating tbuap : pac . the
ectopic copy ( plew100-tbuap ) was then inserted into the rdna region ,
creating tbuap : pac / tbuap . the
second allele of tbuap was then replaced with the hyg gene ,
creating
tbuap : pac / tbuap/tbuap : hyg . genomic dna from the wild type and three mutant cell lines
produced during the creation of the tbuap conditional null mutant was
digested with bglii and nhei and probed with tbuap open reading
frame . genomic dna was from wild type ( lane 1 ) ,
tbuap : pac ( lane 2 ) ,
tbuap : pac / tbuap ( lane
3 ) , and the final
tbuap : pac / tbuap/tbuap : hyg
conditional null mutant ( lane 4 ) . the first endogenous allele of tbuap was replaced
with the pac gene , creating tbuap : pac . the
ectopic copy ( plew100-tbuap ) was then inserted into the rdna region ,
creating tbuap : pac / tbuap . the
second allele of tbuap was then replaced with the hyg gene ,
creating
tbuap : pac / tbuap/tbuap : hyg . genomic dna from the wild type and three mutant cell lines
produced during the creation of the tbuap conditional null mutant was
digested with bglii and nhei and probed with tbuap open reading
frame . genomic dna was from wild type ( lane 1 ) ,
tbuap : pac ( lane 2 ) ,
tbuap : pac / tbuap ( lane
3 ) , and the final
tbuap : pac / tbuap/tbuap : hyg
conditional null mutant ( lane 4 ) . the first tbuap allele was replaced by homologous recombination
following electroporation of the parasites in the presence of linear dna
containing a pac gene flanked by about 500 bp of tbuap
5- and 3-utr . following selection with puromycin , a
tbuap : pac clone was selected and transformed with an
ectopic , tetracycline - inducible , copy of tbuap , introduced into the
ribosomal dna locus under phleomycin selection
( 33 ) . after tetracycline
induction , the second endogenous allele was replaced by a hyg gene to
yield the desired
tbuap : pac / tbuap/tbuap :
hyg clone ( fig . after each round of transformation , genomic dna was
extracted for southern blot analysis using a tbuap orf probe and
genomic dna digested with bglii and nhei . under these conditions , the
endogenous tbuap gene produces a fragment of 5.6 kb , whereas the
ectopic copy produces a fragment of 2.5 kb . the blot
( fig . 2b ) shows the
successful introduction of the ectopic copy and replacement of both endogenous
alleles in the
tbuap : pac / tbua - p/tbuap : hyg
clone used for further studies . this cell line will be referred to from hereon
as the tbuap conditional null mutant . the tbuap gene is essential to bloodstream form t. brucei in vitro and
in vivo triplicate cultures of wild type and tbuap
conditional null mutant cells , under permissive and nonpermissive conditions
( i.e. with and without tetracycline , respectively ) , were inoculated
at 1 10 cells / ml and subcultured every 2 days . the
tbuap conditional null mutant cultures under permissive conditions
had a reduced growth rate and grew to approximately half the cell density of
the wild type but were otherwise healthy
( fig . 3 , a and
b ) . under nonpermissive conditions the cells grew for 2
days but , upon subculturing , failed to grow for more than 5 days
( fig . the
eventual resumption of growth is typical of bloodstream form t.
brucei conditional null mutants for essential genes
( 6 ,
14 ,
18 ,
39 - 42 ) ,
whereby the essential gene becomes constitutively active through loss of
tetracycline control due to deletion of the tetracycline repressor protein
gene ( 14 ) . analysis by reverse
transcription - pcr confirmed that tbuap mrna was undetectable by 12 h
of tetracycline removal but that the cells that resumed growth , 10 days after
tetracycline removal , were reexpressing tbuap mrna
( fig . 3d ) . figure 3.tbuap expression is essential for the growth of bloodstream
form t. brucei in culture . b , growth curves for
tbuap conditional null mutant cells , subcultured every 2 days , grown
under permissive ( plus tetracycline ) conditions . c , growth curves for
tbuap conditional null mutant cells grown under nonpermissive ( minus
tetracycline ) conditions . d , ethidium bromide - stained agarose gel of
reverse transcription - pcr products from rna extracted from tbuap
conditional null mutant cells after 0 , 2 , 6 , 12 , and 48 h and 10 days without
tetracycline , as indicated . the upper panel shows reverse
transcription - pcr products using tbuap primers , the middle
panel is a control without reverse transcriptase , and the lower
panel is a control using dol - p - man synthetase ( dpms ) primers to
show equal rna input . tbuap expression is essential for the growth of bloodstream
form t. brucei in culture . b , growth curves for
tbuap conditional null mutant cells , subcultured every 2 days , grown
under permissive ( plus tetracycline ) conditions . c , growth curves for
tbuap conditional null mutant cells grown under nonpermissive ( minus
tetracycline ) conditions . d , ethidium bromide - stained agarose gel of
reverse transcription - pcr products from rna extracted from tbuap
conditional null mutant cells after 0 , 2 , 6 , 12 , and 48 h and 10 days without
tetracycline , as indicated . the upper panel shows reverse
transcription - pcr products using tbuap primers , the middle
panel is a control without reverse transcriptase , and the lower
panel is a control using dol - p - man synthetase ( dpms ) primers to
show equal rna input . the tbuap conditional null mutant cells were subcultured and grown
for 24 h with and without tetracycline and then introduced into groups of five
mice that were dosed with or without doxycycline in the drinking water . the
animals dosed with the drug showed high blood parasitemias ( > 3
10/ml ) within 4 days and were sacrificed , whereas the
doxycycline - free animals did not show any signs of infection for up to 14
days , when the experiment was terminated . these data show that the
tbuap gene is essential to bloodstream form t. brucei both
in vitro and in vivo . subcellular localization of tbuap to obtain antibodies ,
tbuap - his6 was used to inoculate mice and the resulting anti - serum
was affinity - purified on a column of recombinant prescission protease - treated
( tag - free ) tbuap coupled to cnbr - sepharose beads . the mouse anti - tbuap
affinity - purified antibody was used together with rabbit anti - gapdh antibody
as a glycosomal marker . the secondary antibodies were anti - mouse alexa 594
( red ) and anti - rabbit alexa 488 ( green ) . the fluorescence
micrographs of wild type cells show that the anti - gapdh co - localized with the
anti - tbuap , indicating that tbuap is located in glycosome microbodies in
bloodstream form t. brucei ( fig .
4 , a - c ) . a similar experiment was performed with the
tbuap conditional null mutant grown with and without tetracycline for
48 h. the results show that the tbuap signal co - localizes with the glyosomal
gapdh marker ( as before ) ( fig . 4 ,
d - f ) but that the intensity of the tbuap signal is
greatly reduced after 48 h without tretracycline
( fig . 4 , g - i ) ,
demonstrating the strict specificity of the antibody for tbuap in
immunofluorescence microscopy . sugar nucleotide levels in the tbuap conditional null
mutant to analyze the effect of the selective removal of
tbuap gene expression on parasite udp - glcnac levels , sugar
nucleotides were extracted from the tbuap conditional null mutant
under permissive and nonpermissive conditions , chromatographed as described in
( 35 ) , and quantitated by the
method described in ref . briefly , sugar nucleotides were extracted from t. brucei , separated
by reverse phase hplc and quantitated by multiple reaction monitoring tandem
mass spectrometry using an internal standard ( gdp - glucose ) , a sugar nucleotide
that is not found in trypanosomes . the multiple reaction monitoring approach
exploits characteristic transitions between precursor and product ions to
identify specific metabolites in complex mixtures . for example udp - glcnac
gives rise to an [ m - h ] precursor ion at m / z 606
that fragments to produce a major product ion of
[ udp - h2o ] at m / z 385 . thus , a
chromatogram of the mass transition 606 385 is highly selective for
udp - glcnac . similarly , gdp - man and gdp - glc can be monitored using the
[ gdp - hex ] to [ gdp - h2 o ] transition of
m / z 604 424 . 5 ) illustrate the
dramatic reduction in udp - glcnac levels , relative to the gdp - glc internal
standard , in the tbuap conditional null mutant after 48 h in the
absence of tetracycline . these data and the effects on other sugar nucleotides
are summarized in ( table 2 ) . the levels of udp - glc , udp - gal , and gdp - man in the tbuap conditional
null cell line under permissive conditions agree reasonably well with the wild
type levels determined previously
( 19 ) . however , even under
permissive conditions , the level of udp - glcnac is significantly lower in the
conditional null mutant ( 16 pmol/1 10 cells ) than in the
wild type ( 80 pmol/1 10 cells ) . the reduced level may be
because tbuap expression is no longer under the control of its
endogenous promoter but rather under the control of the procyclin promoter in
the plew100-tbuap ectopic copy . thus , the lower growth rate of the
mutant under permissive conditions may be a result of the reduced level of
udp - glcnac . under nonpermissive conditions , the cells stopped dividing between
48 and 60 h and died , by cell lysis , by around 72 h. at 48 h , the level of
udp - glcnac was 2.9 pmol/1 10 , < 5% of wild type
levels . table 2sugar nucleotide levels in the tbuap conditional null mutant
under permissive and nonpermissive conditionssugar nucleotidewild
typeatbuap cko ( 0
h)btbuap cko ( 48
h)bpmol/10 cellspmol/10 cellspmol/10 cells udp - glcnac
80 20
16
2.9
gdp - man
5.6 3.9
9.5
19
udp - gal
55 3
44
35
udp - glc
123 7
141
70
asugar nucleotide levels for wild type t. brucei taken from ref . sugar nucleotide levels in the tbuap conditional null mutant
under permissive and nonpermissive conditions sugar nucleotide levels for wild type t. brucei taken from ref .
19 . wild type ( a - c ) and
tbuap conditional null mutant cells grown with tetracycline
( d - f ) and without tetracycline for 48 h ( g - i ) were stained
with rabbit anti - gapdh ( a glycosomal marker ) and alexa 488 anti - rabbit
( green channel ; a , d , and g ) and affinity - purified
mouse anti - tbuap and alexa 594 anti - mouse ( red channel ; b ,
e , and h ) . merged images are shown in c , f ,
and i. the merged image in i also includes a
4,6-diamidino-2-phenylindole stain ( blue ) for dna . no
significant signals were obtained when primary antibodies were omitted ( data
not shown ) . wild type ( a - c ) and
tbuap conditional null mutant cells grown with tetracycline
( d - f ) and without tetracycline for 48 h ( g - i ) were stained
with rabbit anti - gapdh ( a glycosomal marker ) and alexa 488 anti - rabbit
( green channel ; a , d , and g ) and affinity - purified
mouse anti - tbuap and alexa 594 anti - mouse ( red channel ; b ,
e , and h ) . merged images are shown in c , f ,
and i. the merged image in i also includes a
4,6-diamidino-2-phenylindole stain ( blue ) for dna . no
significant signals were obtained when primary antibodies were omitted ( data
not shown ) . changes in the levels of other sugar nucleotides in the tbuap
conditional null mutant under nonpermissive conditions were less profound
( table 2 ) . however , when the
level of udp - glcnac falls , there is an accumulation of gdp - man . since the
consumption of gdp - man for protein n - glycosylation and gpi anchor
synthesis depends on the availability of udp - glcnac , an accumulation of
gdp - mannose is not unexpected . the reason for the reduction in udp - glc and
udp - gal levels , to about 60% of wild type , is less clear . however , a
comparable phenomenon was seen in the tbgale ( udp - glc
4-epimerase ) conditional null mutant , where rapid loss of udp - gal under
nonpermissive conditions was followed by a reduction in udp - glc and udp - glcnac
levels ( 16 ) . effects of udp - glcnac starvation on protein
glycosylation ricin , which binds nonreducing terminal galactose
residues , has been shown to bind to the flagellar pocket of t. brucei
( 43 ) and to the
endosomal / lysosomal system of the parasite
( 27 ) . the
poly - n - acetyllactosamine - specific ( gal1 - 4glcnac ) , lectin from
lycopersicon esculentum ( tomato ) has also been shown to bind
exclusively to glycoproteins in the flagellar pocket and endosomal / lysosomal
system in t. brucei
( 26 ) . the ricin and tomato
lectin binding oligosaccharide structures were characterized and were shown to
include a family of unusually large n - linked
poly - n - acetyllactosamine - containing glycans with an average of 54
n - acetyllactosamine repeats / glycan
( 27 ) . the effect of udp - glcnac starvation on these structures was assessed using
western blots of whole cell lysates from wild type and the tbuap
conditional null mutant cell lines probed with tomato lectin . a large smear
was detected in the wild type cell lysate , which decreased progressively from
0 to 48 h in the absence of tetracycline in the tbuap conditional
null mutant ( fig . the decrease in intensity , and a downward shift in apparent molecular weight ,
of the tomato lectin binding high molecular weight glycoproteins indicated a
reduction in total poly - n - acetyllactosamine synthesis as the cellular
levels of udp - glcnac fall . the specificity of the tomato lectin blot for
carbohydrate was confirmed by including chitin hydrolysate , a tomato lectin
inhibitor ( fig . 6b ) .
to distinguish whether this reduction in tomato lectin binding was
specifically caused by udp - glcnac starvation and not just a general phenomenon
in dying cells , the same experiment was performed with a tbgpi12
conditional null mutant ( 6 ) . this is another ( lethal ) glycosylation conditional null mutant but , this time ,
in the gpi biosynthetic pathway . under nonpermissive ( without tetracycline )
conditions , this cell line also ceases cell division and dies , but , in this
case , the cell lysate blots showed relatively little change in tomato lectin
binding ( supplemental fig . 9 ) . to observe the effects of udp - glcnac starvation on a specific glycoprotein , wild type and tbuap conditional null mutant cells , grown in the
presence and absence of tetracycline for 24 and 48 h , were harvested , lysed ,
and analyzed by sds - page and western blotting with the p67-specific monoclonal
antibody mab139 ( supplemental fig . the intensity of the staining was
slightly decreased , compared with wild type , in the tbuap conditional
null mutant under permissive conditions ( 0 h ) , which may reflect the overall
difference in udp - glcnac levels between the mutant and wild type cell line
described earlier . after 48 h in the absence of tetracycline , both the
intensity and the apparent molecular weight of the p67 smear had decreased . figure 5.measurement of sugar nucleotides in the tbuap conditional null
mutant under permissive and nonpermissive conditions . representative
liquid chromatography - tandem mass spectrometry chromatograms of sugar
nucleotides extracted from the tbuap conditional null mutant before
( a ) and after ( b ) withdrawal of tetracycline for 48 h. the
upper chromatogram in each panel shows the peaks
corresponding to gdp - man and the gdp - glc internal standard , and the lower
chromatogram shows the peak corresponding to udp - glcnac . measurement of sugar nucleotides in the tbuap conditional null
mutant under permissive and nonpermissive conditions . representative
liquid chromatography - tandem mass spectrometry chromatograms of sugar
nucleotides extracted from the tbuap conditional null mutant before
( a ) and after ( b ) withdrawal of tetracycline for 48 h. the
upper chromatogram in each panel shows the peaks
corresponding to gdp - man and the gdp - glc internal standard , and the lower
chromatogram shows the peak corresponding to udp - glcnac . to further demonstrate that the poly - n - acetyllactosamine
structures were being affected by udp - glcnac starvation , wild type , and the
tbuap conditional null mutant cells , after 0 , 24 , and 48 h without
tetracycline , were analyzed using fluorescence microscopy
( fig . cells
were fixed onto coverslips and incubated with biotinylated tomato lectin and
fluorescein - conjugated streptavidin . using this approach ,
poly - n - acetyllactosamine structures specific to the flagellar pocket
and endosomal / lysosomal region the
specificity of this reaction was confirmed by including chitin hydrolysate as
a tomato lectin inhibitor ( fig . little difference in tomato lectin staining was seen
in the tbuap conditional null mutant cells after 24 h in the absence
of tetracycline ( fig . 7 , b and
c ) , but after 48 h , almost no signal was seen within the
flagellar pocket or endosomal / lysosomal region
( fig . these
results correlate well with the tomato lectin blot data in
( fig . results using wild type cells and
including 4,6-diamidino-2-phenylindole staining to highlight the
positions of the nuclear and kinetoplast dna , are included
( fig . 7 , f - j ) . to assess the effects of udp - glcnac starvation on gross cellular morphology
and flagellar pocket morphology , including the appearance of the flagellar
pocket lumenal contents , wild type cells and tbuap conditional null
mutant cells , grown in the absence of tetracycline for 48 h , were subjected to
scanning and transmission electron microscopy ( supplemental figs . 11 and 12 ,
respectively ) . however , no significant differences in the images were
recorded . to observe the effects udp - glcnac starvation had on vsg glycosylation , svsg
was purified from wild type and tbuap conditional null mutant cells ,
grown in the presence and absence of tetracycline for 48 h. analysis by
sds - page and coomassie blue staining revealed that svsg from wild type and
tbuap conditional null cells grown under permissive conditions appear
as single bands , whereas svsg from the tbuap conditional null grown
for 48 h in the absence of tetracycline appeared as a doublet
( fig . the formal
possibility that the altered svsg profile may have been caused by the cells
switching vsg expression to a new vsg variant was eliminated by tryptic mass
fingerprinting , by which both bands were positively identified as vsg221 ( data
not shown ) . a , extracts of wild type and tbuap
conditional null mutant ( tbuap cko ) cells 0 , 24 , and 48 h after
tetracycline removal ( as indicated ) were subjected to sds - page , transferred to
nitrocellulose , and probed with biotinylated tomato lectin followed by
horseradish peroxidase - streptavidin . b , as in a , except that
the tomato lectin inhibitor , chitin hydrolysate , was included with the
lectin . tomato lectin blotting of wild type the tbuap conditional null
mutant extracts . a , extracts of wild type and tbuap
conditional null mutant ( tbuap cko ) cells 0 , 24 , and 48 h after
tetracycline removal ( as indicated ) were subjected to sds - page , transferred to
nitrocellulose , and probed with biotinylated tomato lectin followed by
horseradish peroxidase - streptavidin . b , as in a , except that
the tomato lectin inhibitor , chitin hydrolysate , was included with the
lectin . aliquots of svsg from wild type cells and the tbuap conditional
null mutant grown for 48 h in the absence of tetracycline were digested with
the enzymes endo h and pngase f and analyzed by sds - page and coomassie blue
staining . as previously described
( 44 ) , wild type svsg showed a
small shift in molecular weight when digested with endo h , due to the removal
of the c - terminal oligomannose n - linked glycan , and a greater shift
when digested with pngase f , due to the removal of both n - linked
glycans ( fig . when the svsg from the tbuap conditional null mutant was digested
with endo h , the upper band collapsed into the lower band , and when digested
with pngase f , both bands collapsed into a fully deglycosylated form
( fig . these data
suggest that the vsg doublet present in the mutant represents vsg molecules
that are similar to those found in wild type ( i.e. the upper band )
and vsg molecules that have selectively lost an endo h - sensitive
n - linked glycan ( i.e. the lower band ) . to analyze this further , wild type and tbuap conditional null mutant cells grown under
permissive conditions gave the expected glycoform mass ranges for svsg221
( 16 ,
44 )
( table 3 ) . however , svsg from
the tbuap conditional null mutant grown under nonpermissive
conditions for 48 h displayed two discrete sets of svsg glycoforms ,
corresponding to the two bands seen by sds - page . one set was similar to that
of wild type svsg , except that it lacks the higher molecular weight glycoforms
that contain five glcnac residues ( table
3 ) . the other set of glycoforms have masses consistent with the
absence of oligomannose structures at the c - terminal ( asn-428 ) n - glycosylation
site ( consistent with the endo h resistance of the lower vsg band on
sds - page ) . analysis of the glycopeptide fraction of a pronase digest of the
mutant vsg sample , prepared and analyzed by electrospray tandem mass
spectrometry according to ref . 54 , revealed a range of
asn-263 and asn-428 n - linked glycopeptide and ser-433 gpi
glycopeptide species4
similar to those of wild type vsg ( supplemental fig . these results rule
out the possibility that lower molecular weight vsg glycoforms arise from
changes at the n - linked and gpi glycosylation sites that happen to be
equivalent in mass to the loss of the oligomannose structures from
asn-428 . 9 ) for svsg221 samples from wild type ( wt ) and tbuap
conditional null cells grown with ( cko + tet ) or without tetracycline ( cko -
tet ) for 48 h are tabulated in that order , followed by ( in parentheses ) the
theoretical mass of the assigned vsg composition ( theo . ) . the abundance of each isobaric group of vsg molecules is indicated as follows :
+ + + , mass peaks > 70% of the biggest species ; + + , mass peaks > 40% of the
biggest species ; + , mass peaks < 40% of the biggest species.measured ( and theoretical ) molecular mass of wt/+tet/tet ( theo.)proteinaglcn - ino - cpbetnpbhexnachexosewtcko + tetcko tetda 51,542/51,546/na ( 51,531 )
1
1
1
5
23
+
+
51,500/51,505/na ( 51,490 )
1
1
1
4
24
+
+
51,379/51,384/na ( 51,369 )
1
1
1
5
22
+
+
51,338/51,343/na ( 51,328 )
1
1
1
4
23
+
+
51,215/51,219/na ( 51,207 )
1
1
1
5
21
+
+
51,174/51,179/na ( 51,166 )
1
1
1
4
22
+
+
51,053/51,058/na ( 51,045 )
1
1
1
5
20
+
+
51,012/51,017/51,016 ( 51,004 )
1
1
1
4
21
+
+
+
50,891/50,897/50,895 ( 50,883 )
1
1
1
5
19
+
+
50,850/50,856/50,854 ( 50,842 )
1
1
1
4
20
+ + +
+ + +
+ + +
50,688/50,694/50,691 ( 50,680 )
1
1
1
4
19
+ +
+ +
+ +
50,526/50,531/50,530 ( 50,518 )
1
1
1
4
18
+
+
+
50,364/50,369/50,368 ( 50,356 )
1
1
1
4
17
+
+
+
na / na/49,311 ( 49,302 )
1
1
1
2
13
+
na / na/49,149 ( 49,140 )
1
1
1
2
12
+ + +
na / na/48,987 ( 48,978 )
1
1
1
2
11
+ + +
na / na/48,826 ( 48,816 )
1
1
1
2
10
+
na / na/48,663 ( 48,654 )
1
1
1
2
9
+
na / na/48,501 ( 48,492 )
1
1
1
2
8
+
athe average molecular weight of svsg221 polypeptide ( 46,284 da ) minus amino
acids 1 - 27 ( signal peptide ) and residues 460 - 476 ( gpi attachment signal
sequence ) with four disulphide bonds
( 44).bcomponents of gpi common to all glycoforms of svsg221 : glcn - ino - cp ,
glucosamine-1 - 6-myo - inositol-1,2-cyclic phosphate ; etnp ,
ethanolamine phosphate 9 ) for svsg221 samples from wild type ( wt ) and tbuap
conditional null cells grown with ( cko + tet ) or without tetracycline ( cko -
tet ) for 48 h are tabulated in that order , followed by ( in parentheses ) the
theoretical mass of the assigned vsg composition ( theo . ) . the abundance of each isobaric group of vsg molecules is indicated as follows :
+ + + , mass peaks > 70% of the biggest species ; + + , mass peaks > 40% of the
biggest species ; + , mass peaks < 40% of the biggest species . the average molecular weight of svsg221 polypeptide ( 46,284 da ) minus amino
acids 1 - 27 ( signal peptide ) and residues 460 - 476 ( gpi attachment signal
sequence ) with four disulphide bonds
( 44 ) . components of gpi common to all glycoforms of svsg221 : glcn - ino - cp ,
glucosamine-1 - 6-myo - inositol-1,2-cyclic phosphate ; etnp ,
ethanolamine phosphate . taken together , these data show that under udp - glcnac starvation , the
elaboration of the man3glcnac2 and
man4glcnac2 glycans at the fully occupied asn-263
n - glycosylation site glycans with glcnac and gal1 - 4glcnac is
significantly reduced and that the occupancy of the c - terminal asn-428 site is
selectively and dramatically reduced . whereas some enzymes of carbohydrate metabolism in t. brucei are
closer to their prokaryote counterparts
( 45 ,
46 ) , this is not the case for
tbuap , which has 31% sequence identity and 50% sequence similarity to its
human counterpart and , unlike its bacterial counterpart , glmu , is not fused to
a glucosamine-1-phosphate n - acetyltransferase domain
( 47 ) . analysis of the recombinant uap from t. brucei demonstrated that
tbuap is a conventional divalent cation - dependent pyrophosphorylase with
kinetic parameters similar to those reported for other eukaryotic uaps . however , tbuap differs from most other uaps in that it is highly selective for
its sugar phosphate substrate , accepting only glcnac 1-phosphate , and that it
is compartmentalized in a microbody , the glycosome . with respect to the former , the inability of tbuap to accept galnac-1-p
reflects the fact that t. brucei , like all of the trypanosomatids ,
does not incorporate galnac into its glycoconjugates ( reviewed in ref . thus , the organism has no
need to make udp - galnac either from galn or galnac or by epimerization of
udp - glcnac . accordingly , both tbuap ( this study ) and tbgale
( 15 ) are exclusively
glcnac-1-phosphate- and udp - glc / gal - specific , respectively . this is unlike
their mammalian counterparts that can also utilize galnac-1-p
( 36 ) and udp - glcnac / galnac
( 48 ) , respectively . comparison
of the tbuap sequence with those of the human agx1 and agx2 uap splice
variants , for which there are crystal structures
( 36 ) , indicates that tbuap has
a conserved active site , containing only a 2-amino acid difference among the
15 identified substrate - interacting residues ( i.e. arg-313 in place
of pro-288 and ala-489 in place of lys-455 ) . ( 36 ) reported that the
equatorial glcnac c4 hydroxyl of udp - glcnac forms one hydrogen bond with
gly-290 and one with asn-327 of agx1 and that the axial galnac c4 hydroxyl of
udp - galnac forms two hydrogen bonds with asn-327 . however , the context of the key asn residue
( underlined ) in tbuap is kfncanissnlc
whereas in the agx enzymes it is
lfnagnianhff . nonequivalent adjacent
residues ( in italic type ) , particularly the bulkier ca versus ag
sequence immediately before the asn residue , may affect the latter hydrogen
bond network and account for the observed selectivity of tbuap for glcnac-1-p
over galnac-1-p . figure 7.fluorescence microscopy of tomato lectin binding to wild type and
tbuap conditional null mutant cells . wild type ( wt ) and
conditional null mutant ( cko ) cells after 0 , 24 , and 48 h without
tetracycline were visualized by phase - contrast and tomato lectin fluorescence
microscopy , as indicated ( a - e ) . also shown are wild type cells
stained with 4,6-diamidino-2-phenylindole ( dapi ) and tomato
lectin ( tl ) ( f - j ) . fluorescence microscopy of tomato lectin binding to wild type and
tbuap conditional null mutant cells . wild type ( wt ) and
conditional null mutant ( cko ) cells after 0 , 24 , and 48 h without
tetracycline were visualized by phase - contrast and tomato lectin fluorescence
microscopy , as indicated ( a - e ) . also shown are wild type cells
stained with 4,6-diamidino-2-phenylindole ( dapi ) and tomato
lectin ( tl ) ( f - j ) . with respect to the glycosomal , rather than cytosolic , location of the
native enzyme , a recent computational search of the t. brucei , t.
cruzi , and leishmania major genomes did not identify their uaps
as glycosomal enzymes ( 45 ) . however , that study used the pts1 prosite pattern ps00342 of
( sta - gcn)-(rkh)-(livmafy)$ ( where $ denotes the end of the protein )
supplemented with the pattern s - s-(lif)$. thus , the tbuap c - terminal
tripeptide sequence , snm$ , did not pass this filter . on the other hand , the
same authors noted that the most abundant residues , in order of frequency , in
t. brucei pts1 sequences are ( sagpyn)-(khrnq)-(lm - avy)$ , indicating
that although the snm$ permutation has not been identified previously as a
t. brucei pts1 sequence , it appears to be one . significantly , the
c - terminal tripeptide sequences of the t. cruzi and l. major
uaps are gnm$ and anm$ , respectively , which also fit the aforementioned
potential pts1 permutations . it remains to be determined experimentally
whether a c - terminal ( sga)-nm$ sequence is sufficient for glycosomal import . the alternative is that tbuap is targeted to the glycosome through association
with another pts1 or pts2 targeted protein , as has been described previously
for peroxisomal import
( 49 ) . in all trypanosomatids , the two - step conversion of glucose to fructose
6-phosphate , via hexokinase and glucose-6-phosphate isomerase , occurs in the
glycosome , and it is conceivable that the entire udp - glcnac biosynthetic
pathway is located in this organelle . although
glucosamine - fructose-6-phosphate transaminase , glucosamine - phosphate
n - acetyltransferase , and phosphoacetylglucosamine mutase lack obvious
pts1 or pts2 sequences , the t. cruzi and l. major
glucosamine-6-phosphate deaminase sequences do contain pts1 sequences
( 46 ) . our
current hypothesis , which we are currently testing , is that
glucosamine - fructose-6-phosphate transaminase , glucosamine - phosphate
n - acetyltransferase , and phosphoacetylglucosamine mutase are
piggybacked into the glycosome via oligomerization with glucose-6-phosphate
isomerase and/or glucosamine-6-phosphate deaminase and/or tbuap to provide
functional udp - glcnac synthesis machinery in a single location . indeed , it may
be that all sugar nucleotide biosynthesis occurs in this location in
trypanosomes , since both udp - glc 4-epimerase and gdp - man
4,6-dehydratase , required for in udp - gal and gdp - fuc synthesis , respectively ,
have also been shown to be glycosomal in t. brucei
( 15 ,
18 ) . a glycosomal location for
the synthesis of sugar nucleotides further suggests that there may be specific
transporters or antiporters in the glycosome membrane to , for example ,
exchange utp for udp - sugars and gtp for gdp - sugars or ndp - sugars for
ppi . in this regard , it is worth noting that , whereas most sugar
nucleotide antiporters exchange ndp - sugars for nmps
( 50 ) ( requiring nucleoside
diphosphatases to convert ndps to nmps ) , ndp - sugar / ndp and ndp - sugar / ndp - sugar
antiporter activities have also been recently reported
( 51 ) . figure 8.endoglycosidase digestions of svsg221 from wild type and tbuap
conditional null mutant cells . a , svsg221 purified from wild type
and tbuap conditional null mutant ( tbuap cko ) cells before
( 0 h ) and after 48 h without tetracycline ( 48 h ) b , aliquots of wild type svsg221 were
digested with endo h ( that removes only oligomannose n - linked
glycans ) or pngase f ( that removes all n - linked glycans ) , and the
products were subjected to sds - page and coomassie blue staining . species
containing two , one , and no n - linked glycans were resolved , as
indicated . c , aliquots of svsg221 from tbuap conditional
null cells grown for 48 h without tetracycline were digested with endo h or
pngase f , and the products were subjected to sds - page and coomassie blue
staining . species containing two , one , and no n - linked glycans were
resolved , as indicated . a , svsg221 purified from wild type
and tbuap conditional null mutant ( tbuap cko ) cells before
( 0 h ) and after 48 h without tetracycline ( 48 h ) were subjected to sds - page
and coomassie blue staining . b , aliquots of wild type svsg221 were
digested with endo h ( that removes only oligomannose n - linked
glycans ) or pngase f ( that removes all n - linked glycans ) , and the
products were subjected to sds - page and coomassie blue staining . species
containing two , one , and no n - linked glycans were resolved , as
indicated . c , aliquots of svsg221 from tbuap conditional
null cells grown for 48 h without tetracycline were digested with endo h or
pngase f , and the products were subjected to sds - page and coomassie blue
staining . species containing two , one , and no n - linked glycans were
resolved , as indicated . figure 9.electrospray mass spectrometry of svsg221 from wild type and
tbuap conditional null mutant cells . aliquots of svsg221 from
wild type cells ( a ) and tbuap conditional null mutant cells
grown in the presence of tetracycline ( b ) and the absence of
tetracycline for 48 h ( c ) were analyzed by positive ion electrospray
mass spectrometry , and the data were processed by baysian protein
reconstruction to produce mass graphs of isobaric glycoforms . models of some
of the principal glycoforms are indicated , and the compositions of the
detected glycoforms are shown in table
3 . electrospray mass spectrometry of svsg221 from wild type and
tbuap conditional null mutant cells . aliquots of svsg221 from
wild type cells ( a ) and tbuap conditional null mutant cells
grown in the presence of tetracycline ( b ) and the absence of
tetracycline for 48 h ( c ) were analyzed by positive ion electrospray
mass spectrometry , and the data were processed by baysian protein
reconstruction to produce mass graphs of isobaric glycoforms . models of some
of the principal glycoforms are indicated , and the compositions of the
detected glycoforms are shown in table
3 . the phenotype generated through udp - glcnac starvation with regard to tomato
lectin binding is similar to that caused by udp - gal starvation
( 16 ) ( i.e. a
reduction in lectin binding in western blots and fluorescence microscopy ) ,
indicating a reduction in both size and quantity of the giant poly - lacnac
structures normally found throughout the flagellar pocket and
endosomal / lysosomal system
( 26 ,
27 ) . for example , tomato lectin blots remain
unchanged for a tbgpi12 conditional null mutant
( 6 ) under nonpermissive
conditions ( supplemental fig . transmission electron microscopy images of
gold - conjugated ricin binding in the flagellar pocket had suggested that the
bloodstream form - specific fibrous material in the flagellar pocket might
correspond to ricin - binding glycoconjugates
( 43 ) . however , although a
significant reduction in tomato lectin binding was observed in this study ,
transmission electron microscopy images did not show a significant reduction
in the fibrous material when compared with wild type cells . this suggests that
the fibrous material is either unrelated to the poly - lacnac - containing
glycoproteins of the flagellar pocket or that the fibrous appearance of the
glycoproteins in transmission electron microscopy is unaffected by reducing
their poly - lacnac content . hopefully , future studies on the composition of the
lumenal contents of the flagellar pocket will resolve this issue . another way of assessing the glycosylation phenotype of bloodstream form
t. brucei mutants is to use the abundant vsg as a reporter and to
assess the status of its two n - glycosylation sites and gpi anchor
( 16 ,
44 ) . in the case of udp - gal
starvation , the copy number and integrity of the vsg coat is not affected ,
although the gpi anchors , which normally sport an average of five gal side
chain residues , are free of galactose
( 16 ) . the effect on vsg
glycosylation was very different under udp - glcnac starvation . mass
spectrometry and endoglycosidase digestion of vsg221 isolated from the
tbuap conditional null mutants under nonpermissive conditions
revealed the presence of two major species of vsg221 in approximately equal
amounts . one form was almost indistinguishable from wild - type vsg221 , whereas
the other , lower molecular mass form , specifically lacked the c - terminal
( asn-428 ) n - linked glycan . from a hierarchical point of view , it is
not surprising that gpi anchor synthesis is maintained , even when protein
n - glycosylation and n - glycan elaboration are affected , since
gpi synthesis and transfer to protein are clearly essential to bloodstream
form t. brucei
( 5 - 8 ) . furthermore , from a structural point of view , the asn-263 n - linked
glycan may be more important to the correct folding of the vsg . ( 52 ) analyzed the
crystal structures of two vsg variants , vsg iltat.1.24 and vsg221 , and found
that a short -helix in iltat.1.2 is absent in vsg221 but replaced by
the protein - proximal sugars of the asn-263 n - linked glycan . however ,
this does not provide a mechanistic explanation for the hierarchy of asn-263
n - glycosylation in strict preference to the n - glycosylation
of asn-428 under udp - glcnac starvation . radiolabeling studies suggest that
bloodstream form t. brucei contain very low steady - state levels of
man9glcnac2-pp - dolichol and a relatively large pool of
man5glcnac2-pp - dolichol
( 53 ) . despite their
differences in abundance , the organism appears to use both of these
oligosaccharyl - pp - dolichol species for transfer to protein , the former to
sites destined to contain oligomannose glycans and the latter to sites
destined to become complex type structures
( 44 ) . in most eukaryotes , a
single type of dolichol - pp - linked oligosaccharide precursor
( glc3man9glcnac2-pp - dolichol ) is transferred
to the nascent peptide in the er through the action of
oligosaccharyltransferase ( ost ) , a complex of typically eight different
proteins ( 54 ) . trypanosomatids
lack candidate genes for all but the catalytic subunit , stt3 , of this complex .
however , there are three copies of the tbstt3 gene in the t.
brucei genome . two of the tbstt3 genes encode almost identical
proteins , and we speculate that the unique tbstt3 utilizes either exclusively
man5glcnac2-pp - dolichol or
man9glcnac2-pp - dolichol and that one or both of the two
similar tbstt3s utilizes the other donor . under udp - glcnac starvation , gdp - man
levels rise slightly , and presumably dol - p - man is also present at greater than
normal levels . from this point of view , one might expect the ratio of
man9glcnac2-pp - dolichol / man5glcnac2-pp - dolichol
to increase slightly . however , the absolute levels of both precursors are
presumably significantly reduced as udp - glcnac becomes limiting for the
synthesis of the common glcnac2-pp - dolichol core . thus , the
simplest explanation for the selective loss of the c - terminal oligomannose
n - linked glycans from vsg221 under udp - glcnac starvation is that the
effective concentration of man9glcnac2-pp - dolichol falls
so far below the effective km for the relevant tbstt3 that
most vsg molecules appearing in the er fail to receive a c - terminal
n - glycan . comparative studies on the substrate specificities and
kinetic parameters of the two classes of tbstt3 should resolve this issue . meanwhile , additional support for such a dual oligosaccharyltransferase model
comes from the analysis of a t. brucei alg3 mutant that can only make
man5glcnac2 and that also underglycosylates the
c - terminal asn-428 site of vsg221
( 55 ) and from pulse - chase
studies that show , in at least two different vsgs , that endo h - sensitive
oligomannose sites ( but not endo h - resistant sites ) can be
n - glycosylated post - translationaly
( 30 ,
56 ) . finally , through the creation of the conditional null mutant ,
tbuap was shown to be essential for the growth and survival of
bloodstream t. brucei in culture , suggesting that this enzyme might
be considered as a potential therapeutic target for human african sleeping
sickness . further in vivo support for this notion was obtained when
the tbuap conditional null was shown to be infectious to mice dosed
with doxycycline in their drinking water but not to doxycycline - free mice . although the essentiality of udp - glcnac biosynthesis via tbuap was expected ,
experimental validation in vitro and in vivo is ,
nevertheless , important , since it rules out any possible metabolic and/or
nutritional bypasses . compared with other conditional null mutants generated
in this laboratory , the ablation of udp - glcnac synthesis led to more rapid
killing of the parasite ( < 72 h ) than ablation of either udp - gal synthesis
( 14 ,
16 ) or gpi biosynthesis
( 6 ) , which both take 4 - 5 days . the rapidity of cell death presumably reflects the fact that udp - glcnac is
simultaneously required for gpi anchor biosynthesis , core protein
n - glycosylation , and the subsequent decoration of complex
n - glycans with lactosamine units . of course , udp - glcnac is also
essential to mammalian cells
( 57 ) , and selective inhibition
of the parasite uap would be a therapeutic requirement . | a gene encoding trypanosoma brucei
udp - n - acetylglucosamine pyrophosphorylase was identified , and the
recombinant protein was shown to have enzymatic activity . the parasite enzyme
is unusual in having a strict substrate specificity for
n - acetylglucosamine 1-phosphate and in being located inside a
peroxisome - like microbody , the glycosome .
a bloodstream form t.
brucei conditional null mutant was constructed and shown to be unable to
sustain growth in vitro or in vivo under nonpermissive
conditions , demonstrating that there are no alternative metabolic or
nutritional routes to udp - n - acetylglucosamine and providing a genetic
validation for the enzyme as a potential drug target .
the conditional null
mutant was also used to investigate the effects of
n - acetylglucosamine starvation in the parasite .
after 48 h under
nonpermissive conditions , about 24 h before cell lysis , the status of parasite
glycoprotein glycosylation was assessed . under these conditions ,
udp - n - acetylglucosamine levels were less than 5% of wild type .
lectin
blotting and fluorescence microscopy with tomato lectin revealed that
poly - n - acetyllactosamine structures were greatly reduced in the
parasite .
the principal parasite surface coat component , the variant surface
glycoprotein , was also analyzed .
endoglycosidase digestions and mass
spectrometry showed that , under udp - n - acetylglucosamine starvation ,
the variant surface glycoprotein was specifically underglycosylated at its
c - terminal asn-428 n - glycosylation site .
the significance of this
finding , with respect to the hierarchy of site - specific
n - glycosylation in t. brucei , is discussed . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
oral squamous cell carcinoma ( oscc ) accounts for more than 90% of all oral cancers and is the sixth most common type of cancer in the world . the 5-year survival rate of oscc has not improved much over the past 2 decades . in china , evidence has shown that the risk of morbidity for oscc patients is still high and that oscc tends to occur among younger patients . the prognostic value of the traditional tnm system is widely regarded as inadequate in patients with oscc . advances in understanding of the molecular mechanisms underlying oscc have led to an ever - increasing number of molecular markers shown to be related to the behavior of this disease . however , little clear evidence has been provided on the prognostic relevance of these markers . in addition , there is an increased necessity for identification and standardization of sensitive and accurate biomarkers that can identify oscc patients with a poor prognosis . mature mirnas are small ( 2021 nucleotides in length ) endogenous noncoding rnas that regulate the expression of over 50% of human genes at the post - transcriptional level guided by partial complementarities to specific sequences in their target messenger rna . mirnas is indispensable for a variety of basic biological and pathological processes , and mirna signatures are closely correlated with human diseases . deregulations of microrna have been reported in various cancers , and have been showed to play important roles in cancer initiation and progression [ 811 ] . distinct mirna expression profiles in oscc in comparison with matched healthy controls have been demonstrated , indicating mirnas may participate in oscc tumorigenesis . recently , various studies have shown that microrna-1246 ( mir-1246 ) plays a crucial role in regulation of cancer cell biological functions such as proliferation , invasion , and metastasis . in addition , overexpression of mir-1246 has been found in several types of cancers such as liver cancer , pancreatic cancer , and colorectal carcinoma . however , data on the expression of mir-1246 in oscc and its clinical impact on patient survival remain scarce . in the present study we aimed to evaluate the clinical significance of mir-1246 expression in oscc . in addition , the relationship between mir-1246 expression and clinicopathological characters was analyzed and the impact of mir-1246 expression on the prognosis of oscc patients was estimated . this study was approved by the research ethics committee of the second affiliated hospital of nanchang university . written informed consent was obtained from all patients . the selection criteria for patients with oscc were : ( 1 ) histological confirmation of oscc and ( 2 ) no prior history of other cancers . all patients were diagnosed and treated at the second affiliated hospital of nanchang university from march 2009 to june 2014 . tumor stage was classified according to the 7 edition of the classification of malignant tumors of the american joint committee on cancer . tumor grade was classified following the who ( 2005 ) criteria . by last follow - up , 37 patients had died from the disease and the duration of follow - up was 60 months . for qrt - pcr , 106 pairs of fresh oscc and matched adjacent normal tissue specimens were collected from patients who underwent surgery in the second affiliated hospital of nanchang university . the oral squamous cell carcinoma cell lines ( scc25 and cal27 ) were maintained in dulbecco s modified eagle s medium ( gibco , grand island , ny , usa ) containing 10% fetal calf serum ( gibco ) , 100 u / l penicillin , and 10 mg / l streptomycin . hok16e6e7 cells , a human immortalized oral keratinocyte cell line , were cultured in keratinocyte growth medium containing 0.15 mm calcium and supplemented with epidermal growth factor ( gibco ) . total rna was extracted from frozen tissues using trizol ( takara , dalian , china ) according to the manufacturer s instructions . briefly , 2 ug of rna was added to rt reaction , and the cdna served as the template for amplification of pcr with sequence - specific primers ( sangon biotech , shanghai , china ) using sybr primescript mirna rt - pcr kit ( takara ) in the stratagene mx3000p real - time pcr system ( agilent technologies , la jolla , ca , usa ) . the fold change between tumor tissues and noncancerous samples for mir-1246 was calculated with the 2 method . the expression levels of mir-1246 in hok16e6e7 , scc25 , and cal27 were detected using the same method . the normality of data distribution was checked using the shapiro - wilk normality test ( no significant differences from a normal distribution were detected ; p=0.264 ) . the comparison of the expression level of mir-1246 in tissues and cell lines was performed using two - sample student s t - test . the correlation between the expression of mir-1246 and clinicopathological characters was assessed with chi - square test . the overall survival was analyzed by log - rank test , and survival curves were plotted . the univariate cox regression was performed on each clinical covariate to examine its influence on patient survival . a wald statistic of p<0.05 was used as the criterion for inclusion in final multivariate models . multivariate analysis for prognostic factors statistical analysis was performed using spss version 21.0 software ( chicago , il , usa ) and sigmaplot12.5 ( systat software inc . this study was approved by the research ethics committee of the second affiliated hospital of nanchang university . written informed consent was obtained from all patients . the selection criteria for patients with oscc were : ( 1 ) histological confirmation of oscc and ( 2 ) no prior history of other cancers . all patients were diagnosed and treated at the second affiliated hospital of nanchang university from march 2009 to june 2014 . tumor stage was classified according to the 7 edition of the classification of malignant tumors of the american joint committee on cancer . tumor grade was classified following the who ( 2005 ) criteria . by last follow - up , 37 patients had died from the disease and the duration of follow - up was 60 months . for qrt - pcr , 106 pairs of fresh oscc and matched adjacent normal tissue specimens were collected from patients who underwent surgery in the second affiliated hospital of nanchang university . the oral squamous cell carcinoma cell lines ( scc25 and cal27 ) were maintained in dulbecco s modified eagle s medium ( gibco , grand island , ny , usa ) containing 10% fetal calf serum ( gibco ) , 100 u / l penicillin , and 10 mg / l streptomycin . hok16e6e7 cells , a human immortalized oral keratinocyte cell line , were cultured in keratinocyte growth medium containing 0.15 mm calcium and supplemented with epidermal growth factor ( gibco ) . total rna was extracted from frozen tissues using trizol ( takara , dalian , china ) according to the manufacturer s instructions . briefly , 2 ug of rna was added to rt reaction , and the cdna served as the template for amplification of pcr with sequence - specific primers ( sangon biotech , shanghai , china ) using sybr primescript mirna rt - pcr kit ( takara ) in the stratagene mx3000p real - time pcr system ( agilent technologies , la jolla , ca , usa ) . the fold change between tumor tissues and noncancerous samples for mir-1246 was calculated with the 2 method . the expression levels of mir-1246 in hok16e6e7 , scc25 , and cal27 were detected using the same method . the normality of data distribution was checked using the shapiro - wilk normality test ( no significant differences from a normal distribution were detected ; p=0.264 ) . the comparison of the expression level of mir-1246 in tissues and cell lines was performed using two - sample student s t - test . the correlation between the expression of mir-1246 and clinicopathological characters was assessed with chi - square test . the overall survival was analyzed by log - rank test , and survival curves were plotted . the univariate cox regression was performed on each clinical covariate to examine its influence on patient survival . a wald statistic of p<0.05 was used as the criterion for inclusion in final multivariate models . multivariate analysis for prognostic factors statistical analysis was performed using spss version 21.0 software ( chicago , il , usa ) and sigmaplot12.5 ( systat software inc . we examined mir-1246 expression in 106 pairs of oscc tissues and the neighboring healthy controls by qrt - pcr . mir-1246 was expressed significantly higher in tumor tissues compared with corresponding noncancerous tissues ( p<0.01 ; 3.470.78 vs. 1.000.54 ; figure 1 ) . similarly , the mir-1246 expression level in oscc cell lines cal27 ( p<0.01 ; 1.820.12 vs. 1.000.06 ; figure 2 ) and scc25 ( p<0.01 ; 4.150.35 vs. 1.000.06 ; figure 2 ) vastly increased compared to the control cell line . the mean mir-1246 expression level of all oscc was 3.47 , which is utilized to divide oscc patients into 2 groups . forty - nine specimens were assigned to the high - expression group ( equal to or higher than 3.47 ) and the remaining 57 specimens were assigned to the low - expression group ( less than 3.47 ) . the relationship between the relative mir-1246 expression levels and clinicopathological features of oscc are shown in table 1 . there was no correlation between the relative mir-1246 expression levels and age , sex distribution , tumor location , tumor size , local recurrence , or distant organ metastasis ( p>0.05 ) , but the relative mir-1246 expression levels were significantly positively correlated with tnm stage ( p=0.005 ) , nodal status ( p=0.015 ) , and tumor grade ( p=0.002 ) . kaplan - meier analysis showed that patients with higher levels of mir-1246 had significantly poorer survival than patients with lower mir-1246 expression levels ( p=0.002 ; 55.11% vs. 81.71% ; mean survival time 56.401.58 vs. 42.842.63 ; figure 2 ) . a cox proportional hazard regression model was developed to evaluate the association between the potential prognostic markers and overall survival at multivariate level . the variables , including mir-1246 expression , nodal status , tnm stage , tumor grade , and local recurrence , were selected in the final multivariate model because their wald test p values were less than the generally used criterion of 0.05 . the results revealed that mir-1246 expression ( p=0.036 ; hr=2.82 ; 95% ci=1.077.43 ) , tnm stage ( p=0.003 ; hr=2.97 ; 95% ci=1.466.05 ) and tumor grade ( p=0.006 ; hr=3.04 ; 95% ci=1.376.74 ) were independent prognostic parameters for oscc ( table 2 ) . we examined mir-1246 expression in 106 pairs of oscc tissues and the neighboring healthy controls by qrt - pcr . mir-1246 was expressed significantly higher in tumor tissues compared with corresponding noncancerous tissues ( p<0.01 ; 3.470.78 vs. 1.000.54 ; figure 1 ) . similarly , the mir-1246 expression level in oscc cell lines cal27 ( p<0.01 ; 1.820.12 vs. 1.000.06 ; figure 2 ) and scc25 ( p<0.01 ; 4.150.35 vs. 1.000.06 ; figure 2 ) vastly increased compared to the control cell line . the mean mir-1246 expression level of all oscc was 3.47 , which is utilized to divide oscc patients into 2 groups . forty - nine specimens were assigned to the high - expression group ( equal to or higher than 3.47 ) and the remaining 57 specimens were assigned to the low - expression group ( less than 3.47 ) . the relationship between the relative mir-1246 expression levels and clinicopathological features of oscc are shown in table 1 . there was no correlation between the relative mir-1246 expression levels and age , sex distribution , tumor location , tumor size , local recurrence , or distant organ metastasis ( p>0.05 ) , but the relative mir-1246 expression levels were significantly positively correlated with tnm stage ( p=0.005 ) , nodal status ( p=0.015 ) , and tumor grade ( p=0.002 ) . kaplan - meier analysis showed that patients with higher levels of mir-1246 had significantly poorer survival than patients with lower mir-1246 expression levels ( p=0.002 ; 55.11% vs. 81.71% ; mean survival time 56.401.58 vs. 42.842.63 ; figure 2 ) . a cox proportional hazard regression model was developed to evaluate the association between the potential prognostic markers and overall survival at multivariate level . the variables , including mir-1246 expression , nodal status , tnm stage , tumor grade , and local recurrence , were selected in the final multivariate model because their wald test p values were less than the generally used criterion of 0.05 . the results revealed that mir-1246 expression ( p=0.036 ; hr=2.82 ; 95% ci=1.077.43 ) , tnm stage ( p=0.003 ; hr=2.97 ; 95% ci=1.466.05 ) and tumor grade ( p=0.006 ; hr=3.04 ; 95% ci=1.376.74 ) were independent prognostic parameters for oscc ( table 2 ) . oscc are cancers of the mucosal surfaces of the lips , floor of mouth , oral tongue , buccal mucosa , lower and upper gingiva , hard palate , and retromolar trigone . it is becoming a serious and growing problem in many parts of the world because patients with oscc often have disfigurement , loss of ability to eat and speak , and poor quality of life , especially in advanced cases . clinicopathological parameters such as tumor grade of differentiation and clinical stage have been used to evaluate the progression of oscc but their sensitivity is relatively low . therefore , finding novel effective biomarkers is of great significance for improving the outcome of oscc therapy . the expression levels of mirnas are often changed in many cancers , resulting in abnormal increases or decreases [ 1921 ] . these alterations play an important role in almost all facets of cancer development and progression . some mirnas families have been shown to be involved in epithelial - to - mesenchymal transition , which is an important component of cancer metastasis . thus , mirnas are not only regarded as potential markers for cancer diagnosis , but also might serve as potential therapeutic targets by manipulating mirna levels to enhance current cancer therapy outcomes . investigating the correlation between mirna expression profiles and the prognosis of patients with oscc may be beneficial to understanding the underlying molecular mechanisms involved in the cancer progression and enabling the identification of novel targets for oscc treatment . in the present study , mir-1246 expression was shown to be correlated with advanced clinical stage , lymph node metastases , and histological grade , suggesting that mir-1246 might be involved in the carcinogenesis and metastasis of oscc . in addition , we revealed that patients with a higher expression of mir-1246 tended to have much lower survival rates than patients with lower mir-1246 expression , indicating that high mir-1246 level is a potential biomarker for predicting the poor prognosis of oscc . consistent with our study results , overexpression of mir-1246 has been observed in multiple tumor types , suggesting its important role in tumorigenesis . della vittoria scarpati found that the expression level of mir-1246 is enhanced by about 2-fold in colorectal tumor tissue in comparison with stromal tissue . there was no statistical difference between liver cancer and mir-1246 expression level , probably due to the small clinical sample of patients . however , the high expression of mir-1246 combined with low expression of cell adhesion molecule 1 ( cadm1 ) was shown to be a risk factor for early - stage liver cancer . a higher level of mir-1246 expression was correlated with a shorter survival time in patients with pancreatic cancer . used mirna expression profiles to find the difference between mirna expressed in cancer tissues and that in normal tissues . mir-1246 was shown to be significantly up - regulated by 12.01-fold and 9.37-fold in colorectal and pancreatic cancers compared to healthy controls , respectively . some studies have explored the exact molecular mechanism of mir-1246 in cancer development and progression . cancer stem cells have been shown to be closely related with tumor initiation , progression , radioresistance , and chemotherapy resistance . hasegawa found that mir-1246 could increase tumor - initiating potential and maintain cancer stem cell - like properties via ccng2 in pancreatic cancer . mir-1246 has also been shown to specifically target the 3-utr of cadm1 and down - regulate its expression , which leads to enhancing migration and invasion capacity of hepatocellular carcinoma cell lines . enhanced mir-1246 expression can promote cervical cancer cell proliferation , invasion , and migration by suppression of its target gene , thrombospondin 2 ; suggesting that mir-1246 may be closely related with cervical cancer tumorigenesis and progression . however , conflicting results about the expression level of mir-1246 were reported in esophageal squamous cell carcinoma ( escc ) . fu found that mir-1246 was increased in escc tissues , while another study showed that the expression level of mir-1246 was similar between escc tissues and normal controls . further large - scale investigations are needed to address conflicting findings on the expression level of mir-1246 in escc . in addition to the overexpression of mir-1246 in various cancer tissues , mir-1246 is significantly enhanced in serum derived from nude mice implanted with primary human pancreatic ductal adenocarcinoma . moreover , serum mir-1246 has been showed to be up - regulated in patients with early - stage cervical squamous cell carcinoma , indicating that serum mir-1246 may be a promising biomarker for early detection of cervical squamous cell carcinoma . also , serum mir-1246 was significantly higher in primary colorectal cancer patients . recent studies suggest that mir-1246 might also serve as a potential drug target or a biomarker to predict the risk of drug resistance and radioresistance . also , mir-1246 has been reported to be involved in promoting radioresistance of cervical cancer cells . these findings suggest the possibility of a novel method to improve oscc therapy by targeting mir-1246 . in conclusion , the expression level of mir-1246 was increased in oscc tissues and cell lines and it was correlated with tnm stage , nodal status , and tumor grade . in addition , high mir-1246 expression was associated with poorer survival and served as an independent prognostic factor in patients with oscc , suggesting that mir-1246 may be a promising prognostic biomarker for oscc . however , the relatively small sample of this study is a limitation ; a larger sample size would definitely be desirable with longer follow - up to firmly establish the diagnostic value of mir-1246 in oscc . in addition , further studies are needed to elucidate the role of mir-1246 in oscc at the cellular level . | backgroundmicrornas ( mirnas ) are small , non - coding rnas that may function as oncogenes or tumor suppressors .
previous studies have shown that the expression level of mir-1246 was enhanced in multiple types of cancers .
however , the expression of mir-1246 in human oral squamous cell carcinoma ( oscc ) and its prognostic values remain unclear.material/methodsquantitative reverse - transcriptase polymerase chain reaction ( qrt - pcr ) was used to analyze the expression of mir-1246 in 106 pairs of matched normal and tumor tissue samples .
the chi - square test was used to examine the associations between mir-1246 expression and the clinicopathological characters .
the survival curves were constructed by the kaplan - meier method .
the influence of each clinical variable on survival was examined by the cox multivariate regression analysis.resultsthe expression level of mir-1246 was significantly higher in tumor tissues and oral cancer cell lines than in normal controls ( p<0.01 ) .
high expression of mir-1246 was found to significantly correlate with nodal status ( p=0.015 ) , tnm stage ( p=0.005 ) , and tumor grade ( p=0.002 ) . enhanced mir-1246 correlated significantly with patient survival ( p<0.01 ) . in multivariate analysis
, we found that mir-1246 expression was an independent prognostic factor of poor patient survival ( p= 0.036 ; hr=2.82 ; 95% ci=1.077.43).conclusionshigh mir-1246 expression is associated with poor prognosis in oscc and may serve as a novel prognostic marker in oscc . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
may 2006 , a total of 75 patients ( 50 men and 25 women , mean age , 60 years ; age range , 25 - 79 years ) , who had a spn seen on their chest radiography and there was no satellite nodule on the targeted thin - section ct scans ( 0.75 mm collimation , 0.5-second gantry rotation time , 120 kvp , 50 ma ) that were done on the spn , underwent dynamic chest ct with a sixteen - detector row ct scanner ( somatom sensation 16 , siemens , erlangen , germany ) . the nodules were excluded if they contained benign calcifications ( diffuse , laminated , popcorn - like or central ) , ground glass opacity , the spn was less than 5 mm and the nodules were juxtapleura / juxtavascular nodules as seen on the thin - section ct . patients with heart disease or respiratory difficulties during the inspiration period of ct were also excluded . of the 75 patients , 35 patients were excluded : 13 patients were lost to follow - up and 22 patients were followed up without a biopsy . overall , a total of 40 patients with spns ( 29 men and 11 women , mean age , 56.3 years ; age range , 25 - 79 years ) that had proven pathology were analyzed . the average long and short axis diameters of the malignant spns were 20.6 6.4 mm ( 12 - 30 mm ) and 17.0 6.1 mm ( 9 - 30 mm ) , respectively . the average long and short axis diameters of the benign spns were 16.2 7.2 mm ( 7 - 30 mm ) and 12.0 4.9 mm ( 6 - 23 mm ) , respectively . thirtyone patients underwent a percutaneous needle biopsy and nine patients underwent surgery that included a wedge resection . before intravenously injecting the contrast medium , a series of images was obtained throughout the entire nodule along the z - axis with 0.75 mm collimation ( 120 kvp , 170 ma , 0.5 second gantry rotation time ) . thereafter , an additional ten series ( 20s , 40s , 60s , 80s , 120s , 140s , 160s , 180s , 240s and 300s ) of images were obtained at 20-second intervals over a 5-minute period after injecting the contrast medium ( 3 ml / sec , a total of 120 ml of ultravist 300 ; scherring , berlin , germany ) with a power injector ( mct plus ; medrad , pittsburgh , pa ) . immediately after the dynamic imaging , low - dose ( effective ma : 50 - 60 mas , 120 kvp , 0.75 mm collimation ) helical ct scans were obtained from the lung apices to the level of the middle pole of both kidneys for tumor staging . the image data was reconstructed with a thickness of 2.0 mm and using a standard algorithm . all the thin - sections and dynamic ct data were interfaced directly to our picture archiving and communication system ( pacs , m - view ; marotech , korea ) , which displayed all the image data on monitors ( four monitors , 1,5362,048 image matrices , 8-bit viewable gray scale and 60-foot - lambert luminescence ) . both the mediastinal ( window width : 400 hu , window level : 20 hu ) and lung ( window width : 1,500 hu , window level : -700 hu ) window images were viewed on the monitor . the technical adequacy of dynamic ct fulfilled the following criteria that were reported by previous studies ( 14 ) : no extravasated contrast medium at the injection site , appropriate enhancement of the cardiovascular structures during the examination , no reaction to the contrast medium that interfered with image acquisition and satisfactory patient respiratory registration without artifacts on the images . the total organ doses of the thin - section , dynamic and low - dose ct studies were calculated . an effective patient dose from a chest ct examination is calculated by multiplying the dose length product ( dlp ) by the conversion factor ( 25 ) . the dlp is the ct dose index ( ctdivol ) multiplied by the scanned length . to establish an effective dose for an examination from the dlp , the conversion factors that are used are age , the scanned region , and specific ct geometry generally , the conversion factor for adult patients undergoing chest ct examinations is 0.017 msv per mgy cm . the average calculated mean effective dose was 15.7 3.5 msv ( effective dose range : 9.1 - 19.6 msv ) in our study . the measured organ dose was compared with that of the standard thoracic helical ct ( collimation of 1.5 mm , a slice thickeness of 5 mm , a pitch of 1.5 , 0.5 second rotation time , 120 kvp , 200 ma ) at our institution ( 17 ) . the exposed radiation dose on hdct was 30 - 40 mgy and the dose with performing standard thoracic helical ct was 15 - 20 mgy , which is similar to that reported elsewhere ( 15 ) . the attenuation value of the nodule was measured on all the images at each time ( from the unenhanced image to image acquired at 300 seconds ) except for two sections ( the upper most and lowest ) and the maximum diameter of the spn was obtained . an roi covering approximately the full diameter of the nodule , but excluding calcified , cystic or necrotic areas was examined ( fig . all the measurements in hounsfield units were obtained from the mediastinal window images to minimize partial volume averaging . all the measurements were obtained at the time of the ct examination without the radiologist having knowledge of the histologic diagnosis . one radiologist ( prospective ) with seven years experience of directly drawing rois and one technician ( retrospective ) with 20 years experience with chest ct and using a cad system ( lung care , sensation 16 , siemens , germany ) measured the attenuation values of the spns . for each image in which a nodule appeared , an roi was identified by a well trained technician and using a semi - automated procedure in order to get the best possible segmentation . for all nodules , the rois were created , reviewed and edited by a well trained technician and once they were found to be satisfactory , the rois were approved by the thoracic radiologist ( fig . the attenuation value of the nodule obtained from the roi was obtained by averaging the measured values in all section . the enhancement of the spn was evaluated according to the peak enhancement ( pe ) , the net enhancement ( ne ) and the time attenuation curve ( tac ) on the dynamic ct . the pe was defined as the maximum attenuation value of the nodule over the entire duration of the dynamic study . the ne was calculated by subtracting the pre - enhancement attenuation from the pe attenuation . in this study , the benign patterns included persistent enhancement < 15 hu and no wash - out or < 25 hu wash - out . the malignant patterns included early enhancement > 15 hu and early wash - out of 5 - 25 hu . the diagnostic characteristics such as sensitivity , specificity , accuracy , positive predictive value ( ppv ) and negative predictive value ( npv ) were calculated retrospectively by considering the ne that signified a positive test ( malignant with 15 hu or more of ne as a cutoff ) for a dynamic ct . additionally , the diagnostic characteristics were calculated by considering the tac that signified a positive test ( malignant with early enhancement > 15 hu and early wash - out of 5 - 31 hu ) on a dynamic ct scan . fisher 's exact test and paired t - tests were used to analyze statistically significant differences between the malignant and benign nodules . moreover , this study used bland and altman plots ( 26 ) to compare the numerical values of enhancement ( pe and ne ) between using cad systems and those obtained by direct personal drawing . having calculated the mean difference and the 95% limits of agreement , the investigators determined if the methods were sufficiently in agreement for the new method to be used as an alternative . this study was performed retrospectively with the approval of the ethics committee at our institution . may 2006 , a total of 75 patients ( 50 men and 25 women , mean age , 60 years ; age range , 25 - 79 years ) , who had a spn seen on their chest radiography and there was no satellite nodule on the targeted thin - section ct scans ( 0.75 mm collimation , 0.5-second gantry rotation time , 120 kvp , 50 ma ) that were done on the spn , underwent dynamic chest ct with a sixteen - detector row ct scanner ( somatom sensation 16 , siemens , erlangen , germany ) . the nodules were excluded if they contained benign calcifications ( diffuse , laminated , popcorn - like or central ) , ground glass opacity , the spn was less than 5 mm and the nodules were juxtapleura / juxtavascular nodules as seen on the thin - section ct . patients with heart disease or respiratory difficulties during the inspiration period of ct were also excluded . of the 75 patients , 35 patients were excluded : 13 patients were lost to follow - up and 22 patients were followed up without a biopsy . overall , a total of 40 patients with spns ( 29 men and 11 women , mean age , 56.3 years ; age range , 25 - 79 years ) that had proven pathology were analyzed . the average long and short axis diameters of the malignant spns were 20.6 6.4 mm ( 12 - 30 mm ) and 17.0 6.1 mm ( 9 - 30 mm ) , respectively . the average long and short axis diameters of the benign spns were 16.2 7.2 mm ( 7 - 30 mm ) and 12.0 4.9 mm ( 6 - 23 mm ) , respectively . thirtyone patients underwent a percutaneous needle biopsy and nine patients underwent surgery that included a wedge resection . before intravenously injecting the contrast medium , a series of images was obtained throughout the entire nodule along the z - axis with 0.75 mm collimation ( 120 kvp , 170 ma , 0.5 second gantry rotation time ) . thereafter , an additional ten series ( 20s , 40s , 60s , 80s , 120s , 140s , 160s , 180s , 240s and 300s ) of images were obtained at 20-second intervals over a 5-minute period after injecting the contrast medium ( 3 ml / sec , a total of 120 ml of ultravist 300 ; scherring , berlin , germany ) with a power injector ( mct plus ; medrad , pittsburgh , pa ) . immediately after the dynamic imaging , low - dose ( effective ma : 50 - 60 mas , 120 kvp , 0.75 mm collimation ) helical ct scans were obtained from the lung apices to the level of the middle pole of both kidneys for tumor staging . the image data was reconstructed with a thickness of 2.0 mm and using a standard algorithm . all the thin - sections and dynamic ct data were interfaced directly to our picture archiving and communication system ( pacs , m - view ; marotech , korea ) , which displayed all the image data on monitors ( four monitors , 1,5362,048 image matrices , 8-bit viewable gray scale and 60-foot - lambert luminescence ) . both the mediastinal ( window width : 400 hu , window level : 20 hu ) and lung ( window width : 1,500 hu , window level : -700 hu ) window images were viewed on the monitor . the technical adequacy of dynamic ct fulfilled the following criteria that were reported by previous studies ( 14 ) : no extravasated contrast medium at the injection site , appropriate enhancement of the cardiovascular structures during the examination , no reaction to the contrast medium that interfered with image acquisition and satisfactory patient respiratory registration without artifacts on the images . the total organ doses of the thin - section , dynamic and low - dose ct studies were calculated . an effective patient dose from a chest ct examination is calculated by multiplying the dose length product ( dlp ) by the conversion factor ( 25 ) . the dlp is the ct dose index ( ctdivol ) multiplied by the scanned length . to establish an effective dose for an examination from the dlp , the conversion factors that are used are age , the scanned region , and specific ct geometry generally , the conversion factor for adult patients undergoing chest ct examinations is 0.017 msv per mgy cm . the average calculated mean effective dose was 15.7 3.5 msv ( effective dose range : 9.1 - 19.6 msv ) in our study . the measured organ dose was compared with that of the standard thoracic helical ct ( collimation of 1.5 mm , a slice thickeness of 5 mm , a pitch of 1.5 , 0.5 second rotation time , 120 kvp , 200 ma ) at our institution ( 17 ) . the exposed radiation dose on hdct was 30 - 40 mgy and the dose with performing standard thoracic helical ct was 15 - 20 mgy , which is similar to that reported elsewhere ( 15 ) . the attenuation value of the nodule was measured on all the images at each time ( from the unenhanced image to image acquired at 300 seconds ) except for two sections ( the upper most and lowest ) and the maximum diameter of the spn was obtained . an roi covering approximately the full diameter of the nodule , but excluding calcified , cystic or necrotic areas was examined ( fig . all the measurements in hounsfield units were obtained from the mediastinal window images to minimize partial volume averaging . all the measurements were obtained at the time of the ct examination without the radiologist having knowledge of the histologic diagnosis . one radiologist ( prospective ) with seven years experience of directly drawing rois and one technician ( retrospective ) with 20 years experience with chest ct and using a cad system ( lung care , sensation 16 , siemens , germany ) measured the attenuation values of the spns . for each image in which a nodule appeared , an roi was identified by a well trained technician and using a semi - automated procedure in order to get the best possible segmentation . for all nodules , the rois were created , reviewed and edited by a well trained technician and once they were found to be satisfactory , the rois were approved by the thoracic radiologist ( fig . the attenuation value of the nodule obtained from the roi was obtained by averaging the measured values in all section . the enhancement of the spn was evaluated according to the peak enhancement ( pe ) , the net enhancement ( ne ) and the time attenuation curve ( tac ) on the dynamic ct . the pe was defined as the maximum attenuation value of the nodule over the entire duration of the dynamic study . the ne was calculated by subtracting the pre - enhancement attenuation from the pe attenuation . in this study , the benign patterns included persistent enhancement < 15 hu and no wash - out or < 25 hu wash - out . the malignant patterns included early enhancement > 15 hu and early wash - out of 5 - 25 hu . the diagnostic characteristics such as sensitivity , specificity , accuracy , positive predictive value ( ppv ) and negative predictive value ( npv ) were calculated retrospectively by considering the ne that signified a positive test ( malignant with 15 hu or more of ne as a cutoff ) for a dynamic ct . additionally , the diagnostic characteristics were calculated by considering the tac that signified a positive test ( malignant with early enhancement > 15 hu and early wash - out of 5 - 31 hu ) on a dynamic ct scan . fisher 's exact test and paired t - tests were used to analyze statistically significant differences between the malignant and benign nodules . moreover , this study used bland and altman plots ( 26 ) to compare the numerical values of enhancement ( pe and ne ) between using cad systems and those obtained by direct personal drawing . having calculated the mean difference and the 95% limits of agreement , the investigators determined if the methods were sufficiently in agreement for the new method to be used as an alternative . the malignant spns consisted of the following : adenocarcinoma ( n = 8) , squamous cell carcinoma ( n = 2 ) , bronchioloalveolar cell carcinoma ( n = 1 ) , mucoepidermoid carcinoma ( n = 1 ) and carcinoid tumor ( n = 1 ) . the benign spns were tuberculosis ( n = 9 ) , focal bronchopneumonia ( n = 7 ) , hamartoma ( n = 5 ) , fungal infection ( n = 2 ) , intrapulmonary lymph node ( n = 1 ) , fibrotic nodule ( n = 2 ) and sclerosing hemangioma ( n = 1 ) . higher pe and ne were observed by direct personal drawing and by using the cad system ( table 1 ) . as seen from the numerical value of the enhancement of the pe and ne , direct personal drawing showed higher enhancement than did the cad system . there were differences ( mean : 34.9 hu ; 95% ci : 28.0 - 41.8 ) between the pe using cad systems ( mean : 67.2 27.1 hu ) and the pe using the direct personal drawing ( mean : 32.3 28.1 hu ) . in addition , there were differences ( mean : 6.9 hu , 95% ci : 1.1 - 12.7 ) between the ne using the cad systems ( mean : 25.9 27.4 hu ) and the ne using direct personal drawing ( mean : 32.8 24.9 hu ) . on the other hand , as found from the enhancement patterns of pe and ne , the bland and altman plot showed that the mean difference was 95% of the individual differences 2 standard deriation ( sd ) ( fig . 2 ) . with 15 hu or more of ne as a cutoff value for differentiating malignant and benign nodules , the sensitivity for the diagnosis of malignant spns using the cad system was 92% ( 12/13 ) , the specificity was 52% ( 14/27 ) , the ppv was 48% ( 12/25 ) , the npv was 93% ( 14/15 ) and the accuracy was 65% ( 26/40 ) , respectively . however , the sensitivity , specificity , ppv , npv and the accuracy obtained by direct personal drawing of malignant spns was 100% ( 13/13 ) , 37% ( 10/27 ) , 43% ( 13/30 ) , 100% ( 10/10 ) , and 58% ( 23/40 ) , respectively ( table 2 ) . on the other hand , of the 27 benign nodules , the cad system and direct personal drawing of 25 nodules ( 92.6% ) showed a similar tac ( fig . of the 13 malignant nodules , the cad system and direct personal drawing of 10 nodules ( 83.3% ) showed a similar tac ( fig . the sensitivity , specificity , ppv , npv and accuracy obtained by the tac of the malignant spns and using cad was 92% ( 12/13 ) , 85% ( 23/27 ) , 75% ( 12/16 ) , 96% ( 23/24 ) and 88% ( 35/40 ) , respectively . the sensitivity , specificity , ppv , npv and accuracy obtained by the tac of the malignant spns and using direct drawing was 92% ( 12/13 ) , 89% ( 24/27 ) , 79% ( 14/16 ) , 92% ( 24/26 ) and 88% ( 35/40 ) , respectively . in this study , the analysis of the tac using the cad system and the results of the direct personal drawing produced good results for making the diagnosis of malignant spns on hdct , as compared with the using the pe and ne . the increase in attenuation of an spn after contrast media injection , that is , the pe and ne , depends on the blood supply and the volume of the extravascular fluid in the spn . however , the tacs of the spns represented differences in vascularity , the volume of extracellular fluid and the diffuse spread of contrast media . more contrast medium is delivered in malignant spns and the diffusion is faster compared with those of benign spns . for that reason , for better diagnostic accuracy of spns , the assessment of spns should be performed during the washout of contrast medium through the spn in addition to the wash - in , the so called tac . the tac analyses using the cad system showed similar specificity and accuracy to that of direct personal drawing . therefore , the use of the cad system for hdct images to evaluate the tac should help the thoracic radiologist differentiate between benign and malignant spns by reducing the time to calculate the spn enhancement via negating the requirement to draw the rois for all the enhancing nodules on the ct images ( 1 , 6 - 7 , 10 , 15 ) . ( 14 ) reported that cutoff values of 15 hu produced a sensitivity of 98% , a specificity of 58% and an accuracy of 77% for malignant nodules . since then , the cutoff value for diagnosing malignant nodules had been set at 15 hu . however , jeong et al . ( 15 ) reported that when the diagnostic criteria for malignancy of both a wash - in phase of 25 hu or greater and a washout phase of 5 - 31 hu were applied , the sensitivity , specificity and accuracy for malignancy were 94% , 90% and 92% , respectively . higher pe was obtained in the malignant spns for which multi - detector row ct ( mdct ) was used , and thus , higher attenuation values could be used as cutoff values for this differentiation . for differentiating malignant and benign nodules in our study , the cutoff value of the enhancing spns was determined to be 15 hu because the pe with using cad was lower than that of direct personal drawing in spite of performing mdct . we thought that the cutoff value to evaluate malignant spns could change according to the type of ct scan that 's used or the type of measurement that is used for the spns . in the past decade , many studies have reported that cad systems can help the radiologist detect spns on ct images ( 18 - 24 ) . ( 12 ) reported that the radiologists ' performance in interpreting the multi - detector row ct scans can be improved using cad systems . they compared the effect of two cad systems , the image checker ct ( r2 technologies , sunnyvale , ca ) and the nodule enhanced viewing ( nev ) ( siemens medical solutions , forchheim , germany ) for the detection of spn . regardless of the type of cad system used , they showed that the cad system might provide a more accurate diagnosis of spns . ( 21 ) showed that the sensitivities of the cad system for detecting nodules < 5 mm in diameter were higher than those of a radiologist . kim et al . ( 22 ) showed that a cad system may help improve the detection of nodules with ground - glass opacity or localized ground - glass opacity . however , most studies have focused on the size of the spn and not the spn 's enhancement . in this study , the tac using the cad system showed better results for diagnosing a malignant spn than visually evaluating the pe and ne . it is well known that the hemodynamic differences are significant because malignant nodules tend to be more enhanced than benign nodules . there have been some studies designed to evaluate using the enhancement pattern of the hdct images for diagnosing spns with employing a cad system . for this approach , some studies used a number of parameters to evaluate the spns with employing a cad system , e.g. , the volumetric size , shape , enhancement etc ( 12 , 18 - 20 ) . shah et al . ( 18 ) have recently used parameters such as the mean hu value , the median , mode and mean of the 32 highest hu values in the roi etc . the major difficulty with this approach is that the analysis is limited to a single slice and the analysis was restricted to only the two - dimensional features . therefore , in that study , the reviewing radiologist identified the most visually representative slice of the nodule and he drew the rois of the spns , while this experiment was performed helically through the entire nodule . this could result in a difference in diagnostic accuracy between the cad system and the radiologist 's direct drawing . in general , direct personal drawing tended to show higher enhancement than did the cad system because the radiologist tried to perform direct drawing within the spn as much as possible , but the cad system included lung parenchyma for detecting a spn when calculating the enhancement 's properties . as seen from the numerical value of the enhancement of the pe and ne , it seems that there is a difference between cad system and direct personal drawing . on the other hand , the bland - altman plot showed that the analyses of the pe and the ne using the cad system produced similar results to those obtained with direct personal drawing . some studies showed that the sensitivity and specificity of a cad system were lower than those of direct personal drawing because those studies used the morphologic character rather than the enhancement pattern of the spn ( 18 - 20 , 23 , 24 ) . but our study used the enhancement pattern of hdct images with using the cad system to evaluate the spns . therefore , unlike the previous studies , this study showed that the accuracy and specificity of a cad system was similar with that of direct personal drawing . although the cad system is a useful tool for improving the radiologist 's performance , the cad system has a major drawback in that the spn can not be differentiated from small peripheral vessels , most commonly branch points , scarring or other parenchymal heterogeneities . therefore , if the spn is a juxtavascular nodule , then the cad system might calculate the enhancement pattern of both the spn and it vascular component . in the case of a small sized spn , it is difficult to calculate the enhancement pattern of the spn with using a cad system . this might produce a false enhancement pattern of the spn . according , we excluded those cases in this study . first , there was an insufficient the number of cases ( n = 40 ) . therefore , before any type of clinical use could be considered , this proposed method will need to be validated on a larger dataset , i.e. , more cases will be needed to test for generality . accordingly , examination of more nodules of various sizes and shapes by ct will be needed for additional training and testing . second , although the variability in the rois of interest is substantial between radiologists , this was not evaluated in our study . the inadequate placement of the rois of interest on nodules might result in erroneous attenuation values , which can cause false wash - in and washout enhancement calculations . third , 5-minutes was chosen as the length for the delay as a rule of thumb , which we believe is sufficient for the washout of the contrast material from a pulmonary nodule . jeong et al . ( 1 ) reported that the mean time to pe in most malignant nodules was 3.2 minutes . ( 10 ) reported that most spns reached the peak level of enhancement within 3 minutes . they reported that with images acquired during 5 minutes or more , the washout dynamics of the pulmonary nodule enhancement could be obtained . therefore , 5 minutes was chosen as the length for the delay because the pe and ne were calculated and the pattern of the tac differentiated between the malignant nodules and the benign nodules . in addition , there was no attempt to standardize the injection rate and the volume of contrast material according to the cardiac output of each patient nor was there any attempt to standardize the volume of contrast material according to the patients ' weight . moreover , the calculated dose at all sites of the nodule is not same due to a direct influence of the radiation dose , such as the x - ray beam energy , the tube current , rotation of exposure time , the section thickness , the object 's thickness of attenuation , pitch , the sites of nodule etc . therefore , for the larger patient , the exit radiation would be much less intense due to its attenuation through more tissue ( 27 ) . the calculated dose at all sites of the nodules in this study was 30 - 40 mgy and this result was similar to the calculated dose of other reports ( 1 , 10 ) . despite these limitations , the results obtained from the enhancement pattern of the spns using the cad system were similar to those obtained by the direct personal drawing of spns . consequently , the cad system was a useful tool for diagnosing malignant solitary pulmonary nodules . also , in view of the good diagnostic results for a malignant spn , a tac may be more useful than the pe or ne . | objectivewe wanted to investigate the usefulness of a computer - aided diagnosis ( cad ) system in assisting radiologists to diagnosis malignant solitary pulmonary nodules ( spns ) , as compared with diagnosing spns with using direct personal drawing.materials and methodsforty patients with spns were analyzed .
after the pre - contrast scan was performed , an additional ten series of post - contrast images were obtained at 20-second intervals .
two investigators measured the attenuation values of the spns : a radiologist who drew the regions of interest ( rois ) , and a technician who used a cad system .
the bland and altman plots were used to compare the net enhancement between a cad system and direct personal drawing .
the diagnostic characteristics of the malignant spns were calculated by considering the cad and direct personal drawing and with using fisher 's exact test.resultson the bland and altman plot , the net enhancement difference between the cad system and direct personal drawing was not significant ( within 2 standard deriation ) . the sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and accuracy of diagnosing malignant spns using cad was 92% , 85% , 75% , 96% and 88% , respectively .
the sensitivity , specificity , ppv , npv and accuracy of diagnosing malignant spns using direct drawing was 92% , 89% , 79% , 92% and 88% , respectively.conclusionthe cad system was a useful tool for diagnosing malignant spns . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
single photon emission computed tomography ( spect ) is a tomographic technique that allows three - dimensional ( 3d ) visualization of biochemical or physiological processes in the human body by external detection of photons from an administered radiopharmaceutical . spect reveals the function of the body rather than its structure . however , its ability to depict a true activity map depends largely on the imaging properties of the spect system as well as on the methods used for converting the acquired data into values reflecting the activity distribution . the physical factors which severely affect the quantitative accuracy of spect images include attenuation and scatter . the magnitude of the error introduced by the photon attenuation depends upon tissue thickness , tissue path length and the type of the tissue . reconstruction of the tomographic images without attenuation correction can cause erroneously high count densities and reduced image contrast in low attenuation regions ( e.g. lung ) , which introduce errors when images are evaluated quantitatively.- the human body is mainly constituted of low atomic number ( z ) elements , i.e. soft tissue and water ; therefore , the dominant interaction process in the biological system is compton scattering . further , these factors are related to the density and composition of the body tissues , and it is therefore crucial to have access to individual attenuation maps to account for the attenuation and scatter effects when high quantitative accuracy is desired . have studied the impact of scattered photons during spect acquisition using various tc compounds and found that the scattered photons have a relatively minor impact on the reconstructed image quality . however , during ga spect imaging , large number of scattered photons appear in lower energy acquisition window as explained by farncombe et al . in order to solve the problem of photon attenuation , one needs to know the attenuation coefficient or attenuation map for the individual patient being studied . techniques have been developed to correct the attenuation and scattering in spect for single - energy photon emitters such as tc and multienergy emitters such as tl where there is no spillover in the transmission window due to overlapping of the emission and transmission windows . however , these techniques are not satisfactory for generating the patient - specific attenuation maps in the case of a multi - energy emitter such as ga , in which the emitted photon energy window overlaps with the transmission window for gd in addition to the downscatter . owing to the strong influence of the downscatter and the spillover in transmission map during simultaneous emission and transmission tomography , it is necessary to correct the transmission map before reconstruction of the emission images . it is therefore desirable to develop a correction method to produce a corrected attenuation map that can be used for attenuation correction in the emission reconstruction . in the present study , we made an attempt to correct the attenuation map in simultaneous transmission / emission tomography with gd / ga radioisotopes using maximum likelihood method using the expectation maximization ( ml - em ) algorithm . for the proposed study , we used the ml - em algorithm.- there are two steps in this method : ( 1 ) e - step , which computes the expected projection data for the current estimate and is equivalent to a forward projection , and ( 2 ) m - step , in which the expected value is compared to the measured projection data and then the estimated reconstructed image is updated by effectively backprojecting the ratio of the measured to estimated projections . in the algorithm , geometric response , attenuation , and scatter can be accounted for and modeled . attenuation of emitted radiation as it passes through the subject under study is accounted using transmission source and an attenuation map is obtained for image reconstruction . we developed a method to correct the transmission window for both the spillover and downscatter . we used three windows , two for emission , i.e. ga ( 93 and 184 kev ) , and one for transmission , i.e. gd ( 100 kev ) . multiple energy window scatter estimation was not carried out due to constraint in the available acquisition system ( philips / adac ) . the available system for transmission measurement consisted of two gd line sources in a dual head 90 configuration . in order to minimize the depth dependence of resolution , attenuation , spillover fraction and downscatter , the geometric mean ( gm ) of conjugate views the observed image in the transmission window is given by go = gt + ftgl + gut ( 1 ) where gois the observed transmission image , gt is the transmission image without contamination , gl is the lower energy window image , ft is the spillover fraction and gut is the downscatter image in transmission window . the measurements of the spillover fraction were performed using the adac vertex gamma camera . a point source having 130 mbq of ga in a 5-ml syringe a medium energy collimator was fitted to the gamma camera and images were acquired using a 128 128 matrix for 84 seconds . this duration was chosen so that it could accumulate 6 million counts in air to see the spillover fraction in the transmission window . spillover fraction was determined by varying the energy window setting of the lower energy window of ga as shown in [ table 1 ] . this was done by shifting the lower energy window of ga and transmission window ( no gd present ) from their estimated photopeak energy windows , i.e. 90 ( 14% ) and 105 ( 15% ) . spillover fraction ( ft ) ( % ) in the transmission window at different energy window setting experimental setup to measure the spillover fraction in air as a first approximation , the lower energy window was used to estimate the spillover fraction in air in the transmission window . multiplying the lower energy emission image with the spillover fraction ( ft ) the upper energy window was used to estimate the downscatter ( gut ) in the transmission window by convolving the upper energy emission image using bi - exponential function . rather than the monoexponential function used for photopeak scatter correction . gm data were used as first approximation assuming that the scatter function is spatially invariant . therefore , the scatter function can be written as s = 1/n{a * exp(br ) + exp ( cr ) } ( 2 ) where r is radial distance , a , b and c are the parameters for scatter function , and n preserves total counts by normalizing s to unity . then , convolved upper energy window is scaled by a scatter fraction ( kt ) before the subtraction of estimated downscatter from the spillover corrected transmission window . the estimated corrected transmission image ( gt ) { ftgl + kt ( s gu ) } ( 3 ) where gl is the image in 93 kev window , gu is the image in 184 kev window and ktis a scatter fraction ( in transmission window ) . spillover fraction , scatter fraction and a , b and c parameters for the scatter function were determined experimentally and optimized using the optimization program written in idl [ figure 2 ] based on simplex theory . the sum of absolute differences ( sad ) between the measured spillover and downscatter image ( gmt ) and estimated spillover and downscatter image ( mt ) is calculated and can be given by : schematic diagram for determination of a , b , c , kt and ft by using optimization program there were considerable variations in the values for kt and ftwith some degree of interdependency . to reduce the variation and interdependency of kt and ft , we fixed the a , b and c parameters ( a = 49.22 , b = 1.56 , c = 0.122 ) for the scatter function and repeated the optimization procedure . the results for the repeat optimization are given in table 2 . it was found that these parameters have slight variation compared to the results shown in table 3 . however , there was still considerable variation observed for different source depths and materials . these results were evaluated graphically by plotting ktand ft against d ( d is the attenuation path length obtained by multiplying the narrow beam attenuation coefficient by source depth d ) . the results for kt with fixed scatter function parameters could be described by a single buildup relation ( equation ( 4 ) ) which is independent of material : values of kt and ft when a , b and c values are fixed for different materials at various depths values of a , b , c , kt and ft values for different materials at different thicknesses ( cm ) where a , b , and are constants and d is attenuation path length . the correlation between measured kt values and those predicted from the buildup equation was high ( r = 0.976 ) . the chi - squared values obtained during curve fitting reduced from 0.034 to 0.031 for the data obtained with non - fixed versus fixed scatter function parameters . it was evident from the observations that ft had a linear relationship with source depth , which differed significantly for the different materials . the variation of individual values from the best fitted line was reduced when the scatter function parameters were fixed . the pattern of linear slope was similar for different materials [ figure 3 ] . in an attempt to further improve the ftdata , another optimization was performed using fixed values for a , b , c , while ktvalues were obtained from the fitted curve [ figure 4 ] . the results for kt suggest that externally measured transmission data can be used to estimate the scatter fraction pixel by pixel . however , it is not possible to distinguish depth from material using an external transmission measurement . therefore , a single linear equation ( a + b * d ) was used to fit the ft data with moderate success ( r = 0.70 ) as shown in figure 5 . ft versus d(a , b , care fixed ) kt versus d(a , b , c are fixed ) spillover fraction ( ft ) versus d fitted values of ft and kt for different t/2 for different materials if the point source is measured from conjugate views and the geometric mean of those views is calculated , then the spillover fraction ( ft ) and scatter fraction ( kt ) are approximately equivalent to those from a source at the center of the object.[1213 ] therefore , ft and kt in the transmission window are approximately equivalent to those from a source at the center of the object irrespective of the depth . the d can be replaced by t/2 , ( ft= a + b * t/2 ) in the linear equation and buildup equation ( 1 ) can be rewritten as : where t is the total thickness of the object . in most cases , b is set to ( a 1 ) , which results in kt = 0 in air , outside the attenuating object , where e = 1 . these values of ft and kt were used when applying the convolution subtraction method in the phantom study . in air , scattered photons are not present in the beam ; therefore , spillover fraction in the transmission window due to 90 kev would be expected to remain constant . however , in a medium where high - energy photons of ga interact by compton scattering , there is loss of energy and some photons are detected in the transmission window ( 105 kev ) as well as in the lower energy window ( 90 kev ) . scattering depends on the density of the materials ; therefore , downscatter also increases with the density and thickness of the object . the scatter due to the 93 kev photon ( i.e. photopeak scatter ) also increases with thickness and density of material . as a result , the ratio of counts ( spillover ) for the 105 kev to 90 kev windows is a complex mixture of unscattered and scattered photons . the spillover fraction in the transmission window ( gt ) is estimated by the spillover fraction ft multiplied by the lower energy window ( gl ) of ga . to account for regional differences in scattering material , a two - dimensional matrix of spillover fractions ( ft ) which is the set of ft for the projection pixels can be calculated using the linear equation which is derived from the fitted curve ( ft vs. d ) [ figure 5 ] . similarly , the buildup equation establishes a relationship between ktand d as shown in figure 4 . therefore , kt also varies with density and thickness of the object . the scatter fraction kt is replaced by kt to account for variation in scatter fraction pixel by pixel . therefore , the equation for the corrected transmission image can be written as : solution of this equation is only possible by an iterative process since ft and kt are functions of path length as estimated by transmission measurement . the method was validated for ga / gd emission transmission tomography by variable density ( anthropomorphic thorax ) phantom using various parameters for scatter function ( s ) , scatter fraction ( kt ) , spillover fraction ( ft ) and spatial window width ( swt ) 13% ( 13% of the field of view is exposed by the transmission window at any time ) for a more realistic situation . measurements were performed on a vertex gamma camera ( philips dual head ) with the medium energy collimator . the reference scan consists of two static images that are used to calculate the t . the imaging table was removed and the line source of gd was used to acquire the reference scan in air using 105 kev ( 15% window width ) with a 128 128 matrix size . a true transmission scan was acquired using 128 128 matrix size for three photopeak energy windows with centerlines and widths of 90 kev ( 14% ) , 105 kev ( 15% ) and 184 kev ( 20% ) to compare with the corrected transmission scan . simultaneous emission and transmission scans were performed using the ga / gd . the matrix size and window settings were kept same to acquire the true transmission map , uncorrected transmission map and emission image of thorax phantom . two syringes ( 20 ml ) having 50 mbq and 110 mbq of ga in 15 ml solution were placed in the lung and liver of the phantom , respectively . the measurements of the spillover fraction were performed using the adac vertex gamma camera . a point source having 130 mbq of ga in a 5-ml syringe was placed in air at 15 cm from the face of the detector . a medium energy collimator was fitted to the gamma camera and images were acquired using a 128 128 matrix for 84 seconds . this duration was chosen so that it could accumulate 6 million counts in air to see the spillover fraction in the transmission window . spillover fraction was determined by varying the energy window setting of the lower energy window of ga as shown in [ table 1 ] . this was done by shifting the lower energy window of ga and transmission window ( no gd present ) from their estimated photopeak energy windows , i.e. 90 ( 14% ) and 105 ( 15% ) . spillover fraction ( ft ) ( % ) in the transmission window at different energy window setting experimental setup to measure the spillover fraction in air as a first approximation , the lower energy window was used to estimate the spillover fraction in air in the transmission window . multiplying the lower energy emission image with the spillover fraction ( ft ) the upper energy window was used to estimate the downscatter ( gut ) in the transmission window by convolving the upper energy emission image using bi - exponential function . rather than the monoexponential function used for photopeak scatter correction . gm data were used as first approximation assuming that the scatter function is spatially invariant . therefore , the scatter function can be written as s = 1/n{a * exp(br ) + exp ( cr ) } ( 2 ) where r is radial distance , a , b and c are the parameters for scatter function , and n preserves total counts by normalizing s to unity . then , convolved upper energy window is scaled by a scatter fraction ( kt ) before the subtraction of estimated downscatter from the spillover corrected transmission window . the estimated corrected transmission image ( gt ) { ftgl + kt ( s gu ) } ( 3 ) where gl is the image in 93 kev window , gu is the image in 184 kev window and ktis a scatter fraction ( in transmission window ) . spillover fraction , scatter fraction and a , b and c parameters for the scatter function were determined experimentally and optimized using the optimization program written in idl [ figure 2 ] based on simplex theory . the sum of absolute differences ( sad ) between the measured spillover and downscatter image ( gmt ) and estimated spillover and downscatter image ( mt ) is calculated and can be given by : schematic diagram for determination of a , b , c , kt and ft by using optimization program there were considerable variations in the values for kt and ftwith some degree of interdependency . to reduce the variation and interdependency of kt and ft , we fixed the a , b and c parameters ( a = 49.22 , b = 1.56 , c = 0.122 ) for the scatter function and repeated the optimization procedure . it was found that these parameters have slight variation compared to the results shown in table 3 . however , there was still considerable variation observed for different source depths and materials . these results were evaluated graphically by plotting ktand ft against d ( d is the attenuation path length obtained by multiplying the narrow beam attenuation coefficient by source depth d ) . the results for kt with fixed scatter function parameters could be described by a single buildup relation ( equation ( 4 ) ) which is independent of material : values of kt and ft when a , b and c values are fixed for different materials at various depths values of a , b , c , kt and ft values for different materials at different thicknesses ( cm ) where a , b , and are constants and d is attenuation path length . the correlation between measured kt values and those predicted from the buildup equation was high ( r = 0.976 ) . the chi - squared values obtained during curve fitting reduced from 0.034 to 0.031 for the data obtained with non - fixed versus fixed scatter function parameters . it was evident from the observations that ft had a linear relationship with source depth , which differed significantly for the different materials . the variation of individual values from the best fitted line was reduced when the scatter function parameters were fixed . the pattern of linear slope was similar for different materials [ figure 3 ] . in an attempt to further improve the ftdata , another optimization was performed using fixed values for a , b , c , while ktvalues were obtained from the fitted curve [ figure 4 ] . the results for kt suggest that externally measured transmission data can be used to estimate the scatter fraction pixel by pixel . however , it is not possible to distinguish depth from material using an external transmission measurement . therefore , a single linear equation ( a + b * d ) was used to fit the ft data with moderate success ( r = 0.70 ) as shown in figure 5 . ft versus d(a , b , care fixed ) kt versus d(a , b , c are fixed ) spillover fraction ( ft ) versus d fitted values of ft and kt for different t/2 for different materials if the point source is measured from conjugate views and the geometric mean of those views is calculated , then the spillover fraction ( ft ) and scatter fraction ( kt ) are approximately equivalent to those from a source at the center of the object.[1213 ] therefore , ft and kt in the transmission window are approximately equivalent to those from a source at the center of the object irrespective of the depth . the d can be replaced by t/2 , ( ft= a + b * t/2 ) in the linear equation and buildup equation ( 1 ) can be rewritten as : where t is the total thickness of the object . in most cases , b is set to ( a 1 ) , which results in kt = 0 in air , outside the attenuating object , where e = 1 . these values of ft and kt were used when applying the convolution subtraction method in the phantom study . in air , scattered photons are not present in the beam ; therefore , spillover fraction in the transmission window due to 90 kev would be expected to remain constant . however , in a medium where high - energy photons of ga interact by compton scattering , there is loss of energy and some photons are detected in the transmission window ( 105 kev ) as well as in the lower energy window ( 90 kev ) . scattering depends on the density of the materials ; therefore , downscatter also increases with the density and thickness of the object . the scatter due to the 93 kev photon ( i.e. photopeak scatter ) also increases with thickness and density of material . as a result , the ratio of counts ( spillover ) for the 105 kev to 90 kev windows is a complex mixture of unscattered and scattered photons . the spillover fraction in the transmission window ( gt ) is estimated by the spillover fraction ft multiplied by the lower energy window ( gl ) of ga . to account for regional differences in scattering material , ft is replaced by the pixel by pixel spillover fraction ft . a two - dimensional matrix of spillover fractions ( ft ) which is the set of ft for the projection pixels can be calculated using the linear equation which is derived from the fitted curve ( ft vs. d ) [ figure 5 ] . similarly , the buildup equation establishes a relationship between ktand d as shown in figure 4 . the scatter fraction kt is replaced by kt to account for variation in scatter fraction pixel by pixel . therefore , the equation for the corrected transmission image can be written as : solution of this equation is only possible by an iterative process since ft and kt are functions of path length as estimated by transmission measurement . the method was validated for ga / gd emission transmission tomography by variable density ( anthropomorphic thorax ) phantom using various parameters for scatter function ( s ) , scatter fraction ( kt ) , spillover fraction ( ft ) and spatial window width ( swt ) 13% ( 13% of the field of view is exposed by the transmission window at any time ) for a more realistic situation . measurements were performed on a vertex gamma camera ( philips dual head ) with the medium energy collimator . the reference scan consists of two static images that are used to calculate the t . the imaging table was removed and the line source of gd was used to acquire the reference scan in air using 105 kev ( 15% window width ) with a 128 128 matrix size . a true transmission scan was acquired using 128 128 matrix size for three photopeak energy windows with centerlines and widths of 90 kev ( 14% ) , 105 kev ( 15% ) and 184 kev ( 20% ) to compare with the corrected transmission scan . simultaneous emission and transmission scans were performed using the ga / gd . the matrix size and window settings were kept same to acquire the true transmission map , uncorrected transmission map and emission image of thorax phantom . two syringes ( 20 ml ) having 50 mbq and 110 mbq of ga in 15 ml solution were placed in the lung and liver of the phantom , respectively . the aim of using thorax phantom was to assess the transmission - based correction method in a more realistic imaging situation . the results were assessed by defining two rois ( region of interests ) over the contaminated region of lung and liver in the uncorrected transmission scan . these rois were applied to the uncorrected , corrected and the true transmission scans and the mean counts were recorded [ table 5 ] . it was observed from the mean counts that where no spillover and downscatter correction was applied in the transmission scan ( uncorrected ) of liver and lung , the mean counts were 19.29 and 26.90 , respectively . after applying the transmission - based spillover and downscatter corrections , the mean counts were reduced to 3.80 and 15.10 in liver and lung , respectively . these mean counts were close to mean counts of liver 2.15 and lung 14.89 of the true transmission scans . therefore , using the transmission - based correction methods for spillover and downscatter in the uncorrected transmission scan , the quantitative error was reduced by 90.0% and 98.2% in liver and lung regions , respectively . the results were also evaluated by examining the count profiles through lungs and liver of the uncorrected , corrected and true transmission scans as shown in figures 6 and
7 . before the correction , there was significant difference between the profiles of both liver and lungs of uncorrected and true transmission scans . after performing the transmission - based downscatter and spillover corrections , the profiles through the liver and lungs of the corrected transmission scan and true transmission scan showed excellent agreement . the results for the thorax phantom demonstrated the importance of using transmission data for accurate downscatter and spillover correction in the transmission window . the average downscatter and spillover fractions varied considerably from subject to subject and were difficult to correct without transmission data . furthermore , it is inappropriate to use a constant spillover fraction and downscatter fraction when the object has non - uniform density . the relationship between d , ft and kt for this combination of radionuclides ( 67ga/153gd ) can be established and used to implement transmission - based spillover and downscatter correction , which takes into account the effect of variable tissue density and the object thickness on the spillover and downscatter distribution . comparison between uncorrected , corrected and true transmission scans ( liver ) for the anthropomorphic thorax phantom comparison between uncorrected , corrected and true transmission scans ( lung ) for the anthropomorphic thorax phantom quantitative accuracy using transmission - based downscatter and spillover corrections in the anthropomorphic thorax phan in the proposed new method , we introduced the set of ft(spillover ) and kt ( downscatter ) to account for the variations in projection pixels ( ft and kt ) with the density and thickness of the objects . the quantitative error was reduced by 98.0% for lung and 90.0% for liver in thorax phantom when the corrected transmission images were obtained after the subtraction of spillover and downscatter fractions from the contaminated transmission images . it is therefore concluded that the quantitative error in the contaminated transmission image can be considerably reduced using the proposed transmission - based scatter correction method . | reconstruction of the tomographic images without attenuation correction can cause erroneously high count densities and reduced image contrast in low attenuation regions . in order to solve the problem of photon attenuation , one needs to know the attenuation coefficient for the individual patient being studied .
therefore , we made an attempt to correct the attenuation map in simultaneous transmission / emission tomography with 153gd/67ga using maximum likelihood method using the expectation maximization ( ml - em ) algorithm to correct the transmission window for both the spillover and downscatter .
spillover fraction , scatter fraction and parameters for the scatter function ( a , b and c ) were determined experimentally and optimized using the optimization program written in idl based on simplex theory .
all measurements were performed on a vertex gamma camera using the anthropomorphic thorax phantom for validation of data obtained by the proposed method .
it was observed that without spillover and downscatter correction , the mean counts were 19.29 in liver and 26.90 in lung , whereas after after applying the corrections , the mean counts were reduced to 3.80 and 15.10 in liver and lung , respectively , which were close to true mean counts ( liver 2.15 and lung 14.89 ) . in this proposed method , we introduced the set of ft(spillover ) and kt(downscatter ) to account for the variations in projection pixels ( ft and kt ) with the density and thickness .
the ft and kt were determined using the transmission data by an iterative process .
the quantitative error was reduced by 98.0% for lung and 90.0% for liver when the corrected transmission images were obtained after the subtraction of spillover and downscatter fraction . |
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one of the most common chronic diseases associated with aging is type 2 diabetes mellitus ( dm-2 ) . the increasing number of older individuals developing dm-2 in north america is an enormous clinical concern , as this disease is associated with excessive mortality from cardiovascular disease ( cvd ) . the increased cvd - related morbidity and mortality associated with dm-2 may be attributed to the clustering of synergistic co - morbidities such as hypertension , dyslipidemia and obesity . however , recent evidence suggests that cardiac and vascular maladaptations , such as impaired left ventricular ( lv ) filling dynamics and increased arterial stiffness , may also contribute to these observations . exercise training has been shown to reduce cvd mortality and morbidity with senescence possibly by attenuating age - associated declines in lv filling dynamics , arterial compliance and endothelial control of vascular tone . as such , it has been hypothesized that the cardiovascular maladaptations associated with aging are secondary to reduced physical activity patterns rather than senescence . several lines of evidence suggest that the cardiovascular and metabolic maladaptations to dm-2 may also be attenuated with increased physical activity patterns . specifically , remarkably similar improvements in glucose uptake , cardiorespiratory fitness and oxygen uptake kinetics have been documented following exercise training between individuals with dm-2 and age - matched healthy controls . furthermore , data from our laboratory suggests that low cardiorespiratory fitness is associated with a decline in arterial compliance and significantly elevated c - reactive protein expression in postmenopausal women with dm-2 . we therefore proposed that exercise training favorably modulates cardiovascular parameters associated with cvd in individuals with dm-2 . the primary purpose of this investigation was to examine the effects of exercise training on known correlates of cardiorespiratory fitness , mainly lv filling dynamics and arterial compliance , in women with dm-2 . the primary hypothesis was that a 10-week exercise training intervention in women with dm-2 would improve cardiorespiratory fitness subsequent to improvements in lv diastolic filling properties and arterial compliance . a total of 28 postmenopausal women with clinically documented dm-2 , free from diabetic complications , were screened for underlying coronary artery disease with resting and maximal exercise electrocardiograms . exclusion criteria for this investigation included : evidence of ischemic heart disease by history or positive resting or exercise electrocardiogram ; angina or any other cardiac symptoms potentially limiting exercise capacity ; and/or presence of musculoskeletal or peripheral vascular abnormalities that would limit exercise capacity . written informed consent was obtained from all subjects prior to the investigation and the research ethics review board within the faculty of medicine at the university of alberta approved the study protocol . prior to exercise training , subjects were evaluated over the course of two separate visits . initially , participants reported to the university of alberta hospital , division of cardiology for a history and physical examination as well as a resting and exercise electrocardiogram . a graded exercise test was performed on an electronically braked cycle ergometer to determine ventilatory threshold and cardiorespiratory fitness . on the second visit , subjects reported to the metabolic unit following an overnight fast for blood work , applanation tonometry and resting echocardiograms . a period of at least 48 hours separated the two visits , to allow for complete recovery of the acute effects of the exercise test . following baseline testing , participants were assigned to either control ( ct ) or exercise training ( et ) groups . the data reported here are a combination of pilot data collected in 10 subjects and a second study in which 18 patients were randomly distributed into two groups ( exercise or control ) . of the 28 originally screened patients , four women were initially excluded due to either positive stress test or inability to perform the exercise training requirements of the investigation . therefore 24 women were included in the initial investigation ( et = 17 , ct = 7 ) . six women were excluded from the analysis due to poor adherence to the exercise intervention . . therefore data from18 women were included in the final analysis of this investigation ( et = 11 ; ct = 7 ) . the remaining eleven participants in the et group completed 92 3% of the exercise sessions . individuals in this investigation were treated for diabetes through diet and exercise ( n = 5 ) or oral hypoglycemic agents ( n = 13 ) , while no participants were on insulin therapy . oxygen consumption , carbon dioxide production and minute ventilation were sampled every five seconds at rest and during exercise using a parvo medics truemax 2400 metabolic cart ( parvo medics , east sandy , ut ) . the stress test began at an exercise intensity of 30 watts and increased by 20 watts every two minutes . heart rate ( electrocardiogram ) , blood pressure ( auscultation ) and ratings of perceived exertion were recorded at the end of every two - minute stage . peak rate of oxygen consumption ( ) was determined from the average rate of oxygen consumption over the final 60 s of the exercise test . the heart rate response to graded exercise was used to prescribe exercise intensity during the exercise intervention . resting measurements of vascular function were performed in the morning with the participants fasted , lying supine in a dimly lit room , having refrained from vasoactive medications for a period of 48 hours prior to testing . this technique involves 30-s recordings of signal - averaged arterial pulse waves by applanation tonometry using a surface residing pressure transducer on the radial artery ( hypertension diagnostics inc . two components of arterial compliance were calculated based on a modified windkessel model of circulation : capacitive compliance ( large artery ) , and oscillatory ( or reflective ) compliance . imaging of the lv was performed as previously described . in brief , all images were captured using a commercially available ultrasound instrument ( hewlett packard , sonos 5500 ) with a 3.5 mhz transducer by a trained technician . the same technician acquired and analaysed pre and post - intervention images for each participant . subjects were required to lie quietly for a period of 510 minutes , in a dimly lit quiet room prior to imaging . two - dimensional transthoracic images of the lv were obtained from the parasternal short - axis view at the level of the mid - papillary muscles according to american society of echocardiography guidelines . lv diastolic filling dynamics were assessed using pulsed - wave doppler analysis of transmitral and pulmonary venous flow patterns recorded in the apical four - chamber view . the following haematological parameters were determined using common laboratory procedures [ 14 - 17 ] : glucose , insulin , glycosylated hemoglobin ( hba1c ) , total cholesterol , high and density low lipoproteins and triglycerides . plasma lipids [ 15 - 17 ] , glucose and hba1c were determined on a synchron lx20 analyzer ( beckman coulter , fullerton ca ) . the homeostasis model assessment ( homa index ) the 10-week exercise intervention consisted of three supervised exercise sessions per week and included a combination of aerobic and resistance training . the aerobic component of the exercise program was performed on a cycle ergometer for 3055 minutes at intensities between 65 and 75% of heart rate reserve . the intensity and duration of the aerobic component of the exercise intervention increased weekly so that each participant was performing 55 minutes of aerobic exercise at ~75% of heart rate reserve in the final week of the training program . resistance training consisted of three sets of 1015 repetitions at a resistance between 50 and 65% of one - repetition maximum in large muscle groups , using the following exercises : chest press , latissimus dorsi pull down , shoulder press , biceps curls , leg press , leg curl and leg extension . the resistance applied to each apparatus was increased each week so that each participant was performing three sets of 10 repetitions at 6570% of the original one - repetition maximum . control subjects were asked to continue their previous physical activity patterns throughout the 10-week intervention period . all statistical analyses were performed using spss software ( version 11.0 , spss , chicago , il ) . a repeated measures anova was used to compare the effects of an exercise and control condition on lv filling dynamics , arterial compliance and risk factors for cvd . tukey post - hoc analyses were performed to determine group differences . a p value of < 0.05 was considered statistically significant . a total of 28 postmenopausal women with clinically documented dm-2 , free from diabetic complications , were screened for underlying coronary artery disease with resting and maximal exercise electrocardiograms . exclusion criteria for this investigation included : evidence of ischemic heart disease by history or positive resting or exercise electrocardiogram ; angina or any other cardiac symptoms potentially limiting exercise capacity ; and/or presence of musculoskeletal or peripheral vascular abnormalities that would limit exercise capacity . written informed consent was obtained from all subjects prior to the investigation and the research ethics review board within the faculty of medicine at the university of alberta approved the study protocol . prior to exercise training , subjects were evaluated over the course of two separate visits . initially , participants reported to the university of alberta hospital , division of cardiology for a history and physical examination as well as a resting and exercise electrocardiogram . a graded exercise test was performed on an electronically braked cycle ergometer to determine ventilatory threshold and cardiorespiratory fitness . on the second visit , subjects reported to the metabolic unit following an overnight fast for blood work , applanation tonometry and resting echocardiograms . a period of at least 48 hours separated the two visits , to allow for complete recovery of the acute effects of the exercise test . following baseline testing , participants were assigned to either control ( ct ) or exercise training ( et ) groups . the data reported here are a combination of pilot data collected in 10 subjects and a second study in which 18 patients were randomly distributed into two groups ( exercise or control ) . of the 28 originally screened patients , four women were initially excluded due to either positive stress test or inability to perform the exercise training requirements of the investigation . therefore 24 women were included in the initial investigation ( et = 17 , ct = 7 ) . six women were excluded from the analysis due to poor adherence to the exercise intervention . therefore data from18 women were included in the final analysis of this investigation ( et = 11 ; ct = 7 ) . the remaining eleven participants in the et group completed 92 3% of the exercise sessions . individuals in this investigation were treated for diabetes through diet and exercise ( n = 5 ) or oral hypoglycemic agents ( n = 13 ) , while no participants were on insulin therapy . oxygen consumption , carbon dioxide production and minute ventilation were sampled every five seconds at rest and during exercise using a parvo medics truemax 2400 metabolic cart ( parvo medics , east sandy , ut ) . the stress test began at an exercise intensity of 30 watts and increased by 20 watts every two minutes . heart rate ( electrocardiogram ) , blood pressure ( auscultation ) and ratings of perceived exertion were recorded at the end of every two - minute stage . peak rate of oxygen consumption ( ) was determined from the average rate of oxygen consumption over the final 60 s of the exercise test . the heart rate response to graded exercise was used to prescribe exercise intensity during the exercise intervention . resting measurements of vascular function were performed in the morning with the participants fasted , lying supine in a dimly lit room , having refrained from vasoactive medications for a period of 48 hours prior to testing . this technique involves 30-s recordings of signal - averaged arterial pulse waves by applanation tonometry using a surface residing pressure transducer on the radial artery ( hypertension diagnostics inc . two components of arterial compliance were calculated based on a modified windkessel model of circulation : capacitive compliance ( large artery ) , and oscillatory ( or reflective ) compliance . imaging of the lv was performed as previously described . in brief , all images were captured using a commercially available ultrasound instrument ( hewlett packard , sonos 5500 ) with a 3.5 mhz transducer by a trained technician . the same technician acquired and analaysed pre and post - intervention images for each participant . subjects were required to lie quietly for a period of 510 minutes , in a dimly lit quiet room prior to imaging . two - dimensional transthoracic images of the lv were obtained from the parasternal short - axis view at the level of the mid - papillary muscles according to american society of echocardiography guidelines . lv diastolic filling dynamics were assessed using pulsed - wave doppler analysis of transmitral and pulmonary venous flow patterns recorded in the apical four - chamber view . the following haematological parameters were determined using common laboratory procedures [ 14 - 17 ] : glucose , insulin , glycosylated hemoglobin ( hba1c ) , total cholesterol , high and density low lipoproteins and triglycerides . plasma lipids [ 15 - 17 ] , glucose and hba1c were determined on a synchron lx20 analyzer ( beckman coulter , fullerton ca ) . the homeostasis model assessment ( homa index ) the 10-week exercise intervention consisted of three supervised exercise sessions per week and included a combination of aerobic and resistance training . the aerobic component of the exercise program was performed on a cycle ergometer for 3055 minutes at intensities between 65 and 75% of heart rate reserve . the intensity and duration of the aerobic component of the exercise intervention increased weekly so that each participant was performing 55 minutes of aerobic exercise at ~75% of heart rate reserve in the final week of the training program . resistance training consisted of three sets of 1015 repetitions at a resistance between 50 and 65% of one - repetition maximum in large muscle groups , using the following exercises : chest press , latissimus dorsi pull down , shoulder press , biceps curls , leg press , leg curl and leg extension . the resistance applied to each apparatus was increased each week so that each participant was performing three sets of 10 repetitions at 6570% of the original one - repetition maximum . control subjects were asked to continue their previous physical activity patterns throughout the 10-week intervention period . all statistical analyses were performed using spss software ( version 11.0 , spss , chicago , il ) . a repeated measures anova was used to compare the effects of an exercise and control condition on lv filling dynamics , arterial compliance and risk factors for cvd . tukey post - hoc analyses were performed to determine group differences . a p value of < 0.05 was considered statistically significant . , there were no significant differences in conventional risk factors for cvd , such as age , body mass index , systolic blood pressure , lipid profile , hormone replacement therapy or fasting insulin or glucose levels between the two groups . no differences in peak exercise variables were observed between the et and ct groups at baseline . the average respiratory exchange ratio values at peak exercise in both groups did not exceed 1.10 , however they were not significantly different between the groups ( 1.06 0.04 vs 1.05 0.05 in the et and ct groups respectively ) . bmi = body mass index ; tc = total cholesterol ; hdl = high density lipoprotein ; ldl = low density lipoprotein ; hba1c = glycosylated hemoglobin ; homa = homeostasis model assessment of insulin sensitivity ; d / o = diet and exercise / oral diabetic therapy * = p < 0.05 vs pre - training resting hemodynamics prior to and following the exercise intervention are provided in table 2 . after 10 weeks of exercise training , increased by approximately 15% ( 21.3 3.3 to 24.5 4.2 mlkgmin ; p < 0.05 ) in the et group . maximal heart rate and respiratory exchange ratio values at follow - up testing were similar to baseline values , suggesting similar effort for both tests . cardiorespiratory fitness was not assessed in the ct group during follow - up testing . the impact of exercise training on resting hemodynamics in postmenopoausal women with type 2 diabetes . sbp = systolic blood pressure ; dbp = diastolic blood pressure ; = cardiorespiratory fitness ; c1= large artery compliance ; c2 = small artery compliance ; svr = systemic vascular resistance . * = p < 0.05 vs pre = p < 0.05 vs et fasting blood glucose , insulin and lipid profile before and after the 10-week intervention period are presented in table 1 . hematological variables were not significantly different in either group following the intervention time period . the homa index of insulin sensitivity decreased by ~17% in the et while it did not change in ct . lv filling dynamics , estimated from doppler - derived transmitral and pulmonary venous flow profiles , were not significantly different following the intervention time period in either group ( table 2 ) . in the et group , large artery compliance increased by ~16% in response to exercise training ( p < 0.05 ) , while small artery compliance remained unchanged . , there were no significant differences in conventional risk factors for cvd , such as age , body mass index , systolic blood pressure , lipid profile , hormone replacement therapy or fasting insulin or glucose levels between the two groups . no differences in peak exercise variables were observed between the et and ct groups at baseline . the average respiratory exchange ratio values at peak exercise in both groups did not exceed 1.10 , however they were not significantly different between the groups ( 1.06 0.04 vs 1.05 0.05 in the et and ct groups respectively ) . bmi = body mass index ; tc = total cholesterol ; hdl = high density lipoprotein ; ldl = low density lipoprotein ; hba1c = glycosylated hemoglobin ; homa = homeostasis model assessment of insulin sensitivity ; d / o = diet and exercise / oral diabetic therapy * = p < 0.05 vs pre - training resting hemodynamics prior to and following the exercise intervention are provided in table 2 . after 10 weeks of exercise training , increased by approximately 15% ( 21.3 3.3 to 24.5 4.2 mlkgmin ; p < 0.05 ) in the et group . maximal heart rate and respiratory exchange ratio values at follow - up testing were similar to baseline values , suggesting similar effort for both tests . the impact of exercise training on resting hemodynamics in postmenopoausal women with type 2 diabetes . sbp = systolic blood pressure ; dbp = diastolic blood pressure ; = cardiorespiratory fitness ; c1= large artery compliance ; c2 = small artery compliance ; svr = systemic vascular resistance . * = p < 0.05 vs pre = p < 0.05 vs et fasting blood glucose , insulin and lipid profile before and after the 10-week intervention period are presented in table 1 . hematological variables were not significantly different in either group following the intervention time period . the homa index of insulin sensitivity decreased by ~17% in the et while it did not change in ct . these changes in the homa index however , lv filling dynamics , estimated from doppler - derived transmitral and pulmonary venous flow profiles , were not significantly different following the intervention time period in either group ( table 2 ) . in the et group , large artery compliance increased by ~16% in response to exercise training ( p < 0.05 ) , while small artery compliance remained unchanged . the major novel finding of this investigation is that 10 weeks of exercise training elicited an increase in both large artery compliance and cardiorespiratory fitness in women with dm-2 without any appreciable change in lv filling dynamics or conventional cvd risk factors . significant negative correlations have been observed between cardiorespiratory fitness and arterial stiffness , suggesting that exercise training may modulate central arterial compliance in healthy older individuals . more importantly , regular physical activity has been shown to attenuate the age - related decline in arterial compliance in healthy older men and women . the findings from this investigation support the concept that exercise training favorably alters arterial stiffness in older women and extends these findings to women with dm-2 . improvements in arterial compliance have been observed following lipid lowering therapy , however any appreciable changes in lipids following exercise training were not observed in this sample of women with dm-2 . although a slight improvement in the homa index ( a marker of insulin sensitivity ) was observed following exercise training , the changes in the homa index were unrelated to changes in arterial compliance following the exercise intervention . therefore the observed improvement in arterial compliance with exercise training in women with dm-2 in this investigation can not be attributed to changes in lipid or glucose metabolism . improved endothelium - mediated vasodilatation or a reduction in intima - media wall thickness it is possible that the changes in large artery compliance observed with exercise training in this study were observed secondary to an improvement in endothelial dependent dilatation as exercise training - mediated improvements in flow mediated dilatation of the brachial artery have recently been documented in persons with dm-2 . lv diastolic function is believed to be an important determinant of cardiorespiratory fitness in healthy individuals . data from cross - sectional and intervention studies examining the role of exercise training on lv filling dynamics ( a surrogate of lv diastolic function ) in older individuals are inconclusive [ 6,24 - 26 ] . while some demonstrate that exercise training attenuates age - related declines in lv filling dynamics others do not . contrary to the findings reported here , poirier and colleagues have demonstrated a relationship between cardiorespiratory fitness and lv filling dynamics in normotensive men with dm-2 . the data presented here support and extend the findings of spina and associates , demonstrating that lv adaptations to exercise training are limited in post menopausal women and extend them to women with dm-2 . several differences between this investigation and that of others may explain discrepant findings with regards to the lv adaptation to exercise training . first , the training stimulus employed in this investigation may not have been of sufficient duration ( 10 weeks ) or frequency ( three days / week ) to elicit appreciable changes in the structure or cellular properties of the lv detectable with doppler - derived transmitral flow patterns . had the intervention been extended to 12 months , as others have done it is possible that lv filling dynamics may have been altered . it is apparent however that gender differences in the lv adaptation to exercise training exist . the findings presented here support the concept of a limited lv adaptation to aerobic exercise training in older women and extend them to older women with dm-2 . the results presented here have several clinical implications . up to 80% of all deaths in persons with dm-2 are cardiovascular in nature , possibly secondary to an increase in arterial stiffness that has been reported in this population . the results of this study demonstrate that a brief exercise intervention in women with dm-2 enhances arterial compliance . as increased arterial stiffness is considered a risk for cvd , these data suggest that exercise may reduce the risk the risk for cvd in women with dm-2 by reducing arterial stiffness . more importantly , these adaptations occurred despite little or no change in blood pressure , cholesterol profile or insulin sensitivity . therefore , the non - invasive determination of arterial compliance in may be an important adjunct in the assessment of cvd risk and/or treatment efficacy in individuals with dm-2 . finally , low cardiorespiratory fitness in men with dm-2 is associated with increased mortality , independent of conventional cvd risk factors . therefore , the observed increase in cardiorespiratory fitness in women with dm-2 following chronic exercise training could also be interpreted as a reduction in the risk for cvd - related mortality . these data lend support to the need for exercise training in the prevention and treatment of cvd - related morbidity and mortality in persons with dm-2 firstly , although most demographic data were similar between both groups at baseline , a significant difference in arterial compliance was observed ( table 2 ) . these baseline differences may be explained by the baseline differences in disease severity between the groups , as there were more individuals in the et group whose diabetes was being controlled with diet and lifestyle and subsequently had slightly better glycemic control ( table 1 ) . although it is possible that these differences may have affected the results , one would expect the et group to have less adaptive reserve relative to the ct group with lower arterial compliance at baseline . it is therefore unlikely that these baseline differences contributed to the observed improvement in arterial compliance following exercise training . secondly , lv or arterial function was not assessed under physiological stress such as exercise or -adrenergic stimulation . therefore it is unclear whether exercise training elicited improvements in inotropic or lusitropic lv reserve in women studied . several investigations have demonstrated impaired cardiac inotropic responsiveness in both healthy aged and aged individuals with dm-2 . although it is possible that an exercise - induced improvement in cardiorespiratory fitness occurred secondary to enhanced lv inotropic reserve , to our knowledge , this adaptation is limited to the male gender . in conclusion , 10 weeks of exercise training improves both cardiorespiratory fitness and large artery compliance in postmenopausal women with dm-2 . these adaptations were achieved without any significant changes in lv filling dynamics or conventional cvd risk factors , such as blood pressure , lipid profile or insulin sensitivity . cvd cardiovascular disease dm-2 type 2 diabetes mellitus et exercise training group homa index homeostasis model assessment index of insulin sensitivity lv left ventricular vo2peak peak rate of oxygen consumption jm conceived of the study , designed the investigation , participated in data collection , administered the intervention and drafted the manuscript sm participated in the data collection , administered the intervention and preparation of the manuscript . rl participated in the design of the investigation and was involved in the collection of the arterial compliance data . mh was involved in the design and coordination of the investigation and participated in the echocardiographic analysis and interpretation . the impact of exercise training on left ventricular filling dynamics in postmenopoausal women with type 2 diabetes . mean sd e / a = ratio of early to late transmitral filling velocities ; pvs : pvd = ratio of pulmonary venous flow in systole to pulmaonry venous flow in diastole ; pva = pulmonary venous flow during atrial systole ; mad - pad = duration of transmitral atrial wave duration of pulmonary venous atrial wave . | backgroundtype 2 diabetes mellitus ( dm-2 ) is one of the most prevalent chronic diseases of the aged and contributes to a significant amount of cardiovascular disease morbidity and mortality .
exercise training may be beneficial in attenuating the cardiovascular maladaptations associated with dm-2 .
the purpose of this study was to examine the effects of exercise training on left ventricular ( lv ) and vascular function in a sample of postmenopausal women with dm-2.methodstwenty-eight postmenopausal women with dm-2 ( age : 59 7 yrs ) were assigned to either an exercise training ( et ) ( n = 17 ) or control group ( ct ) ( n = 7 ) .
cardiorespiratory fitness ( ) , lv filling dynamics and arterial compliance were assessed at baseline in all participants .
the et group performed a supervised aerobic and resistance training intervention three days per week for a period of 10 weeks , while the ct group continued normal activities of daily living.resultsbody mass index , , age and duration of diabetes were similar between the et and ct groups at baseline .
( 21.3 3.3 to 24.5 4.2 mlkg-1min-1 , p < 0.05 ) and large artery compliance ( 1.0 0.4 to 1.2 0.4 mlmmhg-1 ,
p < 0.05 ) , increased significantly in the et group following training despite no change in lv filling dynamics , blood pressure , lipid profile or insulin sensitivity .
all variables remained unchanged in the ct group.conclusionsexercise training improves large artery compliance and cardiorespiratory fitness in postmenopausal women with dm-2 , without any appreciable changes in lv filling dynamics or conventional risk factors for cardiovascular disease . |
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glaucoma is a multifactorial , progressive optic neuropathy characterized by retinal ganglion cell death , the loss of visual field , and excavation of the optic nerve head . glaucoma , the second leading cause of preventable blindness , is usually caused by the effects of elevated intraocular pressure ( iop ) . as elevated iop is the only treatable risk factor for disease , glaucoma surgery is indicated if target pressures are not achieved , or if the glaucomatous optic neuropathy progresses despite maximally tolerated medical and laser therapies.1 currently , trabeculectomy is still the most reliable and most widely performed surgical procedure to lower iop in the vast majority of patients with uncontrolled glaucoma despite maximal medical therapy . trabeculectomy allows the drainage of aqueous humor from the anterior chamber to the subconjunctival space by creating a half - scleral - thickness limbal fistula . this procedure reduces iop by a filtering bleb.24 the failure of glaucoma filtration surgery ( gfs ) is usually due to excessive wound healing under the conjunctiva or over the scleral flap causing failure of the filtering blebs . thus , suppression of the wound healing response is very important in preventing obstruction of the established fistula.5,6 fibroblasts are the primary cell type to play a role in wound healing in the scleral flap region following gfs . in addition , mononuclear cells ( mncs ) are well known to be the primary source of fibrogenic and angiogenic cytokines such as transforming growth factor ( tgf ) , fibroblast growth factor ( fgf ) , and platelet - derived growth factor ( pdgf ) , which play an authoritative part in initiating wound healing . these growth factors cause the most potent influence on fibroblasts , and they increase the activity of fibroblasts and enhance the secretion of extracellular matrix components.57 thus , the suppression of fibroblasts and mncs in the surgical region increases the success of trabeculectomy . currently , there are many anti - fibrotic agents used in the suppression of wound healing after gfs . mitomycin - c ( mmc ) and fluorouracil are the most used anti - fibrotic agents to increase the success of trabeculectomy . however , they have serious adverse effects such as corneal endothelial toxicity , bleb leaking , hypotonous maculopathy , blebitis , and endophthalmitis.812 thus , new agents that are more selective and less toxic are needed to suppress wound healing after gfs . infliximab is a mouse / human chimeric monoclonal igg1 antibody against tumor necrosis factor ( tnf)-. tnf- is a local paracrine and autocrine regulator for low - density leukocyte and endothelial cells . it stimulates mononuclear phagocytes as well as other cell types that produce various proinflammatory cytokines and chemokines and induces migration of polymorphic nuclear leukocytes . infliximab binds to the tnf- molecule and reduces lymphocyte migration and production of proinflammatory cytokines , including interleukin ( il)-1 , il-6 , and adhesion molecules . it is an agent used in the treatment of disorders such as crohn s disease , ulcerative colitis , rheumatoid arthritis , ankylosing spondylitis , and autoimmune ocular inflammatory diseases such as uveitis , scleritis , and behet s disease.1315 it was also recently reported that infliximab is effective in treating choroidal neovascularization and in the prevention of corneal neovascularization.16,17 in recent studies , it was demonstrated that infliximab downregulated expression and levels of the cytokines that play a role in the wound healing response , including tgf- , basic fgf , and pdgf - b.1820 to the best of our knowledge , there is no previous study in the literature on the suppression of wound healing with topical infliximab following gfs . in our study , we aimed to investigate the effects of infliximab on wound healing in experimental gfs and to compare the anti - fibrotic effects of this agent with those of mmc . the animals were kept under appropriate dietary conditions in special cages in the experimental research center at frat university , elaz , turkey throughout the study . the animals were housed in wire - bottomed individual cages at room temperature ( air - conditioned , 22c26c ) on a 12-hour light dark cycle . all of the rabbits were acclimatized for at least 1 week before the experiments started . the animals were fed standard rabbit chow , but were given only water 12 hours before surgery . the study was approved by the institutional animal care and use ethics committee of frat university . all experiments and the animal - related procedures were performed with strict adherence to the guidelines for animal care and experimentation by the association for research in vision and ophthalmology ( arvo ) as described in their statement for the use of animals in ophthalmic and visual research . only one eye of the rabbits in the study groups was subjected to standard preparation for surgery , anesthesia , and the surgical technique . the rabbits were randomly assigned to four groups , each including seven rabbits : control group , sham group , mmc group , and infliximab group . the rabbits in the control group were not operated on and did not receive any treatment . the rabbits in the sham group underwent trabeculectomy and had one drop of saline instilled four times a day for 14 days . the rabbits in the mmc treatment group underwent trabeculectomy , and a sponge soaked in 0.4 mg / ml mmc was applied intraoperatively to the scleral surgical site for 3 minutes . the rabbits in the infliximab treatment group underwent trabeculectomy , and one drop of 10 mg / ml infliximab was instilled four times a day for 14 days after surgery . a combination of intramuscular 50 mg / kg ketamine hydrochloride ( ketalar ; eczacibasi holding co , istanbul , turkey ) and 6 mg / kg xylazine hydrochloride ( rompun ; bayer ag , leverkusen , germany ) was used in the anesthesia and analgesia . before the operation , a single drop of 0.5% proparacaine hydrochloride ( alcaine ; alcon laboratories , inc . , fort worth , tx , usa ) was instilled in the eyes to be operated on . the surgical procedure was performed using an operating microscope ( carl zeiss meditec ag , jena , germany ) a limbal - based trabeculectomy , as described by cairns,21 was performed on the right eyes of all the rabbits in the study groups , except for the control group . briefly , a lid speculum was placed and a partial - thickness 8 - 0 silk corneal traction suture was applied superiorly to pull down and fix the eyeball . a peritomy was made 5 mm above the limbus to form a limbus - based conjunctival flap . a limbus - based rectangular ( 2.5 2.5 mm ) scleral flap was outlined with a steel blade and carefully dissected . a trephine ( 1.5 mm in diameter ) was then applied to make the entry into the anterior chamber at the surgical limbus , and a peripheral iridectomy was performed . the site of the peripheral iridectomy led to detection of the location of the bleb in the histopathologic examination . the scleral flap was closed by suturing with 10 - 0 nylon at two points , and the conjunctival wound was closed by suturing with two 9 - 0 polyglactin sutures . the rabbits in the sham group received one drop of saline four times a day for 14 days after the surgery . in the mmc group , a sponge soaked in 0.4 mg / ml mmc was applied to the scleral surgical site for 3 minutes , after which the application area was thoroughly irrigated with 10 ml of balanced salt solution . the rabbits in the infliximab treatment group underwent trabeculectomy and one drop of 10 mg / ml infliximab was instilled four times a day for 14 days after surgery . at the end of the 14th postoperative day , the eyes were enucleated , the rabbits were anaesthetized again , and the globes were enucleated together with the conjunctiva , including the bleb , for histologic examination . single ( right ) eyes of the rabbits in the control group were also prepared for histologic examination . the operation site of each enucleated eye was dissected as a blockage , which contained the conjunctiva , tenon s capsule , and sclera . the tissue specimens were fixed in 10% formaldehyde and embedded in paraffin before the sections were cut . serial sections ( 4 m thick ) were cut , dehydrated , and stained with hematoxylin and eosin stain for light microscopy examination . the specimens were stained with masson s trichrome , and microscopic analysis was performed using 400 objective of a standard light microscope ( bx50 photomicroscope ; olympus corporation , tokyo , japan ) . using a specific micrometer attached to the microscope , fibroblasts and mncs in ten randomly determined sections ( area of 50 m ) were counted , and the average numbers of cells in these zones were analyzed . the values obtained from the cell counts of the ten serial sections were presented as arithmetic means and standard deviation . the slides were stained with tgf- , fgf- , and pdgf kits ( bioss inc . , woburn , ma , usa ) in an automated immunostainer device ( benchmark xt ; ventana medical systems , inc . , tucson , az , usa ) according to the manufacturer s instructions . the slides , covered with a special cover substrate , were randomly evaluated under a light microscope ( olympus bx50 photomicroscope ) . digital photographs of the tissues were taken at 40 magnification using the camera on the microscope . immunostaining intensities of tgf- , fgf- , and pdgf were determined semiquantitatively as none ( 0 ) , weak ( 1 ) , moderate ( 2 ) , or intense ( 3 ) . statistical analysis of the study was performed with ibm spss statistics software ( v 21 ; ibm corporation , armonk , ny , usa ) to determine the differences between the study groups . the values obtained from the cell counts of the ten serial sections were presented as arithmetic means and standard deviation . for multiple comparisons , a one - way analysis of variance test tgf- , fgf- , and pdgf immunostaining differences were assessed with the kruskal wallis test . the rabbits were randomly assigned to four groups , each including seven rabbits : control group , sham group , mmc group , and infliximab group . the rabbits in the control group were not operated on and did not receive any treatment . the rabbits in the sham group underwent trabeculectomy and had one drop of saline instilled four times a day for 14 days . the rabbits in the mmc treatment group underwent trabeculectomy , and a sponge soaked in 0.4 mg / ml mmc was applied intraoperatively to the scleral surgical site for 3 minutes . the rabbits in the infliximab treatment group underwent trabeculectomy , and one drop of 10 mg / ml infliximab was instilled four times a day for 14 days after surgery . a combination of intramuscular 50 mg / kg ketamine hydrochloride ( ketalar ; eczacibasi holding co , istanbul , turkey ) and 6 mg / kg xylazine hydrochloride ( rompun ; bayer ag , leverkusen , germany ) was used in the anesthesia and analgesia . before the operation , a single drop of 0.5% proparacaine hydrochloride ( alcaine ; alcon laboratories , inc . , fort worth , tx , usa ) was instilled in the eyes to be operated on . the surgical procedure was performed using an operating microscope ( carl zeiss meditec ag , jena , germany ) a limbal - based trabeculectomy , as described by cairns,21 was performed on the right eyes of all the rabbits in the study groups , except for the control group . briefly , a lid speculum was placed and a partial - thickness 8 - 0 silk corneal traction suture was applied superiorly to pull down and fix the eyeball . a peritomy was made 5 mm above the limbus to form a limbus - based conjunctival flap . a limbus - based rectangular ( 2.5 2.5 mm ) scleral flap was outlined with a steel blade and carefully dissected . a trephine ( 1.5 mm in diameter ) was then applied to make the entry into the anterior chamber at the surgical limbus , and a peripheral iridectomy was performed . the site of the peripheral iridectomy led to detection of the location of the bleb in the histopathologic examination . the scleral flap was closed by suturing with 10 - 0 nylon at two points , and the conjunctival wound was closed by suturing with two 9 - 0 polyglactin sutures . the rabbits in the sham group received one drop of saline four times a day for 14 days after the surgery . in the mmc group , a sponge soaked in 0.4 mg / ml mmc was applied to the scleral surgical site for 3 minutes , after which the application area was thoroughly irrigated with 10 ml of balanced salt solution . the rabbits in the infliximab treatment group underwent trabeculectomy and one drop of 10 mg / ml infliximab was instilled four times a day for 14 days after surgery . at the end of the 14th postoperative day , the eyes were enucleated , the rabbits were anaesthetized again , and the globes were enucleated together with the conjunctiva , including the bleb , for histologic examination . single ( right ) eyes of the rabbits in the control group were also prepared for histologic examination . the operation site of each enucleated eye was dissected as a blockage , which contained the conjunctiva , tenon s capsule , and sclera . the tissue specimens were fixed in 10% formaldehyde and embedded in paraffin before the sections were cut . serial sections ( 4 m thick ) were cut , dehydrated , and stained with hematoxylin and eosin stain for light microscopy examination . the specimens were stained with masson s trichrome , and microscopic analysis was performed using 400 objective of a standard light microscope ( bx50 photomicroscope ; olympus corporation , tokyo , japan ) . using a specific micrometer attached to the microscope , fibroblasts and mncs in ten randomly determined sections ( area of 50 m ) were counted , and the average numbers of cells in these zones were analyzed . the values obtained from the cell counts of the ten serial sections were presented as arithmetic means and standard deviation . the slides were stained with tgf- , fgf- , and pdgf kits ( bioss inc . , woburn , ma , usa ) in an automated immunostainer device ( benchmark xt ; ventana medical systems , inc . , tucson , az , usa ) according to the manufacturer s instructions . the slides , covered with a special cover substrate , were randomly evaluated under a light microscope ( olympus bx50 photomicroscope ) . digital photographs of the tissues were taken at 40 magnification using the camera on the microscope . immunostaining intensities of tgf- , fgf- , and pdgf were determined semiquantitatively as none ( 0 ) , weak ( 1 ) , moderate ( 2 ) , or intense ( 3 ) . statistical analysis of the study was performed with ibm spss statistics software ( v 21 ; ibm corporation , armonk , ny , usa ) to determine the differences between the study groups . the values obtained from the cell counts of the ten serial sections were presented as arithmetic means and standard deviation . for multiple comparisons , a one - way analysis of variance test the means and standard deviations of the fibroblast and mnc numbers in the study groups are presented in table 1 . the means and standard deviations of the tgf- , fgf- , and pdgf immunostaining intensities in the study groups are presented in table 2 . the mean fibroblast and mnc numbers and the mean tgf- , fgf- , and pdgf immunostaining intensities of the sham group were higher than those of the control group ( p<0.01 ) . the mean fibroblast and mnc numbers and the mean of tgf- , fgf- , and pdgf immunostaining intensities of the mmc and infliximab group were statistically significantly lower than those of the sham group ( p<0.01 ) . there was no statistically significant difference between the mean mnc and fibroblast numbers and the mean immunostaining intensities of tgf- , fgf- , and pdgf in the mmc and infliximab groups ( p>0.05 ) . comparative microphotographs showing fibroblast proliferation and mnc infiltration at the bleb site of each of the rabbits in the study groups are shown in figure 1 . microphotographs of the immunostaining of tgf- , fgf- , and pdgf , of one subject from each study group , are shown in figures 24 , respectively . antimetabolites such as mmc and fluorouracil have dramatically augmented the success of gfs , especially in patients at higher risk of surgical failure due to excessive wound healing in the surgical region . however , there is a risk of blindness with antimetabolite use.812 additionally , surgical failure can occur despite the use of these powerful antimetabolites . thus , there is a need for potent agents that have no severe adverse effects . fibroblasts , the main cells responsible for excessive wound healing at the scleral flap region following gfs , synthesize collagen , which is the most important component of granulation tissue . tenon s capsule fibroblast proliferation is stimulated by epidermal growth factor , fgf , tgf- , insulin - like growth factor-1 , pdgf , fibronectin , transferrin , il-1 , and il-6 . tgf- is the most potent stimulator of fibroblast activity.57,22,23 additionally , after conjunctival and scleral surgery , pdgf is released from platelets , which promotes cell migration into the wound site and increases fibroblast proliferation.6,7,2226 thus , the inhibition of fibroblast function after gfs is an important factor in the wound healing process and for the outcome of gfs . in our study , it is possible that infliximab inhibits fibroblast proliferation and migration by toxic effect or by reducing tgf- , fgf- , and pdgf , as seen in our study . mncs are cells coming to the surgical wound region subsequent to polymorph nuclear leukocytes . the chemotaxis of mncs is increased with fibronectin , growth factors , and the complement system . macrophages are mncs derived from monocytes and they secrete various factors that stimulate migration and proliferation of the fibroblasts . they promote early wound healing and play a crucial role in tissue remodeling in the later phases of wound healing.5,22,27,28 macrophages are thought to be the principal source of fibrogenic and angiogenic cytokines such as pdgf and tgf-.29 it has been reported that the conjunctiva of patients whose gfs failed contain more macrophages . mncs also release enzymes that augment tissue degradation , generate chemotactic factors that recruit inflammatory cells , and interact with lymphocytes . they produce cytokines and cause the accumulation and proliferation of fibroblasts and endothelial cells , with subsequent fibrosis and angiogenesis.5,22,27,28 thus , it can be put forward that a decreased number of macrophages parallels a suppressed wound healing reaction and surgical success . in our study , mnc infiltration in the bleb region was significantly suppressed in the mmc and infliximab groups ; however , the differences between these two groups were not statistically significant . the reduction in the number of mncs in both treatment groups was presumably the effect of decreased growth factor levels . tgf- is produced by macrophages , lymphocytes , platelets , and fibroblasts , and it is one of the most important profibrogenic cytokines in the conjunctival wound healing process , causing the proliferation and migration of human tenon s fibroblasts and promoting collagen production.23,3034 tgf- and pdgf have the strongest influence on wound healing , as these growth factors increase fibroblast activity.35,36 reduced levels of tgf- have been correlated with the retardation of the wound healing process in burn injury and with non - scarring fetal skin repair.37,38 similarly , decreased levels of pdgf have been observed in non - healing wounds in diabetic rats.38 on the other hand , elevated levels of tgf- are associated with increased fibrosis in several organs , including the heart , liver , kidney , lung , skin , and bone marrow.3942 tgf- is considered to inhibit the growth of leukocytes and to stimulate the production of fibroblasts.43,44 tgf- enhances resistance of the wound site to tension in corneal incisions in rabbits by enhancing the number of fibroblasts through chemotaxis , reducing the synthesis of matrix metalloprotease , and enhancing the synthesis of metalloprotease tissue inhibitor.42 in mouse models of conjunctival scarring , it has been reported that tgf-2 applied to mouse conjunctiva following mmc application significantly reversed the anti - scarring effects of mmc.31,32 inhibiting tgf- might be an effective means of preventing postoperative scarring after trabeculectomy . in our study , mmc and infliximab similarly reduced the immunostaining density of tgf- at the scleral flap region . although the exact mechanisms underlying this response are not known , it might be that exposure to mmc reduces human tenon s fibroblasts , which are one of the main producers of tgf-. fgf- secreted by macrophages and endothelial cells stimulates fibroblast migration and proliferation . tenon s capsule fibroblasts exposed to mmc have been reported to exhibit a significant decrease in the number of fgf- receptors.4044 in our study , mmc and infliximab equally decreased the immunostaining intensity of fgf- in the bleb region . as both drugs strongly suppressed fibroblast proliferation and reduced fgf- , pdgf also plays a very important role in wound healing by promoting the proliferation and migration of fibroblasts.4044 studies by denk et al strongly support the hypothesis that pdgf isoforms are major stimulators of proliferation of tenon s capsule fibroblasts after gfs , while tgf- isoforms are essential for the transformation of tenon s capsule fibroblasts into myofibroblasts.25,44 in another study , knorr et al demonstrated that pdgf - ab and -bb isoforms are potent stimulators of the proliferation of human tenon s capsule fibroblasts , suggesting that they play an important role in the wound healing response after gfs.26 pdgf is normally present in trace amounts in the aqueous humor and increases the proliferative response of fibroblasts to tgf-.44 pdgf is also a chemoattractant for trabecular meshwork cells . in turn , tgf- regulates tissue growth through pdgf protein and receptor expression in keratocytes , although the exact mechanism by which this takes place is unknown . in other connective tissues , low tgf- concentrations promote pdgf production , whereas , at high tgf- concentrations , the pdgf receptor expression is decreased.4045 in our study , mmc and infliximab equally decreased the immunostaining intensity of pdgf in the scleral flap region . mmc is an antimetabolite and antitumor agent that is isolated from streptomyces caespitosus and which decreases fibroblast proliferation in both the subconjunctival space and tenon s capsule . it is widely used in trabeculectomy to prevent excessive postoperative wound healing in the surgical site in cases at risk of surgical failure , due to its anti - fibrotic and anti - scarring properties . yamamoto et al reported that mmc inhibited the proliferation and motility of cultured rabbit subconjunctival fibroblasts and that its inhibitor effect depends on dose and time of exposure to the drug.46 another experimental study demonstrated that histologic examination of eyes with mmc - treated trabeculectomy revealed hypocellular and well - formed bleb cavities , and that a single intraoperative application of mmc had a marked effect on postoperative wound healing after filtration surgery in monkeys.47 however , topically applied mmc has severe ocular complications , such as necrosis in the cornea and conjunctiva , scleral perforation , choroidal detachment , epithelial downgrowth , filtration bleb failure , and endophthalmitis.1013 although mmc is widely used in gfs with high success rates , it is not possible to use it in every case that has a high risk of failure . in addition , mmc is one of the ten most carcinogenic substances to humans.48 infliximab is a human murine monoclonal antibody that can bind to both soluble and transmembranous forms of tnf- with high affinity . tnf- is a local paracrine and autocrine regulator for small - density leukocyte and endothelial cells . it stimulates mononuclear phagocytes as well as other cell types that produce il-1 , il-6 , and chemokines , and induces migration of polymorphic nuclear leukocytes . tnf- has a synergistic effect with il-6 and plays a crucial role in the development of inflammation in uveitis . furthermore , it contributes to the pathogenesis of uveoretinitis associated with behet s disease.14,15 currently , there is increasing evidence that tnf- is important in the initiation and perpetuation of the fibrotic process.4954 in a recent study , sullivan et al demonstrated that tnf- induces tgf-1 expression in lung fibroblasts.49 battegay et al reported that tnf- stimulates dna synthesis and proliferation of cultured human fibroblasts and that there is a strong relationship between secretion of pdgf and tnf- stimulation.50 therefore , we postulate that tnf- may play a significant role in regulating the expression of the cytokines that themselves play an important role in wound healing after gfs , such as tgf- , fgf , and pdgf . recently , infliximab has been used as a novel immunomodulatory agent , particularly in uveitis refractory to treatment , panuveitis associated with behet s disease , rheumatoid arthritis , and crohn s disease.55 infliximab was shown to inhibit the functional activity of tnf- in several in vitro studies.56,57 it has been suggested that infliximab exerts its clinical effects in patients with uveitis through various mechanisms , which include reducing serum levels of inflammatory mediators by blocking tnf- , preventing lymphocyte migration , and decreasing levels of vascular endothelial growth factor.58 binding of infliximab to soluble tnf- brings about a decrease in bioactivity , while its binding to membrane - bound tnf- results in cytotoxicity through complement - dependent or antibody - dependent cell - mediated mechanisms . nevertheless , it is not known how infliximab acts in in vivo models . secondary to the suppression of tnf- , infliximab lowers serum levels of inflammatory mediators , including il-1 and il-6 and vascular growth factors . in addition , it reduces lymphocyte migration.59 in our study , we observed that topically administered 10 mg / ml infliximab following gfs significantly suppressed fibroblast proliferation and mnc infiltration compared to the sham group , and that it was as effective and strong as mmc . it was also observed that the tgf- , fgf- , and pdgf immunostaining intensities of the infliximab group were significantly lower than those of the sham group . it is possible that infliximab acts via the inhibition of fibroblast proliferation and migration by toxic effect or by reducing tgf- , fgf- , and pdgf , as seen in our study . as far as we know , following a search of the pubmed database , there is no previous study concerning the use of infliximab in an experimental gfs model . we consider that infliximab might reduce fibroblast infiltration via the induction of fibroblast apoptosis or suppression of the effects of tgf- , fgf- , and pdgf , as seen in the present study . the suppressive effect of infliximab on the number of mncs might be related to its cycle arrest effect , apoptosis , or the inhibition of mnc chemotaxis via suppression of the effects of the abovementioned cytokines . the small number of experimental animals used can be regarded as a main limitation of our study . in this study , we used a previously reported experimental dosage and administration rate of infliximab.16,59 we did not observe any corneal or conjunctival toxicity in the histologic sections . taking into consideration the toxic effects of mmc in gfs and the strongly anti - fibroblastic effects of infliximab as well as mmc on conjunctival wound healing , infliximab can be considered a potential alternative agent for the modulation of wound healing after gfs . however , further in vivo and in vitro studies are needed to evaluate its use with gfs . | backgroundthe purpose of this work was to look into the effects of infliximab on wound healing in experimental glaucoma filtration surgery and to compare the antifibrotic effects of this agent to that of mitomycin - c ( mmc).methodstwenty - eight male new zealand white rabbits were randomly assigned to four groups , each including seven rabbits : control group , sham group , mmc group , and infliximab group .
the rabbits in the control group were not operated on and did not receive any treatment .
the rabbits in the sham group underwent trabeculectomy and had one drop of saline instilled four times a day for 14 days . the rabbits in the mmc treatment group underwent trabeculectomy , and a sponge soaked in 0.4 mg / ml mmc was applied intraoperatively to the scleral surgical site for three minutes .
the rabbits in the infliximab treatment group underwent trabeculectomy and one drop of 10 mg / ml infliximab was instilled four times a day for 14 days after surgery . on day 14 of the experiment , the operated and control eyes were enucleated and histologically and immunohistochemically analyzed.resultsthe mean fibroblast and mononuclear cell ( mnc ) numbers and the mean immunostaining intensities of transforming growth factor- ( tgf- ) , fibroblast growth factor- ( fgf- ) , and platelet - derived growth factor ( pdgf ) in the sham group were higher than those of the control group ( p<0.01 ) .
the mean fibroblast and mnc numbers and the mean immunostaining intensities of tgf- , fgf- , and pdgf in the mmc and infliximab groups were statistically significantly lower than those of the sham group ( p<0.01 ) .
the mean fibroblast and mnc numbers and the mean tgf- , fgf- , and pdgf immunostaining intensities of the mmc and infliximab groups were similar ( p>0.05).conclusionour study suggests that topical infliximab effectively suppresses the subconjunctival wound healing response after experimental glaucoma filtration surgery , reducing the mnc and fibroblast numbers and immunostaining intensities of tgf- , fgf- , and pdgf . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
it initially presents with difficulty swallowing and is a chronic progressive condition that usually does not resolve . although the specific cause is unknown , the primary pathology involves the inability of the lower esophagus to relax , resulting in delayed passage of food and liquid into the stomach . clinical symptoms range from initial dysphagia to heartburn , a feeling of fullness in the chest and sometimes weight loss . the objective work - up includes barium radiography , esophagoscopy , and esophageal manometry . in patients with achalasia barium radiography often reveals a classic 1 ) in which the dilation tapers into the lower esophagus , because of the chronic buildup of food contents . long - term sequelae include an increased risk of the development of esophageal cancer in patients diagnosed with achalasia . 1bird s beak appearance of lower esophagus during an upper gastrointestinal x - ray swallow study bird s beak appearance of lower esophagus during an upper gastrointestinal x - ray swallow study the objective of treatment is to resolve symptoms in this usually progressive disease . conservative maneuvers have included the use of muscle relaxing medications or injections and dilation of the lower esophagus with endoscopically guided balloons . these usually result in temporary relief only rather than achieving long - term resolution of the problem . surgical intervention involving splitting of the restrictive external muscle of the lower esophagus and upper stomach with subsequent wrapping of the upper stomach partially around the lower esophagus results in symptom relief in 85% of people 10 years after surgery and in approximately 65% of people 20 years after surgery . achalasia was first described , with its initial treatment , by thomas willis in 1679 . treatment and instrumentation have rapidly evolved since his first description of dilation of the esophagus using a tapered whale bone . in 1887 , russel described initial balloon dilatation using a silk bag over a rubber balloon . this surgical technique involved splitting the outer muscle of the lower esophagus to relieve internal narrowing and associated swallowing difficulties . this procedure was subsequently popularized by approaches through the abdomen or through the chest . in the early 1990s , during the beginning of the era of minimally invasive surgery , thoracoscopic and laparoscopic heller myotomy were described . prompt recovery and good outcomes quickly enabled clinicians to conclude that surgical treatment was the optimum primary treatment of choice for amelioration of symptoms in patients with achalasia . laparoscopic heller myotomy using standard laparoscopic techniques has evolved into an extremely safe and efficient technique of reducing symptoms in the treatment of achalasia . preliminary case series demonstrated that early outcomes were favorable and the perioperative course was much improved compared with an open laparotomy or thoracotomy . perioperative complications were reduced and the complications secondary to the trauma of the incision were largely eliminated . laparoscopic heller myotomy has now been widely reported with large case series with relatively good outcomes in both early and late results [ 68 ] . in addition , favorable outcomes have been achieved in some series in which laparoscopic heller myotomy was offered as the first - line therapy . although patient satisfaction , relief of symptoms , and recovery time were categorized as favorable , these large case series still reported a significant number of intraoperative esophageal perforations ranging between 1 and 15% . in these series , the mucosal perforations were identified to have occurred as a technical failure during the course of the division of the muscle fibers from the underlying esophageal mucosa . the clinical relevance of these complications may be uncertain , and when the perforation is recognized at the time of surgery and repair performed immediately the outcomes are still usually good . these perforations expose the patients to a prolonged hospital stay and the risk of an esophageal leak , however . delayed diagnosis of an esophageal perforation is a potential fatal complication and is one of the most significant and serious risks of heller myotomy . management in this situation requires broad - spectrum antibiotic coverage , proximal bowel decompression and rest , potential operative drainage , debridement and reconstruction of the injured tissues , and sometimes intravenous nutritional support for a prolonged period of time . different surgical techniques have been described to avoid the perforation of the underlying mucosa and have included the injection of epinephrine into the submucosal plane , blunt dissection , sharp dissection , and intraoperative esophagoscopy to evaluate the esophageal mucosa and detect underlying perforations . the da vinci surgical system ( intuitive surgical , inc ; sunnyvale ca , usa ) is currently the only robotic telesurgical device marketed and approved for general surgery in the united states . the device is a computer interface that interacts with the operating surgeon who sits at a control panel connected to a bedside actuator with instrument holders and a camera manipulator ( figs . 2 , 3 ) . motion is enhanced by filtering fine motor tremor and providing motion scaling between the hand - piece and the activator arm , enabling up to 5-to-1 scaling . innovations have now incorporated 5 mm instrumentation enabling reduced abdominal wall trauma and addition of a fourth working arm for enhanced mobility , tissue handling , and retraction . 3the da vinci surgical robot in use with head surgeon operating from the console the da vinci surgical robot in use with assistant surgeons alongside operative table the da vinci surgical robot in use with head surgeon operating from the console the robotic device has the unique ability to enhance surgical technique and , therefore , optimize surgical outcome . these advantages specifically are fine motor control , three - dimensional magnification , and motion scaling . these may be significant in operative procedures such as heller myotomy or in cases when optimum completion of the operation requires these specific surgical techniques . it is this enhancement of surgical performance that may have improved the surgical outcomes mentioned above regarding recent case series comparing robotic telesurgery with historic laparoscopic approaches to heller myotomy . using this model it may be reasonable to identify some surgical procedures and interventions , for example heller myotomy , that can specifically benefit from this type of therapy . at the author s institution robotic telesurgery for achalasia preoperative preparation of patients with achalasia may include a prolonged period of fasting to completely empty the esophagus . clear liquids for 48 h preoperatively are routine , especially in cases of severe esophageal dilatation . preoperative hospital admission may be necessary for patients to enable them to maintain fasting with intravenous hydration . preoperative antibiotics and oral anti - fungal medications should be included to help reduce the amount of contamination within the esophagus . the patient is positioned on a split leg table or in a modified lithotomy ( legs apart and gently angled down ) position after the induction of general anesthesia . the bedside component of the robot is positioned over the patients left shoulder after accessing the abdomen through four ports . the patient is placed with the head of the bed angled upwards and the patient s motion must be completed before docking to the robot ports . two working ports are used with downward traction of the upper part of the stomach by the assistant or the fourth arm of the robot . the esophageal hiatus of the diaphragm is identified and the crura are circumferentially dissected free of peritoneal attachments . the distal esophagus is dissected , and a soft rubber penrose drain is usually placed around the distal esophagus for traction , enabling dissection up into the lower mediastinum . the esophageal myotomy is created using the electrocautery hook of the robotic device ( fig . the articulation of this arm provides precise control and helps keep the end of the hook directly parallel to the esophageal mucosal wall . this dissection is aided by a curved dissector on the opposite arm , to help completely separate the muscle fibers . when the submucosal is plane is developed between the outer esophageal musculature and the inner mucosa , the division of the muscle fibers is carried 810 cm proximal and at least 2 cm distally from the junction of the esophagus and stomach . the gastric dissection is clearly identified by the disordered character of the vasculature of the gastric submucosa . the mucosa is carefully inspected for injury and for complete division of all muscle fibers . the muscular bundles bordering the esophageal diaphragmatic hiatus are reapproximated behind the esophagus using non - absorbable sutures ( fig . 5 ) . a partial gastric fundoplication is then created using a 90 anterior dor or a 270 posterior toupet fundoplication ( fig . patients are allowed liquids post operatively and then are maintained on a soft diet for several days . the rubber penrose drain is used for retraction of the abdominal esophagus during this stepfig . note : the abdominal esophagus is coursing through the diaphragm in the top of the photographfig . 6operative completion of the heller myotomy and posterior 270 toupet fundoplication operative step of longitudinal esophageal myotomy using electrocautery . the rubber penrose drain is used for retraction of the abdominal esophagus during this step operative step of diaphragmatic posterior crural repair using robotic instrumentation . note : the abdominal esophagus is coursing through the diaphragm in the top of the photograph operative completion of the heller myotomy and posterior 270 toupet fundoplication postoperative patients should receive routine follow - up consisting of surveillance endoscopy every 3 years in anticipation of early detection of asymptomatic pathology . patients who complain of recurrent symptoms should undergo formal evaluation and work - up as outlined for initial patients suspected of having achalasia . long - term postoperative results for achalasia patients are good , particularly when patients are selected with preoperative lower esophageal sphincter pressures greater than 35 mmhg . for a group of 174 patients reported overall symptomatic relief of dysphagia in 87% of patients 15 years after heller myotomy and dor fundoplication with 9% of patients requiring medical / surgical treatment of symptomatic reflux and 4% of patients requiring esophagectomy for malignant disease or recurrent disease not amenable to repeat myotomy and fundoplication . in patients who require re - operation for recurrent dysphagia following esophagomyotomy symptoms often present within the first postoperative year and the root cause is usually inadequate or healed myotomy . although success can usually be achieved , results from repeat esophagomyotomy are less impressive than adequate initial operations . it is indicated after failed medical therapy , pneumatic dilation , non - resecting surgical , and redo procedures . although laparoscopic heller myotomy has emerged as the treatment of choice for achalasia , the intraoperative esophageal perforation rate of 115% has remained a significant problem that has prompted investigation of improvements of this operative technique . in 2000 , the da vinci surgical system ( intuitive surgical , inc ; sunnyvale ca , usa ) a robotic telesurgical device , entered the market . the advantages reported include a stable work platform , a magnified three - dimensional image , fine motor control of articulated instruments , and motion scaling , which are uniquely designed to facilitate surgery requiring fine tissue manipulation . they provide a narrower field of view of the operative site and they provide the surgeon with essentially no tactile feedback . with these advantages and hurdles in mind , a variety of surgical procedures have been reported using robotic telesurgical devices . initial data obtained before food and drug administration approval was obtained in clinical trials performing cholecystectomy and gastroesophageal fundoplication . after food and drug administration approval brief clinical reports described cholecystectomy , fundoplication , colon resection , pancreatic resection , heller myotomy , and other general surgery procedures [ 19 , 22 ] . with experience , case series were presented which eventually demonstrated the safety and feasibility of robotic general surgery procedures [ 22 , 23 ] . reports have also been presented that look at traditional laparoscopic procedures compared with robotic telesurgical assistance . reported a comparison case series of gastroesophageal fundoplication performed by standard laparoscopy or with robotic telesurgical assistance . both techniques were safe and associated with low occurrence of complications ; no clear benefit was seen in the robotic group , however . use of the da vinci robotic telesurgical device to perform a heller myotomy was first reported in 2001 and a subsequent small case series was then reported [ 23 , 25 ] . the academic robotic surgery group , comprising surgeons from ohio state university , columbus , oh , usa , johns hopkins university , baltimore , md , usa , and the university of illinois at chicago , chicago , il , usa , has prospectively collected data on 104 robotic telesurgical heller myotomies , the largest reported case series , which were performed without a single esophageal perforation . similar results favoring the robotic telesurgical approach were obtained in three subsequent published series comparing robotic telesugical heller myotomy with laparoscopic heller myotomy [ 2628 ] . this improvement over standard laparoscopic studies which reflected the esophageal perforation rate of 115% may be related to the advantages of the robotic telesurgical technique and surgical enhancement the device offers compared with laparoscopic techniques . one pilot study evaluated the technique of pancreaticojejunostomy after pancreatic head resection using the robotic surgical device . five patients were enrolled and underwent a computer - enhanced pancreaticojejunostomy using a 60 suture in an open abdomen after pancreatic head resection . robotic assistance also has the potential to contribute to the evolving technology of natural orifice transluminal endoscopic surgery ( notes ) in which therapeutic approaches to abdominal surgical diseases are being pursued via an endoscope that is advanced from an intraluminal position within the stomach , vagina , bladder , or rectum into the abdominal cavity to perform a variety of diagnostic and therapeutic maneuvers . this was first attempted with simple peritonoscopy and has recently advanced to organ retrieval , for example appendectomy [ 30 , 31 ] . although no formal treatments for achalasia by a notes approach have been published , a treatment involving transgastric peritonoscopy with retroflexion and endoscopic myotomy with potential intraluminal gastroesophageal plication may prove to be feasible for treatment of achalasia with end results similar to heller myotomy and fundoplication . | the craft of surgery has always relied on the use of instruments .
innovations in surgery have paralleled innovations in instrumentation .
advances in surgical instrumentation continue today and have enabled huge strides in surgical procedures and outcomes during this generation .
computers and related technology are now changing the interface between the surgeon and the patient , and are poised to improve patient outcomes by enhancing the surgeon s skills and training .
the application of computer enhanced telemanipulators , or robots
, may specifically enhance operations , for example heller myotomy , that require good visualization and precise careful dissection of delicate structures .
this review covers the pathophysiology of achalasia and its history of medical and surgical treatment , leading to modern robotic telesurgical approaches .
improvements in outcome from medical to standard surgical to robotic telesurgical approaches are discussed .
current operative technique for robotic telesurgical treatment of achalasia is described and the authors conclude with a glimpse of where , in the future , current research endeavors will lead us in the treatment of achalasia . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
fatigue is the most common , troublesome , and costly side effect of many cancer ( ca ) treatment regimens . not only does it impact patients directly , but it also has significant repercussions on both direct and indirect health economic outcomes.1 ca treatment - related fatigue ( ctrf ) is defined as a subjective sense of tiredness that persists over time , interferes with activities of daily living , and is not relieved by adequate rest.2,3 the majority of ctrf studies are associated with chemotherapy regimens ; however , fatigue during and after external beam radiation therapy ( rt ) is common , increasing in severity during treatment and persisting after rt has been completed.4 ctrf has been reported to be the most distressing symptom reported by patients with non - metastatic prostate ca who receive rt with the greatest negative impact on daily activity , physical well - being / function , and relationships with significant others.5 the trajectory of ctrf is still being defined . during rt , fatigue intensification peaks at midpoint , declines after completion of rt,6 and becomes chronic in a subpopulation of patients . the pathobiology of ctrf , like other toxicities , is complex and is probably attributable to a cascade of events resulting in radiation - induced pro - inflammatory cytokine production , hypothalamic pituitary adrenal ( hpa ) activation dysfunction , and neuromuscular function abnormalities.7,8 ctrf , like other regimen - related toxicities , does not occur in every patient , but rather in a subpopulation of at - risk individuals . in the context of individualizing care , the ability to predict ctrf risk has the potential to help guide treatment choices for patients and providers . for example , one study reported that elevated pre - treatment fatigue , anxiety , and a specific breast cancer diagnosis ( eg , ductal carcinoma in situ , invasive lobular carcinoma ) predicted ctrf during rt in early stage breast cancer.9 another study found dyspnea , pain , lack of appetite , feeling drowsy , feeling sad , and feeling irritable to be forecasters of ctrf among hematology oncology patients.10 however , as it becomes increasingly clear that ctrf is strongly related to a series of underlying genetically controlled biological events , the utility of identifying a group of genes that impact patients risk of the condition seems compelling . we hypothesized that radiation - associated fatigue risk , like other regimen - related toxicities , is determined not by a single gene , but rather a synergistically functioning group of genes . this theory is supported by the finding that clusters of snps , discovered by bayesian network analysis , have been reported to be associated with ctrf risk in patients being treated with cycled chemotherapy for breast and colorectal cancers.11,12 in the current study , we evaluated an alternative analytical method in which genes were identified using a series of hierarchical filters and nearest - neighbor ( nn ) analysis to identify a group of genes that predicted ctrf in men being irradiated for prostate cancer . this proof - of - concept investigation not only demonstrated the utility of the analysis , but also confirmed the observation that focal radiation therapy is capable of inducing gene expression changes in peripheral white blood cell rna.13 this study ( nct00852111 ) was approved by the institutional review board of the national institutes of health ( nih ) , bethesda , maryland , usa . the study involving human participants is in compliance with the declaration of helsinki . men who were 18 years or older , diagnosed with non - metastatic prostate cancer with or without a history of prostatectomy , and scheduled to receive ebrt with or without concurrent androgen deprivation therapy ( adt ) , were enrolled . men with progressive disease causing significant fatigue , those with psychiatric disease within the past five years , uncorrected hypothyroidism and anemia , taking sedatives , steroids , and non - steroidal anti - inflammatory agents , and those with second malignancies , patients were recruited at the magnuson clinical research center , nih , between may 2009 and september 2011 . clinical and demographic data ( eg , age , race , stage of prostate cancer , ebrt dose , type of ebrt technique used , and laboratory values ) were obtained from chart review . questionnaires were completed at baseline ( prior to rt ) and at completion of rt ( day 3842 after ebrt initiation ) . to avoid extraneous influences on their responses , subjects completed the questionnaires in an outpatient setting before clinical procedures were provided . fatigue ( fact - f ) , a frequently used , validated , reliable , stand - alone measure of fatigue in cancer therapy with coefficient alphas in the mid-90s , was used.14 fact - f is scored from 052 , the higher the score , the lower the fatigue symptoms . a greater than three - point decrease in the fact - f score is considered to be a minimally important change that is clinically relevant.15 to optimize the phenotypic characterization of the study participants , subjects were categorized into high - fatigue ( hf ) or low - fatigue ( lf ) groups based on their change in fact - f scores from baseline to completion of ebrt . hf subjects had a decrease of three or more points in fact - f scores , and those who had less than a three - point decrease in fact - f scores between both time points were categorized in the lf group . depressive symptoms were also assessed using the 21-item hamilton depression rating scale ( ham - d ) , a clinician - administered questionnaire with good psychometric properties.16 peripheral blood ( 2.5 ml ) was collected at baseline and on the last day of rt , immediately after fact - f was administered , from each subject using paxgene blood rna tubes ( qiagen , frederick , maryland , usa ) containing red blood cell lysis buffer and a rna - stabilizing solution and stored at 80 c until rna extraction . total rna was extracted using the paxgene blood rna system ( qiagen , frederick , maryland , usa ) according to manufacturer s instructions . the quantity of total rna was measured by a spectrophotometer at an optical density of 260 nm . rna quality was assessed using the rna 6000 nano labchip on a bioanalyzer agilent 2100 ( agilent technologies , palo alto , ca , usa ) . rna purification , cdna and crna synthesis , amplification , hybridization , scanning , and data analyses were conducted by one laboratory technician following standard protocols as previously described.17 affymetrix microarray chips ( hg - u133 plus 2.0 , santa clara , california , usa ) were used for gene expression analysis . the affymetrix hg - u133 plus 2.0 microarray chip is comprised of 47,000 transcripts , including 38,000 well - characterized human genes . affymetrix genechip command console ( agcc , 3.0 v ) was used to scan the images for data acquisition . affymetrix raw data were acquired using comparison expression analysis of gcos software to yield chp files according to the user instructions . peripheral blood has been previously utilized to describe gene expression signature that predicted radiation - related toxicities.18 ingenuity pathway analysis ( ingenuity systems , www.ingenuity.com , redwood city , california , usa ) identified the functional networks of the differentially expressed probe sets from ingenuity s knowledge base . right - tailed fisher s exact test was used to calculate the p - values determining the probability that each biological function and/or disease assigned to these networks is due to chance alone . the one - tailed analysis was used to reduce the random chances of over - representation of focused genes in the relevant pathways.19 descriptive analyses were used to assess the demographic characteristics of the sample . paired t - tests were used to compare fatigue scores and clinical variables between time points . to facilitate the identification of a group of synergistically functioning genes that were associated with ctrf risk , we used an approach that optimized an initial supervised component with a subsequent statistically driven hierarchical ranking . using microarray data from the training set of patients for which the presence or absence of crtf was known , we identified the genes that most discriminated between individuals who developed ctrf from those who did not . those genes were then ranked according to their discriminatory value ( as defined by their fisher s ratio [ fr ] ) , in which the predictive accuracy of the different - ordered reduced sets was determined using a backward recursive feature elimination algorithm ( see flow diagram in fig . 1 below ) . this procedure serves to eliminate redundant or irrelevant genes ( features ) to yield the most precise set of genes with the greatest predictive accuracy . feature selection identified genes with the highest fold change,20,32 fcj(c1,c2 ) , and fr,21frj(c1,c2 ) , using the phenotype information . the fold change and the fr for probe j in a binary classification problem are defined as follows :
fcj(c1,c2)=log2j1j2,(1 )
frj(c1,c2)=(j1j2)2j12+j22,(2 ) where j1,j2 are measures of the center of the distribution ( means ) of gene j in classes 1 and 2 , and
j12 ,
j22 are measures of the dispersion ( variance ) within these classes . the following relationship holds :
frj = k2>1|j1j2|=k.j12+j22,(3 ) that is ,
|j1j2|=k.jt,(4 ) where
|j1j2| is the distance between the centers of the classes , and
jt=j12+j22 is the total variance of the gene j in both classes . the above relationship means that the centers of the distribution are further apart the distance ,
k.jt . also , taking into account that
j1=2fcjj2 , then
|j1j2|=k.jtj2=frj|2fcj1jt.(5 ) this last relationship implies that given a gene characterized by its fr , frj , and fold - change value , fcj , only the most discriminatory genes with means j1 ,
j2 and dispersions j1,j2 in both classes we used the following algorithm to select the smallest and most precise set of discriminatory genes for the lf / hf phenotype :
genes identified by feature selection ( see above ) were ranked in decreasing order according to their fr value.the predictive accuracy of the different sets was iteratively calculated after the sequential elimination of the genes with lowest fr . we termed this novel algorithm , a modification of the technique described by guyon et al ( 2002),22 backward recursive feature elimination . it served to determine the number of helper genes ( genes with the lowest fr ) needed to maximize the leave - one - out - cross - validation ( loocv ) predictive accuracy,23 in a procedure similar to the fourier decomposition of a signal into a sum of harmonics of increasing frequency.24 genes with lower fr provide high frequency details for the discrimination . this procedure yielded the shortest gene set that predicted fatigue risk association with optimum accuracy ( most precise ) . other sets with similar and lower accuracy were also determined by this procedure and were of value , because these sets were also considered as noise buffers ; as the classifier with the highest learning accuracy might not be the one that generalizes ( predicts correctly unseen samples ) better . this approach is appropriate and is especially helpful in designing small - scale signatures that were able to predict hf / lf with a high degree of accuracy . genes identified by feature selection ( see above ) were ranked in decreasing order according to their fr value . the predictive accuracy of the different sets was iteratively calculated after the sequential elimination of the genes with lowest fr . we termed this novel algorithm , a modification of the technique described by guyon et al ( 2002),22 backward recursive feature elimination . it served to determine the number of helper genes ( genes with the lowest fr ) needed to maximize the leave - one - out - cross - validation ( loocv ) predictive accuracy,23 in a procedure similar to the fourier decomposition of a signal into a sum of harmonics of increasing frequency.24 genes with lower fr provide high frequency details for the discrimination . this procedure yielded the shortest gene set that predicted fatigue risk association with optimum accuracy ( most precise ) . other sets with similar and lower accuracy were also determined by this procedure and were of value , because these sets were also considered as noise buffers ; as the classifier with the highest learning accuracy might not be the one that generalizes ( predicts correctly unseen samples ) better . this approach is appropriate and is especially helpful in designing small - scale signatures that were able to predict hf / lf with a high degree of accuracy . the linear separability of the phenotype in the reduced set of genes that is determined in step 2 was checked by performing principal component analysis ( pca ) of the learning dataset expressed in this small - scale signature and projecting these samples in the corresponding 2d pca space . then , the lf / hf phenotype becomes linearly separable by reducing the dimension to the list of most discriminatory genes , if both populations ( hf and lf ) can be linearly separated by a given hyper - plane . the accuracy estimation was based on the loocv method , using the average euclidean distance on the reduced set of features to each training class set . the goal of cross - validation was to estimate how accurately a predictive model ( classifier ) will perform in practice . this procedure , applied to the training dataset , is supervised because the phenotype information of the patients was needed to establish the predictive accuracy of each gene list . loocv implies using a single sample from the original dataset as the validation data ( sample test ) , and the remaining samples as training data . this was repeated such that each sample in the dataset was used once , as a sample test . each sample was characterized by a vector whose dimension was the number of genes that belonged to the reduced base that differentiated between hf and lf . the class with the minimum euclidean distance was assigned to the sample test ( nn classifier),25 and the average accuracy was calculated by iterating over all the samples . for that purpose , all the samples were normalized according to their gene variability ( each attribute or gene separately ) . in this way , all the genes had the same importance in the distance criterion . the distance between a sample and a phenotype class could have been defined in several ways , but the most robust one was using the median distance between the sample test and all the samples in the corresponding class.the legitimacy of the predictive accuracy based on the training set was then tested with the validation set , using the above - mentioned predictive model . it is important to remark that the application of the prediction model , designed in steps 1 to 3 , to the validation set was unsupervised . the final decision was made by consensus ( majority voting ) of the predictions made using the lists of most discriminatory genes . the accuracy estimation was based on the loocv method , using the average euclidean distance on the reduced set of features to each training class set . the goal of cross - validation was to estimate how accurately a predictive model ( classifier ) will perform in practice . this procedure , applied to the training dataset , is supervised because the phenotype information of the patients was needed to establish the predictive accuracy of each gene list . loocv implies using a single sample from the original dataset as the validation data ( sample test ) , and the remaining samples as training data . this was repeated such that each sample in the dataset was used once , as a sample test . each sample was characterized by a vector whose dimension was the number of genes that belonged to the reduced base that differentiated between hf and lf . the class with the minimum euclidean distance was assigned to the sample test ( nn classifier),25 and the average accuracy was calculated by iterating over all the samples . for that purpose , all the samples were normalized according to their gene variability ( each attribute or gene separately ) . in this way the distance between a sample and a phenotype class could have been defined in several ways , but the most robust one was using the median distance between the sample test and all the samples in the corresponding class . the legitimacy of the predictive accuracy based on the training set was then tested with the validation set , using the above - mentioned predictive model . it is important to remark that the application of the prediction model , designed in steps 1 to 3 , to the validation set was unsupervised . the final decision was made by consensus ( majority voting ) of the predictions made using the lists of most discriminatory genes . this study ( nct00852111 ) was approved by the institutional review board of the national institutes of health ( nih ) , bethesda , maryland , usa . the study involving human participants is in compliance with the declaration of helsinki . men who were 18 years or older , diagnosed with non - metastatic prostate cancer with or without a history of prostatectomy , and scheduled to receive ebrt with or without concurrent androgen deprivation therapy ( adt ) , were enrolled . men with progressive disease causing significant fatigue , those with psychiatric disease within the past five years , uncorrected hypothyroidism and anemia , taking sedatives , steroids , and non - steroidal anti - inflammatory agents , and those with second malignancies , patients were recruited at the magnuson clinical research center , nih , between may 2009 and september 2011 . clinical and demographic data ( eg , age , race , stage of prostate cancer , ebrt dose , type of ebrt technique used , and laboratory values ) were obtained from chart review . questionnaires were completed at baseline ( prior to rt ) and at completion of rt ( day 3842 after ebrt initiation ) . to avoid extraneous influences on their responses , subjects completed the questionnaires in an outpatient setting before clinical procedures were provided . fatigue ( fact - f ) , a frequently used , validated , reliable , stand - alone measure of fatigue in cancer therapy with coefficient alphas in the mid-90s , was used.14 fact - f is scored from 052 , the higher the score , the lower the fatigue symptoms . a greater than three - point decrease in the fact - f score is considered to be a minimally important change that is clinically relevant.15 to optimize the phenotypic characterization of the study participants , subjects were categorized into high - fatigue ( hf ) or low - fatigue ( lf ) groups based on their change in fact - f scores from baseline to completion of ebrt . hf subjects had a decrease of three or more points in fact - f scores , and those who had less than a three - point decrease in fact - f scores between both time points were categorized in the lf group . depressive symptoms were also assessed using the 21-item hamilton depression rating scale ( ham - d ) , a clinician - administered questionnaire with good psychometric properties.16 peripheral blood ( 2.5 ml ) was collected at baseline and on the last day of rt , immediately after fact - f was administered , from each subject using paxgene blood rna tubes ( qiagen , frederick , maryland , usa ) containing red blood cell lysis buffer and a rna - stabilizing solution and stored at 80 c until rna extraction . total rna was extracted using the paxgene blood rna system ( qiagen , frederick , maryland , usa ) according to manufacturer s instructions . the quantity of total rna was measured by a spectrophotometer at an optical density of 260 nm . rna quality was assessed using the rna 6000 nano labchip on a bioanalyzer agilent 2100 ( agilent technologies , palo alto , ca , usa ) . rna purification , cdna and crna synthesis , amplification , hybridization , scanning , and data analyses were conducted by one laboratory technician following standard protocols as previously described.17 affymetrix microarray chips ( hg - u133 plus 2.0 , santa clara , california , usa ) were used for gene expression analysis . the affymetrix hg - u133 plus 2.0 microarray chip is comprised of 47,000 transcripts , including 38,000 well - characterized human genes . affymetrix genechip command console ( agcc , 3.0 v ) was used to scan the images for data acquisition . affymetrix raw data were acquired using comparison expression analysis of gcos software to yield chp files according to the user instructions . peripheral blood has been previously utilized to describe gene expression signature that predicted radiation - related toxicities.18 ingenuity pathway analysis ( ingenuity systems , www.ingenuity.com , redwood city , california , usa ) identified the functional networks of the differentially expressed probe sets from ingenuity s knowledge base . right - tailed fisher s exact test was used to calculate the p - values determining the probability that each biological function and/or disease assigned to these networks is due to chance alone . the one - tailed analysis was used to reduce the random chances of over - representation of focused genes in the relevant pathways.19 paired t - tests were used to compare fatigue scores and clinical variables between time points . to facilitate the identification of a group of synergistically functioning genes that were associated with ctrf risk , we used an approach that optimized an initial supervised component with a subsequent statistically driven hierarchical ranking . using microarray data from the training set of patients for which the presence or absence of crtf was known , we identified the genes that most discriminated between individuals who developed ctrf from those who did not . those genes were then ranked according to their discriminatory value ( as defined by their fisher s ratio [ fr ] ) , in which the predictive accuracy of the different - ordered reduced sets was determined using a backward recursive feature elimination algorithm ( see flow diagram in fig . 1 below ) . this procedure serves to eliminate redundant or irrelevant genes ( features ) to yield the most precise set of genes with the greatest predictive accuracy . feature selection identified genes with the highest fold change,20,32 fcj(c1,c2 ) , and fr,21frj(c1,c2 ) , using the phenotype information . the fold change and the fr for probe j in a binary classification problem are defined as follows :
fcj(c1,c2)=log2j1j2,(1 )
frj(c1,c2)=(j1j2)2j12+j22,(2 ) where j1,j2 are measures of the center of the distribution ( means ) of gene j in classes 1 and 2 , and
j12 ,
j22 are measures of the dispersion ( variance ) within these classes . the following relationship holds :
frj = k2>1|j1j2|=k.j12+j22,(3 ) that is ,
|j1j2|=k.jt,(4 ) where
|j1j2| is the distance between the centers of the classes , and
jt=j12+j22 is the total variance of the gene j in both classes . the above relationship means that the centers of the distribution are further apart the distance ,
k.jt . also , taking into account that
j1=2fcjj2 , then
|j1j2|=k.jtj2=frj|2fcj1jt.(5 ) this last relationship implies that given a gene characterized by its fr , frj , and fold - change value , fcj , only the most discriminatory genes with means j1 ,
j2 and dispersions j1,j2 in both classes are selected by this procedure . we used the following algorithm to select the smallest and most precise set of discriminatory genes for the lf / hf phenotype :
genes identified by feature selection ( see above ) were ranked in decreasing order according to their fr value.the predictive accuracy of the different sets was iteratively calculated after the sequential elimination of the genes with lowest fr . we termed this novel algorithm , a modification of the technique described by guyon et al ( 2002),22 backward recursive feature elimination . it served to determine the number of helper genes ( genes with the lowest fr ) needed to maximize the leave - one - out - cross - validation ( loocv ) predictive accuracy,23 in a procedure similar to the fourier decomposition of a signal into a sum of harmonics of increasing frequency.24 genes with lower fr provide high frequency details for the discrimination . this procedure yielded the shortest gene set that predicted fatigue risk association with optimum accuracy ( most precise ) . other sets with similar and lower accuracy were also determined by this procedure and were of value , because these sets were also considered as noise buffers ; as the classifier with the highest learning accuracy might not be the one that generalizes ( predicts correctly unseen samples ) better . this approach is appropriate and is especially helpful in designing small - scale signatures that were able to predict hf / lf with a high degree of accuracy . genes identified by feature selection ( see above ) were ranked in decreasing order according to their fr value . the predictive accuracy of the different sets was iteratively calculated after the sequential elimination of the genes with lowest fr . we termed this novel algorithm , a modification of the technique described by guyon et al ( 2002),22 backward recursive feature elimination . it served to determine the number of helper genes ( genes with the lowest fr ) needed to maximize the leave - one - out - cross - validation ( loocv ) predictive accuracy,23 in a procedure similar to the fourier decomposition of a signal into a sum of harmonics of increasing frequency.24 genes with lower fr provide high frequency details for the discrimination . this procedure yielded the shortest gene set that predicted fatigue risk association with optimum accuracy ( most precise ) . other sets with similar and lower accuracy were also determined by this procedure and were of value , because these sets were also considered as noise buffers ; as the classifier with the highest learning accuracy might not be the one that generalizes ( predicts correctly unseen samples ) better . this approach is appropriate and is especially helpful in designing small - scale signatures that were able to predict hf / lf with a high degree of accuracy . the linear separability of the phenotype in the reduced set of genes that is determined in step 2 was checked by performing principal component analysis ( pca ) of the learning dataset expressed in this small - scale signature and projecting these samples in the corresponding 2d pca space . then , the lf / hf phenotype becomes linearly separable by reducing the dimension to the list of most discriminatory genes , if both populations ( hf and lf ) can be linearly separated by a given hyper - plane . the accuracy estimation was based on the loocv method , using the average euclidean distance on the reduced set of features to each training class set . the goal of cross - validation was to estimate how accurately a predictive model ( classifier ) will perform in practice . this procedure , applied to the training dataset , is supervised because the phenotype information of the patients was needed to establish the predictive accuracy of each gene list . loocv implies using a single sample from the original dataset as the validation data ( sample test ) , and the remaining samples as training data . this was repeated such that each sample in the dataset was used once , as a sample test . each sample was characterized by a vector whose dimension was the number of genes that belonged to the reduced base that differentiated between hf and lf . the class with the minimum euclidean distance was assigned to the sample test ( nn classifier),25 and the average accuracy was calculated by iterating over all the samples . for that purpose , all the samples were normalized according to their gene variability ( each attribute or gene separately ) . in this way the distance between a sample and a phenotype class could have been defined in several ways , but the most robust one was using the median distance between the sample test and all the samples in the corresponding class.the legitimacy of the predictive accuracy based on the training set was then tested with the validation set , using the above - mentioned predictive model . it is important to remark that the application of the prediction model , designed in steps 1 to 3 , to the validation set was unsupervised . the final decision was made by consensus ( majority voting ) of the predictions made using the lists of most discriminatory genes . the accuracy estimation was based on the loocv method , using the average euclidean distance on the reduced set of features to each training class set . the goal of cross - validation was to estimate how accurately a predictive model ( classifier ) will perform in practice . this procedure , applied to the training dataset , is supervised because the phenotype information of the patients was needed to establish the predictive accuracy of each gene list . loocv implies using a single sample from the original dataset as the validation data ( sample test ) , and the remaining samples as training data . this was repeated such that each sample in the dataset was used once , as a sample test . each sample was characterized by a vector whose dimension was the number of genes that belonged to the reduced base that differentiated between hf and lf . the class with the minimum euclidean distance was assigned to the sample test ( nn classifier),25 and the average accuracy was calculated by iterating over all the samples . for that purpose , all the samples were normalized according to their gene variability ( each attribute or gene separately ) . in this way , all the genes had the same importance in the distance criterion . the distance between a sample and a phenotype class could have been defined in several ways , but the most robust one was using the median distance between the sample test and all the samples in the corresponding class . the legitimacy of the predictive accuracy based on the training set was then tested with the validation set , using the above - mentioned predictive model . it is important to remark that the application of the prediction model , designed in steps 1 to 3 , to the validation set was unsupervised . the final decision was made by consensus ( majority voting ) of the predictions made using the lists of most discriminatory genes . subjects were primarily caucasian ( 67% ) , had a mean age of 65.2 6.7 years and were not depressed based on hamilton depression scale ( 1.1 2.2 ) criteria . all subjects received a cumulative radiation dose of at least 68.4 gy and more than 90% received a total dose of 75.6 gy . most ( 64% ) of the subjects had a gleason score of 78 , and 71% had clinical t - stage below t3 . the gleason scoring and clinical staging are unique systems to classify the extent of the prostatic carcinoma.26,27 there was no difference between the clinical and demographic features of subjects in the training and validation sets . in general , ctrf as indicated by a significant decrease in fact - f scores from baseline ( 45.4 7.2 ) to completion of ebrt ( 39.4 10.0 , p < 0.05 ) was found . the training model was developed from the array outputs of 27 subjects ; 18 were hf ( mean fact - f change = 11.8 6.8 ) and 9 were lf ( mean fact - f change = 0.8 3.3 ) . as shown in figure 2 , it was impossible to visually distinguish hf and lf microarray outputs in heat map format using decibels as units of measure ( log2 of gene expression ) . the similarities between the hf and lf groups in the learning dataset were confirmed by further histogram analysis of gene expression . figure 3 shows that the corresponding statistical distributions of gene expressions in both groups were close to lognormal , with the main differences between both phenotypes occurring around the mode of both histograms ( expressions around 24 and 26 ) . a final list of 575 highly discriminatory genes according to expression was noted and defined by the intersection between those genes that were differentially expressed ( located in the 0.05% and 99.5% tails of the fold - change ratio cumulative distribution ) and which had a fr higher than 0.25 ( fig . fr plot for genes in the learning dataset with fold change lower than 0.52 and higher than 0.67 . these values ( of gene under- and over - expression ) corresponded , respectively , to the 0.05% and 99.5% tails of the fold - change distribution . it can be observed that the highest fr was 2.12 , and that genes with the highest fold change did not coincide with those exhibiting the highest fr . figure 6 shows the predictive accuracy curve of the different gene lists , established using the backward feature elimination algorithm . the shortest list with the highest accuracy ( 92.6% ) was composed by the first 14 genes with the highest fr . the lists with the first 15 , and 29 to 35 most discriminatory genes also provide the same maximum accuracy . as the data suggest , continuously adding genes with lower discriminatory power as defined by their fr failed to increase the accuracy of discrimination . when a histogram was used to assess the first 360 most discriminatory genes found by our analysis , we noted a shift of the mode of distribution for the lf patients to higher expressions ( 29210 ) with respect to the hf case ( 2627 ) , suggesting that hf patients show mostly lower expressions of these genes that we hypothesized were responsible for this phenotypic discrimination ( fig . figure 8 shows the pca plots ( unsupervised method ) of the learning dataset expressed in the base of the most 14 ( fig . the following can be observed :
the lf / hf phenotype discrimination became linearly separable in these reduced sets of genes , confirming the fact that the classification problem simplifies when reducing the dimension to the most discriminatory set of genes . both plots have a similar structure.the lf samples lie between samples p1a and xrt28a , which is genetically close to the region of the hf samples.also , sample xrt25a , which belongs to the lf category , is surrounded by hf samples . this sample might be a biological or behavioral outlier.the hf samples lie between samples xrtp2a and 13a . sample xrt20a also seems to mark a transition between lf and hf samples toward the west of the plot . the lf / hf phenotype discrimination became linearly separable in these reduced sets of genes , confirming the fact that the classification problem simplifies when reducing the dimension to the most discriminatory set of genes . the lf samples lie between samples p1a and xrt28a , which is genetically close to the region of the hf samples . also , sample xrt25a , which belongs to the lf category , is surrounded by hf samples . sample xrt20a also seems to mark a transition between lf and hf samples toward the west of the plot . interpretative phenomenological analysis ( ipa ) revealed that the 575 highly discriminatory genes were associated with the following canonical pathways : b cell development , autoimmune thyroid disease signaling , allograft rejection signaling , graft - versus - host disease signaling , and nur77 signaling in t lymphocytes . further , the differentially expressed genes were associated with the following functional networks : cancer and neurological disease . additional ipa was performed on the 360 most predictive genes ( having a learning predictive accuracy higher than 81% ) , a part of the 575 highly discriminatory genes , and it revealed concordance of pathway attributions observed in the initial ipa . the top canonical pathways of the 360 most predictive genes remained to be related to b cell development , but it also revealed other focused pathways related to t helper cell differentiation and interferon signaling . the top functional networks of the 360 genes remained to be related to cancer , followed by neurological disease and psychological disorders , suggesting that the most predictive genes are related to behavior experienced by cancer patients . seventeen subjects , independent of the training set , were used to assess the validity of the learned predictive model . seven were classified as hf ( mean fact - f change = 10.6 6.9 ) and 10 were lf ( mean fact - f change = 0.8 2.2 ) subjects . the prediction was based on majority voting , as follows : we first considered the most predictive gene cluster , a group consisting of the 14 most discriminatory genes deduced from the learning set , and the values of the expressions of these genes on both classes ( lf and hf ) represented in the training dataset . the samples of the training set expressed in the reduced base and their phenotype information were used to define the distance of the nn classifier used in this paper . second , the values of these discriminatory genes in the validation samples were read from the validation dataset . for each sample of the validation set , its predicted class was established using the k - nn algorithm , using the 14 different most discriminatory reduced sets of genes that were defined by the learning dataset . for instance , given the base composed by three first genes of the 14-size reduced set of genes , the k - nn algorithm calculated the distance defined in three - dimensional space between each validation sample and the samples of the training dataset belonging to each phenotype class . the class with the minimum distance was then predicted for the validation sample . this was repeated for the 14 different reduced bases , which yielded 14 different class predictions for each sample in the validation set . the final estimated class was then made by consensus or majority voting classifiers.28 a posterior probability was given to the class prediction , defined as the ratio of the number of votes assigned to the predicted class and the total number of voters . for example , if a validation sample has 12 predictions in the lf class ( and two in the hf class ) , the posterior probability to belong to lf will be 12/14 . three of the four misclassified samples belonged to the lf group ( false positives , patients were predicted to be hf ) and one to the hf ( false negative , patient predicted to be lf ) . these samples are outliers with respect to this classifier , because their expressions in the reduced base of genes are closer to the hf and lf groups , respectively ( tables 2 , 3 , and fig . interestingly , the 14 different predictions for these misclassified samples coincide , that is , the probability of these samples belonging to their predicted class according to the consensus criterion is 1 . this fact also strengthens the argument that these samples are biological or behavioral outliers , that is , their class assignment based on the change in their fact - f scores was ambiguous . subjects were primarily caucasian ( 67% ) , had a mean age of 65.2 6.7 years and were not depressed based on hamilton depression scale ( 1.1 2.2 ) criteria . all subjects received a cumulative radiation dose of at least 68.4 gy and more than 90% received a total dose of 75.6 gy . most ( 64% ) of the subjects had a gleason score of 78 , and 71% had clinical t - stage below t3 . the gleason scoring and clinical staging are unique systems to classify the extent of the prostatic carcinoma.26,27 there was no difference between the clinical and demographic features of subjects in the training and validation sets . in general , ctrf as indicated by a significant decrease in fact - f scores from baseline ( 45.4 7.2 ) to completion of ebrt ( 39.4 10.0 , p < 0.05 ) was found . the training model was developed from the array outputs of 27 subjects ; 18 were hf ( mean fact - f change = 11.8 6.8 ) and 9 were lf ( mean fact - f change = 0.8 3.3 ) . as shown in figure 2 , it was impossible to visually distinguish hf and lf microarray outputs in heat map format using decibels as units of measure ( log2 of gene expression ) . the similarities between the hf and lf groups in the learning dataset were confirmed by further histogram analysis of gene expression . figure 3 shows that the corresponding statistical distributions of gene expressions in both groups were close to lognormal , with the main differences between both phenotypes occurring around the mode of both histograms ( expressions around 24 and 26 ) . a final list of 575 highly discriminatory genes according to expression was noted and defined by the intersection between those genes that were differentially expressed ( located in the 0.05% and 99.5% tails of the fold - change ratio cumulative distribution ) and which had a fr higher than 0.25 ( fig . fr plot for genes in the learning dataset with fold change lower than 0.52 and higher than 0.67 . these values ( of gene under- and over - expression ) corresponded , respectively , to the 0.05% and 99.5% tails of the fold - change distribution . it can be observed that the highest fr was 2.12 , and that genes with the highest fold change did not coincide with those exhibiting the highest fr . figure 6 shows the predictive accuracy curve of the different gene lists , established using the backward feature elimination algorithm . the shortest list with the highest accuracy ( 92.6% ) was composed by the first 14 genes with the highest fr . the lists with the first 15 , and 29 to 35 most discriminatory genes also provide the same maximum accuracy . as the data suggest , continuously adding genes with lower discriminatory power as defined by their fr failed to increase the accuracy of discrimination . when a histogram was used to assess the first 360 most discriminatory genes found by our analysis , we noted a shift of the mode of distribution for the lf patients to higher expressions ( 29210 ) with respect to the hf case ( 2627 ) , suggesting that hf patients show mostly lower expressions of these genes that we hypothesized were responsible for this phenotypic discrimination ( fig . figure 8 shows the pca plots ( unsupervised method ) of the learning dataset expressed in the base of the most 14 ( fig . the following can be observed :
the lf / hf phenotype discrimination became linearly separable in these reduced sets of genes , confirming the fact that the classification problem simplifies when reducing the dimension to the most discriminatory set of genes . both plots have a similar structure.the lf samples lie between samples p1a and xrt28a , which is genetically close to the region of the hf samples.also , sample xrt25a , which belongs to the lf category , is surrounded by hf samples . this sample might be a biological or behavioral outlier.the hf samples lie between samples xrtp2a and 13a . sample xrt20a also seems to mark a transition between lf and hf samples toward the west of the plot . the lf / hf phenotype discrimination became linearly separable in these reduced sets of genes , confirming the fact that the classification problem simplifies when reducing the dimension to the most discriminatory set of genes . the lf samples lie between samples p1a and xrt28a , which is genetically close to the region of the hf samples . also , sample xrt25a , which belongs to the lf category , is surrounded by hf samples . sample xrt20a also seems to mark a transition between lf and hf samples toward the west of the plot . interpretative phenomenological analysis ( ipa ) revealed that the 575 highly discriminatory genes were associated with the following canonical pathways : b cell development , autoimmune thyroid disease signaling , allograft rejection signaling , graft - versus - host disease signaling , and nur77 signaling in t lymphocytes . further , the differentially expressed genes were associated with the following functional networks : cancer and neurological disease . additional ipa was performed on the 360 most predictive genes ( having a learning predictive accuracy higher than 81% ) , a part of the 575 highly discriminatory genes , and it revealed concordance of pathway attributions observed in the initial ipa . the top canonical pathways of the 360 most predictive genes remained to be related to b cell development , but it also revealed other focused pathways related to t helper cell differentiation and interferon signaling . the top functional networks of the 360 genes remained to be related to cancer , followed by neurological disease and psychological disorders , suggesting that the most predictive genes are related to behavior experienced by cancer patients . seventeen subjects , independent of the training set , were used to assess the validity of the learned predictive model . seven were classified as hf ( mean fact - f change = 10.6 6.9 ) and 10 were lf ( mean fact - f change = 0.8 2.2 ) subjects . the prediction was based on majority voting , as follows : we first considered the most predictive gene cluster , a group consisting of the 14 most discriminatory genes deduced from the learning set , and the values of the expressions of these genes on both classes ( lf and hf ) represented in the training dataset . the samples of the training set expressed in the reduced base and their phenotype information were used to define the distance of the nn classifier used in this paper . second , the values of these discriminatory genes in the validation samples were read from the validation dataset . for each sample of the validation set , its predicted class was established using the k - nn algorithm , using the 14 different most discriminatory reduced sets of genes that were defined by the learning dataset . for instance , given the base composed by three first genes of the 14-size reduced set of genes , the k - nn algorithm calculated the distance defined in three - dimensional space between each validation sample and the samples of the training dataset belonging to each phenotype class . the class with the minimum distance was then predicted for the validation sample . this was repeated for the 14 different reduced bases , which yielded 14 different class predictions for each sample in the validation set . the final estimated class was then made by consensus or majority voting classifiers.28 a posterior probability was given to the class prediction , defined as the ratio of the number of votes assigned to the predicted class and the total number of voters . for example , if a validation sample has 12 predictions in the lf class ( and two in the hf class ) , the posterior probability to belong to lf will be 12/14 . three of the four misclassified samples belonged to the lf group ( false positives , patients were predicted to be hf ) and one to the hf ( false negative , patient predicted to be lf ) . these samples are outliers with respect to this classifier , because their expressions in the reduced base of genes are closer to the hf and lf groups , respectively ( tables 2 , 3 , and fig . interestingly , the 14 different predictions for these misclassified samples coincide , that is , the probability of these samples belonging to their predicted class according to the consensus criterion is 1 . this fact also strengthens the argument that these samples are biological or behavioral outliers , that is , their class assignment based on the change in their fact - f scores was ambiguous . while it is efficacious in many cases , its toxicity profile is significant and common , but not ubiquitous . consequently , the ability to predict toxicities of rt has long been of interest . with better understanding of the pathobiology of radiation injury , using genomics as the basis for toxicity risk prediction has been the focus of active research.29 in contrast to the toxicity presented in this paper , the primary toxicity phenotypes studied have been tissue - centric injuries such as mucositis , dermatitis , and pneumonitis and fibrosis.30 and the primary approaches used to try to identify predictive relationships between genes or snps and toxicities have primarily relied on candidate gene or genome - wide association analyses . in both cases , the majority of investigations have sought to identify one or two genes or snps associated with the phenotype of interest . the resulting lack of consistency of results has been disappointing.31 our approach differed in that we proposed that the risk of a complex disease , such as ctrf , could well be more easily defined by identifying groups of simultaneously expressed , synergistically functioning genes . while this hypothesis is supported by studies in which bayesian network development was used to identify snp clusters predictive of chemotherapy - related side effects,1113 we sought to accelerate and simplify the analytical process through the use of a novel method in which we used a sequence of supervised and learned ( unsupervised ) filters to identify the most predictive cluster of genes for ctrf . our finding that the gene cluster so identified was then able to predict ctrf risk with an accuracy of > 75% suggests that the approach has validity . the process of selecting the most predictive cluster of genes revealed informative considerations . for example , the genes with the highest fold change did not coincide with those exhibiting the highest fr because the means of both distributions were different , hence their tails did not overlap . so , in this method we concluded that fr was a better feature selection method than fold change . while , in the case of fold - change analysis , noisy genes are typically penalized by the fr selection method because of an increase of their variance genes with the highest fr and fold change have the biggest discriminatory power and are assumed to be involved in the genesis of fatigue . interestingly , the histogram analysis of the first 360 genes that most discriminated between hf and lf subjects was informative in that the shift of the mode of distribution showed lower expressions of these genes among hf subjects . it seems possible that it is this distributional shift that ultimately is responsible for discriminating the fatigue phenotype in this population . we were unable to correctly predict four samples , based on our phenotypic approach , since the consensus provides the opposite class in all the cases . these classified samples were close to the border of separation between both fatigue classes ( fig . there are three possibilities : ( 1 ) these samples are behavioral outliers , ( 2 ) the phenotypic approach needs further review and improvement , especially dealing with samples that are bordering the cut - off scores set for fatigue grouping , and ( 3 ) possible use of more sophisticated algorithms ( black box neural networks ) to classify the samples may be needed , which could run the risk of losing the clarity in the interpretation . nonetheless , the fact that the analysis was successful in predicting lf / hf in an unrelated population with reasonable accuracy suggests that increasing the number of subjects in the training population would likely improve the predictive model s ability . nevertheless , this analysis confirms that it is possible to separate both classes of the lf / hf phenotype by reducing the dimension to the most discriminatory genes , provided by their fr . the importance of predicting toxicity or adverse event risk associated with cancer treatment regimens can not be understated as the clinical implications in personalizing cancer therapy and prospectively attenuating toxicity risk are significant . furthermore , this type of information provides patients and their care - givers more specific knowledge upon which to make treatment decisions . a novel analytical algorithm introduced in this study that incorporates fold - change differential analysis , linear discriminant analysis , and a k - nn can predict radiation - related fatigue in men with non - metastatic prostate cancer . applicability of this novel algorithm to detect other treatment - related toxicities in other cancer populations would be worthwhile to pursue . | backgroundfatigue is a common side effect of cancer ( ca ) treatment .
we used a novel analytical method to identify and validate a specific gene cluster that is predictive of fatigue risk in prostate cancer patients ( pcp ) treated with radiotherapy ( rt).methodsa total of 44 pcp were categorized into high - fatigue ( hf ) and low - fatigue ( lf ) cohorts based on fatigue score change from baseline to rt completion .
fold - change differential and fisher s linear discriminant analyses ( lda ) from 27 subjects with gene expression data at baseline and rt completion generated a reduced base of most discriminatory genes ( learning phase ) .
a nearest - neighbor risk ( k - nn ) prediction model was developed based on small - scale prognostic signatures .
the predictive model validity was tested in another 17 subjects using baseline gene expression data ( validation phase).resultthe model generated in the learning phase predicted hf classification at rt completion in the validation phase with 76.5% accuracy.conclusionthe results suggest that a novel analytical algorithm that incorporates fold - change differential analysis , lda , and a k - nn may have applicability in predicting regimen - related toxicity in cancer patients with high reliability , if we take into account these results and the limited amount of data that we had at disposal . it is expected that the accuracy will be improved by increasing data sampling in the learning phase . |
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in the united states , pleural effusion is diagnosed in approximately 1.5 million
patients each year , and therapeutic thoracentesis is therefore a common medical
procedure . in addition , thoracentesis is one of the bedside procedures most commonly associated
with iatrogenic complications , particularly pneumothorax . a systematic review and
meta - analysis of 24 studies , including a total of 6,605 thoracenteses , showed a 6.0%
overall incidence of pneumothorax , one third of the cases of pneumothorax requiring
chest tube insertion . the safety of thoracentesis has been directly associated with the technical skills of
the operator . within the past several years , system and procedural
variables , including a lack of real - time ultrasound imaging , operator inexperience ,
drainage of large volumes of fluid , and repeated thoracentesis , have been shown to
increase the likelihood of complications . an optimal thoracentesis protocol should include the
use of the best available techniques ( in order to minimize procedural errors and
complications ) in combination with a system that improves the technical skills of the
operator . various risk factors for complications associated
with thoracentesis ( particularly pneumothorax ) were described in a recent
study and
are displayed in chart 1 . those risk factors
include an inexperienced or poorly trained operator , an inadequate or inexperienced
support team , nonstandardized systems , and lack of ultrasound guidance . in view of these considerations , we sought
to determine whether ultrasound guidance reduces the risk of pneumothorax following
thoracentesis . .
the primary outcome measure of the present study was to determine whether the incidence
of pneumothorax following thoracentesis differed between two conditions : thoracentesis
performed under ultrasound guidance by a team of expert pulmonologists ( with 20 years of
experience , specific physician training in the procedure , more than 500 thoracenteses
performed , and training in chest ultrasound ) using a standardized protocol and standard
equipment ; and thoracentesis performed without ultrasound guidance by the same team
using the same protocol and equipment . secondary outcome measures included the number of
procedures during which no fluid was removed , the volume of fluid removed during each
procedure , and the need for chest tube placement . this was a prospective randomized study conducted between may of 2012 and october of
2012 , involving consecutive inpatients and outpatients with pleural effusion treated in
the department of respiratory diseases ( respiratory monitoring section , respiratory
ward , or day hospital ) of the sestri levante general hospital , in the municipality of
sestri levante , italy . the inclusion criteria were as follows : presenting with pleural
effusion visible on chest x - rays and requiring pleural puncture ( diagnostic or
therapeutic thoracentesis ) on the basis of previously published
criteria ;
and being in the 18 - 85 year age bracket . we enrolled a total of 197 patients with
pleural effusion due to various causes , including neoplasms , chronic heart failure ,
rheumatic diseases , pneumonia , and tuberculosis . of those 197 patients , 34 declined to
participate and 3 were excluded for other reasons ( not recorded ) . therefore , 160
patients were randomly allocated to undergo thoracentesis with ultrasound guidance
( ultrasound - guided thoracentesis group ) or without ultrasound guidance ( control group ) . a statistician who was not involved in the present study devised a randomization plan
using a computerized random number generator . the patients underwent thoracentesis with or
without ultrasound guidance on the basis of the randomization plan . ultrasound was not
used for real - time guidance ; rather , it was used immediately before the procedure in
order to identify the appropriate site . we employed a portable ultrasound system ( logiq p5 ; ge
healthcare , chalfont , uk ) with a convex probe ( 3.5 - 5.0 mhz ) . thoracentesis was performed by one of three pulmonologists , with the patient in the
sitting position , following a local protocol written in accordance with the 2010 british
thoracic society pleural disease guideline . , livorno , italy ) consisting of a three - way stopcock , a large ( 60-ml ) syringe , a
2,000-ml vacuum - free collection bag , and a 50-mm , 18-g needle . fluid was drained either
by passive drainage or by active drainage with the 60-ml syringe connected to the
three - way stopcock . fluid removal was terminated when there was chest pain , excessive cough , a vasovagal
event , shortness of breath , or air suction . drainage was stopped when 1.5 l of pleural
fluid had been removed . within 60 min after the procedure , posteroanterior and lateral
decubitus chest x - rays were performed . the occurrence of pneumothorax was determined on
the basis of the british thoracic society guidelines . the investigators involved in the analysis
of the data were blinded to the complications . the primary outcome measure of the
present study was the incidence of pneumothorax following thoracentesis . secondary
outcome measures included the need for chest tube drainage and the volume of fluid
drained . the study was conducted in accordance with the declaration of helsinki and was
approved by the research ethics committee of azienda sanitaria locale
no the main objectives of the present study were to compare the incidence of pneumothorax
following thoracentesis performed under ultrasound guidance with that of pneumothorax
following thoracentesis performed without ultrasound guidance and to determine the
diagnostic value ( i.e. , the number of procedures during which fluid was removed and the
volume of fluid removed ) of ultrasound - guided thoracentesis . we used logistic regression
for categorical variables ( incidence of pneumothorax and secondary outcomes of
thoracentesis ) and analysis of covariance for continuous variables . all data were
analyzed with the r software , version 2.13.2 ( the r foundation for statistical
computing , vienna , austria ) . all of the
patients were in the 32 - 84 year age bracket ( mean age , 67.8 14.9 years ) . the causes of pleural effusion were as follows : pleural mesothelioma , in 11 patients ;
metastatic pleural neoplasm , in 90 patients ; rheumatic disease , in 6 patients ; chronic
heart failure , in 23 patients ; pneumonia , in 20 patients ; and tuberculosis , in 10
patients ( table 1 ) . 8 were suspended because of
complications : chest pain ( in 4 ) ; air suction ( in 3 ) ; and excessive cough with dyspnea
( in 1 ) . of the 80 thoracenteses performed under ultrasound guidance , only 1 was
suspended ( because of chest pain and shortness of breath ) . pneumothorax following thoracentesis occurred in 1 of the 80 patients submitted to the
procedure performed under ultrasound guidance and in 10 of the 80 patients submitted to
the procedure performed without ultrasound guidance , the difference being statistically
significant ( p = 0.009 ) . none of the 11 cases of pneumothorax required chest tube
placement . as can be seen in table 2 , 79 of the 80
ultrasound - guided procedures were successful , as were 72 of the 80 procedures performed
without ultrasound guidance . as can be seen in figure
2 , the mean volume of fluid drained during the former procedures was larger
than was that drained during the latter procedures ( 960 500 ml vs. 770 480 ml ) , the
difference being statistically significant ( p = 0.03 ) . table 2patient data and results ( including statistics ) in the patients submitted
to thoracentesis performed under ultrasound guidance ( the ultrasound - guided
thoracentesis group ) and in those submitted to thoracentesis performed without
ultrasound guidance ( the control group ) . figure 2mean volume of pleural fluid removed during thoracentesis in the patients
who underwent the procedure performed under ultrasound guidance
( ultrasound - guided thoracentesis group ) and in those who underwent the
procedure performed without ultrasound guidance ( control group ) . the difference
between the two groups was statistically significant ( p = 0.014 ) .
in 8 of the patients in the control group , an
ultrasound - guided thoracentesis was performed in 4 of those 8 patients , pleural fluid
being successfully removed ( 240 30 ml ) . the probability of success ( i.e. , fluid removal ) was approximately nine times higher in
the patients who underwent ultrasound - guided thoracentesis than in those who underwent
thoracentesis without ultrasound guidance ( or = 8.8 ) . in addition , the risk of
pneumothorax was 90% lower in the former than in the latter ( or = 0.09 ) . the purpose of the present study was to determine whether the incidence of pneumothorax
following thoracentesis decreases when most of the risk factors ( mechanical ventilation
use , an inexperienced team , and an inexperienced operator ) are absent and ultrasound
guidance is used . we have demonstrated that ultrasound - guided thoracentesis is a very
safe procedure , which was associated with a very low ( 1.25% ) incidence of pneumothorax
in our sample ( 1 case among the 80 patients in the ultrasound - guided thoracentesis
group ) . in fact , ultrasound guidance reduced the risk of pneumothorax by 90% ( or = 0.09 ;
95% ci : 0.005 - 0.5 ; p = 0.009 ) . although there was no difference between the two groups
in terms of the need for chest tube drainage , the amount of fluid drained was
significantly greater in the ultrasound - guided thoracentesis group than in the control
group ( p = 0.0014 ) . the strength of the present study lies in the fact that it was performed by skilled
pulmonologists using carefully standardized procedures to evaluate the benefit of
ultrasound guidance without any bias . to our knowledge we are aware of the limitations of our study , which include the fact that patients
on mechanical ventilation were not enrolled and the fact that real - time ultrasound
guidance was not performed ( ultrasound being used only to mark the site before
thoracentesis was performed ) . real - time ultrasound guidance allows clinicians to
visualize the needle and important adjacent structures and therefore avoid accidental
punctures , as well as allowing a further reduction in the risk of pneumothorax following
thoracentesis . nevertheless , its routine use at the bedside remains low , although it is
considered the standard of care in safely locating , characterizing , and draining pleural
fluid . ultrasound is highly sensitive for detecting pleural effusions , even when chest
x - rays are normal . chest x - rays can miss up to 500 ml of pleural effusion in cases of
interlobar and loculated pleural effusion . lung ultrasound can detect as little as 20 ml
of pleural fluid , whereas an upright posteroanterior chest x - ray can not detect blunting
of the costophrenic angle unless there is at least 100 ml of fluid . ultrasound - guided thoracentesis has been
associated with lower total hospital costs and a lower incidence of pneumothorax and
hemorrhage . the overall incidence of pneumothorax varies from
4.0% to 30.3% , and chest tube insertion is required in 1.7% of all
thoracenteses , in order to evacuate symptomatic pneumothoraces ; therefore , 20 - 50% of all
pneumothoraces following thoracentesis require chest tube insertion . the reported incidence of pneumothorax is
significantly lower in studies published after 2000 than in earlier studies ( 4.6% vs.
8.7% ) . first , air can flow from the atmosphere into the pleural space , as occurs when the
negative pressure of the pleural space communicates freely with the atmosphere . this
most often occurs as the syringe is removed from a needle or catheter , particularly when
the individual performing the procedure is inexperienced . second , the thoracentesis
needle can lacerate the lung and allow air to enter the pleural space from the
alveoli . third , the decrease in pleural pressure can lead to
a rupture of the visceral pleura . fourth , trapped lung or lung entrapment develops as
a result of transitory pleuropulmonary fistula . multiple risk factors for pneumothorax following
thoracentesis have been identified , including the type of needle used , operator inexperience , the presence of
emphysema , having previously undergone
thoracentesis , being on mechanical ventilation , and even the lack of ultrasound
guidance . the training of chest ultrasound technicians is
task - specific and is aimed at developing the skill of identifying pleural fluid and
surrounding organs , as well as that of providing an unobstructed view of the pleural
fluid . these simple and well - defined skills can be readily acquired by pulmonologists
and can avoid other complications during most thoracentesis procedures . in addition , ultrasound - guided
thoracentesis is a procedure that most pulmonologists can perform after short - term
training . we believe that meticulous adherence to sonographic
criteria and avoidance of patient movement during the time elapsed between ultrasound
examination and fluid removal are key factors responsible for the low rate of
pneumothorax following thoracentesis and the larger volume of fluid removed during the
procedure .
in conclusion , ultrasound guidance can increase patient safety and the amount of fluid
removed during thoracentesis . ultrasonography is an easily learned technique that not
only enhances the physical examination but also has the distinct advantage of being a
portable imaging technique for the evaluation of the pleural space . ultrasound is
currently used in a limited number of thoracenteses , and the present study provides
evidence supporting the more widespread use of this technique . dentre os procedimentos realizados beira do leito , a toracocentese um dos
mais comumente associados a complicaes iatrognicas , particularmente
pneumotrax . foram recentemente identificados vrios fatores de risco de
complicaes associadas toracocentese : a inexperincia do operador , a
inadequao ou inexperincia da equipe de apoio , a ausncia de um protocolo
padronizado e a ausncia de ultrassonografia para guiar o procedimento . nosso
objetivo foi determinar se a toracocentese guiada por ultrassonografia pode
reduzir o risco de pneumotrax e melhorar os desfechos ( menos procedimentos sem
remoo de lquido e maior volume de lquido removido durante os procedimentos ) . para compararmos a toracocentese guiada por ultrassonografia toracocentese sem
ultrassonografia , todos os procedimentos foram realizados pela mesma equipe de
pneumologistas especialistas , os quais usaram o mesmo protocolo padronizado em
ambas as condies . cento e sessenta pacientes foram aleatoriamente divididos em dois grupos :
toracocentese guiada por ultrassonografia e toracocentese sem ultrassonografia ( n
= 80 por grupo ) . o desfecho primrio foi pneumotrax aps a toracocentese . os
desfechos secundrios foram o nmero de procedimentos sem remoo de lquido e o
volume de lquido drenado durante o procedimento . houve pneumotrax em 1 dos 80 pacientes submetidos a toracocentese guiada por
ultrassonografia e em 10 dos 80 submetidos a toracocentese sem ultrassonografia ; a
diferena foi estatisticamente significante ( p = 0,009 ) . lquido foi removido em
79 dos 80 procedimentos guiados por ultrassonografia e em 72 dos 80 que no o
foram . a mdia do volume de lquido drenado foi maior nos procedimentos guiados
por ultrassonografia do que naqueles que no o foram ( 960 500 ml vs. 770 480
ml ) ; a diferena foi estatisticamente significante ( p = 0,03 ) . a ultrassonografia aumenta o rendimento da toracocentese e reduz o risco de
pneumotrax aps o procedimento . nos estados unidos , o derrame pleural diagnosticado em aproximadamente 1,5 milhes
de pacientes por ano ; portanto , a toracocentese teraputica um procedimento mdico
comum . alm disso , dentre os procedimentos realizados beira do leito , a toracocentese um
dos mais comumente associados a complicaes iatrognicas , particularmente
pneumotrax . uma reviso sistemtica e meta - anlise de 24
estudos , incluindo um total de 6.605 toracocenteses , mostrou uma incidncia global de
pneumotrax de 6,0% ; houve necessidade de insero de dreno torcico em um tero dos
casos de pneumotrax . estabeleceu - se uma associao direta entre a segurana da toracocentese e as
habilidades tcnicas do operador . nos ltimos anos , demonstrou - se que variveis
relacionadas ao sistema e ao procedimento , tais como a ausncia de ultrassonografia
em tempo real , a inexperincia do operador , a drenagem de grandes volumes de lquido
e a repetio da toracocentese , aumentam a probabilidade de
complicaes . o protocolo ideal de toracocentese deveria
incluir as melhores tcnicas disponveis ( a fim de minimizar erros de procedimento e
complicaes ) e um sistema que melhorasse as habilidades tcnicas do
operador . vrios fatores de risco de complicaes
associadas toracocentese ( particularmente pneumotrax ) foram descritos em um estudo
recente
e so apresentados no quadro 1 . esses fatores
de risco incluem um operador inexperiente ou mal treinado , uma equipe de apoio
inadequada ou inexperiente , sistemas no padronizados e a ausncia de orientao
ultrassonogrfica . diante dessas consideraes , procuramos
determinar se a ultrassonografia reduz o risco de pneumotrax aps a
toracocentese . o objetivo primrio ( ou desfecho primrio ) do presente estudo foi determinar se a
incidncia de pneumotrax aps a toracocentese guiada por ultrassonografia e
realizada por uma equipe de pneumologistas especialistas ( com 20 anos de experincia ,
treinamento mdico especfico para o procedimento , mais de 500 toracocenteses
realizadas e treinamento em ultrassonografia de trax ) , com protocolo e
equipamento padronizados , seria diferente da incidncia de pneumotrax aps a
toracocentese realizada pela mesma equipe , com o mesmo protocolo e o mesmo
equipamento , porm sem a ultrassonografia para guiar o procedimento . de procedimentos sem retirada de lquido , o volume de
lquido retirado durante cada procedimento e a necessidade de colocao de dreno
torcico . trata - se de um estudo prospectivo randomizado , realizado entre maio de 2012 e outubro
de 2012 , envolvendo pacientes internados e ambulatoriais consecutivos , com derrame
pleural , tratados no departamento de doenas respiratrias ( unidade de monitorao
respiratria , enfermaria respiratria ou hospital - dia ) do hospital geral de sestri
levante , no municpio de sestri levante , itlia . os critrios de incluso foram os
seguintes : apresentar derrame pleural que fosse visvel na radiografia de trax e
necessitasse de puno pleural ( toracocentese diagnstica ou teraputica ) com base em
critrios previamente publicados ; e estar na faixa etria de 18 - 85 anos . foram
selecionados 197 pacientes com derrame pleural de diversas causas , tais como
neoplasias , insuficincia cardaca crnica , doenas reumticas , pneumonia e
tuberculose . dos 197 pacientes selecionados , 34 recusaram - se a participar e 3 foram
excludos por outros motivos ( no registrados ) . portanto , 160 pacientes foram
aleatoriamente divididos em dois grupos : toracocentese guiada por ultrassonografia e
toracocentese sem ultrassonografia ( grupo controle ) . um estatstico que no se envolveu no presente estudo elaborou um plano de
randomizao por meio de um gerador computadorizado de nmeros aleatrios . cada um
dos mdicos responsveis pelo recrutamento recebeu o plano de randomizao em um
envelope lacrado . os pacientes foram submetidos toracocentese com ou sem orientao
ultrassonogrfica com base no plano de randomizao . a ultrassonografia no foi usada
para orientao em tempo real , mas sim imediatamente antes do procedimento , para
identificar o local apropriado . a toracocentese foi realizada imediatamente aps o
local ter sido marcado . empregamos um aparelho de ultrassom porttil ( logiq p5 ; ge
healthcare , chalfont , reino unido ) com uma sonda convexa ( 3,5 - 5,0 mhz ) . a toracocentese foi realizada por um dos trs pneumologistas de nosso grupo , com o
paciente na posio sentada , conforme um protocolo local , baseado na british
thoracic society pleural disease guideline , de 2010 . , livorno , itlia ) composto por uma torneira de
trs vias , uma seringa grande ( 60 ml ) , uma bolsa coletora de 2.000 ml ( sem vcuo ) e
uma agulha de 18 g e 50 mm . o lquido foi drenado por meio de drenagem passiva ou de
drenagem ativa com a seringa de 60 ml ligada torneira de trs vias . a retirada de lquido era interrompida caso houvesse dor torcica , tosse excessiva ,
um evento vasovagal , falta de ar ou suco de ar . determinou - se a ocorrncia de pneumotrax com base nas diretrizes da british
thoracic society . os investigadores envolvidos na anlise dos dados
no estavam cientes das complicaes . o desfecho primrio do presente estudo foi a
incidncia de pneumotrax aps a toracocentese . desfechos secundrios incluram a
necessidade de drenagem torcica e o volume de lquido drenado . o estudo foi
conduzido de acordo com a declarao de helsinki e foi aprovado pelo comit de tica
em pesquisa da azienda sanitaria locale n 4 chiavarese , em ligria ,
na itlia . todos os pacientes que participaram do estudo assinaram um termo de
consentimento livre e esclarecido . os principais objetivos do presente estudo foram comparar a incidncia de pneumotrax
aps a toracocentese guiada por ultrassonografia incidncia de pneumotrax aps a
toracocentese realizada sem ultrassonografia e determinar o valor diagnstico ( isto
, o nmero de procedimentos nos quais se retirou lquido e o volume de lquido
retirado ) da toracocentese guiada por ultrassonografia . usamos regresso logstica
para as variveis categricas ( incidncia de pneumotrax e desfechos secundrios da
toracocentese ) e anlise de covarincia para as variveis contnuas . todos os dados
foram analisados com o programa r , verso 2.13.2 ( the r foundation for
statistical computing , viena , ustria ) . todos os 160 pacientes avaliados ( 122 homens e 38 mulheres ) completaram o estudo . todos os pacientes estavam na faixa etria de 32 - 84 anos ( mdia de idade : 67,8 14,9
anos ) . as causas de derrame pleural foram as seguintes : mesotelioma pleural , em 11
pacientes ; neoplasia pleural metasttica , em 90 pacientes ; doena reumtica , em 6
pacientes ; insuficincia cardaca crnica , em 23 pacientes ; pneumonia , em 20
pacientes e tuberculose , em 10 pacientes ( tabela
1 ) . desses 80
procedimentos , 8 foram suspensos por causa de complicaes : dor torcica ( em 4 ) ;
suco de ar ( em 3 ) e tosse excessiva com dispneia ( em 1 ) . das 80 toracocenteses
guiadas por ultrassonografia , apenas 1 foi suspensa ( por causa de dor torcica e
falta de ar ) . houve pneumotrax em 1 dos 80 pacientes submetidos toracocentese guiada por
ultrassonografia e em 10 dos 80 submetidos toracocentese sem ultrassonografia ; a
diferena foi estatisticamente significante ( p = 0,009 ) . como se pode ver na tabela 2 , 79 dos 80
procedimentos guiados por ultrassonografia foram bem sucedidos , assim como o foram 72
dos 80 procedimentos realizados sem orientao ultrassonogrfica . como se pode ver na
figura 2 , a mdia do volume de lquido
drenado foi maior nos procedimentos guiados por ultrassonografia do que naqueles que
no o foram ( 960 500 ml vs. 770 480 ml ) ; a diferena foi estatisticamente
significante ( p = 0,03 ) . tabela 2dados dos pacientes e resultados ( incluindo estatsticas ) nos pacientes
submetidos a toracocentese guiada por ultrassonografia e naqueles submetidos
a toracocentese sem ultrassonografia ( grupo controle ) . figura 2mdia do volume de lquido pleural retirado durante a toracocentese nos
pacientes submetidos ao procedimento guiado por ultrassonografia e naqueles
submetidos ao procedimento realizado sem orientao ultrassonogrfica ( grupo
controle ) . a diferena entre em 8 dos pacientes do grupo controle , no houve retirada de lquido durante o
procedimento . desses 8 pacientes , 4 foram submetidos a toracocentese guiada por
ultrassonografia , em que se logrou xito em retirar lquido pleural ( 240 30
ml ) . a probabilidade de sucesso ( ou seja , retirada de lquido ) foi aproximadamente nove
vezes maior nos pacientes submetidos toracocentese guiada por ultrassonografia do
que naqueles submetidos toracocentese sem orientao ultrassonogrfica ( or = 8,8 ) . alm disso , o risco de pneumotrax foi 90% mais baixo naqueles do que nestes ( or =
0,09 ) . o objetivo do presente estudo foi determinar se a incidncia de pneumotrax aps a
toracocentese diminui quando a maioria dos fatores de risco ( uso de ventilao
mecnica , uma equipe inexperiente e um operador inexperiente ) est ausente e o
procedimento guiado por ultrassonografia . ns demonstramos que a toracocentese
guiada por ultrassonografia um procedimento muito seguro , associado a uma
incidncia muito baixa ( 1,25% ) de pneumotrax em nossa amostra ( 1 caso dentre os 80
pacientes submetidos toracocentese guiada por ultrassonografia ) . de fato , a
ultrassonografia reduziu o risco de pneumotrax em 90% ( or = 0,09 ; ic95% : 0,005 - 0,5 ;
p = 0,009 ) . embora no tenha havido diferena entre os dois grupos no tocante
necessidade de drenagem torcica , a quantidade de lquido drenado foi
significativamente maior no grupo de pacientes submetidos toracocentese guiada por
ultrassonografia do que no grupo controle ( p = 0,0014 ) . a fora do presente estudo reside no fato de que foi realizado por pneumologistas
qualificados , os quais usaram procedimentos cuidadosamente padronizados para avaliar
de maneira imparcial o benefcio da orientao ultrassonogrfica . estamos cientes das limitaes de
nosso estudo , tais como o fato de no termos includo pacientes que recebiam
ventilao mecnica e o fato de no termos usado a ultrassonografia em tempo real ( a
ultrassonografia foi usada antes da toracocentese , apenas para marcar o local ) . a
orientao ultrassonogrfica em tempo real permite que os clnicos visualizem a
agulha e estruturas adjacentes importantes e , portanto , evitem perfuraes
acidentais , alm de permitir maior reduo do risco de pneumotrax aps a
toracocentese . no obstante , seu uso rotineiro beira do leito permanece baixo ,
embora seja o padro de tratamento para localizar com segurana , caracterizar e
drenar o lquido pleural . o ultrassom apresenta grande sensibilidade no tocante
deteco de derrames pleurais , mesmo quando a radiografia de trax normal . possvel que a radiografia de trax no detecte at 500 ml de derrame pleural em
casos de derrame pleural interlobar e loculado . a ultrassonografia de pulmo detecta
apenas 20 ml de lquido pleural , ao passo que a radiografia de trax em incidncia
posteroanterior na posio vertical no capaz de detectar a obliterao do ngulo
costofrnico a menos que haja no mnimo 100 ml de lquido . a toracocentese guiada por
ultrassonografia j foi associada a menores custos hospitalares totais e menor
incidncia de pneumotrax e hemorragia . a incidncia global de pneumotrax varia de
4,0% a 30,3% , e 1,7% de todos os indivduos submetidos a
toracocentese necessitam de dreno torcico para a evacuao de pneumotrax
sintomtico ; portanto , a insero de dreno torcico necessria em 20 - 50% de todos
os casos de pneumotrax aps toracocentese . a incidncia de pneumotrax
significativamente menor em estudos publicados aps 2000 do que em estudos anteriores
( 4,6% vs. 8,7% ) . primeiro , o ar pode fluir da atmosfera para o espao pleural , como
ocorre quando a presso negativa no espao pleural comunica - se livremente com a
atmosfera . isso ocorre mais comumente quando a seringa retirada de uma agulha ou
cateter , particularmente quando o indivduo que realiza o procedimento
inexperiente . segundo , a agulha de toracocentese pode
dilacerar o pulmo e permitir que o ar vindo dos alvolos entre no espao pleural . terceiro , a diminuio
da presso pleural pode levar a uma ruptura da pleura visceral . foram identificados mltiplos fatores
de risco de pneumotrax aps toracocentese , tais como o tipo de agulha
usada , a inexperincia do operador , a presena de
enfisema , toracocentese prvia , ventilao
mecnica e at mesmo a ausncia de orientao
ultrassonogrfica . o treinamento de ultrassonografistas de trax
voltado prtica de tarefas especficas e seu objetivo desenvolver as habilidades
de identificar lquido pleural e rgos circunjacentes e propiciar visualizao
desobstruda do lquido pleural . essas habilidades simples e bem definidas podem ser
prontamente adquiridas por pneumologistas e podem evitar outras complicaes durante
a maioria das toracocenteses . alm disso , a toracocentese guiada por
ultrassonografia um procedimento que a maioria dos pneumologistas pode realizar
aps um treinamento de curta durao . acreditamos que observar rigorosamente os
critrios ultrassonogrficos e evitar mover o paciente entre a ultrassonografia e a
retirada do lquido sejam os principais fatores responsveis pela baixa incidncia de
pneumotrax aps a toracocentese e pelo maior volume de lquido retirado durante o
procedimento . em suma , a ultrassonografia pode aumentar a segurana do paciente e a quantidade de
lquido retirado durante a toracocentese . a ultrassonografia uma tcnica que se
aprende facilmente e que no s melhora a qualidade do exame fsico , mas tambm tem a
clara vantagem de ser a ultrassonografia atualmente usada em um nmero limitado de
toracocenteses , e o presente estudo fornece evidncias a favor do uso mais amplo
dessa tcnica . | objective : thoracentesis is one of the bedside procedures most commonly associated with
iatrogenic complications , particularly pneumothorax . various risk factors for
complications associated with thoracentesis have recently been identified ,
including an inexperienced operator ; an inadequate or inexperienced support team ;
the lack of a standardized protocol ; and the lack of ultrasound guidance . we
sought to determine whether ultrasound - guided thoracentesis can reduce the risk of
pneumothorax and improve outcomes ( fewer procedures without fluid removal and
greater volumes of fluid removed during the procedures ) . in our comparison of
thoracentesis with and without ultrasound guidance , all procedures were performed
by a team of expert pulmonologists , using the same standardized protocol in both
conditions.methods : a total of 160 participants were randomly allocated to undergo thoracentesis with
or without ultrasound guidance ( n = 80 per group ) .
the primary outcome was
pneumothorax following thoracentesis .
secondary outcomes included the number of
procedures without fluid removal and the volume of fluid drained during the
procedure.results : pneumothorax occurred in 1 of the 80 patients who underwent ultrasound - guided
thoracentesis and in 10 of the 80 patients who underwent thoracentesis without
ultrasound guidance , the difference being statistically significant ( p = 0.009 ) .
fluid was removed in 79 of the 80 procedures performed with ultrasound guidance
and in 72 of the 80 procedures performed without it .
the mean volume of fluid
drained was larger during the former than during the latter ( 960 500 ml vs. 770
480 ml ) , the difference being statistically significant ( p = 0.03).conclusions : ultrasound guidance increases the yield of thoracentesis and reduces the risk of
post - procedure pneumothorax .
( chinese clinical trial registry identifier :
chictr - trc-12002174 [ http://www.chictr.org/en/ ] ) |
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cerebrospinal fluid ( csf ) leakage is a potential complication of cranial and spinal surgery11 ) . duraseal ( covidien , waltham , ma , usa ) is a synthetic product which has been increasingly used to facilitate watertight repair of dural defects after cranial and spinal surgery . the duraseal has been demonstrated to be both safe and effective in clinical studies2,4,7,10 ) and was approved in 2005 by the food and drug administration ( fda ) . the authors report a patient who developed cord compression following the use of duraseal in cervical spine surgery in which the expansion of the duraseal was believed to be the causative factor . a 59-year - old man presented to the outpatient department with a 2-year history of progressive leg numbness . mri ( magnetic resonance image ) had shown intradural mass broad - based on the left side of the dura that compresses the cord severely , displacing it to the right posterior aspect at c7 level ( fig . 1 ) . opening the lamina and dura at c7 level , the mass removal was performed . the mass was totally removed and the histopathologic findings of frozen biopsy were consistent with those of a meningioma . when repairing dura , duraseal was applied to dural surface that was incompletely approximated with sutures . eight hours after surgery , the patient developed left - side weakness to motor grade i. a mri scan was urgently performed , which demonstrated a large extradural mass which was causing severe cord compression at the level of tumor removal ( fig . on reoperating wound , a thick layer of gelatinous material consistent with duraseal was initially encountered . also the minimal amount of surgicel was removed , which was separate and not interfering with the duraseal. after surgery the patient is showing slightly improvement with his power increasing to motor grade ii . leakage of csf can lead to delayed healing of surrounding tissues such as skin , muscle , and bone due to its caustic effect11 ) . the aim therefore once a csf leak is identified , is to stem the leak . hydrogel sealant ( duraseal ) is a synthetic sealant approved by the fda , which has been increasingly used to facilitate watertight repair of dural defects after cranial and spinal surgery , and has been demonstrated to be both safe and effective in clinical studies2,4,7,10 ) . duraseal is a self - polymerizing agent which rapidly forms a firm , watertight hydrogel layer within several seconds of application over the dural surface . in comparison to fibrin glue the firm layer is robust enough to withstand irrigation and gentle suction maneuvers without threat of dislodgement . these properties are advantageous in affecting a watertight barrier to minimize the risk of csf leakage8 ) . reported no evidence of postapplication csf leaks or adverse events in 46 patients who had a spontaneous dural tear treated with duraseal2 ) . cosgrove et al . reported similar findings with no evidence of postapplication csf leaks in 95.5% of patients and no adverse events with the use of duraseal4 ) . after these reports , the use of duraseal was extended as an adjunct to suture closure of an iatrogenic dural tear . duraseal is believed to be an ideal sealant for spine procedures because it is a nontoxic bioabsorbable synthetic hydrogel composed of 90% water with similar properties to tissue2,9 ) . however , one of the drawbacks of using duraseal is its potential to swell up to 50% and slow absorption rate . according to the manufacturers ( covidien , waltham , ma , usa ) , product application to " confined bony structures where nerves are present is contraindicated . " a study in dog models has demonstrated that hydrogel shows in vivo expansion , and mass effect within the first 2 weeks , after insertion characterized by computerized tomography ( ct ) and mri imaging6,11 ) . the first reported complication of duraseal was reported when used as a dural sealant after a posterior fossa decompression for a chiari malformation1 ) . a 13-year - old girl developed a worsening quadriparesis after the mass effect of hydrdogel sealant on the cervicomedullary region following foramen magnum decompression . a case of cervical cord compression has also been reported in a patient who underwent a c5-c6 anterior cervical decompression and fusion who sustained a csf leak when excising the posterior longitudinal ligament with duraseal being used as the primary sealant11 ) . three hours postoperatively , the patient developed progressive upper and lower extremity motor weakness . during emergent exploration and decompression , the surgeons determined the expanded hydrogel was the cause of the compression . also a case has been reported of duraseal causing cauda equina compression after a patient underwent a lumbar laminotomy and discectomy complicated by a dural tear9 ) . this patient 's dural tear was treated with duraseal and the patient developed symptoms of cauda equina compression 9 days postoperatively . the patient was taken emergently to the operating room and during exploration and decompression , a large amount of blue duraseal was visualized . the volume of duraseal removed was measured as 10ml , which is more than three times the amount used primarily , suggesting considerable hydrogel expansion after implantation . duraseal has now been approved for spinal usage by the fda . to date , the time profile and radiographic appearances of this dural sealant has previously only been systematically characterized in a dog craniotomy model6,8 ) . in a dog model , duraseal 's volume expansion was reported to peak between day 3 and 2 weeks after implantation6,9 ) . to avoid such a complication , we would recommend judicious application of the duraseal with thin - layer application to the defective area only . we would also caution against applying duraseal in a manners such that it extends over and beyond the bone edges bordering the dural surface after bony decompression . our case demonstrates the use of duraseal over a site of a dural repair is associated with the risk of developing postoperative cord compression as a result of swelling of the duraseal. duraseal tendency to expand is documented both in the instructions for its use and published case reports , and this is also confirmed by our experience . a very thin layer of duraseal should be delivered over the site of dural repair and the possibility of postoperative expansion of it should be recognized by the surgeon . | cerebrospinal fluid ( csf ) leakage is a potential complication of cranial and spinal surgery .
postoperative csf leakage can induce delayed healing , wound infection and meningitis .
duraseal ( covidien , waltham , ma , usa ) is a synthetic product which has been increasingly used to facilitate watertight repair of dural defects after cranial and spinal surgery . despite some advantages of duraseal ,
the authors report a patient who developed cord compression following the use of duraseal in cervical spine surgery in which the expansion of the duraseal was believed to be the causative factor . |
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it is characterized by a horizontal skin fold across the edge of the eyelid , occurring more often in the lower lid [ 15 ] . the two known causes of lower eyelid epiblepharon are inadequate lower lid retractor development , marked by the absence of adhesion to the skin , and a pretarsal orbicularis muscle inserted too closely to the lid margin ; subsequently , the muscle and skin anterior to the tarsal plate are pushed forward over the tarsal plate , resulting in muscle and skin hypertrophy . in contrast , the causes of congenital entropion , which differs from epiblepharon , are a lack of a tarsus , hypertrophy of the pretarsal orbicularis muscle in the eyelid , and an aponeurosis retractor attachment rupture [ 6 , 7 ] . with redundant lower eyelid skin and insufficient adhesion of the orbicularis oculi muscle to the tarsal plate this skin causes the cilia to be inverted toward the eyeballs , which may touch and irritate the cornea , particularly in the downward gaze [ 1 , 2 ] . associated symptoms of this condition include ocular irritation ( e.g. , epiphora , photophobia , eye rubbing , increased discharge , frequent blinking , and foreign body sensation ) and corneal erosion , both of which can result in vision impairment [ 8 , 9 ] . in most cases , symptoms are not serious and are outgrown with facial bone growth and the accompanying skin and muscle extension . however , in serious cases or when the condition persists , surgical correction is often necessary [ 10 , 11 ] . compared to caucasians , asians generally have nasal bones that tend to rise at a later age , and it has been reported that 12.6% of asian children aged 7 to 14 years old have epiblepharon . the condition does not always correct itself with age , and surgical correction should be performed once symptoms appear . despite numerous studies examining the prevalence of epiblepharon in asian populations [ 1214 ] , no large - scale , long - term studies on epiblepharon in korean infants and children have been previously performed . here , we report on the prevalence , symptoms and surgical success , recurrence , and complication rates observed in korean children undergoing epiblepharon surgical correction . among our patients , 768 patients were diagnosed with epiblepharon , underwent corrective surgery between january 2005 and december 2013 , and were followed for at least 6 months following surgery . patient medical records were retrospectively reviewed for the age , gender , chief complaint , presence of keratitis , location of epiblepharon , concomitant disorders , postoperative location of epiblepharon , complications , and recurrence rate . epiblepharon was classified by the location into the upper lid and the lower lid groups . additionally lower cilia - touching lesions were classified into nasal , center , and temporal groups , according to the corneal region involved . surgery was performed when significant corneal damage occurred , as confirmed by slit lamp examination or serious irritation complaints . the surgeries were performed by the same surgeon and involved skin resection and full - thickness rotating suturing in the lower lid corrections and simple - interrupted buried suturing in the upper lid corrections . skin resection and full - thickness rotating suture placement on the lower lid involved marking a skin incision line with the aid of bishop forceps . after an incision was made with a number 15 bard - parker blade , skin and subcutaneous tissue resection was performed with stevens scissors . part of the orbicularis oculi was also removed using electrocautery to minimize bleeding . after exposing the tarsal plate , one or two simple - interrupted buried sutures ( 6 - 0 nylon ) were placed in the subcutaneous tissues of the incision 's upper end and on the lower end of the tarsal plate , with the resected skin inserted into the incision . for the remaining skin , a continuous suture ( 6 - 0 fast absorbing plain gut ) was placed . the simple - interrupted buried suture was also placed for the upper lid correction , paying close attention to bilateral lid symmetry . four points were marked with a marking pen where the double fold was to be formed and the stitches were to be placed ( figure 2 ) . epinephrine mixed with 2% lidocaine was injected into the subcutaneous space and the upper subconjunctival layers . several 2 - 3 mm incisions were then made with a number 11 blade for the suture entry . the upper lid was everted and a single - interrupted buried suture ( 4 - 0 vicryl ) was placed on the 4 marked points . all postoperative follow - up exams were performed in an outpatient setting in which the position of the cilia and cilia - corneal touch were monitored by slit lamp examinations . during the follow - up period , the patients were carefully monitored for surgical complications and epiblepharon recurrence . among the 768 epiblepharon patients included , 302 ( 39.3% ) were male and 466 ( 60.7% ) were female . the mean patient age at the time of presentation was 6.55 2.37 years ( table 1 ) . four - year - olds composed the largest patient group ( n = 107 , 13.9% ) , with the majority of patients aged between 3 and 7 years ( n = 475 , 61.8% , figure 1 and table 2 ) . at the time of presentation , 712 patients ( 92.8% ) reported epiblepharon symptoms , but 56 patients ( 7.3% ) were incidentally discovered while undergoing visual acuity and/or ophthalmologic examinations for other reasons . the most common complaints among patients were foreign body sensation due to cilia - corneal touch ( n = 209 , 27.2% ) , ocular discharge ( n = 97 , 12.6% ) , photophobia ( n = 80 , 10.4% ) , and decreased visual acuity ( n = 69 , 9.0% , table 3 ) . lower lid epiblepharon patients had cornea - cilia touch most often in the nasal area ( n = 408 , 58.6% ) , followed by the central area ( n = 264 , 37.9% ) , and , finally , the temporal area ( n = 24 , 3.4% , table 1 ) . the concomitant eye disorders were ptosis ( n = 62 , 8.1% ) , strabismus ( n = 48 , 6.3% ) , floppy eyelid syndrome ( n = 44 , 5.8% ) , trichiasis ( n = 37 , 4.8% ) , and amblyopia ( n = 10 , 1.3% , table 4 ) . epiblepharon correction surgery was performed on both the upper and lower lids of 82 patients ( 10.5% ) , on the lower lids of 629 patients ( 80.3% ) , and on the upper lids of 72 patients ( 9.2% , table 1 ) . eyelid shape and function were maintained in 740 patients ( 96.4% ) with no epiblepharon recurrence at the final follow - up examination . the remaining 28 patients ( 3.6% ) had epiblepharon recurrence involving the lower lid , 15 ( 1.9% ) of whom were operated on a second time . following the second procedure , no complications ( e.g. , ectropion , scar formation , and wound dehiscence ) the 13 patients who did not undergo a second correction procedure showed improvement without surgical intervention through the follow - up period . statistically , the recurrence rate of lower cilia - corneal touch was significantly higher in the nasal area compared to the other corneal regions ( table 5 ) . it is generally a bilateral anomaly of the lower lid and is characterized by a skin fold that runs horizontally across the eyelid edge [ 1 , 2 , 15 ] . in their study of japanese children , noda et al . reported that 81% of epiblepharon cases involved the lower lid and that only 7% and 12% of cases involved the upper lid or both lids , respectively . fortunately , most infants outgrow epiblepharon , and symptoms spontaneously improve as facial bones , skin , and muscles grow . only approximately 2% of infants with epiblepharon show no symptomatic improvement by the time they reach school age [ 1517 ] . because epiblepharon pushes the cilia toward the cornea and/or conjunctiva , symptoms include conjunctival irritation and injury , eye rubbing , and epiphora . it has also been associated with astigmatism and decreased visual acuity [ 9 , 18 ] . in this study , the most common symptom at the time of presentation was cilia - induced corneal irritation , even in patients in whom severe epiblepharon was incidentally discovered ( 7.3% ) . epiblepharon was observed most frequently in patients aged 4 to 7 years , which was indicative of the high prevalence rate for this age group . therefore , even in infants without noticeable symptoms , careful examination is necessary to detect epiblepharon and prevent subsequent refractive error and decreased visual acuity caused by corneal irritation . epiblepharon that is mild or in an early stage may be conservatively addressed either with medication or by retracting the skin fold downward with adhesive or cellophane tape [ 16 , 20 ] . surgical intervention criteria vary slightly between surgeons , but , in most cases , the presence of symptomatic cilia - induced corneal irritation is the decisive factor [ 12 , 21 ] . however , some controversy remains regarding this issue because even in the presence of corneal irritation , some infants and children do not complain , and more conservative therapies can be used successfully . in the current study , the severity of corneal irritation was subjectively assessed by a single surgeon to determine whether surgical intervention was needed . no objective criteria for this evaluation have been established , although such criteria would have been of great use and an interesting area for further study . nonincisional methods result in less postoperative edema , can easily be repeated , and leave minimal scars ; unfortunately , these methods result in occasional recurrence [ 25 , 26 ] . in this study , the nonincisional simple - interrupted buried suture technique was used to correct upper lid epiblepharon , and no recurrence was observed during the follow - up . the nonincisional suture technique , first presented by quickert et al . , is simple and rapid with a reported success rate of 100% . nevertheless , the disadvantages of this technique make it impractical for severe epiblepharon correction and include frequent suture infection and a recurrence rate of up to 29% . the modified hotz procedure ( i.e. , hotz - celsus procedure ) was widely used until recently . it involves lower lid redundant skin and partial orbicularis oculi resection and placing an anchoring suture of skin to the tarsal plate . it is a relatively simple procedure with a high success rate but requires an unnecessarily large skin resection , which can result in ectropion and lid retraction [ 4 , 25 , 26 ] . this procedure involves exposing the tarsal plate and burying it together with subcutaneous tissues [ 21 , 27 ] . our surgical method for lower lid epiblepharon correction involved only little skin resection , and when further correction was needed , the full - thickness rotating suture technique was used . thus , lower lid crease , scar formation , and complication rates ( e.g. , ectropion and lid retraction ) could be reduced , all while maximizing surgical success rate and minimizing recurrence rate . our recurrence and reoperation rates were highest in patients with lower lid epiblepharon with nasal cilia - cornea touch . this result was in agreement with swan , who found that , in severe epiblepharon , creasing at the nasal end of the eyelid occurs substantially more frequently . . reported on an additional epiblepharon surgical technique that , coupled with z - epicanthoplasty , positively affected defect correction . in our study , in an attempt to overcome the defect , an additional full - thickness rotating suture was placed in cases of severe epiblepharon . therefore , further research is necessary to identify the most effective intervention method that reduces recurrence and reoperation rates by sufficiently improving cases of severe epiblepharon at high risk of recurrence . the greatest limitation of this study was the subjective assessment for the need of surgery , but the influence of this limitation was minimized by having only one evaluating surgeon . unfortunately , no objective system to evaluate cilia - induced corneal irritation symptoms and epiblepharon severity is available , and this system is a needed area of future research . to conclude , epiblepharon is frequently found in korean infants and children and should be addressed with caution and care , even in the absence of symptoms . many surgical interventions carry the risks of general anesthesia and of postoperative complications , such as postoperative scar formation and ectropion . here , we reinforce the idea that effective epiblepharon correction can be achieved with a simple surgery , alleviating symptoms and preventing keratitis - induced decreases in visual acuity . nevertheless , age and symptom severity should be considered when determining which surgical correction method to use . | purpose . to examine the demographic characteristics , clinical features , surgical outcomes , and long - term prognoses of epiblepharon in korean children
.
methods .
epiblepharon patients who were followed for 6 month following surgical correction between january 2005 and december 2013 .
the patient demographics , clinical features , concomitant disorders , surgical outcomes , and complications were retrospectively reviewed .
results .
a total of 768 epiblepharon records were included in the analysis .
the mean patient age was 6.55 2.37 years . at presentation , 712 patients ( 92.8% ) complained of typical epiblepharon symptoms .
the mean patient age at surgery was 6.95 2.52 years , with 629 patients ( 81.9% ) on the lower lid and 72 patients ( 9.4% ) on the upper lid and 82 patients ( 10.7% ) undergoing surgery on both lids .
the eyelid was well everted with no recurrence in 740 patients ( 96.4% ) .
conclusion .
epiblepharon frequently occurs in korean children and is correctable with a simple surgery .
recurrence and serious complications do not occur often , and any suspicions of epiblepharon should be investigated .
a thorough ocular examination can lead to a correct diagnosis and timely corrective surgery .
most procedures are successful and prevent secondary complications that often occur with uncorrected epiblepharon . |
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pemphigus vulgaris ( pv ) is an uncommon ( 0.53.2 cases per 100,000 ) , potentially life - threatening ( 5%15% ) , autoimmune bullous mucocutaneous disease,1 which can rarely affect the eyes . autoantibodies are formed against desmoglein , the glue that attaches adjacent epidermal cells . when autoantibodies attack desmogleins , the cells become separated from each other , a phenomenon called acantholysis . this causes blisters and erosions.2,3 we present a case of a patient with ocular manifestations of pv resistant to systemic and topical medication , which was successfully treated with subconjunctival injections of triamcinolone acetonide ( ta ) . a 76-year - old woman , suffering from pv , was referred to our department due to acute onset of intense burning and foreign body sensation in her right eye , despite her treatment with systemic methylprednisolone 15 mg / day and azathioprine 100 mg / day due to pv . pv was diagnosed clinically and by performing ( a ) histopathology from the edge of a blister , ( b ) direct immunofluorescence ( dif ) on normal - appearing perilesional skin , and ( c ) indirect immunofluorescence ( idif ) . acantholytic cells were found in a tzanck smear , taken from the base of an erosion , which helped to differentiate pv from pemphigus foliaceous . slitlamp examination revealed pronounced conjunctival hyperaemia , mucus secretions , ulcerative erosions involving the palpebral conjunctiva at the right lower eyelid margin ( figure 1 ) and punctate corneal epithelial erosions involving the inferior part of the cornea of the same eye . schirmer s test without anesthetic was markedly reduced ( 3 mm ) as well as the tear break up time ( but ) ( 3 seconds ) . therefore the dose of methylprednisolone was increased to 40 mg / day , and the azathioprine was replaced by cyclophosphamide 100 mg daily . in addition , the patient was treated with topical dexamethasone 0.1% daily and frequent instillation of unpreserved carbomer 0.3% . hence , a subconjunctival injection of ta 20 mg in 0.5 ml ( kenacort 40 mg / ml ; bristol - myers squibb ) was given after informed consent was obtained , but without stopping the systemic treatment . two weeks following the ta injection the patient s symptoms were reduced significantly the eye was less inflamed and the conjunctival ulcers were reduced in size . due to the initial positive response to the ta injection three months later the symptoms had improved significantly and the ulcers had disappeared ( figure 2 ) , enabling us to taper further the systemic treatment . the schirmer s test was also increased to 6 mm as well as the but was prolonged to 6 seconds . one year after the second injection the patient is still asymptomatic without signs of recurrence . ocular pemphigus is probably underdiagnosed and its frequency appears underestimated.3 pv is recognized by a dermatologist from the appearance and distribution of the skin lesions . definitive diagnosis requires examination of a skin biopsy from the edge of a blister by a pathologist . definitive diagnosis also requires the demonstration of anti - desmoglein autoantibodies by direct immunofluorescence on the skin biopsy . these antibodies appear as igg deposits along the desmosomes between epidermal cells , a pattern reminiscent of chicken wire . anti - desmoglein antibodies can also be detected in a blood sample using the elisa technique . a high titer of canca is claimed to be an important feature of the disease.3,4 ocular involvement with pv is very rare.2 the most characteristic f indings are conjunctival hyperemia , conjunctivitis , eye irritation and mucus secretions.5 catarrhal conjunctivitis is very common in pv , while bullae and conjunctival erosions are seen rarely.5,6 conjunctival fibrosis is very rare2 as is corneal involvement.7 the therapeutic approach for the treatment of pv requires a systemic therapy with steroids and immunosuppressive drugs . in our case , neither the increase of the initial systemic doses , nor the replacement of azathioprine by cyclophosphamide , had any effect on the ocular signs of the disease . even topical steroids ( drops ) and lubricants were used , but with no symptomatic effect , probably due to persistent erosion of the lid margins , affecting the quality of the tear film and further causing severe dryness of the eye . a considerable improvement was seen following 2 subconjunctival injections of ta 20 mg with an interval of 2 weeks , probably due to normalization of the lid margin anatomy , which improved ocular surface dryness . the subconjunctival ta injections provided sustained release of steroids at the side of greatest need , ie , the palpebral conjunctiva , with no systemic adverse effects . this was of particular importance in our patient who started to present signs of cushing s syndrome from the long - term use of systemic steroids . to the best of our knowledge , this is the first reported case of subconjunctival injections of ta for the treatment of ocular manifestations of pv . daouds et al7 reported 11 cases of ocular manifestations of pv treated mainly with systemic steroids , and in 1 case they used ta . the authors concluded that the disease could recur ( mean follow up 48.9 months ) , but that finally all patients eventually recovered fully without sequelae . although no long case series are available , ta could be regarded as an effective alternative for patients with serious ophthalmic manifestations of pv unresponsive to systemic therapy or those with serious adverse effects to systemic treatment . | we present an interesting case of pemphigus vulgaris with severe and rare eye manifestations .
an old lady with systemic and ocular manifestations of pemphigus vulgaris was treated with systemic corticosteroids and immunosuppressants , with no effect .
subconjunctival injection of triamcinolone acetate reduced the ocular signs and led to gradual reduction of the systemic medications . |
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over the last three decades closed intramedullary nailing has become the gold standard technique for the treatment of femoral shaft fractures . its value has been proven by a high union rate , low number of complications , and low invasiveness to the soft tissues around the femur . the procedure is still technically demanding , so modern intramedullary nails with their insertion instruments are increasingly surgeon and patient friendly . between 1995 and 2007 , over 400 patients with femoral fractures were treated in our department with various types of antegrade cannulated locked femoral nails , but until recently we did not recognize any femoral neck fracture ( fnf ) associated with this procedure . since 2005 we have followed two cases with unusual complications attributed , in our opinion , to technical errors during the end stage of the procedure end cup placement . the two senior authors fully recollect the procedure due to technical intraoperative complications . in both cases immediately postoperative x - rays did not reveal signs of fnf , but on first follow - up after two weeks both patients were symptomatic , and anteroposterior ( ap ) radiograms revealed misplaced end cups and fnf . both patients presented below were informed about the nature of this report and gave their consent to its publication . a 76-year - old obese woman ( bmi 32 ) was referred to our department after infected destabilization of a plate fixation of the left femoral shaft . on the next day she underwent removal of the implant with debridement and fixation of the bone with zespol internal fixator a polish harbinger for the locked plates ( fig . 1 ) . on the third postoperative day she sustained another fracture of the left femur while the wound dressings were changed . during an operation carried out on the next day , the fixator was lengthened proximally to incorporate the proximal fragment ( fig . 2 ) . after three months , despite non - weight - bearing , recurrent destabilization occurred without any microbiological symptoms of infection . the implant was removed and open intramedullary nailing was performed . during this procedure significant bone fragility was evident , and the end cup was misplaced behind the femoral neck . multiple efforts to remove it ( using a finger and vascular clamps under the image intensifier ) failed . after two weeks , during the first follow - up visit , a fnf ( ao 31-b2 ) was diagnosed but the patient refused the proposed operative treatment . she came back after three months with significant shortening of the left femur and nonunions of both the neck and diaphysis ( fig . the end cup was replaced , and fixation of the femoral neck was attempted with two screws , one cortical interlocking through the dynamic slot of the nail and another cancellous with the miss a nail technique . we were not able to place additional screws due to the width of the proximal nail . the soft bone of the proximal fragment did not cause any noticeable resistance during screw placement . the patient remains severely disabled , walking around the house with a walker and occasionally using a thomas splint based on her pelvis.fig . 2case 1 : extended zespol fixator after additional fracture was sustained during in - bed patient care . apparently healthy femoral neckfig . 3case 1 : nonunion of the femoral neck and femoral diaphysis case 1 : zespol internal fixator after removal of an infected plate . proximal fragment periosteal elevation ( black arrow ) suggesting an inflammatory reaction case 1 : extended zespol fixator after additional fracture was sustained during in - bed patient care . apparently healthy femoral neck case 1 : nonunion of the femoral neck and femoral diaphysis a 68-year - old obese woman ( bmi 30 ) was admitted after a fall at home causing fracture of the femoral shaft ( figs . 4 , 5 ) . on the same day the end cup was placed inside the femoral neck and efforts to remove it with a vascular clamp failed . medial cortex of the femoral neck apparently intact case 2 : broken femoral shaft magnified view of the femoral neck region ( same radiogram as in fig . medial cortex of the femoral neck apparently intact the patient presented with symptoms two weeks after discharge . the patient was reoperated after seven weeks with symptoms of bone union of the femoral shaft . the nail was removed and bipolar arthroplasty of the hip was performed ( fig . 8case 2 : bipolar arthroplasty of the hip after intramedullary nail removal case 2 : iatrogenic femoral neck fracture case 2 : bipolar arthroplasty of the hip after intramedullary nail removal a 76-year - old obese woman ( bmi 32 ) was referred to our department after infected destabilization of a plate fixation of the left femoral shaft . on the next day she underwent removal of the implant with debridement and fixation of the bone with zespol internal fixator a polish harbinger for the locked plates ( fig . 1 ) . on the third postoperative day she sustained another fracture of the left femur while the wound dressings were changed . during an operation carried out on the next day , the fixator was lengthened proximally to incorporate the proximal fragment ( fig . 2 ) . after three months , despite non - weight - bearing , recurrent destabilization occurred without any microbiological symptoms of infection . the implant was removed and open intramedullary nailing was performed . during this procedure significant bone fragility was evident , and the end cup was misplaced behind the femoral neck . multiple efforts to remove it ( using a finger and vascular clamps under the image intensifier ) failed . after two weeks , during the first follow - up visit , a fnf ( ao 31-b2 ) was diagnosed but the patient refused the proposed operative treatment . she came back after three months with significant shortening of the left femur and nonunions of both the neck and diaphysis ( fig . the end cup was replaced , and fixation of the femoral neck was attempted with two screws , one cortical interlocking through the dynamic slot of the nail and another cancellous with the miss a nail technique . we were not able to place additional screws due to the width of the proximal nail . the soft bone of the proximal fragment did not cause any noticeable resistance during screw placement . the patient remains severely disabled , walking around the house with a walker and occasionally using a thomas splint based on her pelvis.fig . 2case 1 : extended zespol fixator after additional fracture was sustained during in - bed patient care . apparently healthy femoral neckfig . 3case 1 : nonunion of the femoral neck and femoral diaphysis case 1 : zespol internal fixator after removal of an infected plate . proximal fragment periosteal elevation ( black arrow ) suggesting an inflammatory reaction case 1 : extended zespol fixator after additional fracture was sustained during in - bed patient care . a 68-year - old obese woman ( bmi 30 ) was admitted after a fall at home causing fracture of the femoral shaft ( figs . 4 , 5 ) . the end cup was placed inside the femoral neck and efforts to remove it with a vascular clamp failed . medial cortex of the femoral neck apparently intact case 2 : broken femoral shaft magnified view of the femoral neck region ( same radiogram as in fig . medial cortex of the femoral neck apparently intact the patient presented with symptoms two weeks after discharge . the patient was reoperated after seven weeks with symptoms of bone union of the femoral shaft . the nail was removed and bipolar arthroplasty of the hip was performed ( fig . 8case 2 : bipolar arthroplasty of the hip after intramedullary nail removal case 2 : iatrogenic femoral neck fracture case 2 : bipolar arthroplasty of the hip after intramedullary nail removal we assume that both of the above - described fnfs were iatrogenic and related to harmful maneuvers during efforts to remove the misplaced end cup . the damage to circumflex vessels in case 1 and to endosteal blood supply and bone stock itself in case 2 resulted in late fnf . apart from this suggestion , in case 1 the weakened , ischemic bone failed during physiologic stresses at bed rest after first fixation with zespol , so we also consider the possibility of a similar cause for fnf . the proposed causes vary from misplacement of the entry point to forceful removal of a drill jammed into the femoral shaft . various types of fractures are reported , but the classification is inconsistent ( pauwels iii , comminuted transverse , comminuted segmental , etc . ) . all were treated conservatively ( due to minimal displacement ) and resulted in various malunions in two cases . he attributed the fracture to forceful insertion of the initiating awl in the wrong direction and the use of multiple entry points ( like harper ) . christie and court - brown reported four ( of 143 treated ) extracapsular fnfs during closed femoral intramedullary nailing , which were attributed to an excessively lateral insertion point in the trochanteric area and to the oblique insertion of the nail . in a laboratory study of strain gauge locations on cadaver bones , researchers from the ao research institute found that antegrade femoral nails , introduced both into the piriformis entry point or laterally , do not induce higher strains if correspondingly designed nails are implanted the correct way . deviations from these ideal insertion points are associated with higher stains and sometimes fractures in the proximal femur . accordingly , in obese patients , difficult access to the piriformis entry point might cause additional stress to the femoral neck if the ideal site for nail insertion is missed . simonian had four cases of iatrogenic fnf which he attributed to overhanging of the medial prong of the old type of ao insertion jig that may impinge on the superior aspect of a valgus femoral neck during the final stage of a deep nail impaction . another unusual case was due to nail insertion with a reversed curvature ( posterior bowing ) . we currently use synthes reamed femoral nails , where the introductory device fits the nail s diameter perfectly , eliminating the circumferential overhanging that was blamed for the fracture in simonian s cases . end cup misplacement errors are common , especially in obese individuals if the piriformis entry site is used , but consequences such as those described in this manuscript have not been reported previously in the literature . we currently tie the end cup with an absorbable suture prior to its placement , so that it is easy to recover the device in the event of misplacement without the need for any additional use of fluoroscopy , loss of time and injuries to adjacent structures , which could end up with fnf . | femoral neck fracture is an unusual complication of intramedullary fixation of a broken femur .
we report on two cases of femoral neck fractures attributed to misplacement of an end cup and subsequent invasive maneuvers in an effort to remove it .
iatrogenic fractures of the femoral neck during or after intramedullary nailing are reported in the medical literature .
authors associate it with many possible technical mistakes performed during the procedure , yet no complications after missed end cup placement were noted .
we suggest that the fractures described below were a consequence of injury to the vascular supply and bone stock of the initially intact femoral neck . |
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total hip replacement is one of the most successful orthopedic surgeries . annually , about 800 000 total hip replacement is done around the world . unfortunately , this number is increasing , because prevalence osteoarthritis of hip joint is increasing around the world including iran . quality of life is personal idea and is determined by oneself . to determine quality of life , general and specific instruments exist for each disease . general instruments like short - form 36 health survey ( sf-36 ) evaluate situation of person broadly . on the other hand , specific instruments for each disease have created to focus on those aspects of life that are affected by a particular disease ; also , we can utilize these tools to evaluate the effectiveness of various treatments . in recent years , the outcome of orthopedic surgeries has increasingly been evaluated according to patient - reported questionnaires . so , self - report questionnaires should be used to achieve more information on patients situations . self - report questionnaires generally should not include many questions so that the response rate is increased and the risk of data loss is decreased . the questionnaires should be adapted cross - culturally to maintain the content and construct validity of the original instrument and to prevent population - related and culture - related bias in assessment . the oxford hip score ( ohs ) , which is a self - administered questionnaire , has been studied extensively and is a reliable , valid , and responsive instrument for evaluating hip pain and disability in patients suffering from osteoarthritis of hip joint , especially those undergoing total hip arthroplasty.[1116 ] it is a 12-item instrument with each item scored by the patient on a 1- to 5-point likert scale . the global score is given by the sum of the scores for all 12 items resulting in values between 12 and 60 . permission was obtained from the original questionnaire developer and the technology transfer company of the university of oxford ( oxford , england ) to develop this study . we followed the recommended process of the mapi research trust as well as the principles ordered by the translation and cultural adaptation working group . the standard forward - backward translation ( linguistic validation ) was done ; two health professionals ( orthopedic surgeons ) did the forward translation ( english to persian ) independently . after receiving forward translations of the ohs questionnaire from our translators , we decided to arrange meetings with two forward translators and an orthopedic surgeon to integrate the contents of the translations . so , we made the opportunity for them to meet each other and we had a discussion on translations . the session was actually a peaceful settlement about the differences existed between the translations of each of the two translators . we matched their translations and they tried to convince each other for choosing the most suitable word for each case . we did the exact things for the backward translations ( persian to english ) as well . for the next step , in a common meeting in the presence of all four translators and orthopedic surgeon , the original questionnaire of ohs was shown and the backward translated questionnaire was compared with the original one . we have sent differences we found between our backward translated questionnaire and the original ohs questionnaire to the original questionnaire developer accompanying with the backward translators explanations for each case of difference . at the end , we established the pre final persian version of ohs . after translation process , the persian version of the ohs was tested in a pilot study by administering to 30 adult patients suffering from osteoarthritis of hip joint ( 30 adult inpatients admitted for hip replacement in alzahra and kashani hospitals ) for cognitive debriefing and to two orthopedic surgeons for clinician 's review . according to the results of the pilot study and feedbacks from the developer , the questionnaire was edited more and the final persian version of the ohs was produced . the study involved 130 adult persian - speaking patients suffering osteoarthritis of hip joint admitted for primary total hip arthroplasty in two university hospitals ( alzahra and kashani hospitals ) in isfahan in iran from september 2009 until april 2011 . of these 130 patients , 25 were excluded , five patients did not accept to participate in the study , 15 patients underwent other operative procedures , and five persons were not operated on during the study period . thus , preoperative data were available for 105 patients with hip osteoarthritis . all of them filled out persian ohs , womac , and sf12 in preoperative examination two weeks before their surgery . retest was performed on 39 patients two weeks later , when they were admitted for surgery . access to those hospitals was open to every patient , and our routine patients are a mixture of urban and rural inhabitants from different states of iran . we used cronbach 's alpha and intra - class correlation coefficient ( icc ) , bland and altman method , respectively , to evaluate the internal consistency and reproducibility of data from the persian ohs . cronbach 's alpha indicates the average correlation between all items of a scale and the correlation between each item and the whole scale . we anticipated cronbach 's alpha values greater than 0.9 and 0.8 , which were considered excellent and good , respectively . to analyze the discriminant and convergent validity , the persian ohs was correlated to the persian version of western ontario and mcmaster universities osteoarthritis index ( womac ) and the persian version of 12-item short form health survey ( sf-12 ) . the ohs is a 12-item instrument with each item scored by the patient on a 1-to 5-point likert scale . the global score is generated by summing the scores for all 12 items resulting in values between 12 and 60 . the distribution of floor and ceiling effects of the persian ohs was determined by calculating the proportion of individuals obtaining the lowest and highest scores , respectively . the womac is a self - administered , disease - specific instrument that includes subscales for pain , stiffness , and physical function . scores range from 0 to 20 ( pain ) , 0 to 8 ( stiffness ) , and 0 to 68 ( function ) . the higher the score , the worse is the health state . the sf-12 is a self - administered generic measure for evaluating the quality of life . scores are transformed into two weighted summary scores for physical function ( physical component scale [ pcs ] ) and mental health ( mental component scale [ mcs ] ) which can score between 0 and 100 . the higher the score , the better is the health state . we recoded the scores of ohs and womac into a 0 to 100 point scale with 100 being the best score . the persian womac and sf12 were recorded in preoperative examination two weeks before their surgery accompanying the ohs . to examine convergent validity , we assumed that the correlation coefficients describing the relationship between the ohs and the womac and the pcs of the sf-12 would be moderate to high ( r > 0.50 ) . to examine divergent validity , we anticipated the correlation coefficients describing the relationship between the ohs and the mcs of the sf-12 which would be lower than those between the ohs and pcs ( r < 0.50 ) . pearson correlation was also applied to assess the validity of the persian ohs with respect to the womac and sf12 . most discrepancies concerned synonyms for specific expressions , for example , the translators have written the pain of your hip instead of the pain from your hip , or have used the words sharp and burning instead of the words shooting and stabbing . similarly , the phrase walking stick has been used instead of walking aid . we found no floor or ceiling effects for the persian ohs that were determined by calculating the proportion of respectively . the persian ohs overall score demonstrated high reproducibility ( icc , 0.93 , p < 0.001 ) and internal consistency ( cronbach 's alpha , 0.94 ) . mean scores for the first and second administrations were similar ( p = 0.83 ) ( 42.7 12.7 vs 41.4 13.9 , respectively ) . convergent validity for the persian ohs was observed by the moderate to high correlations between persian ohs scores and the other questionnaire scores [ table 1 ] . the strongest correlations were between the persian ohs and the womac total score ( r = 0.86 ) , the persian ohs and the womac function score ( r = 0.86 ) , and then between persian ohs and the womac pain ( r = 0.79 ) . divergent validity for the persian ohs was observed by the low correlation between the persian ohs and the mcs of the sf-12 ( r = 0.40 ) . our study presents the results of the trans - cultural adaptation and validation of the ohs for iranian patients with persian language , who suffered from hip osteoarthritis . according to the results , items on the persian ohs were well understood by iranian patients with hip osteoarthritis demonstrating that the translation process was acceptable . the ohs has been evaluated extensively and is a reliable , valid , and responsive instrument for assessing hip pain and disability in patients undergoing total hip arthroplasty . unlike the studies of wood and mclauchlan and mcmurray et al . we observed no floor or ceiling effects for the persian ohs , similar to the findings for preoperative patients reported by garbuz et al . the persian ohs overall score demonstrated high reproducibility ( icc , 0.93 , p < 0.001 ) and internal consistency ( cronbach 's alpha , 0.94 ) . we found good internal consistency for the persian ohs more than the value reported by dawson et al . convergent validity for the persian ohs was observed by the high correlations between persian ohs and the womac total score ( r = 0.86 ) and the womac function score ( r = 0.86 ) and then between persian ohs and the womac pain ( r = 0.79 ) . correlation coefficients between ohs and womac pain and function subscales were 0.76 and 0.88 in a cohort study of 147 patients with a mean age of 68 years . the correlation coefficients were also ( r = 0.81 - 0.87 ) in a prospective cohort study on 402 patients ( mean age , 61 years ) of ( 29 ) . we observed that the correlation coefficient describing the relationship between the persian ohs and the womac stiffness subscale was somewhat lower ( r = 0.69 ) , which was consistent with those of ostendorf et al . divergent validity for the persian ohs was observed by the low correlations between the persian ohs and the mcs of the sf-12 ( r = 0.40 ) , which was slightly lower than the values of ostendorf et al . ( r = 0.49 ) . similar to the values reported by ostendorf et al . and garbuz et al . ( r = 0.53 ; r = 0.60 ) , the correlation coefficient between the persian ohs and the pcs of the sf-12 in our study was moderate ( r = 0.58 ) . in our patient samples , when using the original scoring system , mean score for the first administrations was 42.7 , similar to the mean preoperative ohs score reported by dawson et al . , which was 43.6 ; field et al . reported a mean preoperative value of 41.0 and ostendorf et al . reported a value of 42.5 for dutch patients our study demonstrated that the trans - cultural adaptation and validation of the persian ohs is a reliable and practicable instrument for self - assessment of function and pain in iranian patients with hip osteoarthritis . we believe further investigation of the persian ohs in patients after tha is needed to assess the sensitivity to change of this questionnaire . | introduction : in recent years , outcome assessment related to orthopedic surgeries has increasingly focused on patient - reported questionnaires .
the oxford hip score ( ohs ) , self - administered questionnaire , is a reliable , valid , and responsive instrument for assessing hip in patients undergoing arthroplasty.methods:the study involved 105 adult persian - speaking patients admitted for primary total hip arthroplasty in two hospitals in isfahan in iran from september 2009 until april 2011 .
all of them filled out their scales ( persian ohs , womac , and sf12 ) in preoperative examination.results:mean scores of ohs in first administrations was 42.7 12.7 . the persian ohs overall score demonstrated high reproducibility ( icc,0.93 , p < 0.001 ) and internal consistency ( ca , 0.94 ) .
persianohs had high correlations with womac total score ( r = 0.86 ) , function score ( r = 0.86 ) , and pain score ( r = 0.79 ) , the relationship between the persian ohs and the womac stiffness subscale was somewhat lower ( r = 0.69 ) .
the correlation coefficient between the persian ohs and the pcs of the sf-12 in our study was moderate ( r = 0.58 ) .
persian ohs had low correlation with mcs of the sf-12 ( r = 0.40).discussion : persian ohs had high correlations with womac total score , function score , and pain score .
it had moderate correlation with pcs of the sf-12 and low correlation with mcs of the sf-12.conclusions:our study demonstrated the trans - cultural adaptation and validation of the persian ohs is a reliable and practicable instrument for assessment of function and pain in iranian patients with hip osteoarthritis . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
cervical cancer is the seventh most common malignancy in developed countries , and the second most common cancer in developing countries . in 2004 , 30,570 new cases of invasive cervical cancer were diagnosed in the european union . in 2012 , the estimated new cases in the usa are 12,170 , and the estimated deaths 4,220 . higher incidence occurs in countries where an effective screening program is not present . in the usa , the incidence of cervical cancer is about 6.8 per 100,000 person and the mortality 2.4/100,000 . gynecological malignancies often affect women in reproductive age and about 28% of all cervical cancers is diagnosed prior to 40 years of age . where a screening program is present , the disease is often diagnosed in early stages with high survival rates . in the usa , between 2001 and 2007 the 5-year survival for localized disease in white women under 50 years old was 94.2% . the golden standard treatment of early stage disease ranges from simple hysterectomy ( stage ia1 ) to a radical hysterectomy ( rh ) and pelvic lymphadenectomy ( ia2 to ib1 ) . nevertheless , the high survival rates and the delayed childbearing in our society result in more cervical cancer patients who desire preserving their fertility . luckily , fertility sparing treatment approaches are available for a large part of cases . cervical conization is a feasible treatment for stage ia1 and radical trachelectomy with laparoscopic lymphadenectomy has become a surgical alternative for stages ia2 and ib1 . the aim of this paper was to review available literature on fertility preserving surgery in early cervical cancer , focusing on safety and reproductive outcomes . nodal and parametrial tissue involvement is rare in very early stage disease ( stage ia1 ) and the standard treatment is a simple hysterectomy . candidates for conization are patients with stage ia1 cervical cancer without lymphovascular space involvement at the pathological examination , negative margins , and normal endocervical curettage . although lymphovascular space invasion does not affect staging , its presence increases the risk of lymph node metastasis and the standard treatment has been rh and pelvic node dissection . radical trachelectomy with pelvic lymph node dissection is the treatment of choice when patient desires to preserve fertility . several authors have reported absence of node metastasis when stromal invasion is less than 4 mm [ 57 ] . these lesions have less than 1% incidence of lymph nodal metastasis , and , within this group , lymph vascular invasion increases the risk . the lymph vascular space involvement ( lvsi ) in patients with early stage disease is associated with pelvic nodal metastasis and the quantity of lvsi , as defined by the percentage of all cervical histopathologic sections containing lvsi , correlates significantly with the risk of nodal metastases . if lymph vascular invasion is present radical trachelectomy with lymph node dissection should be considered . conization is controversial in cases of adenocarcinoma because of the difficulty of establishing a pathologic diagnosis of microinvasion from a glandular lesion . reports suggest that conization may be performed on patients with both squamous carcinoma and adenocarcinoma . report no recurrence at 72 months follow - up in 16 patients with stage ia1 adenocarcinoma who underwent conization alone . the same authors recommend conization and pelvic node dissection where lymph vascular space involvement is present . the efficacy of laser conization , loop electrosurgical excision procedure ( leep ) , and cold - knife conization is similar . however , it is known that frequently a diathermy loop excision can produce a thermal effect at the margin , making evaluation of the margins difficult . conization appears safe in ia1 stage , as no difference in survival rates between this approach and hysterectomy has been reported in many studies . small series have shown a risk of recurrent invasive cancer below 0.5% , and a risk of recurrence of preinvasive disease between 7 and 10% [ 12 , 13 ] . reported 12 cases of stage ia1 cervical cancer treated with conization with an overall survival rate of 100% at 6.7 years . report 4 conizations in patients with adenocarcinoma stage ia1 . at 1672 months of follow - up , study the survival rate after a five - year follow - up was 99% ( 95% ci 9699% ) following hysterectomy and 98% ( 95% ci 9799% ) following conization . conization is associated with an increased risk of preterm delivery but no major differences in obstetrical outcome among different techniques of conization are demonstrated . two meta - analyses [ 16 , 17 ] evaluate the obstetric outcome after cervical surgery . both studies reported an increased risk of preterm delivery ( relative risk [ rr ] , 2.59 ; 95% ci , 1.83.72 ; rr , 2.78 ; 95% ci , 1.724.51 ) and low birth weight ( rr , 2.53 ; 95% ci , 1.195.36 ; rr , 2.86 ; 95% ci , 1.375.97 ) in patients with a history of knife conization . patients with cervical cancer at stages ia2 and ib1 have a high incidence of pelvic lymph node metastases and pelvic node dissection is necessary . the conventional treatment in these stages consists of rh and bilateral pelvic lymph node dissection . vaginal radical trachelectomy ( vrt ) with pelvic lymphadenectomy , as a fertility sparing treatment of early - stage cervical carcinoma , was first described in 1987 by dargent . refer that 40% of patients who underwent a rh at their institution would have been candidates for a vrt . during surgery spread to the lymph nodes should always be assessed therefore a laparoscopic pelvic lymph node dissection is performed before trachelectomy . the lymph nodes from the external and internal iliac and obturator region are removed and the presence of metastasis is evaluated by a frozen section . if lymph nodes are negative , the trachelectomy is performed . in case of positive lymph nodes an rh followed by chemo - radiotherapy or definitive chemo - radiotherapy is the treatment of choice . trachelectomy can be performed vaginally or abdominally with open or laparoscopic technique and it is usually accompanied by cervical cerclage placement . because of the limitations such as cervical stenosis , crypto - menorrhea and dysmenorrheal , cerclage is also recommended in second trimester of pregnancy . the sensitivity and specificity for the detection of lymph node metastases for sentinel node biopsy is 91% and 100% , respectively . trachelectomy has been adopted by many oncological centers all over the world with good oncological and obstetrical results . the selection of patients by adequate preoperative evaluation is an important process before a decision regarding conservative treatment is taken . the extension of the lesion is of great importance ; the tumor should be small in size and confined to the cervix without parametrial invasion or spread to the uterine corpus . a recurrence rate of 19% has been reported for patients with lesions > 2 cm and 25% for those with lesions > 2 cm and depth of invasion > 1 cm [ 20 , 21 ] . it is controversial whether imaging studies are more useful than clinical examination to assess tumor size and local spread . colposcopy can also be performed and it is useful in assessing the exocervical diameter and the extension to the vagina . some data suggest that a preoperative assessment with magnetic resonance imaging ( mri ) is the most adequate diagnostic option of measurement of lesion diameters , amount of cervical stroma infiltration , and parametrial invasion . a prospective study including 208 women who underwent mri and ct prior to surgery reports that mri correlated more closely with surgical pathologic findings than ct or physical examination . mri is also useful for the detection of the endocervical extension of the lesion in relation to the isthmus . many centers suggest that infiltration of less than half of the stroma is a safe limit because it allows the free margin of 1 cm . one cm cervical stroma is necessary to reduce the risk for premature delivery [ 2325 ] . in cases where the upper margin of the lesion is less than 1 cm from the isthmus , vaginal and rectal ultrasound can also be used to assess the size of the tumor . the preoperative evaluation of lymph nodes is commonly performed with ct , but pet and pet / ct are the most adequate imaging modalities and they have replaced mri and lymphangiography . a meta - analysis of 72 studies including 5042 women with cervical cancer found that pet has a better sensitivity and specificity for the detection of lymph node metastases ( sensitivity : 75% , specificity : 98% ) ; than mri ( 56% and 93% ) or ct ( 58% and 92% ) . integrated pet / ct may be more sensitive than pet alone for detection of nodal metastases , particularly for pelvic lymph nodes . it is important that patients have a strong desire to preserve fertility and have been adequately informed . when lymph vascular space involvement is present in stage ia1 , conization is not an adequate treatment and radical trachelectomy should be performed . the lesion should be less than 2 cm and confined to the cervix with stromal infiltration less than 10 mm . the procedure is not recommended for small cell carcinoma or sarcoma such as in cases with capillary space involvement . although lymph vascular space involvement is a negative prognostic factor when present alone it is not an absolute contraindication there is a general controversy whether adenocarcinoma histology is associated with worse prognosis than squamous cell carcinoma [ 2932 ] . adenocarcinoma often involves the endocervix and it can be difficult to eliminate the entire lesion . although all series included cases of adenocarcinoma , none have addressed the influence of histology on outcome . helpman et al . in a recent large series , where the influence of histology on outcome is determinate , report no significant difference in recurrence - free survival between adenocarcinoma and squamous cell carcinoma . the radical trachelectomy present a risk for intraoperative complications to ureter , bladder , and rectum . report a 5.6% of intraoperative complications in a large series which includes 125 patients underwent radical trachelectomy . in this study a total of 7 complications are described , four related to laparoscopy and only 3 to the trachelectomy , two bladder lesions and one uncontrollable bleeding . fewer blood transfusions , less total blood loss , and shortened hospital stays are associated with vrt . specific problems are associated with radical trachelectomy as dysmenorrhea , dysplastic smears , irregular bleeding , excessive vaginal discharge , problem with cerclage suture , isthmic stenosis , amenorrhea , and sexual inactivity . whether trachelectomy is effective and safe as rh is of great importance . a randomized study comparing this two surgical procedures is not feasible because it is difficult to have enough patients to reach statistical significance and because of ethical reasons . found no significant differences in 5-year overall survival rate and 5-year progression - free rate between radical trachelectomy and rh . report a 2.7% recurrence rate in a case series including 112 patients with early cervical cancer underwent vrt . recurrence occurred in only 3 patients after 18 months follow - up . in the first patient for technical problems carcinoma in situ was found in the endocervical margin of the trachelectomy specimen in the second patient but she did not accept hysterectomy and invasive carcinoma was found four months later . the third patient was diagnosed with cancer of 22 mm in diameter and insisted on vrt . we reviewed the case series regarding vrt as fertility sparing option ( tables 1 and 2 ) . trachelectomy has been abandoned because of different reasons as metastatic nodes detected during lymphadenectomy or more extensive lesions and no adequate margins discovered at the time of surgery . the median follow - up was from 21 to 95 months in different studies with a total of 35 recurrences ( 4.3% ) and 20 deaths ( 2.5% ) reported . abdominal radical trachelectomy ( art ) has a low intraoperative complication rate , making it possible to operate on cases with distorted cervicovaginal anatomy , providing larger parametrial resection . in addition it seems to be more familiar to gynecologic oncologists and it is easier to perform in nulliparous patients where advanced vaginal surgical skills are required to perform a vaginal operation . in 1997 , smith et al . the largest series is reported by li et al . , in 2011 , and include 62 patients treated with art . in this series there are no recurrence in 22.8 months of median follow - up . although open art offers better resection of the margins , it is associated with longer hospitality stay , more blood loss , and wound complications compared with the vrt . laparoscopic radical trachelectomy ( lrt ) is , in principle , identical to the art and it has all the advantages of an abdominal approach combined to a mini invasive surgery . there are only a few small case series published ; however the available data suggests that the laparoscopic way to perform a trachelectomy can be a safe alternative [ 4046 ] . the largest series published by kim et al . in 2010 regarding 32 patients reports one recurrence and one death after a median follow - up of 31 months ( table 5 ) . the dissection is extremely difficult and requires advanced surgical skills and expertise when performed with laparoscopic approach . the robotic approach offers better motion with finer instruments , precision , and three - dimensional image . furthermore contrary to the art the uterine artery can be preserved and only the descending braches are transected maintaining the blood supply to the uterus . a total of six reports are published with overall 25 patients underwent robotic radical trachelectomy ( rrt ) ( table 6 ) . the first reported case series was published by persson in 2008 , describing two patients with stage ib1 who underwent rrt without complications . report a series of 6 patients who operated with robotic laparoscopically assisted trachelectomy with preservation of fertility in 5 and one case of conversion in hysterectomy because of positive margins . study the robotic surgical approach resulted in less blood loss and decreased length of hospital stay with no compromise in histopathological outcome . the evaluable studies have not reported on obstetrical results because of a short follow - up time . a total of 359 pregnancies in 217 patients have been reported , resulting in 229 births . at the moment of the studies 58 pregnancies have miscarried in the first trimester ( 17% ) and this is comparable to the rate in the general population ( 16 to 20 percent ) . 24 patients ( 7% ) had a second trimester miscarriage versus 4% of the general population . a total of 229 live births are reported and 67% gave birth after 36 weeks . only 8 of the 10 published reports report the exact number of pregnancies before 32 weeks . second trimester miscarriage and premature rupture of membrane ( prom ) with following premature labor are complications related to trachelectomy . the shortened cervix with absence of mucus facilitats the ascending infections and can result in chorioamnionitis and premature rupture of the membranes [ 51 , 52 ] . pregnancies after trachelectomy should be considered as high risk but no definitive guidelines regarding the management of these patients are published . some authors suggest regular vaginal swabs every two weeks or the use of prophylactic antibiotics at 16 and 24 weeks , bed rest , and routine administration of steroids routine cytologies in asymptomatic women and sexual intercourse from the 20th week should be avoided because of the increase risk of infection . follow - up with serial cervical ultrasound measurement has a predictive value of preterm labor and could be useful to decide the time for steroid administration . a vaginal delivery could be dangerous because of the possibility of a lateral cervical tear extending to the uterine arteries . . demonstrated that the uterine perfusion after trachelectomy is unchanged and support no risk for intrauterine growth restriction . infertility has been reported in 2530% of patients after trachelectomy and possible causes include cervical cervical stenosis , decreased cervical mucus , and subclinical salpingitis [ 51 , 55 ] . cervical stenosis occur in 15% of patients who underwent trachelectomy and dilatation of the cervix can resolve the problem in the majority of the cases [ 55 , 56 ] . report infertility in 15 patients of 111 underwent trachelectomy but patients fertility was not proved before operation . of those 40% were due to cervical factor , and the remaining 60% were unrelated to the trachelectomy . patients with infertility after trachelectomy can be pregnant with iui or ivf but particular attention should be made to avoid multiple gestations considering the preexisting high risk of preterm delivery in this patients . intraperitoneal insemination in the pouch of douglas can be an option when tubes are patent and semen quality is sufficient . if it is impossible to perform transcervical embryo transfer with a severely stenotic cervical opening , a transmyometrial embryo transfer under ultrasound guidance can be performed . [ 57 , 58 ] gametes or zygotes intrafallopian transfer are also a therapeutic option . patient who underwent trachelectomy should been seen every 3 to 4 months for the first two years , then every 6 months . some centers continue the follow - up every year after the first five years . a follow - up with clinical and colposcopic examination and cervical cytology problematic can be also the interpretation of cytology . in about 58%60% of smears atypical cells all cases reported as atypical glandular cells were endometrial stromal cells , tubal metaplasia , and lower uterine segment glandular cells . there are different prognostic factors for early stage cervical cancer and they are classified in intermediate- and high - risk factors for recurrent disease . if high risk factors ( positive or close resection margins , positive lymph nodes , parametrial involvement ) are identified an adjuvant radiation or chemo - radiotherapy is needed . beiner and covens in a review report that approximately 10% of patients who underwent vrt would be candidates for adjuvant treatment . the included patients had either positive nodes , positive margins , or parametrial involvement on final pathology . deep stromal invasion , large tumor size , and lymph vascular invasion are intermediate risk factors and if any of these are identified , adjuvant radiation decreases the risk of recurrence . whether adjuvant therapy can be avoided in some of these patients has not been determined . a conservative approach with close follow - up has a potential high risk of recurrence and the patient should be informed . ovarian transposition can be performed to avoid damage of ovarian tissue when radiation is needed after surgery . laparoscopic lateral ovarian transposition is the simplest and most effective technique that can be used in patients with cervical cancer who will be undergoing pelvic radiation . ovarian transposition is beneficial not only for preservation of fertility but also to prevent premature menopause . the preserving of ovarian function has successful rates about 90% after vaginal brachytherapy and 60% in patients undergoing pelvic radiation . ovarian failure rates reported after ovarian transposition were due to various factors , such as vascular compromise , type of radiation , external or brachytherapy , and dose . complications of ovarian transposition are chronic ovarian pain , ovarian cysts , and infraction of the fallopian tubes . patients with cervical cancer stage ia1 with lvsi , stage ia2 , and ib1 are treated with radical trachelectomy and lymphadenectomy . radical trachelectomy is associated with complications due to the removal of the parametrium which contains nerve fibers implicated in the innervations of bowel and bladder . fertility outcomes are good but not as good as the results after conization . on the other hand , the utility of parametrial resection is controversial [ 84 , 85 ] . approximately 60% of patients undergoing trachelectomy have no residual disease in the final pathologic specimen after a diagnostic cone and less than 1% of patients with favorable pathologic characteristics have parametrial involvement . several retrospective studies report a low incidence of parametrial involvement ( 0 to 0.6% ) in patients , with early stages of cervical cancer and favorable histopathological characteristics , who underwent rh [ 8588 ] . in the largest study of 536 patients with tumor size < 2 cm , depth invasion < 10 mm , and negative pelvic lymph nodes , including any histology , only 0.6% had parametrial involvement . report 0% of parametrial involvement in 101 patients underwent rh . in this study patients with squamous , adenocarcinoma , adenosquamous , or clear cell histology , tumor size < 2 cm , depth invasion < 10 mm , no lvsi , and negative pelvic lymph nodes were included . the resection of part of the parametrium is based on the presence of small lymph nodes in the tissue . lanowska et al . , in a recent study , found an incidence of metastasis less than 1% . the presence of lymph node in the parametria was 7.1% and the authors found a statistically significant difference in the thickness of the parametrium between patients with and without lymph nodes . a radiological preoperative evaluation with measurement of parametrium volume using rmn could help for the selection of patients who could benefit of a less invasive surgery . in the recent years some centers have adopted more conservative surgery using large cone or simply trachelectomy with a high successful pregnancy rate and encouraging oncologic results [ 89 , 90 ] . there are only a few case reports about less radical fertility sparing surgery in early stages . in a recent small case series by fagotti et al . , conization was performed in patients with cervical cancer stage ia2-ib1 and tumor < 20 mm . no recurrences were observed after a median follow - up of 16 months ( range 8101 months ) . two of five patients , attempting to conceive , had a spontaneous pregnancy and delivery . report the results of 40 patients with early stage cervical cancer ( stage ia1 to ib1 ) who underwent laparoscopic sentinel node identification followed by conization or trachelectomy . a cone ( stage ia1 with lvsi and stage ia2 ) or simple trachelectomy ( stage ib1 less than 2 cm ) was performed some days after definitive histopathologic confirmation of negative nodes . only one recurrence has been reported after a mean follow - up of 47 months . these recent studies report conization or simple trachelectomy and pelvic lymphadenectomy as a safe conservative approach but more studies are needed to confirm the safety of this method . neoadjuvant chemotherapy before conservative surgery in early cervical cancer can reduce the tumor size prior trachelectomy or conization . neoadjuvant chemotherapy in patients who did not meet the favorable pathological criteria has been reported by small series suggesting that this approach may be an option . report 9 patients who underwent three cycles with isofosfamide and cisplatin or cisplatin and adriamycin . cervical conization or simple trachelectomy and pelvic lymphadenectomy was performed after chemotherapy and no recurrences have been reported . report 21 patients with larger tumors <3 cm , in stage ib1 , who underwent neoadjuvant chemotherapy ( three cycles of isofosfamide , paclitaxel , and cisplatin ) followed by conization and pelvic lymphadenectomy . no residual disease was found in five patients and no recurrence is reported after a median follow - up of 69 months . recent studies show that there are interesting treatment alternatives to the golden standard for patients with early stage of cervical cancer . the available data support the safety of trachelectomy , with a low and acceptable rate of recurrence similar to the traditional options . vaginal trachelectomy has been the most common approach but abdominal and laparoscopic procedures are now being favored in view of the radicality of excision of parametria . the obstetrical outcome is excellent and the possibility to conceive is high . possible infertility problems due to cervical stenosis can be overcome with dilation or assisted reproductive technologies . recent studies show that conization or simple trachelectomy with pelvic lymphadenectomy could be an alternative in stage ia2 and ib1 with favorable pathologic characteristics , which would reduce the obstetrical risks and perioperative complications . the role of parametrium thickness and development of imagine technique to measure parametrium volume before surgery is important for the selection of patients who probably benefit from resection of parametrium . all young patients with diagnosis of cervical cancer should be encouraged to discuss conservative treatment with their gynecologist . gynecologists should be informed about the fertility sparing options and referred to appropriate gynecologic oncologist . guidelines and clinical protocols for the management of these patients should be created and followed by the gynecologist involved in the counseling and treatment of these patients . | fertility preservation is an important issue for patients in reproductive age with early stage cervical cancer . in view of recent developments ,
our purpose was to review and discuss available surgical alternatives .
a literature search was conducted using pubmed , including papers between 1980 and december 2011 . in patients with stage ia1 cervical cancer
, conization is a valid alternative .
patients with stage ia2-ib1 disease can be conservatively treated by radical trachelectomy .
this is as well - established conservative approach and appears to be safe and effective in allowing a high chance of conception .
prematurity is the most serious issue in pregnancies following trachelectomy .
less invasive options such as simple trachelectomy or conization seem to be feasible for stages ia2-ib1 , but more and better evidence is needed .
neoadjuvant therapy might allow conservative surgery to be performed also in patients with more extensive lesions .
ovarian transposition is important when adjuvant radiation is needed . in conclusion ,
available literature shows that there are interesting fertility - sparing treatment alternatives to the golden standard for the management of early cervical cancer in young women . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
stress fractures are common injuries most often associated with the participation in sports involving running , jumping , or repetitive stress . in comparison with other locations of stress fractures , a patellar stress fracture was first described in a german journal by muller in 1943 and in an english journal by devas in 1960 . stress fractures of the patella have been described to be represented by 2 different types ; those with a longitudinal fracture of the lateral part and those with a transverse fracture of the distal or middle part . to our knowledge , no report about a stress fracture of the patella has described a transverse fracture of the proximal part . in this report , we describe a case of an extremely rare type of patellar stress fracture , a proximal transverse fracture , in a 9-year - old athlete . informed consent was obtained from the patient and his parents . a 9-year - old boy participated in various sports , including running sprints twice per week for 1.5 years , and engaging in kendo twice per week for 3 years . he presented at a nearby orthopedic clinic 1 month after experiencing left knee pain while playing these sports , without any injury event . he had no subcutaneous hemorrhage and no external injury at the first visit to the clinic . his left knee radiograph showed a transverse fracture of the left proximal patella and a right knee radiograph showed no evidence of fracture ( figure 1 ) , and he was therefore diagnosed to have a transverse stress fracture of the left proximal patella . the femorotibial angle of the left knee was 174 and that of the right knee was 176. the insall and salvati ratio of the left knee was 0.91 and that of the right knee was 0.97 . the roentgenographic patellofemoral congruence was almost normal , because the sulcus angle of the left knee was 138 and the congruence angle was 5. he was treated conservatively for 7 months , with inhibition from playing sports , but without immobilization , such as cast fixation or bracing . however , his left knee pain did not disappear , so he presented to our hospital for treatment . a plain radiograph taken 7 months before the first visit of our hospital . the left knee radiographs ( a , d , f ) revealed a transverse fracture of the proximal patella , whereas the right knee radiographs ( a , c , e ) revealed no evidence of a fracture . the anterior - posterior view ( a , b ) showed that the knees were neither valgus nor varus . the lateral view ( c , d ) showed that neither of the knees had patella alta nor patella infera . the axial view ( e , f ) showed that the patellofemoral congruence was almost normal for the knees . a physical examination revealed no ligamentous laxity , no effusion , no range of motion deficit in his left knee and mildly localized tenderness at the left proximal patella . his left knee radiograph showed a transverse fracture of the proximal patella with nonunion ( figure 2 ) . we made a diagnosis of nonunion of a proximal transverse patellar stress fracture and performed surgery . the left proximal patellar stress fracture was drilled with a 0.88-mm k - wire for freshening of the nonunion portion and was internally fixed using acutrak mini screws ( acumed , hillsboro , or ) . three months after the operation , he had returned to his previous level of sports activity without any complaints . six months after the operation , the left patella fracture was completely united ( figure 3 ) . at 2 years a left knee radiograph taken at the first visit to our hospital revealed nonunion of a proximal transverse patella stress fracture . a left knee radiograph taken 6 months after the operation revealed that the patellar stress fracture was completely united . a 9-year - old boy participated in various sports , including running sprints twice per week for 1.5 years , and engaging in kendo twice per week for 3 years . he presented at a nearby orthopedic clinic 1 month after experiencing left knee pain while playing these sports , without any injury event . he had no subcutaneous hemorrhage and no external injury at the first visit to the clinic . his left knee radiograph showed a transverse fracture of the left proximal patella and a right knee radiograph showed no evidence of fracture ( figure 1 ) , and he was therefore diagnosed to have a transverse stress fracture of the left proximal patella . the femorotibial angle of the left knee was 174 and that of the right knee was 176. the insall and salvati ratio of the left knee was 0.91 and that of the right knee was 0.97 . the roentgenographic patellofemoral congruence was almost normal , because the sulcus angle of the left knee was 138 and the congruence angle was 5. he was treated conservatively for 7 months , with inhibition from playing sports , but without immobilization , such as cast fixation or bracing . however , his left knee pain did not disappear , so he presented to our hospital for treatment . a plain radiograph taken 7 months before the first visit of our hospital . the left knee radiographs ( a , d , f ) revealed a transverse fracture of the proximal patella , whereas the right knee radiographs ( a , c , e ) revealed no evidence of a fracture . the anterior - posterior view ( a , b ) showed that the knees were neither valgus nor varus . the lateral view ( c , d ) showed that neither of the knees had patella alta nor patella infera . the axial view ( e , f ) showed that the patellofemoral congruence was almost normal for the knees . a physical examination revealed no ligamentous laxity , no effusion , no range of motion deficit in his left knee and mildly localized tenderness at the left proximal patella . his left knee radiograph showed a transverse fracture of the proximal patella with nonunion ( figure 2 ) . we made a diagnosis of nonunion of a proximal transverse patellar stress fracture and performed surgery . the left proximal patellar stress fracture was drilled with a 0.88-mm k - wire for freshening of the nonunion portion and was internally fixed using acutrak mini screws ( acumed , hillsboro , or ) . three months after the operation , he had returned to his previous level of sports activity without any complaints . six months after the operation , the left patella fracture was completely united ( figure 3 ) . at 2 years a left knee radiograph taken at the first visit to our hospital revealed nonunion of a proximal transverse patella stress fracture . a left knee radiograph taken 6 months after the operation revealed that the patellar stress fracture was completely united . the location of stress fractures in adult athletes was reported to be the following : 49.1% in the tibia , 25.3% in the tarsal bones , 8.8% in the metatarsal bones , 7.2% in the femur , 6.6% in the fibula , 1.6% in the pelvis , 0.9% in the sesamoids , and 0.6% in the spine ; in children , these were 47% in the tibia , 21% in the fibula , 12% in the femur , 9% in the radius , 9% in the metatarsals , and 3% in the humerus . . there have been 22 case reports of patellar stress fractures in athletes published in the english literature since 1960 . the previously reported cases in the english literature have described 2 different types : a longitudinal fracture type and a transverse fracture type . the longitudinal fractures were described in 6 case reports , all of which were located in the lateral part . transverse fractures were described in the other 16 case reports , 14 of these cases were located in the distal 1/3 and 2 cases were located in the middle 1/3 . in our case , patellar stress fractures have been reported in athletes including soccer players , basketball players , and runners . these sports activities involve the high frequency use of the knee extensor mechanism , repetitively increasing the patellofemoral joint reaction force and patellofemoral contact pressure . as it was demonstrated that an increased patellofemoral joint reaction force was dependent on the increase of the knee flexion angle , we think that the repetitive increase in the patellofemoral joint reaction force due to the kendo , in addition to the sprints , is 1 factor associated with the development of the transverse stress fracture of his left patella . it was previously reported that the contact area of the patella articular surface against the femoral surface was different from the flexion angle of the knee . for example , if the knee was flexed at 30 , 60 , and > 90 to 120 , the patella surface contact , respectively , was in the distal 1/3 area , middle 1/3 area , and proximal 1/3 area . in the present case , the transverse stress fracture occurred in the proximal patella , whereas most reported locations of the transverse stress fractures of the patella were in the distal 1/3 . the radiograph of left lateral patellar view taken 6 months after the operation ( figure 3 ) revealed a subtle , shallow radiolucent area at proximal patella , which may have been the contact point for the creation of the stress fracture . we suggest that the repetitive motion with > 90 of left knee flexion during sprinting and while performing kendo caused the transverse stress fracture of his left proximal patella , because these motion increased the patellofemoral contact pressure for the proximal patellar articular surface , in addition to repetitively increasing the patellofemoral joint reaction force , including the longitudinal traction force for the patella . some methods of surgical treatment for patellar stress fractures have been reported . internal fixation with the tension band technique , the cannulated screw technique , curettage , bone grafting , or drilling have all been reported . in our case , 7 months of conservative treatment , including no sports activities , did not improve the left knee pain . we therefore performed surgical treatment by means of drilling and internal fixation using the cannulated screw technique with acutrak screws for the left knee , because the patient had a nonunion of the proximal patellar transverse stress fracture . postoperatively , the patient was able to return to his previous level of athletic sports activity . therefore , we think that we could successfully treat the nonunion of a transverse stress fracture of the proximal patella with internal fixation using acutrak screws . we herein reported an extremely rare case of transverse stress fracture of the proximal patella . we believe that the repetitive motion with 90 left knee flexion induced by running sprints and participating in kendo caused the transverse stress fracture of the patient 's left proximal patella , because these motions increased the patellofemoral contact pressure for the proximal patellar articular surface , in addition to repetitively increasing the patellofemoral joint reaction force , including the longitudinal traction force on the patella . the nonunion of the transverse stress fracture of his proximal patella was successfully treated with internal fixation using acutrak screws , and he was able to return to his previous level of athletic sports activity postoperatively . | abstractamong stress fractures associated with sports activities , patellar stress fracture is rare . regarding patella stress fractures , so far only distal transverse or lateral longitudinal fractures have been reported , but there are no reports of transverse fractures occurring in the proximal patella .
we describe an extremely rare case of transverse stress fracture of proximal patella in a 9-year - old athlete.a 9-year old boy , who participated in sports ( sprints and kendo ) presented with left knee pain without any external injury . in plain radiographs
, a fracture line was observed in the proximal 1/3 of the left patella , and a patella stress fracture was diagnosed . for treatment , because 7 months of conservative therapy showed no improvement , internal fixation was carried out using acutrak screws , and bone union was thus achieved .
three months after the operation , he was able to return to his previous level of athletic sports activity.regarding the mechanism of onset , it is believed that the causes are longitudinal traction force and patellofemoral contact pressure . on the other hand ,
the contact region of the patella with the femur changes with the flexion angle of the knee . in the current case ,
the fracture occurred at a site where the patella was in contact with the femur at a flexion angle of > 90 , so it is believed that it occurred as a clinical condition from being subjected to repeated longitudinal traction force and patellofemoral contact pressure at a flexion angle of > 90 , during the sports activities of sprints and kendo .
the nonunion of the transverse stress fracture of his proximal patella was successfully treated with internal fixation using acutrak screws . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
there has been increasing interest in an immune - based etiology for adult - onset epilepsy . this disorder , which is also called steroid - responsive encephalopathy associated with autoimmune thyroiditis ( sreat ) , was first described by brain et al . as an association between antithyroid antibodies and encephalopathy . its prevalence is estimated to be 2:100,000 , with a predominance of females in their 5th6th decades of life and with varying presentations , such as stroke - like events or behavioral changes with or without epilepsy . one of the proposed mechanisms for antiepileptic drug hypersensitivity is also an immune - mediated process . we report two cases of hashimoto 's encephalopathy manifesting as adult - onset temporal epilepsy , cognitive decline , and multi - antiepileptic drug ( aed ) hypersensitivity . a 60-year - old female with a medical history remarkable for satisfactorily treated hypothyroidism and dyslipidemia was first admitted because of a dissociative event that lasted for 1 h , in which she described viewing herself outside of her body . no abnormal movements , amnesia , focal neurological deficits , fever , or systemic signs were noted . her neurological examination was normal , as were her head computerized tomogram ( ct ) and routine laboratory workup ( complete blood count , electrolytes , kidney and liver function , and thyroid functions ) . a lumbar puncture demonstrated high opening pressure ( 285 mm h2o ) with normal content and no detectable oligoclonal bands . she was discharged from the hospital on a regimen of lamotrigine treatment ( 25 mg / day , with instructions for a slow , gradual increase in the dosage ) . she returned one week later because of a severe rash over her entire body for which she received prednisone ( 60 mg / day ) and antihistamines ( chlorpheniramine maleate , 2 mg bid ) . the lamotrigine was discontinued , and carbamazepine ( 100 mg bid , with instructions of a 100-mg / week increase ) was started . two weeks later , she presented with worsening of the rash and reported that she had stopped the steroid therapy without tapering down the dose as instructed . she was admitted to the hospital and treated with iv hydrocortisone and then oral prednisone ( 1 mg / kg ) , following a stepwise decrease of the steroid dosage . the carbamazepine was replaced by valproate ( with an initial dosage of 250 mg bid ) . the valproate dose was increased gradually , but the aed treatment was switched to levetiracetam ( with an initial oral dosage of 250 mg bid ) when she developed thrombocytopenia ( down to 25,000/ml ) that began 48 h after the first dosage . her condition subsequently improved , and she was discharged , ending a total of 17 days with either iv or oral steroid therapy . she was instructed to reduce the steroid treatment for several weeks until it was stopped . a few months later , the patient was readmitted to our department because of cognitive changes , insomnia , and agitation . a brain magnetic resonance imaging ( mri ) serology results for hiv , hepatitis b and c , vdrl , tpha , and hhv6 were all negative . blood samples were sent to test neoplastic markers as well as the following paraneoplastic antibodies : anti - yo ( anti - purkinje cell cytoplasmic antibody , associated with ovarian and breast cancer ) and anti - hu ( antineuronal nuclear antibody type i , associated with small - cell lung cancer ) . she also underwent colonoscopy , which revealed a single polyp whose histology was consistent with high - grade dysplastic adenoma , as well as chest - abdominal and pelvic ct , the results of which were normal . thus , the only positive finding was extremely high titers of antithyroglobulin ( 5318 u / ml ) and of antithyroid peroxidase ( tpo , > 3000 u / ml ) , corresponding to the diagnosis of hashimoto 's encephalitis . since steroid therapy had been previously administered for the diagnosis of severe drug eruption and found to be ineffective for the clinical presentation of hashimoto 's encephalitis as cognitive changes were added to the clinical picture , she was treated with plasma exchange ( 5 courses over 10 days ) . although no clinical improvement was noted at first , eeg monitoring demonstrated significant improvement after 14 days , with only one eeg - verified electroencephalographic seizure per night , compared to 34 electroencephalographic seizures per night before the plasma exchange ( fig . 1e ) . cognitive assessment ( montreal cognitive assessment [ moca ] ) performed before and after plasma exchange revealed a mild cognitive impairment ( 24/30 , mainly attention deficit and memory ) that improved by two points after receiving treatment ( figs . there was no further improvement in the moca score at the 1-month follow - up , but the patient reported that she felt that her cognitive function improved . at that time , she complained of side effects related to the aed treatment ( mainly fatigue ) . one month later , she returned for another scheduled outpatient visit , after having stopped all antiepileptic therapy , and felt that she had returned to her regular levels of function , an observation supported by her spouse . further follow - up demonstrated clinical and electroencephalographic relapses every few months , with elevated antithyroid antibodies at similar levels , which responded to an additional course of plasma exchange with improvement in her clinical state . our suggestion was therefore to continue treatment with plasma exchange on a regular basis , and because of the good response , there was no need for more aggressive immunomodulatory treatment . a 70-year - old woman with a past medical history of well - controlled hypothyroidism and hypertension was admitted to our service because of events manifested by a detachment from her surroundings lasting for about 1 min each that had started 18 months earlier . some of these began with a scream , following a tonic phase with loss of consciousness . her eeg and head ct were unremarkable , and oral lamotrigine treatment beginning at 25 mg / day ( with instructions for a slow increase ) was initiated . a hypersensitivity reaction presenting as a rash all over her body appeared about 10 days later , and the treatment was switched to oral valproate ( beginning at a dosage of 250 mg bid with a slow increase ) . the valproate treatment did not lead to any significant reduction in the seizure frequency , and oral topiramate ( beginning at 25 mg / day ) was added . she was then hospitalized because of two seizure events , each lasting up to 15 min , in which she collapsed and was nonresponsive . the neurological examination on admission was notable for disorientation and evident cognitive abnormality as demonstrated by a moca score of 12/30 ( specifically , abnormal serial seven , immediate memory , and abstraction ) . the lumbar puncture had normal opening pressure and content , and the tau protein level was within the normal range . routine blood work revealed polycythemia and mild leukocytosis , while the electrolyte levels , liver and kidney functions , thyroid functions , c - reactive protein levels , and neoplastic markers were all normal . the only positive relevant result was very high titers of antithyroglobulin ( 5564 u / ml ) and antithyroid microsomal antibody ( > 3000 u / ml ) . a repeated brain mri revealed progressive mild atrophy and a hyperintense temporal cortical signal restricted in diffusion . methylprednisolone ( iv , 500 mg / day for 5 days ) was administered , and the valproate was switched to levetiracetam ( 500 mg bid ) because of extrapyramidal side effects . she returned three weeks later with an allergic drug reaction , manifested through a skin rash and pruritus , while being treated with 30-mg prednisone ( during the tapering down ) that was attributed to the levetiracetam . the repeated moca score was 16/30 , suggesting mild improvement . at a follow - up visit several months later , the patient and her family reported significant improvement . she had returned to her usual levels of function and normal lifestyle , and she was seizure - free . although we did not notice a prominent cognitive improvement , she did return to her baseline daily function , and , therefore , we did not suggest further immunomodulatory treatment , except for low prednisone dosage ( 10 mg daily ) . further measurement of the antithyroid antibodies demonstrated a marked reduction ( anti - tpo : 349 u / ml , antithyroglobulin : 461 u / ml , fig . 2 ) . we describe here two cases of adult - onset temporal lobe epilepsy with cognitive decline and antithyroid antibodies ( hashimoto 's encephalopathy ) , as well as multi - aed hypersensitivity and clinical improvement with immunomodulatory therapy . hashimoto 's encephalopathy has been previously linked to new - onset epilepsy [ 57 ] and to cognitive changes [ 68 ] . however , to our knowledge , this is the first report describing multi - aed hypersensitivity coexisting with hashimoto 's encephalopathy . hashimoto 's encephalopathy is a controversial syndrome , since it is defined as an association of autoantibodies ( anti - tpo and antithyroglobulin ) and encephalopathy , a weak connection between two common entities without any known causal relationship . proposed pathogenesis includes autoimmune central nervous system ( cns ) vasculitis and an autoimmune reaction to antigens shared by the thyroid and the cns . this syndrome can be manifested by a wide range of symptoms and signs , including behavioral changes , confusion , cognitive decline , epileptic seizures , and stroke - like episodes . there is little evidence for delineating the relevant target of antithyroid antibodies in hashimoto 's encephalopathy , although blanchin et al . demonstrated how anti - tpo antibodies adhere to human astrocytes and monkey - derived cerebellar slices , and ochi et al . hashimoto 's encephalopathy is probably an immune - based syndrome , not only because of its correlation with autoantibodies but also because of its response to immune - mediated treatment . it is possible that the antithyroid antibodies are markers of a predisposition to the syndrome and not necessarily active participants in the encephalopathic process . one research study demonstrates that patients with multidrug hypersensitivity have a subpopulation of regulatory t - cells which are in a preactivation state , as opposed to healthy controls . another recent study demonstrated how specific human leukocyte antigens ( hlas ) known to predispose to specific drug reactions ( e.g. , for carbamazepine ) activate the immunological system when exposed to the drug . however , no connection between multidrug hypersensitivity and antithyroid autoantibodies has been reported to date . these case reports are intended to raise the level of awareness on immune - based epilepsy and/or dementia , especially since these entities may respond to immunomodulatory therapies . we suggest that an immune - based etiology should be considered in the differential diagnosis of patients who present with adult - onset temporal epilepsy , especially in patients with idiosyncratic drug reactions . | hashimoto 's encephalopathy is defined by the coexistence of encephalopathy and antithyroid antibodies .
we report two cases of adult - onset temporal lobe epilepsy with subacute cognitive decline , high titers of antithyroid antibodies , multi - antiepileptic drug hypersensitivity , and good response to immunomodulatory treatment .
the relevance of multidrug hypersensitivity in the setting of adult - onset epilepsy and the importance of searching for autoimmune causes for epilepsy are discussed . |
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tumor angiogenesis , the development of new blood vessels within the primary tumor or in metastasis hotspots , is an essential process for the growth and progression of metastases . both innate and adaptive immune systems are involved in positive and negative regulations of this process . all over the world various research teams are conducting studies to find agents , which could potentially be able to inhibit the process . despite the discovery of many such agents , the need for further research exists , because the angiogenesis inhibitors obtained so far are either very costly to synthesize , or they exhibit a number of side effects . angiogenesis is a complex process with many contributing factors and one regulated by endogenic stimulators and inhibitors . among the scientists involved in such studies a common conviction exists thus the need to recourse to low - toxic vegetal compounds and studies of combined effects of a few low - dosage compounds with different modes of action on the processes of angiogenesis in tumor growth . many phytochemicals and diet derivatives are able to exert chemopreventive and antitumor activity targeting the tumor environment and inflammatory angiogenesis [ 13 ] . pervivo is a digestive herbal remedy , mixture of 27 herbs alcoholic extracts dissolved in 32% ethyl alcohol , some of them traditionally used as anti - inflammatory agents also possessing antimicrobial or anti - tumor properties . we were the first to show angioinhibitory effect of this remedy in local cutaneous model of tumor angiogenesis . the most important anti - tumor substance in pervivo is radix zingiberis ( ginger ) . the anticancer properties of ginger are connected mainly with gingerols , shogaols , and zingerone . 6-gingerol , a natural component of ginger , was shown to inhibit growth of colon cancer cells via induction of g2/m arrest . the second ginger component , 6-shogaol , induced apoptosis in human hepatocellular carcinoma cells and exhibited anti - tumor activity in vivo through endoplasmic reticulum stress . their results suggest that 6-gingerol has potential to bind with dna and induce cell death by autophagy and caspase-3-mediated apoptosis . silva et al . demonstrated specific antiproliferative activities of gingerols against mda - mb-231 tumor cell line . shogaols have stronger inhibitory effect than gingerols on growth of cancer cells , arachidonic acid release , and nitric oxide ( no ) synthesis . in experiments of weng et al . both 6-shogaol and 6-gingerol effectively inhibited invasion and metastasis of hepatocellular carcinoma but through diverse molecular mechanisms . 6-gingerol directly decreased expression of urokinase plasminogen activator and 6-shogaol indirectly by upregulation plasminogen activator inhibitor and via blockade of nuclear factor-b activation . it was also documented that ginger and its compounds inhibit angiogenesis in vitro and in vivo [ 1315 ] . the next pervivo ingredient , artemisia absinthium , inhibited tnf alpha production and accelerated healing of patients with crohn 's disease . artemisinin , active antimalarial compound isolated from herbs belonging to artemisia species , dihydroartemisinin ( dha ) , a semisynthetic derivative of artemisinin , and hispidulin , small flavonoid from artemisia vestita , inhibited growth of pancreatic , prostate , and ovarian cancer cells and were shown to be cytotoxic to cancer cells through induction of apoptosis . the antiangiogenic effect of artemisinin in vitro and in vivo was also described [ 1725 ] . the claims concerning anti - tumor activity of some other pervivo compounds are not largely supported by scientific evidence as yet . radix angelicae sinensis is a medicinal herb and health food supplement that has been widely used in asia for centuries . cytotoxicity against tumor cell lines of its extracts , epigenetic modifications of cancer oncogenes , and tumor suppressor genes were described . it was also reported that radix angelicae sinensis may be a potential source of glutathione s - transferase inhibitors and counteract multidrug resistance [ 2630 ] . one report described antiproliferative activity of gentiana triflora root extract on cultured and implanted tumor cells . fruit oils of litsea cubeba from taiwan exhibited cytotoxic activity against human lung , liver , and oral cancer cells in vitro [ 30 , 31 ] . galangin , flavonol present in alpinia galanga rhizome , induced apoptosis of cancer cells [ 32 , 33 ] . extract of radix liquiritiae ( licoricidin from glycyrrhiza uralensis ) inhibited the metastatic potential of human prostate cancer cells . essential oil and decoction of carlinae radix ( carlina acanthifolia l. ) showed significant antimicrobial effect against staphylococcus aureus . compounds from artemisia are antiplasmodial and antitrypanosomal drugs , and could be an alternative drug against trichinellosis [ 3638 ] . compounds from alpinia galanga are potent inhibitors for the influenza virus replication and exhibit significant activity in vitro against promastigotes of leishmania donovani . essential oils from litsea cubeba contain fungicidal and antibacterial terpenoids [ 42 , 43 ] . epidemiological studies have suggested that the use of nonsteroidal anti - inflammatory drugs ( nsaids ) may reduce the risk of cancer . numerous studies have been devoted to the action of such anti - inflammatory agents as aspirin , indomethacin , piroxicam , and sulindac . the anticancer effect of nsaids was mainly found to result from their proapoptotic and antiproliferative effects which , therefore , restrict tumorigenesis [ 4549 ] . we were the first to show that sulindac and its metabolites ( sulindac sulfone and sulindac sulphide ) , described previously by other authors as pro - apoptotic anticancer drugs [ 50 , 51 ] , inhibit tumor growth and angiogenesis induced in mice skin by cells isolated from murine l-1 sarcoma as well as angiogenesis induced by cells collected from human kidney and pulmonary cancers [ 5255 ] . the aim of the present work was to study the combined in vivo effect of multicomponent herbal remedy pervivo and non - steroidal anti - inflammatory drug sulindac on tumor growth , tumor angiogenesis , and tumor volume in balb / c mice . these effects were checked after grafting of cells collected from syngeneic sarcoma l-1 tumors into mice skin . pervivo ( richard bittner gmbh , weitensfeld , austria ) is herbal remedy composed of 27 herbal extracts dissolved in 32% ethyl alcohol ( table 1 ) . sulindac ( sudaklin , polpharma sa , starogard gdaski , poland ) is nonsteroidal anti - inflammatory drug . the study was performed on female , 810-week old inbred balb / c mice , about 20 g of body mass , delivered from the polish academy of sciences breeding colony . for all performed experiments animals were handled according to the polish law on the protection of animals and nih ( national institutes of health ) standards . mice were housed 4 - 5 per cage and maintained under conventional conditions ( room temperature 22.523.0c , relative humidity 5070% , and 12 h day / night cycle ) with free access to standard rodent diet and water . l-1 sarcoma cells from in vitro culture stock were delivered from warsaw 's oncology center collection , passaged in vivo , and grafted subcutaneously ( for evaluation of tumor growth ) or intradermally ( for evaluation of angiogenic activity ) to syngeneic balb / c mice . mice received orally by eppendorff pipette 20 l of pervivo with 20 l of water , or sulindac 0.6 mg , both drugs , for 3 days in cutaneous tumor - induced angiogenesis ( tia ) test or for 14 days in evaluation of the effect of drugs on tumor growth . these doses correspond to 10 ml of pervivo and 300 mg of sulindac given to 70 kg person ( applying the counter 7 for differences between mouse and human in relation of the surface to body mass ) . briefly , sarcoma l-1 cells from in vitro stock were grafted ( 10/0.1 ml ) subcutaneously into subscapular region of balb / c mice . after 14 days , the tumors were excised , cut to smaller pieces , rubbed through sieve , and suspended in 5 ml of pbs . after sedimentation , the supernatant was collected and centrifuged for 10 min at 1500 rpm . obtained sarcoma cells were washed once with pbs for 10 min , then centrifuged at 1500 rpm , and resuspended in parker medium in concentration of 4 10 cells / ml ( for tumor - induced angiogenesis ) or 10 cells / ml ( for experiments with tumor growth ) . viability of cells was about 95% of living cells as estimated by trypan - blue method . multiple 0.05 ml samples of 200 thousands of cells were injected intradermally into partly shaved , narcotised balb / c mice ( at least 3 - 4 mice per group ) . in order to facilitate the localization of cell injection sites later on , after 72 hours , mice were sacrificed with lethal dose of morbital ( biowet , puawy ) . all newly formed blood vessels were identified and counted in dissection microscope , on the inner skin surface , at magnification of 6x , in 1/3 central area of microscopic field . identification was based on the fact that the new blood vessels are thin , directed to the point of cells injection and ( or ) differ from the background vasculature in their tortuosity and divarications ( figure 1 ) . all experiments were performed in anaesthesia ( 3.6% chloral hydrate , sigma - aldrich ) , 0.1 ml per 10 g of body mass . suspensions of sarcoma cells were grafted ( 2 millions of cells ) subcutaneously into mice . on the day of cells grafting and on the following 13 days mice were fed pervivo , sulindac , pervivo and sulindac , or diluted ethyl alcohol as a control . mice were observed during these days , with number of appearing tumors noted at 7 , 9 , and 11 days after grafting . at days 9 and 14 the tumors volume was measured with electronic caliper ( the fowler ultra - cal mark iii caliper ) . morphological examination was done using light - microscopic analysis . immediately after resection , tumor specimens after fixation the specimens were dehydrated in increased concentration of alcohol and embedded in paraffin . evaluation of the results was performed by chi - square test and one - way anova with bartlett 's test for equal variances , and the significance of differences between the groups was verified with a bonferroni multiple posttest ( graphpad prism ) . histological examination revealed no major differences between tumors collected from control and experimental groups of mice . as shown in figure 2 and table 2 the number of newly formed vessels that were induced by the tumor presence was decreased both by pervivo and sulindac , with the effect of sulindac comparatively stronger . joint application of both drugs resulted in even stronger effect than treatment with separate compounds . the influence of the study drugs on measurable tumor emergence in respective days after sarcoma load inoculation is shown in figure 3 and table 3 . similar to the antiangiogenic effect , the influence of sulindac alone was greater than this by pervivo , and the common appliance of both drugs resulted in even greater delay in measurable tumor appearance . the time of the strongest impediment of tumor growth was similar in case of all study settings ( day 7 ) . the effect was intermittent , and only after application of both drugs together , the measurable tumors have not developed in all animals . the influence of the study drugs on the mean tumor volume at the respective study time points is shown in figures 4 and 5 . the mean volume was statistically significantly lower at day 9 after administration of sulindac and , to the lesser extent , by both drugs ( table 4 ) . the mean tumor volume was lower than in placebo group but the difference was not significant . at day 14 only in the group that received both drugs the mean tumor volume was significantly lower compared to the placebo ( figure 5 and table 5 ) . l-1 sarcoma tumor arose spontaneously in the lung of balb / c mouse and was described by przemysaw janik from warsaw oncology center ( 58 ) . this tumor has been maintained since then by subcutaneous serial passages in balb / c mice and frozen and stored in warsaw oncology center tissue collection . l-1 sarcoma cells from culture , after grafting to animals , form tumors in in vivo conditions . l-1 sarcoma is a perfect experimental model for assessing the impact of various substances upon the tumor growth and activity of its cells . previously , we used l-1 sarcoma cells for evaluation of pro- and antiangiogenic activity of various substances of synthetic and natural origin . we were the first to report the anti - angiogenic activity of sulindac and its metabolites , as well as that of theobromine , catechins in the cacao tree seeds , salidroside and rosavin isolated from the rhodiola rosea and the rhodiola quadrifida , convallamaroside isolated from the convallaria majalis rhizome , alkylglycerols found in the shark liver oil , and other substances of natural origin [ 5662 ] . we also demonstrated inhibitory effect of these substances in the angiogenic reaction induced in the mouse skin by cells or tissue homogenates obtained from surgically removed human cancers of the lung , kidney , ovary , and urinary bladder . we also proved the synergic anti - angiogenic activity of particular combinations of synthetic and natural inhibitors in relation to the reaction induced in mice skin with the human serum . we demonstrated the synergic activity of small doses of sulindac , convallamaroside , ursolic acid and epigallocatechin gallate ( egcg ) in inhibition of cutaneous angiogenesis induced in mice by intracutaneous administration of serum obtained from patients with diabetic retinopathy . additionally , we demonstrated that human recombined cytokines ( vegf , bfgf , il-8 , and il-18 ) induced the cutaneous angiogenesis inhibited by natural and synthetic inhibitors . sulindac and its metabolites inhibited angiogenesis induced by bfgf and il-18 and did not affect the angiogenic vegf and il-8 activity [ 63 , 64 ] . our experience , dealing with the question of angiogenic inhibitors , indicates that it is crucial to use simultaneously multiple anti - angiogenic factors of different handle points in the treatment . many inhibitors display a synergic activity ; therefore , their appropriate combinations may significantly reduce drugs doses and their possible side effects . one of the examples of remarkable synergistic effect is inhibition of both the neovascularization and growth of tumor in head and neck squamous cell carcinoma by retinoic acid and interferon alfa by different mechanisms of action . tumor cells treated by interferon alfa stopped secretion of interleukin 8 , the major angiogenic factor , while retinoic acid caused them to secrete an inhibitor of angiogenesis . there is also a proof for synergism between cyclooxygenase inhibitors and cytotoxic chemotherapy drugs in inhibition of angiogenesis . both celecoxib and 5-fluorouracil impaired angiogenesis by inhibiting vascular endothelial growth factor ( vegf ) . it was shown that this effect is related to antiangiogenic influence of thalidomide on different tumor - related mediators : vegf , basic fibroblast growth factor , hepatocyte growth factor , and il-8 . all evidence , of which examples are noted above , may lead to the concept of integrative approach of managing a patient with cancer . cytotoxic drugs that are used as golden standard in cancer chemotherapy currently have high toxicity in therapeutic doses . the research is ongoing on combining them with compounds that target multiple biochemical pathways in processes that promote different aspects of cancer development , angiogenesis being one of the most important . natural health products that may be used as biological response modifiers and adaptogens , providing quality assurance of extracts is assured , and the effectiveness of combinations is proved in clinical trials . we described inhibitory in vivo effect of two combinations of natural substances on angiogenesis and l-1 sarcoma growth in balb / c mice . first of them was a composition of two scandinavian folk medicine products , greenland shark liver oil ( rich in alkoxyglycerols ) and arctic birch ashes , supplemented with squalene . all these substances alone and in combination significantly diminished cutaneous angiogenesis induced by tumor cells and tumor growth . the second remedy composed of echinacea purpurea again , a significant inhibitory effect on tumor angiogenesis and l-1 sarcoma growth was observed . other authors reported enhancing effect of theanine , a component of green tea leaves , on the antitumor activity of adriamycin , inhibition of liver metastasis of human pancreatic carcinoma by angiogenesis inhibitor tnp-470 ( analog of fumagillin derived from aspergillus fumigatus ) in combination with cisplatin in nude mice , and augmented antitumor effects of combination therapy cisplatin / tnp-470/il-12 in melanoma ( b6d2f1 mice ) and colon carcinoma ( balb / c mice ) . recently , anticancer activity of noscapine , an opioid alkaloid in combination with cisplatin in human nonsmall cell lung cancer , in vitro and in vivo , in murine xenograft model was reported . selvendiran et al . reported the results of in vitro and in vivo experiments with ho-3867 , a curcumin analog , combined with cisplatin . this compound sensitized cisplatin - resistant ovarian carcinoma , leading to therapeutic synergy through stat3 inhibition . similar effect was obtained for dihydroartemisinin ( derivative of artemisia compound artemisinin , one of the pervivo components ) which induced apoptosis and sensitized human ovarian cancer cells to carboplatin therapy and improved the efficiency of chemotherapeutics ( cisplatin , cyclophosphamide ) in lung carcinomas in vivo and in vitro . reported synergistic action of the c - jun n - terminal kinase ( jnk ) inhibitor and dihydroartemisinin in apoptosis induction through accelerating bax translocation into mitochondria in human lung adenocarcinoma cells . described synergistic proapoptotic effects of epigallocatechin gallate and epicatechin on human lung cancer cell line pc-9 in vitro . recently , beneficial effect of sulindac , in combination with dimethylamino parthenolide ( nuclear factor-b inhibitor ) and gemcitabine , in genetically engineered mouse model of pancreatic cancer was described . it was revealed that sulindac sulfide can inhibit tumor cell invasion in vitro at concentrations less than those required to inhibit tumor cells growth , by suppressing nf-b - mediated transcription of micrornas . novel sulindac derivatives that do not inhibit cox-1 and cox 2 suppressed colon tumor cell growth in vitro by inhibiting cgmp phosphodiesterase and -catenin transcriptional activity and inhibited in vivo malignant pleural adenocarcinoma dissemination in mice . | anticancer activity of many herbs was observed for hundreds of years .
they act as modifiers of biologic response , and their effectiveness may be increased by combining multiple herbal extracts .
pervivo , traditional digestive herbal remedy , contains some of them , and we previously described its antiangiogenic activity .
numerous studies documented anticancer effects of nonsteroidal anti - inflammatory drugs .
we were the first to show that sulindac and its metabolites inhibit angiogenesis . in the present paper
the combined in vivo effect of multicomponent herbal remedy pervivo and nonsteroidal anti - inflammatory drug sulindac on tumor growth , tumor angiogenesis , and tumor volume in balb / c mice was studied .
these effects were checked after grafting cells collected from syngeneic sarcoma l-1 tumors into mice skin .
the strongest inhibitory effect was observed in experimental groups treated with pervivo and sulindac together .
the results of our investigation showed that combined effect of examined drugs may be the best way to get the strongest antiangiogenic and antitumor effect . |
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although targeted endoscopic ultrasound - guided fine - needle aspiration ( eus - fna ) of the cyst wall following fluid aspiration has demonstrated improved overall diagnostic sensitivity for potentially malignant mucinous cysts 1 , there are reports concerning a dissemination risk from eus - fna . the enhanced diagnostic capability of eus - fna must be balanced against the risk of tumor seeding . a 75-year - old man was admitted to our hospital due to a pancreatic cystic lesion accompanying a solid mass , and with liver metastasis . eus observation ( uc240p - al5 , olympus co , tokyo , japan ) demonstrated a pancreatic cyst of 30-mm maximum diameter , and a solid mass beside the cystic lesion . using a 25-gauge needle ( expect , boston scientific japan , tokyo ) with a 20-ml syringe suction , we performed eus - fna on the solid mass , which also had a 30-mm maximum diameter through the posterior gastric wall . during the procedure , no early adverse events occurred . the pathological findings showed the lesion to be an adenocarcinoma that was diagnosed as intraductal papillary mucinous carcinoma ( ipmc ) ( fig . 1 ) . eus - fna on a mass beside cystic lesions through the posterior gastric wall using a 25-gauge needle ( expect , boston scientific japan , tokyo ) . because the patient s condition was inoperable , gemcitabine - based chemotherapy was started at a dose of 1600 mg on days 1 , 8 , and 15 on a 4-week cycle . approximately 3 months after eus - fna , follow - up computed tomography scan showed a cystic mass in the posterior gastric wall ( fig . 2 ) . the cystic mass was visualized by esophagogastroduodenoscopy ( egd ) as a subepithelial lesion ( sel ) ( fig . 3 ) , and eus suggested that this sel was primarily localized in the third layer of the gastric wall . needle tract seeding of eus - fna for intraductal papillary mucinous carcinoma ( ipmc ) was suspected as the cause of this new lesion . eus - fna was thus performed on the sel and a definite diagnosis of ipmc that was similar to previous pathological findings was obtained ( fig . 4 ) . because the cystic mass was located where the eus - fna had been performed , the sel was therefore compatible with needle tract seeding from the initial eus - fna . as a second - line treatment , the patient underwent combined chemotherapy with one course comprising intravenous infusion of gemcitabine 1600 mg on day 1 and s-1 ( tegafur + gimeracil + oteracil ) 500 mg twice a day for 14 days with a 1-week rest period in between courses . despite chemotherapy , he died from the cancer 29 months after the initial eus - fna . a follow - up ct scan 3 months after eus - fna showed a new cystic lesion in the posterior gastric wall ( arrow ) . eus showed an echoic cystic mass measuring 24 mm in diameter , primarily located in the third layer of the gastric wall . eus - fna for the gastric cystic lesion revealed a differentiated tubular adenocarcinoma that was consistent with the primary tumor in the pancreas . eus - fna for pancreatic cystic lesions is increasingly being used to differentiate benign cysts from cysts that have malignant potential or those with frank malignancy that may have otherwise been misdiagnosed as benign 2
3 . 3 reported that the sensitivity , specificity and accuracy of eus - fna for the diagnosis of malignancy in ipmc were 75 % , 91 % , and 86 % , respectively . although a retrospective study reported no associated increased rate of gastric or peritoneal cancer recurrence following eus - fna 4 , levy et al . 5 demonstrated the potential for tumor cell displacement during eus - fna . in the study by levy et al . , the authors assessed the prevalence of luminal fluid - positive cytology among patients with luminal , extraluminal , and benign disease , and positive cytology was detected within post eus - fna luminal fluid in 3 of 26 patients ( 11.5 % ) with pancreatic cancer . cytological examination of luminal fluid aspirates did not demonstrate malignant cells in patients with nonmalignant disease , suggesting that the process of eus - fna may withdraw malignant cells from a pancreatic cancer into the gastrointestinal luminal tract . this is likely an analogous method by which needle tract seeding occurs , and is concordant with reports on a dissemination risk from eus - fna . with respect to ipmc , 6 reported a case of ipmc dissemination to the peritoneum that was caused by eus - fna . due to copious mucus production in ipmc , mucus production after needle tract seeding may lead to widespread dissemination . in cases where eus - fna is performed for a cystic solid mass , therefore eus - fna should be performed only after the risks and benefits have been thoroughly evaluated . | background and study aims : we report on a 75-year - old man who was admitted due to pancreatic cystic lesion accompanied by a solid mass with liver metastasis .
endoscopic ultrasound - guided fine - needle aspiration ( eus - fna ) was performed on the solid mass , and pathological findings revealed the lesion to be an adenocarcinoma diagnosed as intraductal papillary mucinous carcinoma ( ipmc ) . approximately 3 months after , a cystic subepithelial lesion appeared in the posterior gastric wall where the eus - fna had been performed .
we performed eus - fna again , which revealed that the cystic mass was ipmc with pathology similar to the original lesion .
this is a rare case demonstrating needle tract seeding of eus - fna for ipmc . |
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customer 's satisfaction , quality , and care are global subjects that affect all the organizations . most of the organizations , for improving customer 's satisfaction and , consequently , their own survival , are willing to evaluate the strategies of their service quality , and so , the customer is considered the key indicator in this evaluation . the quality of care is a judgment that is made by the customer after receiving the service and comparing their expectations with the received service . one of the principle characteristics of desirable management is accepting customer 's perceptions and expectations as the main factor for determining the quality . expectations are customers ' demands , meaning that what they feel a service provider must present . perceptions are a costumer 's experiences or , in other words , the current status . the difference between costumer 's expectations and perceptions is called service quality gap . by determining the gap between costumers ' perceptions and expectations of the quality of provided services , the process of service provision could be improved and costumer 's satisfaction , as a very sensitive element in today 's field of competition , could be provided . zithmal believes that expectations of services is affected by different factors such as individual needs , communication with others , past experiences , and society 's environmental conditions ; therefore , customers in different countries have different levels of expectations . parasurman considers the following factors effective in determining the quality of service : the ability to provide right , in - time , and reliable services ; the ability of responding to customer 's problems and complaints rapidly and increase the speed of services ; creating confidence and trust in costumers ; having sufficient professional skills and qualification ; paying attention to and regarding human dignity in behaviors ; and neat and tidy appearance for tangible elements like facilities and staff clothing . one of the methods to determine the quality of services is to evaluate costumers ' satisfaction , and one of the tools for evaluating and analyzing service quality gap is to study the difference between costumer 's expectations and perceptions using servqual method . this tool evaluates the quality of services by comparing costumer 's expectations and perceptions in different dimensions . this model has a questionnaire that measures the quality of services in five quality dimensions of tangibles , reliability of services , responding , assurance , and empathy . quality in the field of health services has a special place because the critical duty and mission of protecting the health and lives of the society is the responsibility of this part and any mistake could be irreparable ; on the other hand , health services are related to a vast part of the society ad providing flawless and professionally standard services must be common in the society . most of the clients and patients , because of increased level of knowledge about health and medicine and also to prevent the increase in health service expenses , demand services with desirable quality . different studies have been conducted to evaluate costumers ' satisfaction for improving the quality of provided services using servqual model in different sections including industry , service department , restaurants , municipalities , higher education , and banks . inside and outside iran , in the field of health service also , this model has been used in some sections , including the following places : tehran , zahedan , zanjan , yazd , and isfahan in iran and singapore , korea , malaysia , sana of yemen , ghana , and england outside iran ; but less attention has been paid to evaluation of service quality in different sections of primary health care of health centers , including the midwifery section . in this regard , some cases could be mentioned : sharifi rad et al . evaluated the service quality gap of primary health services in health centers of isfahan . results showed that patients ' expectations were way much beyond their perception of the current condition . agha mollaei et al . conducted a study on the expectations and perceptions of primary health service receivers about the quality of services in health centers of bandar abbas . results showed that patients did not have similar perception of different dimensions of quality of service and these differences should be considered in the programs for improving the quality of services . aron et al . conducted a study about the quality of medical services in health care centers of ghana . their results revealed that to increase the loyalty of patients , improving the quality of provided services was necessary . the aim of the present study was to evaluate the quality of services of midwifery in the health centers of isfahan , because women , as the mothers of families , are the pillar of health in families and their mortality , disability , and behaviors would affect all the health dimensions of the family , and the threats for the health of mothers would be a threat for the health of other age groups too . by determining the expectations and perceptions of midwifery service receivers , the policy makers , planners , and service providers could be informed about the strengths and weaknesses of the quality of midwifery services and this could be a starting point for improving the quality of services . the present study was a descriptive , comparative , cross - sectional study that was conducted on women who referred to the midwifery section of the health centers of isfahan . inclusion criteria were being able to at least read and write , having the ability to answer the questionnaire , having a medical file at the health center , and willingness to participate in the study , and the exclusion criterion was unwillingness to participate in the study . in the present study , considering the formula for sample size ( z= 95% , z= 80% , d = 2s ) , the number of samples required was calculated to be 218 . for sampling , from the main two health centers of isfahan , based on the covered population , 15 centers were randomly selected , and then , at each center , random sampling was conducted . for gathering data , the standard servqual questionnaire has 22 questions that evaluate the quality of provided services in five dimensions of tangibles ( 4 questions ) , reliability ( 5 questions ) , responding ( 4 questions ) , assurance ( 4 questions ) , and empathy ( 5 questions ) . this questionnaire was completed by the participants , once before receiving the service to evaluate their expectations ( evaluation of desirable situation ) and once after receiving the service to evaluate their perceptions ( evaluation of current situation ) . the questionnaire had two parts ; the first part contained demographic characteristics of the service receiver and the second part was the standard servqual questionnaire for evaluating the expectations and perceptions of participants about the quality of the provided service . questions were scored based on 5-point likert scale from completely agreed ( 5 ) to completely disagreed various studies have used servqual questionnaire in iran previously and its reliability and validity have been approved . in one of the studies , chronbach 's of expectation questions was 0.8 and of perception questions was 0.9 . data were analyzed using spss 20 ; frequency distribution , mean , standard deviation , paired t , pearson correlation , spearman correlation , and independent t tests were used for analysis . to determine the quality of the provide care ( quality gap ) , negative score ( negative gap ) indicated a gap between the current status and desirable status ( low quality of services ) and positive score indicated a positive gap that means providing ideal services ( high - quality services ) . the present study was approved by the ethics committee of isfahan university of medical sciences before execution and all the ethical issues were regarded at all the stages of the study . the present study was approved by the ethics committee of isfahan university of medical sciences before execution and all the ethical issues were regarded at all the stages of the study . in this study , 201 women who referred to the midwifery section of selected health centers of isfahan were selected and their average age was 31.8 7.3 years , which ranged from 18 to 61 years . the mean scores of expectations , perceptions , and quality gap in the midwifery section of isfahan health centers separately for each dimension are shown in table 1 . the mean score of expectations of all the participants was higher than their score of perception in all the aspects ; the highest score of expectations belonged to reliability dimension and the lowest score belonged to responding dimension . in the perception part , the highest score belonged to reliability and the lowest score belonged to tangibles . dimensions of midwifery service quality and mean scores for clients expectations and perceptions as shown in table 1 , the highest difference in the mean scores ( quality gap ) was in the field of tangible quality of services and the least difference was in the field of service quality assurance . as shown in table 2 , comparing the age and educational level in five dimensions of service quality by spearman and pearson correlation coefficients showed no significant relation between the scores of expectations and perceptions of the quality of midwifery services , and age and educational level . mean scores for clients expectations and perceptions and demographic characteristics ( age and educational level ) as can be seen in table 3 , independent t - test showed no significant relation between the mean scores of expectations and perceptions , and employment and marital status . the main aim of the present study was to evaluate the quality of provided services at the midwifery sections of isfahan health centers using servqual model . results showed that negative gap existed in all the five dimensions of service quality ( tangibles , reliability , responding , assurance , and empathy ) ; meaning that the performance of midwifery staff has not been satisfactory for patients . in this regard , noor'ain et al . reported a significant negative relation between patients ' satisfaction and the quality of provided services in malaysia . another study revealed a gap between the expectations and perceptions of icu patients and the gap was positive . patients ' score of perceptions was higher than their score of expectations ; however , this positive difference could be due to short time of their hospitalization in the icu ( 24 h ) or their special condition . in the present study , , also in isfahan , reported that the greatest gap belonged to the tangibles dimension . the greatest gap in the study of roohi et al . from gorgan , agha mollaei et al . from bandar abbas , and papanikolaou and zygiaris from greece was in the empathy dimension , in the study of mohammadi and shoghli from zanjan and lee and yom in korean hospitals was in the reliability dimension , in the study of tarig et al . from khartoum , sudan was in the assurance dimension , and in the study of kebriaei et al . de cyril conducted in malaysia , the highest negative score of service quality belonged to responding and reliability dimensions . , in a study conducted in hospitals of sanaa , found that the greatest service quality gap belonged to responding , assurance , and empathy dimensions . in this study , the least service quality gap or , in other words , the most appropriate dimension of service quality was the assurance dimension . agha mollaei et al . also reported that the least service quality gap belonged to the assurance dimension . also , the least service quality gap in the studies of tarahi et al . and roohi et al . belonged to the reliability dimension , in the studies of mohammadi and shoghli and lee and yom to the tangibles dimension , and in the study of anbori et al . to the tangibles and reliability dimensions . in this study , there was no significant relation between the scores of expectations and perceptions and some demographic characteristics like age , marital status , employment status , and educational level . in the study of agha mollaei et al . also , the relation between service quality gap and age , educational level , and employment was not significant . in the study of tarahi et al . , the mean score of service quality gap had a reverse correlation with the age of patients , but showed no significant relation with their educational level . in the study conducted by gholami et al . , there was a significant relation between patients ' age and educational level , and their expectations and perceptions ; meaning that as the patients ' mean of age and educational level increased , the negative service quality gap also increased and patients with higher education believed that negative gap existed in all dimensions except empathy . also , the study of kebriaei et al . found a significant relation between patients ' expectations and perceptions , and their age and educational level and the relation was reverse . reported no significant relation between patients ' expectations and perceptions , and their age , marital status , employment status , and educational level , but the relation between educational level and expectations was significant . on comparing the results of the present study and other similar studies , it was found that different characteristics of patients , different facilities of health organizations , and the diverse nature of services have led to dissimilarity of the results . health organization administrators could not lean on traditional methods and personal knowledge and experiences for planning and governing their own organizations and providing quality services for their customers . therefore , it is necessary for the administrators to prioritize the programs for different sections of their organizations for increased customer satisfaction and improving the quality of their organization 's services by discovering the lowest and highest expectations and perceptions of customers about the current and the desirable condition . considering that the mentioned studies have been conducted on different groups with different organizational conditions and also knowing that the study has been conducted on a special group , the results of this study are not generalizable to other fields of health system and medical services and organizations . it is recommended that in every city , for improving the quality of their organization , all the administrators of health centers should try to determine the expectations of patients at different sections of their center and conduct periodical studies based on their organizational condition and improve the quality of their organization by insisting on patients ' ideas and expectations and receiving their feedback . negative gap between the expectations and perceptions of patients indicates that patients ' perception of the current situation is lower than their expectations and there is a long distance to satisfaction of patients of midwifery section and reaching the desirable condition . on the other hand , this negative gap was not similar in all the dimensions of quality , and since the greatest service quality gap in the midwifery section of isfahan health centers belonged to the tangibles dimension , the administrators of this organization should reform the physical environment , provide up - to - date and sufficient facilities , and pay special attention to the hygiene of equipment . this study was financially sponsored by the research deputy of isfahan university of medical sciences . this study was financially sponsored by the research deputy of isfahan university of medical sciences . | background : quality of care is of great importance in health services as these services have the important mission to preserve health , and to give care to the society . the present study aimed to investigate the quality of midwifery services from the viewpoint of the clients , under coverage of health care centers in isfahan , through servqual model.materials and methods : this descriptive and analytical study was conducted on 218 subjects in 2014 .
study population comprised the women referring to midwifery services clinics in health care centers in isfahan .
data of the subjects ( n = 218 ) were collected by servqual model containing the dimensions of tangibles , reliability , responsiveness , assurance , and empathy .
data were analyzed by paired t - test , spearman and pearson correlation coefficients , and independent t - test through spss 20.results:there was a negative gap in all five relevant dimensions of giving services .
the widest gap was in the mean of dimension of tangibles ( 1.5 ) , and the narrowest gap was in the dimension of assurance ( 0.9 ) .
there was no significant association between the scores of expectations and perceptions , and age , education level , occupation , and marital status.conclusions:the obtained negative gap showed that the level of service receivers ' perception from existing condition was far from their expectation , and there was a wide gap between attaining their satisfaction with midwifery services and their expectation and reaching their appropriate level of services . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
ayurvedic medicine ( am ) is in number three in position in terms of worldwide popularity . am derives from plants which contain various phytochemicals and these phytochemicals make relief from disease by removing metabolic toxins from our body , boost up immunity and purify the blood . many traditional healing herbs and their parts have been shown to have medicinal value and can be used to prevent , alleviate , or cure several human diseases . presently , there is a resurgence of herbal medicine as people want more control in their personal healthcare . bangladesh has approximately 170 million population and is now facing and tackling public health problems such as deficiency of iron and iodine , especially for women , and the contamination of arsenic in drinking water . we examined the frequency of iron and iodine deficiencies and associations of iron and iodine deficiencies with common diseases among 395 under-2 children , 355 adolescent girls , and 263 pregnant women of bangladesh . anemia was found in 49.1% of children , 24.8% of adolescent girls , and 44.4% of pregnant women . mu g / l ) was 38.4% in adolescent girls and 39.4% in pregnant women . the ministry of health and family welfare of bangladesh makes strong countermeasures to enhance the health conditions for the nation . furthermore , with the viewpoint of disease treatment , a larger contribution of ayurveda should be focused on , conducted by medical doctors or doctors of am . the traditional medical practitioners perform a central role in providing primary healthcare to the rural inhabitants of bangladesh . there are 86,000 villages in the country and almost every village has one or two traditional practitioners . they are the providers of primary healthcare to village populations in bangladesh . on the other hand , the declaration of alma - ata of international conference on primary health care , alma - ata , ussr , on 612 september 1978 declared that the conference strongly reaffirmed that health , which is a state of complete physical , mental and social well - being , and not merely the absence of disease or infirmity , was a fundamental human right and that the attainment of the highest possible level of health is the most important world - wide social goal whose realization required the action of many other social and economic sectors in addition to the health sector . based on the concept of the declaration of alma - ata , complementary and alternative medicine ( cam ) therefore , the purpose of this study is to confirm the perceptions of am by citizens in dhaka , bangladesh . we hope this research will contribute to promoting and enhancing the effective usage of am , including herbal medicines as medical resources in bangladesh , asia , and the world . this study was conducted by face - to - face interview by trained staffs in dhaka , bangladesh using a structured questionnaire during december 2010 to january 2011 . the respondents were randomly selected by the trained staffs of the research team of dhaka , bangladesh . the research team of bangladesh initially obtained the name of villages and coded them using digital numbers . the subjects were informed that they were free to decline answering any question with which they were not comfortable . the questionnaire was translated into bengali and changes were made to make it understandable for the respondents before data collection in the field and back translated into english . to respond the questions of situation and perception of use of am and satisfaction on am use among citizens , an index of yes or no was applied . for questions of attitudes toward am use among citizens , a 5-point likert scale ranging from 1 = strongly disagree to 5 = strongly agree was applied . was not involved in experimental research involving human subjects and also anonymization of personal identity was preserved , the concept of exemption of ethical approval was accepted at that time . table 1 shows the demographics of the survey respondents of citizens in dhaka , bangladesh . statistically significant gender differences of citizens were observed in the residence , marital status , education , occupation , monthly income , and health status . though a statistically significant difference was observed in the residence by gender , the majority of male citizens ( 53.0% ) were in rural areas , as were the majority of female citizens ( 61.7% ) . though a statistically significant difference was observed in marital status by gender , the majority of males ( 59.1% ) were married , as were the majority of females ( 70.4% ) . a significant difference was observed in education by gender , the majority of males ( 49.1% ) had an education period of 11 years and more , 18.0% having 6 to 11 years education period , whereas the majority of females ( 36.4% ) had an education period of 11 years and more and 22.9% had 6 to 11 years education period . the majority occupation of males ( 35.0% ) was others which mainly consisted of retired , day laborer , rickshaw / van puller , and students , whereas the majority occupation of females ( 60.3% ) was housewife . on the other hand , though a statistically significant difference was observed in the monthly income by gender , the majority of males ( 63.9% ) received less than 7000 ( taka ) , as did the majority of females ( 84.4% ) . a significant difference was observed in health status by gender , the majority of males ( 43.0% ) had good health status , as did the majority of females ( 44.6% ) . demographic data of citizens in dhaka , bangladesh a : mann - whitney u - test , b : 1usd = 70 taka table 2 shows satisfaction on am use among citizens . 61.1% of citizens aged 1534 years old and 52.0% of citizens aged 35 years old or elder did not receive benefits from am . on the other hand , 76.8% of citizens aged 1534 years old and 70.1% of citizens aged 35 years old or elder did not get harm from am . 73.6% of citizens aged 1534 years old and 64.7% of citizens aged 35 years old or elder were satisfied with am , respectively . 72.9% of citizens aged 1534 years old and 62.1% of citizens aged 35 years old or elder recommended am to others . on the other hand , in terms of gender , there was no statistically significant difference . satisfaction on ayurvedic medicine ( am ) use among citizens in dhaka , bangladesh table 3 shows attitudes of citizens on am . though 41.5% of citizens aged 1534 years old agreed that their am provider gives good information on maintaining a healthy lifestyle , 43.1% of citizens aged 35 years old or elder strongly agreed it . though 56.1% of citizens aged 1534 years old and 52.9% of citizens aged 35 years old or elder agreed that herbal medicine has less side effects , 21.1% of citizens aged 1534 years old had not decided it , while 32.3% of citizens aged 35 years old or elder strongly agreed . though 53.5% of citizens aged 1534 years old and 58.8% of citizens aged 35 years old or elder agreed that am involves natural plant formulas which were healthier than taking western medicines , 26.1% of citizens aged 1534 years old had not decided it , while 19.6% of citizens aged 35 years old or elder strongly agreed it . 55.4% of citizens aged 1534 years old and 47.1% of citizens aged 35 years old or elder agreed that teacher can influence youths am use by exposing them to it . though 29.0% of citizens aged 1534 years old had not decided whether people who believe in the physical , mental and spiritual aspects of health were more likely to use am , 33.6% of citizens aged 35 years old or elder agreed it . 29.5% of citizens aged 1534 years old and 36.6% of citizens aged 35 years old or elder agreed that those who fear the discomfort of treatment from medical doctors were more likely to use am . 43.2% of citizens aged 1534 years old and 48.5% of citizens aged 35 years old or elder agreed that am was not harmful . attitudes of the citizens on ayurvedic medicine ( am ) in dhaka , bangladesh a : mann - whitney u - test on the other hand , in terms of gender , although 29.5% of male citizens agreed that people who believe in the physical , mental and spiritual aspects of health were more likely to use am and 28.2% had not decided , 22.2% disagreed , 29.5% of female citizens agreed and 26.9% had not decided , 17.5% strongly agreed . 29.0% of male citizens and 38.6% of female citizens agreed that those who fear the discomfort of treatment from medical doctors were more likely to use am , respectively . as a whole 42.7% of respondents agreed that people would be more likely to use am if there were more am clinics , with 31.3% strongly agreeing . 46.6% of respondents agreed that am built up the body s own defenses , with 17.9% strongly agreeing . 41.6% of respondents agreed that the more knowledge a person had of am , the more likely he / she would use it , with 30.7% strongly agreeing . 48.3% of respondents agreed that parents could influence youths am use by exposing them to it , with 15.3% strongly agreeing . 51.3% of respondents agreed that people could be influenced to use am if friends were using it , with 17.2% strongly agreeing . 50.3% of respondents agreed that people were mostly motivated to use am by television , radio and mass media , with 28.5% strongly agreeing . therefore , this kind of study should be conducted continuously to confirm precisely the perceptions of am by citizens in dhaka , bangladesh . we think this is one of the limitations of the study . to the best of the authors ' knowledge , this is the first study on the perceptions of am , including herbal medicine by citizens in dhaka , bangladesh . this study showed elder citizens had better impressions of am than younger citizens especially in terms of adverse drug reactions . this study also showed that younger citizens did not get more benefit from am than elder citizens . on the other hand younger citizens were more satisfied with am and also recommended am to others more , with statistically significant differences . we think that in terms of effectiveness of am , the younger generation in dhaka seems to be more skeptical of am than the elder generation , even though the younger generation are more satisfied with am than elder generation . from the viewpoint of enhancement of usage of am in dhaka , we think that appropriate information should be delivered vigorously in order to remove the skeptical feeling of am from younger citizen in dhaka . this study also showed that elder citizens thought more that am providers gave good information on maintaining a healthy lifestyle than younger citizens did . in terms of side effects , in addition , elder citizens thought more that am involved natural plant formulas which were healthier than taking western medicines . elder citizens seemed more to believe in the physical , mental and spiritual aspects of health , were more likely to use am and also agreed more that those who feared the discomfort of treatment from medical doctors were more likely to use am . on the other hand , in japan herbal medicine is used to treat patients as not only over - the - counter drugs but also ethical drugs covered by universal health insurance . recently a lot of studies regarding the safety and efficacy of herbal medicines have been vigorously conducted . the studies regarding safety and efficacy of herbal medicines do n't always show that herbal medicines are much safer than western medicine . but we think every kind of information should be delivered , because any kinds of scientific data may become one of the strong tools to enhance the usage of herbal medicine in bangladesh as well as asia and the world as the sound evidence . in addition , the previous studies we conducted in japan showed that there was a perception gap in medical terms between healthcare worker and citizens . the perception gap also becomes an obstacle to ensuring the healthy lives of citizens with medical care , including am . therefore , this issue is also considered by the healthcare workers of dhaka , bangladesh . the results of this study confirmed that the more am was familiar to the citizens in dhaka , the more the usage of am was enhanced and improved . in addition , this study also confirmed that television , radio and mass media had huge impact on the promotion of usage of am , including herbal medicine . from the viewpoint of effective utilization of limited medical resource , therefore , scientifically sound information should be collected rigorously and brought to the citizens vigorously . | abstractbangladesh is now facing the public health problems of deficiency of iron and iodine , especially for women .
the ministry of health and family welfare of bangladesh has implemented strong countermeasures to enhance the health condition of the nation . on the other hand , based on the concept of the declaration of alma - ata
, complementary and alternative medicine should be used more vigorously to enhance public health in the world .
the usage of complementary and alternative medicine such as ayurvedic medicine ( am ) should be increased in bangladesh .
therefore we conducted the study on perceptions of am by citizens in dhaka , bangladesh in order to promote and enhance the effective usage of am , including herbal medicines as medical resources , from december 2010 to january 2011 .
this study showed younger citizens ( 61.1% ) did not get more benefit from am than elder citizens ( 48.0% ) .
on the other hand , younger citizens ( 76.8% ) did not get more harm from am than elder citizens ( 70.1% ) .
we think that in terms of effectiveness of am , the younger generation in dhaka seems to be more skeptical to am than the elder generation in dhaka , even though the younger generation are more satisfied with am than the elder generation . with viewpoint of enhancement of usage of am in dhaka
, we think that scientifically sound information on am should be collected rigorously and brought to the citizens vigorously to remove the skeptical feeling of am from younger citizen in dhaka . in terms of the effective utilization of limited medical resources ,
am should be used appropriately in bangladesh , asia and the world . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
posterior reversible encephalopathy syndrome ( pres ) is a clinico - radiological syndrome , comprising symptoms such as headache , seizures , visual disturbances , lethargy , confusion , stupor , and focal neurologic findings . in addition , radiological findings of bilateral gray and white matter abnormalities suggestive of edema in the posterior regions of the cerebral hemispheres are essential for the diagnosis . since hinchey et al first reported , pres has been seen in various clinical settings , including renal disorders such as acute glomerulonephritis , lupus nephritis , and nephrotic syndrome [ 3 , 4 ] . the two theories regarding the pathophysiology of onset of vasogenic edema are hyperperfusion due to autoregulatory failure of the cerebral vasculature and hypoperfusion due to vasoconstriction of the cerebral artery . when blood pressure exceeds a certain set point in cases of significant systemic hypertension , autoregulation failure may occur . this results in cerebral hyperperfusion and breakdown of the blood - brain barrier , which in turn lead to vasogenic edema . pres is a relatively in the renal desorders but potentially a serious complication . in this report , we present two patients with pres in whom the primary diagnosis was hemolytic uremic syndrome ( hus ) and henoch - schonlein purpura ( hsp ) . a previously healthy 6-year - old girl presented with abdominal pain , vomiting , and macroscopic hematuria . at the time of application , her vital signs except blood pressure were normal . a full blood count revealed anemia ( hg 7.4 g / dl ) and thrombocytopenia ( 42,000/mm ) . blood chemistry results were as follows : serum creatinine 4.6 mg / dl and lactic dehydrogenase ( ldh ) 1,200 iu / l . she was immediately treated with both plasmapheresis and hemodialysis . on the eighth day of her hospitalization , she had a generalized tonic - clonic seizure , became confused and complained of bilateral complete loss of vision . neurologic examination showed normal findings , including her pupillary reflexes to light and normal accommodation . her contrast - enhanced computed tomography showed no abnormality but cranial magnetic resonance image ( mri ) revealed high signal abnormality . brain mri including diffusion - weighted images ( dwi ) showed faint signal intensities on bilateral parietal subcortical white matter without diffusion restriction ( fig . axial flair image ( a ) of case 1 shows faint signal intensities on both parietal white matter . diffusion image ( b ) fails to show any restriction which is consistent with pres . axial flair image ( c ) of case 2 and corresponding dwi image ( d ) show increased signal intensities at bilateral occipital and parietal lobes consistent with pres . a previously healthy 8-year - old girl presented with abdominal pain , hematochezia , rash on her legs , and macroscopic hematuria . with the diagnosis of hsp , she was put on oral prednisolone therapy . on the 10th day of her hospitalization the rest of her physical and neurological examinations and her vital sign were all within normal limits . laboratory findings showed anemia ( hg 9.6 g / dl ) , proteinuria 3 + , and hematuria . renal ultrasonography revealed normal sized , moderately echogenic kidneys ( grade 2 - 3 ) . the renal biopsy due to the underlying hsp nephritic disease stage showed focal necrotizing cresentic glomerulonephritis . due to fourteen days later , she suddenly complained of loss of vision ; thereafter , she had generalized tonic seizure . blood pressure was found to be 130/90 mm hg at the time and amlodipine was started and followed by enalapril and propanolol for persistant hypertension . the contrast - enhanced cranial computed tomography was normal but the cranial mri revealed bilateral hyperintense white matter lesions in the occipital and posterior parietal regions on t2-weighted fluid - attenuated inversion recovery ( flair ) and dwi ( fig . an eeg showed diffuse slow waves at all leads and there were focal isolated temporooccipital sharp wave discharges . a previously healthy 6-year - old girl presented with abdominal pain , vomiting , and macroscopic hematuria . at the time of application , her vital signs except blood pressure were normal . a full blood count revealed anemia ( hg 7.4 g / dl ) and thrombocytopenia ( 42,000/mm ) . blood chemistry results were as follows : serum creatinine 4.6 mg / dl and lactic dehydrogenase ( ldh ) 1,200 iu / l . she was immediately treated with both plasmapheresis and hemodialysis . on the eighth day of her hospitalization , she had a generalized tonic - clonic seizure , became confused and complained of bilateral complete loss of vision . neurologic examination showed normal findings , including her pupillary reflexes to light and normal accommodation . her contrast - enhanced computed tomography showed no abnormality but cranial magnetic resonance image ( mri ) revealed high signal abnormality . brain mri including diffusion - weighted images ( dwi ) showed faint signal intensities on bilateral parietal subcortical white matter without diffusion restriction ( fig . axial flair image ( a ) of case 1 shows faint signal intensities on both parietal white matter . diffusion image ( b ) fails to show any restriction which is consistent with pres . axial flair image ( c ) of case 2 and corresponding dwi image ( d ) show increased signal intensities at bilateral occipital and parietal lobes consistent with pres . a previously healthy 8-year - old girl presented with abdominal pain , hematochezia , rash on her legs , and macroscopic hematuria . with the diagnosis of hsp , she was put on oral prednisolone therapy . on the 10th day of her hospitalization the rest of her physical and neurological examinations and her vital sign were all within normal limits . laboratory findings showed anemia ( hg 9.6 g / dl ) , proteinuria 3 + , and hematuria . renal ultrasonography revealed normal sized , moderately echogenic kidneys ( grade 2 - 3 ) . the renal biopsy due to the underlying hsp nephritic disease stage showed focal necrotizing cresentic glomerulonephritis . due to both renal and gastrointestinal involvement of hsp , prednisolone was started . fourteen days later , she suddenly complained of loss of vision ; thereafter , she had generalized tonic seizure . blood pressure was found to be 130/90 mm hg at the time and amlodipine was started and followed by enalapril and propanolol for persistant hypertension . the contrast - enhanced cranial computed tomography was normal but the cranial mri revealed bilateral hyperintense white matter lesions in the occipital and posterior parietal regions on t2-weighted fluid - attenuated inversion recovery ( flair ) and dwi ( fig . an eeg showed diffuse slow waves at all leads and there were focal isolated temporooccipital sharp wave discharges . for the primary disease of hsp , she was given oral steroid and cyclophosphamide for the last 4 months . although they are completely different diseases with different etiologies , the vascular systems are affected in both diseases and hypertension was the common finding in two children . pres was originally defined in 1996 , with the clinical signs of renal insufficiency , hypertension , or immunosuppressive status , exhibiting headaches , altered mental functioning , seizures , and visual disturbance . the neuroimaging findings were characteristic of posterior brain edema without infarction , suggesting capillary leakage and breakdown of blood - brain barrier . the clinical symptoms and neuroradiological findings of pres are typically indistinguishable , regardless of etiology and underlying factors . beside its specific clinical setting which comprises mostly nephrologic patients to date , there is no evidence supporting genetic susceptibility to pres . although there are some limitations , cranial mri is the gold standard of imaging studies for detecting the lesions of pres . typical cranial images show focal regions of relatively symmetric hemispheric edema and the parietal and occipital lobes are most commonly affected . the differential diagnosis of pres includes cerebral vasculitis , cerebral venous trombosis , encephalitis , hypertensive encephalopathy , cerebral findings secondary to hypoglycemia and hyponatremia . the mainstays of the management of pres are control of blood pressure , treatment of seizures , and removal or reduction of other causative factors . after ruling out cerebral infarction , blood pressure should be lowered to the mean arterial pressure by 25% within the first hour . long - term prognosis is an important issue in children with pres , but evidence is scarce . these may suggest the necessity of long - term follow - up even including those who show complete clinical and radiological recovery in the short term period . subtle neurological disabilities , including subclinical developmental delay and learning problems , are of particular significance in some children . in the literature , there are also some cases diagnosed as hsp and hus [ 8 , 9 ] who developed pres during the natural course of their diseases . our patients demonstrated the characteristic radiologic signs and clinical features of pres , including headache , altered sensorium , seizures and visual disturbances . so that both of them were treated with anticonvulsant and proper antihypertensive drugs . a repeated mri scan of the head , an ophthalmologic assessment , and a follow - up eeg produced normal results with no sequelae . hemodynamic changes due to severe hypertension and renal insufficiency may be involved in the pathogenesis of pres associated with hsp . platelet - fibrin thrombus by occluding predominantly renal but rarely brain vessels subsequently may induce endothelial cell damages . stimulated endothelial cells can produce cytokines and chemokines , i.e. , interleukin-1 , 6 , 8 , tnf - alpha , and von - willebrand factor in situ . these substrates can induce vasospasms in cerebral vessels , which may lead to additional microemboli and breakdown of blood - brain barrier , which resemble to the underlying pathogenesis of pres . although pres was initially supposed to be a reversible and treatable disease , unusual pres cases associated with irreversible brain lesions have been accumulated . however , that was not the case in our patients . clinical symptoms leading to consideration of pres should be first evaluated for the primary disease especially in nephrologic patients and patients with hypertension . afterwards if there is no suspicion of the complication of the primary disease , cerebral infarction and hypertensive encephalopathy , pres should be considered for the diagnosis . in that cases mri becomes important and should be performed without hesitation before the resolution of the edema . morever , the eeg findings also should be assessed , in order to exclude the later development of epilepsy . | posterior reversible encephalopathy syndrome ( pres ) is a clinico - radiological syndrome , composed of symptoms such as headache , seizures , visual disturbances , lethargy , confusion , stupor , focal neurologic findings and radiological findings of bilateral gray and white matter abnormalities suggestive of edema in the posterior regions of the cerebral hemispheres .
pres is associated with significant morbidity and mortality if it is not expeditiously recognized . magnetic resonance image ( mri )
represents the most sensitive imaging technique for recognizing pres .
pres has been seen in various clinical settings including renal disorders such as acute glomerulonephritis , lupus nephritis , nephrotic syndrome , and drug usage such as calcineurin inhibitors .
we aimed to present two study cases for such clinical setting . in this report , we present two patients with pres in whom the primary diagnosis was hemolytic uremic syndrome ( hus ) and henoch - schonlein purpura ( hsp ) .
both of them were treated with anticonvulsant and proper antihypertensive drugs . a repeated mri scan of the head , an ophthalmologic assessment , and a follow - up electroencephalogram produced normal results with no sequelae .
early recognition of pres as a complication during different diseases and therapies in childhood may facilitate the appropriate treatment , so that intensive treatment should be performed as soon as possible to avoid neurological sequelae . |
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the carotid body ( cb ) , a neural - crest - derived paired organ located in the carotid bifurcation , is the main arterial chemoreceptor in mammals and facilitates reflex hyperventilation during hypoxia ( lpez - barneo et al . , 2001 ) . in addition , the cb is able to undergo neurogenesis to contribute to acclimatization of the organism during a persistent hypoxia ( edwards et al . , 1971 , arias - stella and valcarcel , 1976 , heath et al . , 1982 , we have previously shown that glia - like sustentacular , or type ii , cells surrounding chemosensory neuron - like glomus cells are neural crest stem cells ( ncscs ) and contribute to cb growth in hypoxia by differentiating into new glomus cells ( pardal et al . in addition to this neurogenic activity , a profound angiogenic process takes place in the hypoxic cb ( wang and bisgard , 2002 , chen et al . , 2007a ) . whether neural - crest - derived adult cb stem cells ( cbscs ) are able to differentiate into vascular cells , contributing to angiogenesis in vivo , in addition to neurogenesis , is largely unknown we have recently described the presence of mesectoderm - restricted progenitors within cb parenchyma and have shown that these cells are able to convert into endothelial cells in vitro ( navarro - guerrero et al . , 2016 ) . therefore , the cb constitutes an ideal model where to study the potential plasticity of an adult population of ncscs . utilizing both in vitro assays and in vivo cell - fate mapping experiments , we show that adult cbscs do indeed retain multipotent differentiation capacity , being able to convert into endothelial cells ( ecs ) contributing to hypoxia - induced angiogenesis during acclimatization of the organ . moreover , we provide mechanistic insight into this process by showing that cbscs differentiate into ecs via a hypoxia inducible factor ( hif)-dependent mechanism and in response to hypoxia - released molecules such as erythropoietin . thus , cbscs use plasticity to facilitate physiological adaptation of the cb , and hence the organism , to hypoxia . in order to test the ability of cbscs to contribute to vascular cell types , we decided to perform in vivo cell - fate mapping using gfap - cre / floxed lacz or yfp transgenic mice . these animals express the -galactosidase enzyme ( -gal ) , or the yellow fluorescent protein ( yfp ) , in gfap cbscs and their derivatives ( pardal et al . , 2007 ) . however , the human gfap promoter in this type of construct is not expressed in all gfap mouse cells , as previously revealed in brain astrocytes ( malatesta et al . , 2003 ) . in the case of cb , a first exposure to hypoxia ( 7 days ; figure s1 ) needs to be performed in order to activate the construct ( pardal et al . , 2007 ) , and only around 50% of gfap cbscs get labeled ( figure s1f ) . after this first exposure to hypoxia , only gfap cb stem cells and their derivatives in resting conditions , i.e. , some nestin progenitor cells , appear labeled ( figures s1 g and s1h ) , confirming the specificity of the construct . after construct activation , we exposed gfap - cre / floxed lacz mice to hypoxia for 7 days ( for hypoxic stimulus confirmation , see figures 4k and s5c ) and interrogated vascular cells for the expression of lacz . by using an antibody against -gal , and gsa i , an endothelium - specific lectin , we found that 16.3% 2.7% of ecs were positive for -gal ( 39 out of 229 gsa i cells analyzed ; figures 1a and s1j ) . no single -gal gsa i cell was found in the cbs of normoxic gfap - cre / floxed lacz littermate mice ( 99 gsa i cells analyzed ) , a result that was confirmed with yfp transgenic mice ( figure s1d ) . differentiation of gfap cbscs into ecs was confirmed in cb dispersed cells from hypoxic gfap - cre / floxed lacz mice , by using classical x - gal staining to reveal the activity of -gal , and fluorescent cytochemistry to detect gsa i ( figure 1b ) , or the endothelial markers cd31 ( figure 1i ) , or von willebrand factor ( vwf ; figures s1k and s1l ) . moreover , by using this cb ex vivo preparation , we confirmed neuronal differentiation capacity of gfap cbscs in the same mice ( figure 1c ) . interestingly , by using a smooth muscle cell marker ( sma ; figure 1d ) and a pericyte marker ( ng2 ; figure 1e ) , we also uncovered the ability of cb neural progenitors to convert into other mesenchymal cell types forming the vascular compartment , suggesting that cb progenitors can give rise to complete blood vessels . the use of a second type of reporter mouse strain ( gfap - cre / floxed yfp mice ) corroborated the cell - fate mapping results , both at the immunochemical ( figures 1f1h ) and flow cytometry ( fluorescence - activated cell sorting [ facs ] ; figure s1i ) levels . the percentage of yfp gsa i cells , obtained from gfap - cre / floxed yfp mouse cbs , was smaller ( 9.31% 0.59% ) than from the lacz mouse model , probably due to differences in cre - mediated excision efficiencies between the two reporter genes used . the use of a different ec marker , such as cd31 , for cell - fate mapping ( figures 1h , 1i , and s1i ) yielded qualitatively very similar results . no single ec , labeled with the reporter gene , was found when analyzing the cb of th - cre / floxed lacz mice ( figure s1 m ) , ruling out an unspecific expression of cre recombinase by ecs , and confirming the differentiation of gfap cbscs into vascular cells . we corroborated the multipotent differentiation capacity of cbscs in vivo , by performing bluo - gal staining and electron microscopy ( em ) on the cb of hypoxic gfap - cre / floxed lacz mice ( see figure s2 and its legend ) . altogether , in vivo cell - fate mapping experiments identify cbscs as multipotent neural - crest - derived adult stem cells contributing to both neurogenesis and angiogenesis during hypoxic acclimatization of the organ . to directly prove the multipotential capacity of cbscs to differentiate into both neuronal and vascular cell types , we performed single - cell deposition experiments in vitro ( figure 2 ) . out of 1,536 dispersed primary neurosphere ( ns ) cells plated , 200 of them gave rise to secondary ns . upon plating onto adherent conditions , 15 of those ns ( 7.5% ) contained both neuronal and mesectodermal cells within the colony ( figures 2a2d and s3a s3f ) , confirming differentiation into both cell lineages from a single progenitor cell . the percentage of multipotent progenitor cells is likely being underestimated due to the lack of appropriate neuronal differentiation conditions in the single - cell plating assay . in addition to tuj1 and gsa i markers ( figure 2b ) , we have corroborated the presence of neuronal and mesectodermal cells with different widely used markers : tyrosine hydroxylase ( th ) for dopaminergic neuronal cells and vwf , ng2 , and cd34 for vascular cells ( figures 2d and s3 ) . we also performed single - cell plating analysis with facs - sorted rat cb cells previously transfected with a pgfap - egfp construct , where 100% of gfp - sorted cells were gfap cbscs ( figures 2e and 2f ) . although the frequencies of ns formation and neuronal differentiation were quite low , likely due to transfection- and facs - induced damage , and lack of optimal neuronal differentiation conditions , we were able to find some examples of multipotent differentiation from single cbscs ( figures 2 g and 2h ) . consistently , we also found multipotent differentiation when plating single gfp cells from cbs of gfap - egfp transgenic mice ( figure 2i ) . we next decided to explore the mechanisms by which cbscs differentiate into ecs by using in vitro assays and rat cb cells , since the organ in rats is more manageable than in mice and the cells grow better in culture . rat cbsc - derived ns can be plated onto adherent conditions where , upon mitogen withdrawal , cells are induced to differentiate . using this assay , we found that cb progenitors differentiate into ecs ( gsa i ) when exposed to adherent substrate ( control in figures 3a and 3b ) . however , this differentiation was dramatically enhanced when the cells were exposed to hypoxia ( see figures 3c and 3d for hypoxia confirmation in vitro ) or vascular factors ( figures 3a and 3b ) , such as erythropoietin ( epo ) , endothelin-1 ( et-1 ) , platelet - derived growth factor ( pdgf ) , vascular endothelial growth factor ( vegf ) , or adenosine ( ado ) . this differentiation effect appeared to be specific for vascular factors since the addition of typical cb neuronal factors , such as dopamine ( da ) or methionine - enkephalin ( met - enk ) ( gonzlez - guerrero et al . , 1993 , gauda and gerfen , 1996 ) , did not increase endothelial differentiation ( figures 3a and 3b ) . interestingly , most vascular factors studied are consistently expressed in cb ecs , as assessed by rt - pcr on facs - sorted cb cells ( figure s3h ) . we next evaluated the expression of mature ec markers in gsa i ns cells , using gsa i cb dispersed cells and liver cells as positive controls ( figure 3e ) . we confirmed the expression of vwf , endothelial no synthase ( enos ) , and cadherin v , all of them specific for mature endothelium . we also confirmed the expression of the receptors for the different vascular factors that activate endothelial differentiation in cbscs ( figure s3i ) . both cb- and ns - derived gsa i cells lack expression of the receptor for da ( drd2 ; figure s3j ) , a neurotransmitter that did not increase endothelial differentiation ( figures 3a and 3b ) . as a control , we tested the expression of a cb neuronal marker ( th ; figure 3f ) , corroborating the lack of neuronal cell contamination in gsa i populations . moreover , we studied the functionality of cbsc - derived ecs obtained in vitro , by performing a low - density lipoprotein ( ldl ) uptake assay ( voyta et al . , 1984 ) . out of 216 gsa i cells analyzed from three independent cultures , 95% 1.5% of them were also positive for dii - conjugated ac - ldl ( figure 3 g ) , confirming the uptake and intracellular storage of lipoproteins , and hence the functionality of ecs differentiated from cbscs in vitro . finally , we decided to confirm the vasculogenic capacity of cb progenitor cells by using matrigel and a subcutaneous vasculogenesis assay ( see figure s4 ) . taken together , in vitro and in vivo functional assays and endothelial marker expression analysis indicate that neural - crest - derived cbscs are able to differentiate into mature and functional ecs , supporting their capacity to participate in cb angiogenesis . interestingly , in vitro differentiation assays indicate that cbsc conversion into ecs is directly favored by low oxygen ( see figure 3a ) . therefore , we decided to check whether cbscs were intrinsically sensitive to hypoxia , and whether prolyl hydroxylase ( phd)/hif pathway had a role in the vascular differentiation process . our data ( see figure s5 ) clearly establish an important , although partial , role for hif2 in the endothelial differentiation process . in addition , we decided to further study the role of a relevant hypoxia - induced vascular cytokine , epo . epo is a hematopoietic factor , finely regulated by oxygen tension ( franke et al . , 2013 ) , that has been described to participate in angiogenesis ( heeschen et al . , 2003 ) . in vitro results described above show that epo increases the number of ecs obtained from cbscs ( see figures 3a and 3b ) , and this is achieved without affecting neuronal differentiation ( figures 4a and 4b ) . moreover , epo receptor ( epor ) is expressed by both ns- and cb - derived nestin - activated progenitors ( figure 4c ) , suggesting a direct role of this cytokine on cbsc fate determination . therefore , we decided to further examine the role of epo on cb angiogenesis both in vitro and in vivo . the addition to adherent ns cultures of an antibody against epo , that sequesters the hematopoietic cytokine , completely abrogated the increase in endothelial differentiation ( figures 4d and 4e ) . ag490 is an inhibitor of epor - associated janus kinase ( jak ) 2 that has been described to block epo intracellular signaling ( lester et al . , 2005 ) . addition of ag490 to epo - treated adherent ns provoked a significant decrease in the number of ecs obtained ( figures 4d and 4e ) . thus , our results suggest a specific role for epo in the induction of endothelial differentiation from cbscs in vitro . we then decided to corroborate that epo was being endogenously produced in the cb ( lam et al . , 2009 ) . as assessed by rt - pcr , cb cells produce epo especially in hypoxic conditions ( figure 4f ) . in order to test whether cell - released epo was able to direct vascular differentiation from cb progenitors , we performed hypoxic co - cultures of ns progenitor cells and ecs ( figure 4 g ) . for these co - culture experiments , we used a well - characterized human ec line ( huvec ) , which produces epo ( figure 4h ) . the presence of huvecs in the surrounding wells increased the amount of functional ecs in cb ns , an effect that was abrogated after the addition of a blocking antibody against epo ( figures 4i and 4j ) , further corroborating the role of epo on cb progenitor endothelial differentiation in vitro . finally , we wanted to test whether epo was having a relevant role in hypoxia - induced cb angiogenesis in vivo . to this end , we used gfap - cre / floxed lacz transgenic mice exposed to hypoxia . systemic injection of epo signaling blocker ag490 in these mice significantly attenuated the typical hypoxia - induced epo - mediated hematocrit increase ( figure 4k ) , corroborating the systemic action of the inhibitor . cbsc - derived newly formed ecs are labeled by -galactosidase expression and hence appear with blue precipitate after x - gal staining ( figure 4l ) . quantification of x - gal cells in dispersed cb of these ag490-treated hypoxic mice revealed a decrease in the number of gsa i ecs derived from cbscs ( figure 4 m ) , confirming the importance of epo release and epo signaling in cbsc - dependent angiogenesis . in the present study , we show that adult cbscs display multipotent differentiation capacity , giving rise to both neural and non - neural derivatives during physiological acclimatization of cb to hypoxia . our results show that around 10%15% of total ecs , after a single exposure of the organ to chronic hypoxia , are derived from cbscs , and this value is likely being underestimated due to inefficient expression of transgenic constructs ( figure s1 ) . considering that ecs are described to double their number in the organ in response to hypoxia ( chen et al . , 2007a ) , we therefore estimate that around one every three or four newly formed ecs is cbsc derived . the ability of nscs to differentiate into ecs has been previously described only in some in vitro studies ( wurmser et al . , 2004 , ii et al . , 2009 ) or in the context of nervous system oncological disease ( ricci - vitiani et al . , 2010 , wang et al . , 2010 , however , to the best of our knowledge , no other example has been reported about adult neural progenitors participating in a physiological process of angiogenesis in vivo . in addition to the direct effect of hypoxia ( see the role of hif2 in the supplemental information ) , conversion of cbscs into ecs is also clearly enhanced by the exposition of cells to vascular factors , which are particularly released during hypoxia ( fandrey , 1995 ) . among these factors , epo has been described to regulate cns progenitor behavior ( shingo et al . , 2001 , chen et al . , 2007b ) and has a clear role enhancing vasculogenesis ( bahlmann et al . , 2004 , wang et al . , 2004 ) . our results show that cb cells produce this vascular factor mainly during hypoxia , indicating that locally produced epo might play a role in angiogenesis in the cb . this result is consistent with the described production of epo by neuronal glomus cells in the organ ( lam et al . moreover , we have recently described a role for cb neuronal cells on the proliferative activation of cbscs through a different cytokine , et-1 ( platero - luengo et al . , 2014 ) . the additional release of epo by cb glomus cells might represent another example of neuronal activity - dependent regulation of stem cell behavior , in this case promoting progenitors to participate in angiogenesis . nevertheless , we can not formally discard an additional role for circulating epo in cb angiogenesis . altogether , our results suggest that cbscs retain mesectodermal differentiation potential , resembling neural progenitors in the peripheral nervous system during development ( le douarin et al . , 2008 ) . this ability allows them to participate in physiological angiogenesis , in addition to neurogenesis , during adaptation of the organ to hypoxia . this finding might be relevant to understand the pathophysiology of this chemoreceptor organ and to improve the therapeutic use of these stem cells . male and female transgenic mice ( 46 weeks old or 89 months old for dimethyloxalylglycine [ dmog ] in vivo experiments ) and wistar rats ( 6 weeks old ) were housed and treated according to the animal care guidelines of the european community council ( 86/609/eec ) . all experimental procedures were approved by the committee on animal research at the university of seville . rat and transgenic mouse cbs were dissociated by enzymatic treatment and cultured to form ns , following protocols previously described ( pardal et al . , 2007 ) . immunostainings were performed following standard procedures described elsewhere ( pardal et al . , 2007 ) . dispersed cb cells from gfap - cre / floxed lacz and th - cre / floxed lacz mice were plated in ultralow binding petri dishes and allowed to recover superficial epitopes , lost upon enzymatic dispersion , in an incubator overnight . after that , standard protocols for x - gal staining were applied , as previously described ( pardal et al . , 2007 ) . once x - gal staining was performed , we processed the cells for antibody staining as indicated previously . carotid bifurcations were embedded in gelatin ( 80100 bloom ; panreac ) supplemented with 2% glutaraldehyde ( electron microscopy sciences ) , and 50-m - thick slices were cut on a vibratome ( vt1000s ; leica microsystems ) . primary ns were collected in 15-ml conical tubes and centrifuged at 100 g for 1 min . following aspiration of supernatant , ns were dispersed by accutase ( sigma ) , gently pipetting up and down during 10 min . . dissociated ns cells were centrifuged for 5 min at 300 g at 4c . afterward , cells were resuspended and cultured at clonal density on an ultralow binding 96-well plate . secondary ns were re - plated onto adherent conditions using fibronectin pre - treated plates , as described before . after 3 days in culture , adherent ns colonies were fixed in 4% paraformaldehyde ( pfa ) and processed for immunolabeling and gsa i staining ( see above ) . ns obtained from dispersed rat cbs were re - plated onto adherent conditions using 5 g / ml fibronectin ( biomedical technologies ) pre - treated 24-well plates , at a density of 4 or 5 ns / well . drug treatments are described in the supplemental information . after growing for 3 days in adherent conditions , flat ns colonies were fixed and stained with rodhamine or fluorescein - conjugated gsa i ( see immunocytochemistry section ) . total rna was extracted from gsa i positive and negative cell fractions from dispersed adherent ns or dispersed rat cbs , using a commercial kit ( rneasy micro kit ; qiagen ) , following manufacturer s instructions . for retrotranscription , cdna was synthesized with quantitec reverse transcription kit ( qiagen ) , as indicated by manufacturer s instructions . all sorts and analysis were performed in a moflo three - laser flow cytometer ( dako cytomation ) and a lsr fortessa ( bd biosciences ) , respectively . whole - cell protein extracts were obtained from huvec cell line , cultured during 3 days in hypoxic conditions ( 3% o2 ) in ec basal medium-2 ( ebm-2 , lonza ) for epo protein detection . analyses of significant differences between means were carried out using the unpaired or paired two - tailed student s t test . newman - keuls post hoc or tukey s multiple comparison tests were used whenever a significant difference between three or more sample means was revealed by a one - way anova . tukey or newman - keuls test was used depending upon whether or not additional power is required to detect significant differences between means . | summaryadult stem cell plasticity , or the ability of somatic stem cells to cross boundaries and differentiate into unrelated cell types , has been a matter of debate in the last decade .
neural - crest - derived stem cells ( ncscs ) display a remarkable plasticity during development .
whether adult populations of ncscs retain this plasticity is largely unknown .
herein , we describe that neural - crest - derived adult carotid body stem cells ( cbscs ) are able to undergo endothelial differentiation in addition to their reported role in neurogenesis , contributing to both neurogenic and angiogenic processes taking place in the organ during acclimatization to hypoxia . moreover , cbsc conversion into vascular cell types is hypoxia inducible factor ( hif ) dependent and sensitive to hypoxia - released vascular cytokines such as erythropoietin .
our data highlight a remarkable physiological plasticity in an adult population of tissue - specific stem cells and could have impact on the use of these cells for cell therapy . |
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the use of broad - spectrum antibiotics , parenteral nutrition , indwelling catheters , or the presence of immunosuppression , or disruption of mucosal barriers due to surgery , chemotherapy , and radiotherapy are among the most important predisposing factors for invasive fungal infection . candida bloodstream infection is the third most common cause of nosocomial bacteremia in patients requiring intensive care and the most common etiologic agent of fungal - related biofilm infection . c. albicans , a normal commensal of human mucosal surfaces and opportunistic pathogen in immunocompromised patients , is most frequently associated with biofilm formation . indwelling medical devices , such as intravascular catheters , can become colonized with candida spp . allowing the development of adherent biofilm structures from which cells can then detach and cause an acute fungemia and/or disseminated infection . it has recently been shown that the cells that detach from the biofilm have a greater association with mortality than equivalent planktonic yeasts . these implant - associated infections are inherently difficult to resolve and may require both long - term antifungal therapy and the physical removal of the implant to control the infection . other nonalbicans candida species associated with biofilm formation and catheter - related bloodstream or device - related infections include c. glabrata , c. parapsilosis , c. dubliniensis , c. krusei , and c. tropicalis [ 35 ] . yeasts and filamentous fungi biofilm - related infections have also been increasingly described , including pneumocystis , coccidioides , aspergillus , zygomycetes , blastoschizomyces , saccharomyces , malassezia , trichosporon , and cryptococcus . cryptococcus neoformans has been shown to colonize and subsequently form biofilms on ventricular shunts , peritoneal dialysis fistulas , prosthetic hip joints , and cardiac valves . different trichosporon species can cause disseminated life - threatening infections associated with biofilm - related infections [ 14 , 19 , 20 ] , including cardiac grafts , catheters , and breast implants . malassezia pachydermatis has been isolated from patients undergoing parenteral nutrition , blastoschizomyces capitatus has been associated with catheter - related fungemia , saccharomyces cerevisiae has been detected from dentures of stomatitis patients , and recurrent meningitis has been associated with a coccidioides immitis biofilm at the tip of a ventriculoperitoneal shunt tubing . there are also growing reports of the filamentous mould aspergillus fumigatus being involved in biofilm infections . for example , in the respiratory tract , it can cause an aspergilloma , which is a localised infection consisting of a spherical mass of hyphae . aspergillary bronchitis has also been reported , which is characterized by bronchial casts containing mucus and mycelia . bronchopulmonary lavage ( bal ) of patients with aspergillosis may also reveal the presence of numerous hyphae in the form of a complex multicellular mycetoma structure samples when examined histologically . in addition to this , it has been reported to cause serious biomaterial - related infections of joint replacements , catheters , heart valves , cardiac pace makers , and breast augmentation implants [ 2730 ] . the urinary tract , whilst less frequently associated with a. fumigatus , has been reported to support an aspergilloma [ 31 , 32 ] . it is also frequently associated with complex sinus infections , which in canines have been described as superficial mucosal fungal plaque [ 3336 ] . it is increasingly clear that a diverse panel of fungi have the capacity to form biofilms , and as such our knowledge of fungal biofilms has improved dramatically . through work primarily with c. albicans , we now have a clearer perspective on the molecular characteristics of fungal biofilm development [ 3 , 6 , 37 , 38 ] . clinically , these are important as they are refractory to antifungal treatment , which poses a major problem to clinicians as the dose required to eradicate the biofilm can exceed the highest therapeutically attainable concentrations of antibiotics . the focus of this paper is to provide an up - to - date understating of the key factors responsible for the failure of antifungal agents against fungal biofilms . microbiologists have historically studied planktonic ( free floating and homogeneous cells ) in pure culture . however , there has been a paradigm shift as the link between sessile ( surface attached and heterogeneous cells ) and microbial pathogenesis and human infection is now widely accepted . it is apparent that a wide range of bacteria and fungi are able to alternate between planktonic growth and sessile multicellular communities , commonly referred to as biofilms . estimates suggest that up to 80% of all microorganisms in the environment exist in biofilm communities . biofilms are defined as highly structured communities of microorganisms that are either surface associated or attached to one another and are enclosed within a self - produced protective extracellular matrix ( ecm ) . the advantages to an organism of forming a biofilm include protection from the environment , resistance of physical and chemical stress , metabolic cooperation , and a community - based regulation of gene expression . in recent years , there has been an increased appreciation of the role that fungal biofilms play in human disease as microbes growing within biofilms exhibit unique phenotypic characteristics compared to their planktonic counterpart cells , particularly increased resistance to antimicrobial agents . in addition to providing safe sanctuary for microorganisms , biofilms may also act as reservoirs for persistent sources of infection in a patient and as such adversely effect the health of an increasing number of individuals , including patients with hiv - infection , cancer , transplants , patients requiring surgery or intensive care , and newborn infants [ 3 , 42 ] . the adhesion and colonization of complex fungal populations onto biological and innate surfaces , such as the oral mucosa or denture acrylic substrates , is commonplace for clinically relevant fungi [ 4346 ] . a wide variety of environmental factors contribute to the initial surface attachment , including the flow of the surrounding medium ( urine , blood , saliva , and mucus ) , ph , temperature , osmolarity , bacteria , presence of antimicrobial agents , and host immune factors [ 4752 ] . fungal biofilms have defined phases of development that have been described through the use of defined model systems [ 51 , 5358 ] . these key phases include arriving at an appropriate substratum , adhesion , colonisation , extracellular matrix ( ecm ) production , biofilm maturation , and dispersal [ 37 , 55 , 59 ] . understanding this entire process has enabled us to begin to unravel how some of the mechanisms are involved in resistance . initial studies that began to investigate biofilm resistance were basic , investigating antifungal effects purely at the phenotypic level through descriptive analyses . the pioneering work by julia douglas 's group working on c. albicans biofilms utilised some of the earliest models , from which quantitative assessment using dry weight measurements , tetrazolium salt ( mtt ) reduction assays , and incorporation of [ h ] leucine were described [ 60 , 61 ] . these simple static models were expanded to include flow , which was shown to alter antifungal susceptibility . however , typically these models were cumbersome , requiring expert handling , longer processing times , and the use of specialized equipment not generally available . therefore , methods for rapid high - throughput testing were preferable , and around this time , the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2h - tetrazolium hydroxide ( xtt ) colorimetric method was described for investigating yeast adhesion and susceptibility [ 62 , 63 ] . this assay measures the collective metabolic activity of the cells within biofilm and is used as the basis for developing a standardized high - throughput susceptibility screen on candida biofilms , which is now widely used by the research community [ 55 , 58 ] . the xtt assay is noninvasive and nondestructive , requiring minimal postprocessing of samples compared to other methods , such as viable cell counts . using this technique , a caveat to its use is that it does not quantify biofilm - dependant characteristics , such as biomass or morphological status , and caution must be exercised when evaluating xtt data from different isolates as there is often dramatic variability between strains . therefore , it should only be used for direct comparison of a treated isolate to an untreated control rather than absolute quantification of biofilm formation per se . the next breakthrough in high - throughput biofilm testing has recently been described where nanoproduction of c. albicans biofilms is achievable , creating 768 equivalent and spatially distinct nanobiofilms on a single glass microarray . however , it remains to be determined whether there is an assay system sensitive enough to quantify the metabolic activity of each nanobiofilm . the recent interest in aspergillus and cryptococcus biofilms , as well as others like pneumocystis , has led to further modifications of these , but in essence the platforms and techniques remain largely similar . for example , martinez and casadevall ( 2006 ) developed a microtiter plate biofilm assay for c. neoformans to determine the susceptibility profiles of in vitro sessile structures . similarly , a 96-well - based biofilm model for a. fumigatus has been described and used to determine the susceptibility profiles and resistance mechanisms of conidia and adherent hyphal biofilms using an xtt - based reduction and alamar blue assays [ 6971 ] . the oxidation reduction indicator alamar blue has also been shown to be a reproducible and cheaper alternative to xtt in recent years , which merits further study [ 72 , 73 ] . as mentioned , the presence of flowing liquid over the biofilm can alter antifungal sensitivity . there is a growing range of flow systems utilized to model biofilm development [ 51 , 56 , 7476 ] . seed and feed modified robin 's device that permits multiple biofilms to be formed under constant flow conditions , cylindrical cellulose filters , constant depth film fermenters , perfusion fermenters , flow chambers , and a robbin 's device have all been described [ 51 , 52 , 54 , 55 , 77 , 78 ] . the lopez - ribot group recently described a simple flow model based on a gravity - fed flow method that enabled the group to demonstrate that biofilms were thicker and more resistant to polyenes and echinocandins by 4- and 2-fold , respectively . interestingly , perfusion of biofilms created under flow with these two antifungal agents showed time- and dose - dependant activity , which were potent against dispersed cells . these systems will prove useful for future investigations of invasive candidiasis where biofilms are common , particularly for catheter - related infections in the icu , where there is a growing interest in catheter - lock therapy [ 81 , 82 ] . in addition , there are now also a significant number of biofilm models available for in vivo investigations , and many of these have been utilised to elucidate biofilm resistance mechanisms , including an implanted chamber under the skin , catheter models [ 84 , 85 ] , vaginal model , and denture model . fungi have been reported to be up to 1000-fold more resistant to antifungal agents than planktonic free - floating cells , yet this recalcitrance to antimicrobial therapy has yet to be fully elucidated [ 14 , 58 , 88 ] . despite some antifungal agents being efficacious against fungal biofilms , particularly the echinocandins and liposomal amphotericin b formulations , the intrinsic resistance exhibited by these complex structures has promoted detailed investigation [ 70 , 8992 ] . . it can be inducible in response to a compound , or an irreversible genetic change resulting from prolonged exposure . specifically , these include alterations or overexpression of target molecules , active extrusion through efflux pumps , limited diffusion , tolerance , and cell density , which are all characterised mechanisms utilised by fungi to combat the effects of antifungal treatment . planktonic cells generally rely on irreversible genetic changes to maintain a resistant phenotype , whereas biofilms are able to persist due to their physical presence and the density of the population , which provides an almost inducible resistant phenotype irrespective of defined genetic alterations . the following section will now describe some of the pivotal factors that play a role in fungal biofilm resistance , which are summarised in figures 1 and 2 . the general physiological state of cells in sessile populations has also been implicated to influence the susceptibility profiles of biofilms . metabolic dyes assays ( e.g. , xtt - based assays ) confirm that cells within biofilms are undergoing mitochondrial respiration during development [ 55 , 58 , 62 , 65 , 70 ] . other factors including the effect of growth rate on c. albicans biofilm resistance have also been studied , where varying the rates was shown to play no role in resistance to amphotericin b . similarly , biofilms of c. albicans grown under glucose- and iron - limited conditions were shown to both be highly resistant to amphotericin b . furthermore , studies of biofilms grown under anaerobic conditions demonstrated that c. albicans biofilms were resistant to the high levels of amphotericin b and different azole antifungals . nevertheless , factors including ph , temperature , oxygen availability , and other environmental stresses will alter the biofilm architecture , and possibly antifungal sensitivity [ 96 , 97 ] . therefore , whilst the physiological state of the cell may have a minor role in resistance ( e.g. , dormancy ) , it is more likely that more complex factors are involved . the architecture of biofilms is highly ordered to enable the perfusion of nutrients and expulsion of waste products . mature biofilms , whilst densely populated , exhibit spatial heterogeneity with microcolonies and water channels being present , and features are common to both bacterial and fungal biofilms [ 55 , 98 , 99 ] . cell density is therefore an important resistance factor within complex biofilm populations of yeast and filamentous fungal biofilms , particularly towards azoles . it was demonstrated that both planktonic and resuspended biofilm cells exhibited azole sensitivity at low cell numbers ( 10 cells / ml ) , which became increasingly resistant as the density of the cells increased tenfold , a phenomenon also been demonstrated in a. fumigatus . both our group and others have shown phase - dependant increased antifungal resistance in a. fumigatus and c. albicans , respectively [ 70 , 102 ] , which support the idea that the physical density of the cells within the biofilm produces recalcitrance to antifungal agents . within dense biofilms , there is cooperation between individual cells through quorum sensing processes , which provides the ability of microorganisms to communicate and coordinate their behaviour via the secretion of signalling molecules in a population - dependent manner . in fungi , this was first described in c. albicans when hornby and colleagues identified farnesol trans , trans-3,7,11-trimethyl-2,6,10-dodecatrien-1-ol . exposing c. albicans to exogenous farnesol results in genome wide responses , including activation of genes involved in drug resistance ( cafcr1 and capdr16 ) [ 105 , 106 ] . it has now been shown that quorum sensing in c. albicans is likely driven by the two - component regulatory system of chk1p . however , when deleted the chk1 strain shows a similar azole resistance profile to that of wild type , indicating that the regulatory circuit controlling biofilm resistance may be yet to be discovered , or cell density is not a defined biofilm resistance factor . however , given that echinocandins are highly effective against biofilms suggests cell density has a limited effect against this compound . in addition , previous work has shown that disrupted biofilms that are resuspended and tested using the clsi methodology in comparison to planktonic cells retain a resistant phenotype [ 71 , 108 ] , indicating alternative mechanisms of resistance . the azoles are generally fungistatic against yeasts , including candida species , and fungicidal against moulds , such as aspergillus species . the fungistatic nature of the azoles towards c. albicans induces a strong directional selection on the surviving population to evolve drug resistance [ 109 , 110 ] . in fact , high levels of azole resistance in c. albicans clinical isolates often accumulate through multiple mechanisms including the alteration of erg11 . azoles actively target the 14-demethylase enzyme encoded by erg11 , blocking ergosterol biosynthesis and leads to depletion of the ergosterol content of membranes and results in the accumulation of toxic sterol pathway intermediates , such as 14 -methylergosta-8,24(28)-dien-3b,6a - diol , which inhibits growth [ 111 , 112 ] . the principle drug target , erg11p , can develop point mutations or be overexpressed [ 111113 ] . common mutations in the erg11p that confer moderate azole resistance are s405f , y132h , r467k , and g464s [ 114116 ] . given the importance of ergosterol as a target of azoles and the high level resistance exhibited by these structures , then the sterol composition of c. albicans biofilms has been investigated . sterol analyses showed that ergosterol levels were significantly decreased in intermediate ( 12 h ) and mature phases ( 48 h ) , compared to those in early - phase biofilms ( 6 h ) . in contrast , in one of the first c. albicans biofilm studies to use microarray analysis , overexpression of caerg25 and caerg11 was reported . alteration of ergosterols in biofilm membranes may explain their resistance to both azole and polyene - derived antifungal agents . for example , c. albicans biofilms cultured in a flow cell for 36 h were compared to planktonic cells , where it was shown that a subpopulation of blastospores from the biofilm were 10 times more resistant to amphotericin b than planktonic populations . transcriptional analysis of this biofilm subpopulation for genes from the beta-1,6-glucan pathways indicated a possible association between the high level of resistance and upregulation of caskn1 , and cakre1 in the biofilm blastospore population compared to exponential and stationary phase planktonic c. albicans cells . therefore , changes in both the cell membrane and the cell wall may be important determinants of resistance in the biofilm . subsequent work in c. albicans has shown that transcriptional responses in young and mature biofilms after exposure to high doses of fluconazole or amphotericin b demonstrated differential antifungal drug responses . exposure of both young and mature biofilms to fluconazole induced upregulation of genes encoding enzymes involved in ergosterol biosynthesis ( caerg1 , caerg3 , caerg11 , and caerg25 ) , particularly biofilms exposed for longer periods ( 22 h ) , whereas treatment of both young and mature biofilms with amphotericin b resulted in an overexpression of predominantly caskn1 , with a modest upregulation of cakre1 . removal of the antifungal in this study depleted further transcriptional changes , except for caskn1 , which was impacted by prior fluconazole exposure . it was speculated that this is related to biofilm regrowth . increased ergosterol genes have also been reported in vivo in a c. albicans central venous catheter biofilm model , demonstrating the importance of the molecule within the biofilm . induction of ergosterol genes has also been described in c. dubliniensis , where incubation with fluconazole and formation of biofilm was coupled with upregulation of the cderg3 and cderg25 . moreover , upregulation of genes involved with ergosterol biosynthesis has been described in c. parapsilosis biofilms , which are also resistant to azole antifungal therapy . overall , these data highlight the importance of ergosterol in biofilm resistance , particularly with respect to azoles , which indirectly inhibit their biosynthesis . recent studies have shown that simvastatin , which impairs cholesterol metabolism in humans , is capable of inhibiting c. albicans biofilms , thus providing a potential novel strategy of combating these tenacious infections . the primary molecular mechanism leading to high - level azole resistance in c. albicans , that is , increased efflux of drug mediated mostly by the atp - binding cassette ( abc ) and the major facilitator superfamily ( mfs ) transporters [ 123125 ] . the abc transporters in c. albicans constitute a multigene family , which includes several cdr genes ( cdr1 - 4 ) [ 126 , 127 ] . the abc transporters include a membrane pore composed of transmembrane segments and two abcs on the cytosolic side of the membrane , which provide the energy source for the pump [ 128 , 129 ] . importantly , multiple antifungal agents can be substrates for these transporters , and thus , their overexpression can lead to cross - resistance among different drugs , particularly azoles . among members of the mfs , which are secondary transporters and use proton - motive force across the plasma membrane , the mdr1 gene encodes a major facilitator that has been implicated in c. albicans azole resistance , and its overexpression leads to exclusive fluconazole resistance [ 46 , 113 ] . genes encoding for drug efflux pumps have been reported in biofilms to be differentially regulated during development and upon exposure to antimicrobial agents include cacdr1 , cacdr2 , and camdr1 [ 102 , 108 , 131 , 132 ] . in the first study to investigate the role of efflux pumps , it was demonstrated that expression of genes encoding both types of efflux pump were upregulated during the course of biofilm formation and development . both cacdr1 and cacdr2 were upregulated in 24 and 48 h biofilms , whereas camdr1 was transiently upregulated at 24 h . however , their contribution to resistance in the biofilm phenotype was placed in doubt when a set of c. albicans isogenic strains deficient in efflux pumps carrying single- and double - deletion mutations ( cdr1 , cdr2 , mdr1 , cdr1/cdr2 , and mdr/cdr1 ) that rendered the cells hypersusceptible to fluconazole when planktonic retained the resistant phenotype during biofilm growth . in a subsequent investigation , c. albicans biofilms were formed through three distinct developmental phases that were associated with high fluconazole resistance . again , the same set of isogenic c. albicans strains were utilised where it was shown that 6 h old biofilms formed by double and triple mutants were > 4- to 16-fold more susceptible to fluconazole than the wild - type strain . at 12 and 48 h , all the strains became highly resistant to this azole , indicating lack of involvement of efflux pumps in resistance at late stages of biofilm formation . in cell density studies of the efflux pump isogenic strains , these remain hypersensitive at low cell concentrations yet resistant at high cell concentrations and in biofilm , indicating a contributory resistance role of cell density . nevertheless , similar to the study by ramage and coworkers , c. albicans biofilms were shown to express cacdr and camdr1 genes in all three phases ( 6 , 12 , and 48 h ) , whilst planktonic cells expressed these genes transiently . in fact , gfp promoter studies have shown induction of efflux pumps after 15 min adherence to provide a tolerant biofilm phenotype . animal studies have also shown that biofilms formed on implanted catheters express efflux pumps [ 84 , 118 ] . transcript upregulation of cacdr2 at 12 h ( 1.5-fold ) and camdr1 at both 12 h ( 2.1-fold ) and 24 h ( 1.9-fold ) was demonstrated . in c. glabrata , similar results are reported , where expression of cgcdr1 and cgcdr2 was investigated during the early ( 6 h ) , intermediate ( 15 h ) , and mature ( 48 h ) phases of biofilm development . at 6 h and 15 h , the biofilms exhibited approximately 1.5- and 3.3-fold upregulation of cgcdr1 and 0.5- and 3.1-fold upregulation of cgcdr2 , respectively , in comparison to planktonic cells . collectively , these studies suggest that multifaceted , phase - specific mechanisms are functional in resistance of fungal biofilms . mutations within the cyp51a gene , which alters the ergosterol biosynthesis pathway , have been reported to cause azole resistance in a. fumigatus [ 135138 ] . however , a recent study reported that 43% of azole - resistant isolates did not carry the cyp51a mutation , indicating that other mechanisms of resistance were responsible . it was hypothesised that efflux - mediated mechanisms of resistance may explain this clinical resistance , which may also be important in biofilms . a. fumigatus mdr pumps have previously been shown to be associated with increased resistance to itraconazole [ 141 , 142 ] . currently , however , there is little evidence to suggest that these play an active role in clinical resistance . phase - specific analysis of resistance in a. fumigatus biofilms revealed increased resistance to azoles , polyenes , and echinocandins as each biofilm matured from 8 to 12 to 24 h [ 70 , 71 ] . biochemical analysis of efflux pump activity showed a significant increase in efflux pump activity in the 12 h and 24 h phases , and upregulation in 8 h germlings when treated with voriconazole . moreover , inhibition of efflux pump activity with the competitive substrate ( mc-207 , 110 ) reduced the susceptibility to voriconazole by 5-fold . quantitative expression analysis of afumdr4 mrna transcripts revealed a biphasic increase as the mycelial complexity increased ( maximal at 12 h ) , which was coincidental with strain - dependant increase in azole resistance . global transcriptional analyses of voriconazole - treated a. fumigatus mycelia of over 2000 genes were shown to be differentially expressed , and amongst these was a cluster of 15 different transporters mrna at significantly increased levels , including mdr proteins of the abc and mfs classes , such as afmdr1 and afmdr2 . also , unpublished microarray studies of the different phases of a. fumigatus biofilms from our group revealed a cluster of pumps and transporters , which is similar to studies of c. albicans . collectively , this data in addition to the available literature support the hypothesis that efflux pumps are an important , but not exclusive , determinant of fungal biofilm resistance to azoles [ 143 , 145 ] . their primary role may be for homeostasis within complex environments to protect themselves from acute toxicity , but within clinical environments exposure to azoles dugs may enhance the levels of efflux pump expression , therefore either contributing towards or inducing clinical resistance . however , it is likely that they play a greater protective ( resistance ) role in the early phases of biofilm growth until the production of ecm , one of the primary mechanisms of biofilm resistance . ecm is a defining characteristic of fungal biofilms , providing the cells protection from hostile factors such as host immunity and antifungal agents . in some of the pioneering works by the douglas group , c. albicans ecm was shown to increase when biofilms are grown under dynamic flow conditions [ 47 , 48 , 53 ] . however , subsequent work has shown that while diffusion is hampered by ecm , penetration of antifungal drugs is not thought to play a key role in biofilm resistance . recent studies have provided new insights that suggest the chemical composition of ecm and its regulation may play a central role in resistance . the composition of the ecm of these biofilms in c. albicans and c. tropicalis is complex , comprising of protein , hexosamine , phosphorus , uronic acid , and carbohydrates . recently , it has been shown that extracellular dna is another important component of the ecm in c. albicans , as the addition of dnase improves the efficacy of polyenes and echinocandins , but not to azoles . one of the principle carbohydrate components is beta-1,3 glucans , as treatment of c. albicans biofilms with beta-1,3 glucanase helps detach biofilms from a substrate . its contribution is confirmed in a series of investigations by the andes group where it was shown to increase in c. albicans biofilm cell walls compared to planktonic organisms and was also detected in the surrounding biofilm milieu and as part of the ecm . beta-1,3 glucans have also been shown to increase in investigations of three specific phases of biofilm development grown on both denture acrylic and catheter substrates . its contribution to resistance was realized when it was also shown that biofilm cells walls bound 4- to 5-fold more azole than equivalent planktonic cells , and culture supernatant bound a quantifiable amount of this antifungal agent . moreover , beta-1,3 glucanase markedly improved the activity of both fluconazole and amphotericin b. addition of exogenous biofilm ecm and commercial beta-1,3 glucan also reduced the activity of fluconazole against planktonic c. albicans in vitro . the group has recently shown that the ecm -1,3 glucan is synthesised from fks1p using a defined knockout and overexpressing strain . this study demonstrated that beta-1,3 , glucan is responsible for sequestering azoles , acting as a drug sponge and conferring resistance on c. albicans biofilms . further studies have shown that they are also responsible for sequestering echinocandins , pyrimidines , and polyenes . this has been confirmed independently where amb was shown to physically bind c. albicans biofilms and beta - glucans . subsequent studies have identified a role for the casmi1 , a gene involved in cell wall glucans , in biofilm ecm production and development of a drug - resistant phenotype , which appears to act through transcription factor carlmp and glucan synthase fks1p . in addition to cafks1 , a zinc - response transcription factor cazap1 has been shown to be a negative regulator of ecm soluble beta-1,3 glucan in both in vitro and in vivo c. albicans biofilm models through expression profiling and full genome chromatin immunoprecipitation . conversely , two glucoamylases , cagca1 and cagca2 , are thought to have positive roles in matrix production . a group of alcohol dehydrogenases caadh5 , cacsh1 , and calfd6 also have roles in matrix production , with caadh5 acting positively , and cacsh1 and calfd6 acting negatively . it is thought that these alcohol dehydrogenases generate quorum - sensing aryl and acyl alcohols , which coordinate biofilm maturation . collectively , it appears that c. albicans ecm production is highly regulated and is a key resistance factor . , including c. glabrata , c. parapsilosis , c. tropicalis , and c. dubliniensis [ 157 , 158 ] . significantly less is known regarding the role of a. fumigatus biofilm ecm in antifungal resistance . in an aerial static model , the presence of extracellular hydrophobic ecm is composed of galactomannan , alpha-1,3 glucans , monosaccharides , polyols , melanin , and proteins , including major antigens and hydrophobins . this study demonstrated that hydrophobic matrix cohesively bound hyphae together , and that the matrix increased with maturity of the developing structure . further studies report that a new galactosaminogalactan and the galactomannan were the major polysaccharides of the in vivo a. fumigatus ecm . for a. niger , after germination upon a support , the new hyphae also produce an ecm . the production of ecm has also been reported elsewhere , where it has been shown to be produced on both polystyrene and cystic fibrosis ( cf ) bronchial epithelial cells by a. fumigatus , that were resistant to antifungal agents . martinez and casadevall ( 2006 ) reported that c. neoformans also have the ability to form biofilm structures in vitro and produce ecm . in pneumocystis spp . intense monoclonal antibody staining to the major surface glycoproteins and an increase in ( 1,3)-beta - d - glucan content were also evident , suggesting that these components contributed to resistance . blastoschizomyces capitatus , malassezia pachydermatis , saccharomyces cerevisiae , rhizopus oryzae , lichtheimia corymbifera , rhizomucor pusillus , and apophysomyces elegans are all reported to produce ecm in their biofilms [ 10 , 11 , 13 , 163 ] . therefore , ecm clearly plays a critical role in fungal biofilm resistance , particularly for c. albicans , from which we currently understand the greatest . it is one of the most significant and regulated resistance mechanisms utilized in the biofilm phenotype . persister cells are an important mechanism of resistance in chronic infections , and a mechanism of resistance that has gathered some attention recently in fungal biofilms [ 165167 ] . persister cells are dormant variants of regular cells that form stochastically in microbial populations and are highly tolerant to antibiotics . in c. albicans biofilms , a small subset of yeast cells have been described that are highly resistant to amphotericin b following adhesion , which is independent of upregulation of efflux pumps and cell membrane composition [ 76 , 166 ] . the first study in fungi was to describe persister cells in fungi , described as a subpopulation of highly tolerant cells . in this study , c. albicans persisters were detected only in biofilms and not in different planktonic populations . reinoculation of cells that survived killing of the biofilm by amphotericin b produced a new biofilm with a new subpopulation of persisters , suggesting that these were not mutants but phenotypic variants of the wild type , and that attachment to a substratum initiated dormancy . the presence of persisters in c. albicans , c. krusei , and c. parapsilosis biofilms treated with amphotericin b was also described . it was further hypothesized that the periodic application of antimicrobial agents may select for strains with increased levels of persister cells , so 150 isolates of c. albicans and c. glabrata were obtained from cancer patients who were at high risk for the development of oral candidiasis and who had been treated with topical chlorhexidine once a day . it was shown that the persister levels of the isolates varied from 0.2 to 9% , and strains isolated from patients with long - term carriage had high levels of persisters , whereas those from transient carriage did not . therefore , in this clinically relevant scenario , prolonged and ineffectual antifungal treatment may be beneficial to the biofilm population , which may be responsible for antimicrobial drug failure and relapsing infections . the role of reactive oxygen species ( ros ) in sessile c. albicans cells was investigated as they are known to be induced by high concentrations of miconazole , allowing 1 - 2% of miconazole - tolerant cells to persist . superoxide dismutases ( sods ) were found to be differentially expressed by miconazole - treated sessile c. albicans cells compared to untreated cells . inhibition of superoxide dismutase resulted in an 18-fold reduction of the miconazole - tolerant persister cells and increased endogenous ros levels in these cells . in biofilms from strains lacking sod4/sod5 , at least 3-fold less miconazole - tolerant persisters were observed , and ros levels were increased compared to the isogenic wild type . therefore , miconazole - tolerant persisters are linked to the ros - detoxifying activity of sods . whether this is the definitive molecular basis for c. albicans persister cells or a tolerance mechanism still remains to be determined , but these subpopulations are clearly another important fungal biofilm resistance mechanism . pathogenic fungi encounter a range of physiological stresses from different environments , including temperature changes , ionic stress , changes in osmolarity , and oxidative stress , such as that experienced in the phagosomes of neutrophils . these stresses are sensed through various receptors , which elicit responses through conserved signaling pathways . one of the most important is the mitogen - activated protein kinase ( mapk ) signal transduction network , and the many others are subject to review . it was first shown that the mitogen - activated protein kinase ( mapk ) mck1p , which is activated by contact stress , is involved in biofilm development . moreover , the null mutant ( mck1 ) biofilms were azole sensitive , in contrast to the sessile wild type and both planktonic strains . calcineurin is a ca - calmodulin - activated serine / threonine - specific protein phosphatase that plays many critical stress roles in the fungal cell , including amongst other things antifungal drug responses . in planktonic cells , calcineurin is critical for c. albicans survival during azole treatment . inhibiting calcineurin pharmacologically or impairing calcineurin function genetically has synergistic activity with fluconazole and renders the azoles fungicidal against c. albicans . calcineurin has also been implicated in mediating resistance to the azoles in both in vitro and in vivo models of biofilm formation . c. albicans cells in biofilms are up to 1,000-fold more resistant to fluconazole than planktonic cells , indicating that inhibitors could be used in combinations as novel therapeutic interventions to treat or prevent biofilms , whereas c. dubliniensis calcineurin inhibitors were unable to form biofilms . similar studies have evaluated the efficacy of a voriconazole - micafungin combination against c. albicans biofilms . voriconazole significantly antagonized the fungicidal effect of micafungin against biofilms . to investigate the mechanism of antagonism , an inhibitor of calcineurin this study also suggested that heat shock protein 90 ( hsp90 ) molecular chaperone played a role in this antagonism . hsp90 regulates complex cellular circuitry in eukaryotes and potentiates the emergence and maintenance of resistance to azoles and echinocandins in c. albicans , at least in part via calcineurin . it physically interacts with the catalytic subunit of calcineurin , keeping it stable and poised for activation . a recent study led by the cowen group demonstrated that genetic depletion of hsp90 reduced c. albicans biofilm growth and maturation in vitro and interestingly impaired dispersal of biofilm cells . it also abrogated resistance of c. albicans biofilms to the azoles , which was also shown in vivo . furthermore , depletion of hsp90 led to reduction of calcineurin and mkc1 in planktonic but not biofilm conditions , suggesting that hsp90 regulates drug resistance through different mechanisms . a marked decrease in matrix glucan levels was observed , providing a mechanism through which hsp90 might regulate biofilm azole resistance . in a. fumigatus , moreover , a recent investigation of the c. glabrata biofilm proteome demonstrated upregulation of a heat shock protein ( hsp12p ) and other stress proteins ( trx1p , pep4p ) . therefore , fungal biofilm resistance is multifaceted , involving some basic physical barriers and some complex regulatory processes . the evidence that has been collected over the past decade would suggest that as the biofilm changes from an adherent phenotype into a complex biofilm , then different mechanisms of resistance are utilised , that is , phase - specific mechanisms . clearly , efflux pumps are utilised during the early to intermediate phases of biofilm development but are relinquished towards maturity as ecm is produced to the density of the mature biofilm may act as a physical barrier , and reduction of growth rates under different environmental pressures can benefit the biofilm . moreover , during hyphal growth , ergosterol biosynthesis is regulated by antifungal treatment , the direct and indirect targets of polyenes and azoles , respectively . within the dense biofilms , where penetration of antifungal drugs is possible , persister cells phenotypes ensure survival , and in these stressed environments , global stress proteins kick into action to protect and maintain . overall , the evidence highlights that fungal biofilm resistance is an inducible phenotype that is a part of a highly evolved series of molecular pathways that regulate biofilm development and homeostasis . | fungal biofilm infections have become increasingly recognised as a significant clinical problem .
one of the major reasons behind this is the impact that these have upon treatment , as antifungal therapy often fails and surgical intervention is required .
this places a large financial burden on health care providers .
this paper aims to illustrate the importance of fungal biofilms , particularly candida albicans , and discusses some of the key fungal biofilm resistance mechanisms that include , extracellular matrix ( ecm ) , efflux pump activity , persisters , cell density , overexpression of drug targets , stress responses , and the general physiology of the cell .
the paper demonstrates the multifaceted nature of fungal biofilm resistance , which encompasses some of the newest data and ideas in the field . |
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on - site wastewater treatment systems ( owts ) are widely utilized to treat and dispose of household wastewater in areas where centralised reticulated sewerage systems are unavailable . septic tanks , aerobic treatment units ( atus ) , and composting toilets with greywater treatment are among the more commonly used owts in australia and elsewhere . in the 1990s it was estimated that approximately 20% of australian households rely on septic systems for domestic wastewater management . by 2001 , it was estimated that more than 1 million owts were installed in australia , with the greatest distribution in new south wales ( 300,000 ) , followed by victoria ( 250,000 ) and queensland ( 250,000 ) . in western australia ( wa ) itself figure 1 represents the numbers of owts installations while the percentages on the figure indicate the proportion of unsewered properties in each state across australia . these figures are likely to increase along with australian population growth , the continuous development of rural and unsewered urban regions , and the acceptance that owts are a cost effective and long - term option for meeting public health and environmental health goals if properly installed , operated , and maintained . current average in australia is 2.6 people per household . assuming each person produces approximately 200 l of wastewater per day a total of approximately 31 tons of nitrogen , 8.4 tons of phosphorus , and 70 10 faecal coliform organisms will be discharged to the owts . nutrients and pathogens are released into the environment when owts fail and the public health and environmental consequences could be significant considering that owts failure in australia has been reported high , reaching up to 40% . undoubtedly , poorly performing or failing owts have economic impacts on the owners and pose environmental impacts due to nutrients that may disrupt ecosystem balance . furthermore , poorly performed or failed owts can contribute to eutrophication of sensitive water bodies and create algae blooms . failing owts are one of the main causes of streams , lakes , rivers , wetlands , and groundwater contamination due to the release of nutrients and pathogens into the environment . various studies worldwide and in australia have shown that poorly performing or failing owts have resulted in degradation of soil and water quality [ 3 , 6 , 7 ] . furthermore , effluent from underperformed or failed owts may pose a public health risk , particularly to susceptible population group such as children , the elderly , or people with compromised immune system . contamination of drinking water or recreational waters by failing owts may increase concentration of pathogens that may impact on human health . untreated effluent lying in gutters or pooling in public places provides an environment for the transmission of diseases , such as those listed below : gastroenteritis from bacteria such as salmonella or viruses such as rotavirus or protozoa ( namely giardia),bacterial dysentery , typhoid fever from salmonella , polio from enterovirus , infectious hepatitis ( hepatitis a ) causing liver disease , skin infections from viruses , bacteria , and fungi . gastroenteritis from bacteria such as salmonella or viruses such as rotavirus or protozoa ( namely giardia ) , typhoid fever from salmonella , polio from enterovirus , infectious hepatitis ( hepatitis a ) causing liver disease , skin infections from viruses , bacteria , and fungi . pooling off effluent creates a nuisance by emitting offensive odours thus affecting the amenity of an area . spray irrigation of untreated greywater or septic tank effluent can result in aerosol transmission of diseases causing organisms . irrigation of effluent or greywater water onto food crops can cause diseases such as gastroenteritis or serious life - threatening diseases . contact with lawn areas recently irrigated with untreated wastewater may also result in skin infections or gastroenteritis via direct or indirect ingestion of organisms . swimming in water contaminated with effluent may result in skin , ear , and eye infections or gastroenteritis . similarly , consuming shellfish from effluent contaminated water may result in diseases such as hepatitis a . however the main chemical hazard of poorly performing owts is nitrate . elevated nitrogen levels in drinking water supplies impacted by wastewater contamination may result in irregular haemoglobin levels or methemoglobinemia in children . poor performance or failures of owts are often associated with systems not situated and constructed correctly . in particular , areas with low permeability soils and/or high water tables present the greatest challenge in wa . failure of many onsite systems is generally not due to inherent flaws in system technology but rather due to inappropriate sitting and construction design or their operation and management . other contributor factors to poor performance or failure of owts are poor public awareness , poor local authority guidance , continuous wastewater management issues , poor maintenance procedures , and inadequate governance . the following causes have been identified as the major reasons for owts failure : lack of area on the properties for the treatment and disposal of wastewater , undersized or inadequate wastewater treatment units or absorption trenches , poor maintenance , such as failure to desludge septic tanks , a change in occupancy from occasional to permanent , inappropriate location of absorption trenches , that is , in areas that are poorly drained , subject to flooding or high seasonal water table , poor knowledge of operation and maintenance procedures by homeowners / occupiers . lack of area on the properties for the treatment and disposal of wastewater , undersized or inadequate wastewater treatment units or absorption trenches , poor maintenance , such as failure to desludge septic tanks , a change in occupancy from occasional to permanent , inappropriate location of absorption trenches , that is , in areas that are poorly drained , subject to flooding or high seasonal water table , poor knowledge of operation and maintenance procedures by homeowners / occupiers . there is an increasing concern with the cumulative environmental impacts and local public health risk of failing or inadequately designed owts in unsewered areas . therefore , at the planning stage , it is important to ensure that inappropriate development does not proceed with an expectation that reticulated sewerage will come at a later date to solve problems being incurred . in areas where reticulated sewerage is not available , on - site disposal of wastewater is required . an application to construct or install an apparatus for the treatment of sewage in accordance with the health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 is required to be lodged to the local government ( lg ) in the first instance . if it is intended that the proposed owts will handle less than 540 l / day of wastewater and the building to be serviced is a single dwelling , the lg will process the application . if the wastewater volume generated is greater than 540 l / day and the building being serviced is not a single dwelling , the lg will assess the application , prepare a local government report , and forward the application to the dohwa for approval . the dohwa is also responsible for the approval of apparatus for sewage treatment within the state and it provides technical information to assist lgs and the community . these legislations and guidelines for owts in unsewered areas in wa are listed and briefly described below . sewage in the act includes sewage from faecal waste , urine from toilets , and water from shower , bath , laundry , and kitchen . it necessitates the collection , treatment , and disposal of sewage and greywater to ensure public health safety . health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 . applications for the installation of owts are only approved in the cases where sewer connections are not available . this code of practice supports the long - term reuse of greywater in both sewered and unsewered areas in wa . dohwa assesses and approves applications when multiple dwellings or commercial premises intend to use greywater for in - house end - uses , premises to reuse greywater via spray irrigation , or premises to reuse greywater via subsoil irrigation that produce more than 5,000 l of greywater daily . code of practice for the design , manufacture , installation , and operation of aerobic treatment units ( atus ) . this code of practice is published under the provisions of the health act 1911 ( wa ) and to be used simultaneously with the health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 . it provides guidance on the design , construction , installation , and operation of atu to serve single dwellings . the code of practice specifies the conditions necessary for on - site wastewater systems ( ows ) manufacturers who wish to sell their systems to the public or to use the systems for treating and disposing sewage . applicants are required to demonstrate that the ows comply with relevant australian standards and other regulations through documentations submitted to the dohwa . the dohwa is currently adopting standards australia and standards new zealand ( as / nzs ) 1547:2000 on - site domestic wastewater management ( as / nzs 1547.2012 ) . this standard is performance based and it guides system design based on site and soil assessment , with the aim of achieving sustainable performance . the dohwa is currently preparing the code of practice for on - site wastewater management with endorsement of as / nzs 1547 . it is expected that this code of practice will supersede the current codes and regulations . the policies encourage all new developments and subdivisions in wa to connect to reticulated sewerage as the most efficient , socially equitable , and safe method of wastewater disposal . however , in situations where connection to reticulated sewerage is not possible or practicable , the policies set out the on - site wastewater disposal requirements that proposals for developments and subdivisions must meet , in order to obtain the dohwa approval [ 17 , 18 ] . sewage in the act includes sewage from faecal waste , urine from toilets , and water from shower , bath , laundry , and kitchen . it necessitates the collection , treatment , and disposal of sewage and greywater to ensure public health safety . health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 . applications for the installation of owts are only approved in the cases where sewer connections are not available . this code of practice supports the long - term reuse of greywater in both sewered and unsewered areas in wa . dohwa assesses and approves applications when multiple dwellings or commercial premises intend to use greywater for in - house end - uses , premises to reuse greywater via spray irrigation , or premises to reuse greywater via subsoil irrigation that produce more than 5,000 code of practice for the design , manufacture , installation , and operation of aerobic treatment units ( atus ) . this code of practice is published under the provisions of the health act 1911 ( wa ) and to be used simultaneously with the health ( treatment of sewage and disposal of effluent and liquid waste ) regulations 1974 . it provides guidance on the design , construction , installation , and operation of atu to serve single dwellings . the code of practice specifies the conditions necessary for on - site wastewater systems ( ows ) manufacturers who wish to sell their systems to the public or to use the systems for treating and disposing sewage . applicants are required to demonstrate that the ows comply with relevant australian standards and other regulations through documentations submitted to the dohwa . the dohwa is currently adopting standards australia and standards new zealand ( as / nzs ) 1547:2000 on - site domestic wastewater management ( as / nzs 1547.2012 ) . this standard is performance based and it guides system design based on site and soil assessment , with the aim of achieving sustainable performance . the dohwa is currently preparing the code of practice for on - site wastewater management with endorsement of as / nzs 1547 . it is expected that this code of practice will supersede the current codes and regulations . the policies encourage all new developments and subdivisions in wa to connect to reticulated sewerage as the most efficient , socially equitable , and safe method of wastewater disposal . however , in situations where connection to reticulated sewerage is not possible or practicable , the policies set out the on - site wastewater disposal requirements that proposals for developments and subdivisions must meet , in order to obtain the dohwa approval [ 17 , 18 ] . this paper aims to review the installations and failures of owts in wa from 1997 to 2011 . recommendations to the dohwa and local government in regard to the administration of owts are also presented . a literature review related to owts legislation , standards , and approval process was undertaken . following on from this , data on the numbers of owts installed in wa was collected from two sources : centralised dohwa data management system and data provided by lg from their own data systems . as explained above , the lg is responsible for the approval of owts producing less than 540 l / day of wastewater while nondomestic and commercial owts producing more than 540 l / day are referred to the dohwa for approval . inspections of installed owts are the responsibility of the lg environmental health officer ( eho ) . the eho is required to inspect the owts and ensure that installation complies with the approval and conditions issued either by lg or by dohwa . the dohwa database collected the following information : applicant 's details , date of application , dwelling address and purpose , type of apparatus installed , and date of inspection . the database was created in 1997 and in order to match the information with the dohwa database , data from 1997 onwards was requested to lg . data related to the numbers of owts approved by lg was obtained via emails that were distributed in august 2012 to 140 lgs across wa . there were 140 lgs across wa at the time the information was requested . attached with the email was a microsoft excel 2007 spreadsheet containing two tables that lg environmental health officer ( eho ) filled out in order to record the number of installations approved . tables 1 and 2 contain overall numbers of owts by year for periods 19972011 approved by lg and dohwa , respectively . all data was checked and analysed in microsoft excel 2007 . at the end of the analysed period in 2011 , the dohwa conducted a survey to gather data from various lgs in wa on the primary causes of owts failures . the survey in the form of microsoft excel 2007 spreadsheet was sent out to 140 lgs across wa . the information collected in the survey included the type of owts and the reasons for system failures . follow - up interviews via phone calls were conducted to correct any errors in the data . overall , 28 lgs ( out of 140 ) provided data on the numbers of owts installations ( table 3 ) . nine of the lgs were located within perth urban areas while 19 others were in the outer suburban areas . based on the data obtained , more than 12,500 applications were approved between the periods of 1997 to 2011 . the highest number of applications was recorded in 2008 with over 1,000 applications , while the lowest was observed in 1997 with less than 500 applications . based on the data collected from the dohwa database from 1997 to 2011 , the dohwa approved over 3,700 owts applications , with an average of 247 applications annually . the highest number of applications was noted in 1999 , with over 350 applications whereas the lowest was observed in 1997 , with 128 applications . the number of owts approved by the dohwa from january 1997 to december 2011 is shown in figure 2 . east pilbara and derby were the two regions with the highest numbers of owts approvals , with 304 and 293 approvals , respectively , whereas most of perth urban areas ( e.g. , 73 applications in stirling ) recorded relatively low number of installations ( figure 2 ) . this is expected because communities with higher population density in metropolitan and urban areas are more likely to be connected to reticulated sewerage . the proportions of owts installed in wa regional areas are larger than in urban areas . however , the risks of contamination due to failed systems are higher in urban / metro areas due to the increased density of owts . moreover , increasing population density increases the likelihood of release of contaminants such as nitrogen to the environment in owts failure . east pilbara , for example , had the highest number of owts approvals between the periods of 1997 and 2011 , with a total of 304 installations , whereas city of stirling has a total of 73 approvals . however , according to the australian bureau of statistics ( abs ) the population density of these two lgs is quite different . east pilbara with an area of over 379,000 km had a total population of 11,950 in 2011 , giving it a population density of 0.031 people per km . on the other hand , city of stirling had a total population of over 200,000 with a relatively smaller area of 105.2 km , giving it a population density of 1,958 people per km . data collected from 1997 to 2011 have revealed that conventional septic tanks account for 76.5% and 63.7% of all wastewater systems approved by lg and dohwa , respectively . due to its simplicity and affordable cost , this is despite the fact that atu is becoming more prevalent . as evident from figure 3 , atu has become more popular in wa over the last decade due to its ability to produce high quality effluent , as well as its capacity to contain and reuse treated effluent on lawns or garden beds via surface or subsurface irrigation . figures 4 and 5 indicate that lg and dohwa have approved more than 1,800 ( 15% ) and over 800 ( 23% ) of atu installations , respectively , during the study period of 1997 to 2011 . the proportions of owts types approved by lg and dohwa are shown in figures 4 and 5 . more than 80% of the other systems were simply described as other without any description or explanation , whereas the remaining 10% corresponds to 2% holding tanks , 4% greywater system , and 4% nutrient removal system . on the other hand , composting was the type described as other in the owts approved by dohwa during the study period of 1997 to 2011 . as mentioned in section 4 , the dohwa conducted a survey in 2011 to collect information from various lgs in wa on the major causes of owts failures . out of the 21 lgs that responded back to the survey , 6 were located within perth metro areas while the remaining 15 lgs were in the outer suburban areas . based on the data collected , there were a total of 53 owts failures , with shire of dundas and shire of derby / west kimberley reporting the highest number of failures at 11 and 6 owts failures , respectively . the most common reasons for owts failures in the survey include groundwater and surface water ingress , systems not installed properly , unsuitable soil type , and undersized systems . several lgs also mentioned other factors that contributed to owts failures , including increase in wastewater volume , root invasion , illegal installation , unauthorised tampering , cross connection to stormwater disposal , undersized systems , and unauthorised materials ( such as fats , oils , and yeasts ) . septic systems remain a preferred option for onsite wastewater management in unsewered urban and rural regions of wa . however , the use of other types of owts such as atu and composting toilets with greywater treatment has increased over the years . given that wa climate is characterised by mild winters and very hot dry summers , there is an increased tendency to improve wastewater quality to achieve fit for purpose nonpotable quality to alleviate the pressure on scarce water resources . as significant amount of effluent is generated , owts have to be installed and monitored properly in order to prevent biological and nutrient contaminants from entering surface water and groundwater . throughout wa , over 12,500 owts applications were approved by 28 lgs during the period of 1997 to 2011 , representing 20% of the total number of existing lgs . while 20% of lgs managed to provide statistics of their owts approvals , the remaining could not produce any information mainly due to the absence of and difficulties retrieving data . additionally , more than 3,700 applications were approved through the dohwa within the same time period , with east pilbara and derby being the two regions with the highest number of approvals . as expected , the number of applications was generally higher in regional areas compared to urban areas as most urban areas are connected to centralized sewerage systems . currently , national and state guidelines are available to ensure adequate construction , installation , and management of owts in order to protect public health . however , the survey conducted in 2011 indicated that a number of owts are either performing poorly or failing with about 48% of owts failures in wa , which is due to groundwater and surface water ingress . other reasons include incorrect system installation , unsuitable soil type , undersized system , increasing wastewater volume , hydrostatic pressure , high groundwater , root invasion , cross connection to stormwater disposal , and a small percentage of unauthorized tampering . although technology is improving over time and some owts are capable of producing better effluent quality , the problems identified in the dohwa survey are also reported elsewhere . as such , there is a need to assess site and soil conditions rather than relying solely on technology . failing or poorly performing systems often lead to various environmental and public health issues [ 1 , 4 , 5 ] . it is common that a slow passage of raw septic tank effluent through 0.5 to 0.6 m of soil results in a high degree of purification . therefore it is necessary to ensure that there is a minimum of 0.5 to 0.6 m of soil below the base of a trench before high permeable fissured rock , soil , or a groundwater table . in accordance with the as / nzs 1547:2000 , the 600 mm soil absorptive zone is required for filtering , isolating , and absorbing wastewater , microorganisms , nutrients , and partials . thus , to reduce the potential for owts failure due to ground and surface water ingress , implementation of 600 mm absorptive zone is recommended . population density and number of installed owts need to be addressed in more detail . identifying and comparing areas with high owts installations in wa in addition , it is essential to ensure that a fair and consistent approach to dealing with malfunctioning owts is taken by lgs under the guidance from the dohwa . inadequate management strategies and the lack of coordinate institutional arrangements in relation to owts have been previously reported in australia . for instance , in a survey of 48 septic tanks , it was found that 72% of dispersal fields were soggy and ( by inference ) ineffective , 67% required solids removal , 8% needed structural repairs , 6% had insufficient capacity , and 4% were incorrectly sited . also , a study of owts performance in south australia ( sa ) indicates that 22% of atus contains indicator bacteria at levels higher than the maximum acceptable levels specified in the sa 's regulatory guidelines . collecting data of owts installations was conducted by the dohwa via emails , relying on individual lg across wa to provide data on the number of owts installed during the study period . however , as previously mentioned , data regarding the number of approved owts applications by lg was only collected from 20% of the existing lgs in wa ( 28 out of 140 ) . while responses were not expected from many of the metropolitan lgs as they have infill sewerage systems it is acknowledged that response was not received from some lgs with owts . a follow - up on the lg that failed to respond to the email sent out by the dohwa staff enquiring about the numbers of owts approvals indicates inadequate number of staff to review the applications or lack of adequate records to respond to the survey . among those who responded , however , several ehos found it difficult to complete the task due to lack of simple and efficient methods to collate / retrieve the information . several reasons for the lack of available data from lgs include the following.records were not available , due to lack of general file , lg in remote locations , and lack of full time ehos.records were not easily retrieved as data stored in offsite storage.information on owts was not recorded in the early years.for some lgs approval , inspection , and ongoing management for owts were conducted in a reactive way rather than on a proactive manner.ehos do not have the time to collate information because owts are only one part of their several duties.systems described as records were not available , due to lack of general file , lg in remote locations , and lack of full time ehos . information on owts was not recorded in the early years . for some lgs approval , inspection , and ongoing management for owts ehos do not have the time to collate information because owts are only one part of their several duties . lack of appropriate administration system to record owts approvals and installations is a significant drawback in the management of owts in wa . without any feasible management strategies to assess and maintain the performance of owts in each region , the resulting environmental and public health risks associated with poor system performance would increase . hence , there is a need to provide a generic approach for a standardized , common system that will allow each lg to record the number of owts applications that have been approved and to store information related to the approvals in a clear and standard record system . furthermore , there is a need for a multigovernance approach that can address the importance of an effective and robust planning and development of sites . government agencies need to understand their roles and responsibilities in the management of owts when assessing applications for land - use or other developments to ensure that all potential effects arising from such developments are considered prior to approvals . it is important that the site assessment for on - site wastewater management be carried out in the early stage of planning phase . this site assessment procedure should be applied at the rezoning or subdivision stages of the planning process . this will help to develop a management regime for owts that can minimize health risks and environmental impacts and enhance owts 's long - term sustainability . based on the findings reported above , to improve water quality from owts , the dohwa should do the following.it should provide a standard management tool to all lgs for record of owts including gps location . database should be able to record information on installation , operation , maintenance , and any other inspection requirements particularly for atus or systems achieving a secondary treated quality effluent.it should implement a soil absorption zone path length of at least 600 mm to reduce the potential for owts failure due to ground and surface water ingress.it should provide ehos with on - site wastewater management training based on as 1547 and current legislations as part of their professional development and an important measure of quality assurance for the industry , government , and general public.it should produce educational materials to owners of owts to facilitate the understanding of how owts work , how to identify signs of systems failures , and why inadequate maintenance can increase the system failures and the risk to humans and the environment . it should provide a standard management tool to all lgs for record of owts including gps location . database should be able to record information on installation , operation , maintenance , and any other inspection requirements particularly for atus or systems achieving a secondary treated quality effluent . it should implement a soil absorption zone path length of at least 600 mm to reduce the potential for owts failure due to ground and surface water ingress . it should provide ehos with on - site wastewater management training based on as 1547 and current legislations as part of their professional development and an important measure of quality assurance for the industry , government , and general public . it should produce educational materials to owners of owts to facilitate the understanding of how owts work , how to identify signs of systems failures , and why inadequate maintenance can increase the system failures and the risk to humans and the environment . as recommendations , the lgs should do the following.they should implement a risk management program to minimise failures , to rapidly identify and to address failures when problems occur . it is considered that a tolerable rate of system failure is smaller than 5% annually.they should implement a planning strategy for owts in the early stage of any development to identify the resources that are required to ensure adequate owts performance.they should implement site inspection strategies that are predominantly risk based , taking account of sensitive receptors , and should be integrated with other regulatory inspections as appropriate.they should distribute educational materials produced by the dohwa or lgs to owners of owts and provide advices to applicants on owts - related matters when required.they should update the register of installed owts in each lg jurisdiction and implement an inspection regime to identify legacy sites that will require remediation work . inspection can initially be targeted at the highest risk areas or owts with previous history of failures and complaints.in the case of low - risk sites , a variety of nonroutine inspection strategies and in particular proxy ( e.g. , monitoring of water quality ) , third party , engagement , and incentive strategies may be implemented . these strategies can involve working closely with home owners or stakeholders to ensure that those who are responsible for owts understand how to comply with the regulations and are encouraged to do so.they should embark on a series of activities to raise awareness and compliance related to the operation and maintenance of owts . information campaigns may include the communication of international best practice to water services australia ( wsa ) and specific local guidance to nongovernment organizations ( ngos ) and homeowners.they should conduct follow - up site inspections of approved owts that focus specifically on operation and maintenance . priority should be given to known areas of risk or environmental sensitive areas such as owts close to rivers and lakes . inspections will vary in both type and frequency as the inspection program develops over time . they should implement a risk management program to minimise failures , to rapidly identify and to address failures when problems occur . it is considered that a tolerable rate of system failure is smaller than 5% annually . they should implement a planning strategy for owts in the early stage of any development to identify the resources that are required to ensure adequate owts performance . they should implement site inspection strategies that are predominantly risk based , taking account of sensitive receptors , and should be integrated with other regulatory inspections as appropriate . they should distribute educational materials produced by the dohwa or lgs to owners of owts and provide advices to applicants on owts - related matters when required . they should update the register of installed owts in each lg jurisdiction and implement an inspection regime to identify legacy sites that will require remediation work . inspection can initially be targeted at the highest risk areas or owts with previous history of failures and complaints . in the case of low - risk sites , a variety of nonroutine inspection strategies and in particular proxy ( e.g. , monitoring of water quality ) , third party , engagement , and incentive strategies may be implemented . these strategies can involve working closely with home owners or stakeholders to ensure that those who are responsible for owts understand how to comply with the regulations and are encouraged to do so . they should embark on a series of activities to raise awareness and compliance related to the operation and maintenance of owts . information campaigns may include the communication of international best practice to water services australia ( wsa ) and specific local guidance to nongovernment organizations ( ngos ) and homeowners . they should conduct follow - up site inspections of approved owts that focus specifically on operation and maintenance . priority should be given to known areas of risk or environmental sensitive areas such as owts close to rivers and lakes . inspections will vary in both type and frequency as the inspection program develops over time . | on - site wastewater treatment systems ( owts ) are widely used in western australia ( wa ) to treat and dispose of household wastewater in areas where centralized sewerage systems are unavailable .
septic tanks , aerobic treatment units ( atus ) , and composting toilets with greywater systems are among the most well established and commonly used owts . however , there are concerns that some owts installed in wa are either performing below expected standards or failing . poorly performing owts
are often attributed to inadequate installation , inadequate maintenance , poor public awareness , insufficient local authority resources , ongoing wastewater management issues , or inadequate adoption of standards , procedures , and guidelines .
this paper is to review the installations and failures of owts in wa .
recommendations to the department of health western australia ( dohwa ) and local government ( lg ) in regard to management strategies and institutional arrangements of owts are also highlighted . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
neuraxial blockade , a commonly performed , landmark - based anaesthetic or analgesic technique , can be difficult in certain patients for as yet unclear reasons . technical challenges in the performance of this blind technique warranted an investigation into the reasons for difficulty in its performance . the quality of anatomical landmarks has been traditionally considered the single most important predictor of technical difficulty for neuraxial access . anatomic landmarks have so far been subjectively graded by different studies as either none ( if the spinous processes were non - palpable ) , poor ( if hardly palpable ) and good ( if easily palpable ) or grade 1 ( spinous processes are visible ) , grade 2 ( spinous processes are not seen but easily palpated ) , grade 3 ( spinous processes are not seen but barely palpable under the thumb ) and grade 4 ( none of the previous ) . while significant differences were found between easily palpable or grade 1 anatomy , and the ease of a spinal , no differences were found between hardly palpable and poorly palpable or grades 2 , 3 and 4 anatomy , introducing the difficulty of clinically applying these results . the percutaneous window to access the interlaminar space is defined by the bony confines of the spinous process and the lamina . our study was designed with the specific aim of using the spinous process dimensions ( the spinous process width ( spw ) and the interspinous gap ( isg ) ) , which serve as the only palpable landmark , in predicting the ease of neuraxial blockade . the palpable spinous process and the isg traditionally have determined the needle entry point at a selected level . assessment of the vertical length of the spinous process does not aid neuraxial needle placement . hence , we hypothesised that the isg and the spw are likely predictors of ease or difficulty in accessing the neuraxial space . spinal anaesthesia was selected in this preliminary study as a better modality for assessing technical success as there is a clear end point with intrathecal access ( free flow of cerebrospinal fluid ) as compared with epidural , where the possibility that the loss of resistance used in epidural needle placement may , erroneously , occur at locations other than entry to the epidural space . after obtaining institutional approvals , consecutive patients scheduled for surgery under spinal anaesthesia were prospectively enrolled from a population in tamilnadu , india belonging to the same ethnicity . inclusion criteria were patients scheduled for surgery under spinal anaesthesia , ability to provide informed consent , no evidence of infection or on anticoagulants . patients not able to provide informed consent , on anticoagulants , having an ongoing infection , either locally or bacteraemic , were excluded . the study was concluded at approximately 12 months as the anaesthesiologist moved from the location . demographic data including age , gender , height and weight were recorded prior to performing the spinal anaesthetic . the patient was held by an assistant until the spinal was completed after actively flexing the spine in the lateral decubitus position to the maximum extent possible by drawing the knees to the chest and flexing the neck [ figure 1 ] . the spinous processes above and below the level determined by the anaesthesiologist for accessing the intrathecal space was then palpated and the edges marked on the skin with a thin skin marker [ figure 2 ] . the isg between adjacent spinous processes and the spw at these locations were measured with a caliper from the surface markings . as there was no appreciable difference in spws above and below the levels , patients were classified as having difficult spinal anatomy if they had any palpable spinal deformity , e.g. lordosis , kyphosis , scoliosis or prior back surgery . photograph showing how the patient was positioned and held by an assistant prior to the measurement and until completion of the spinal anaesthetic administration photograph showing the interspinous gap marked with a skin marker after patient positioning an attempt was defined as every withdrawal of the needle either out of the skin or to less than approximately 1 cm from the skin prior to advancing again . all new skin punctures were considered an attempt , whether at the initial spinal level or at a second level . withdrawal of the needle beyond approximately 1 cm from the skin to change the direction of approach was regarded as a redirection . three outcome measures were used to assess the difficulty encountered in performing the spinal , namely 3 redirections , > 1 attempt and > 1 level , although these cut - offs were chosen based on an earlier study , where increasing number of attempts and more than one level were considered difficult neuraxial placement . finally , the success or need for additional anaesthesia was also recorded . data collection and anaesthetic management was performed by an experienced anaesthesiologist ( hs ) with over a decade of clinical experience at the time of data collection . the responsible anaesthesiologist also determined the level / s of access , the needle type and gauge , depending on availability . 1-sample kolmogorov smirnov z test and visual assessment of distribution curves was conducted to assess deviation from gaussian distribution and guide application of parametric ( t - test ) versus non - parametric ( mann - whitney u test ) tests to compare the two groups . for descriptive purposes , normally distributed variables were expressed as mean 1 sd and non - gaussian data as median ( inter - quartile range ) . p - value < 0.05 was considered significant . logistic regression was used to identify significant independent predictors for the three outcomes . pearson 's chi square test was used to evaluate whether distributions of categorical variables differed from one another . the median age of the study population was 30 ( 2448.5 ) years and the mean weight was 59 12.5 kg . all neuraxial blocks were performed using a midline approach with the subject in the lateral decubitus position , except in six subjects where a sitting position was used . during the placement in the sitting position , the l3-l4 spinal level , based on the intercristal line , was accessed in 69% of the subjects and the l4-l5 level in 21% of the subjects for the first attempt . depending on availability , a 26 g ( 74% patients ) or a 25 g ( 16% of patients ) quincke spinal needle was used . an introducer was used in 13 patients based on perceived difficulty in needle placement by the anaesthesiologist . free flowing cerebrospinal fluid was obtained in all subjects before the injection of local anaesthetic . 28.3% of the patients required 3 redirections , 22.8% required > 1 attempt and 15.9% required > 1 level . patient features ( continuous variables ) associated with 3 redirections , > 1 attempt and > 1 level are listed in table 2 . weight , isg and difficult spinal anatomy were individually predictive of difficulty , as assessed by the three outcome variables , while age and spw were not . patient characteristics predictors of a difficult spinal based on three outcome variables ( 3redirections , > 1 attempt and > 1 level ) using logistic regression analysis of the three outcome variables with the three independent predictive variables ( weight , isg , difficult spinal anatomy ) , isg alone significantly predicted all three outcomes [ table 3 ] . in other words , with increasing isg , there were increased 1 attempt and 1 level successes and fewer redirections , as shown by the odds ratio [ table 4 ] . there was a decreasing trend of difficulty with increase in the isg [ figure 3 ] . logistic regression model for predicting difficulty of spinal as defined by the three outcome variables using three independent predictive variables odds ratios from logistic regression model for predicting ease of spinal in relation to isg in mm chart comparing the interspinous gap measurements with the number of attempts and redirections there was no statistically significant difference in the isg or spw between subjects < 60 and 60 years of age ( p=0.99 and 0.25 , respectively ) . our study , with a specific focus on the spinous processes , being an offshoot from prior studies that had suggested landmarks as predictors of difficult neuraxial blockade may be clinically more applicable . proper patient positioning is important for the success of neuraxial blockade , and is impeded by pregnancy , spinal deformities and advanced age . decision to use the sitting position was made at random and on a trial basis by the anaesthesiologist . according to de oliveira filho et al . , anthropometric impediments to proper patient positioning are age > 65 years , body mass index ( bmi ) > 30 kg / m , abnormal spinal anatomy and short , broad biotype ( brevilineal , based on the xipho - costal angle > 90 ) . their report also highlighted that anatomical landmarks are poor with age > 40 years , bmi > 25 kg / m , abnormal spinal anatomy and those with brevilineal biotype . the depth to the neuraxial space and bmi was shown to be a predictor of success , along with the practitioner 's experience in a more recent study . our analysis did not find body weight or height to predict a successful outcome when isg and spinal difficulty were held constant . bmi could not be calculated in all our subjects due to missing height data in 119 subjects . our study also did not find a significant difference in the isg between patients younger and older than 60 years of age . chien et al . reported that age and gender were not associated with the first - level success or first attempt success , which was similar to our study . contrary to our hypothesis , spw was not found to be a predictor of ease or difficulty . surprisingly , the use of an introducer increased the number of attempts and levels . our data does not allow interpretation of this finding to determine a subjective assessment of difficulty felt by the anaesthesiologist . prior studies have reported that neither needle type nor gauge affects the difficulty of subarachnoid blocks , and our data does not permit a comparison of the different gauges used . not surprisingly , difficult spinal anatomy was associated with requirement of 3 redirections , > 1 attempt and > 1 level . difficult spinal anatomy as a description included patients with visible or palpable deformity as well as prior spine surgery . our results are in contrast to an earlier report in which first - level success was not affected by spinal anatomy , age , sex or body habitus for either spinal or epidural blocks . there are a few limitations to our study , including the overall low body weight of the study population . this may also be the reason for the absence of grade 4 anatomic landmarks in our subjects , although prior studies had shown poor correlation between landmarks and bmi . as this was a preliminary study , no power analysis was conducted prior to the study . the translation of palpatory observations of the spinous process dimensions for the determination of exact dimensions may pose a challenge . skin turgor and movements could potentially alter these dimensions and affect the precision of the final measurements . to decrease its impact , the patient was held in place by an assistant until the spinal anaesthetic was completed and to limit aberrancies in data collection . one could argue that the measurement of isg with skin markings and calipers may be subjective , preventing an objective assessment of fine differences . our results demonstrate an obvious trend of increasing difficulty in spinal needle placement with decrease in isg . the final limitation is the performance of the spinal anaesthetic by the investigator collecting the data and measurements introducing an observer bias . although prior knowledge of the spinous process dimensions can introduce a bias during the subsequent performance of the spinal anaesthetic , our data shows that few patients with larger interspinous gaps also required more than one attempt , suggesting that this bias was minimal . in conclusion , the spw measured by palpation does not correlate with ease or difficulty of spinal needle placement . | background : neuraxial anaesthesia , despite being a common technique , may pose some technical challenges leading to complications such as post - dural puncture headache , trauma to neural structures and neuraxial haematoma .
we hypothesised that the interspinous gap ( isg ) and the spinous process width ( spw ) could be used as objective measures to predict ease of access to the neuraxial space.methods:two hundred and two consecutive patients scheduled to have spinal anaesthesia for various surgical procedures were enrolled prospectively after institutional approval .
following proper positioning for the neuraxial blockade , the isg and spw at the intended level were measured with calipers .
the number of attempts , and redirections at the selected spinal level , and the number of levels required for successful needle placement were also recorded.results:group-wise analysis of the data into patients requiring > 1 attempt , > 1 level and 3 redirections showed that the single independent predictor of a difficult neuraxial block was the isg .
twenty - three percent of the patients required more than one attempt , with a mean gap of 6.35 ( 1.2 ) mm , in contrast to 8.15 ( 2.4 ) mm in those with a single attempt ( p=0.000 ) .
in addition , 16% of the patients needed more than one level , with a mean gap of 6.03 ( 2.01 ) mm in contrast to 8.07 ( 2.37 ) mm for a single level ( p=0.000).conclusions : the single independent predictor of ease or difficulty during spinal anaesthesia was the isg ( p=0.000 ) . |
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we selected a prospective cohort from the hong kong diabetes registry enrolled between 1 december 1996 and 8 january 2005 because drug dispensary data became fully computerized and available for analysis purposes in 1996 . a detailed description of the hong kong diabetes registry is available elsewhere ( 1113 ) . briefly , the registry was established at the prince of wales hospital , the teaching hospital of the chinese university of hong kong , which serves a population of > 1.2 million . the referral sources of the cohort included general practitioners , community clinics , other specialty clinics , the prince of wales hospital , and other hospitals . enrolled patients with hospital admissions within 68 weeks prior to assessment accounted for < 10% of all referrals . a 4-h assessment of complications and risk factors was performed on an outpatient basis , modified from the european diabcare protocol ( 14 ) . once a patient had undergone this comprehensive assessment , he / she was considered to have entered this study cohort and would be followed until the time of death . ethical approval was obtained from the chinese university of hong kong clinical research ethics committee . this study adhered to the declaration of helsinki , and written informed consent was obtained from all patients at the time of assessment , for research purposes . by 2005 , 7,387 diabetic patients were enrolled in the registry since december 1996 . we sequentially excluded 1 ) 328 patients with type 1 diabetes or missing data on types of diabetes ; 2 ) 45 patients with non - chinese or unknown nationality ; 3 ) 175 patients with a known history of cancer or receiving cancer treatment at enrollment ; 4 ) 736 patients with missing values on any variables used in the analysis ( see table 1 for a list of variables ) ; and 5 ) 3,445 patients who used metformin during 2.5 years before enrollment . the cutoff point of 2.5 years was chosen because any duration longer than that did not lead to any noticeable changes in the hazard ratios ( hrs ) and 95% cis of metformin use for cancer ( supplementary table 1 ) . clinical and biochemical characteristics of the study cohort stratified according to occurrence of cancer during follow - up period data are median ( 25th to 75th percentile ) or n ( % ) . derived from fisher exact test . from enrollment to the earliest date of cancer , death , or censoring . patients attended the center after an 8-h fast and underwent a 4-h structured clinical assessment that included laboratory investigations . a sterile , random - spot urinary sample was collected to measure albumin - to - creatinine ratio ( acr ) . in this study , albuminuria was defined as an acr 2.5 mg / mmol in men and 3.5 mg / mmol in women . the abbreviated modification of diet in renal disease study formula recalibrated for chinese subjects ( 15 ) was used to estimate glomerular filtration rate ( gfr ) ( expressed in ml / min per 1.73 m ) : estimated gfr = 186 ( scr 0.011 ) ( age ) ( 0.742 if female ) 1.233 , where scr is serum creatinine expressed as mol / l ( original mg / dl converted to mol / l ) , and 1.233 is the adjusting coefficient for chinese subjects . total cholesterol , triglycerides , and hdl cholesterol were measured by enzymatic methods on a hitachi 911 automated analyzer ( boehringer mannheim , mannheim , germany ) using reagent kits supplied by the manufacturer of the analyzer , whereas ldl cholesterol was calculated using the friedewald equation ( 16 ) . drug usage data were extracted from the hong kong hospital authority central computer system , which recorded all drug dispensary data in public hospitals , including the start dates and end dates for each of the drugs of interest . in hong kong , all medications are dispensed on site in both inpatient and outpatient settings . these databases were matched by a unique identification number , the hong kong identity card number , which is compulsory for all residents in hong kong . all medical admissions of the cohort from enrollment to 30 july 2005 were retrieved from the hong kong hospital authority central computer system , which recorded admissions to all public hospitals in hong kong . collectively , these hospitals provide 95% of the total hospital bed - days in hong kong ( 17 ) . additionally , mortality data from the hong kong death registry during the period were retrieved and cross - checked with hospital discharge status . hospital discharge principle diagnoses , coded by the international statistical classification of diseases and related health problems 9th revision ( icd-9 ) , were used to identify cancer events . the outcome measure of this study was incident cancer ( fatal or nonfatal : codes 140208 ) during the follow - up period . we used biological interactions to test whether metformin use was associated with a greater cancer risk reduction in patients with low hdl cholesterol than in those with normal or high hdl cholesterol . the statistical analysis system ( release 9.10 ) was used to perform the statistical analysis ( sas institute , cary , nc ) , unless otherwise specified . follow - up time was calculated as the period in years from the first enrollment since 1 december 1996 to the date of the first cancer event , death , or censoring , whichever came first . cox proportional hazard regression was used to obtain the hrs and 95% cis of the variables of interest . we first plotted the full - range association of hdl cholesterol and cancer and further refined cutoff points of hdl cholesterol for cancer risk in the cohort without prevalent metformin users , using restricted cubic spline cox models ( 11 ) . then , we examined the biological interaction for cancer risk between low hdl cholesterol and nonuse of metformin using three measures : 1 ) relative excess risk caused by interaction ( reri ) ; 2 ) attributable proportion ( ap ) caused by interaction ; and 3 ) the synergy index ( s ) ( 18 ) . a detailed calculation method of additive interaction , including the definition of three indicator variables , an sas program , and a calculator in microsoft excel ( www.epinet.se ) , was described by the authors . the reri is the excess risk attributed to interaction relative to the risk without exposure . ap refers to the attributable proportion of disease , which is caused by the interaction in subjects with both exposures . s is the excess risk from both exposures when there is biological interaction relative to the risk from both exposures without interaction . a simulation study showed that reri performed best and ap performed fairly well , but s was problematic in the measure of additivity in the proportional hazard model ( 19 ) . the current study refined the criteria as either a statistically significant reri > 0 or ap > 0 to indicate biological interactions . the following two - step adjustment scheme was used in these analyses : 1 ) only adjusting for ldl cholesterol related cancer risk indicators ( ldl cholesterol 3.80 mmol / l and ldl cholesterol < 2.80 mmol / l plus albuminuria ) ( 11,12 ) , triglycerides ( 2 ) , and high hdl cholesterol , where appropriate ( 2 ) , and 2 ) further adjusting for age , sex , employment status , smoking status , alcohol intake , duration of diabetes , bmi ( 27.6 and < 24.0 kg / m ) ( 2 ) , systolic blood pressure ( sbp ) , and a1c ( 20 ) at enrollment and use of statins ( 13 ) , fibrates , other lipid - lowering drugs , ace inhibitors / angiotensin ii receptor blockers ( arbs ) ( 13 ) , and insulin ( 20 ) during follow - up . use of drugs during follow - up was defined as use of the drugs from enrollment to cancer , death , or censoring date , whichever came first . by definition , the use of any drugs after the first cancer event was coded as nonuse of these drugs , and any drug users had been given at least one prescription of the drug during follow - up . the total metformin dosage divided by the total number of days during which metformin was prescribed was used as daily metformin dosage . we also used propensity score to adjust for the likelihood of initiation of metformin during the follow - up period ( 21 ) . the former was obtained using a logistic regression procedure that includes the following independent variables : age ; sex ; bmi ; ldl cholesterol ; hdl cholesterol ; triglycerides ; tobacco and alcohol intake ; a1c ; sbp ; ln ( acr + 1 ) ; estimated gfr ; peripheral arterial disease ; retinopathy ; sensory neuropathy ; and history of cardiovascular disease ( coronary heart disease , myocardial infarction , and stroke ) at baseline ( c statistics = 0.73 ) . we then used stratified cox models on deciles of the score to adjust for the likelihood of metformin use . sensitivity analyses were performed to address 1 ) the impacts of undiagnosed cancer by limiting the analysis to patients who were followed for 2.5 years ( n = 2170 ) ; 2 ) the impact of incomplete exclusion of patients who used metformin during 2.5 years before enrollment by limiting the analysis to patients who were enrolled on or after 1 july 1998 ( n = 1707 ) ; 3 ) the impact of prevalent bias by reinclusion of 3,445 patients who used metformin during 2.5 years before enrollment ; and 4 ) inclusion of subjects with missing values in univariable analysis and without adjusting for the propensity score to maximize the valid sample size ( n of the valid sample size = 2,996 and n of the sample size with missing values in hdl cholesterol = 53 [ i.e. , 1.74% missing - value rate ] ) . patients attended the center after an 8-h fast and underwent a 4-h structured clinical assessment that included laboratory investigations . a sterile , random - spot urinary sample was collected to measure albumin - to - creatinine ratio ( acr ) . in this study , albuminuria was defined as an acr 2.5 mg / mmol in men and 3.5 mg / mmol in women . the abbreviated modification of diet in renal disease study formula recalibrated for chinese subjects ( 15 ) was used to estimate glomerular filtration rate ( gfr ) ( expressed in ml / min per 1.73 m ) : estimated gfr = 186 ( scr 0.011 ) ( age ) ( 0.742 if female ) 1.233 , where scr is serum creatinine expressed as mol / l ( original mg / dl converted to mol / l ) , and 1.233 is the adjusting coefficient for chinese subjects . total cholesterol , triglycerides , and hdl cholesterol were measured by enzymatic methods on a hitachi 911 automated analyzer ( boehringer mannheim , mannheim , germany ) using reagent kits supplied by the manufacturer of the analyzer , whereas ldl cholesterol was calculated using the friedewald equation ( 16 ) . drug usage data were extracted from the hong kong hospital authority central computer system , which recorded all drug dispensary data in public hospitals , including the start dates and end dates for each of the drugs of interest . in hong kong , these databases were matched by a unique identification number , the hong kong identity card number , which is compulsory for all residents in hong kong . all medical admissions of the cohort from enrollment to 30 july 2005 were retrieved from the hong kong hospital authority central computer system , which recorded admissions to all public hospitals in hong kong . collectively , these hospitals provide 95% of the total hospital bed - days in hong kong ( 17 ) . additionally , mortality data from the hong kong death registry during the period were retrieved and cross - checked with hospital discharge status . hospital discharge principle diagnoses , coded by the international statistical classification of diseases and related health problems 9th revision ( icd-9 ) , were used to identify cancer events . the outcome measure of this study was incident cancer ( fatal or nonfatal : codes 140208 ) during the follow - up period . we used biological interactions to test whether metformin use was associated with a greater cancer risk reduction in patients with low hdl cholesterol than in those with normal or high hdl cholesterol . the statistical analysis system ( release 9.10 ) was used to perform the statistical analysis ( sas institute , cary , nc ) , unless otherwise specified . follow - up time was calculated as the period in years from the first enrollment since 1 december 1996 to the date of the first cancer event , death , or censoring , whichever came first . cox proportional hazard regression was used to obtain the hrs and 95% cis of the variables of interest . we first plotted the full - range association of hdl cholesterol and cancer and further refined cutoff points of hdl cholesterol for cancer risk in the cohort without prevalent metformin users , using restricted cubic spline cox models ( 11 ) . then , we examined the biological interaction for cancer risk between low hdl cholesterol and nonuse of metformin using three measures : 1 ) relative excess risk caused by interaction ( reri ) ; 2 ) attributable proportion ( ap ) caused by interaction ; and 3 ) the synergy index ( s ) ( 18 ) . a detailed calculation method of additive interaction , including the definition of three indicator variables , an sas program , and a calculator in microsoft excel ( www.epinet.se ) , the reri is the excess risk attributed to interaction relative to the risk without exposure . ap refers to the attributable proportion of disease , which is caused by the interaction in subjects with both exposures . s is the excess risk from both exposures when there is biological interaction relative to the risk from both exposures without interaction . a simulation study showed that reri performed best and ap performed fairly well , but s was problematic in the measure of additivity in the proportional hazard model ( 19 ) . the current study refined the criteria as either a statistically significant reri > 0 or ap > 0 to indicate biological interactions . the following two - step adjustment scheme was used in these analyses : 1 ) only adjusting for ldl cholesterol related cancer risk indicators ( ldl cholesterol 3.80 mmol / l and ldl cholesterol < 2.80 mmol / l plus albuminuria ) ( 11,12 ) , triglycerides ( 2 ) , and high hdl cholesterol , where appropriate ( 2 ) , and 2 ) further adjusting for age , sex , employment status , smoking status , alcohol intake , duration of diabetes , bmi ( 27.6 and < 24.0 kg / m ) ( 2 ) , systolic blood pressure ( sbp ) , and a1c ( 20 ) at enrollment and use of statins ( 13 ) , fibrates , other lipid - lowering drugs , ace inhibitors / angiotensin ii receptor blockers ( arbs ) ( 13 ) , and insulin ( 20 ) during follow - up . use of drugs during follow - up was defined as use of the drugs from enrollment to cancer , death , or censoring date , whichever came first . by definition , the use of any drugs after the first cancer event was coded as nonuse of these drugs , and any drug users had been given at least one prescription of the drug during follow - up . the total metformin dosage divided by the total number of days during which metformin was prescribed was used as daily metformin dosage . we also used propensity score to adjust for the likelihood of initiation of metformin during the follow - up period ( 21 ) . the former was obtained using a logistic regression procedure that includes the following independent variables : age ; sex ; bmi ; ldl cholesterol ; hdl cholesterol ; triglycerides ; tobacco and alcohol intake ; a1c ; sbp ; ln ( acr + 1 ) ; estimated gfr ; peripheral arterial disease ; retinopathy ; sensory neuropathy ; and history of cardiovascular disease ( coronary heart disease , myocardial infarction , and stroke ) at baseline ( c statistics = 0.73 ) . we then used stratified cox models on deciles of the score to adjust for the likelihood of metformin use . sensitivity analyses were performed to address 1 ) the impacts of undiagnosed cancer by limiting the analysis to patients who were followed for 2.5 years ( n = 2170 ) ; 2 ) the impact of incomplete exclusion of patients who used metformin during 2.5 years before enrollment by limiting the analysis to patients who were enrolled on or after 1 july 1998 ( n = 1707 ) ; 3 ) the impact of prevalent bias by reinclusion of 3,445 patients who used metformin during 2.5 years before enrollment ; and 4 ) inclusion of subjects with missing values in univariable analysis and without adjusting for the propensity score to maximize the valid sample size ( n of the valid sample size = 2,996 and n of the sample size with missing values in hdl cholesterol = 53 [ i.e. , 1.74% missing - value rate ] ) . the median age of the cohort was 56 years ( 25th to 75th percentiles [ interquartile range { iqr } 4567 ] ) at enrollment . during 13,808 person - years of follow - up ( 5.51 years [ 3.087.39 ] ) , 129 patients developed cancer . in the cohort , 16.3% ( n = 433 ) of patients had low hdl cholesterol < 1.0 mmol / l and 46.7% ( n = 1,243 ) had hdl cholesterol 1.30 patients with low hdl cholesterol were more likely to use insulin , develop cancer , and die prematurely . patients who developed cancer were older , more likely to use tobacco and alcohol , and had longer disease duration . they had high ldl cholesterol , low hdl cholesterol , and albuminuria and were more likely to have premature death than those free of cancer . patients who developed cancer were less likely to use statins and metformin during the follow - up period than patients without cancer ( table 1 ) . compared with patients with hdl cholesterol 1.0 but < 1.3 mmol / l , patients with hdl cholesterol < 1.0 mmol / l ( hr 2.22 [ 95% ci 1.383.58 ] ) and those with hdl cholesterol 1.3 mmol / l ( 1.61 [ 1.052.46 ] ) had increased cancer risk in univariable analysis . after adjusting for other covariates ( supplementary fig . 1 ) , hdl cholesterol < 1.0 mmol / l for cancer remained significant ( 2.41 [ 1.463.96 ] ) but not hdl cholesterol 1.3 mmol / l ( p = 0.1197 ) . additional subgroup univariable and multivariable analyses indicate that low hdl cholesterol was associated with increased cancer risk only among those who did not use metformin but not among those who did ( power = 0.37 ) ( table 2 ) . hrs of different combinations of low hdl cholesterol and metformin use for cancer risk in type 2 diabetes * adjusted for ldl cholesterol related risk indicators ( ldl cholesterol 3.8 mmol / l and ldl cholesterol < 2.8 mmol / l plus albuminuria ) , hdl cholesterol 1.30 mmol / l ( not for models 7 and 8) , and the nonlinear association of triglycerides with cancer . further adjusted for age , sex , employment status , smoking status , alcohol intake , duration of diabetes , bmi ( 27.6 or < 24.0 kg / m ) , a1c , and sbp at enrollment and use of statins , fibrates , other lipid - lowering drugs , ace inhibitors / arbs , and insulin during follow - up . stratified cox model analyses on deciles of the propensity score of metformin use were included in models 512 to control for the likelihood of starting metformin therapy during follow - up . use of metformin was associated with a decreased risk of cancer in a dose - response manner . after adjusting for covariates , patients with hdl cholesterol < 1.0 mmol / l and who were not treated with metformin had a 5.8-fold risk of cancer compared with the referent group , who had hdl cholesterol 1.0 and used metformin . mmol / l but who were not treated with metformin also had higher cancer risk than the referent group . however , the cancer risk associated with hdl cholesterol < 1.0 mmol / l was rendered nonsignificant among those who used metformin ( table 2 and supplementary fig . 2 ) . there was a significant interaction between low hdl cholesterol and nonuse of metformin for cancer risk , after adjusting for covariates ( ap 0.44 [ 95% ci 0.110.78 ] ) ( table 3 ) . measures for estimation of biological interaction between low hdl cholesterol and nonuse of metformin for the risk of cancer in type 2 diabetes * adjusted for ldl cholesterol related risk indicators ( ldl cholesterol 3.8 mmol / l and ldl cholesterol < 2.8 mmol / l plus albuminuria ) , hdl cholesterol 1.30 mmol / l , and the nonlinear association of triglyceride with cancer . further adjusted for age , sex , employment status , smoking status , alcohol intake , duration of diabetes , bmi ( 27.6 or < 24.0 kg / m ) , a1c , and systolic blood pressure at enrollment and use of statins , fibrates , other lipid - lowering drugs , ace inhibitors / arbs , and insulin during follow - up . stratified cox model analyses on deciles of the propensity score of use of metformin were used to control for likelihood of starting metformin therapy during follow - up . consistently , the copresence of hdl cholesterol < 1.0 mmol / l and nonuse of metformin was associated with an increased risk of cancer at sites other than the digestive organs and peritoneum and , to a lesser degree , cancers of the digestive organs and peritoneum . copresence of both factors also was associated with an increased risk of fatal cancer and , to a lesser degree , nonfatal cancer ( table 4 ) . hrs of the copresence of hdl cholesterol < 1.0 mmol / l and nonuse of metformin during follow - up versus all other groups for site - specific cancers and fatal and nonfatal cancers * univariable cox models with stratification on deciles of the propensity score of use of metformin during follow - up were used to obtain the hrs . classification was based on the icd-9 ( there are overlaps among site - specific cancers ) . 46 nonfatal cancer events developed before fatal cancer . the series of sensitivity analyses showed a consistent trend toward an interactive effect of nonuse of metformin and hdl cholesterol < 1.0 mmol / l on the risk of cancer , although not all interactions in these sensitivity analyses reached statistical significance ( supplementary tables 2 and 3 ) . the median age of the cohort was 56 years ( 25th to 75th percentiles [ interquartile range { iqr } 4567 ] ) at enrollment . during 13,808 person - years of follow - up ( 5.51 years [ 3.087.39 ] ) , 129 patients developed cancer . in the cohort , 16.3% ( n = 433 ) of patients had low hdl cholesterol < 1.0 mmol / l and 46.7% ( n = 1,243 ) had hdl cholesterol 1.30 patients with low hdl cholesterol were more likely to use insulin , develop cancer , and die prematurely . patients who developed cancer were older , more likely to use tobacco and alcohol , and had longer disease duration . they had high ldl cholesterol , low hdl cholesterol , and albuminuria and were more likely to have premature death than those free of cancer . patients who developed cancer were less likely to use statins and metformin during the follow - up period than patients without cancer ( table 1 ) . compared with patients with hdl cholesterol 1.0 but < 1.3 mmol / l , patients with hdl cholesterol < 1.0 mmol / l ( hr 2.22 [ 95% ci 1.383.58 ] ) and those with hdl cholesterol 1.3 mmol / l ( 1.61 [ 1.052.46 ] ) had increased cancer risk in univariable analysis . after adjusting for other covariates ( supplementary fig . < 1.0 mmol / l for cancer remained significant ( 2.41 [ 1.463.96 ] ) but not hdl cholesterol 1.3 mmol / l ( p = 0.1197 ) . additional subgroup univariable and multivariable analyses indicate that low hdl cholesterol was associated with increased cancer risk only among those who did not use metformin but not among those who did ( power = 0.37 ) ( table 2 ) . hrs of different combinations of low hdl cholesterol and metformin use for cancer risk in type 2 diabetes * adjusted for ldl cholesterol related risk indicators ( ldl cholesterol 3.8 mmol / l and ldl cholesterol < 2.8 mmol / l plus albuminuria ) , hdl cholesterol 1.30 mmol / l ( not for models 7 and 8) , and the nonlinear association of triglycerides with cancer . further adjusted for age , sex , employment status , smoking status , alcohol intake , duration of diabetes , bmi ( 27.6 or < 24.0 kg / m ) , a1c , and sbp at enrollment and use of statins , fibrates , other lipid - lowering drugs , ace inhibitors / arbs , and insulin during follow - up . stratified cox model analyses on deciles of the propensity score of metformin use were included in models 512 to control for the likelihood of starting metformin therapy during follow - up . use of metformin was associated with a decreased risk of cancer in a dose - response manner . after adjusting for covariates , patients with hdl cholesterol < 1.0 mmol / l and who were not treated with metformin had a 5.8-fold risk of cancer compared with the referent group , who had hdl cholesterol 1.0 and used metformin . mmol / l but who were not treated with metformin also had higher cancer risk than the referent group . however , the cancer risk associated with hdl cholesterol < 1.0 mmol / l was rendered nonsignificant among those who used metformin ( table 2 and supplementary fig . 2 ) . there was a significant interaction between low hdl cholesterol and nonuse of metformin for cancer risk , after adjusting for covariates ( ap 0.44 [ 95% ci 0.110.78 ] ) ( table 3 ) . measures for estimation of biological interaction between low hdl cholesterol and nonuse of metformin for the risk of cancer in type 2 diabetes * adjusted for ldl cholesterol related risk indicators ( ldl cholesterol 3.8 mmol / l and ldl cholesterol < 2.8 mmol / l plus albuminuria ) , hdl cholesterol 1.30 mmol / l , and the nonlinear association of triglyceride with cancer . further adjusted for age , sex , employment status , smoking status , alcohol intake , duration of diabetes , bmi ( 27.6 or < 24.0 kg / m ) , a1c , and systolic blood pressure at enrollment and use of statins , fibrates , other lipid - lowering drugs , ace inhibitors / arbs , and insulin during follow - up . stratified cox model analyses on deciles of the propensity score of use of metformin were used to control for likelihood of starting metformin therapy during follow - up . consistently , the copresence of hdl cholesterol < 1.0 mmol / l and nonuse of metformin was associated with an increased risk of cancer at sites other than the digestive organs and peritoneum and , to a lesser degree , cancers of the digestive organs and peritoneum . copresence of both factors also was associated with an increased risk of fatal cancer and , to a lesser degree , nonfatal cancer ( table 4 ) . hrs of the copresence of hdl cholesterol < 1.0 mmol / l and nonuse of metformin during follow - up versus all other groups for site - specific cancers and fatal and nonfatal cancers * univariable cox models with stratification on deciles of the propensity score of use of metformin during follow - up were used to obtain the hrs . classification was based on the icd-9 ( there are overlaps among site - specific cancers ) . 46 nonfatal cancer events developed before fatal cancer . the series of sensitivity analyses showed a consistent trend toward an interactive effect of nonuse of metformin and hdl cholesterol < 1.0 mmol / l on the risk of cancer , although not all interactions in these sensitivity analyses reached statistical significance ( supplementary tables 2 and 3 ) . in this study , we observed that hdl cholesterol < 1.0 mmol / l and nonuse of metformin was associated with a 5.8-fold cancer risk compared with metformin users with hdl cholesterol 1.0 the significant additive interaction indicates that the increased cancer risk as a result of a combination of nonuse of metformin and hdl cholesterol < 1.0 mmol / l was more than the addition of the risks attributed to the presence of either nonuse of metformin or low hdl cholesterol alone . in other words , the significant interaction suggests that the use of metformin may confer an extra cancer benefit in type 2 diabetic patients with low hdl cholesterol . although there are ongoing debates about the associations between insulin usage and cancer in diabetes , epidemiological studies have consistently found that the use of metformin is associated with reduced cancer risk . among these studies , libby et al . ( 9 ) reported that metformin use was associated with a 54% ( 95% ci 4760 ) lower crude incidence and a 37% ( 2547 ) lower adjusted incidence of cancer than metformin nonusers over a period of 10 years . in support of these findings , we also found a 50% lower adjusted cancer risk among metformin users with hdl cholesterol 1.0 several lines of evidence support the pivotal role of ampk , which can be triggered by a large number of upstream signals , in maintaining energy homeostasis by providing a balance between energy expenditure through lipolysis and energy storage through protein and glycogen synthesis . activation of ampk by the tumor suppressor , lkb1 , promotes glucose uptake , increases fatty acid oxidation , and reduces protein and lipid synthesis . metformin is known to activate the ampk pathway , possibly through the activation of the lkb1 suppressor gene ( 22 ) . on the other hand , hyperglycemia can downregulate apoa - i gene transcription , which is the major lipoprotein component of hdl lipid particles ( 23 ) . in this regard , apoa - i has been shown to stimulate phosphorylation of ampk and acc ( 5 ) . more recently , kimura et al . ( 24 ) reported that hdl can activate ampk through binding to both sphingosine 1-phosphate receptors / gi proteins and scavenger receptor class b type i ( sr - bi)/protein pdzk1 , with lkb1 being involved in the sr - bi signaling . given the close relationship between hdl cholesterol and the ampk pathway , the interactive effects between metformin use and hdl cholesterol on cancer risk is thus plausible . 2 ) hospital principle discharge diagnosis was used to retrieve cancer events in the cohort , and this approach may have missed a small number of cancer events . 3 ) the use of drug dispensary data are an indirect method and may overestimate exposure because drug acquisition is only a surrogate marker for actual drug consumption . although our definition of drug use should not introduce major bias ( 25 ) , some unmeasured confounding factors may exist . 4 ) the sample size of the study was not large enough to address whether there were sex - specific cutoff points of hdl cholesterol for the risk of cancer . 5 ) there were insufficient numbers of patients / events to explore the relationships between hdl cholesterol status , metformin exposure , and risk of specific cancers . 6 ) reri did not reach statistical significance . however , reris were significant in sensitivity analyses 3 and 4 with larger sample sizes , suggesting that the marginally significant reri in the analysis is possibly attributed to insufficient power . 7 ) these findings were only derived from a chinese cohort and need to be replicated in other ethnic populations . in conclusion , the use of metformin might confer stronger benefits in reducing cancer risk in patients with hdl cholesterol < 1.0 mmol / l . although low hdl cholesterol is not an indication for metformin usage , if our findings can be independently replicated , patients with low hdl cholesterol with or without type 2 diabetes might be candidate subjects for clinical trials that formally test the anticancer effects of metformin or agents that modulate the apoa - i lkb1ampk pathway . | objectivethe amp - activated protein kinase ( ampk ) pathway is a master regulator in energy metabolism and may be related to cancer . in type 2 diabetes ,
low hdl cholesterol predicts cancer , whereas metformin usage is associated with reduced cancer risk .
both metformin and apolipoprotein a1 activate the ampk signaling pathway .
we hypothesize that the anticancer effects of metformin may be particularly evident in type 2 diabetic patients with low hdl cholesterol.research design and methodsin a consecutive cohort of 2,658 chinese type 2 diabetic patients enrolled in the study between 1996 and 2005 , who were free of cancer and not using metformin at enrollment or during 2.5 years before enrollment and who were followed until 2005 , we measured biological interactions for cancer risk using relative excess risk as a result of interaction ( reri ) and attributable proportion ( ap ) as a result of interaction .
a statistically significant reri > 0 or ap > 0 indicates biological interaction.resultsduring 13,808 person - years of follow - up ( median 5.51 years ) , 129 patients developed cancer .
hdl cholesterol < 1.0 mmol / l was associated with increased cancer risk among those who did not use metformin , but the association was not significant among those who did .
use of metformin was associated with reduced cancer risk in patients with hdl cholesterol < 1.0 mmol / l and , to a lesser extent , in patients with hdl cholesterol 1.0
mmol / l .
hdl cholesterol < 1.0 mmol / l plus nonuse of metformin was associated with an adjusted hazard ratio of 5.75 ( 95% ci 3.0310.90 ) compared with hdl cholesterol 1.0 mmol / l plus use of metformin , with a significant interaction ( ap 0.44 [ 95% ci 0.110.78]).conclusionsthe anticancer effect of metformin was most evident in type 2 diabetic patients with low hdl cholesterol . |
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we systematically reviewed medical records and extracted information about : age , sex , breed , month and year of hospitalization , presenting complaint , clinical signs before hospital admittance , and lactation stage . data from clinical signs , complete blood count ( cbc ) , serum biochemistry profile ( sbp ) , and ultrasonographic findings from the date immediately before liver biopsy were performed or when cholangiohepatitis was included as a differential diagnosis was recorded . information on liver biopsy , bacteriological culture , treatments , duration of hospitalization , and outcome was collected . at chuv , 12 of 5,519 cases met inclusion criteria , whereas at avc , 1 of 245 patients was included . the diagnosis of cholangiohepatitis was confirmed antemortem from a liver biopsy in 12 cases and during postmortem examination in 1 case . six cows were in the first 150 days of lactation , 1 cow was over 150 days of lactation , and 4 cows were dry . reasons for veterinary consult included the following : fever ( n = 5 ) , decreased appetite ( n = 4 ) , colic ( n = 2 ) , stranguria ( n = 1 ) , sunburns ( n = 1 ) , right displacement of the abomasum ( n = 1 ) , and decreased milk and fecal output after omentopexy ( n = 1 ) . at the time of diagnosis of cholangiohepatitis , 6 cows were anorectic and 7 had decreased appetite . the median rectal temperature was 39c ( range : 37.740c ) and 5 cows were febrile ( temperature > 39.2c ) . median heart rate was 92 beats per minute ( bpm ) ( range : 60110 bpm ) , and 8 cows were tachycardic ( hr > 80 bpm ) . yellow discoloration was identified on the skin of the udder ( n = 3 ) , conjunctiva ( n = 2 ) , and vulva ( n = 1 ) . hydration status was recorded for eleven cows : 7 cows were mildly ( 57% ) , 3 moderately ( 810% ) , and 1 severely ( > 10% ) dehydrated . characteristics of the feces were recorded in twelve cows ; 6 cows had absent or scant feces , 3 had diarrhea , 1 had diarrhea with hematochezia , 1 had feces with partially digested fibers , and 1 had normal feces . 10 cells / l ) in 6 cases ( median : 11 10 cells / 10 cells / l ) , neutrophilia ( > 4 10 cells / l ) in 9 cases ( median : 8 10 cells / l , range : 2.319 10 cells / l ) , toxic changes identified in the neutrophil morphology in 4 cases , and hyperfibrinogenemia ( > 5 g / l ) in 8 cases ( median : 7 g / l , range : 311 g / l ) in 8 cases ( median : 48 g / l , range : 2065 g / l ) , hypoalbuminemia ( < 30 g / dl ) in 10 cases ( median : 28 iu / l ) in 8 cases ( median : 156 iu / l , range : 401411 iu / l ) in 10 cases ( median : 165 iu / l , range 34385 sdh activity values were available for 7 cases with 5 cases having increased sdh activity ( > 12 iu / l , median : 24 , range 42,309 hyperbilirubinemia ( > 14 mol / l ) was detected in 10 cases ( median 53 mol / l , range 7188 mol / l ) . in this study , 4 of 5 cases with icterus had a bilirubin concentration > 77 mol / l ( range : 77188 mol / l ) , whereas 1 of 5 icteric cases had a bilirubin concentration of 9 mol / l . on the other hand , 1 of 8 without icterus had a bilirubin concentration of 124 mol / l , whereas 7 of 8 cases without icterus had a bilirubin concentration < 56 mol / l ( range : 756 mol / l ) . serum bilirubin concentrations of all cases with icteric serum were > 77 mol / l ( range : 77188 mol / l ) . serial serum liver enzyme activity and bilirubin concentration from the time that cholangiohepatitis was suspected to the day of discharge or euthanasia were available in 6 cases ( 4 survivors and 2 nonsurvivors ) . despite the clinical improvement of surviving cases , a marked improvement in the serum activity of hepatobiliary enzymes or hyperbilirubinemia subjective abnormal findings included distension of the gallbladder ( n = 4 ) , increased thickness of the wall of the gallbladder ( n = 3 ) , presence of sediment in the gallbladder occupying twothirds of the volume of the gallbladder ( n = 2 ) , and increased diameter of the bile duct ( n = 3 ) . final histopathological diagnosis , using the description suggested previously,2 included mild , subacute , nonsuppurative cholangiohepatitis ( n = 2 ) , mild , chronic , nonsuppurative cholangiohepatitis ( n = 2 ) , mild or severe , subacute , suppurative cholangiohepatitis ( n = 4 ) , mild mixed ( neutrophilic and lymphocytic ) cholangiohepatitis ( n = 3 ) , and moderate , chronic , suppurative cholangiohepatitis ( n = 1 ) . bacteriological culture from liver biopsy tissue yielded growth in 3 of twelve cases in which it was performed . ( n = 1 ) , e. coli ( n = 1 ) , e. coli , and trueperella pyogenes ( n = 1 ) . in 1 animal , culture of the liver tissue obtained on necropsy yielded growth of a multidrug resistant e. coli . in 2 animals in which bacteriological cultures from liver biopsy were negative , however , salmonella spp . was cultured from a fecal sample submitted during their hospitalization . in addition to cholangiohepatitis , other diseases diagnosed included peritonitis ( n = 3 ) , photosensitization ( n = 2 ) , salmonellosis ( n = 2 ) , abomasal dilatation / displacement ( n = 2 ) , glomerulonephritis ( n = 1 ) , pyelonephritis ( n = 1 ) , enteritis of unknown origin ( n = 1 ) , and bronchopneumonia ( n = 1 ) . treatment consisted of administration of broadspectrum antibiotic treatment and supportive treatment with intravenous or oral fluids ( 12/13 ) . antimicrobial therapy included procaine benzyl penicillin ( 22,000 u / kg , i m q12h ) ( n = 10 ) , trimethoprimsulfadoxine ( tms , 16 mg / kg iv q12h ) ( n = 4 ) , oxytetracycline ( 11 mg / kg iv or i m q12h ) ( n = 3 ) , ampicillin ( 10 mg / kg iv q8h ) ( n = 2 ) , ceftiofur ( 1 mg / kg i m q24h ) ( n = 2 ) , and lincomycin ( dose was not recorded ) ( n = 1 ) . the number of days ( median ) of treatment with antimicrobials was 10 ( range : 135 days ) . for surviving cases , two cows underwent a laparotomy1 was to correct a right abomasal displacement , and the second was an exploratory laparotomy . in the exploratory laparotomy , a large distended gallbladder was palpated and no other abnormal findings were detected . after surgery , the cow developed suppurative peritonitis and bilirubin and biliary acids were detected on abdominocentesis . six cows were discharged from the hospital , whereas 7 were euthanized most likely due to poor prognosis or poor response to the instituted therapy . three of 7 euthanized cows had chronic changes on the histopathology examination , and 1 had severe suppurative cholangiohepatitis . the median time of hospitalization for the surviving cows was 9.5 days ( range : 812 days ) . the median time of hospitalization for nonsurviving cows was 24 hours ( range 110 days ) . the most common macroscopic lesion was a mildtosevere increase in thickness of the gallbladder wall ( n = 6 ) . the mucosal surface of the gallbladder showed different grades of multifocal ulceration and coalesced areas of ulceration with or without hemorrhage . fibrin plaques admixed with necrotic material were commonly identified in the gallbladder ( n = 4 ) . in 2 cases , peritonitis and adhesions from the duodenum to the liver leading to obstruction of the bile flow were confirmed . peritonitis of unknown origin was identified in 2 cases , but in both cases , peritonitis was suspected to have occurred after bile flow obstruction . at chuv , 12 of 5,519 cases met inclusion criteria , whereas at avc , 1 of 245 patients was included . the diagnosis of cholangiohepatitis was confirmed antemortem from a liver biopsy in 12 cases and during postmortem examination in 1 case . six cows were in the first 150 days of lactation , 1 cow was over 150 days of lactation , and 4 cows were dry . reasons for veterinary consult included the following : fever ( n = 5 ) , decreased appetite ( n = 4 ) , colic ( n = 2 ) , stranguria ( n = 1 ) , sunburns ( n = 1 ) , right displacement of the abomasum ( n = 1 ) , and decreased milk and fecal output after omentopexy ( n = 1 ) . at the time of diagnosis of cholangiohepatitis , 6 cows were anorectic and 7 had decreased appetite . the median rectal temperature was 39c ( range : 37.740c ) and 5 cows were febrile ( temperature > 39.2c ) . median heart rate was 92 beats per minute ( bpm ) ( range : 60110 bpm ) , and 8 cows were tachycardic ( hr > 80 bpm ) . yellow discoloration was identified on the skin of the udder ( n = 3 ) , conjunctiva ( n = 2 ) , and vulva ( n = 1 ) . hydration status was recorded for eleven cows : 7 cows were mildly ( 57% ) , 3 moderately ( 810% ) , and 1 severely ( > 10% ) dehydrated . characteristics of the feces were recorded in twelve cows ; 6 cows had absent or scant feces , 3 had diarrhea , 1 had diarrhea with hematochezia , 1 had feces with partially digested fibers , and 1 had normal feces . hematologic findings included leucocytosis ( > 12 10 cells / l ) in 6 cases ( median : 11 10 cells / l , range : 626 10 cells / l ) , neutrophilia ( > 4 10 cells / l ) in 9 cases ( median : 8 10 cells / l , range : 2.319 10 cells / l ) , toxic changes identified in the neutrophil morphology in 4 cases , and hyperfibrinogenemia ( > 5 g / l ) in 8 cases ( median : 7 g / l , range : 311 g / l ) in 8 cases ( median : 48 g / l , range : 2065 g / l ) , hypoalbuminemia ( < 30 g / dl ) in 10 cases ( median : 28 g / l , range : 1935 iu / l ) in twelve cases ( median : 156 iu / l , range : 302,137 iu / l ) in 10 cases ( median : 165 iu / l , range 34385 sdh activity values were available for 7 cases with 5 cases having increased sdh activity ( > 12 iu / l , median : 24 , range 42,309 mol / l ) was detected in 10 cases ( median 53 mol / l , range 7188 mol / l ) . in this study , 4 of 5 cases with icterus had a bilirubin concentration > 77 mol / l ( range : 77188 mol / l ) , whereas 1 of 5 icteric cases had a bilirubin concentration of 9 mol / l . on the other hand , 1 of 8 without icterus had a bilirubin concentration of 124 mol / l , whereas 7 of 8 cases without icterus had a bilirubin concentration < 56 mol / l ( range : 756 mol / l ) . serum bilirubin concentrations of all cases with icteric serum were > 77 mol / l ( range : 77188 mol / l ) . serial serum liver enzyme activity and bilirubin concentration from the time that cholangiohepatitis was suspected to the day of discharge or euthanasia were available in 6 cases ( 4 survivors and 2 nonsurvivors ) . despite the clinical improvement of surviving cases , a marked improvement in the serum activity of hepatobiliary enzymes or hyperbilirubinemia subjective abnormal findings included distension of the gallbladder ( n = 4 ) , increased thickness of the wall of the gallbladder ( n = 3 ) , presence of sediment in the gallbladder occupying twothirds of the volume of the gallbladder ( n = 2 ) , and increased diameter of the bile duct ( n = 3 ) . liver biopsy and bacteriological culture were performed in twelve of thirteen cases . final histopathological diagnosis , using the description suggested previously,2 included mild , subacute , nonsuppurative cholangiohepatitis ( n = 2 ) , mild , chronic , nonsuppurative cholangiohepatitis ( n = 2 ) , mild or severe , subacute , suppurative cholangiohepatitis ( n = 4 ) , mild mixed ( neutrophilic and lymphocytic ) cholangiohepatitis ( n = 3 ) , and moderate , chronic , suppurative cholangiohepatitis ( n = 1 ) . bacteriological culture from liver biopsy tissue yielded growth in 3 of twelve cases in which it was performed . ( n = 1 ) , e. coli ( n = 1 ) , e. coli , and trueperella pyogenes ( n = 1 ) . in 1 animal , culture of the liver tissue obtained on necropsy yielded growth of a multidrug resistant e. coli . in 2 animals in which bacteriological cultures from liver biopsy were negative , however , salmonella spp . was cultured from a fecal sample submitted during their hospitalization . in addition to cholangiohepatitis , other diseases diagnosed included peritonitis ( n = 3 ) , photosensitization ( n = 2 ) , salmonellosis ( n = 2 ) , abomasal dilatation / displacement ( n = 2 ) , glomerulonephritis ( n = 1 ) , pyelonephritis ( n = 1 ) , enteritis of unknown origin ( n = 1 ) , and bronchopneumonia ( n = 1 ) . treatment consisted of administration of broadspectrum antibiotic treatment and supportive treatment with intravenous or oral fluids ( 12/13 ) . antimicrobial therapy included procaine benzyl penicillin ( 22,000 u / kg , i m q12h ) ( n = 10 ) , trimethoprimsulfadoxine ( tms , 16 mg / kg iv q12h ) ( n = 4 ) , oxytetracycline ( 11 mg / kg iv or i m q12h ) ( n = 3 ) , ampicillin ( 10 mg / kg iv q8h ) ( n = 2 ) , ceftiofur ( 1 mg / kg i m q24h ) ( n = 2 ) , and lincomycin ( dose was not recorded ) ( n = 1 ) . the number of days ( median ) of treatment with antimicrobials was 10 ( range : 135 days ) . for surviving cases , two cows underwent a laparotomy1 was to correct a right abomasal displacement , and the second was an exploratory laparotomy . in the exploratory laparotomy , a large distended gallbladder was palpated and no other abnormal findings were detected . after surgery , the cow developed suppurative peritonitis and bilirubin and biliary acids were detected on abdominocentesis . six cows were discharged from the hospital , whereas 7 were euthanized most likely due to poor prognosis or poor response to the instituted therapy . three of 7 euthanized cows had chronic changes on the histopathology examination , and 1 had severe suppurative cholangiohepatitis . the median time of hospitalization for the surviving cows was 9.5 days ( range : 812 days ) . the median time of hospitalization for nonsurviving cows was 24 hours ( range 110 days ) . the most common macroscopic lesion was a mildtosevere increase in thickness of the gallbladder wall ( n = 6 ) . the mucosal surface of the gallbladder showed different grades of multifocal ulceration and coalesced areas of ulceration with or without hemorrhage . fibrin plaques admixed with necrotic material were commonly identified in the gallbladder ( n = 4 ) . in 2 cases , peritonitis and adhesions from the duodenum to the liver leading to obstruction of the bile flow were confirmed . peritonitis of unknown origin was identified in 2 cases , but in both cases , peritonitis was suspected to have occurred after bile flow obstruction . the proportion of cattle diagnosed with cholangiohepatitis in this study confirmed that the disease appears to be rare . it is possible that the low prevalence of cholangiohepatitis found in this retrospective study could have been biased by the chosen case definition.7 the diagnosis of cholangiohepatitis was established based on the histopathological findings on a liver biopsy or postmortem examination . some cases of suspected cholangiohepatitis based on history , clinicopathologic examination , ancillary test , and ultrasonographic examination could have been treated symptomatically without a liver biopsy being performed , and therefore , some potential cases were likely missed . also , postmortem examination archives at the teaching hospitals compile only the 2 or 3 most important diagnoses in each case . those cases in which cholangiohepatitis was an incidental finding cholangiohepatitis can be a primary or secondary disease.3 in healthy cattle , a relatively sterile biliary duct system is preserved by the continuous flow of bile from the gallbladder and biliary tree through the sphincter of oddi into the duodenum.8 primary cholangiohepatitis results from alteration of the normal bile flow , which leads to the ascending bacterial colonization of the biliary and hepatic tissue and results in cholangitis and cholangiohepatitis.1 secondary cholangiohepatitis results from obstruction of normal bile flow due to external mechanical pressure exerted on the common bile duct by liver abscesses , by extensive adhesions in the area of the cystic and common bile ducts , or by severe inflammatory lesions of the common duct near the hilus or the duodenal papilla.1
due to the retrospective nature of this study , it was not possible to accurately determine in which cases primary or secondary cholangiohepatitis occurred . however , primary cholangiohepatitis was suspected in 4 cows in which comorbidities were not identified on physical or postmortem examination . two of those cows had photosensitization as the primary complaint and was considered to be secondary to cholangiohepatitis and liver insult . the other 2 cows had acute septic peritonitis as the principal diagnosis , suspected to have resulted from a primary biliary obstruction . secondary cholangiohepatitis was suspected in 9 cases where comorbidities were identified on physical examination , postmortem examination , or both . comorbidities that could have resulted in secondary cholangiohepatitis included enteritis , adhesions of the small intestine blocking the biliary duct , renal infections , and abomasal displacement.1
the clinical signs of the cows with cholangiohepatitis reported in this study included anorexia , obtundation , fever , and colic . although these signs are nonspecific , they are observed consistently with cholangiohepatitis in different species.9 , 10 , 11 icterus is also commonly reported in cases of cholangiohepatitis.9 , 10 it has been suggested that clinical icterus in cattle develops when serum bilirubin is greater than 51 mol / l.8 in this study , icterus was present in 5 of 13 cows and serum bilirubin concentrations were greater than 77 further , icteric serum was identified in 5 cows in which serum bilirubin was > 77 these results support the current understanding that a marked increase in serum bilirubin occurs before jaundice becomes easily recognized.8
in this study , the majority of cows had increased activity of at least 5 variables ( bilirubin , ggt , ast , alp , and sdh ) . ggt , alp , and bilirubin were consistently increased , demonstrating insult of the biliary tree . neutrophilia , hyperfibrinogenemia , and hyperglobulinemia were also common findings consistent with an active inflammatory process . these findings were expected as it was likely that those changes prompted the clinicians to proceed with a liver biopsy to confirm the diagnosis of cholangiohepatitis . increased serum activity of hepatobiliary enzymes or hyperbilirubinemia or both conditions are commonly reported in cases of cholangiohepatitis . similar results have been reported in both horses and cattle with cholangiohepatitis.3 , 11 , 12 , 13 it is possible that comorbidities such as peritonitis , pyelonephritis , or glomerulonephritis can also contribute to the increase in inflammatory markers . importantly , cows suffering with hepatic lipidosis in combination with an inflammatory process ( e.g , acute metritis or mastitis ) can have similar laboratory results to the cattle with cholangiohepatitis found in this study . therefore , the changes in cbc and sbp are not specific to cholangiohepatitis . during ultrasonographic examination , distension of the gallbladder alone is not an indication of cholestasis as the volume of the gallbladder increases without any impairment of bile flow in anorexic cattle.14 abnormal gallbladder content , such as sediment or concretion , without thickening of the wall of the gallbladder can also be observed in anorexic cows with various diseases not specific to the liver.15 however , distension of the gall bladder in combination with thickening of the wall and sediment due to inflammation suggests disease such as cholangiohepatitis.14 , 16 , 17 ultrasonographic findings compatible with cholangiohepatitis in cattle included distension and increased thickness of the wall of the gallbladder with the presence of sediment and increased diameter of the bile canaliculi . in cattle with suspected cholestasis , the diagnostic approach should also include the histologic and bacteriological examination of a liver biopsy specimen . all the liver biopsy samples in this study were processed for aerobic and anaerobic culture , but only 3 samples yielded a positive result . for instance , a previous study in horses with cholangiohepatitis revealed that only 2 of 9 liver biopsy samples submitted for both aerobic and anaerobic culture yielded a positive result . negative bacteriological cultures were obtained in horses with or without receiving prebiopsy antimicrobials.13 it is possible that antimicrobial therapy administered before the biopsy procedure prevents positive results . however , culture can be negative in horses with cholangiohepatitis even in the absence of prior antimicrobial therapy . in the present study , liver samples and gallbladder content yielded growth of e. coli , trueperella pyogenes and bacteroides spp . several studies in humans , horses , dogs , and cattle have reported that enteric gramnegative bacteria are frequently encountered in patients with cholangiohepatitis , cholelithiasis or both . bile cultures are positive in 80100% of humans who have cholangitis.18 enteric gramnegative organisms including e. coli , klebsiella spp . , and enterococcus spp . are commonly isolated from bile , and anaerobic bacteria such as clostridium spp . and bacteroides spp . are isolated more commonly in polymicrobial infections in humans who have severe disease.13 , 19 , 20 , 21 gramnegative enteric bacteria , such as e. coli , salmonella spp . , are associated with cases of cholangiohepatitis and cholelithiasis in horses.11 , 13 consistently , bacteria involved in previous reports of cholangiohepatitis in dogs were e. coli , klebsiella spp . , and clostridium spp . , which are all normal constituents of the intestinal flora.22 in this study , salmonella spp . was isolated from the feces , but not from liver tissue , of 2 cows suffering from enteritis , however , whether salmonella was the cause of cholangiohepatitis remains unclear . treatment in all cases was similar and consisted of antimicrobial therapy and supportive medical care . medical treatment without surgery is effective in approximately 80% of patients.21 there are 6 cases in the literature regarding cholangitis and cholangiohepatitis in dogs in which 4 of 6 responded to medical treatment alone.9 , 22 , 23 in human medicine , antibiotics are given early when acute cholangitis is suspected . in this study , in surviving cases , the number of days on antimicrobial therapy ranged from 4 to 35 days . in humans , broadspectrum antibiotics with adequate biliary excretion such as ampicillin , potentiated sulphas , and third or fourthgeneration cephalosporins and quinolones are widely used in empirical antimicrobial therapy of biliary tract infection.24 , 25 , 26 antimicrobial therapy for cattle suffering from cholangiohepatitis should be selected based on culture and sensitivity results from liver biopsy . however , in clinical practice , empirical antimicrobial therapy often needs to be instituted before culture results are available and must respect the judicious use of antibiotics . initial treatment options for cattle with cholangiohepatitis include aminopenicillins ( ampicillin ) , potentiated sulphas ( trimethoprimsulfadoxine , where allowed ) , thirdgeneration cephalosporins ( ceftiofur ) as biliary excretion is one of the major routes of elimination for these antimicrobials.27 tetracycline can also be used for treatment of cholangiohepatitis . tetracyclines are excreted in bile , they are reabsorbed from the intestine and returned to the liver ( enterohepatic cycle ) for reentry into systemic circulation.27 once the microorganisms have been identified and their susceptibility has been determined , the antimicrobials should be adjusted to cover the identified microorganism.28 the duration of antimicrobial therapy for surviving cows ranged from 4 to 35 days . in humans , the duration of antimicrobial therapy is based on the clinical response and the presence of bacteremia . for mild disease , antibiotics are generally continued for 57 days and for patients who have a positive blood culture , a 1014 days course of antibiotics is recommended.21 , 24 longterm parenteral antimicrobials were associated with survival in horses13 and dogs.9 , 22 in this study , clinical improvement in surviving cattle during hospitalization was noted despite of a persistent increased in hepatobiliary enzyme activity and hyperbilirubinemia . therefore , extended antimicrobial could be beneficial for cattle with cholangiohepatitis ; however , the optimal duration of antimicrobial therapy in cattle can not be withdrawn from the present study and remains to be established . the pathological grading system failed to demonstrate any association between the severity and chronicity of the lesion and prognosis . similar observations have been reported in horses.13 it appeared that the diagnosis of cholangiohepatitis carried a guarded prognosis , at least in the small group of hospitalized cattle included in this study . it is possible that this study had a bias toward sicker patients , but such cattle seem to be representative of the population presented to referral teaching hospitals . the prognosis for cholangiohepatitis in dogs9 , 22 and horses13 appeared to be good , with most of the cases making a full recovery . based on the information registered in the medical records used in this study , it was not possible to determine whether the reason for euthanasia was primarily cholangiohepatitis or comorbidities or both , and further deductions could not be drawn . in conclusion , the results of this study revealed that cholangiohepatitis is a rare condition in adult cattle . diagnostic processes should include clinical history collection , physical examination , hematologic and serum biochemistry profile , ultrasonography imaging , and liver biopsy with bacteriological culture and histopathology examination . | backgroundthe signalment , clinicopathologic , bacteriological , histopathological , ultrasonographic characteristics , and the treatment and outcomes of adult cattle with cholangiohepatitis are poorly defined.animalsthirteen holstein cows with cholangiohepatitis.methodsretrospective study of medical records of cattle admitted to the chuv and the avc between 1992 and 2012 and 2000 and 2012 , respectively , for cattle older than 3 months of age with a histopathological diagnosis of cholangiohepatitis .
cholangiohepatitis was defined as the presence of portal inflammation surrounding or infiltrating bile ducts , with or without epithelial damage , and extending into the adjacent lobules.resultsat the time of diagnosis of cholangiohepatitis , cows had decreased appetite ( n = 7 ) or were anorectic ( n = 6 ) , had fever ( n = 5 ) , and had tachycardia ( n = 8) .
icterus was detected in 5 cows .
yellow discoloration was identified on the skin of the udder ( n = 3 ) , conjunctiva ( n = 2 ) , and vulva ( n = 1 ) .
there was leukocytosis ( n = 6 ) , neutrophilia ( n = 9 ) , and hyperfibrinogenemia ( n = 8) .
alteration in the serum biochemistry profile included hyperglobulinemia ( n = 8) , hypoalbuminemia ( n = 10 ) , increased activity of ggt ( n = 12 ) , ast ( n = 8) , and alp ( n = 10 ) , and hyperbilirubinemia ( n = 10 ) .
histopathological diagnosis included mild , subacute , nonsuppurative cholangiohepatitis ( n = 4 ) , mild suppurative cholangiohepatitis ( n = 4 ) , mild mixed ( neutrophilic and lymphocytic ) cholangiohepatitis ( n = 3 ) , and moderate , chronic , nonsuppurative cholangiohepatitis ( n = 1 ) .
six cows were discharged from the hospital , and 7 were euthanized.conclusions and clinical importancecholangiohepatitis is a rare condition in adult cattle .
antemortem diagnosis can be challenging because clinical signs are unspecific . |
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the regional postal processing center for the patient is the southern connecticut processing and distribution center ( scpdc ) in wallingford . with a floor area of 350,000 square feet and the capacity to process up to 3 million pieces of mail a day , the center is equipped with 6 advanced - facer canceller machines , 5 optical character reader machines , 5 bar - code sorting machines , and 13 digital bar - code sorting ( dbcs ) machines for processing letters . in addition , automated flat sorting machines , linear integrated parcel sorters , and small bundle and parcel sorters are used to process flats ( large flat mail that is not a package ) and parcels ( wrapped packages ) . although all these machines are part of the facility , they differ in function , speed of processing , and location within the facility . the advanced facer - canceller machines cancel letters originating from southern connecticut and apply two bar codes that are used to identify and sort letters for their final destination . the identification tag , a fluorescent orange bar code on the back of the envelope , records the time and date that the letter was canceled . the postnet barcode , a series of vertical full and half bars applied to the front of an envelope , contains zip code and delivery point information in machine - readable format . advanced facer - canceller machines are used primarily to process stamped mail ; bulk letters are not processed on canceling machines because they already have barcodes applied by the mailers and are presorted . the high - speed computerized dbcs machines are used for preliminary and final sorting of the mail by barcode . during the preliminary sort , letters can be processed on any one of the 13 dbcs machines at the facility . this step arranges the letters by the 5-digit zip code of the delivery address , usually requiring < 2 passes to sort a batch of mail . once this step is accomplished , mail is transported for final processing to a designated dbcs machine , which sorts the letters to the 9- or 11-digit zip code , usually requiring <3 passes . therefore , letters addressed to the patient could have been processed initially on any of the 13 dbcs machines . 6 , and specific bins were designated for the carrier route . in october and november 2001 , independent contractors working for the u.s postal service ( usps ) tested postal processing and distribution plants nationwide to determine if any had become contaminated with b. anthracis following the bioterrorism events . as part of this effort , scpdc was tested on november 11 , 2001 ; all results were negative for b. anthracis contamination . following the report of the inhalational anthrax case in oxford , connecticut , the facility was tested again extensively . the advanced facer - canceller machines cancel letters originating from southern connecticut and apply two bar codes that are used to identify and sort letters for their final destination . the identification tag , a fluorescent orange bar code on the back of the envelope , records the time and date that the letter was canceled . the postnet barcode , a series of vertical full and half bars applied to the front of an envelope , contains zip code and delivery point information in machine - readable format . advanced facer - canceller machines are used primarily to process stamped mail ; bulk letters are not processed on canceling machines because they already have barcodes applied by the mailers and are presorted . the high - speed computerized dbcs machines are used for preliminary and final sorting of the mail by barcode . during the preliminary sort , letters can be processed on any one of the 13 dbcs machines at the facility . this step arranges the letters by the 5-digit zip code of the delivery address , usually requiring < 2 passes to sort a batch of mail . once this step is accomplished , mail is transported for final processing to a designated dbcs machine , which sorts the letters to the 9- or 11-digit zip code , usually requiring <3 passes . therefore , letters addressed to the patient could have been processed initially on any of the 13 dbcs machines . 6 , and specific bins were designated for the carrier route . in october and november 2001 , independent contractors working for the u.s . postal service ( usps ) tested postal processing and distribution plants nationwide to determine if any had become contaminated with b. anthracis following the bioterrorism events . as part of this effort , scpdc was tested on november 11 , 2001 ; all results were negative for b. anthracis contamination . following the report of the inhalational anthrax case in oxford , connecticut , the facility was tested again extensively . samples were obtained from scpdc on november 11 , 21 , 25 , and 28 and december 2 . sampling methods included dry swabs , wet swabs , wet wipes , and hepa vacuum . on november 11 , a contracting company working for usps obtained samples from scpdc as part of the nationwide testing of postal facilities for anthrax spores . the contractor took dry synthetic swabs from random sites in the facility and sent them to be analyzed at the connecticut department of public health laboratory . on november 21 , 2001 , after the report of the 94-year - old woman with anthrax in connecticut , a second independent contractor hired by usps collected additional dry swab samples from surfaces where letters , flats , and parcels were processed . these samples , along with others collected from air circulating units , were analyzed by the connecticut department of public health laboratory . on november 25 , the investigation team obtained samples from the facility using wet synthetic swabs and processed them by methods recommended by cdc ( 7,8 ) . samples were taken from the letter canceling and sorting machines , flat and parcel sorting machines , and five facility vacuum filters in use since october 27 , 2001 . samples taken on november 28 were more extensive . guided by additional epidemiologic data , we collected samples from carefully selected sites ( the canceling and sorting machines ) by using wet synthetic 2x2-inch wipes and hepa vacuum . wipe and vacuum samples were cultured and analyzed at a cdc - contracted laboratory . on december 2 , following the first report of anthrax - positive results in the facility , we collected follow - up samples . a composite sample from the vertical column of four bins was taken from all columns on the four dbcs machines that were presumptively positive based on sampling done on november 28 . these wet wipe samples , taken to determine the extent of contamination on the machines , were analyzed by a cdc - contracted laboratory . a total of 589 samples were collected from november 11 to december 2 , 2001 . three hundred forty - six ( 59% ) of these were from the dbcs machines . of the 589 samples , 117 ( 20% ) were dry swabs , 60 ( 10% ) wet swabs , 300 ( 51% ) wet wipes , and 112 ( 19% ) hepa vacuum samples . of these , only one ( 2% ) sample was from a dbcs machine ( no . dbcs , digital bar - code sorting ; usps , united states postal service ; cdc , centers for disease control and prevention ; atsdr , agency for toxic substances and disease registry . on november 21 six ( 10% ) were from the dbcs machines , two each from dbcs nos . all samples were negative for b. anthracis ( tables 1,2 ) . on november 25 , the investigation team took a total of 60 wet synthetic swab samples ; 8 ( 13% ) were from the dbcs machines . of the eight samples taken from the dbcs machines , one sample each was taken from dbcs nos . 1 , 2 , 9 , 11 , and 13 and three from dbcs no . on november 28 , the most extensive sampling was conducted , with 212 samples collected . of these , we used wet wipes for sampling the stacker bins ( hard surfaces ) and the hepa vacuum for sampling the machines , including the inaccessible parts . we focused our sample collection on machines likely to have processed mail delivered to the patient 's address . although all machines were tested , 131 ( 62% ) samples were from dbcs machines , which processed both stamped mail and nearly all the bulk presorted mail ; approximately 80% of the mail recovered from the patient 's home was bulk mail . of 212 samples , 6 ( 3% ) yielded b. anthracis , and all positive samples were from dbcs machines . of the six anthrax - positive samples , two were vacuum samples from dbcs nos . 4 and 10 , and four were wet wipe samples from the bins of dbcs machines nos . one vacuum sample ( 0.55 g of specimen ) from the feeder part of machine no . 10 had 2.9x10 cfu of b. anthracis , equal to 5.5x10 cfu of b. anthracis per gram of sample material . this machine has not been sampled before november 28 , the fourth round of sampling . following the results of the sampling on november 28 , we collected follow - up samples on december 2 . 4 , 10 , and 11 , the machines from which results were positive for b. anthracis on the november 28 sampling . 6 because preliminary positive results from the november 28 sampling were reported and because this machine was used for final processing of mail to the address of the patient . the 200 wet wipe samples taken on december 2 were composite wipes from a vertical column of four bins from each machine ( each machine has 4852 columns of four bins ) . we collected composite samples to allow complete sampling of all bins from all suspect machines without taking an excessive number of samples ( table 2 ) . of 200 composite column samples from dbcs machines nos . 4 , 6 , 10 , and 11 , ( 2% ) of 48 columns on both machines no . 4 and 6 were positive . 6 was used for final mail sorting for several zip codes including the town where the patient lived . supplemented by the findings of the epidemiologic investigation team , our investigation identified cross - contaminated mail as a possible source of anthrax for the connecticut patient ( 4 ) . no other source of contamination in her community was identified after extensive sampling of her home and areas she visited ; no other cases of anthrax were reported . we identified a contaminated sorting machine that was used to sort most of the mail delivered to the patient , including bulk mail ; the specific column of bins that held mail for her carrier route was positive ( 4 ) . positive results were obtained following sample collection based on information learned during the epidemiologic investigation . all positive results were obtained from samples collected by using wet wipes and vacuum sampling . environmental sampling during an anthrax investigation is critical in determining the likely source of infection and the extent and degree of environmental contamination , to support decisions on the need for prophylaxis with antibiotics or clean - up , and to provide guidance about when clean - up is adequate to permit reentry into an area . during this investigation , we lacked data on the effectiveness of the sample collection media ( swabs , wipes , and vacuum ) for typical porous and nonporous surfaces encountered in indoor environments . the effect of varying concentrations of b.
anthracis containing particles and dust loading on sampling efficiency had also not been studied . furthermore , recovery efficiency of the analytical methods ( efficiency of removal of b. anthracis spores from the sample collection media ) had not been adequately evaluated , and limits of detection have not been established ( 8) . although our investigation showed that different sample collection techniques and sampling sites and numbers of samples yielded different findings , results are based on observation and can not be used to specifically compare the different approaches . however , exploring the reasons for the different results may be useful for future investigations . on november 11 , all samples were collected by using dry swabs from random sites in the facility with the intent of finding contamination anywhere in the facility . only one sample from the dbcs machines was taken . on november 21 , more samples were taken from the dbcs machines , but still only three machines were sampled . this sampling was performed with emphasis on the oxford mail route because the illness had been reported in that community . however , whether the patient s mail was predominantly bulk mail and whether letters could have been sorted preliminarily on any dbcs machine were not known at the time . the november 25 sampling was similar to the november 21 sampling except that investigators used wet swabs instead of dry swabs . on november 28 , more extensive and directed sampling was conducted , and epidemiologic information was available to guide us to the appropriate sites . using wet wipes and hepa vacuum led to the first positive results for anthrax in the facility . a recent study , conducted after the connecticut investigation , has confirmed our findings ( wt sanderson et al . side - by - side surface swabs , wipes , and hepa vacuum samples were taken at the brentwood processing and distribution center in washington , d.c . wet wipes and vacuum sampling were found to be more effective methods than surface swabs ; results from wet wipes and vacuum samples were highly concordant . of 28 sample locations tested , 4 ( 13% ) were positive with dry swabs , compared with 13 ( 46% ) wet swabs , 23 ( 82% ) wet wipes , and 23 ( 82% ) vacuum samples ( wt sanderson et al . although the effectiveness of sampling techniques influences which are used , other factors that determine the choice of sampling techniques include the site of sampling , size of the surface to be sampled , character of the surface ( porous or nonporous ) , need to quantify the results , and preference and specialization of the laboratory where the test is done . swab samples may still be the best method to sample small hard surfaces not easily accessible for wiping or vacuum sampling ( e.g. , a keyboard ) . in addition , sampling all surfaces within a building by using surface wipes is not feasible . therefore , vacuum samples provide an important tool for maximizing the surfaces that can be evaluated during an investigation ( 8) . sampling methods and number of samples are also influenced by the circumstances of the potential contamination . a sufficient number of samples must be taken to increase the probability that the sampling is representative , given the likely extent of contamination . in an initial investigation where a known or suspected release of potentially contaminated material has occurred , the first priority should be to collect samples near the suspected release source ( often called directed or targeted sampling ) . in determining the extent of contamination , investigators should include coverage of areas along an anticipated contaminant pathway , i.e. , those associated with air movement or dust collection , as well as activities that result in re - aerosolization or cross - contamination . when sampling to identify contamination in a facility , the length of time between the suspected contamination of the facility and the time that sampling occurs is also important in determining where and how to collect samples . for example , since the sampling on november 11 was conducted > 3 weeks after contamination was probably introduced into the facility , any aerosolized spores of b.
anthracis had likely already settled on surfaces , and therefore surface sampling , as opposed to air sampling , was reasonable . the environmental investigation did not identify anthrax spores in the patient s home , possibly because her house was routinely cleaned thoroughly or because the piece of mail that was the source for her infection was not identified . one resident of her community is known to have received an envelope from which b. anthracis spores were isolated that was likely to have become cross - contaminated as it passed through the postal system , although no one in that household became ill ( 2 ) . the patient also probably became ill following exposure to a low number of b. anthracis spores , which may explain why she had a relatively long incubation period compared with the other cases reported ( 9,10 ) . other host factors , including advanced age , underlying lung disease , medication use ( 2 ) , and the practice of tearing up bulk mail ( 4 ) , may have increased her chances of acquiring the disease . the results of our investigation influenced the adherence and compliance of postal workers on postexposure prophylaxis at scpdc . a study conducted there showed that 13% of the postal workers stopped taking postexposure prophylaxis because of the initial report of negative environmental cultures in the facility . an increase in postexposure prophylaxis adherence occurred , however , following the positive results in the facility ( 11 ) . the reasons why no postal workers at scpdc became ill during this event are unknown . perhaps host factors were important or anthrax spores were not aerosolized in sufficient concentration . the finding that spores were not widespread in the facility suggests that the dispersion was likely not due to substantial aerosolization . following the experience from the brentwood facility in october 2001 , cleaning practices in postal facilities nationwide changed from use of compressed air , which easily aerosolized small particulate materials such as anthrax spores , to use of hepa vacuums for cleaning ( 12 ) . at scpdc , maintenance workers stopped using forced air to clean equipment on october 27 , 2001 , which may have reduced the time when spores could have been aerosolized . the highly contaminated dbcs machine could have been a source of exposure to postal workers if the cleaning measures had not been changed . the environmental investigation was central in demonstrating a possible source of infection for the case of inhalational anthrax in connecticut . our investigation showed that extensive sampling was required and that epidemiologic investigation was essential in identifying sites for sampling . for future investigations of large facilities , we recommend the use of wet wipes and vacuum . further research is needed to clarify the sensitivity of the sampling and analytical methods for known or suspected b. anthracis and to develop clear algorithms for sampling if future investigations are needed . this investigation also demonstrated that illness associated with cross - contaminated mail is a rare but possible phenomenon . | on november 11 , 2001 , following the bioterrorism - related anthrax attacks , the u.s .
postal service collected samples at the southern connecticut processing and distribution center ; all samples were negative for bacillus anthracis .
after a patient in connecticut died from inhalational anthrax on november 19 , the center was sampled again on november 21 and 25 by using dry and wet swabs .
all samples were again negative for b. anthracis . on november 28 , guided by information from epidemiologic investigation , we sampled the site extensively with wet wipes and surface vacuum sock samples ( using hepa vacuum ) . of 212 samples , 6 ( 3% )
were positive , including one from a highly contaminated sorter .
subsequently b. anthracis was also detected in mail - sorting bins used for the patient s carrier route .
these results suggest cross - contaminated mail as a possible source of anthrax for the inhalational anthrax patient in connecticut . in future such investigations , extensive sampling guided by epidemiologic data
is imperative . |
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human immunodeficiency virus ( hiv ) is the most devastating plague facing us as the 21st century begins . in india , nearly 22.7 lakh people are living with hiv infection . progressive destruction of immune system by chronic hiv infection leading to progressive fall in the level of cd4 cells ( < 200/l to < 50 / l ) is known to be responsible for the occurrence of infections by a variety of opportunistic microorganisms . this is also responsible for the recurrent , prolonged , intractable and severe nature of infection in hiv seropositive individuals . knowledge of the pattern of opportunistic infections can often guide therapy when resource limitations hamper the exact diagnosis of the etiological agent . hiv / aids may not be curable but most of the opportunistic infections can be effectively treated . prophylaxis against some of these infections will not only prolong the life of an hiv infected individual but also improve the quality of life . national aids control organization ( naco ) data reveal that tuberculosis is the commonest infection in aids patients , followed by candidiasis , cryptosporidiosis and others . considering this fact , the present study was conducted to determine the prevalence of bacterial , parasitic , fungal infections in hiv seropositive patients . the present study was carried out on 100 hiv seropositive patients in department of microbiology , s. s. g. hospital , vadodara , during the period from jan 2006 to jan 2007 . randomly selected 100 hiv seroreactive patients from in - patients and out - patients department of the hospital were included . their hiv status was confirmed by three tests with different principles / antigens ( enzaids elisa , i.e. enzyme - linked immunosorbent assay , combaids - rs , hiv tridot ) in the integrated counselling and testing centre attached to the department as per naco guidelines . antibodies against immunodominant regions of hiv 1 ( gp 120 , gp 41 ) and hiv 2 ( gp 36 ) and p24 antigens were detected . necessary pre and post - test counseling of the patients was carried out and detailed history was taken . sputum samples , stool samples , oral swabs and cerebrospinal fluid ( csf ) from patients were collected taking all aseptic precautions . ( saline and iodine ) and wet preparation from formal ether concentrated samples were used for the detection of protozoan trophozoites , cysts and helminthic eggs and larva . for the detection of coccidian parasites , smears of stool samples were prepared and stained with modified acid fast method . early morning expectorated sputum samples were collected in sterile containers . for the detection of bacterial pathogens , samples were processed for gram 's stain , zeihl and nelson ( zn ) stain and aerobic culture on selective and enrichment media ( lowenstein jenson media , chocolate agar , macconkey agar ) as per the standard methods . one swab was used for wet mount ( 10% koh , i.e. potassium hydroxide ) and gram stain . the other swab was inoculated on sabouraud 's dextrose agar ( sda ) with antibiotics . the growth obtained was identified for yeasts as per the standard protocol including germ tube test , chlamydospore formation , sugar assimilation , sugar fermentation and urease test . india ink preparation , wet mount , gram stain and culture on sda , chocolate agar and macconkey agar were carried out in csf samples . randomly selected 100 hiv seroreactive patients from in - patients and out - patients department of the hospital were included . their hiv status was confirmed by three tests with different principles / antigens ( enzaids elisa , i.e. enzyme - linked immunosorbent assay , combaids - rs , hiv tridot ) in the integrated counselling and testing centre attached to the department as per naco guidelines . antibodies against immunodominant regions of hiv 1 ( gp 120 , gp 41 ) and hiv 2 ( gp 36 ) and p24 antigens were detected . necessary pre and post - test counseling of the patients was carried out and detailed history was taken . sputum samples , stool samples , oral swabs and cerebrospinal fluid ( csf ) from patients were collected taking all aseptic precautions . saline and iodine ) and wet preparation from formal ether concentrated samples were used for the detection of protozoan trophozoites , cysts and helminthic eggs and larva . for the detection of coccidian parasites , smears of stool samples were prepared and stained with modified acid fast method . early morning expectorated sputum samples were collected in sterile containers . for the detection of bacterial pathogens , samples were processed for gram 's stain , zeihl and nelson ( zn ) stain and aerobic culture on selective and enrichment media ( lowenstein jenson media , chocolate agar , macconkey agar ) as per the standard methods . one swab was used for wet mount ( 10% koh , i.e. potassium hydroxide ) and gram stain . the other swab was inoculated on sabouraud 's dextrose agar ( sda ) with antibiotics . the growth obtained was identified for yeasts as per the standard protocol including germ tube test , chlamydospore formation , sugar assimilation , sugar fermentation and urease test . india ink preparation , wet mount , gram stain and culture on sda , chocolate agar and macconkey agar were carried out in csf samples . out of the total 100 patients in the study , majority were males ( 75% , 75/100 ) . the preponderant age group affected from hiv was 20 - 29 years ( 61% , 61/100 ) . other routes included homosexual contact ( 1% , 1/100 ) , blood transfusion ( 3% , 3/100 ) and vertical transmission ( 2% , 2/100 ) . fever , cough , weight loss , diarrhea and oral thrush were the most common symptoms . candida ( 32.67% , 32/101 ) was the commonest pathogen identified , followed by mycobacterium tuberculosis ( 22.77% , 23/101 ) and cryptosporidium parvum ( 19.8% , 20/101 ) . out of the four patients who presented with meningitis , cryptococcus neoformans was isolated in two patients and in the remaining two patients no fungal pathogen was identified . clinical presentation of study population microbial profile of hiv seropositive patients polymicrobial profile of hiv seropositive patients ( n=30 ) in the present study , out of 100 patients , majority were in the reproductive age group , i.e. 2029 years ( 61% , 61/100 ) , with a male preponderance ( 75% , 75/100 ) . rangnathan and his colleagues reported 77.4% male patients with 81% patients in the age group of 2140 years in their study done at south india . reported the highest number of hiv patients to be in the age group of 2130 years ( 63% ) with majority of male patients ( 94% ) in their study done at manipur . majority of the patients in the study group were laborers ( 42% ) , followed by truck drivers ( 18% ) . majority of them were migrants staying away from home , thereby increasing the risk of hiv infection . heterosexual contact ( 95% ) was the commonest route of transmission in our study which correlates well with other studies . more than 80% rate of heterosexual transmission was reported by naco ( 87.1% ) and rangnathan ( 95% ) . in the present study , majority of patients had more than one symptom and 27% patients had multiple system involvement . fever and weight loss were the predominant symptoms , followed by cough , oral lesions and diarrhea . the clinical profile reported in the present study was similar to that reported in lucknow by ayyagari et al . and in amritsar by aruna aggarwal et al . polymicrobial etiology in 30 of the hiv reactive patients is a significant finding , indicating severity of the infection in this group . in the present study , 43% ( 43/100 ) diseases like bacterial pneumonia occur at a rate many times higher in the hiv infected population than in the general population . m. tuberculosis ( 60.5% , 23/38 ) was the commonest organism among the bacterial respiratory pathogens , followed by klebsiella pneumoniae ( 15.7% , 6/38 ) , pseudomonas aeruginosa ( 10.5% , 4/38 ) , staphylococcus aureus ( 7.8% , 3/38 ) and escherichia coli ( 1.9% , 2/38 ) . aruna aggarwal et al . in their study at amritsar reported m. tuberculosis ( 32.75% ) as the commonest bacterial respiratory pathogen , followed by klebsiella spp . ( 23.8% ) , p. aeruginosa ( 12.69% ) and s. aureus ( 12.69% ) . reported m. tuberculosis ( 28% ) , followed by k. pneumoniae ( 10% ) , streptococcus pneumoniae ( 8% ) and s. aureus ( 4% ) . this difference may be due to geographical differences and also possible hospital acquired superadded infections . oral lesions were present in 40% ( 40/100 ) of patients in the present study . out of 33 isolates from oral lesions , 27 were candida albicans ( 81.8% ) and 6 were non - albicans candida spp . 23.08% ) . oral and esophageal candidiasis ( 57.5% ) is reported as the second most common opportunistic infection among hiv patients from india . it is the second most common fungal infection and the fourth commonest cause of life - threatening illness in aids patients in india . singh et al . reported 7% rate of cryptococcal meningitis in their study done at manipal . an incidence of 3% of cryptococcal meningitis has been reported in hiv infected patients in india . in the present study parvum ( 71.4% , 20/28 ) was the commonest parasite found , followed by isospora belli ( 10.7% , 3/28 ) , giardia lamblia ( 7.1% , 2/28 ) and entamoeba histolytica ( 7.1% , 2/28 ) parvum was reported as the most common enteric parasite by aruna aggarwal ( 51.42% ) and singh ( 43% ) . i. belli was found as the commonest parasite by gupta at jamnagar ( 17.24% ) and kumar at chennai ( 18% ) . differences in the incidence of intestinal parasitic infections can be attributed to the differences in geographical distribution of parasites , sanitary practices and different selection of cases . it is concluded that oral candidiasis , pulmonary tuberculosis and cryptosporidiosis are the commonest opportunistic infections in the hiv seropositive patients in the present study group . improved hygienic practices , regular examination and appropriate antimicrobial prophylaxis can reduce the substantial morbidity and mortality caused by opportunistic infections in patients with hiv infections . with better knowledge and diagnosis of the opportunistic infections , | objectives : the present study was undertaken to determine the prevalence of respiratory , gastrointestinal and other pathogens in 100 human immunodeficiency virus ( hiv ) seropositive patients.settings:this study was carried out on randomly selected 100 hiv seropositive patients from s. s. g. hospital during the period from jan 2006 to jan 2007.materials and methods : sputum samples , stool samples and oral swabs were collected from all the patients and cerebrospinal fluid ( csf ) was collected from symptomatic patients and processed as per the standard protocol .
sputum samples were examined by microscopy and cultured for bacterial respiratory pathogens .
stool samples were concentrated and examined by microscopy for enteric parasites .
oral swabs and csf were also examined microscopically and cultured for fungal pathogens .
csf was also examined for bacterial pathogens.results:a total of 101 pathogens were detected in 60 patients . more than one pathogen was observed in 30 patients .
candida was the commonest isolate ( 32.67% ) , followed by mycobacterium tuberculosis ( 22.71% ) and cryptosporidium parvum ( 19.8%).conclusions : since opportunistic infections are a major cause of mortality and morbidity in hiv seropositive patients , an early diagnosis and effective treatment are required to tackle them .
the type of pathogens infecting hiv patients varies from region to region , and therefore such patients should be constantly screened for these pathogens . |
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leishmaniasis is a group of diseases , which is caused by intracellular protozoan parasites , leishmania and is transmitted through sand flies . cutaneous leishmaniasis which is known as salak in iran is a common disorder in several provinces of the country and is a major health problem in some provinces , such as fars , kerman , and isfahan . it is estimated that about 12 million people are infected with leishmania in various regions of the world , and 350 million people are at risk of leishmaniasis . first - line therapy in the treatment of this disease is pentavalent antimony derivatives , but drug failure and drug resistance are increasing in many regions of the world , especially in iran . in addition to different clinical responses of the patients to the treatment , side effects such as accumulation of drugs in tissues such as spleen and liver , myalgia , pancreatitis , cardiac arrhythmia , and hepatitis are the main problems of using these medicines in the treatment of leishmaniasis . therefore , it seems necessary to study and evaluate the new drugs for the treatment of leishmaniasis . one of the common methods regarding the appropriate treatment of this disease is the use of combination therapy . simultaneous stimulation of the immune system , with stimulating compounds of the immune system , along with treatment with drugs might improve the treatment of this disease . one of the compounds which have attracted researchers attention during the recent years is compounds from purple coneflower plant by scientific name , echinacea purpurea . this plant which is known as the plant to strengthen the immune system is a plant of the asteraceae family , and it had been considered a lot since a long time ago , and it has been used , widely , in traditional medicine . recent studies showed that extracts of this plant increase the resistance of the body against the infectious diseases . it seems that combination therapy of the extract of this plant along with the first - line drug for the treatment of leishmaniasis ( meglumine antimoniate ) might improve the treatment of this disease . the extracts of echinacea plant induce the stimulation of immune system by its immunomodulatory effect , and this might increase the effect of meglumine antimoniate . considering the aforementioned points , this study was conducted to find out the effects of hydroalcoholic extract of e. purpurea in combination with glucantime on the treatment of leishmania major - induced cutaneous leishmaniasis in balb / c mice . e. purpurea plant was provided by zardband company of yasouj from medicinal herb garden in yasouj , in the south of iran . dried e. purpurea plant , including leaf , aerial parts , and root the species of the plants was confirmed in the school of pharmacy of shiraz university of medical sciences , and a herbarium specimens number ( pm189 ) was assigned to it . to prepare the extract , the plant was powered by the electric mill . since the extract should be taken orally , ethanol at 30c , without bitrex , two liters of solvent ( ethanol at 30c ) was added to 900 g of the powder of the plant and was stirred thoroughly . then , another 2 l of solvent was added to the container of extracting after 24 h and finally , another 2 l of solvent ( a total of 6 l ) was added to the specimen for the third times after 24 h. in next stage , obtained extract was concentrated using rotary device after smoothing extract . to facilitate the conversion of extract to dry powder , the extract was concentrated in rotary device for several times and finally , it was reformed to dry powder by freeze dryer machine . at first , thirty 56-week - old female balb / c mice were obtained from the animal house of shiraz university of medical sciences . l. major ( mrho / ir/75/er ) was used in this study . with consideration to this fact that available parasite in culture medium loses its pathogenesis due to various passages , the method of direct transmission of parasites from mouse to mouse was used to infect mice . 0.2 ml of a suspension containing 4 10 amastigotes was injected in the top of mice 's tail base by insulin syringe , subcutaneously . lesions were developed after 25 days of inoculating parasites to the mice , in all mice . the second group received the extract of e. purpurea plant orally with dose of 100 mg / kg . the third group was treated by meglumine antimoniate with dose of 20 mg / kg . combination therapy with the extract of e. purpurea plant along with meglumine antimoniate drug was used in fourth , fifth , and sixth groups where the fourth group received 100 mg / kg of extract and 10 mg / kg of meglumine antimoniate , the fifth group received extract with dose of 100 mg / kg , and meglumine antimoniate with dose of 20 mg / kg and the sixth group received the extract with dose of 200 mg / kg , and meglumine antimoniate with dose of 20 mg / kg . meglumine antimoniate drug in all groups was used by intralesional injection of the drug for 20 days . detection and confirmation of leishmania infection in all of the studied mice group was based on parasitological detection of amastigotes in geimsa - stained smear of the lesions . the length of the major and minor axes of the lesion at the base of the mouse 's tail perpendicular to each other were measured with a direct - reading vernier caliper and the area of the lesion in square millimeters was achieved by calculating ellipse area formula . wallis test were used to study the mean difference of the size of wounds in six groups of studied mice . moreover , the trends of skin lesion changes in each mice group were compared , using anova for repeated measures and post hoc multiple comparison test . the preintervention measures ( time = 0 ) were used for adjustment of trend and grouping comparison . kruskal wallis test were used to study the mean difference of the size of wounds in six groups of studied mice . moreover , the trends of skin lesion changes in each mice group were compared , using anova for repeated measures and post hoc multiple comparison test . the preintervention measures ( time = 0 ) were used for adjustment of trend and grouping comparison . the sizes of the lesion were increased in all mice of the control group by the time . figure 1 shows the mean size of lesions in various periods of time in five groups of studied mice . series 1 : received normal saline ; 2 : echinacea 100 mg / kg ; 3 : echinacea 100 mg / kg and meglumine antimoniate 10 mg / kg ; 4 : echinacea 100 mg / kg and meglumine antimoniate 20 mg / kg ; 5 : echinacea 200 mg / kg and meglumine antimoniate 20 mg / kg ; 6 : meglumine antimoniate 20 mg / kg statistical analysis of the mean size of lesions did not show any significant difference among groups of mice receiving the extract along with meglumine antimoniate with negative control group ( p > 0.05 ) . also , comparing the trend of changes in each mice group , using anova for repeated measures analysis , did not show a significant difference between groups of mice receiving the extract along with meglumine antimoniate with negative control group ( p > 0.05 ) . however , the differences between the group of mice which received meglumine antimoniate alone and the rest of groups were statistically significant ( p < 0.05 ) . although pentavalent antimony derivatives are first - line therapy in the treatment of cutaneous leishmaniasis , but drug failure and drug resistance are increasing in many regions of the world . in some cases , other drugs such as pentamidine , amphotricine b , and paramomycin are used as the second option in cases of resistance to pentavalent antimony derivatives , despite their high toxicity for the patient . resistance to meglumine antimoniate in patients with leishmaniasis has been reported in iran , in recent years . it seems necessary to study about finding alternative medicines for the treatment of leishmaniasis . in this study , a significant difference was not obtained between the size of wounds in groups receiving the extract and drug and control group after studying mean size of created wounds in various groups of studied mice . this means that the combination of the drug with the extract has not improved the treatment of leishmaniasis in infected mice . the reason for the lack of difference in the size of wounds in various groups of studied mice might be the interference of the extract of the plant with meglumine antimoniate drug in the treatment of the disease . support for this statement comes from the observation that the group of mice which received meglumine antimoniate alone had smaller lesion size in comparison to the rest of the groups and that difference was statistically significant . this can be explained by the stronger effect of this compound alone , in comparison to the combination therapy . finally , inadequate consumed dose of the plant can be another reason in the lack of the effect of an extract of this plant in the treatment of cutaneous leishmaniasis in this study . application of topical gel prepared with hydroalcoholic extract of e. purpurea ( 10 or 20% ) for treatment of l. major - induced cutaneous leishmaniasis in balb / c mice has not been encouraging . the authors suggested that dose of extract is one of the reasons for obtaining such unfavorable results . taken together , in this study , the extract of e. purpurea plant at different concentrations along with meglumine antimoniate had not effectively been able to control the growth of l. major parasite in balb / c mice . using the extract of the plant in higher doses and also separating the nonhydroalcoholic compound of this plant is recommended in future studies with consideration to the importance of leishmaniasis and needs for new drugs in its treatment . the study was financially supported by the office of vice - chancellor for research of shiraz university of medical sciences ( grant no . the study was financially supported by the office of vice - chancellor for research of shiraz university of medical sciences ( grant no . | context : progressive resistance of leishmania parasite to available drugs including , meglumine antimoniate , has been reported from various regions of the world , especially iran.aims:this study was conducted to evaluate the effect of hydroalcoholic extract of echinacea purpurea in a combination therapy with glucantime in the treatment of cutaneous leishmaniasis caused by leishmania major.materials and methods : hydroalcoholic extract of e. purpurea was prepared from the plant .
amastigote form of l. major was inoculated to the tail base of thirty mice .
after their tails became wounded , mice were divided into six groups .
the first group was used as control and the second group received 100 mg / kg of echinacea extract ( orally ) .
the third group was treated by meglumine antimoniate with dose of 20 mg / kg .
combination therapy was used for group four , five , and six where the mice received a different concentration of extract ( 100200 mg / kg ) and glucantime ( 1020 mg / kg ) .
the size of the cutaneous lesion on tail base was measured regularly .
findings were analyzed by spss software and using kruskal - wallis test.results:the sizes of the lesion were increased in all mice of control group by the time .
the mean size of lesions in mice receiving the extract and/or receiving the extract along with meglumine antimoniate was lower than those of control mice , but the differences were not statistically significant ( p > 0.05 ) . on the other hand , the differences between the group of mice which received meglumine antimoniate alone , and the rest of groups were statistically significant ( p < 0.05).conclusion : e .
purpurea extract in doses which have been used in this study and combination with meglumine antimoniate was not much effective against l. major in balb / c mice . |
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during recent years , the trend toward laparoscopic approach for hernia repair in children has been increasingly justified . the ability to detect and repair the contralateral opening of internal rings simultaneously , along with safe high ligation of the hernia sac without injury of the vas deference or the spermatic vessels , make laparoscopic approach a reliable alternative to the conventional open techniques ( 1 ) most of these methods employ a laparoscope inserted via an umbilical incision and two lateral ports for instruments to ligate the hernia defect . the necessity for intra - abdominal skills , such as intracorporeal suturing , knot tying , and ( 2 ) recurrence rate after laparoscopic surgery is generally known to be higher than after open surgery . with the increase in laparoscopic inguinal hernia repair , several treatment techniques have developed over the past two decades , aimed at improving the outcome ; the various techniques differ in their approach to the inguinal internal ring , suturing and knotting techniques , number of ports used in the procedures , endoscopic instruments used , mode of dissection of the hernia sac , and extracorporeal and intracorporeal suturing and knotting techniques . i used two 5 mm ports which reduce the port numbers and size , the purse string suturing , and extracorporeal knotting of the hernia sac . the main outcome measurements were correlation between clinical diagnosis , abdominal ultrasound results and the laparoscopic evaluation of contralateral hernia , feasibility of two ports laparoscopic repair of the inguinal hernia , conversion rate , need for additional port , operative time , postoperative pain and requirement of analgesia , hospital stay , recurrence rate , and fat of nonexcised hernia sac and development of postoperative hydrocele . written consent was obtained from the family after getting full information about the surgery and the postoperative squeal . all patients received one dose of antibiotic prophylaxis in the form of ceftriaxone 50 mg / kg at the time of induction of anesthesia . under general endotracheal anesthesia , endopath xcel port ( 5 mm ) with 00 scope inserted supra umbilical by close technique . seconded working port 5 mm was inserted according to the unilaterally and bilaterally hernia under visualization ( figure 1 ) . patient position was changed to trendelenburg position 45 which helped in reduction of the hernia contents and gave better visualization of the rings . proline 3/0 suture mounted needle was passed from the outside through skin and abdominal wall muscles to the peritoneal cavity under visualization ( figure 2 ) . the hernial sac neck was closed by purse string 3/0 non- absorbable proline suture as high as possible ( figure 3 ) . the suture was placed between the peritoneum and the fascia with preservation of the testicular vessels and the vas deference . the needle pushed out the abdomen by the laparoscopic needle holder until it appears outside the abdomen . the knot was fixed deep in the abdominal wall by small snip skin incision by number 11 knife . full inspection of the abdominal wall and the closed defect was done ( figure 4 ) . all patients were asked to come for follow - up at outpatient clinic after 7 days , 2 weeks , 6 months , and 1 year . 90 children including 75 males and 15 females underwent two ports laparoscopic inguinal hernia repair . 70 ( 77.7% ) cases were suffering unilateral inguinal hernia and 20 ( 22.2% ) patients had bilateral inguinal hernia . out of the 20 cases these cases were diagnosed as left congenital inguinal hernia by clinical and ultrasound examinations while the laparoscope shows the metachronous hernia at the time of surgery . the mean operative time for unilateral repairs was ( 15 to 20 minutes ) and bilateral hernia repair was ( 21 to 30 minutes ) . the scars on the abdominal wall were small and minute ( 5 mm incision for umbilical port , 5 mm working port and stitch site ) . there was one patient who got noncomplicated right recurrent inguinal hernia after two weeks from surgery . this patient underwent open right herniotomy where the hernia sac was not closed and proline stitch did not surround all the circumferences of the sac . another case gets stitch sinuses at the proline knot site , which required open surgical removal of the stitch and open herniotomy at the same time . in this case the hernia sac was closed completely by the proline stitch . once the proline stitch removed the sac opened , so , herniotomy and trans fixation was done . inguinal hernia in pediatric age group is a common problem and all the pediatric surgeons are fully familiar with the various aspects of its traditional surgical repair through the groin incision which has a high success rate and acceptable cosmetic results with few complications . by far one of the drawbacks of this conventional technique is inability to rule out the contralateral patent processes vaginalis and synchronous hernia . with the advent of minimal access surgery , many pediatric surgeons accepted it , as a suitable and reliable alternative to the open techniques , considering its superiority for handling tissues during repair of recurrent inguinal hernias and also for its capabilities in regard to justifying and managing the synchronous subtle contralateral hernia . however , there are still some issues regarding the introduction of laparoscopic inguinal hernia repair as the gold standard method , especially taking into consideration the possible longer operative time and the inevitable need for three separate ports which is the case in routine laparoscopic herniotomy techniques . in these works only two ports were used to do laparoscopic hernia repair in children , which increases the benefit of the laparoscopic herniotomy . the modified , new laparoscopic technique improved the diagnosis of ipsilateral hernia , using extracorporeal tying , that yields excellent cosmetic result . out of the 20 cases 5 ( 25% ) case these cases were diagnosed as left congenital inguinal hernia clinically and ultrasound examinations can not detect the metachronous hernia in the right side prior to surgery . laparoscope was superior to ultrasound in diagnosis of the metachronous hernia , while in other studies significant number of children ( 20% ) presenting with unilateral hernias have contralateral patent processes vaginalis [ 5 , 6 ] . the options for detection of contralateral patent processes vaginalis are many , namely , routine bilateral open surgery explorations , use of ultrasonography , laparoscopy , and the wait and watch policy . although laparoscopy proves advantageous over open surgery by precise detection and simultaneous repair of contralateral patent processes vaginalis , its management remains a contentious issue . the current consensus amongst surgeons practicing open surgery favors operating on the symptomatic side alone as the rate of metachronous hernia is significant that it necessitates subsequent surgery in a twentieth of patients . therefore , this advantage of laparoscopic hernia repair may be significant in clinical practice as it gives good diagnosis and also repair for the metachronous hernia . in open surgery , time is consumed in gaining access , obtaining adequate exposure , identification of the hernia sac , and dissection of the cord structures from the sac without harming the important cord structures . in laparoscopic surgery , approaching from within makes the area of interest bloodless , and the magnification renders anatomy splendidly clear , making surgery precise . but the time limiting step remains intracorporeal suturing that places considerable demands on the requirement of hand eye coordination , especially while negotiating the posterior and medial hemicircumference of the internal ring , over the iliac and inferior epigastric vessels . with growing experience and in this study the subfascia purse string suture and extracorporeal subcutaneous knotting with two ports markedly reduce the operative time over the traditional three ports laparoscopic hernia repair with intracorporeal suture ligation . in this study the mean operative time for unilateral repairs was ( 15 to 20 minutes ) and bilateral hernia repair was ( 21 to 30 minutes ) ; the mean duration of surgery was markedly less than the operative time noted in lukong study . the extracorporeal suturing was effective easy not need for special tool or surgical skills which also proved by other studys [ 11 , 12 ] . the difference in postoperative pain following open surgery and laparoscopic surgery is subject to controversy . some report less pain while others report greater pain in the immediate postoperative period following laparoscopic surgery compared with open surgery . parietal pain predominates in open surgery can well be controlled by caudal analgesia . on the other hand , pain perception is multimodal and multifactorial in laparoscopic surgery . in addition to parietal pain caused by port placement , capnoperitoneum causes visceral pain due to stretching ( peritoneal and diaphragmatic ) and acidosis . neither the use of smaller ports nor the use of caudal analgesia would completely obliterate pain following laparoscopy . therefore , the decrease in the size of the incision does not necessarily translate into a proportionate decrease in pain . hence , the difference in postoperative pain between laparoscopic surgery and open surgery is not significant enough to rate either surgery superior . in this study , the intraperitoneal gas pressure was located less than in other studies due to the less number of ports and no need for intra - abdominal surgical maneuvers . which reduced the post - operative pain . lee and liang introduced the endo - needle designed specifically for laparoscopic extraperitoneal closure of the patent processus vaginalis . they reported no complications related to the procedure and a remarkably low recurrence rate ( 0.88% ) . prasad et al . used a stainless steel curved awl and a 1.7 mm telescope to safely perform needlescopic inguinal herniorrhaphy . shalaby et al . used rn for closure of iir in 150 patients successfully with excellent cosmetic results without any recurrence . chan and tam introduced the saline injection and needle sign to reduce the recurrence rate in his series . used the tnh technique in 67 repairs , and 146 repairs were performed using the seal technique . they stated that seal resulted in marked reduction of operative time when compared to the tnh technique ( unilateral : 15 versus 25 minutes ; bilateral : 25 versus 40 minutes ) . they added that avoiding the vas deferens and gonadal vessels during the seal repair in boys may leave a small gap at the internal ring as well as leaving the hernia sac in situ , which has the potential to contribute to a higher incidence of recurrence in male patients . they believe that ligation of the internal ring leads to scarring and obliteration of the space distally . fluid accumulating in the distal sac postoperatively often reabsorbs spontaneously and does not necessitate additional intervention . in this study there was no hydrocele detected as a postoperative complication in spite of no excision of the sac and the recurrence rate was 1% recurrence . several piercings of the peritoneum by rn around the neck of the sac may add fixation of the suture at this level that prevents migration of the suture distally initiating recurrence . this may explain the high incidence of recurrence in the series of manoharan et al . and bharathi et al . however , chan and tam reported a very low recurrence rate ( 1% ) after refinement of the technique by using tnh and injecting saline around the vas and vessels and using the needle sign to avoid damage to the testicular vessel and vas . prasad et al . reported no recurrence in their early small series of 8 cases . the results of this study confirm the safety and efficacy of laparoscopic hernia repair with two ports in congenital inguinal hernia in children . it resulted in good diagnosis and management of metachronous hernia and marked reduction of operative time , less recurrence , no hydrocele formation , excellent cosmetic , and no postoperative pain results . | introduction .
several laparoscopic treatment techniques were designed for improving the outcome over the last decade .
the various techniques differ in their approach to the inguinal internal ring , suturing and knotting techniques , number of ports used in the procedures , and mode of dissection of the hernia sac .
patients and surgical technique .
90 children were subjected to surgery and they undergone two - port laparoscopic repair of inguinal hernia in children . technique feasibility in relation to other modalities of repair was the aim of this work .
90 children including 75 males and 15 females underwent surgery . hernia in 55 cases was right - sided and in 15 left - sided .
two patients had recurrent hernia following open hernia repair .
70 ( 77.7% ) cases were suffering unilateral hernia and 20 ( 22.2% ) patients had bilateral hernia . out of the 20 cases
5 cases were diagnosed by laparoscope ( 25% ) .
the patients ' median age was 18 months .
the mean operative time for unilateral repairs was 15 to 20 minutes and bilateral was 21 to 30 minutes .
there was no conversion .
the complications were as follows : one case was recurrent right inguinal hernia and the second was stitch sinus . discussion .
the results confirm the safety and efficacy of two ports laparoscopic hernia repair in congenital inguinal hernia in relation to other modalities of treatment . |
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both eukaryotic and prokaryotic cells contain important gene - regulating elements , located within promoter regions . it is known that promoter sequences possess special signatures to distinguish them from the rest of the genome sequences . a few core promoter elements have been detected , of which the most common elements are the cpg island , tata box , initiator ( inr ) , downstream promoter element ( dpe ) , and tfiib recognition element ( bre ) , but these promoter elements may not be universal . recently , a group from the encyclopedia of dna elements ( encode ) project published chromatic maps to mark histone modifications within nine cells of different types from the human genome , drawn from the results from chromatin immunoprecipitation sequencing ( chip - seq ) analyses of these same cells . from each of these nine cells , ernst et al . published 15 chromatin states of the human genome . among them , the first three states represent promoter regions : state 1 ( active_promoter ) , state 2 ( weak_promoter ) , and state 3 ( poised_promoter ) . the information gained from the exploration of promoter elements and transcription starting sites will enable researchers to produce more accurate promoter prediction algorithms , which in turn will yield deeper insight into the process of transcription initiation and gene functions . here , we publish frequency profiles of the chromhmm promoter states of the human genome , at 200bp intervals , paying particular attention to the existence of promoter elements , such as cpg islands , tata boxes , inr , dpe , and recognition elements from the transcription factors that bind with polymerase ii ( bre ) ( signal features ) , together with transcription starting sites , in terms of size , overlapping patterns , and locations . ernst et al . applied unsupervised learning methods to convert the broad histone track 's results from chip - seq analyses into specific points that indicate promoter regions on chromatin maps for 15 chromatin elements within the genome . these 15 chromatin states were chosen because of their rich biological content , as well their well - established presence in previous research . the resulting files are hosted by the encode analysis data hub , which is available for public download under the encode data release policy ( http://genome.ucsc.edu/encode/downloads.html ) . we downloaded the bed files for the chromatin signal tracks of the nine encode cell types . within them , chromatin states 1 , 2 , and 3 represent active , weak , and posed promoters , respectively , which differ only in regard to the levels of expression in various biological assays . these states were defined , based purely on 9 chromatin marks , and their input controls across 9 cell types . depending upon the cell type , slight variances were found , as seen in table 1 . for example , the gm12878 cell line has an active promoter consisting of 14,674 blocks and occupies 0.80% of the whole genome , whereas k562 has 14,951 blocks and occupies 0.68% of the whole genome , for chromatin state 1 . to overcome this slight variance in cell lines , we divided the promoter regions uniformly at 200-bp resolution , regardless of the 9 cell line block boundaries . 1 shows our java code , which relies on java 's oracle interface ( oracle database 12c standard edition ) and implements regular expressions , based on predefined classes from the java.util.regex package , in order to pattern - match different promoter elements , such as cpg islands , tata boxs , inr , dpe , and bre , for each 200-bp window . the code also builds upon formal grammar rules based on the structural properties seen in promoter sequences , though these patterns usually apply only to the core promoters . our java code produces an output profile consisting of the 18 required fields as follows ( fig . the first three fields are : the name of the chromosome , the starting position of the feature in the chromosome , and the ending position . 2 shows that the nucleotide sequences starting from 28337 to 23537 ( 200 bp ) in chromosome 1 contains 1 occurrence of a tata box , 6 occurrences of inr , 0 occurrences of bre , and 6 occurrences of dpe . the next 9 fields indicate the chromhmm annotation of 9 cell lines ( k562 , gm12878 , h1hesc , hepg2 , hmec , hsmm , huvec , nhek , and nhlf ) . the last two fields indicate the existence of database of human transcription start sites ( dbtss ) data , and the gc ratio , respectively , where the hg19promoter file for dbtss data was used for indexing ( ftp://ftp.hgc.jp/pub/hgc/db/dbtss/dbtss_ver8/sanger_data/ ) . it is due to the fact that we divided the regions into 200 bp lengths whereas the core promoter elements are known to exist typically within 50 bp ( base pairs ) upstream to 50 bp downstream of the tss , and that we are including the poised_promoter state of the chromhmm . from the chromhmm human promoter blocks , the results of our statistical survey on 176,579 units show that dpe sequences have the highest frequency among the four chosen motifs in promoter elements , appearing in about 97.1% of the chromhmm promoter regions , meanwhile inr is seen in 92% of promoter regions , and bre is seen in only 17.3% of the promoters ( it is known that inr usually occurs only about half of the human promoters ) . for example , we can expect to find bres within gc - rich areas , meanwhile areas with tata boxes are typically gc - poor . in total , there are 9,435 chromhmm promoter blocks in the dbtss locations ( approximately 5.34% ) . the number of 200-bp blocks that contain all four promoter elements was 176,579 . among them , only 9,436 of the blocks contain dbtss data . for example , the promoter blocks that do not contain any of the promoter elements tend to have a higher gc ratio ( 0.68 ) where a dbtss location exists . one of the strengths of our work versus previous studies is that we profiled the core elements separately , based on the three classes of promoter states ( active_promoter , weak_promoter , and poised_promoter of the chromhmm ) . a more detailed analysis of frequency profiles in regard to these three states is beyond the scope of this paper . however , the complete frequency profiles and java code are published through github ( https://github.com/kyungeunlee/motif ) . we believe that the frequency profiles presented of the three promoter states can be helpful for bioinformaticians to gain a deeper understanding of the characteristics of the various promoter regions and to improve upon old methods of core promoter analysis algorithms for their specific research needs . | recently , the encyclopedia of dna elements ( encode ) analysis working group converted data from chip - seq analyses from the broad histone track into 15 corresponding chromatic maps that label sequences with different kinds of histone modifications in promoter regions . here
, we publish a frequency profile of the three chromhmm promoter states , at 200-bp intervals , with particular reference to the existence of sequence patterns of promoter elements , gc - richness , and transcription starting sites . through detailed and diligent analysis of promoter regions
, researchers will be able to uncover new and significant information about transcription initiation and gene function . |
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sleep is essential for everybody and any disturbance in the natural sleep patterns can cause mental problems , and diminish the function of the brain . although sleep disorder may be seen at any age , the elderly usually experience more problems . as a matter of fact , the prevalence of sleep disorder is estimated to be about 10 - 20% in the general population . sleep disorders significantly influence the individuals quality of life resulting in a noticeable decrease in the physical , emotional and social aspects of their daily functions . the effect of sleep disorders and the illnesses they cause are seen more amongst the elderly compared to young people . the elderly often complain of problems such as difficulty in going to sleep , disintegration of sleep , short duration of sleep , low quality of sleep and drowsiness during daytime . the most common sleep disorder in the elderly is insomnia from which 50% of the elderly suffer . sleep disorder in the elderly results in short memory , concentration disorder , chronic fatigue , visual auditory and behavioral disorders , higher risk of falling down , disability in daily life activities , napping during daytime , depression , and high intake of sleep medications . diseases , medications , environmental factors , depression and anxiety are among the most common causes for hospitalized clients sleep disorders . studies have shown that insomnia can result in fatigue , irritability , aggression , and low tolerance to pain . since the elderly have the highest rate of incidence of hospitalization ( 38% ) , and the longest duration of stay in hospitals ( 45% ) , they experience the highest level of sleep disorder . sleep related changes are among the main concerns of the elderly and should not be underestimated . although the prevalence of sleep disorders grows by age , these problems are not inevitable . investigation of individuals sleep health necessitates close detection of sleep habits and behaviors as well as environmental factors affecting sleep . a comprehensive study of sleep behavior and appropriate interventions can help the elderly obtain their needed sleep and rest in order to have their highest function . therefore , the researcher decided to conduct a study on sleep related behaviors of hospitalized elderly people . the population studied comprised 300 elderly subjects , selected through convenient sampling , who were 60 years old and over , and were hospitalized in different hospitals in zahedan . the researcher frequently visited all wards of hospitals in zahedan , after obtaining the necessary official permissions , and selected the subjects based on the inclusion criteria until reaching the needed sample size . the data were collected with a questionnaire structured by the researcher . the validity ( content and face ) and reliability ( test - retest ) of the questionnaire were confirmed . the questionnaire , completed through interviews , included two sections of demographic characteristics , and sleep related behavior . descriptive statistical tests such as concrete and relative frequency , mean , sd and inferential tests ( x ) were employed to detect the association between the qualitative variables . about 54% of the elderly were male and 46% female with the mean age of 68.8(5.7 ) years . about 69.6% lived in urban areas and 30.4% in rural areas . with regard to marital status , 69.7% were married and 29.3% were widowed . about 70% of the subjects were illiterate , 4.3% had reading and writing literacy , 12% primary school , 5.7% a high - school education , and 3% an associate and bachelor 's degree . about 76.3% of the subjects suffered from more than one chronic disease , 17% from one , and 6.7% suffered from no chronic diseases . the hospitalization was due to a chronic disease for 72.4% and acute diseases for 27.6% . in the findings of this study showed that 62% of the subjects suffered from sleep disorder with a higher prevalence among female elderly subjects ( 68.1% ) compared to male subjects ( 56.8% ) ( table 1 ) . some other studies reported the prevalence of sleep disorder in the iranian elderly to be 67% which is consistent with the result of the present study . epidemiologic data show that 12 - 54% of the elderly suffer from sleep disorder which is more prevalent among women compared to men . in a study by mehra et al . on about 2400 elderly subjects , prevalence of insomnia was reported to be 54% in women and 36% in men . in this study just as the current study the percentage of women suffering from insomnia is higher than the men . association of sleep disorder with demographic variables of the hospitalized subjects in zahedan the study by foly et al . on over 9000 elderly people at ages of 65 and over found that , more than 42% of the subjects complained of a delay in going to sleep and sleep disintegration . ( 2006 ) showed that 390 hospitalized elderly in kashan had low sleep quality , with women having lower sleep quality than men . ( 2008 ) studied 115 subjects over the age of 18 and reported that 59% had insomnia . about 67.9% of those aged 41 - 65 had insomnia with the highest frequency being in women ( 74.4% ) . they reported 60.9% sleep disorder in people over 65 years of age . in a study conducted by ohayon and lemoine , the prevalence of insomnia was estimated to be 20 - 40% in west europe with the highest level being in women and the elderly . in another study in japan , the level of sleep disorder amongst the hospitalized elderly in japan was reported to be 18.7% in men and 20.3% in women . the results of the above researches show a higher prevalence of sleep disorders among the iranian elderly possibly due to life style , socioeconomic and cultural factors , and low awareness of the importance of sleep and its role in elderly health . in a survey conducted by gallup on 1000 americans at the age of 50 and over ( 43% were 65 or over ) showed that 80% of the subjects believed sleep is important for elderly people 's health . about 45% believed they needed more sleep compared to the youth , and 25% had sleep problems . various studies as well as the present study showed higher rates of sleep disorder among women compared to men . based on former studies , women experience sleep disorder twice as much as men due to various factors such as stress , diseases , sleeping place and being over weight . sleep disorder in women can also be due to post hormonal changes especially estrogen . as women have more familial and social responsibilities , compared to men , the stress caused as a result of this can be another reason for higher prevalence of sleep disorder among women . impaired sleep patterns caused during pregnancy and breast feeding may cause disordered sleep patterns even at the age of menopause . the results of this study showed that there is a significant association between education and sleep disorders ( p = 0.048 ) ( table 1 ) . this means that less sleep disorders are seen among the elderly with a higher education compared to those with lower education . showed that the quality of sleep is poorer among the elderly with less literacy compared to those of other age groups . also concluded that there is a higher rate of sleep disorder among those with lower education . higher rate of sleep disorders among illiterate or low educated elderly people may be due to the effect of education on the level of awareness and efficient problem solving methods . low educated elderly people also face more socioeconomic problems and stress with a negative effect on their natural sleep habits , compared to those with higher education . based on the present study this finding is not in accordance with the results of izadi onji et al . which reported sleep quality to be better among the married compared to those divorced and widowed . this difference can possibly be due to having more family support that the widowed elderly receive after losing their spouses . the results of the present study showed that there is a significant association between residential area and sleep disorder ( p = 0.014 ) so that those who live in urban areas ( 66.3% ) complained more of sleep disorders compared to those who live in rural areas . this is possibly due to the difference between the urban life style of the elderly and its related stress versus rustic elderly life and its simple and low expectations ( table 1 ) . although sleep disorders increase by age , they are not necessarily a part of the aging process but are a result of secondary physical and mental diseases as well as their related medications . with regard to the high prevalence of sleep disorders and their complications and health related outcomes in the elderly , the main concern is the bilateral association between sleep disorder and physical diseases among them . hypertension , depression , cardiovascular diseases and cva put people at a higher risk of sleep disorders and vice versa ; therefore , one of the major reasons for sleep disorders of the studied elderly in this study can be chronic diseases . the results of the present study showed a significant association between hospitalization due to chronic and acute diseases and sleep disorders ( p = 0.01 ) so that the elderly hospitalized due to a chronic disease were reported to have more sleep disorders compared to those due to acute diseases . another finding of the present study is less complaint of sleep disorder among those with one chronic disease ( 51.1% ) compared to those with two or more ( 66.5% ) ( table 2 ) . association of sleep disorder with occupation , type of disease , number of chronic diseases in the elderly in hospitals of zahedan most of the elderly regularly take several medications for their chronic diseases which worsen their sleep disorder . these medications are : beta blocker , bronchial vasodilators , corticosteroids , anti congestive , diuretics and other cardiovascular medications , especially anti depressants . on the other hand , sleep disorder itself requires the elderly to take even more medication ( table 3 ) . frequency distribution of the subjects sleep and rest behaviors the results of the present study showed that 16% of the studied elderly always , 28.7% often , and only 16.7% never used to take sleep medications to go to sleep , reflecting the high intake of sleep medications among them . it increased by 60% in us within 2000 - 2006 , and american and european studies report 50% of sleep medications to be taken by those of 60 years of age and over . numerous studies have shown a vast number of side effects of sleep medications that can even increase mortality . therefore , physicians should avoid excessive prescription of sleep medications and the elderly should be given necessary education about their side effects , and the prevention of self medications . sleep health includes sleeping habits and behaviors as well as environmental factors that can affect sleep . having a regular schedule for sleeping and waking up is among the healthy behaviors affecting sleep . the findings of the present study showed that 16% of the studied elderly always , 28.7% often and 23.7% sometimes have a certain schedule for their sleeping and waking up while 15% seldom and 16.7% never had such a schedule . going to bed and getting up at a certain time is recommended ( table 3 ) . this seems to set ones sleep and waking up to a certain time and to decrease the delay in going to sleep . the findings of the present study show that from the 300 elderly people studied , 27.3% always , 27% often , 25.3% sometimes , 12% seldom , and 8.3% never took a nap during the day ( table 3 ) . foley states that an increase in age is not a direct cause of napping but it gives the elderly more chance and time to nap . a study showed that a nap during the day can complete night sleep with the only negative effect of causing a delay in going to sleep at night . izadi onji et al . reported no association between napping during the day and sleep quality , which needs further investigation . finally , the elderly are recommended against frequent and long naps during the day , and are advised to go to bed just for sleeping and having sex . the present study showed that 56.7% of the subjects were not involved in drug abuse , did not have a big diner , drink alcohol , or smoking cigarettes before sleep . however 21.3% used drugs , 7.7% smoked cigarettes and 4.7% had a big diner before sleeping ( table 4 ) . however , giving up this habit is very hard for the elderly , and needs education and a great deal of time . with regard to the important role of sleep in the quality of life of the elderly , it is crucial that we detect the causes of sleep disorders amongst them , and educate and motivate them to obtain appropriate behavior , and prevent them from inappropriate sleep behaviors . therefore , strategies should be taken so that the elderly regularly assess their sleep habits and behaviors as a part of their health and try to promote the quality of their sleep . since , nurses spend more time with the patients in hospitals compared to other health team members , they are in a better position to detect sleep disorders and educate the patients about healthy sleep behaviors . inappropriate sleep behavior seems to be due to lack of awareness which can be decreased or amended through the necessary education | background : sleep is an essential need in every individual 's life . a disorder in the natural sleep can cause physical and mental problems .
the elderly are usually faced with more sleep problems .
therefore , the present study aimed to define sleep behavior among the elderly hospitalized in zahedan.materials and methods : this is a descriptive analytical study conducted on 300 elderly people aged 60 years and over who were hospitalized in zahedan . in this research convenience sampling method was used and the research tool was a questionnaire .
the data were collected through interviews .
descriptive ( frequency distribution ) and inferential ( x2 ) statistical tests were employed to analyze the data.findings:the results showed that 62% of the hospitalized elderly people ( total of 300 ) had sleep disorder .
about 44.7%took sleep medication in order to sleep , and only 16.7% did not take sleeping medications .
about 44.7% had no special schedule for the time of their sleeping and waking up , and 4.3% were involved in drug abuse and smoking , and had a big dinner before sleeping .
there was a significant association between sleep disorder , gender , education , living in urban or rural areas , the cause of hospitalization , and suffering from a chronic diseaseconclusions : sleep disorder and inappropriate sleep related behaviors had a high prevalence among the elderly .
with regard to the important role of sleep in the quality of life of the elderly , detection of the reasons of sleep disorder , motivating them to practice an appropriate sleep behavior , and preventing them from having inappropriate sleep related behaviors are crucial issues . |
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substance which has at least one characteristic dimension between 1 - 100 nm is termed as nanomaterial . nanotechnology can be defined as the science and engineering involved in design , synthesis , characterization and application of materials and devices whose smallest functional organization in at least one dimension is on nanometer scale ( one billionth of meter).1 constituents of dental implant and its surface treatment determine electrical charge and chemical nature of implant surface which directly influence osteoblast adhesion and protein adsorption . grade 5 ( ti6al4v ) is an alloyed grade specially made for dental implant procedures because of its superior fatigue properties and yield strength . hydrophilic property is influenced by chemical composition of dental implant . in light of interactions with cells , tissues and biological fluids hydrophilic surfaces are preferred over hydrophobic surfaces.2 after dental implantation two different types of reactions can occur . the first reaction includes the development of a soft tissue fibrous capsule around the metal implant termed as fibro osseous integration . the second reaction related to direct contact of bone to implant surface without interposing soft tissue between bone and dental implant . implant surface composition , topography , hydrophilicity , geometry and surface roughness determine rate and quality of osseointegration.3 rapid and early osseointegration is significantly associated with long term success of oral implant . cochran et al.4 did a histometric analysis in foxhound dog 's canine mandible and demonstrated that the surface roughness of oral implant is a determining factor for biomechanical fixation and osseointegration . wennberg et al.5 performed evaluation of screw shaped implants histomorphometrically and concluded that bone implant contact ( bic ) is dependent on the rough surface . surface roughness increases specific surface area due to increase in thermodynamic reaction potential.6 increase in specific surface area will increase surface active sites for interaction with cells , tissues and biological fluid . depending on the scale features , surface roughness can be divided into three levels : a ) macro level topography b ) micro level topography c ) nano level topography . macro and micro level surface features demonstrated to be efficient in enhancing in vitro and in vivo7 biological events . a variety of techniques has been employed to create micron scale surface characteristics on the surface of biocompatible metals but macro and micron scale features can only have an indirect influence on cellular activity so these features have limited actions and performances . now it is documented that interaction between material and body tissue is principally governed by nanometric implant surface modifications.8 various approaches have been devised and implemented to create nano rough surfaces that can directly influence the biological performance of oral implant.9 this systematic review aimed to summarize the role of nanoscale surface modifications of titanium dental implants for the purpose of improving osseointegration and various techniques that can impart nanoscale topographic features to dental implants . a search of online studies was conducted using pubmed , medline and google scholar databases . a systematic search of english language dental literature was performed for articles published between december 1987 to january 2012 . the search was conducted to identify studies related to response of nanotopography on the bone implant contact ( bic ) , osteoblast response , changes occurring on the titanium implant surface . the aim was to identify different techniques used to generate nano topography on dental implants up to and including january 2012 . the systematic search was further enhanced by hand search through following journals : nanostructured materials , journal of biomedical materials research part a , dental material , journal of biomedical materials research , clinical oral implants research , trends in biomaterials & artificial organs , journal of long - term effects of medical implants , biomaterials , materials and technology , journal of materials science : materials in medicine , small , nano letters , acta biomaterialia , advanced engineering materials , international journal of nanomedicine , nanotechnology , progress in materials science , journal of biomedical materials research part b : applied biomaterials , materials science and engineering : c , european journal of oral implantology . electronic search was conducted by applying the following free text and medical subject headings ( mesh )
# search 1 : dental implant , dental implants , dental implantation , endosseous implant , endosseous dental implantation , titanium implants . titanium implant surfaces . # search 2 : nanotechnology , nano surface , nanotopography , nano surface characteristics , nano roughness , nano surface modification . # search 3 : combination of # and # ( not including case reports ) . # search 1 : dental implant , dental implants , dental implantation , endosseous implant , endosseous dental implantation , titanium implants . # search 2 : nanotechnology , nano surface , nanotopography , nano surface characteristics , nano roughness , nano surface modification . # search 3 : combination of # and # ( not including case reports ) . all studies were screened according to inclusion criteria :
physicochemical studies of novel dental implant surfaces.in vitro studies illustrating cellular responses in different implant surfaces such as smooth or rough surfaces.in vivo action of various implant surfaces.studies related to osseointegration and clinical performance from different implant surface modification techniques.studies of which complete text could be assessedpublished in english language . physicochemical studies of novel dental implant surfaces . in vitro studies illustrating cellular responses in different implant surfaces such as smooth or rough surfaces . in vivo action of various implant surfaces . studies related to osseointegration and clinical performance from different implant surface modification techniques . studies of which complete text could be assessed published in english language . case reportscase studiesbook chaptersconference proceedingsin vivo studies including sample size less than 5 were excludedin vitro studies include agents that do not use bone related cellsstudies including evaluation of currently commercially available surfacesstudies whose title did not meet aim of present review
conference proceedings in vivo studies including sample size less than 5 were excluded in vitro studies include agents that do not use bone related cells studies including evaluation of currently commercially available surfaces studies whose title did not meet aim of present review after applying search strategy , total of 101 studies displaying the used terms were found . selection of relevant studies was conducted as follows : initial assessment of each publication for relevance using information presented in abstract . when the abstract failed to provide sufficient information , a print of full paper was obtained . data extraction was done by one reviewer . after checking studies that did not fulfill selection criteria , the final outcome of the search using search strategy in pubmed , medline and google scholar databases yielded 101 titles using search terms like dental implants and surface modifications or surface modifications on dental implant surfaces . the articles in which nanotechnology and dental implants were described along with the illustration of techniques to produce nanosurface were selected and they were 39 in number . out of 39 studies seven4,5,6,7,10,11,12 studies demonstrated that bone implant contact increases with increase in surface roughness . two10,11
in vitro and one6 review article concluded that amount of bone contact to implant is dependent on surface roughness . two10,13
in vitro studies showed difference in bone implant contact between acid - etched and machined surfaces , influence of electrical stimulation on anodized surfaces , calcium ion coated on titanium showed better bone response than titanium alone ( uncoated ) . eight studies9,16,17,18,19,20,21,22 concluded that osteoblasts preferably adhere to nano structure as compared to smooth surface . seven9,16,17,19,20,21,22
in vitro studies and one18
in vivo study concluded that nano topography influence cell adhesion and osteoblastic differentiation . four16,25,26,27
in vitro studies , one2
in vivo study and one review24 described role of nano topography in influencing dental implant surface . nine28,29,31,33,34,35,36,38,40
in vitro studies and four30,32,37,39
in vivo studies described various techniques such as electrochemical method , ion implantation , fluoride modification , non lithographic method , chemical oxidation , sol - gel technique , plasma spray , magnetron sputtering and ultra violet treatment . nanotopography can determine various cellular processes like cell orientation , alignment , differentiation , migration and proliferation by regulating cell behavior . nano topographic modifications are described as surface nano roughness produced by adding nano features to the implant surface . oh et al.23 illustrated that nanoscale technology can alter oral implant surface at an atomic level and may influence the chemical composition of these implant surfaces . dental implant surface structure , morphology and chemistry can be changed by two ways : chemical methods , physical methods . principal function of these techniques is to modify the implant surface characteristics such as increasing bone formation to improve periimplant osteogenesis , improvement of corrosion and wear resistance and removal of surface contaminants.10 chemical method is the most commonly used method to modify titanium surface at nanoscale . chemical method includes i ) anodic oxidation ( anodization ) ii ) acid treatment iii ) alkali treatment iv ) combination of anodization and chemical etching v ) hydrogen peroxide treatment vi ) sol - gel treatment vii ) chemical vapor deposition viii ) combination of chemical vapor deposition and sol - gel method.13 according to bagno and di bello41 surface roughness , composition and surface wettability are altered by chemical surface modification of titanium . i ) anodic oxidation : smooth titanium surfaces can be successfully transformed into nano tubular structures with diameter less than 100 nm with the help of anodization.42 by modifying parameters like voltage , current density and chemistry of electrolyte one can control physicochemical properties of surfaces,24 spacing and diameter of nano tubes.17 on titanium surfaces , anodization also form pillar like nanostructures with tunable size as well as deposition of long nano tube arrays ( 10 m).18 for example , multi walled nano tubes and nano hydroxyapatite coatings ( 15 - 25 nm ) have been deposited on titanium that results in improved bioactivity.28 ii ) acid treatment : acid treatment serves to produce uniform and clean implant surface by removing contamination and oxide layer formed on implant surface . the acids commonly used are hydrochloric acid , hydrofluoric acid , nitric acid and sulfuric acid . takeuchi et al.25 evaluated the efficiency of three acids h2so4 , na2s2o8 , hcl decontamination to ti surfaces as pretreatment for implant surface modifications and concluded that decontamination done by hydrochloric acid was an excellent treatment . by combining strong acids or bases and oxidants nano pits networks ( pit diameter 20 - 100 nm ) can effectively be generated on titanium , ti6al4v , crcomo alloys and tantalum.19 various parameters such as nano roughness , wettability , surface topography as well as thickness of protective oxide layer can be controlled by modulating temperature , length of exposure and composition of etching solution.26 moreover by making variation in nature of etching solution it is possible to incorporate selected elements ( fluorine , which has antibacterial effects29 and also contributes to bone formation30 on nano surface of titanium . iii ) alkali treatment : it is the method in which the titanium implant is immersed in either sodium or potassium hydroxide followed by heat treatment at 800 for 20 minutes that is followed by rinsing in distilled water . this method results in the growth of a nano structured and bioactive sodium titanate layer on dental implant surface . on immersion in simulated body fluid ( sbf ) this bioactive surface acts as a site for nucleation of calcium phosphate . through ion exchange there is release of sodium ion from sodium titanate results in formation of ti - oh . when negatively charged ti - oh reacts with positively charged calcium ion from sbf leads to the formation of calcium titanate . in calcium titanate , phosphate and calcium ion are present which develop into apatite crystal that can provide favorable conditions for bone marrow cell differentiation . iv ) combination of anodization and chemical etching : these two methods are combined to create metal or polymer composites with improved biological properties . anodized nano tubular titanium has been coated with sodium hydroxide treated nano porous poly ( lactic - co - glycolic acid ) ( plga ) that results in stimulation of cell activity but when compared with anodized titanium no significant difference was seen.11 a combination of hydrothermal treatments ( tuning concentration , temperature , reaction medium composition and time duration ) and sodium hydroxide has been employed to titanium to create wide variety of unique nano structures such as octahedral bipyramids , nano flowers , nano needles , nano rods and mesoporous nanoscaffolds.31 v ) hydrogen peroxide treatment : this method leads to oxidation and chemical dissolution of the titanium dental implant surface . reaction takes place between hydrogen peroxide and titanium dental surfaces leads to formation of titanium peroxy gels . control on treatment time tavares et al.32 concluded that immersion in simulated body fluid lead to development of thicker layers of titania gel which is beneficial for deposition of apatite crystals . vi ) sol - gel method : it is one of the widely used methods for depositing cap , tio2 , tio2-cap composite and silica - based coatings on implant surface materials . this method leads to the formation of sol which is a uniform suspension of submicroscopic oxide particles in liquid by the procedure of controlled hydrolysis and condensation . factors such as surface roughness , chemical pretreatment and sintering temperature determines the degree of adhesion of tio2 sol gel coatings on substrate ( implant surface ) . bajgai et al.33 studied bioactivity of titanium coating and concluded that when substrate immersed in simulated body fluid for 1 to 15 days results in faster growth of apatite crystals in gel containing titania . nishimura et al.20 illustrated a process of dual acid - etching on titanium dental implant surfaces in a rat model in order to deposit capo4 nano scale features . by the deposition of discrete 20 - 40 nm nanoparticles on a dual acid - etched implant surface led to early bone healing and enhanced mechanical interlocking with bone . gutwein and webster21 investigated osteoblast viability and its proliferation in presence of nano phase titania and alumina particle and concluded that nanoparticles impose positive impact on osteoblast viability and its proliferation . high bond strength between implant surface and nanoscale coating is due to high electron density at atomic level . vii ) chemical vapor deposition : in this procedure chemical reaction takes place between implant surface and chemicals present in the gas phase that will lead to deposition of compound ( non - volatile ) on the oral implant substrate . popescu et al.34 did comparative study and concluded that by the process of chemical vapor deposition , metallic surface properties can be modified at the nanoscale level . viii ) combination of chemical vapor deposition and sol - gel method : metallic surface properties can also be improved by these methods.34 with the help of these techniques niobium oxide and diamond like carbon nano topographies has been deposited on titanium and other substrates which improve the bioactivity of implantable metals.35 various physical techniques had been employed to create bioactive nanosurface on biocompatible metal surface . i ) plasma spraying : this method is able to create a nano engineered surface with less than 100 nm in dimensions . this process includes elimination of surface contaminants with the help of vacuum followed by deposition of charged metallic ions or plasma guided by kinetic energy on dental implant surface . this process allows coating of various materials like au , ti , ag on wide range of materials such as polymers , metals or ceramics . this process is widely used for deposition of calcium phosphate coatings ( ha ) onto dental implant surfaces to modify its bioactivity . regardless of clinical success there are disadvantages of plasma - spraying method such as variation in composition of coatings , non - uniform thickness of deposited layer and lack of long - term adherence of the coating to the substrate material that can pose health hazards and affect the long term stability of dental implants.43 ii ) sputtering : this process involves ejection of atoms or molecules by bombardment of high - energy ions that results in deposition of bio ceramic thin films . this method provides greater adhesion between the coating and substrate along with greater control on properties of coating . this method also improves mechanical properties like wear and corrosion resistance , biological activity and bio compatibility . vercaigne et al.14 did histological evaluation on trabecular bone of goat using tio2 grit blasted and sputtered cap implants . the main drawback of this technique is that this process is very slow as well as deposition rate is also very low . the slow deposition rate is improved by radio frequency magnetron sputtering.37
magnetron sputtering : this is a method of depositing viable thin - film coating . this process maintains the bioactivity of hydroxyapatite coating along with the preservation of mechanical properties of metal . custom - built sputter deposition chamber is used for deposition of coatings at room temperature . wolke et al.38 concluded that hydroxyapatite coating deposited by magnetron sputtering was dense and uniform.radio frequency sputtering : this method involves deposition of thin films of calcium phosphate on substrate metal surface . the main advantage of this method is strong adhesion of coating to metal surface , crystalline nature of coating and ca : p ratio can be altered easily . this process maintains the bioactivity of hydroxyapatite coating along with the preservation of mechanical properties of metal . custom - built sputter deposition chamber is used for deposition of coatings at room temperature . wolke et al.38 concluded that hydroxyapatite coating deposited by magnetron sputtering was dense and uniform . radio frequency sputtering : this method involves deposition of thin films of calcium phosphate on substrate metal surface . the main advantage of this method is strong adhesion of coating to metal surface , crystalline nature of coating and ca : p ratio can be altered easily . ion implantation method enables to inject any element on the near - surface region of any substrate like hydroxyapatite layer on the titanium surface is synthesized by implantation of calcium and phosphorous ion . this method includes using beam of high energy ( 10 kev ) ions to fall on metal surface under vacuum chamber . due to the collision between incident ions and substrate ions , incident ions loses energy and come to rest on near - surface region of metal . following are advantages of this technique :
it is an ultra clean process so synthesis of high purity layers is possible.depth and concentration of impurities are easily controlled and determined.adhesion between implanted surface and substrate is excellent.process is performed at low substrate level so no effect on bulk properties of substrate.method is easily reproducible and controllable.39
it is an ultra clean process so synthesis of high purity layers is possible . process is performed at low substrate level so no effect on bulk properties of substrate . method is easily reproducible and controllable.39 when using ion implantation method , modification of preexisting nanometric features and creation of superficial stresses must be carefully considered.15,44 this approach offers possible insertion of biologically effective ions such as calcium ion ( ca ) , fluoride ion ( f ) , sodium ion ( na ) . thermal oxidation method act by changing crystalline structure of nanometric oxide layer.12,45 recent advancements that will modify surface chemistry of dental implant surface without altering bioactive surface topography includes effect of laser procedure , pico to nanometer thin tio2 coatings and synergistic effect of micro - nano hybrid topography on titanium surface with ultraviolet ( uv ) photo functionalization . laser is used as a micromachining device to produce a three dimensional structure at micrometer and nanometer scale level . this technique enables the energy to focus on one spot by generation of short pulses of single wavelength . advantages of laser technology : ( i ) allows generation of complex feature with high resolution ( ii ) extremely clean ( iii ) rapid ( iv ) suitable for the selective changes in implant surfaces ( v ) precise , targeted and guided surface roughening.22 thomsson and esposito40 performed retrospective case series and developed laser micromachining procedure to create micro and nano scale surface roughness only in the inner part of the thread of dental implant as inner part is considered suitable for osteogenesis . sugita et al.27 directed the possibility of pico to nanometer thin tio2 coatings on micro roughened metal surfaces . using slow rate sputter deposition method a thin titanium oxide coating ( 300 pm - 6.3 nm ) was effectively deposited on implant surface . these biological activities were exponentially correlated with the thickness of tio2 coating and oxygen saturation on the surface . this suggests that biological response of titanium can effectively be enhanced by even picometer super thin coatings . another surface modification is through synergistic effect of nanotopography with ultraviolet ( uv ) photofunctionalization . the effect of micro - nano hybrid surface topography with uv photofunctionalization on implant surface is synergistic . this method involves removal of surface contaminants of hydrocarbon with the help of titanium oxide mediated by photo catalysis and decomposition by ultra violet light.46 tsukimura et al.47 addressed the synergistic effects of uv photofunctionalization and surface factors responsible for synergistic effect . they concluded that high biological activity occurred with the micro pits having 300 nm nodules after ultra violet treatment . advantages of nanoroughness on titanium implants
increase surface area of implant adjacent to bone.improved cell attachment to the implant surface.increase bone present at the implant surface.improve biomechanical interaction of implant with bone . several techniques have been widely studied and developed to modify dental implant surfaces to promote rapid osseointegration and faster bone healing . several in vivo and in vitro studies demonstrated various novel dental implant surfaces mostly consisting in modifications of commercial available ones . nano surface modification methods are likely to enhance surface properties of titanium dental implant that increase peri - implant osteogenesis . the main shortcoming in dental implant surfaces is empirical nature of manufacturing process as it lacks generalized consensus to make one standard for obtaining controlled topographies . in order to overcome this matter , several in vitro and in vivo studies | purposedental implant has gained clinical success over last decade with the major drawback related to osseointegration as properties of metal ( titanium ) are different from human bone .
currently implant procedures include endosseous type of dental implants with nanoscale surface characteristics .
the objective of this review article is to summarize the role of nanotopography on titanium dental implant surfaces in order to improve osseointegration and various techniques that can generate nanoscale topographic features to titanium implants.materials and methodsa systematic electronic search of english language peer reviewed dental literature was performed for articles published between december 1987 to january 2012 .
search was conducted in medline , pubmed and google scholar supplemented by hand searching of selected journals .
101 articles were assigned to full text analysis .
articles were selected according to inclusion and exclusion criterion .
all articles were screened according to inclusion standard .
39 articles were included in the analysis.resultsout of 39 studies , seven studies demonstrated that bone implant contact increases with increase in surface roughness .
five studies showed comparative evaluation of techniques producing microtopography and nanotopography .
eight studies concluded that osteoblasts preferably adhere to nano structure as compared to smooth surface .
six studies illustrated that nanotopography modify implant surface and their properties .
thirteen studies described techniques to produce nano roughness.conclusionmodification of dental osseous implants at nanoscale level produced by various techniques can alter biological responses that may improve osseointegration and dental implant procedures . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
postoperative pain is defined as pain of any degree that occurs after initiation of root canal therapy . although the success of endodontic treatment is highly related to the elimination or reduction of postendodontic pain , many clinical studies have reported varying degrees of postoperative pain , ranging from 25% to 40% . even though microorganisms are usually regarded as the most common cause of postoperative pain , other causes include mechanical or chemical injury to the periradicular tissues . during root canal preparation , irrigants , dentin chips , pulp tissue , and microorganisms may get extruded into the periradicular tissues , triggering an acute inflammatory reaction . this leads to the release of chemical mediators and changes in local adaptation and periapical tissue pressure causing postoperative pain . studies evaluating the apical extrusion of debris have stated that instrumentation techniques and instruments used are the main contributing factor associated with the extrusion of debris . reciprocating file systems were developed as a single file system with m - wire technology which uses a reciprocating motion to cut and proceed into the root canal . data on reciprocating files proved that these files have a better resistance to file separation and less incidence of dentinal damage . variability has been observed between different rotary systems in terms of debris extrusion which can be due to differences in cross - sectional geometry , cutting blade design , taper , flute depth , tip , and sequence of files , kinematics , and cutting efficacy . furthermore , it has previously been suggested that the reciprocating movement per se might play a role in packing the debris into the irregularities of the root canal space , pushing them beyond the apical foramen . ti rotary systems may yield different patterns of neurogenic inflammation response in the periodontal ligament as a consequence of different amounts of irritant extruded from the root canal space . however , the threshold amount of debris required for inducing pain in a patient is unknown . on literature search , there is a lack of clinical evidence comparing the postoperative pain after using different ni ti systems employing varied file design and motion kinematics ( waveone , protaper , and mtwo ) . the objective of this study was to understand the influence of different motion kinematics on postinstrumentation pain . the null hypothesis of this study was that there was no difference in postinstrumentation pain after using any file systems . hence , the aim of this study was to compare the postinstrumentation pain following the use of waveone , protaper , and mtwo ni the study protocol was approved by the ethics in clinical research committee of meenakshi university , chennai , india ( ref no : madc / irb/2014/000 ) . the sample size was calculated as 49 in each group ( allocation ratio 1:1:1 ) using g * power 3.1.2 software ( spss version 0.17 ; spss inc . , chicago , il , usa ) with alpha error left at 5% and statistical power of 80% with a dropout rate of 10% in each group . a total of 183 patients were screened for selection criteria in the dental outpatient unit at the department of conservative dentistry and endodontics , meenakshi ammal dental college and hospitals . patients aged between 18 and 55 , diagnosed of asymptomatic irreversible pulpitis , symptomatic irreversible pulpitis , or pulp necrosis with or without apical periodontitis ( acute or chronic ) in premolars and molars with fully matured roots were enrolled in this study . curved root canals of more than 25 , calcified root canals , periapical radiolucency of more than 2 mm , complex root canal morphology , teeth with abscess / cellulitis , and medically compromised patients were excluded from the study . pulp vitality was diagnosed using thermal tests and electronic pulp tester ( parkell , edgewood , ny , usa ) . the extent and dimensions of periapical radiolucency ( paralleling technique ) were assessed along the greatest diameter using schick cdr software ( sirona , long island city , ny , usa ) . the level of pre- and post - instrumentation pain was recorded as a continuous data using simple visual analog scale ( vas ) scoring . the vas consisted of a line of 10 cm length anchored by two extremes with 0 cm signifying no pain and 10 cm representing the worst pain imaginable . patients who experienced a preinstrumentation pain score of more than 6 cm were excluded from this trial . baseline parameters such as age , gender , vitality status of the tooth , and preinstrumentation pain scores were recorded prior to treatment . the design used in this study was a randomized , triple - blinded , triple arm parallel trial . the confounding factors identified in the study were vitality status and the presence of lesion . computer generated randomization sequence was carried out by the statistician who was not involved in the study . bias in the trial was further reduced by masking the patients and the outcome assessmentor to the intervention . parallel groups included group wo ( waveone ; dentsply maillefer , ballaigues , switzerland ) , group pu ( protaper universal ; dentsply maillefer , ballaigues , switzerland ) , and group mt ( mtwo ; vdw , munich , germany ) where the new intervention was group wo . the randomized order of interventions with ni ti files was concealed in opaque individual envelopes by the staff nurse , which were later opened by the operator at the time of endodontic treatment . root canal procedure was done for patients under local anesthetic administration ( lidocaine hydrochloride and epinephrine 1:80,000 ; xilonibsa , inibsa , spain ) and rubber dam isolation . access cavity was prepared and working length was determined with root zx apex locator ( j morita europe gvbh , frankfurt , germany ) and confirmed with digital radiographs . apical patency and glide path were maintained in all the cases with a 10 size k file ( mani , utsunomiya , tochigi , japan ) . cleaning and shaping in group wo were done with waveone primary or large reciprocating file as a single file system . protaper rotary files and mtwo rotary files were used in a sequential order until master apical file in group pu and group mt , respectively . irrigation with 5% naocl was performed using a side vented needle ( maxi - i - probe ; dentsply , tulsa , ok , usa ) , at 1 mm short of working length between each file . based on the initial apical binding files , the master apical file ranged from iso sizes # 20 to # 30 in narrow canals and from iso sizes # 30 to # 40 in wide canals . ethylenediaminetetraacetic acid ( 17% ) solution was used for smear layer clearance . a final flush with 2% chlorhexidine solution calcium hydroxide ( ca[oh]2 ) was given as an intracanal medicament for all the patients and temporized with cavit ( 3 m deutschland gmbh , neuss - germany ) . the patients were treated by 4 final year postgraduate students who are calibrated and trained to use different shaping systems . all patients were prescribed ibuprofen 400 mg as an escape medicine in the event of severe pain . the patients were contacted through phone calls by the outcome assessor and the vas scoring was recorded at 2 , 4 , 6 , 12 , 24 , 36 , and 48 h , respectively . subgroup analysis for independent variables ( sex , vitality status , and presence of lesion ) was done using bonferroni - corrected mann whitney test ( p < 0.05 ) . relative risk ( rr ) of experiencing pain postinstrumentation was calculated for 12 , 24 , and 48 h. root canal procedure was done for patients under local anesthetic administration ( lidocaine hydrochloride and epinephrine 1:80,000 ; xilonibsa , inibsa , spain ) and rubber dam isolation . access cavity was prepared and working length was determined with root zx apex locator ( j morita europe gvbh , frankfurt , germany ) and confirmed with digital radiographs . apical patency and glide path were maintained in all the cases with a 10 size k file ( mani , utsunomiya , tochigi , japan ) . cleaning and shaping in group wo were done with waveone primary or large reciprocating file as a single file system . protaper rotary files and mtwo rotary files were used in a sequential order until master apical file in group pu and group mt , respectively . irrigation with 5% naocl was performed using a side vented needle ( maxi - i - probe ; dentsply , tulsa , ok , usa ) , at 1 mm short of working length between each file . based on the initial apical binding files , the master apical file ranged from iso sizes # 20 to # 30 in narrow canals and from iso sizes # 30 to # 40 in wide canals . ethylenediaminetetraacetic acid ( 17% ) solution was used for smear layer clearance . a final flush with 2% chlorhexidine solution calcium hydroxide ( ca[oh]2 ) was given as an intracanal medicament for all the patients and temporized with cavit ( 3 m deutschland gmbh , neuss - germany ) . the patients were treated by 4 final year postgraduate students who are calibrated and trained to use different shaping systems . all patients were prescribed ibuprofen 400 mg as an escape medicine in the event of severe pain . the patients were contacted through phone calls by the outcome assessor and the vas scoring was recorded at 2 , 4 , 6 , 12 , 24 , 36 , and 48 h , respectively . subgroup analysis for independent variables ( sex , vitality status , and presence of lesion ) was done using bonferroni - corrected mann whitney test ( p < 0.05 ) . relative risk ( rr ) of experiencing pain postinstrumentation was calculated for 12 , 24 , and 48 h. a total of 183 patients were screened , out of which 31 patients did not meet the inclusion criteria and 152 patients participated in the study [ figure 1 ] . the study was conducted from september 2013 to june 2014 . statistical analysis of baseline parameters had no significant difference among them [ table 1 ] . the overall incidence of postinstrumentation pain at various time periods is shown in table 2 . the postinstrumentation pain values were lower than preinstrumentation values and the difference was statistically significant ( p < 0.05 ) . there was no statistically significant difference in postinstrumentation pain between groups with regards to gender . the pain scores at 48 h were significantly higher in group wo when compared to group mt and pu ( p < 0.05 ) . there was no statistical difference between group mt and pu at 48 h. pulp vitality was not a significant factor leading to pain at the end of 2 and 4 h between groups . however , there was a significant difference in pain between vital and nonvital pulp status at 6 h and the same trend continued till 48 h , irrespective of file systems used ( p < 0.05 ) . pain scores of nonvital tooth in group wo had comparatively high pain value that started at 6 h and continued till 48 h , whereas the pain scores of nonvital teeth in group pu and mt started at 12 h and lasted till 48 h and 36 h , respectively ( p < 0.05 ) . the rr of developing postinstrumentation pain was higher with waveone when compared to mtwo ( rr = 1.8 95% ci 1.32.7 ) and protaper ( rr = 1.7 95% ci 1.22.5 ) at the end of 48 h [ table 3 ] . a consolidated standards of reporting trial diagram showing the flow of participants through each stage of trial preendodontic baseline parameters overall incidence of postinstrumentation pain at different time intervals the relative risk of developing postinstrumentation pain at different time intervals for the groups studied the success of endodontic therapy depends not merely on their efficacy and proper completion but also on minimal patient discomfort . in recent decades , few in - vitro studies proved that the extrusion of debris is more with reciprocating files when compared to continuous rotary files . however , no attempt was made to simulate the presence of vital pulp or periapical tissues when determining the debris extrusion . results could change in a clinical situation because of the presence of periapical tissues , which act as a natural barrier to prevent debris extrusion . hence , this study compared the postinstrumentation pain on using two different rotary file systems ( protaper , mtwo ) and one reciprocating file system ( waveone ) . this study rejects the null hypothesis and has shown that there is a difference of pain values among the groups . waveone reciprocating files were selected for this trial as a new intervention to evaluate the influence of motion kinematics for reduction of postinstrumentation pain . protaper was selected in this study to assess the influence of variable taper along the length of the file in causing postinstrumentation discomfort . mtwo has been proved to have a better cyclic fatigue resistance , shaping ability in curved root canals , and less debris extrusion when compared to other conventional ni waveone rotary files extruded more debris when compared to other continuous rotary files in in - vitro studies . however , the clinical data on reciprocating systems does not compare this file system with constant taper files . moreover , the published data on postoperative pain using waveone reciprocating files were recorded following endodontic treatment in a single visit . neglecting apical patency may cause risk of loss of length , canal transportation , other accidents such as ledges and improper irrigation and disinfection in the apical third of the canal . compared the incidence of postoperative pain with or without apical patency and reported that the apical patency has no role in postoperative pain . a similar finding hence , apical patency was maintained with k 10 # file in all the patients to ensure disinfection of apical one - third . concomitantly , side - vented needles have shown to cause minimal debris extrusion and hence employed in this study . walton et al . have reported that the use of ca[oh]2 as an intracanal medication was unrelated to the incidence and/or severity of posttreatment pain . pain perception is purely subjective depending on patient 's threshold and variable experience modulated by multiple physical and psychological factors . in the current study , simple vas was used as a result of its ease , valid , and reliable scale to measure pain . statistical analysis of the baseline parameter showed no significant difference among the groups , thus confirming equal and homogeneous distribution of the samples among the three groups ( successful randomization ) . the principal investigator , outcome assessor , and the patients were blinded in this clinical trial . the operators were not blinded in this trial but were not aware of the instrumentation system until the moment of cleaning and shaping ( allocation concealment ) . gender has been suggested to play a role in other pain studies . in the endodontic literature , females have shown to experience higher levels of postoperative pain compared to males . however , in our study , gender did not play a role in postinstrumentation pain . the results of this study showed a significant reduction in postinstrumentation pain score values when compared to preoperative pain values irrespective of file system used . this concept is supported by many previous studies proving that endodontic procedure of a diseased tooth helps in alleviation of symptomatic pulpitis . at 48 h , patients in group wo experienced more discomfort when compared to patients in group mt followed by group pu . the risk of developing postinstrumentation pain was almost twice with waveone file system compared to other two file systems . the possible reasons for the debris extrusion may be due to the cross - sectional geometry of the file , instrumentation technique , motion of the file , irrigation system , or anatomy of the root . protaper and waveone files are characterized by a triangular or modified triangular cross - section resulting in a lower cutting efficiency with smaller chip space . this design may enhance debris transportation toward the apex when used in combination with a reciprocal motion . protaper and waveone systems have a similar file cross - section ; however , patients in group wo experienced more pain compared to group pu . the difference in motion of kinematics between the two file systems could be the underlying reason . on comparison of teeth with regards to vitality , nonvital teeth experienced more pain compared to vital teeth across all groups . , the direct interaction of extruded bacteria and host cells can result in postinstrumentation pain . at 2 and 4 h the lack of pain initially could be attributed to the influence of anesthesia . for nonvital teeth , among the three experimental groups , patients in wo group experienced early onset of pain at 6 h and lasted until 48 h implying that this file system can complicate postinstrumentation pain . patients with nonvital teeth in group mt and group pu had the onset of pain at 12 h and lasted up to 36 and 48 h , respectively . patients with nonvital teeth in group mt experienced lesser pain following instrumentation as compared to other groups . even though the influence of several factors was studied in this trial , multivariate comparison was not possible due to insignificant odd 's ratio for a majority of factors . the results of the study can be extrapolated to posterior teeth with mild to moderately curved canals and in the absence periapical lesion . the influence of root canals with curvature > 25 degrees , effect on anterior teeth , and presence of periapical lesion needs be explored in future trials . under the limitations of this study , it can be concluded that waveone reciprocating files cause more postinstrumentation pain followed by protaper and mtwo . caution should be exercised on instrument selection for appropriate cases such as nonvital pulp with curved root canals . | aim : this randomized , controlled trial evaluated the incidence of postinstrumentation pain on using two different rotary ni ti and one reciprocating ni ti cleaning and shaping systems.materials and methods : asfter sample size estimation , a total of 152 patients were randomized to three groups ( group wo [ waveone ] , group pu [ protaper universal ] , and group mt [ mtwo ] ) .
selection of patients was based on root curvature , periapical lesion , vitality , gender , and diagnosis of the patient .
a similar clinical protocol was followed and endodontic treatment was performed .
pre- and post - instrumentation pain scores ( 2 , 4 , 6 , 8 , 12 , 36 , and 48 h ) were recorded using visual analog scale score .
kruskal
wallis test was done for overall comparison of the groups .
bonferroni - corrected mann whitney test was done for pair - wise comparisons and friedman test was done to compare pain between time points in each file system ( p < 0.05).results : all preinstrumentation pain values were higher than postinstrumentation pain values ( p < 0.05 ) . at 48 h ,
patients in group wo experienced more pain compared to the other two groups .
pain scores of nonvital teeth were significantly higher when compared to vital teeth . among nonvital teeth , patients in group wo had early onset of pain at 6 h and lasted till 48 h , whereas pain started at 12 h and lasted till 36 and 48 h for group mt and group pu , respectively.conclusion:patients treated with waveone files experienced more pain when compared to the other files .
nonvital teeth experienced more pain compared to vital teeth .
group mt had the lowest postinstrumentation pain . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the interest in the teaching of orofacial pain and tmds issue in scientific literature
is reported since the seventies .
since then , considerable advances in this subject were observed , but they are still far
from what can be considered ideal . only few universities offer in their graduation
curricular program the subject or a graduate program in orofacial pain and tmds , either
as an elective or as a compulsory . this deficit implies in losses in the professional training of the dentist , who often
feels disqualified to deal with tmd / orofacial pain patients . currently , there is accreditation for advanced general
dentistry education programs in orofacial pain , and initiatives have been implemented to
encourage institutions to embrace new interprofessional models . the interprofessional nature of this field makes it
difficult to establish the role of the dentist , which results in important implications
for the whole process of teaching , research and clinical care . temporomandibular disorders ( tmd ) can be defined as a series of signs and symptoms
involving the temporomandibular joint ( tmj ) , masticatory muscles , or both . historically , tmds were often associated with the study of
occlusion ; however , this association has been increasingly questioned by the current
scientific advances . therefore , the study of tmds has ceased to be seen as
traditional dentistry and focused on an interdisciplinary involvement with other areas
of health for the management of patients in a biopsychosocial paradigm . within the context of dentistry , it is essential that dentists may be able to recognize ,
evaluate and manage patients with tmds . although the disorders present as highly
prevalent in the general population ,
for many years this field of knowledge remained a gray area in dentistry , full of
controversies and poorly discussed and taught in the academic milieu . in brazil , specifically , the year of 2002 represents a milestone in the recognition of
the specialty of temporomandibular disorders and orofacial pain ( tmd / op ) . this
recognition led to greater technical and scientific improvement by the dentists and
contributed in raising the level of knowledge of the area . thus , this increased capacity
comes to reflect directly in teaching and research , disseminating scientific knowledge
and increasing the number of people involved with the specialty . considering that tmd / op was recognized as a dental specialty over a decade ago , it is
extremely important to trace the current situation of the field of temporomandibular
disorders in brazil , specifically in the areas of research and training . the objective
of this study was to perform a bibliometric survey to investigate the scientific works
on tmd produced in the country , determine the number of specialization courses on tmd
and orofacial pain and the number of specialists in the area . by describing this
panorama , we hope to discuss the importance of the recognition of this specialty and the
inclusion of these subjects in undergraduate programs in dentistry . this study can be divided in three stages : bibliometric survey in the theses database of
coordination for the improvement of higher education personnel ( capes ) ; survey on the
involvement of brazilian researchers in the publication of articles indexed by pubmed ;
and panorama of tmd / op field in brazil . the bibliometric survey was conducted by means of a careful search in the theses
database of coordination for the improvement of higher education personnel ( capes )
( http://capesdw.capes.gov.br ) . we adopted the word " temporomandibular "
as the search term , and the search was delimited to the period from 2000 to 2012 . unfortunately , information about the year of 2013 was not available at the time of
the submission of this manuscript . for each result of the search conducted on the
capes portal , we collected the following information : the works registered on the capes database are required in order to be a candidate
for a phd or a masters degree , therefore they could be doctoral theses , masters
dissertations or professional masters dissertations . according to capes , the
professional masters programs are subjected to the same criteria as the academic
master 's programs and , additionally , emphasize studies and techniques aimed at
professional performance . the works were classified according to the following fields of knowledge : dental
sciences , medical sciences , basic sciences , and other . thematic classification of the work , subdivided into : associated themes ( when the research is related to two or more themes , for
instance , diagnosis an intervention ) data were independently collected by the authors ffl and nagm . discrepancies were
discussed in a final revision with all the authors . additionally , the authors ffl and nagm carried out a thorough search in the pubmed
database to verify the participation of brazilian researchers in the publication of
scientific articles indexed to this basis . for each result , we confirmed the participation of
brazilian researchers either as first author or co - author of the study . discrepancies
were discussed in a final revision with all the authors . to determine the number of specialization courses and the number of specialists in
temporomandibular disorders and orofacial pain in brazil , a survey was conducted on
the website of federal council of dentistry ( cfo ) , a brazilian agency responsible for
the regulation of specialized courses and specialist licenses in brazil . the survey
was conducted on the website ( http://cfo.org.b ) , accessed on 01/15/2014
by the author nagm . the bibliometric survey was conducted by means of a careful search in the theses
database of coordination for the improvement of higher education personnel ( capes )
( http://capesdw.capes.gov.br ) . we adopted the word " temporomandibular "
as the search term , and the search was delimited to the period from 2000 to 2012 .
unfortunately , information about the year of 2013 was not available at the time of
the submission of this manuscript . for each result of the search conducted on the
capes portal , we collected the following information : the works registered on the capes database are required in order to be a candidate
for a phd or a masters degree , therefore they could be doctoral theses , masters
dissertations or professional masters dissertations . according to capes , the
professional masters programs are subjected to the same criteria as the academic
master 's programs and , additionally , emphasize studies and techniques aimed at
professional performance . the works were classified according to the following fields of knowledge : dental
sciences , medical sciences , basic sciences , and other . thematic classification of the work , subdivided into : associated themes ( when the research is related to two or more themes , for
instance , diagnosis an intervention ) data were independently collected by the authors ffl and nagm . additionally , the authors ffl and nagm carried out a thorough search in the pubmed
database to verify the participation of brazilian researchers in the publication of
scientific articles indexed to this basis . for each result , we confirmed the participation of
brazilian researchers either as first author or co - author of the study . to determine the number of specialization courses and the number of specialists in
temporomandibular disorders and orofacial pain in brazil , a survey was conducted on
the website of federal council of dentistry ( cfo ) , a brazilian agency responsible for
the regulation of specialized courses and specialist licenses in brazil . the survey
was conducted on the website ( http://cfo.org.b ) , accessed on 01/15/2014
by the author nagm . according to the bibliometric survey on the capes portal , 731 scientific works were
registered with tmd / op as the main subject . from this total , 421 were masters
theses , 195 were doctoral theses and 115 were professional masters theses . regarding the state where the scientific works were performed , we found that 73% were
from the state of so paulo , 5% from minas gerais , 4% from rio de janeiro , 4% from
rio grande do sul , 4% from paraba , and 10% from other brazilian states . distribution of scientific works available in capes theses database according
to field of knowledge from 2001 to 2012 the thematic approaches found in the capes theses database are shown in figure 2 . thematic approaches of scientific works registered in capes theses database
from 2001 to 2012 search in the pubmed database yielded 576 articles published with the participation
of brazilian researchers as first authors and/or co - authors in the period from 2000
to 2013 . from this amount , only 5 were published in portuguese , while all the others
were published in english . search on the federal council of dentistry ( cfo ) website found that until january
2014 there were : - 254,380 registered dentists in brazil ; - 111 accredited / recognized specialization courses in orofacial pain and
temporomandibular disorder concluded ; - 8 accredited / recognized specialization courses in orofacial pain and
temporomandibular disorder in progress ; - 1,064 registered specialists in temporomandibular disorders and orofacial pain in
brazil . according to the bibliometric survey on the capes portal , 731 scientific works were
registered with tmd / op as the main subject . from this total , 421 were masters
theses , 195 were doctoral theses and 115 were professional masters theses . regarding the state where the scientific works were performed , we found that 73% were
from the state of so paulo , 5% from minas gerais , 4% from rio de janeiro , 4% from
rio grande do sul , 4% from paraba , and 10% from other brazilian states . distribution of scientific works available in capes theses database according
to field of knowledge from 2001 to 2012 the thematic approaches found in the capes theses database are shown in figure 2 . search in the pubmed database yielded 576 articles published with the participation
of brazilian researchers as first authors and/or co - authors in the period from 2000
to 2013 . from this amount , only 5 were published in portuguese , while all the others
were published in english . search on the federal council of dentistry ( cfo ) website found that until january
2014 there were : - 254,380 registered dentists in brazil ; - 111 accredited / recognized specialization courses in orofacial pain and
temporomandibular disorder concluded ; - 8 accredited / recognized specialization courses in orofacial pain and
temporomandibular disorder in progress ; - 1,064 registered specialists in temporomandibular disorders and orofacial pain in
brazil . the control of temporomandibular disorders and orofacial pain is a complex issue that
requires the performance of professionals who demonstrate expertise capable of directing
the diagnosis and appropriate treatment . some studies have shown
the great deficiency of education in the field of tmd/ op leading to frequent
discussions on strategies for curricular restructuring and search for alternatives that
disorders and orofacial pain as a specialty represents an important advance in general . a specialist should be a licensed professional who has acquired special knowledge about
a particular subject through study and/or experience . according to the resolution of the
federal council of dentistry , the specialty of temporomandibular disorders and orofacial
pain can be defined as the specialty that aims to promote and develop scientific
knowledge for better understanding of the diagnosis and treatment of pain and disorders
of the masticatory system , orofacial region and related structures . the orofacial pain specialist is responsible for diagnosing and treating patients with
pain that is often chronic , multifactorial and complex . failure in understanding the
mechanisms of pain can lead to delayed diagnosis and/or incorrect , inaccurate
treatments , which are ineffective or even harmful . thus , it is the responsibility of
orofacial pain specialist to accurately diagnose the causes of pain and decide whether
treatment procedures may include dental , medical interrelated areas , counseling or even
a combination of all these treatment approaches . initially , this study
used the term " orofacial pain " as search term ; however , the results were inconclusive . thus , we chose to use the term " temporomandibular " for the following reasons : ( 1 ) to
perform a comprehensive search , since mesh terms " temporomandibular joint " or
" temporomandibular joint dysfunctions syndrome " could limit the results to research
works related only to the temporomandibular joint or only to a disease - related scenario .
( 2 ) to facilitate the search , due to the similarity of the spelling in both english and
portuguese languages , we could cluster the scientific works retrieved in a unique
result . despite the limitation of restricting the evaluation to tmd and not encompassing
other forms of orofacial pain , the results provide a clear and detailed discussion of
the tmd area specifically . capes theses database is an important channel of access to knowledge production
specifically aimed at disseminating information on theses and dissertations produced in
brazilian universities since 1987 . annually , this database is updated based on new
information submitted by graduate programs in the country . capes theses database still
has some structural limitations , for example : information about the year of 2013 was not
available until the submission of this manuscript . however , this database is an
important tool for open access dissemination of results of academic research ,
contributing for greater visibility of the work carried out in the country . when considering scientific works registered in capes theses database , we observed that ,
from 2000 to 2013 , the total number of registered works doubled . the reasons for this
increase can be attributed to several factors such as the recognition of the specialty
associated with increased interest of the dental class in the study of tmd , greater
dissemination of graduate programs in the country , government incentives for research
activities , among other factors . despite national policies to encourage the teaching and research , there is a notable
disparity between the performance of research and production of scientific knowledge
among the different brazilian states , which suggests a need for reassessment of current
policies and government action to improve this situation . the southeast region of the
country accounted for more than 82% of capes theses database , while the northern region
under 2% . ( 2009 ) also
emphasized the highest concentration of scientific research in the southeast region of
the country and especially in the state of so paulo , with the largest number of
colleges and graduate programs . regarding the fields of knowledge involved in research activities related to tmd ,
greater participation of graduate programs on dentistry was expected , considering that
dentistry is the area of health that has been the most devoted to the subject . within
the interdisciplinary context , cooperation of other academic areas such as basic
sciences and medical sciences is increasingly required , contributing to a better
understanding of tmd in view of its multidisciplinary nature . research in the medical
and basic sciences have shown that many patients with temporomandibular disorders
present structural and generalized symptoms such as mood disorder , generalized pain and
significant systemic changes . thus , dentistry has taken a leadership role in healthcare
in order to solve the problems in the area of tmd , though its performance is not unique and distinct , requires the
involvement of other groups of health professionals , including physicians , pharmacists ,
psychologists , physiotherapists , occupational therapists and speech therapists . another important point relates to the greatest achievement of clinical research
involving diagnostic parameters , which can be attributed to the ease of conducting this
relatively short term studies when compared to other modalities involving thematic
treatment , etiology and epidemiology . another reason for prioritizing this approach is
associated to the possibility of performing methodologies with the fewest bias , greater
availability of sampling and the need for less costly resources . from a scientific point of view , we had significant changes in publishing articles due
to the increase in the amount of online publications and open access journals , new
funding policies , and also to the fact that universities increasingly require the
participation of their teachers in research activities . the considerable growth in the
number of articles published from 2000 to 2009 with the participation of brazilian
researchers as authors / co - authors can be attributed to the increasing interest of the
dental community in the field of tmd and also to the capes policy of prioritizing the
publication of articles when evaluating the scientific quality of graduate programs . this growth can also be attributed to the spread of postgraduate programs all over the
country , the establishment of research groups , the great number of qualified researchers
and the consolidation of scientific production . with the expansion of scientific and
human resources training through postgraduate studies , there was a significant rise in
demand for resources for funding research projects and grants for research productivity ,
as well as application of post - doctoral and phd scholarships abroad . on the other hand , the slight decline
observed in the participation of brazilian researchers in articles published between
2009 and 2011 can be attributed to a greater demand for quality on the part of journals
for publishing the papers . in 2012 , the brazilian society of temporomandibular disorders and orofacial pain was
founded , representing a milestone which will favor the systematization and dissemination
of practice , teaching and research in this specialty in brazil . we expect this society
can promote the enrichment and integration of teaching programs in temporomandibular
disorders / orofacial pain with related subjects and professions . this society will be
responsible for providing information and recommendations related to the diagnosis and
treatment of tmd / op , not only to specialists but also to general practioners and , thus ,
assist the dentists in offering primary care for patients affected by these
conditions . given the rapidly changing demography of populations worldwide , dental professionals of
the future need to be able to meet the challenge posed by the evolving landscape in
health care needs . according to the latest demographic data from the brazilian institute
of geography and statistics ( ibge ) for the year 2012 , brazil has 199.3 million
inhabitants . considering the number of inhabitants in the country and the high
prevalence of orofacial pain and tmd , we can say the number of specialists in the field
is small . we have to point that to be a specialist in brazil you must complete a
specialization course . it is necessary to fit the new educational realities to the
population needs , to develop guidelines for the expansion of theoretical and practical
knowledge in the care of patients with orofacial pain and tmd . we suggest extending the
specialization titles to highly qualified professionals in the fields , like researchers
with several works published in the area . considering it is a problem with high occurrence among the population and the loss
caused ( financial onus , the patient suffer ) , the temporomandibular disorders demand the
dental community 's special attention in order to provide better physical , social and
emotional welfare to tmd patients . although a large number of people suffer from
orofacial pain or temporomandibular disorders , dentistry has not developed adequate
educational guidelines for dealing with this problem . thus , recognition of specialty in
the area will contribute to the development of a scientifically - supported knowledge base
for improved understanding of the diagnosis , management of pain and dysfunction of the
masticatory system and related structures , as well as development of the clinicians '
skill in order to provide quality treatment for patients . the recognition of the specialty in brazil presents many challenges and hard work , but
the perspectives are promising . given the complexity of temporomandibular disorders and
orofacial pain , it is necessary to emphasize attention on the education and the
development of this area . new models should be implemented , surpassing the prevailing
mechanistic models of the past and preparing professionals for a broader and
comprehensive care . professionals should be prepared for interaction between the
different areas of health and adopt the biopsychosocial model in understanding patients .
based on the results analyzed , we can conclude that the number of scientific works
related to temporomandibular disorders increased significantly in recent years in
brazil , as well as the number of specialization courses and the number of
specialists . | in 2012 , the recognition of the specialty of temporomandibular disorders and
orofacial pain completed ten years . given this scenario ,
it is extremely important to
track the current situation of this field of knowledge in brazil , specifically in the
area of research and training .
we hope to discuss the importance of the recognition
of this specialty and the inclusion of these subjects in undergraduate programs in
dentistry.objectivethe objective of this study is to perform a bibliometric survey of researches
regarding temporomandibular disorders and orofacial pain conducted in the country ,
determine the number of specialization courses in orofacial pain and the number of
specialists in the field.methodsthe bibliometric survey was conducted based on the dissertations portal of
coordination for the improvement of higher education personnel ( capes ) and on
pubmed .
the panorama of the field of orofacial pain and temporomandibular
disorders in brazil was determined by searching on the website of the brazilian
council of dentistry.resultswe found 731 theses and dissertations with temporomandibular disorders and
orofacial pain as the main subjects ; 81 accredited / recognized courses on orofacial
pain and temporomandibular dysfunction completed ; 8 accredited / recognized
specialization courses on orofacial pain and temporomandibular dysfunction still
in progress , and 1,064 registered specialists in orofacial pain and
temporomandibular dysfunction in the brazilian council of dentistry .
search in the
pubmed database yielded 576 articles published with the participation of brazilian
researchers as first authors and/or co - authors in the period from 2000 to 2013 .
from this amount ,
only 5 were published in portuguese , while all the others were
published in english .
we can also notice that the number of published articles
increases over time.conclusionthe number of researches related to temporomandibular disorders has increased over
the last ten years , as well as the number of specialization courses and the number
of specialists , which represents a major breakthrough for this field of
knowledge . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
hospital - acquired pneumonia ( hap ) is defined as a low respiratory tract infection , which develops 48 h after hospital admission in a patient without infection at admission . hap currently ranks second among nosocomial infections and accounts for 25% of the infections in intensive care units . hap has a significant impact on the financial burden of health care , and new cases drive the increasing emergence of pathogens with multi- or pan - antibiotic resistance . therefore , identifying the infectious etiology in different settings is the key step for mitigating or obviating the severe infection in time . early identification of specific pathogens could significantly improve the morbidity and mortality of hap , and lower the cost of treatment as well . so far , the most common method used to implement hap etiology is still tied to sputum culture . in addition to sputum culture 's relatively low sensitivity and the difficulty with which it identifies atypical pathogens , the time required to obtain results always leads to empirical antibiotic therapies rather than target therapies for patients with hap . an rapid innovative method for etiology identification of hap , the quantitative loop - mediated isothermal amplification ( qlamp ) , has already been used in the diagnosis of virus , fungus , parasite , and tuberculosis infections and is now commercially available . it is a novel assay that focuses on the genetics of pathogens based on rapid nucleic acid amplification method . therefore , this technique has two important advantages such as rapid diagnosis and high sensitivity . in addition , qlamp is also a high specific assay , which could detect different bacteria with quantified copies . since the excellent timeliness and accuracy of qlamp for etiological diagnosis to the lower respiratory tract infection has been confirmed by our group , we initiated a pilot , prospective , and interventional study to investigate the value of qlamp to guide target antibiotics therapies in a small group of patients with hap . patients with suspected hap from august 2011 to march 2014 at peking university people 's hospital ( beijing , china ) were recruited in the study , which were approved by the ethical committee of peking university people 's hospital ( no . these patients were initially diagnosed as suspected cases of hap occurring more than 48 h after admission and were not incubated at the time of admission , having typical characteristics of pneumonia , which were firmly inferred from chest x - rays and the following criteria : [ i ] at least one of the following : ( 1 ) fever ( > 38.5c ) , ( 2 ) leukopenia ( peripheral white blood cell count [ wbc ] < 4.0 10/l ) or leukocytosis ( wbc > 10.0 10/l ) , and ( 3 ) for adults 70 years old or older , mental status changes with no other recognized cause ; [ ii ] at least two of the following : ( 1 ) new - onset of purulent sputum , or change in character of sputum , or increased respiratory secretion , or increased suctioning requirements , ( 2 ) new - onset or worsening cough , or dyspnea , or tachycardia , ( 3 ) rales or bronchial breath sounds , and ( 4 ) worsening gas exchange ( pao2/fio2 240 ) , increased oxygen requirements , or increased ventilation demand . patients with noninfectious diseases , viral infection , fungal infection , or tuberculosis were subsequently excluded from the study . once patients were enrolled , lower respiratory secretion samples were collected on the 1 day for both routine culture and qlamp assays , of which the results were reported to the clinicians . data of each patient were also collected from the medical records , with particular attention to clinical manifestations and treatment strategies before and after the qlamp results reporting . to determine the final diagnosis and assess the treatment response for each patient , 2 independent pulmonologists blinded to qlamp results reviewed all available medical records ( including patient history , physical examination , and results of laboratory tests , including blood routine examination , biochemical indicators , plasma electrolytes , blood gas analysis , and chest radiograph ) pertaining to the patient from the time of hap presentation to discharge / death . after liquefied in an equal volume of 10% naoh , dna specimen of each sample was isolated using the universal kit for bacterial dna extraction ( capitalbio corporation , china ) . the specimens were then prepared for qlamp using a set of specific primers for streptococcus pneumonia ( sp ) , staphylococcus aureus ( sa ) , escherichia coli ( ec ) , klebsiella pneumonia ( kp ) , pseudomonas aeruginosa ( pa ) , acinetobacter baumannii ( ab ) , stenotrophomonas maltophilia ( sm ) , haemophilus influenzae ( hi ) , legionella pneumophila ( lp ) , mycoplasma pneumonia ( mp ) , and chlamydophila pneumoniae ( cp ) . qlamp primer system of each species of pathogen is composed of six primers recognizing eight distinct regions on the target dna , termed a forward outer primer ( f3 ) , a backward internal primer ( bip ) ( b3 ) , a forward internal primer ( fip ) , a bip , and loop primers ( lf and lb ) . eight - pathogen primer sequences are used same as we did before , including sp , sa , ec , kp , pa , ab , sm , and hi . those for atypical pathogens were redesigned as shown in table 1 , and both their sensitivity and specificity were ensured by quantified dna isolated in 27 bacterial species as we did before . primers for atypical pathogens used in this study fip : forward internal primer ; bip : backward internal primer ; lp : legionella pneumophila ; mp : mycoplasma pneumonia ; cp : chlamydophila pneumonia . the reaction was performed at 65c for 45 min in a 25-l reaction mixture consisting of 1.6 mol / l each of fip and bip , 0.2 mol / l each of f3 and b3 , 0.4 mol / l each of lf and lb , 8 u of the bst dna polymerase large fragment ( new england biolabs inc . , beverly , mass . , usa ) , 0.4 mmol / l dntp , 0.1 mmol / l dutp , 0.8 mol / l betaine , 6 mmol / l mgso4 , 0.5 mg / ml bsa , 0.6 evagreen ( biotium , inc . , ca , usa ) , 0.1 u / ml uracil - dna glycosylase ( fermentas inc . , md , usa ) , 20 mmol / l tris - hcl ( ph 8.8 at 25c ) , 10 mmol / l kci , 10 mmol / l ( nh4)2 so4 , 0.1% triton x-100 , and 2 l template dna or pcr grade h2 o as negative control , and then heated at 80c for 5 min to terminate the reaction . all amplifications were performed with an rt - cycler real - time fluorescence quantitative pcr instrument ( capitalbio corporation , beijing , china ) . the titer was quantified according to the standard curves obtained from prequantified dna templates as described previously . biochip technology was introduced in january 2013 at peking university people 's hospital , and the reaction was performed on a microfluidic device after then . the qlamp tests and routine cultures were conducted by two experienced technicians awareness of the sample identities in two separated laboratories of peking university people 's hospital . we constructed a contingency table and used mcnemar test to evaluate the congruence of qlamp and culture results . the differences between patients with or without treatment strategies adjustment based on qlamp results were tested with fisher 's exact test or t test . baseline data of these patients were expressed as mean standard deviation ( sd ) for normally distributed values . all analyses were performed with the use of spss statistics software , version 19.0 ( ibm , usa ) . a p < 0.05 is considered statistically significant difference . patients with suspected hap from august 2011 to march 2014 at peking university people 's hospital ( beijing , china ) were recruited in the study , which were approved by the ethical committee of peking university people 's hospital ( no . these patients were initially diagnosed as suspected cases of hap occurring more than 48 h after admission and were not incubated at the time of admission , having typical characteristics of pneumonia , which were firmly inferred from chest x - rays and the following criteria : [ i ] at least one of the following : ( 1 ) fever ( > 38.5c ) , ( 2 ) leukopenia ( peripheral white blood cell count [ wbc ] < 4.0 10/l ) or leukocytosis ( wbc > 10.0 10/l ) , and ( 3 ) for adults 70 years old or older , mental status changes with no other recognized cause ; [ ii ] at least two of the following : ( 1 ) new - onset of purulent sputum , or change in character of sputum , or increased respiratory secretion , or increased suctioning requirements , ( 2 ) new - onset or worsening cough , or dyspnea , or tachycardia , ( 3 ) rales or bronchial breath sounds , and ( 4 ) worsening gas exchange ( pao2/fio2 240 ) , increased oxygen requirements , or increased ventilation demand . patients with noninfectious diseases , viral infection , fungal infection , or tuberculosis were subsequently excluded from the study . once patients were enrolled , lower respiratory secretion samples were collected on the 1 day for both routine culture and qlamp assays , of which the results were reported to the clinicians . data of each patient were also collected from the medical records , with particular attention to clinical manifestations and treatment strategies before and after the qlamp results reporting . to determine the final diagnosis and assess the treatment response for each patient , 2 independent pulmonologists blinded to qlamp results reviewed all available medical records ( including patient history , physical examination , and results of laboratory tests , including blood routine examination , biochemical indicators , plasma electrolytes , blood gas analysis , and chest radiograph ) pertaining to the patient from the time of hap presentation to discharge / death . after liquefied in an equal volume of 10% naoh , dna specimen of each sample was isolated using the universal kit for bacterial dna extraction ( capitalbio corporation , china ) . the specimens were then prepared for qlamp using a set of specific primers for streptococcus pneumonia ( sp ) , staphylococcus aureus ( sa ) , escherichia coli ( ec ) , klebsiella pneumonia ( kp ) , pseudomonas aeruginosa ( pa ) , acinetobacter baumannii ( ab ) , stenotrophomonas maltophilia ( sm ) , haemophilus influenzae ( hi ) , legionella pneumophila ( lp ) , mycoplasma pneumonia ( mp ) , and chlamydophila pneumoniae ( cp ) . qlamp primer system of each species of pathogen is composed of six primers recognizing eight distinct regions on the target dna , termed a forward outer primer ( f3 ) , a backward internal primer ( bip ) ( b3 ) , a forward internal primer ( fip ) , a bip , and loop primers ( lf and lb ) . eight - pathogen primer sequences are used same as we did before , including sp , sa , ec , kp , pa , ab , sm , and hi . those for atypical pathogens were redesigned as shown in table 1 , and both their sensitivity and specificity were ensured by quantified dna isolated in 27 bacterial species as we did before . primers for atypical pathogens used in this study fip : forward internal primer ; bip : backward internal primer ; lp : legionella pneumophila ; mp : mycoplasma pneumonia ; cp : chlamydophila pneumonia . the reaction was performed at 65c for 45 min in a 25-l reaction mixture consisting of 1.6 mol / l each of fip and bip , 0.2 mol / l each of f3 and b3 , 0.4 mol / l each of lf and lb , 8 u of the bst dna polymerase large fragment ( new england biolabs inc . , usa ) , 0.4 mmol / l dntp , 0.1 mmol / l dutp , 0.8 mol / l betaine , 6 mmol / l mgso4 , 0.5 mg / ml bsa , 0.6 evagreen ( biotium , inc . , ca , usa ) , 0.1 u / ml uracil - dna glycosylase ( fermentas inc . , md , usa ) , 20 mmol / l tris - hcl ( ph 8.8 at 25c ) , 10 mmol / l kci , 10 mmol / l ( nh4)2 so4 , 0.1% triton x-100 , and 2 l template dna or pcr grade h2 o as negative control , and then heated at 80c for 5 min to terminate the reaction . all amplifications were performed with an rt - cycler real - time fluorescence quantitative pcr instrument ( capitalbio corporation , beijing , china ) . the titer was quantified according to the standard curves obtained from prequantified dna templates as described previously . biochip technology was introduced in january 2013 at peking university people 's hospital , and the reaction was performed on a microfluidic device after then . the qlamp tests and routine cultures were conducted by two experienced technicians awareness of the sample identities in two separated laboratories of peking university people 's hospital . we constructed a contingency table and used mcnemar test to evaluate the congruence of qlamp and culture results . the differences between patients with or without treatment strategies adjustment based on qlamp results were tested with fisher 's exact test or t test . baseline data of these patients were expressed as mean standard deviation ( sd ) for normally distributed values . all analyses were performed with the use of spss statistics software , version 19.0 ( ibm , usa ) . a p < 0.05 is considered statistically significant difference . totally , 76 samples were recruited from 110 eligible samples overall in our study [ figure 1 ] . none of the samples were collected from the same onset of hap . as shown in table 2 , there were 70 samples with qlamp results greater than 10 copies / ml , 23 samples with qlamp results between 10 and 10 copies / ml , and 16 samples with qlamp results below 10 copies / ml . study profile . for each eligible patient , we collected lower respiratory secretion samples on the 1 day for routine culture and quantitative loop - mediated isothermal amplification tests and reported the results to the clinicians . we also collected the clinical data and treatment strategies before and after reporting the quantitative loop - mediated isothermal amplification results . qlamp results of specimen from patients with hospital - acquired pneumonia data are presented as number , unless otherwise indicated . qlamp : quantitative loop - mediated isothermal amplification ; lp : legionella pneumophila ; mp : mycoplasma pneumonia ; cp : chlamydophila pneumoniae ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia ; hi : haemophilus influenza . the concordance rates of the two assays for detecting sa , ec , pa , kp , sm , sp , and ab are 90.79% , 98.68% , 89.47% , 93.42% , 93.42% , 100.00% , and 77.63% , respectively [ table 3 ] . we also evaluated the difference between qlamp and culture results by mcnemar test , in which no significant difference was found ( p > 0.05 ) [ table 3 and supplementary tables 17 ] . the qlamp results of 4 samples for hi , lp , or mp were positive , while the culture results for these specimens were negative probably because of their low detectable rates in culture . we then studied the clinical data of these 4 samples and found that the qlamp results were all reliable except 1 mp positive sample due to the lack of specific species identified in the final diagnosis . no cp positive results were reported in the 76 samples either by qlamp or culture assay . congruence of qlamp and culture results in patients with hospital - acquired pneumonia qlamp : quantitative loop - mediated isothermal amplification ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for escherichia coli * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for pseudomonas aeruginosa * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for klebsiella pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for stenotrophomonas maltophilia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for streptococcus pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for acinetobacter baumannii * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification a total of 44 qlamp - positive samples were identified in the study . eight samples were subsequently excluded from analysis because of discharge or death within 3 days after admission . the final analysis group of 36 patients consisted of 19 with treatment established or adjusted to target antibiotics therapies according to qlamp results ( pathogen target - driven therapy group ) and 17 without treatment strategies adjustment whose treatment strategy was inconsistent with qlamp results ( empirical therapy group ) . demographic and clinical characteristics of the two groups demographic and clinical characteristics of the two groups are shown in table 4 . there is no significant difference ( p > 0.05 ) in the characteristics of the patients between the two groups , including gender , age , complications , clinical manifestation ( temperature , cough , sputum , and rales ) , blood routine examination , biochemical indicators , blood gas analysis , blood coagulation index , and chest radiograph infiltration . demographic and clinical characteristics of patients in the two groups data are presented as mean sd unless otherwise indicated . : data not applicable ; sd : standard deviation ; wbc : white blood cell ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; alb : albumin ; cre : creatinine ; bun : blood urea nitrogen ; pt : prothrombin time ; aptt : activated partial thromboplastin time ; ne : neutrophil count . patients taking an advantage for clinical condition with pathogen target - driven therapy there are 16 patients from the pathogen target - driven therapy group with clinical condition improvement 3 days after qlamp results reported , while there is only 1 from the empirical therapy group . the remission rate is 84.2% in the group with pathogen target - driven therapy , and 5.9% in the group with empirical therapy . the differences in the remission rates between these two groups evaluated by fisher 's exact tests are statistically significant ( p = 2 10 ) . daily mean temperature of the group with pathogen target - driven therapy shows a more obvious tendency of improvement than the group with empirical therapy [ figure 2 ] . on the 1 day when samples were collected , the mean temperature of target - driven therapy group and empirical therapy group were 37.95c and 37.68c , respectively , while the mean temperature of those two groups changed to 37.42c and 38.02c 3 days later . the decrease of daily mean temperature was 0.53c in the group with pathogen target - driven therapy while the decrease in the group with empirical therapy was 0.34c . similarly , decrease of total wbc number in group with pathogen target - driven therapy is more significant than the group with empirical therapy ( 2.15 10/l with pathogen target - driven therapy vs. 0.70 10/l with empirical therapy ) [ figure 3 ] . the body temperature of the group with pathogen target - driven therapy decreased while the group with empirical therapy had no significant improvement in body temperature . the wbc count of the group with pathogen target - driven therapy decreased while the group with empirical therapy fluctuated in wbc count . totally , 76 samples were recruited from 110 eligible samples overall in our study [ figure 1 ] . none of the samples were collected from the same onset of hap . as shown in table 2 , there were 70 samples with qlamp results greater than 10 copies / ml , 23 samples with qlamp results between 10 and 10 copies / ml , and 16 samples with qlamp results below 10 copies / ml . study profile . for each eligible patient , we collected lower respiratory secretion samples on the 1 day for routine culture and quantitative loop - mediated isothermal amplification tests and reported the results to the clinicians . we also collected the clinical data and treatment strategies before and after reporting the quantitative loop - mediated isothermal amplification results . qlamp results of specimen from patients with hospital - acquired pneumonia data are presented as number , unless otherwise indicated . qlamp : quantitative loop - mediated isothermal amplification ; lp : legionella pneumophila ; mp : mycoplasma pneumonia ; cp : chlamydophila pneumoniae ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia ; hi : haemophilus influenza . the concordance rates of the two assays for detecting sa , ec , pa , kp , sm , sp , and ab are 90.79% , 98.68% , 89.47% , 93.42% , 93.42% , 100.00% , and 77.63% , respectively [ table 3 ] . we also evaluated the difference between qlamp and culture results by mcnemar test , in which no significant difference was found ( p > 0.05 ) [ table 3 and supplementary tables 17 ] . the qlamp results of 4 samples for hi , lp , or mp were positive , while the culture results for these specimens were negative probably because of their low detectable rates in culture . we then studied the clinical data of these 4 samples and found that the qlamp results were all reliable except 1 mp positive sample due to the lack of specific species identified in the final diagnosis . no cp positive results were reported in the 76 samples either by qlamp or culture assay . congruence of qlamp and culture results in patients with hospital - acquired pneumonia qlamp : quantitative loop - mediated isothermal amplification ; sp : streptococcus pneumonia ; sa : staphylococcus aureus ; ec : escherichia coli ; kp : klebsiella pneumonia ; pa : pseudomonas aeruginosa ; ab : acinetobacter baumannii ; sm : stenotrophomonas maltophilia . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for escherichia coli * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for pseudomonas aeruginosa * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for klebsiella pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for stenotrophomonas maltophilia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for streptococcus pneumonia * * data are presented as no . qlamp : quantitative loopmediated isothermal amplification qlamp vs culture results for acinetobacter baumannii * * data are presented as no . eight samples were subsequently excluded from analysis because of discharge or death within 3 days after admission . the final analysis group of 36 patients consisted of 19 with treatment established or adjusted to target antibiotics therapies according to qlamp results ( pathogen target - driven therapy group ) and 17 without treatment strategies adjustment whose treatment strategy was inconsistent with qlamp results ( empirical therapy group ) . demographic and clinical characteristics of the two groups demographic and clinical characteristics of the two groups are shown in table 4 . there is no significant difference ( p > 0.05 ) in the characteristics of the patients between the two groups , including gender , age , complications , clinical manifestation ( temperature , cough , sputum , and rales ) , blood routine examination , biochemical indicators , blood gas analysis , blood coagulation index , and chest radiograph infiltration . demographic and clinical characteristics of patients in the two groups data are presented as mean sd unless otherwise indicated . : data not applicable ; sd : standard deviation ; wbc : white blood cell ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; alb : albumin ; cre : creatinine ; bun : blood urea nitrogen ; pt : prothrombin time ; aptt : activated partial thromboplastin time ; ne : neutrophil count . patients taking an advantage for clinical condition with pathogen target - driven therapy there are 16 patients from the pathogen target - driven therapy group with clinical condition improvement 3 days after qlamp results reported , while there is only 1 from the empirical therapy group . the remission rate is 84.2% in the group with pathogen target - driven therapy , and 5.9% in the group with empirical therapy . the differences in the remission rates between these two groups evaluated by fisher 's exact tests are statistically significant ( p = 2 10 ) . daily mean temperature of the group with pathogen target - driven therapy shows a more obvious tendency of improvement than the group with empirical therapy [ figure 2 ] . on the 1 day when samples were collected , the mean temperature of target - driven therapy group and empirical therapy group were 37.95c and 37.68c , respectively , while the mean temperature of those two groups changed to 37.42c and 38.02c 3 days later . the decrease of daily mean temperature was 0.53c in the group with pathogen target - driven therapy while the decrease in the group with empirical therapy was 0.34c . similarly , decrease of total wbc number in group with pathogen target - driven therapy is more significant than the group with empirical therapy ( 2.15 10/l with pathogen target - driven therapy vs. 0.70 10/l with empirical therapy ) [ figure 3 ] . the body temperature of the group with pathogen target - driven therapy decreased while the group with empirical therapy had no significant improvement in body temperature . the wbc count of the group with pathogen target - driven therapy decreased while the group with empirical therapy fluctuated in wbc count . this is a pilot study to assess the value of qlamp in guiding early target antibiotic therapies of hap , which may have significant effects on the mortality of hap and reduce the cost . although bacterial pneumonia is a kind of curable diseases due to the advent of the antibiotics , the mortality of bacterial hap is still high , which may contribute to the delay of target antibiotics therapies according to the results of sputum culture . as a new manner of detecting the etiology of different kinds of infections , qlamp is now commercially available . with the availability of this rapid ( results are available within 12 h ) , sensitive , and specific therefore , we apply qlamp for the decision - making regarding whether we selected empirical antibiotic therapies or the target antibiotic therapies for hap patients . since we would investigate the value of qlamp steering therapies , the first important issue was whether qlamp can etiologically diagnose hap in time . as qlamp assay was much more rapid than sputum culture , the most common assay in recent clinical practice , we first focus on the congruency of the results of qlamp and sputum culture . fortunately , there was no significance between qlamp and culture results of hap patients with infections of sa , ec , pa , kp , sm , sp , and ab . in addition , qlamp can detect hi , lp , and mp , which were not detectable by culture . besides , qlamp was a candidate method which could differentiate the pathogens between colonized and infectious status . after that , we prospectively enrolled 36 patients with hap with the same baseline data to evaluate the value of qlamp steering early target therapies . among these patients , the qlamp results were all positive based on the cut - off value ( > 1.0 10 copies / ml ) which was established in our former work ( data not shown ) . we randomly adjusted the regimen of these patients with empirical therapies according to the 2005 american thoracic society / infectious diseases society of america hap guideline or target therapies based on the results of qlamp . interestingly , we found that the clinical condition was significantly improved in the group with pathogen target - driven therapy compared to the group with empirical therapies . firstly , it was performed with a small sample size , and the stochastic effects were too big to drive a definite conclusion . secondly , we did not test the infection of fungus and virus of hap , which may contribute a small number of hap infections . thirdly , we did not assess the immunological conditions and nutritional statuses of the patients in these two groups , which may influence the effects of antibiotic therapies . perhaps , we could combine qlamp and sputum drug sensitivity test to individualize the hap regimens . however , the definition of conclusion can only be driven after multi - centered , randomized , and large sample sized research . since qlamp can not test the drug sensitivity , the combination of qlamp and sputum culture is the good choice for guiding early target therapies in hap patients . in conclusion , supplementary information is linked to the online version of the paper on the chinese medical journal website . this study was supported by grants from capital medical research and development foundation ( no . 2009 - 1018 ) , special fund for health - scientific research in the public interest program ( no . 201202011 ) by ministry of health of china , national high - tech research and development program ( no . 2006aa02z4a9 ) by ministry of science and technology of china , national 12 five - year plan major scientific and technological program ( no . 2012zx10004 - 206 ) by ministry of science and technology of china . this study was supported by grants from capital medical research and development foundation ( no . 2009 - 1018 ) , special fund for health - scientific research in the public interest program ( no . 201202011 ) by ministry of health of china , national high - tech research and development program ( no . 2006aa02z4a9 ) by ministry of science and technology of china , national 12 five - year plan major scientific and technological program ( no . 2012zx10004 - 206 ) by ministry of science and technology of china . | background : it is important to achieve the definitive pathogen identification in hospital - acquired pneumonia ( hap ) , but the traditional culture results always delay the target antibiotic therapy . we assessed the method called quantitative loop - mediated isothermal amplification ( qlamp ) as a new implement for steering of the antibiotic decision - making in hap.methods:totally , 76 respiratory tract aspiration samples were prospectively collected from 60 hap patients .
dna was isolated from these samples .
specific dna fragments for identifying 11 pneumonia - related bacteria were amplified by qlamp assay .
culture results of these patients were compared with the qlamp results .
clinical data and treatment strategies were analyzed to evaluate the effects of qlamp results on clinical data .
mcnemar test and fisher 's exact test were used for statistical analysis.results:the detection of staphylococcus aureus , escherichia coli , pseudomonas aeruginosa , klebsiella pneumonia , stenotrophomonas maltophilia , streptococcus pneumonia , and acinetobacter baumannii by qlamp was consistent with sputum culture ( p > 0.05 ) .
the qlamp results of 4 samples for haemophilus influenzae , legionella pneumophila , or mycoplasma pneumonia ( mp ) were inconsistent with culture results ; however , clinical data revealed that the qlamp results were all reliable except 1 mp positive sample due to the lack of specific species identified in the final diagnosis .
the improvement of clinical condition was more significant ( p < 0.001 ) in patients with pathogen target - driven therapy based on qlamp results than those with empirical therapy.conclusion:qlamp is a more promising method for detection of pathogens in an early , rapid , sensitive , and specific manner than culture . |
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, rural health issues have attracted much attention , and providing rural health care has become a concern and topic for international discussions . the health of these societies is a major concern for nurses , midwives , other health care providers and health care systems , and governments . if half of the world 's population lives in rural areas then considerable services are needed in those places . the primary health care program is used as a strategy to meet the needs of rural areas across the world . most of the primary health care programs in low income countries have used community health workers who were not competent for providing health services in poor rural areas . many of these services have not been widely appreciated , therefore they were mostly considered as unsuccessful . the quality , effectiveness , and performance of health care services are mostly related to the people in charge of providing them . only skilled and well trained professionals are able to provide high quality health care services . iran has a population of more than 75 million people , 25% of which are in rural areas according to 2011 census . after the revolution the priority of rural and underserved areas has been the basic policy . iran 's primary health care system has been established to improve access to health care for the deprived , and reduce the gap between health consequences of urban and rural areas . the health house , as the most basic unit of the primary health care network , is usually the only health facility for rural areas in iran . these health houses are equipped with health staff known as behvarz that are employed from local communities . behvarz have an essential role in the primary health care system of iran . in the past two decades the health indicators in iran have been significantly improved due to the execution of primary health care programs . however , the health care system is still facing many challenges in dealing with the society 's needs in rural areas . studies show that in addition to the primary problems , the primary health care system is facing new challenges . iran is experiencing rapid changes in all aspects of life : lifestyle changes , demographical changes , environmental , economic , social changes , and changes in the health and disease patterns , which is due to epidemiological transition . evidence related to international studies in health and health care in rural areas suggest that there are problems and challenges in these fields . strasser believes that despite the differences between developed and developing countries they all face challenges in terms of primary health care in rural areas . moreover , despite the claim to the efficiency of primary health care this approach has not been actually evaluated . sadrizadeh has mentioned some weaknesses in the primary health care system in iran , including weak inter- and intra - sector coordination , dissatisfied clients and suppliers , limited resources , and centralized decisions . malekafzali has also noted some other issues in iran 's health care system such as lack of attention to primary health care in medical education , incompatibility of the health data collection system with the new technology , lack of an evidence - based decision making culture , and lack of organization and community participation in decision making . these challenges are mostly related to the structure of the health care system , and are the result of a quantitative perspective toward the issues of health care in rural areas . there have been no studies with a qualitative approach on health care of rural areas , whereas understanding the process of health care in terms of social and cultural complexities is not possible within the proof oriented paradigm with its scientific and experimental approach . therefore , the best method to understand it is to study it in its natural environment . this view is compatible with the nature - oriented approach or an interpretation which attempts to study phenomena and processes in their natural environment . qualitative research methods have been specified as suitable methods of studying health and other areas of social researches in rural areas . the qualitative methods have the capacity to produce data that can determine the effects of social context on health . application of qualitative approaches in primary health care research is essential when the researcher wants to focus on the experiences of the participants . considering these view points and the limited understanding of health care phenomena , due to its social and cultural complexities , this research aimed to describe the experiences of health care workers and people of the process of health care in rural areas . this study was a qualitative research with content analysis and aimed to describe the process of health care consequences in rural areas of iran in 2011 - 2012 . research environment was the rural areas of arsanjan in fars province of iran . 26 participants were chosen including 21 health care providers ( 13 behvarz , 2 family physicians , 2 midwives and 4 nurses in the rural area ) , and 5 patients from that area . they were chosen based on the aim of the study and having experience in providing health care in rural areas ( at least 2 years of experience ) and the tendency to express their experience . the main participants of the study were behvarzs , who had a key role in the process of health care in rural areas , and according to need their experience was used to complete the data . data collection was conducted through semi - structured interview with open questions and small group discussion ( one session with four nurses ) . the interview took place in the health houses , health care centers , and the people 's houses in rural areas . the questions started with from your experience what are the processes of rural health care ? and all the ethical considerations were followed including written consent , right to withdraw from the study , maintaining anonymity , and confidentiality . the form about the mentioned facts was given to the participants before the study and the process of the study was explained , then the forms were signed by the participants . explanation about recording their voices during the interview were given and permission was obtained . the time and place of the interviews were agreed between them . data analysis was performed using qualitative content analysis approach based on the descriptive method by granheme and landman . the interviews were read sentence by sentence and freely coded by the researcher so that all the aspects of the content were described by the respondents . next the codes which were semantically similar were placed in the same level and these levels were grouped based on bigger categories . the aim of categorizing them was to reduce the levels , and finally they were interpreted in order to reveal their implications [ table 1 ] . an example of the data analysis process different methods were used to determine the accuracy of the study . the researcher performed member checking , prolonged engagement , and triangulation to increase the study 's credibility . after coding the interview , some parts of it were returned to the participants so they could check its accuracy . the researcher tried to have more meetings and engage more with the participants to increase his experience . for combining the sources and methods of data collection the researcher tried to choose the samples from all the related groups associated with the process of health care in rural areas . he also used different methods such as interviews and group discussion to collect the data . the interviews , codes , and emerged levels were sent to supervisors , consultants , and lecturers , who were familiar with the qualitative approach , to check the accuracy of the analysis and the interpretation for at least one - third of the interviews . to increase the transmission of the study , the researcher tried to make it possible for others to follow the research process by giving detailed and targeted descriptions of the research process . the participants in this study were 26 people ; including 21 people from health care team and 5 rural patients . their demographic characteristics are given in table 2 . by analyzing the data a level was formed and named inefficiency of care process in rural area , and this level had sub - classes such as self - arbitrary treatment , slow process of care , burnout and stress , dissatisfaction with care process , superficial view to care , and ineffective care . participant 's demographical characteristics the results showed that self - treatment and excessive drug use were very common among rural participants . behvarz number 3 stated that self - treatment can be seen very often in a way that in every house you go to you can see lots of drugs without prescriptions . the participants explained the reason for this was not having enough access to physicians and the inability of the health houses in providing drugs . the health house does not have medications when there is no physician and people will go to drug stores and get cold stops and painkillers . furthermore , cultural factors are another reason for self - treatment . people demand medication due to their wrong culture and we do not have that medication , so they go to the drugstore and buy it . the results indicated that another reason for inefficient care process is the slow process of care and this is from both the patients and the health care providers . the quality of care registration system ( old registration system , recording one problem in many places , and the many forms and frequent replacement ) and behvarz 's inadequate health capabilities are the reasons for delays of health care providers . the behvarz 's are very slow , for example they would delay a pregnant lady 's checkup by two to three hours just because it is time consuming . participant number 16 stated that he has to spend a whole day for a simple matter in the health care center since they are very slow in performing their duties . behvarz number 8 stated that when a child has vaccination they delay coming to the center so we have to go to their house . in many treatment cases patients do not refer to the health center on time . a nurse in the group discussion argued that in rural areas people go to the health center very late , for example a man had chest pain for three days and did not know that the pain was related to his heart . results showed that job stress is another reason for inefficient performance of health care providers . behvarz number 6 stated that since we have lots of work to do the pressure is very high and it influences our personal life . the large increase in population over time has also affected this matter . behvarz number 7 said that we have to deal with all kinds of people from their birth to the day they die , and it is really frustrating . other factors such as the patients and behvarz number 10 stated that we are trapped between the patients and the authorities , on the one hand the patients expect from us and on the other hand the authorities want us to warn them and this causes a lot of discomfort for us . dissatisfaction is one of the consequences of inefficient care process which can be seen among patients and care providers . patients dissatisfaction has various reasons such as behvarz 's inadequate capabilities , their disability in meeting patients needs and providing medications , and the quality of existing services and its accessibility . participant number 18 stated that the health house does not perform what they should do ; they only measure people 's blood pressure and check their height and weight , which we can do ourselves , therefore we are not satisfied . behvarz number 8 said for a simple illness they refer to the center and they complain why there is no medication in the center . people are not satisfied with the referral system ; they expect us to do something which we can not since it is set by the ministry . behvarz 's are also dissatisfied due to the high workload , and authorities not respecting and understanding their work . behvarz number 1 said : we do all this work and no one even sees and respects it ; we do not even receive any appreciation for what we do . behvarz number 12 declared : you have to continuously track the patients which is very difficult since the work is too much . behvarz number 13 stated : the written work is too much and everyone is complaining about it . results showed that there is a superficial view to the care process and care is not performed the way it should be in rural areas . the findings showed that another consequence of inefficient care process was quantity oriented and paying attention to the appearances and figurative numbers . number 1 behvarz stated : as i said it 's all about quantity not quality , we should just make files , our section has become a statistical section . the care providers feel that the aim of the health care system is not about providing care in its actual meaning , but it is about paying attention to the appearances and extracting statistical and figurative numbers . a nurse in the group discussion declared that most of the work is paper work and the statistical information does not help the patient . behvarz number 10 stated : it does not matter how much training we provide , behvarz number 2 said : what kind of care is this , everything is paper work , and nothing is effective . findings showed that an effective relationship does not exist in the health care process of rural areas . behvarz number 2 stated : people do not trust the health house ; therefore , they do not refer to it . this distrust exists among the people and care providers , and also the authorities do not trust the behvarzs . midwife number 5 said : all the statistical forms are in my handwriting , i do nt trust the behvarz , and they always have problems . misunderstanding also exists among people and behvarz , or between the specialist and behvarz . i keep explaining to the specialist that people in rural areas have problems , they can not even provide food for their families , how can you expect them to have a sanitized bathroom , they wo nt understand . care providers believe that the authorities do not fully understand the issues that exist in rural areas . a couple of people in the ministry provide programs without considering the issues , they do not know how rural areas are and they wo nt listen . the results showed that self - treatment and excessive drug use were very common among rural participants . behvarz number 3 stated that self - treatment can be seen very often in a way that in every house you go to you can see lots of drugs without prescriptions . the participants explained the reason for this was not having enough access to physicians and the inability of the health houses in providing drugs . the health house does not have medications when there is no physician and people will go to drug stores and get cold stops and painkillers . furthermore , cultural factors are another reason for self - treatment . people demand medication due to their wrong culture and we do not have that medication , so they go to the drugstore and buy it . the results indicated that another reason for inefficient care process is the slow process of care and this is from both the patients and the health care providers . the quality of care registration system ( old registration system , recording one problem in many places , and the many forms and frequent replacement ) and behvarz 's inadequate health capabilities are the reasons for delays of health care providers . the behvarz 's are very slow , for example they would delay a pregnant lady 's checkup by two to three hours just because it is time consuming . participant number 16 stated that he has to spend a whole day for a simple matter in the health care center since they are very slow in performing their duties . behvarz number 8 stated that when a child has vaccination they delay coming to the center so we have to go to their house . in many treatment cases patients do not refer to the health center on time . a nurse in the group discussion argued that in rural areas people go to the health center very late , for example a man had chest pain for three days and did not know that the pain was related to his heart . results showed that job stress is another reason for inefficient performance of health care providers . behvarz number 6 stated that since we have lots of work to do the pressure is very high and it influences our personal life . the large increase in population over time has also affected this matter . behvarz number 7 said that we have to deal with all kinds of people from their birth to the day they die , and it is really frustrating . other factors such as the patients and behvarz number 10 stated that we are trapped between the patients and the authorities , on the one hand the patients expect from us and on the other hand the authorities want us to warn them and this causes a lot of discomfort for us . i am tired of this job , every day there is a new problem . dissatisfaction is one of the consequences of inefficient care process which can be seen among patients and care providers . patients dissatisfaction has various reasons such as behvarz 's inadequate capabilities , their disability in meeting patients needs and providing medications , and the quality of existing services and its accessibility . participant number 18 stated that the health house does not perform what they should do ; they only measure people 's blood pressure and check their height and weight , which we can do ourselves , therefore we are not satisfied . behvarz number 8 said for a simple illness they refer to the center and they complain why there is no medication in the center . people are not satisfied with the referral system ; they expect us to do something which we can not since it is set by the ministry . behvarz 's are also dissatisfied due to the high workload , and authorities not respecting and understanding their work . behvarz number 1 said : we do all this work and no one even sees and respects it ; we do not even receive any appreciation for what we do . behvarz number 12 declared : you have to continuously track the patients which is very difficult since the work is too much . behvarz number 13 stated : the written work is too much and everyone is complaining about it . results showed that there is a superficial view to the care process and care is not performed the way it should be in rural areas . the findings showed that another consequence of inefficient care process was quantity oriented and paying attention to the appearances and figurative numbers . number 1 behvarz stated : as i said it 's all about quantity not quality , we should just make files , our section has become a statistical section . the care providers feel that the aim of the health care system is not about providing care in its actual meaning , but it is about paying attention to the appearances and extracting statistical and figurative numbers . a nurse in the group discussion declared that most of the work is paper work and the statistical information does not help the patient . behvarz number 10 stated : it does not matter how much training we provide , behvarz number 2 said : what kind of care is this , everything is paper work , and nothing is effective . the findings showed that another consequence of inefficient care process was quantity oriented and paying attention to the appearances and figurative numbers . number 1 behvarz stated : as i said it 's all about quantity not quality , we should just make files , our section has become a statistical section . the care providers feel that the aim of the health care system is not about providing care in its actual meaning , but it is about paying attention to the appearances and extracting statistical and figurative numbers . a nurse in the group discussion declared that most of the work is paper work and the statistical information does not help the patient . behvarz number 10 stated : it does not matter how much training we provide , behvarz number 2 said : what kind of care is this , everything is paper work , and nothing is effective . findings showed that an effective relationship does not exist in the health care process of rural areas . behvarz number 2 stated : people do not trust the health house ; therefore , they do not refer to it . this distrust exists among the people and care providers , and also the authorities do not trust the behvarzs . midwife number 5 said : all the statistical forms are in my handwriting , i do nt trust the behvarz , and they always have problems . misunderstanding also exists among people and behvarz , or between the specialist and behvarz . i keep explaining to the specialist that people in rural areas have problems , they can not even provide food for their families , how can you expect them to have a sanitized bathroom , they wo nt understand . care providers believe that the authorities do not fully understand the issues that exist in rural areas . a couple of people in the ministry provide programs without considering the issues , they do not know how rural areas are and they wo nt listen . the findings of the present study showed the inefficiency of the process of health care in rural areas . the consequences of this inefficiency can be seen in different aspects of the care process for people and care providers . one aspect of these consequences was the self - treatment of patients and using excessive medications . this matter was due to the culture of rural areas and the value of self - reliance and independence . but the main reason was not having access to care facilities , disability of health houses in providing care , and medication and the patient 's financial problems . arbitrary self - treatment in rural areas is associated with the structure of the health system and the cultural context of rural populations . the findings also indicated that there was delay in the process of health care system from the staff and also from the patients . delay from the patients was related to their not following treatment and failure to refer to the health center on time . other reasons such as culture , economic status , and health care system 's quality and accessibility were also effective . these findings are consistent with the study by mason that showed people in rural areas do not follow their treatment , and by the time they refer to the health center their illness has become sever . rass also indicated in his study that people do not refer to health care center unless their illness is sever and then they need to be hospitalized . the delay in health care system from the staff was related to the traditional registration of health care system such as the huge amount of files and information that need to be recorded , the time that it takes and being inadequate . they showed that registering all the information takes most of the health providers time and it is the main obstacle for the service quality . overall , the results of this study indicated that both health care structure and economic and cultural factors are involved in delaying the people 's treatment follow up . however , for the care providers this delay is mainly due to the health care system structure especially the health care system registration . according to the study , the outcome of inefficiency in the process of health care in rural areas is the stress and burnout of the staff . other factors such as the patient 's and authorities expectations from the care providers and the contradictory expectations of the care system also influence behvarzs stress and depression . their study also showed that many of the health care providers in rural areas had medium to high stress level due to the increase of workload and role confusion . the stress from workload was associated with the high population that each care provider had to cover compared with the standards , and the new health care programs which were added to their duties over time . their role confusion was related to the lack of unity of commands from the health centers in rural areas . lloyd et al . also noted in their study that organizational factors such as work pressure , workload , role confusion , and communication with supervisors are the predictors of social workers stress and exhaustion . on the other hand malakouti indicated in their study that despite the increase in the behvarzs workload during 30 years their stress and burnout has not increased . the results of the present study reveal dissatisfaction on both sides of the care process . these results were consistent with the study of arab et al . which showed that the model of overall job satisfaction of primary health care providers indicated that they are dissatisfied with their job . stated in their research that the number of care providers who were dissatisfied with their job was more than the number who were satisfied . the dissatisfaction of the patients in the present study was related to insufficient capabilities of care providers , the quality , and accessibility of services , and the obligation to comply with the referral system . they believe that people in developing countries , such as iran , will not be satisfied by the health care services due to the care system structure , characteristics of care providers , distribution of resources and payment methods , the mechanism of the referral system , and health information management . the results of both studies confirm the effects of the characteristics of health care providers and referral system on the people 's satisfaction with the care process . the results showed that the care process in rural areas was superficial and due to the care system being quantity oriented and its paper works has been detached from its aim which was to improve people 's health . most of the care providers believe that the care actions are not effective . they also noted in their study that there was a common complaint among the participants about the high amount of paper work ; most of the records were unnecessary and redundant , lack of coordination between different parts of a client 's case , the care providers had to record all the provided services in different parts including the patient 's file , different papers , and report tables . some of the care providers stated that the high amount of recording was the reason that they could not provide their services efficiently and it reduced the quality their work . lack of provided care in relation to distrust and lack of mutual understanding in the process of care in rural areas was another outcome of the inefficient health care process . gilson showed in his study that ensuring health and care depends on the patient 's cooperation , and care providers and health system factors . patient 's confidence in the health care system and health care staff is a prerequisite for the effectiveness of health care system , and an important requirement for a good communication , treatment success , and acceptance . the results also showed the lack of mutual understanding among caregivers , authorities , and expert supervisors . they reported conflict between the values of managers and caregivers as a source for stress , and that care givers felt frustration due to the misunderstanding of their role by others , lack of understanding of their skills and values , and not being appreciated for what they do . overall , the findings of the present study indicated dysfunction and inefficiency in the current health care process of rural areas in iran . this inefficiency has been found in different aspects of the care process such as the care quality , quality of interactions , people and caregivers satisfaction , stress and burnout , and arbitrary self - treatment . inefficient health care process outcomes showed difficulties in the current health care system in rural areas of iran . these consequences are rooted in the cultural and social context of rural areas , and are associated with the constituent elements of the health system such as the quality of care system registration , the ability of health care providers , the service quality and accessibility , and the referral system . | background : health care systems in rural areas face numerous challenges in meeting the community 's needs and adequate attention has not been paid to this problem .
the aim of this study was to describe the outcomes of health care process in rural society.materials and methods : twenty - six participants including twenty - one rural health care providers and five clients were selected according to purposive sampling .
the data were collected via semi - structured individual interviews and a mini focus group .
data were analyzed by using qualitative content analysis based on methods described by granheme and landman.results:data analysis eventually led to formation of one category of inefficiency in health care process in rural society including subcategories such as arbitrary self - therapy , slow care process , dissatisfaction with the care process , superficial caring , job stress and burn out of caregivers , and ineffective caring relationship.conclusion:outcomes in health care in rural society of iran represents inefficiency of the current health care process .
these outcomes are related to the cultural and social context of rural communities and the structure of the health system .
these outcomes in health care in the rural society of iran represent impairment of the current health care process .
the necessity of modifying the existing care trend with new models designed to improve the health care process is felt . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
glaucoma , a neurodegenerative disease , is characterized by gradual loss of retinal ganglion cells ( rgcs ) and optic nerve atrophy.1 multiple factors are related to glaucoma , such as high intraocular pressure ( iop)2 and low cerebral spinal fluid pressure.3 mller cells are the major type of glial cells in the mammalian retina that can support and nourish retinal neurons , maintain extracellular ion homeostasis , glutamate recycling , and interaction in synaptic transmission.4,5 therefore , mller cells are involved in retinal function . investigations indicate that mller cells not only play an important physiological role , but they are also involved in multiple pathological retinal diseases such as glaucoma.68 it has been reported that reactive mller cells can aggravate retinal damage by releasing cytokines such as tumor necrosis factor- ( tnf-).9 growing evidence supports that increased glial production of tnf- contributes to the neurodegeneration in glaucoma.10 tnf- is a secreted inflammatory cytokine that is responsible for apoptosis , necrosis , and inflammation.11,12 tnf- is increased in the retina following ischemia or damage and other neurodegenerative disorders.1317 -aminoadipic acid ( aaa ) , a six - carbon homolog of glutamate , is a well - known compound that induces specific glial toxicity through blocking the glutamate uptake.1820 the use of aaa to interfere with glial influence on neuronal tissues is reported.21,22 however , the effects of aaa inhibition on mller cell gliosis in glaucoma are unknown . the current study aimed to determine if aaa treatment inhibits mller cell gliosis and protects against retinal abnormalities induced in a rat model of acute ocular hypertension ( aoh ) mimicking glaucoma adult male sprague dawley rats ( weight range , 200250 g , 810 weeks ) were used in this study in accordance with the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research and followed the people s republic of china animal care guidelines . this study was monitored and approved by the animal care committee of the capital medical university , scxk ( jing ) 2012 - 0001 . the animals were housed with a 12-hour light/12-hour dark cycle with standard chow and water . all surgical procedures were performed under anesthesia with intraperitoneal chloral hydrate ( 450 mg / kg ) and topical 0.5% proparacaine hydrochloride eye drops ( alcon , inc . , the iop was increased to 110 mmhg ( 14.63 kpa ) for 60 minutes by using an elevated 500 ml plastic container of sterile physiological saline connected to a 27-gauge needle placed in the anterior chamber of the eye . sham procedure eyes were treated similarly but without the elevation of the bottle ; hence , the normal iop was maintained . time - course examination was performed at 1 day , 3 days , and 5 days after acute iop elevation . animals were divided into sham operation ( ctrl ) group , aoh group , aoh + phosphate - buffered saline ( pbs ) control group , and aoh + aaa - treated group . aaa ( 250 g ) was injected into vitreous humor in 5 l at 50 g / l concentration after 3 hours of acute iop elevation according to the literature.23,24 tissues were fixed with 4% paraformaldehyde . eyeballs were cryoprotected in 30% sucrose overnight at 4c and then embedded in optimal cutting temperature compound ( sakura finetechinical co. ltd . , sections ( 14 m ) were incubated in 3% bovine serum albumin and 0.3% triton x-100 ( sigma - aldrich co. , st louis , mo , usa ) to block nonspecific binding and then incubated with primary mouse monoclonal antibodies against thy-1 ( 1:200 , rgc marker ; abcam , cambridge , uk ) , primary mouse monoclonal antibodies against glutamine synthetase ( gs ; 1:100 , mller glial cell maker ; abcam ) , rabbit polyclonal antibodies against glial fibrillary acidic protein ( gfap ; 1:200 , gliosis maker ; cell signaling technology inc . , danvers , ma , usa ) overnight at 4c , followed by incubation with secondary antibodies for 1 hour at room temperature . sections were mounted with fluoromount - g ( southernbiotech , birmingham , al , usa ) . slides were visualized using a confocal microscope ( leica microsystems , wetzlar , germany ) . the intensity of immunoreactivity from photographs was analyzed using image - proplus 6.0 ( media cybernetics inc . , silver spring , md , usa ) optical densities obtained from immunohistochemistry images were corrected by subtracting the average value of background noise from five image inputs . the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay was conducted according to the manufacturer s protocol . briefly , tissue sections were fixed in paraformaldehyde and were subsequently incubated in a tunel reaction medium . the sections were then mounted on microscope slides and examined under ultraviolet light using an epifluorescence microscope . retinas from sham operation group ( ctrl ) , aoh group , aoh + pbs control group , and aoh + aaa group were homogenized in lysis buffer and sonicated to dissolve the tissue completely . in all , 40 g of total proteins from each sample were loaded per lane for sodium dodecyl sulfate polyacrylamide gel electrophoresis . the proteins were transferred onto the nitrocellulose membrane ( schleicher & schuell , dassel , germany ) and the blocked nitrocellulose membrane was incubated with a primary rabbit polyclonal antibody against tnf- ( 1:1,000 ; abcam ) for 3 hours ( room temperature ) . after washing in tris buffered saline , with tween-20 ( tbst ) , the membranes were incubated with horseradish peroxidase - conjugated goat anti - rabbit or mouse igg prior to detection of the labeled proteins by using ecl - plus kit ( perkinelmer inc . , waltham , ma , usa ) followed by exposure of blots to x - omat ( kodak , rochester , ny , usa ) imaging film . the images were quantified using imagej software ( national institutes of health , maryland , usa ) . the gel doc-2000 imaging system ( bio - rad laboratories inc . , hercules , ca , usa ) was used to perform the quantitative analysis of western blot results . the -actin band density was expressed as 100% , and the other group was expressed as percentage of that from the control group . analysis of variance was used to determine the difference between tnf- and fluorescence intensity among three or more independent ( unrelated ) groups , followed by multiple comparisons using the student newman wallis test was used to determine the difference between tunel - positive cells and thy-1-positive rgcs among three or more independent ( unrelated ) groups , followed by multiple comparisons using the nemenyi test . statistical analysis was performed using sas 9.2 ( sas institute inc . , cary , nc , usa ) . adult male sprague dawley rats ( weight range , 200250 g , 810 weeks ) were used in this study in accordance with the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research and followed the people s republic of china animal care guidelines . this study was monitored and approved by the animal care committee of the capital medical university , scxk ( jing ) 2012 - 0001 . the animals were housed with a 12-hour light/12-hour dark cycle with standard chow and water . all surgical procedures were performed under anesthesia with intraperitoneal chloral hydrate ( 450 mg / kg ) and topical 0.5% proparacaine hydrochloride eye drops ( alcon , inc . , hnenberg , switzerland ) . the iop was increased to 110 mmhg ( 14.63 kpa ) for 60 minutes by using an elevated 500 ml plastic container of sterile physiological saline connected to a 27-gauge needle placed in the anterior chamber of the eye . sham procedure eyes were treated similarly but without the elevation of the bottle ; hence , the normal iop was maintained . time - course examination was performed at 1 day , 3 days , and 5 days after acute iop elevation . animals were divided into sham operation ( ctrl ) group , aoh group , aoh + phosphate - buffered saline ( pbs ) control group , and aoh + aaa - treated group . aaa ( 250 g ) was injected into vitreous humor in 5 l at 50 g / l concentration after 3 hours of acute iop elevation according to the literature.23,24 eyeballs were cryoprotected in 30% sucrose overnight at 4c and then embedded in optimal cutting temperature compound ( sakura finetechinical co. ltd . , tokyo , japan ) . sections ( 14 m ) were incubated in 3% bovine serum albumin and 0.3% triton x-100 ( sigma - aldrich co. , st louis , mo , usa ) to block nonspecific binding and then incubated with primary mouse monoclonal antibodies against thy-1 ( 1:200 , rgc marker ; abcam , cambridge , uk ) , primary mouse monoclonal antibodies against glutamine synthetase ( gs ; 1:100 , mller glial cell maker ; abcam ) , rabbit polyclonal antibodies against glial fibrillary acidic protein ( gfap ; 1:200 , gliosis maker ; cell signaling technology inc . , danvers , ma , usa ) overnight at 4c , followed by incubation with secondary antibodies for 1 hour at room temperature . sections were mounted with fluoromount - g ( southernbiotech , birmingham , al , usa ) . slides were visualized using a confocal microscope ( leica microsystems , wetzlar , germany ) . the intensity of immunoreactivity from photographs was analyzed using image - proplus 6.0 ( media cybernetics inc . , silver spring , md , usa ) . optical densities obtained from immunohistochemistry images were corrected by subtracting the average value of background noise from five image inputs . the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay was conducted according to the manufacturer s protocol . briefly , tissue sections were fixed in paraformaldehyde and were subsequently incubated in a tunel reaction medium . the sections were then mounted on microscope slides and examined under ultraviolet light using an epifluorescence microscope . retinas from sham operation group ( ctrl ) , aoh group , aoh + pbs control group , and aoh + aaa group were homogenized in lysis buffer and sonicated to dissolve the tissue completely . in all , 40 g of total proteins from each sample were loaded per lane for sodium dodecyl sulfate polyacrylamide gel electrophoresis . the proteins were transferred onto the nitrocellulose membrane ( schleicher & schuell , dassel , germany ) and the blocked nitrocellulose membrane was incubated with a primary rabbit polyclonal antibody against tnf- ( 1:1,000 ; abcam ) for 3 hours ( room temperature ) . after washing in tris buffered saline , with tween-20 ( tbst ) , the membranes were incubated with horseradish peroxidase - conjugated goat anti - rabbit or mouse igg prior to detection of the labeled proteins by using ecl - plus kit ( perkinelmer inc . , waltham , ma , usa ) followed by exposure of blots to x - omat ( kodak , rochester , ny , usa ) imaging film . the images were quantified using imagej software ( national institutes of health , maryland , usa ) . the gel doc-2000 imaging system ( bio - rad laboratories inc . , hercules , ca , usa ) was used to perform the quantitative analysis of western blot results . the -actin band density was expressed as 100% , and the other group was expressed as percentage of that from the control group . analysis of variance was used to determine the difference between tnf- and fluorescence intensity among three or more independent ( unrelated ) groups , followed by multiple comparisons using the student newman wallis test was used to determine the difference between tunel - positive cells and thy-1-positive rgcs among three or more independent ( unrelated ) groups , followed by multiple comparisons using the nemenyi test . statistical analysis was performed using sas 9.2 ( sas institute inc . , cary , nc , usa ) . values were reported as mean sd . a value of p<0.05 was considered as statistically significant . to determine the effects of aaa on rgcs in the aoh model , aoh was induced in sd rats and rats were intravitreally treated with pbs , aaa , aoh + pbs , and aoh + aaa . significantly reduced retinal thickness in the inner plexiform layer ( ipl ) and inner nuclear layer was observed after aoh induction versus control ( figure s1 ) . the mean values of tunel - positive cells per visual field were 0.10.3 in control ( figure 1a ) , 0.20.4 in aaa(figure 1b ) , 4.60.8 in aoh + pbs , and 1.70.7 in aoh + aaa . aoh induced the apoptotic death of cells in the ganglion cell layer(gcl ; figure 1c ) . in the aoh model , intravitreal treatment with aaa attenuated the tunel - positive rgcs more significantly than the intravitreal treatment with pbs ( p<0.05 ; figure 1d ) , indicating that aaa treatment rescued rgcs from the cell apoptosis induced in the aoh model . in addition , retinal sections were immunostained with antibody against thy-1 , a marker for rgcs.25 the mean values of thy-1-positive rgcs per visual field in ctrl , aaa , aoh + pbs , and aoh + aaa groups were 8.20.9 , 8.10.8 , 3.90.7 , and 60.6 , respectively . aaa treatment did not change the number of rgcs in normal conditions ( figure 2a and b ) . when compared with the ctrl group ( figure 2a ) , the number of thy-1-positive rgcs was decreased in the aoh + pbs group ( figure 2c ) , and aaa treatment significantly rescued thy-1-positive rgcs in aoh ( p<0.05 ; figure 2d ) , corresponding to aaa - attenuated apoptosis of rgcs in aoh ( aoh + pbs versus aoh + aaa , p<0.05 ; figure 1 ) . in the rat model of aoh , aaa rescued rgcs . to determine if aaa protects against aoh - induced rgc death through modulating mller cell gliosis , immonohistochemistry for gfap ( a marker for activated glial cells ) and gs ( mller cell - specific marker ) was conducted.26 mller cells cover the whole retina from the nerve fiber layer to the photoreceptor layer,27 and gfap was mainly expressed along nerve fiber layer and gcl and was less or absent at other layers in the control group ( figure s2 ) . in the rat model of aoh , colocalization of gfap and gs was observed across ipl ( figure s2 ) , indicating mller cell activation . after 1 day , 3 days , and 5 days of aoh induction in rat , gfap expression was also observed across ipl ( figure s3 ) , indicating mller cell gliosis . aaa treatment versus pbs control markedly reduced gfap immunoreactivity across ipl ( figure 3 ) , indicating attenuation in mller cell gliosis . as mentioned earlier , aaa rescued rgcs likely through the inhibition of mller cells gliosis in the rat model with aoh . activation of mller cells may induce tnf- production , leading to neurodegeneration in glaucoma.10 to determine if aaa modulates tnf- production , western blot was performed for the investigation of retinal protein levels of tnf-. significantly upregulated protein levels of retinal tnf- were observed in aoh versus sham operation ( ctrl ) group ( p<0.05 ; figure 4 ) . importantly , aaa treatment versus pbs control significantly attenuated tnf- levels in the retinas of the rat model of aoh ( p<0.05 ; figure 4 ) . to determine the effects of aaa on rgcs in the aoh model , aoh was induced in sd rats and rats were intravitreally treated with pbs , aaa , aoh + pbs , and aoh + aaa . significantly reduced retinal thickness in the inner plexiform layer ( ipl ) and inner nuclear layer was observed after aoh induction versus control ( figure s1 ) . the mean values of tunel - positive cells per visual field were 0.10.3 in control ( figure 1a ) , 0.20.4 in aaa(figure 1b ) , 4.60.8 in aoh + pbs , and 1.70.7 in aoh + aaa . aoh induced the apoptotic death of cells in the ganglion cell layer(gcl ; figure 1c ) . in the aoh model , intravitreal treatment with aaa attenuated the tunel - positive rgcs more significantly than the intravitreal treatment with pbs ( p<0.05 ; figure 1d ) , indicating that aaa treatment rescued rgcs from the cell apoptosis induced in the aoh model . in addition , retinal sections were immunostained with antibody against thy-1 , a marker for rgcs.25 the mean values of thy-1-positive rgcs per visual field in ctrl , aaa , aoh + pbs , and aoh + aaa groups were 8.20.9 , 8.10.8 , 3.90.7 , and 60.6 , respectively . aaa treatment did not change the number of rgcs in normal conditions ( figure 2a and b ) . when compared with the ctrl group ( figure 2a ) , the number of thy-1-positive rgcs was decreased in the aoh + pbs group ( figure 2c ) , and aaa treatment significantly rescued thy-1-positive rgcs in aoh ( p<0.05 ; figure 2d ) , corresponding to aaa - attenuated apoptosis of rgcs in aoh ( aoh + pbs versus aoh + aaa , p<0.05 ; figure 1 ) . in the rat model of aoh , aaa rescued rgcs . to determine if aaa protects against aoh - induced rgc death through modulating mller cell gliosis , immonohistochemistry for gfap ( a marker for activated glial cells ) and gs ( mller cell - specific marker ) was conducted.26 mller cells cover the whole retina from the nerve fiber layer to the photoreceptor layer,27 and astrocytes mainly locate along gcl . gfap was mainly expressed along nerve fiber layer and gcl and was less or absent at other layers in the control group ( figure s2 ) . in the rat model of aoh , colocalization of gfap and gs was observed across ipl ( figure s2 ) , indicating mller cell activation . after 1 day , 3 days , and 5 days of aoh induction in rat , gfap expression was also observed across ipl ( figure s3 ) , indicating mller cell gliosis . aaa treatment versus pbs control markedly reduced gfap immunoreactivity across ipl ( figure 3 ) , indicating attenuation in mller cell gliosis . as mentioned earlier , aaa rescued rgcs likely through the inhibition of mller cells gliosis in the rat model with aoh . activation of mller cells may induce tnf- production , leading to neurodegeneration in glaucoma.10 to determine if aaa modulates tnf- production , western blot was performed for the investigation of retinal protein levels of tnf-. significantly upregulated protein levels of retinal tnf- were observed in aoh versus sham operation ( ctrl ) group ( p<0.05 ; figure 4 ) . importantly , aaa treatment versus pbs control significantly attenuated tnf- levels in the retinas of the rat model of aoh ( p<0.05 ; figure 4 ) . glaucoma is a leading cause of irreversible vision loss characterized by progressive death of rgcs , and elevated iop is a major risk factor.28 a rodent model of aoh is well established for acute angle closure glaucoma , and it has been widely used to investigate the pathogenesis of death of rgcs.29 in the current rat model of aoh , we found induced cell apoptosis and decreased number of rgcs with iop induction versus sham operation control . importantly , treatment of aaa versus pbs control significantly attenuated rgc apoptosis and rescued the reduced number of rgcs , demonstrating protective effects on aoh retinas ( figure 5 ) . activated mller cells contribute to the progression of glaucoma.30 mller cells perform a multitude of important regulatory and supportive roles , including secretion of trophic factors , removal of metabolic waste , and neurotransmitter recycling.31,32 we found that mller cells were activated with induced gfap immunoreactivity in the rat model of aoh versus control retinas . gfap , an intermediate filament protein , is considered as a marker of reactive mller cell gliosis,27 which is not or less expressed in mller cells in normal retinas and expressed highly at ischemic,33 light - induced retinal degeneration,34 and retinal detachment.35 activation of mller cells so far was demonstrated to have both protective and detrimental effects.3638 especially early after injury , mller cell gliosis is believed to be neuroprotective and promotes the repair of neurons in response to injury,36 with gfap upregulation to provide additional structural integrity to the retina at the site of injury.39 however , activated mller cells also express tnf-,40 monocyte chemoattractant protein,41 and nitric oxide42 to induce death of rgcs . tnf- induction contributes to inflammation , apoptosis , and necrosis , leading to cell death . we observed that aaa treatment attenuated gfap immunoreactivity in mller cell processes across ipl and reduced retinal tnf- levels in the rat model of aoh . these findings suggested that aaa promoted survival of rgcs in the rat model of aoh , likely through the inhibition of aoh - induced mller cell gliosis and in turn downregulation of tnf- protein production . we found that aaa could effectively protect retina against loss of rgcs and apoptosis in aoh retinas through attenuating mller cell gliosis and downregulating tnf- production . these observations suggested that aaa might be a potential therapeutic target in the treatment of neurodegeneration in glaucoma . ( a ) reprehensive results of hoechst staining in rat retina after 1 d , 3 d , and 5 d of aoh ; ( b ) quantitative analysis of the retinal thickness . notes : the results showed that the thickness of ipl and inl layers decreased significantly following the reperfusion time after aoh treatment ( scale bar = 50 m ) . * p<0.05 compared with the control group ( n=4 per group ) . abbreviations : aoh , acute ocular hypertension ; ipl , inner plexiform layer ; inl , inner nuclear layer ; ctrl , control ; d , day ; gcl , ganglion cell layer ; onl , outer nuclear layer . note : strong expression of gfap was shown in the gs - positive mller cell after 5 d of aoh compared with the control group ( scale bar = 50 m ) . abbreviations : gs , glutamine synthetase ; gfap , glial fibrillary acidic protein ; aoh , acute ocular hypertension ; ctrl , control ; gcl , ganglion cell layer ; ipl , inner plexiform layer ; inl , inner nuclear layer ; onl , outer nuclear layer ; d , days . increased gfap immunoreactivity after aoh induction in rat retinas . notes : ( a ) immunofluorescent staining showed that aoh increased gfap immunoreactivity in the mller cell processes across ipl . abbreviations : gfap , glial fibrillary acidic protein ; aoh , acute ocular hypertension ; ipl , inner plexiform layer ; d , day ; pbs , phosphate - buffered saline ; aaa , -aminoadipic acid . | objectiveocular hypertension is an important risk factor for glaucoma .
the purpose of this study was to investigate the gliotoxic effects of -aminoadipic acid ( aaa ) in a rat model of aoh and its underlying mechanisms.materials and methodsin the rat model of acute ocular hypertension ( aoh ) , intraocular pressure was increased to 110 mmhg for 60 minutes .
animals were divided into four groups : sham operation ( ctrl ) , aoh , aoh + phosphate - buffered saline ( pbs ) , and aoh + aaa .
cell apoptosis in the ganglion cell layer was detected with the terminal deoxynucleotidyl transferase - mediated uridine 5-triphosphate - biotin nick end labeling ( tunel ) assay , and retinal ganglion cells ( rgcs ) immunostained with thy-1 were counted .
mller cell activation was detected using immunostaining with glutamine synthetase and glial fibrillary acidic protein .
tumor necrosis factor- ( tnf- ) was examined using western blot.resultsin the rat model of aoh , cell apoptosis was induced in the ganglion cell layer and the number of rgcs was decreased .
mller cell gliosis in the retinas of rats was induced , and retinal protein levels of tnf- were increased .
intravitreal treatment of aaa versus pbs control attenuated these retinal abnormalities to show protective effects in the rat model of aoh.conclusionin the retinas of the rat model of aoh , aaa treatment attenuated retinal apoptosis in the ganglion cell layer and preserved the number of rgcs , likely through the attenuation of mller cell gliosis and suppression of tnf- induction .
our observations suggest that aaa might be a potential therapeutic target in glaucoma . |
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the term adhesion molecules refers to those cell surface structures that allow cells to adhere to each other and the extracellular matrix . cell adhesion molecules ( cams ) enable cancer - related biological processes like survival , detachment , migration , extravasation , and metastasis , and thus play a crucial role in tumorigenesis , tumor progression , and metastasis ( 1 , 2 ) . apart from regulating cell - cell and cell - matrix interactions , cams also influence cell motility , signalling , and differentiation , apoptosis , and gene transcription ( 3 ) . five families of cell adhesion molecules ( cams ) have been identified , which include cadherins , integrins , immunoglobulin superfamily , selectins , and cd44 ( 4 ) . to date , reduced , absent , or disorganized expression of cams has been observed in a variety of human tumor , including breast , lung , gastric , bladder , prostate , head and neck , and colorectal cancer ( 5 , 6 ) . icam-1 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of cams ( 7 ) . icam-1 is normally expressed on the surface of various types of cells : leukocytes , endothelial cells , and fibroblasts ( 8 , 9 ) . there is mounting evidence demonstrating that icam-1 is also expressed on the surface of many cancer cell types ( 1013 ) . it has also been proposed that icam-1 may be involved in the process of cancer metastases , facilitating the spread of metastatic cancer cells to secondary sites ( 9 ) . moreover , increased icam-1 expression enhances tumor growth , while altered icam-1 expression could be caused by genetic variation ( 14 ) . the icam-1 gene , located in chromosome 19p13 , has at least two functional biallelic polymorphisms . these two single nucleotide polymorphisms ( snps ) , previously described in icam-1 gene at codons 241 ( glycine to arginine substitution ; g to a ; rs1799969 ) in exon 4 and 469 ( a lysine to glutamic acid substitution ; a to g ; rs5498 ) in exon 6 , were also shown to modulate the susceptibility for several types of cancers including prostate ( 15 ) , colorectal ( 16 ) and breast cancers ( 17 ) . recent genome - wide association study has demonstrated a strong correlation between k469e polymorphism and icam-1 levels ( 18 ) . icam-1 g241r polymorphism has been demonstrated to be of importance in binding to the mac-1 form of leucocyte integrin ( 19 ) , and therefore affect the adhesive function of icam-1 . a growing number of studies have studied the relationship between icam-1 gene polymorphisms and tumor susceptibility , but their results remain inconsistent . this lack of consistency might be attributable to the presence of genetic heterogeneity across ethnic populations , small sample size limitations , and publication bias . therefore , to confirm the role of the icam-1 k469e and g241r polymorphisms in tumorigenesis , we conducted a comprehensive meta - analysis on eligible case - control studies published to date . to the best of our knowledge , this is the most comprehensive meta - analysis regarding the icam-1 k469e and g241r polymorphisms and their association with cancer risk . a comprehensive literature search of pubmed , embase , web of science , science direct , springerlink , ebsco , wanfang , and chinese national knowledge infrastructure databases ( last search updated in november 2013 ) was conducted to identify case - control studies that investigated the association between icam-1 k469e and g241r polymorphisms and cancer risk . the search terms were as follows : cancer or carcinoma or neoplasm or tumor in combination with there was no restriction on period , sample size , population , language , or type of report for minimizing potential publication bias . we evaluated potentially relevant genetic association studies by examining their titles and abstracts , and all published studies matching with the eligible criteria were retrieved . studies included in the meta - analysis were required to meet the following criteria : 1 ) case - control studies which evaluated the association between icam-1 k469e and/or g241r polymorphisms and cancer risk ; 2 ) study design : either retrospective or nested case - control design ; 3 ) any diagnoses of patients with cancer had to be confirmed by pathological examinations ; and 4 ) independent variables : the genotype and/or allele counts of icam-1 k469e polymorphism or g241r polymorphism . the exclusion criteria of the meta - analysis were : 1 ) case - control studies not focusing on the correlation between icam-1 k469e and g241r polymorphisms and cancer risk ; 2 ) studies with duplicate data ; 3 ) studies based on incomplete data ; and 4 ) meta - analyses , letters , reviews and editorial articles . when an individual author published several articles obtained from the same patient population , only the newest or most complete article was included in the analysis . the data from the published studies were extracted independently by two reviewers ( d cheng and b liang ) . the following information was collected from each study : first author s name , year of publication , country of origin , ethnicity , cancer type , genotyping method , source of controls , number of cases and controls , genotype frequency in cases and controls , and hardy - winberg equilibrium ( hwe ) . in case of discrepancies , a consensus on each item crude odds ratios ( ors ) together with their corresponding 95% cis were used to assess the strength of association between icam-1 k469e or g241r polymorphisms and the risk of cancer . allele model ( mutation ( m ) allele versus wild ( w ) allele ) , dominant model ( wm+mm versus ww ) , recessive model ( mm versus wm+ww ) , homozygote comparison ( mm versus ww ) , and heterozygote comparison ( wm versus ww ) were evaluated , respectively . subgroup analyses were done by ethnicity ( asian , european , and america ) . between - study heterogeneity was assessed by calculating q - statistic ( heterogeneity was considered statistically significant if p < 0.10 ) ( 20 ) and quantified using the i value ( i < 25% represents no heterogeneity , i= 2550% represents moderate heterogeneity , i= 5075% represents large heterogeneity , and i > if results were not heterogeneous , the pooled ors were calculated by a fixed - effect model ; otherwise , a random - effect model was used . the significance of the combined ors was determined by the z - test , in which p<0.05 was considered significant . moreover , relative influence of each study on the pooled estimate was assessed by excluding a single study each time for sensitivity analysis . begg s funnel plots ( 22 ) and egger s linear regression test ( 23 ) were used to evaluate publication bias . all statistical analyses were performed using stata version 12.0 ( stata corporation , college station , tx ) . a comprehensive literature search of pubmed , embase , web of science , science direct , springerlink , ebsco , wanfang , and chinese national knowledge infrastructure databases ( last search updated in november 2013 ) was conducted to identify case - control studies that investigated the association between icam-1 k469e and g241r polymorphisms and cancer risk . the search terms were as follows : cancer or carcinoma or neoplasm or tumor in combination with there was no restriction on period , sample size , population , language , or type of report for minimizing potential publication bias . we evaluated potentially relevant genetic association studies by examining their titles and abstracts , and all published studies matching with the eligible criteria were retrieved . studies included in the meta - analysis were required to meet the following criteria : 1 ) case - control studies which evaluated the association between icam-1 k469e and/or g241r polymorphisms and cancer risk ; 2 ) study design : either retrospective or nested case - control design ; 3 ) any diagnoses of patients with cancer had to be confirmed by pathological examinations ; and 4 ) independent variables : the genotype and/or allele counts of icam-1 k469e polymorphism or g241r polymorphism . the exclusion criteria of the meta - analysis were : 1 ) case - control studies not focusing on the correlation between icam-1 k469e and g241r polymorphisms and cancer risk ; 2 ) studies with duplicate data ; 3 ) studies based on incomplete data ; and 4 ) meta - analyses , letters , reviews and editorial articles . when an individual author published several articles obtained from the same patient population , only the newest or most complete article was included in the analysis . the data from the published studies were extracted independently by two reviewers ( d cheng and b liang ) . the following information was collected from each study : first author s name , year of publication , country of origin , ethnicity , cancer type , genotyping method , source of controls , number of cases and controls , genotype frequency in cases and controls , and hardy - winberg equilibrium ( hwe ) . in case of discrepancies , a consensus on each item crude odds ratios ( ors ) together with their corresponding 95% cis were used to assess the strength of association between icam-1 k469e or g241r polymorphisms and the risk of cancer . allele model ( mutation ( m ) allele versus wild ( w ) allele ) , dominant model ( wm+mm versus ww ) , recessive model ( mm versus wm+ww ) , homozygote comparison ( mm versus ww ) , and heterozygote comparison ( wm versus ww ) were evaluated , respectively . subgroup analyses were done by ethnicity ( asian , european , and america ) . between - study heterogeneity was assessed by calculating q - statistic ( heterogeneity was considered statistically significant if p < 0.10 ) ( 20 ) and quantified using the i value ( i < 25% represents no heterogeneity , i= 2550% represents moderate heterogeneity , i= 5075% represents large heterogeneity , and i > if results were not heterogeneous , the pooled ors were calculated by a fixed - effect model ; otherwise , a random - effect model was used . the significance of the combined ors was determined by the z - test , in which p<0.05 was considered significant . moreover , relative influence of each study on the pooled estimate was assessed by excluding a single study each time for sensitivity analysis . begg s funnel plots ( 22 ) and egger s linear regression test ( 23 ) were used to evaluate publication bias . all statistical analyses were performed using stata version 12.0 ( stata corporation , college station , tx ) . 16 articles containing 18 case - control studies were included in the meta - analysis , including 4,844 cancer cases and 5,618 healthy controls . there were 18 case - control studies concerning icam-1 k469e polymorphism ( 1517 , 2435 ) , and 6 case - control studies concerning icam-1 g241r ( 24 , 2730 , 33 ) . for icam-1 k469e polymorphism , seven studies were conducted in european populations , six in asian populations , four in america populations , and one in oceania populations . moreover , there were four studies of european populations ( 2830 , 33 ) , one study of asian populations ( 24 ) , and one study of america populations ( 27 ) for icam-1 g241r polymorphism . the genotype distributions among the controls of all studies were in agreement with hwe except for two studies for k469e ( 27 , 31 ) and one study for g241r ( 33 ) . the detailed characteristics of the eligible studies included in this meta - analysis were shown in table 1 . flow chart showing study selection procedure baseline characteristics of the 18 eligible studies for the analysis of icam-1 k469e and g241r polymorphisms pb , population - based controls , hb , hospital - based controls . pcr , polymerase chain reaction ; ssp , sequence - specific primers ; rflp , restriction fragment length polymorphism results of this meta - analysis were shown in table 2 . icam-1 k469e and g241r polymorphisms and cancer risk in the overall analysis , we did not find any significant association between icam-1 k469e polymorphism and the risk of cancers in all comparison models ( g allele vs. a allele : or = 1.03 , 95% ci = 0.891.19 , p=0.66 ; gg+ga vs. aa : or= 0.98 , 95% ci = 0.831.17 , p=0.86 ; gg vs. ga+aa : or = 1.09 , 95% ci = 0.871.37 , p=0.44 ; gg vs. aa : or = 1.15 , 95% ci = 0.841.56 , p=0.38 ; ga vs. aa : or= 0.95 , 95% ci = 0.821.11 , p=0.53 ) ( fig . the results suggested that the icam-1 k469e polymorphism may be not associated with overall cancer risk . meta - analysis with a random - effects model for the association between the risk of cancer and icam-1 k469e polymorphism ( dominant model : gg + ga vs. aa ) as shown in table 2 , in the subgroup analysis by ethnicity , we found a significant association under homozygous comparison ( gg vs. aa : or=1.53 , 95 % ci=1.032.27 , p=0.03 ) in asian subgroup . moreover , there was significant association under dominant model ( gg+ga vs. aa : or=0.77 , 95 % ci=0.620.96 , p=0.02 ) and heterozygous comparison ( ga vs. aa : or=0.77 , 95 % ci=0.640.93 , p<0.01 ) in european subgroup . no significant associations were found in the other models in asian subgroup and european subgroup . there was no significant association between icam-1 k469e polymorphism and cancer risk under all models in america subgroup ( all p>0.05 ) . in addition , there was a tendency that the gg genotype was associated with a higher risk for cancer in asian subgroup and america subgroup ( or>1.0 under all comparison models ) ; however , g - containing genotypes , gg / ga , were associated with decreased risk for cancer in european subgroup ( or<1.0 under all comparison models ) . a total of 921 cases and 955 controls from 6 case - control studies on the correlation of icam-1 g241r polymorphism and cancer risk were included for data synthesis . in general , the overall analysis revealed icam-1 g241r polymorphism seemed to be associated with cancer risk ( a allele vs. g allele : or= 2.21 , 95% ci = 1.313.74 , p<0.01 ; aa+ag vs. gg : or = 2.16 , 95% ci = 1.373.43 , p<0.01 ; aa vs. gg : or = 2.45 , 95% ci = 1.125.35 , p=0.02 ; ag vs. gg : or= 2.03 , meta - analysis with a random - effects model for the association between the risk of cancer and icam-1 g241r polymorphism ( dominant model : aa + ag vs. gg ) subgroup analysis by ethnicity showed that icam-1 g241r polymorphism was associated with the risk of cancer in european populations ( a allele vs. g allele : or = 2.46 , 95% ci = 1.065.69 , p=0.04 ; aa+ga vs. gg : or = 2.38 , 95% ci = 1.125.08 , p=0.02 ; ga vs. gg : or = 1.95 , 95% ci = 1.412.70 , p<0.01 ) . in order to assess the stability of the results of the meta - analysis , sensitivity analysis was performed by sequentially excluding each study . statistically similar results were obtained after sequentially excluding each study , suggesting the stability of this meta - analysis . in this meta - analysis , we performed begg s funnel plot and egger s test to access the publication bias . the shape of the funnel plots did not reveal any evidence of obvious asymmetry under all contrast models for icam-1 k469e and g241r ( fig . , the p value of egger s test was 0.633 for icam-1 k469e polymorphism , and 0.704 for icam-1 g241r polymorphism under the allele model , respectively , providing statistical evidence of funnel plot s symmetry . therefore , the results revealed that publication bias was not significant in this meta - analysis . 16 articles containing 18 case - control studies were included in the meta - analysis , including 4,844 cancer cases and 5,618 healthy controls . there were 18 case - control studies concerning icam-1 k469e polymorphism ( 1517 , 2435 ) , and 6 case - control studies concerning icam-1 g241r ( 24 , 2730 , 33 ) . for icam-1 k469e polymorphism , seven studies were conducted in european populations , six in asian populations , four in america populations , and one in oceania populations . moreover , there were four studies of european populations ( 2830 , 33 ) , one study of asian populations ( 24 ) , and one study of america populations ( 27 ) for icam-1 g241r polymorphism . the genotype distributions among the controls of all studies were in agreement with hwe except for two studies for k469e ( 27 , 31 ) and one study for g241r ( 33 ) . the detailed characteristics of the eligible studies included in this meta - analysis were shown in table 1 . flow chart showing study selection procedure baseline characteristics of the 18 eligible studies for the analysis of icam-1 k469e and g241r polymorphisms pb , population - based controls , hb , hospital - based controls . pcr , polymerase chain reaction ; ssp , sequence - specific primers ; rflp , restriction fragment length polymorphism in the overall analysis , we did not find any significant association between icam-1 k469e polymorphism and the risk of cancers in all comparison models ( g allele vs. a allele : or = 1.03 , 95% ci = 0.891.19 , p=0.66 ; gg+ga vs. aa : or= 0.98 , 95% ci = 0.831.17 , p=0.86 ; gg vs. ga+aa : or = 1.09 , 95% ci = 0.871.37 , p=0.44 ; gg vs. aa : or = 1.15 , 95% ci = 0.841.56 , p=0.38 ; ga vs. aa : or= 0.95 , 95% ci = 0.821.11 , p=0.53 ) ( fig . the results suggested that the icam-1 k469e polymorphism may be not associated with overall cancer risk . meta - analysis with a random - effects model for the association between the risk of cancer and icam-1 k469e polymorphism ( dominant model : gg + ga vs. aa ) as shown in table 2 , in the subgroup analysis by ethnicity , we found a significant association under homozygous comparison ( gg vs. aa : or=1.53 , 95 % ci=1.032.27 , p=0.03 ) in asian subgroup . moreover , there was significant association under dominant model ( gg+ga vs. aa : or=0.77 , 95 % ci=0.620.96 , p=0.02 ) and heterozygous comparison ( ga vs. aa : or=0.77 , 95 % ci=0.640.93 , p<0.01 ) in european subgroup . no significant associations were found in the other models in asian subgroup and european subgroup . there was no significant association between icam-1 k469e polymorphism and cancer risk under all models in america subgroup ( all p>0.05 ) . in addition , there was a tendency that the gg genotype was associated with a higher risk for cancer in asian subgroup and america subgroup ( or>1.0 under all comparison models ) ; however , g - containing genotypes , gg / ga , were associated with decreased risk for cancer in european subgroup ( or<1.0 under all comparison models ) . a total of 921 cases and 955 controls from 6 case - control studies on the correlation of icam-1 g241r polymorphism and cancer risk were included for data synthesis . in general , the overall analysis revealed icam-1 g241r polymorphism seemed to be associated with cancer risk ( a allele vs. g allele : or= 2.21 , 95% ci = 1.313.74 , p<0.01 ; aa+ag vs. gg : or = 2.16 , 95% ci = 1.373.43 , p<0.01 ; aa vs. gg : or = 2.45 , 95% ci = 1.125.35 , p=0.02 ; ag vs. gg : or= 2.03 , meta - analysis with a random - effects model for the association between the risk of cancer and icam-1 g241r polymorphism ( dominant model : aa + ag vs. gg ) subgroup analysis by ethnicity showed that icam-1 g241r polymorphism was associated with the risk of cancer in european populations ( a allele vs. g allele : or = 2.46 , 95% ci = 1.065.69 , p=0.04 ; aa+ga vs. gg : or = 2.38 , 95% ci = 1.125.08 , p=0.02 ; ga vs. gg : or = 1.95 , 95% ci = 1.412.70 , p<0.01 ) . in order to assess the stability of the results of the meta - analysis , sensitivity analysis was performed by sequentially excluding each study . statistically similar results were obtained after sequentially excluding each study , suggesting the stability of this meta - analysis . in this meta - analysis , we performed begg s funnel plot and egger s test to access the publication bias . the shape of the funnel plots did not reveal any evidence of obvious asymmetry under all contrast models for icam-1 k469e and g241r ( fig . 4 ) . in addition , the p value of egger s test was 0.633 for icam-1 k469e polymorphism , and 0.704 for icam-1 g241r polymorphism under the allele model , respectively , providing statistical evidence of funnel plot s symmetry . therefore , the results revealed that publication bias was not significant in this meta - analysis . to the best of our knowledge , our meta - analysis represents the most comprehensive investigation on the association between icam-1 k469e and g241r polymorphisms and cancer risk . since demographic characteristics influence genotype frequencies , different races have different gene - environment interaction models . therefore , we conducted a subgroup analysis according to ethnic differences , and the results indicated that there was a significant association between k469e polymorphism and decreased cancer risk in european populations , while there were not significant associations in asian populations and america population . moreover , icam-1 g241r polymorphism displayed significant association with cancer risk , especially in european populations . recent studies demonstrated that icam-1 possibly contributes to tumorigenesis and metastasis ( 12 , 36 , 37 ) . the potential involvement of icam-1 expression in cancer invasion and metastasis was reported in melanomas , pancreatic , lung , and oral cancers ( 34 ) . conversely , some studies indicated that increased icam-1 expression was correlated with a more favorable prognosis in gastric , breast , and colorectal cancers under the influence of the host immuno surveillance system ( 3840 ) . the progression of most cancers is resulted from the interaction of environmental and genetic factors . it is well known that genetic variants in the pathway of the pathogenesis of cancer may alter protein function and individual s susceptibility to cancer . thus , polymorphism within the icam-1 gene likely played a significant role in the susceptibility to and development of cancer . while several genetic polymorphisms have been indentified in icam-1 , the frequently investigated two polymorphisms in several types of cancer involve the k469e and g242r polymorphisms , due to their functional implications in the icam-1 protein . icam-1 k469e polymorphism is located three bases upstream of icam-1 mrna splicing site that influence rna splicing patterns ( 31 ) . compared to icam-1 monomers , icam-1 dimers exhibit enhanced binding to lymphocyte function - associated protein-1 therefore , the amino acid exchange might diminish icam-1 dimerization and in turn lead to decreased integrin receptor binding , thus affecting icam-1 function ( 41 ) . g241r polymorphism is located in exon 4 , which has been shown to be of importance in binding to the mac-1 form of the leukocyte integrin . the interaction between mac-1 and icam-1 makes an important contribution to leukocyte adhesion in the execution of immunological and inflammatory functions and may play a role in regulating localization of leukocytes ( 42 ) . g241r polymorphism can modify the functional activity of the icam-1 molecule leading to a different recruitment and activation of the inflammatory cells ( 43 ) . in our study , given the difference , it is reasonable to speculate that the substitution ( g to a ) in g241r site could have functional significance in the progression of cancer by affecting the adhesive function of icam-1 . the result in the subgroup analysis demonstrated that icam-1 g241r polymorphism was associated with cancer risk in european populations . as for icam-1 k469e , the results indicated that gg genotype was associated with a higher risk of cancer in asian subgroup ; however , g - containing genotypes , gg / ga , were associated with decreased risk for cancer in european subgroup . the differences may be explained by genetic diversities , different risk factors in life styles , and the exposure to different environmental factors ( 44 ) . the identification of susceptibility genes in cancer patients of different ethnicities provides an opportunity to explore new mechanisms of disease that are specific in different populations . on the other hand , limitation of this meta - analysis first , heterogeneity can interfere with the interpretation of the results of a meta - analysis , which was unavoidable when combing many studies . variation in the environmental and genetic background of study participants may contribute to the heterogeneity . second , small number of included studies may decrease statistical power and even may produce a fluctuated risk estimate . therefore , this relationship needs to be further confirmed in larger size , well - designed prospective studies . third , the interaction of different susceptibility genes and environment factors leaded to the disease , but our study could not assess gene - gene and gene - environment interactions due to the limited information of included studies . forth , only studies published were included in the meta - analysis , and non - significant or negative findings may be unpublished . hence , some inevitable publication biases might exist in the results . this meta - analysis suggested that icam-1 g241r polymorphism might be a genetic risk factor for the development of cancer , especially in european populations . in addition , icam-1 k469e polymorphism might not act as a cancer risk factor among all subjects . however , subgroup analysis revealed one genetic model ( gg vs. aa ) presented the relationship with cancer risk in asian subgroup , and two genetic models ( gg+ga vs. aa and ga vs. aa ) in european subgroup , respectively . further studies with large sample size , standardized unbiased genotyping methods , homogeneous cancer patients , well - matched controls and multiethnic groups would be warranted . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors . | background : intercellular adhesion molecule-1 ( icam-1 ) lys469glu ( k469e ) polymorphism and gly 241arg ( g241r ) polymorphism might play important roles in cancer development and progression .
however , the results of previous studies are inconsistent .
the aim of this study was to evaluate the association between icam-1 k469e and g241r polymorphisms and the risk of cancer by meta-analysis.methods:a comprehensive literature search ( last search updated in november 2013 ) was conducted to identify case - control studies that investigated the association between icam-1 k469e and g241r polymorphisms and cancer risk.results:a total of 18 case - control studies for icam-1 polymorphisms were included in the meta - analysis , including 4,844 cancer cases and 5,618 healthy controls . for k469e polymorphism , no significant association was found between k469e polymorphism and cancer risk . however , subgroup analysis by ethnicity revealed one genetic comparison ( gg vs. aa ) presented the relationship with cancer risk in asian subgroup , and two genetic models ( gg+ga vs. aa and ga vs. aa ) in european subgroup , respectively . for g241r polymorphism ,
g241r polymorphism was significantly association with cancer risk in overall analysis .
the subgroup analysis by ethnicity showed that g241r polymorphism was significantly associated with cancer risk in european subgroup.conclusion:icam-1 g241r polymorphism might be associated with cancer risk , especially in european populations , but the results does nt support icam-1 k469e polymorphism as a risk factor for cancer . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
stroke is the second most common cause of mortality and the most common cause of long - term
neurological disability worldwide1 . in
developing countries , the survival rate of stroke patients has increased with the
improvement of medical technology , and as a result , relevant costs are increasing2 . therefore , many studies on the
limitations of activity and participation in daily life have been conducted . physical activity is a key factor in the improvement of personal health , and increasing
physical activity is associated with a decreased mortality rate4 . in addition , physical activity is associated with improved
quality of life and decreased levels of depression5 . according to bandura6 , 7 , low physical activity levels lead to the
loss of self - efficacy and a lack of self - confidence in daily life . if medical parameters are stable , it is critical to start physical activity in stroke
patients as soon as possible8 . according
to lacroix et al.9 , 62% of stroke patients
in hospitals and rehabilitation institutions have insufficient physical activity . furthermore , in a study by touillet et al.10 in which nine stroke patients were trained at home , after 3 months ,
only one performed physical activity in accordance with the guidelines . therefore ,
post - stroke physical activity remains an ongoing issue , and research is still required to
address this problem . studies on the relationship between stroke and physical activity have been conducted , but
most included small samples ( about 300 participants)11,12,13,14 . however , the korean
community health survey ( kchs ) is a study conducted in large samples of all south korean
residents , and various studies have used the raw data , including those for depressive
symptoms , sleep duration , risk factors , and healthcare utilization15,16,17,18 . the kchs is
conducted annually ; the raw data are provided two years after completion of the
investigation . the 2012 kchs was conducted with 228,921 participants , and more than 4,000
were stroke patients . furthermore , kchs is not conducted in
a controlled laboratory setting , but rather is a personalized survey at home . therefore , the
present study investigated a wide range of stroke patients living in south korea by using
kchs raw data to determine the correlation between stroke and physical activity . this study used raw data from the 2012 kchs , which was conducted under the supervision of
the korea centers for disease control and prevention ( kcdc ) . the kchs was conducted to
monitor the health level of local residents between august 15 , 2012 and october 31 , 2012 .
among all participants , an average of 900 were selected from a sample representative of the
population in a total of 17 areas , including smoking , exercise , medical service use ,
limitation of activity , quality of life , and heart problems . a sample representative of the
population data is selected annually by the ministry of public administration and security
in cooperation with the ministry of land , transport and maritime affairs . sample households
were extracted from these data to aid in the investigation of an average 900 individuals for
each health center19,20,21,22 . trained surveyors personally visited the sampled households and
performed a one - on - one electronic survey with a notebook pc on which a survey program was
installed . the total number of participants was 228,921 ; of the 4,475 stroke patients who
had been diagnosed by a medical doctor or an oriental medical doctor , the data for 4,460
patients , excluding 15 whose amount of physical activity was unclear , were used in the
analysis . the protocol of the kchs was reviewed and approved by the institutional review
board of the kcdc ( 2012 - 07con-01 - 2c ) . the general characteristics of the patients are listed in table 1table 1.characteristics of stroke patientscharacteristicsn%%setotal stroke patientsyes 4,4751.30.0no224,44698.70.0currently undergoing treatment for strokeno1,44333.81.1yes 3,01766.21.1gendermale2,25053.71.1female2,21046.31.1age ( years)19641,17134.01.1653,28966.01.1educational levelelementary school or less2,92353.71.1middle school57515.40.8high school67419.80.9college and over28111.10.8monthly household income ( 10,000 won)1002,47548.01.210120078022.41.020130040711.30.73014001886.50.640133111.80.8living with familyyes3,65686.30.7no ( alone)80413.70.7occupationyes1,29225.61.0no3,16474.41.0residential areaurban1,86071.81.1rural2,60028.21.1physical activityyes 1,85544.11.1no2,60555.91.1 * % : estimated percent of the population .
physical activity : more than moderate levels of physical activity
( moderate physical activity more than 3 days a week , strenuous physical activity for
more than 20 minutes a day or moderate levels of physical activity more than 5 days a
week , or strenuous physical activity for more than 30 minutes at a time ) , or walking
for more than 30 minutes , 5 days a week , or over 30 minutes at a time .. * n : sample size . % : estimated percent of the population .
physical activity : more than moderate levels of physical activity
( moderate physical activity more than 3 days a week , strenuous physical activity for
more than 20 minutes a day or moderate levels of physical activity more than 5 days a
week , or strenuous physical activity for more than 30 minutes at a time ) , or walking
for more than 30 minutes , 5 days a week , or over 30 minutes at a time . to investigate the correlations between the factors related to the sequelae of stroke and
physical activity in stroke patients , the types of sequelae were classified as : had no
sequelae , recovered after experiencing sequelae , and currently experiencing sequelae . for
the type and number of sequelae in patients who were currently experiencing sequelae or had
recovered after experiencing sequelae , palsy in the arms and legs , facial palsy ,
communication disability such as poor pronunciation , swallowing or eating disorder , and
visual disabilities such as poor vision , were used . among these five types of sequelae , the
total number that each patient had was calculated ( range : 05 ) , and these were additionally
classified as 0 , 1 , 2 , and 3 or more sequelae of each type . physical activity was classified
as strenuous , moderate , and walking , and each question asked the number of days per week and
time spent in daily physical activity . moderate physical activity was defined as that
performed more than 3 days a week for more than 20 minutes per day ; strenuous physical
activity , as that performed more than 5 days a week for more than 30 minutes per day ; and
walking , as walking 5 days per week for more than 30 minutes per day . in this study , physical activity was defined as performing any physical activity , including moderate
physical activity , strenuous physical activity , and walking . , chicago , il , usa ) for the
complex sampling design , and individual weights were applied to estimate the population . a
frequency analysis was performed to examine the patient distribution , and a chi - square test
was performed to examine the sociodemographic characteristics and compare stroke sequelae
and physical activity . finally , to examine the correlations between the factors related to
the sequelae of stroke and physical activity , multiple logistic regression analysis was
performed under the control of gender , age , educational level , monthly household income ,
living with family , occupation , and residential area . for the complex sampling design , and individual weights were applied to estimate the
population . a frequency analysis was performed to examine the patient distribution , and a
test was performed to examine the sociodemographic characteristics and
compare stroke sequelae and physical activity . finally , to examine the correlations between
the factors related to the sequelae of stroke and physical activity , multiple logistic
regression analysis was performed under the control of gender , age , educational level ,
monthly household income , living with family , occupation , and residential area . the stroke prevalence among all patients was 1.3% , and the current stroke treatment rate
was 66.2% . of all patients , 53.7% were male , 66.0% were 65 years or older , 53.7% were
elementary school graduates or lower , 48.0% had one million won or less monthly household
income , 86.3% lived with family members , 74.4% had no occupation , 71.8% lived in cities , and
44.1% performed physical activity ( table 1 ) . the
comparison between the factors and physical activity showed significant differences based on
gender , age , education level , monthly household income , living with family , occupation ,
residential area , and whether or not currently being treated for stroke ( table 2table 2.sociodemographic characteristics of stroke patients and physical activitycharacteristicsnoyesgendermale50.4 ( 1.5)49.6 ( 1.5)female62.2 ( 1.6)37.8 ( 1.6)age ( years)196448.6 ( 2.0)51.4 ( 2.0)6559.6 ( 1.3)40.4 ( 1.3)educational levelelementary school or less59.8 ( 1.4)40.2 ( 1.4)middle school53.9 ( 2.8)46.1 ( 2.8)high school52.2 ( 2.5)47.8 ( 2.5)college and over46.3 ( 4.1)53.7 ( 4.1)monthly household income ( 10,000 won)10059.1 ( 1.5)40.9 ( 1.5)10120048.9 ( 2.5)51.1 ( 2.5)20130051.6 ( 3.5)48.4 ( 3.5)30140051.3 ( 4.9)48.7 ( 4.9)40154.9 ( 3.7)45.1 ( 3.7)living with familyyes54.8 ( 1.2)45.2 ( 1.2)no ( alone)63.0 ( 2.4)37.0 ( 2.4)occupationyes41.9 ( 2.2)58.1 ( 2.2)no60.7 ( 1.3)39.3 ( 1.3)residential areaurban54.7 ( 1.4)45.3 ( 1.4)rural59.0 ( 1.4)41.0 ( 1.4)currently undergoing treatment for
strokeno51.5 ( 1.9)48.5 ( 1.9)yes 58.1 ( 1.3)41.9 ( 1.3)* test ( p<0.05 ) ) . as a result of the multiple logistic regression analysis with adjustment for gender , age ,
educational level , monthly household income , living with family , occupation , and residential
area to examine the correlation between the factors related to the sequelae of stroke and
physical activity , the ratio of physical activity performed by patients who were currently
experiencing sequelae compared to that of patients who were no longer experiencing sequelae
was 0.58 ( 95% confidence interval [ ci ] : 0.470.71 ) . thus , the amount of physical activity
performed by patients who had sequelae was significantly lower . compared to patients who did
not have sequelae , the ratio of physical activity performed by patients who had palsy in the
arms and legs was 0.57 ( 95% ci : 0.470.69 ) ; facial palsy , 0.70 ( 95% ci : 0.540.91 ) ;
communication disability , 0.73 ( 95% ci : 0.590.90 ) ; swallowing or eating disability , 0.53
( 95% ci : 0.390.72 ) ; and visual disability , 0.63 ( 95% ci : 0.490.80 ) . thus , the amount of
physical activity performed by patients who had stroke sequelae was significantly lower than
that performed by patients who had not . furthermore , compared to patients who had suffered
no sequelae , the ratio of physical activity performed by patients who suffered 1 sequela was
0.76 ( 95% ci : 0.600.96 ) ; 2 sequelae , 0.72 ( 95% ci : 0.540.95 ) ; and 3 or more sequelae , 0.42
( 95% ci : 0.320.56 ) . thus , as the number of sequelae increased , patients performed
significantly less physical activity ( table
3table 3.the association between sequelae in stroke patients and physical activitynoyesaor ( 95% ci)occurrence of sequelaeno48.6 ( 1.7)51.4 ( 1.7)1.00recovery51.6 ( 2.9)48.4 ( 2.9)0.93 ( 0.711.22)yes 63.6 ( 1.5)36.4 ( 1.5)0.58 ( 0.470.71)*type of sequelaepalsy in the arms and legsno50.5 ( 1.4)49.5 ( 1.4)1.00yes 63.7 ( 1.6)36.3 ( 1.6)0.57 ( 0.470.69)*facial palsyno54.7 ( 1.2)45.3 ( 1.2)1.00yes 62.7 ( 2.8)37.3 ( 2.8)0.70 ( 0.540.91)*communication disabilitiesno53.4 ( 1.3)46.6 ( 1.3)1.00yes 62.1 ( 2.0)37.9 ( 2.0)0.73 ( 0.590.90)*swallowing or eating disordersno54.4 ( 1.1)45.6 ( 1.1)1.00yes 70.8 ( 3.1)29.2 ( 3.1)0.53 ( 0.390.72)*visual disabilityno53.4 ( 1.2)46.6 ( 1.2)1.00yes 67.6 ( 2.3)32.4 ( 2.3)0.63 ( 0.490.80)*the number of sequelae049.3 ( 1.6)50.7 ( 1.6)1.00157.8 ( 2.2)42.2 ( 2.2)0.76 ( 0.600.96)*258.1 ( 2.9)41.9 ( 2.9)0.72 ( 0.540.95)*370.1 ( 2.4)29.9 ( 2.4)0.42 ( 0.320.56)**tested with multiple logistic regression analysis ( p<0.05 ) . aor , odds
ratio adjusted for gender , age , educational level , monthly house hold income , living
with family , occupation , and residential area ) . * aor , odds
ratio adjusted for gender , age , educational level , monthly house hold income , living
with family , occupation , and residential area compared to patients who were not currently experiencing sequelae , the amount of physical
activity performed by patients who were currently experiencing sequelae was significantly
lower . this result corresponds to the results of a previous study that showed that stroke
limited movement23 . in particular , no
significant difference in physical activity was found in patients who had recovered from
stroke sequelae in this study . based on the type of sequela , the amount of physical activity performed by patients who had
palsy in the arms and legs was significantly lower than that performed by patients who did
not . previous studies reported that significantly decreased activity of the extensor muscles
of the upper arm and the hand muscles would not be adequate for efficient
rehabilitation24 , 25 . in addition , physical activity is reduced due to limitations in
walking when leg movement is not natural . furthermore , as sequelae in the arm have a
profound effect on the performance of delicate movements and work , palsy likely contributes
to decreased physical activity . the amount of physical activity performed by patients who had facial palsy was
significantly lower than that performed by patients who did not . according to kim et
al.19 , facial palsy decreases the
quality of life . facial palsy causes social phobia , which narrows the radius of activity ,
and complex palsy , including facial palsy , reduces physical activity . another stroke sequela , aphasia , has a profound effect on daily living , and many studies
have been conducted on the difficulty of treating this sequela19 , 26 . in the present
study , the amount of physical activity performed by patients who had communication
disability was significantly lower than that performed by patients who did not . such
communication disabilities decrease quality of life and can have other negative sequelae ,
such as depression . stroke in the cerebral hemispheres or brain stem leads to dysphagia in 3080% of cases , and
dysphagia is directly associated with swallowing27,28,29,30,31 . in the present study , the amount of physical activity performed by
patients had swallowing or eating disability sequelae was significantly lower than that
performed by patients who did not . this result is similar to that reported in a previous
study that patients with parkinson s disease had worsened swallowing disorders as their
disease progressed , and this lowered their quality of life32 . therefore , more active interventions based on the prescription of
a rehabilitation doctor , such as electrotherapy of the muscles related to dysphagia , are
required . the amount of physical activity performed by patients who had visual disability sequelae
was significantly lower than that performed by patients who did not . a previous study
reported that unilateral spatial neglect is a serious disability that can result from
stroke , and this sequela can lead to a poor rehabilitation outcome . the present study showed
that visual disability narrows the radius of the living environment and increases the amount
of time patients remain at home , which results in reduced physical activity . therefore ,
decreased physical activity due to visual disability should be improved through various
methods , such as neuropsychological rehabilitation as well as cognitive rehabilitation ,
which is currently being applied in the clinical setting33 . the amount of physical activity decreased significantly as the number of sequelae increased
to 1 , 2 , and 3 compared to no sequelae . this result is related to the finding that brain
atrophy is associated with reduced physical activity34 . therefore , even though post - stroke survival through surgery is
very important , interventions to reduce the number of sequelae must be applied throughout
the post - stroke recovery period . data for all south korean residents were
presented in the raw kchs data . if the data had been presented by region , detailed
differences according to the regional environment could have been provided . in future
studies , the correlations between stroke and physical activity according to regional
characteristics of south korea should be presented for a better understanding of this
subject . in conclusion , in this study , the correlations between stroke and physical activity were
investigated using data from the kchs , a nationwide survey of south korean residents . as a
result , significant differences were found between the amount of physical activity and
stroke sequelae , including palsy in the arms and legs and facial palsy , communication
disability , swallowing or eating disorders , visual disability , and the number of sequelae . therefore , when decisions on national policies and budgets are made , methods for increasing
the physical activity of patients with a history of stroke should be considered . | [ purpose ] the present study investigated a wide range of stroke patients living in south
korea using the korean community health survey raw data to determine the correlation
between stroke and physical activity .
[ subjects and methods ] this study used raw data from
the 2012 korean community health survey .
the total number of participants was 228,921 ; of
the 4,475 stroke patients who had been diagnosed by a medical doctor or an oriental
medical doctor , the data for 4,460 patients , excluding 15 whose amount of physical
activity was unclear , were used in the analysis . [ results ] the amount of physical activity
performed by patients who had sequelae was significantly lower than that performed by
patients who no longer had sequelae
.
similarly , for the type of sequelae , palsy in the
arms and legs , facial palsy , communication disability , swallowing or eating disability ,
and visual disability were associated with lower physical activity .
furthermore , as the
number of sequelae increased , patients performed significantly less physical activity .
[ conclusion ] the findings suggest that when decisions on national policies and budgets are
made , methods for increasing the physical activity of patients with a history of stroke
should be considered . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
ischaemic heart disease is among the leading causes of death worldwide and thus cardiac regeneration has caught an ever - increasing interest among scientists . the ability of the heart to regenerate has been studied since the mid - nineteenth century and the consistent conclusion has been that the heart has little or no regenerative capacity [ 2 , 3 ] . recently , however , an increasing number of studies have reported induction of cardiomyocyte proliferation and cardiac regeneration . nevertheless , it is still disputed to what extent cardiomyocyte proliferation can be induced and whether induction of proliferation can be utilized therapeutically . mammalian cardiomyocytes in vivo lose their ability to proliferate and exit the cell cycle during the first weeks after birth [ 3 , 4 ] . therefore , the adult mammalian heart is considered incapable of regeneration after ischaemic or other forms of injury . natural compensatory processes of the injured heart are limited to hypertrophy of the remaining cardiomyocytes and replacement of necrotic regions with fibrotic scar tissue . cardiac scarring and loss of contractile tissue can cause arrhythmias , dilation , heart failure and other complications , contributing significantly to morbidity and mortality [ 1 , 5 ] . however these treatment regimens fail to correct the primary cause of impaired heart function : the loss of cardiomyocytes . therefore , two strategies have been employed in recent years : cell replacement through cell therapy and induction of endogenous cardiomyocyte proliferation . multiple cell types have been tested for repopulation of the injured myocardium to improve heart function . at first , it had been demonstrated that transplanted foetal and neonatal cardiomyocytes could functionally integrate and enhance recipient cardiac function . therefore , scientists enhanced their efforts to identify a cell type that can be obtained in large quantities and can be differentiated into cardiomyocytes . skeletal myoblasts seemed a good alternative , since they are autologous , easy to obtain and they have a high proliferative potential . it has been demonstrated that they incorporate in the myocardium and improve myocardial function upon transplantation after myocardial infarction . however , they do not convert into cardiomyocytes and do not establish connections with the host cardiomyocytes . on the other hand , clinical studies in patients with chronic myocardial disease several studies have reported improved cardiac function after transplantation of bone marrow - derived stem cells , i.e. mesenchymal stem cells ( msc ) and haematopoeietic stem cells ( hsc ) , in animal models of myocardial injury [ 9 , 10 ] as well as in clinical trials [ 11 , 12 ] . however , the improvement is rather minor and , as recently reported , might be only transient [ 13 , 14 ] . many stem cell types can be forced in vitroto differentiate into cardiomyocytes . however , various experiments using several stem / progenitor cell types indicate that there is little or no differentiation of the engrafted cells into cardiomyocytes in vivo and that their survival rate is low , with the exception of embryonic stem cells [ 1618 ] . therefore , the mechanism of stem cell therapy is still controversial . the main current opinion is that stem cells as well as skeletal myoblasts secrete cytokines and/or growth factors that cause the functional improvement by stimulating neovascularization , cardiomyocyte proliferation and/or preventing apoptosis [ 15 , 1923 ] . recently , it has been reported that the mammalian heart contains resident cardiac stem cells ( csc ) . the possibilities of expanding autologous cscs ex vivo or stimulating the regeneration capacity of these cells in vivo are exciting options for therapeutic regeneration . cardiac regeneration through cardiomyocyte proliferation is appealing because mammalian heart growth during foetal development as well as newt heart regeneration is mediated by cardiomyocyte proliferation [ 3 , 23 ] . the mechanism of cardiac regeneration in zebrafish remains unclear but it appears at least to be partially based on cardiomyocyte proliferation . cell proliferation is an orderly sequence of events in which cells duplicate their contents and then divide . this cycle of duplication and cell division is known as the cell cycle ( fig.1 ) . the cell cycle consists of four distinct phases : g1 ( gap 1 ) phase , s ( synthesis ) phase , g2 ( gap 2 ) phase ( collectively known as interphase ) and m ( mitotic ) phase . in s phase , the dna is replicated . during gap phases , the cell grows and progression to the next cell cycle stage cells that have temporarily or reversibly stopped proliferating enter a state of quiescence called go . m phase is composed of two tightly coupled processes : nuclear division ( karyokinesis ) in which the chromosomes are segregated , and cell division ( cytokinesis ) , in which the cytoplasm divides forming two distinct daughter cells . the cell cycle consists of four distinct phases : g1 phase , s phase , g2 phase and m phase . in this review , finally , we critically analyse the recent literature about induction of mammalian cardiomyocyte proliferation and cardiac regeneration . urodele amphibians are generally regarded as the champions of regeneration among vertebrates , as first reported by spallanzani in 1768 . an adult newt is able to regenerate its tail and limbs , upper and lower jaws , ocular tissue such as the retina and lens , and even substantial sections of the heart [ 2730 ] . the cardiac muscle of amphibians is capable of regenerating up to 50% of the ventricle after mechanical excision . this process involves dedifferentiation of the remaining cardiac muscle cells and proliferation of both cardiomyocytes and connective tissue cells [ 3032 ] . to increase the proliferative response , the apical portion of the newt ventricle was amputated , minced and placed onto the amputation gap . this procedure was followed by a period of proliferation that peaked at 16 days after amputation . these data suggest that in urodele amphibians most of the mature cardiomyocytes retain the ability to proliferate . to further understand the process of newt cardiac regeneration , ventricular cardiomyocytes were placed into cell culture . dna synthetic and mitotic activity in these cultures was studied using 24-hr exposures to tritiated thymidine and time lapse videotaping . nuclear labelling was first seen after 6 days ( 7.5% ) , increasing to 30.5% after 10 days and subsequently declined . the majority of cardiomyocytes ( 75% ) were found to replicate dna and 76% of those entered mitosis . mitotic indices near the peak time were 0.48% ( 8 days ) , 1.7% ( 10 days ) and 1.55% ( 12 days ) . a total of 81% of the observed mitotic cardiomyocytes underwent cytokinesis . taken together , approximately one - third of the initial cardiomyocyte population progressed through mitosis and entered successive cell divisions [ 3234 ] . interestingly , also multinucleated cells were found to replicate dna and to complete mitosis and cytokinesis . initially , it has been reported that it occurs through robust proliferation of cardiomyocytes after amputation of the apex . the time course of cardiac regeneration as well as the number of cardiomyocytes undergoing dna synthesis and mitosis highly resembled cardiac regeneration in newt . unfortunately , zebrafish cardiomyocyte proliferation has so far not been achieved in vitro . recent data suggest that stem / progenitor cells might also be involved in this process . in conclusion , these data strongly suggest that naturally occurring heart regeneration in lower vertebrates is based on cardiomyocyte proliferation . this transition is characterized by maturation of the contractile apparatus , a cytoplasmic structure that is thought to preclude cytokinesis . historically , this conclusion was derived from the evidence that during histogenesis of skeletal muscle in vertebrates , only myoblast and/or satellite cells are able to synthesize dna and to divide mitotically . cell fusion and the onset of contractile protein synthesis are accompanied by a stable repression of genes controlling the multiplication of the muscle cell . thus , differentiation and proliferation are mutually exclusive in skeletal muscle [ 38 , 39 ] . in contrast to skeletal muscle , differentiation of cardiomyocytes proceeds gradually during the course of embryonic and postnatal development . it happens in a step - by - step increase in size , number , and structural complexity of myofibrils , mitochondria and sarcoplasmic reticulum , along with the formation of intercalated disks , desmosomes and tight junctions . at the same time , the relative amount of non - sarcomeric filaments , microtubules , free ribosomes , golgi and rough endoplasmatic reticulum elements diminish visibly , especially during the postnatal stages of cardiac myogenesis . interestingly , as they differentiate , foetal beating cardiomyocytes still undergo cytokinesis [ 40 , 41 ] . in chick the mitotic index even increases from 1.5% at 30 hrs after incubation to a peak of 3.2% by 4 days when myofibrils accumulate [ 4244 ] . this demonstrates that differentiation and proliferation are compatible in cardiomyocytes . naturally occurring proliferation of cardiomyocytes from newt and developing chick , rat and mouse myocardium have been studied with several techniques including high laser confocal microscopy and live cell imaging [ 3 , 31 , 4046 ] . all these studies observed in essence the same changes in cardiomyocytes during the course of cell division : cardiomyocytes in prophase do not differ in their appearance from non - dividing cardiomyocytes except for the condensation of the chromosomes . the myofibrils remain unchanged up to the late prophase whereas the nucleolus persists frequently up to mid - prophase . after the metaphase plate has formed almost all the myofibrils are seen subdivided into isolated sarcomeres and myofilament bundles as a result of the progressive z - disk degradation . this is followed by a progressive isolation and scattering of the myofilament bundles and of the whole sarcomeres during the subsequent phases of mitosis . z - disk and thin - filament associated proteins appear in a diffuse localization pattern before m - band and thick - filament - associated proteins . importantly , no myocardial cell has been observed in these studies that underwent mitosis without these disruptive changes of the myofibril striation patterns [ 40 , 41 , 44 ] . cleavage furrow formation begins in late telophase and continues until complete separation of both daughter cells . when each daughter nucleus attains around half of its volume of the interkinetic nucleus the first signs of gradual restoration of contrast rich z - bands interconnecting the previously isolated sarcomeres are observed . the re - assembled myofibrils are first disorganized , crossing one another . throughout the subsequent growth and maturation , myofibrils become ultimately oriented parallel to the long axis of the cell . in 1977 , it was concluded , from the observation of mitotic figures in injured adult hearts , that a limited percentage of adult mammalian cardiomyocytes can pass through all phases of the mitotic cycle and that this was associated with the transient disassembly of the contractile apparatus ( fig.2 ) . suggested scheme of myofibril changes throughout mitosis and cell division of adult mammalian cardiomyocytes in vivo . after the metaphase plate has formed almost all z - disks are degraded and the myofibrils are subdivided into isolated sarcomeres and myofilament bundles . the first signs of gradual restoration of z - disks interconnecting the previously isolated sarcomeres are observed . these early myofibrils are often in disorder , crossing one another . throughout the subsequent growth and maturation myofibrils become rearranged and oriented parallel to the long axis of the cell . interestingly , dividing cardiomyocytes in vitro and in whole mount preparations retain close contact with their non - dividing neighbours throughout all phases of mitosis . persistence of desmosome - like structures prevents the mitotic cell from a completely rounding off and from the loss of its association with the neighbouring cells [ 40 , 44 ] . this evidence from static pictures has also been observed in live cell imaging studies depicting actively contracting rat cardiomyocytes in a state of division . the authors noted that the myocardial cell , in contrast to non - myocytes , did not round up prior to division . taken together , a plethora of characteristic features of dividing cardiomyocytes has been collected over the last decades . these data provide a framework for careful analysis of recent data reporting the induction of proliferation of cardiomyocytes and cardiac regeneration . cardiac injury is followed by down - regulation of cell cycle inhibitors like p21 and p27 and up - regulation of cell cycle perpetuating factors like pcna and cdk / cyclin complexes in cardiomyocytes . several studies have concluded from these observations and the occurrence of mitotic figures that fully matured mammalian cardiomyocytes can undergo cell division after injury as a normal , although limited , physiological response to injury [ 3 , 4753 ] . however , induction of dna synthesis or mitosis after injury could simply result in endoreduplication or poly - nucleation ( increased dna per nuclei or increased nuclei per myocyte ) as cell cycle activity during development leads to cell division but also to poly - nucleation . thus , re - expression of cell cycle genes , the detection of dna synthesis and mitotic figures are no proof of cardiomyocyte cell division . this phenomenon has been reported for human myocardium [ 3 , 54 , 55 ] and for the myocardium of c - myc transgenic mice . recently , a study concluded from ki-67 stainings and mitotic figures that human cardiomyocytes undergo cell division after acute myocardial infarction . however , ki-67 stainings and mitotic figures can not be used to distinguish between cell division and poly - nucleation / endoreduplication . however , they showed that metaphase and late anaphase chromosomes were always located within a preserved nuclear envelope , a clear sign of endoreduplication . in contrast , the nuclear membrane disintegrates during cell division at the end of prophase . this demonstrated that expression of ki67 , usually a marker for proliferating cells , and mitotic figures alone are inadequate to distinguish between cell division , endoreduplication and poly - nucleation in cardiomyocytes . considerable effort has been invested to achieve mammalian cardiomyocyte proliferation to prove that it can be utilized for cardiac regeneration . various growth factors ( e.g. fgf1 , neuregulin ) , small molecules ( e.g. bio , sb203580 ) , viral oncoproteins ( e.g. e1a , sv40 t antigen ) , an extracellular matrix protein ( periostin ) and cell cycle activators ( e.g. cyclin a2 , cyclin d2 , e2f1 , e2f2 , c - myc ) were studied [ 4 , 5860 ] . these studies proved beyond doubt that cell cycle can be re - induced in postnatal cardiomyocytes . to what extent postnatal cardiomyocytes can undergo mitosis , however , is controversial . mitosis is rare and it is not always simple to determine whether a mitotic nucleus actually belongs to a cardiomyocyte or non - myocyte . as shown in figure 3 , it is not obvious whether the mitotic nuclei are cardiomyocyte nuclei even when using phase contrast microscopy and dna- as well as phosphorylated histone h3 ( h3p)-staining . this is also a problem in vivo as in the injured heart large amounts of proliferating inflammatory cells and ( myo)fibroblasts infiltrate the myocardium laying in close proximity to the cardiomyocytes . moreover , in vivo cardiomyocytes display a highly irregular shape and non - cardiomyocytes are often found in the indentations in the cardiomyocytes ( fig.4 ) . similar problems have been described in an editorial questioning the occurrence of cardiac chimerism and in a review on stem cell - based cardiac regeneration . thus , cardiomyocyte - specific cytoplasmic markers are not suitable to distinguish cardiomyocyte from non - myocyte nuclei [ 63 , 64 ] . ( a , c ) the h3p - positive nuclei ( black ) appear to be mitotic cardiomyocyte nuclei indicating proliferation . however , dna stain ( dapi , black , b , d ) reveals in each cell two h3p - negative nuclei , which argue against proliferation as does the presence of intact myofibrils . possibly , the h3p - positive nuclei belong to overlaying non - myocytes explaining the membrane separating the chromosomes from the cardiomyocytes . another explanation for c , d is that the membrane is a nuclear membrane separating an endomi - totic cardiomyocyte nucleus . examples of the problem of identifying cardiomyocyte nuclei in transversal tissue sections of murine myocardium ( a d , 5 m - thick ) . cardiomyocyte - specific cytoplasmic markers ( troponin i , red ) are often used to determine the identity of nuclei ( dapi , blue ) . cardiomyocyte nuclei ( asterices ) are embedded in cardiomyocyte cytoplasm whereas non - myocyte nuclei ( dots ) are not . a muscle - specific membrane marker ( caveolin 3 , green ) , however , shows that some of the embedded nuclei are separated from the cardiomyocyte cytoplasm by a muscle - membrane ( arrows ) . this indicates that these nuclei belong to non - myocytes located in between two cardiomyocytes . the drawings give a schematic overview of this phenomenon ( e and f ) . in case , that this nucleus would indeed belong to a cardiomyocyte one should see a double muscle - membrane . even a 3d reconstruction of cytoplasmic - stained cardiomyocytes appears to be inconclusive [ 59 , 65 ] . to avoid false - positives , markers that stain the cell membrane of cardiomyocytes , such as caveolin 3 , could be used [ 66 , 67 ] . non - myocyte but not cardiomyocyte nuclei will be separated from the cardiomyocte cytoplasm by cell membrane ( fig.4 ) . another elegant way of detecting cardiomyocyte nuclei is labelling the cardiomyocyte nuclei with markers like nkx2.5 or utilizing genetic models like the mhc - nlac reporter mice introduced by loren field 's group [ 68 , 69 ] . however , nuclear markers can not be used to identify mitotic cardiomyocytes as the nuclear membrane disintegrates during prophase . however , they allow distinguishing endoreduplication from karyokinesis . some examples of mitotic cardiomyocytes presented in the literature do not match the features of naturally occurring cardiomyocyte proliferation . for instance , mitotic figures within striated cardiomyocytes have been shown after bcl2 overexpression as well as in mirna-12 deficient mice [ 64 , 70 ] . this is unusual , as disassembly of the myofibrils has to take place to allow cell division . another atypical observation has been reported after cyclin a2 overexpression : cardiomyocytes detach and round up during mitosis . finally , to define cardiomyocyte cytokinesis it is important to observe the precise localization of cytokinetic markers and not simply evaluate the generalized expression of these markers . aurora b , for example , associates with centromeric het - erochromatin early in mitosis , transfers to the central spindle and finally localizes to the contractile ring and midbody . however , in cardiomyocytes it can localize at the midbody during proliferation as well as binucleation . furthermore , it has been suggested that contraction of the contractile ring and cleavage furrow ingres - sion is disturbed during binucleation . in a recent study , several examples of aurora b - positive cardiomyocytes were presented . whereas some of the examples indicate cell division others are rather indicative for binucleation as the cardiomyocytes undergo a one - sided furrow ingression . although it is impossible to confer from brdu - labelling , h3p - or aurora b staining that cells proliferate the combination of these results can indicate problems in cell cycle progression . this method allows detecting all cells that have entered s phase during the labelling period . in contrast , mitosis can only be detected at the moment it occurs , usually utilizing antibodies against h3p . therefore , even fast proliferating cells like foetal cardiomyocytes in vivo or hela cells show a relatively low percentage of h3p - positive cells ( 3.7% or 7% , respectively ) . thus , a fast proliferating cell population will have a mitotic / cytokinetic index of approximately 7% and a brdu - labelling index of 100% , when labelled for 24 hrs , giving a ratio of 14.29 . if there are problems in executing or in progressing through mitosis / cytokinesis this ratio will decrease . in a study describing the effect of periostin , the authors reported a brdu - labelling index of 1.1% ( 3 days brdu - labelling ) and a cytokinesis index of 0.5% resulting in a surprisingly low ratio of 2.2 . another study describing the effect of gsk-3 inhibitor bio reports for adult cardiomyocytes a higher mitotic index than for neonatal cardiomyocytes ( 2.41%versus 2.25% ) although the brdu - labelling index was lower ( 13.1%versus 47.2% ) . there are several possible explanations for those data : ( 1 ) the cells accumulate during cytokinesis due to a cell division defect or ( 2 ) the cells have been induced to enter the cell cycle and almost synchronously progressed through the cell cycle into cytokinesis . very few studies have combined detection of dna synthesis , mitosis and cytokinesis and added cell count assays to their analyses . this is important as detection of proteins involved in cytokinesis does not unequivocally prove proliferation . for example , it has been demonstrated that septins , another protein family involved in contractile ring formation , are re - expressed during pathological hypertrophy . furthermore , it has been shown that cells in the end stage of cytokinesis can fail to undergo cell division and reverse their cytoplasmic constriction resulting in poly - nucleated cells . a molecular marker that has been described to indicate whether a cytokinetic cardiomyocyte will undergo binucleation rather than cell division is anillin . it localizes to the cleavage furrow , binds septins , associates with mysosin ii , and is required for cytokinesis . expression of t antigen was associated with mitotic figures and a 3-fold increase in the number of myocardial cells 72 hrs after infection . within a week however , the number of poly - nucleated cells also increased by almost 4-fold ( 8.6%versus 2.3% ) . busk and co - workers used similar approaches to demonstrate that cyclin d2 overexpression results in an increase in cell number of neonatal cardiomyocytes ( 1.8-fold ) and of 21 days old cardiomyocytes in the presence of serum ( 2-fold ) at 6 days after infection . this approach increased the mitotic index in neonatal cardiomyocytes after 72 hrs from 0.06% to 3.72% . in addition , cell division was visualized using aurora b and anillin staining as well as facs analysis to monitor binucleation . fgf1/p38 inhibitor stimulation induced also cell division in cultured adult cardiomyocytes demonstrated by transient dediffer - entiation combined with cleavage furrow formation and separation of the cytoplasm . the authors utilized aurora b staining to detect cardiomyocyte cytokinesis and reported that stimulation resulted in 0.5% aurora b - positive adult cardiomyocytes in vitro and 0.1% in healthy myocardium . an interesting technique has recently been introduced that follows a very different approach . instead of concentrating on markers for the different cell cycle phases this study utilized the so - called flowcytometric brdu - hoechst assay . in the absence of brdu , all cells within go/1 -phase show the same intensity of fluorescence for the dna staining using both propidium iodide and hoechst 33258 . when brdu is incorporated into the dna , it selectively quenches the hoechst fluorescence but does not interfere with that from pi . hence , cardiomyocytes that have progressed through s - phase and subsequently divided will have the same pi fluorescence but a reduced hoechst fluorescence compared with go/1 cells , which did not proliferate . using this method the authors demonstrated that expression of e2f1 and e2f2 significantly increased the number of dividing newborn cardiomyocytes . taken together , many studies have shown that dna synthesis , mitosis and karyokinesis can be induced in cardiomyocytes . however , whether induction results in cardiomyocyte proliferation is still a matter of interpretation . it will be important to understand why it is easier to induce neonatal than adult cardiomyocyte proliferation . an obvious reason is the increased maturity of the contractile apparatus in adult cardiomyocytes and thus the need for dedifferentiation , i.e. disassembly of the myofibrils . another reason might be changes in the distribution and composition of adherence junctions , gap junctions and desmosomes ( combined intercalated discs ) between embryonic and adult cardiomyocytes . early embryonic cardiomyocytes have this structures distributed circumferentially at the peripheral membrane whereas these components are restricted to the bipolar ends in adult cardiomyocytes . while adherence junctions and desmosomes seem to play mainly a mechanical role , gap junctions maintain the ionic and thus electrical coupling of individual cardiomyocytes . interestingly , the postnatal gap junction protein connexin 43 but not the embryonic isoform connexin 40 has been demonstrated to be involved in cell cycle control in cardiomyocytes as well as in other cell types [ 82 , 83 ] . finally , the recent finding that an extracellular matrix protein can induce cardiomyocyte proliferation highlights the importance of the microenvironment . previously , it had already been suggested that extracellular matrix proteins play an important role in peripheral nerve regeneration and cancer development [ 84 , 85 ] . whether cardiac dedifferentiation can be induced in vivo or whether the re - expression of the foetal gene program during hypertrophy or cardiac injury is comparable to the observed cardiac dedifferentiation during natural occurring regeneration remains unclear . studies to date have shown that cyclin d2 , cyclin a2 , fgf5 [ 87 , 88 ] , vegf165 , periostin and fgf1/p38 inhibitor treatment promote cardiac regeneration due to induction of cardiomyocyte proliferation . most of these studies have in addition shown improved heart pump function ( ejection fraction or fractional shortening ) after myocardial infarction . the question is whether the newly formed cardiomyocytes can directly account for the improved heart function . the mentioned studies demonstrate a mitotic index between 0.2% and 1.2% , which is in the same range as for the regenerating newt and zebrafish heart ( < 2% ) . the only study that directly demonstrated that cardiomyocyte proliferation can be utilized to improve heart function has been conducted by the group of loren field . other studies are dependent on estimations by combining quantitative data obtained by histological techniques to suggest formation of new myocardium . for example , kuhn and colleagues calculated that treatment of the infarcted heart with periostin resulted in the regeneration of 7.2 10 cardiomyocytes based on brdu incorporation , or 5.5 10 cardiomyocytes based on quantification of cardiomyocytes with metaphase chromosomes over a period of 12 weeks . the total number of cardiomyocytes in the left ventricle of the rat heart was previously calculated to be 18.5 10 . based on these numbers periostin treatment resulted in a surprisingly high amount of cardiomyocyte restoration of approximately 30% . interestingly , both periostin treatment and periostin deficiency lead to decreased fibrosis and improved cardiac function after myocardial infarction [ 92 , 93 ] . as indicated above , improved cardiac function after treatment does not directly indicate that cardiomyocytes have proliferated . for example , growth factors could improve cardiac function by other mechanisms as they display pleiotropic actions . first , there could be a direct effect of the treatment on the remaining cardiomyocytes after injury , such as protection against irreversible injury [ 95 , 96 ] , increased compensatory hypertrophy , or alterations in calcium handling that could improve contractility . second , the genetic or therapeutic manoeuvres could have additional indirect positive effects , e.g. on neovascularization promoting cell survival and contractility of surviving cardiomyocytes or on scar formation preventing ventricular rupture . there are many examples that improvement of the latter parameters alone can have a substantial effect on heart function . it has been shown that induction of vascularization after myocardial infarction by administration of cd34 + cells enabled survival of the remaining cardiomyocytes and led to a sustained improvement in heart function . fazel and co - workers showed that bone marrow - derived c - kit+ cells can improve heart function after myocardial infarction , which is independent of transdifferen - tiation of these cells into cardiomyocytes . instead , c - kit+ cells release cytokines , which improve vascularization and potentiate the formation of myofibroblast - rich scar tissue . hearts treated with periostin after myocardial infarction had a 4-fold increase in capillary and a1.5-fold increase in arteriolar density in the infarct and in the border zone . intramyocardial injection of a plasmid encoding the 165 isoform of human vascular endothelial growth factor ( pvegf165 ) in sheep hearts after myocardial infarction increased arteriol density 6.4-fold and capillary density 2.6-fold and decreased myofibroblast proliferation and fibrosis . in this study , all of these effects may be involved in the observed reduction in infarct size together with car - diomyocyte proliferation [ 99101 ] . intracoronary injection of an adenoviral construct overexpressing fgf-5 into hibernating myocardium led to significant myocardial hypertrophy with a 30% increase in lv mass within 2 weeks . the authors concluded that stimulation of hypertrophy together with induction of cell cycle activity in a small number of cardiomyocytes was responsible for the improvement in heart function . taken together , there are studies that demonstrate cardiomy - ocyte proliferation together with functional improvement after myocardial injury . however , it is unclear how much of the beneficial effects were related to cardiomyocyte proliferation or to secondary effects like vascularization and survival . an important advance would be to perform proliferation inductive therapies as a control on animals in which cardiomyocytes were genetically disabled to undergo proliferation . however , it is rather unlikely that providing new cardiomyocytes alone is enough to regenerate the injured heart . cardiac fibroblasts , myocytes , endothelial cells and vascular smooth muscle cells are the major cellular constituents of the heart . the relationship of these cellular populations depends on developmental stage and is species - specific . cardiomyocytes make up 30% ( rat ) to 56% ( mouse ) of the total number of cells in the adult ventricle and thus a large part of the myocardium consists of other cell types . it has been previously discussed for other organs that the induction of proliferation of one cell type will be corrected during development , for example , by apoptosis . it appears that cells can sense whether the appropriate number and type of cells as well as extracellular matrix surround them [ 103 , 104 ] . therefore , the ratio between cardiomyocytes , supporting cells and extracellular matrix provided by the cells in the myocardium has to be kept in balance . following myocardial injury cardiomyocytes but also other necessary cells , such as endothelial cells , fibroblasts , smooth muscle cells and purkinje cells die . many of these cells have , in contrast to cardiomyocytes , high proliferation capacity and are restored to some extent during wound healing without any need of therapeutical intervention . for example , blood vessels re - form after myocardial infarction . however , mostly large arterioles were found in the infarct area , and not capillaries , which usually supply the cardiomyocytes with oxygen and nutrients . regeneration of the infarct area through cardiomyocyte proliferation without increasing capillary density is impossible . in the peri - infarct region , insufficient capillary perfusion leads to apoptosis of the hypertrophied but viable cardiomyocytes that remain after infarction [ 106 , 107 ] . therefore , induction of cardiomyocyte proliferation in the peri - infarct region might just have a transient effect on heart regeneration if the vas - culature and other supporting cells are not restored . this might explain why p38 inhibition that has no effect on capillary density causes only a transient effect in improving heart function despite induction of cardiomyocyte proliferation . insufficient revas - cularization might also be the reason why overexpression of c - myc [ 108110 ] and cdk2 resulted only in a transient induction of proliferation as well as smaller cardiomyocytes . another challenge is that the contraction of all cardiomyocytes within the myocardium has to be coordinated to guarantee maximal possible cardiac output . therefore , it has to be taken into account that dividing cardiomyocytes cease to contract during anaphase . thus , for optimal myocardial regeneration the organization and orientation of the new cardiomyocytes , their proper timely contraction as well as a slow proliferation rate is important . this might be the reason why newt and zebrafish heart regeneration takes several weeks [ 30 , 35 ] . it remains a challenge to understand precisely how the combination of tissue repair mechanisms with reactivation of embryonic programs in urodele and zebrafish can generate growth , pattern formation , and morphogenesis in an adult animal . whether this kind of regeneration can be achieved in mammals however , full regeneration might not be necessary . functional regeneration to improve cardiac output might suffice in most clinical settings to restore a satisfactory functional performance without achievement of complete tissue repair . a good example is the liver , which regenerates very efficiently although the original tissue architecture is not rebuild completely . after excision of one of the five liver lobes the liver does not restore the original morphology re - growing the excised lobe . in fact , after injury the remaining four lobes grow to compensate for the loss in liver mass . the correct question regarding regeneration is : can we induce proliferation of postnatal cardiomyocytes after injury and cause improvement of heart function ? as outlined in this review , neonatal mammalian cardiomyocytes are endowed with the full cell cycle program and can be efficiently induced to proliferate . however , so far it has not been tested whether this can be used to improve heart function , for example in patients with congenital heart defect ( chd ) ; the most frequently occurring birth defect and leading cause of birth - defect - related death [ 113 , 114 ] . recently , it has also been demonstrated that complete cell division can be induced in adult cardiomyocytes . the main problem hereby appears to be the transition from mitosis to cytokinesis and its completion . taken together , the scientific community has gathered a plethora of data that allows the encouraging conclusion that repopulation of the heart by inducing cardiomyocyte proliferation is a realistic future option for cardiac repair . at the same time , we urge to a critical analysis of data regarding this hot topic to avoid misinterpretations and too fast conclusions . | the human heart does not regenerate . instead , following injury , human hearts scar .
the loss of contractile tissue contributes significantly to morbidity and mortality . in contrast to humans , zebrafish and newts
faithfully regenerate their hearts .
interestingly , regeneration is in both cases based on cardiomyocyte proliferation . in addition ,
mammalian cardiomyocytes proliferate during foetal development .
their proliferation reaches its maximum around chamber formation , stops shortly after birth , and subsequent heart growth is mostly achieved by an increase in cardiomyocyte size ( hypertrophy ) .
the underlying mechanisms that regulate cell cycle arrest and the switch from proliferation to hypertrophy are unclear . in this review ,
we highlight features of dividing cardiomyocytes , summarize the attempts to induce mammalian cardiomyocyte proliferation , critically discuss methods commonly used for its detection , and explore the potential and problems of inducing cardiomyocyte proliferation to improve function in diseased hearts . |
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gluteal muscle contracture ( gmc ) is a clinical syndrome characterized by gait abnormality and limb dysfunction , as well as secondary deformities of pelvis and femur . at present , arthroscopic release , a minimally invasive surgery that was reported by zhang et al for the first time , femoroacetabular impingement ( fai ) typically could be diagnosed on the basis of computed tomography ( ct ) such as the equatorial - edge angle ( ee angle ) . although ee angle may be sufficiently sensitive to identify and differentiate impingement morphologies in all cases and the diagnostic challenges have been previously reported , the judgment method did not work well in gmc patients . in this study , we retrospected all image data and found small ee angles in gmcs , which meant retroverted acetabulum ; however , none of them showed symptoms and signs of fai . therefore , we had reasons to think that , some normal hips with unbalanced hip myodynamia as same as gmcs , may be incorrectly diagnosed as fai through measuring ee angle only . in consequence , the paper was designed to assess the use of the ee angle in the assessment of fai in the diagnosis , as described by werner . twenty - three people of those gmc patients coming for arthroscopic treatment agreed to join in the study . pelvic ct scans of patients admitted to the peking university shenzhen hospital , shenzhen , china , were retrieved from the electronic radiological archive . there were 23 patients ( 46 hips ) who met the premise of having images available ( fig . 1 ) , and all data were further analyzed . before the ct scans were included , it was required to validate correct patient position and radiographic technique to eliminate any measurement error as possible . ( a c ) the 3d reconstruction ct shows a gmc patient with severe gluteus maximus and gluteus medius atrophy for ( a ) posterior view , and ( b ) left side , and ( c ) right side , which have the contracture bands ( white arrow ) in left and right side , respectively . msct scans were performed on ct scanners ( discovery 750hd ; ge healthcare , waukesha , america ) with slice thickness varying between 0.625 and 2.5 mm and a gantry tilt of 0. all scans ranged from the pelvic bone to the proximal femur . the indicator of acetabular retroversion on the ct scans was measured using an adapted version of the method originally described by werner et al and reynolds et al : the ee angle was the angle of the acetabular opening at the maximum diameter of the femoral head ( fig . 2 ) , which was represented by the sagittal plane and a line drawn between the anterior and posterior acetabular rim on the appropriate ct slice . in light of reynolds et al , ee angle was defined as negative if opening posteriorly ( fig . ( a , b ) the ct slice at the level of the maximum diameter of the femoral head . a line was drawn from the anterior to the posterior border of the acetabulum , as well as a line representing the sagittal plane . the graph shows 2 positive angles of ( a ) , one positive and another negative of ( b ) as well . distributions of angles were examined using histogram , and reported as mean standard deviation if distributions were symmetrical . we used independent groups t tests for comparisons between groups ( sex and side ) if assumptions of approximate normality and equal variances were met . review of the hips showed a mean ee angle was 12.93 , with a minimum of -3.42 and a maximum of 24.08 ( table 1 ) . the mean value for males and females were 13.52 and 12.40 , respectively , without statistical significance ( table 1 ) , although the mean value of left hips and right sides reached 13.32 and 12.54 individually , not having statistical differences neither ( table 1 ) . thus , the reduced ee angle could suggest the local excessive coverage of the femoral head by the anterior acetabular edge , but might not be a reasonably good predictor of fai . gmc mainly affects the patient 's tensor fasciae latae , gluteus maximus , gluteus medius , and gluteus minimus , what 's more , sometimes even involves the lateral rotator group and the joint capsule of hip . these involved muscles may be found with muscle fibers degeneration and necrosis , fibrous connective tissue proliferation , and myofascial and fascia hypertrophy , leading to a sequence of secondary pathological changes , such as the hip muscles out - of - balance , which may cause abduction and external rotation deformities of the hip joint , pelvic tilt , and acetabular dysplasia . fai is an abnormal condition where chronic pains occur in the hip joint resulted from degenerative changes in the acetabular labrum and articular cartilage , due to the impingement between the femoral head and acetabulum caused by anatomical abnormalities in the hip joint . in these cases , the hip joint 's range of motions , especially flexion and internal rotation , are limited , eventually resulting in the hip osteoarthritis . as the good compatibility between the acetabulum and the femoral head is the functional basis for normal movement of the hip joint , morphological changes in any one of them will affect the hip functions . the normal border of the femoral head neck junction is a concave arc , which can smoothly move into the acetabulum when the hip joint is flexing or internally rotating . when the femoral offset is reduced or vanished , or the acetabular edge excessively covers the femoral head , the acetabular edge and the head neck junction can be more likely to squeeze and impinge on each other at the end stage of the motion , resulting in fai . it is traditionally held that fai is divided into the cam - type and the pincer - type . the cam - type fai is distinguished by a noncircular femoral head but an anatomically normal acetabulum . the noncircular femoral head may collide with the acetabular edge , leading to cartilage damage and labrum tear and even causing bone cystic degeneration and labrum ossification of the acetabulum . on the contrary , in the pincer - type fai , the femoral head is anatomically normal , but the abnormal acetabulum is characterized by the acetabular retroversion or the excessive coverage on the femoral head , causing linear contact between the acetabular labrum and the femoral head neck junction during the joint motions , thereby leading to the degeneration , hardening , or ossification of the acetabular labrum . such changes aggravate the wrapping of the femoral head by the acetabulum and further exacerbate the range and severity of the lesion . actually , some fai patients can be observed to show the anatomical abnormalities in both the femoral head and the acetabulum , and some researchers have defined this condition as the mixed - type fai . msct has been widely applied in the examination of bone and joint diseases , and its diagnostic value has been recognized as an effective imaging method . as the isotropic advantage of msct allows the 3-dimensional reconstruction in any orientation after the scanning , it is more conducive to observe and measure the structural abnormalities in order to evaluate the severity of the disease . it is reported that msct is more sensitive than x - ray in displaying many aspects , including the hardening of the impacted area on the acetabular edge , early changes in subchondral bone density , tiny synovial hernia , and acetabular rim ossicles on the acetabular edge . there were still some shortcomings in this study , such as the relatively small sample size , the absence of a healthy control group , and the lack of a the previously reported x - ray findings , such as the pistol grip deformity and the cross - over sign are nonquantitative description . in this study , the ee angle was selected to quantify the abnormal bone structure of the hip joint in fai . the ee angle reflects the degree of the acetabular retroversion in the pincer - type fai , which is one of the features of pincer - type fai , typically manifesting as decreased ee angle . the study of a large sample by werner et al showed that the ee angle of the acetabular edge crossing negative group was 21.0 , while that of the crossing positive group was 17.3 . similarly , in chinese populations , chen et al discovered it was 20.4 in 59 fai patients . although in this study , the ee angle was measured as 13.1 3.1 on the left side and 12.5 3.9 on the right side , significantly smaller than previous studies . the reduced ee angle suggested the local excessive coverage of the femoral head by the anterior acetabular edge . according to werner 's study , the participants in our study should be diagnosed as fai ; actually , there was not any meaningful discovery of fai in all patients . in a word , we discovered that gmcs have a higher degree of retroversion ( excessive acetabular retroversion ) than fai hips , seemingly suggesting that they should be attributed to fais . however , in this study , all patients showed no symptoms and positive signs of fai . possible reasons might be that most symptoms of the fai patients occur during flexion , adduction , and internal rotation , but the gmc is a disease manifested as the abduction and external rotation deformities of the hip joint , and thus the impingement is impossible to happen . the first , ee angle can be used for diagnosing the people with balanced hip muscle forces whether have fai or not , while those populations with imbalanced myodynamia , such as gmcs , were not suitable . another probability is that , perhaps ee angle is worthy for everyone , but it has its own limitation . in other words , at least , it can not be a single imaging diagnostic basis ; the muscular factor also plays a significant role in fai pathogenesis . apart from these 2 points , this study also reminded us that , even with the presence of abnormal bone structure , as long as the muscle strength of the hip joint 's abduction and external rotation is greater than that of the adduction and internal rotation , the acetabular impingement would not happen . therefore , some studies indicated that , in the conservative treatment of fai , the excessive hip flexion , adduction , and internal rotation should be prevented , and most patients symptoms could be alleviated by doing so . emara et al reported that 33 of 37 patients showed alleviative symptoms in follow - up , and 6 of them showed recurrence of symptoms , but the pain could be tolerated and no surgery was required . gmc patient 's acetabular deformity mainly manifests as increased retroversion , which may be the anatomical basis for fai and lead to high risks of the acetabular impingement . however , all patients in this study showed no symptoms and signs of fai , suggesting that the measurement of ee angle can only be applied to assessing those people with normal hip myodynamia , and the bone deformity and the muscular disorder should be both considered in the diagnosis of fai . | abstractgluteal muscle contracture ( gmc ) is a clinical syndrome characterized by gait abnormality and limb dysfunction , as well as secondary deformities of pelvis and femur .
femoroacetabular impingement ( fai ) typically could be diagnosed on the basis of computed tomography ( ct ) such as the equatorial - edge angle ( ee angle ) , but it did not work well in gmc patients . in this study , we retrospected all image data and found small ee angles in gmcs , which meant retroverted acetabulum ; however , none of them showed no symptoms and signs of fai .
therefore , we had reasons to think that , some normal hips with unbalanced hip myodynamia as same as gmcs , may be incorrectly diagnosed as fai through measuring ee angle only.in consequence , the paper was designed to assess the use of the ee angle in the assessment of fai in the diagnosis , as described by werner.twenty-three patients ( 46 hips ) were collected and calculated with the equatorial - edge angle ( ee angle ) by ct scans .
all of them were excluded from fai.review of the hips showed a mean ee angle was 12.93 , with a minimum of -3.42 and a maximum of 24.08. the mean value for males and females were 13.52 and 12.40 , respectively , without statistical significance , although the mean value of left hips and right sides reached 13.32 and 12.54 individually , not having statistical differences neither .
there were not any symptoms or signs of fai in all patients .
thus , the reduced ee angle could suggest the local excessive coverage of the femoral head by the anterior acetabular edge , but might not be a reasonably good predictor of fai.gmc patient 's acetabular deformity mainly manifests as increased retroversion , which may be the anatomical basis for fai and lead to high risks of the acetabular impingement .
however , all patients in this study showed no symptoms and signs of fai , suggesting that the measurement of ee angle can only be applied to assessing those people with normal hip myodynamia , and the bone deformity and the muscular disorder should be both considered in the diagnosis of fai . |
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retaining coronal restoration in endodontically treated teeth usually requires the use of intra - canal posts.1 - 6 although high success rates have been reported for fiber - reinforced composite ( frc ) post restorations,7,8 root fracture and loss of retention are still the most common reasons for failing these restorations.9,10 due to the similar elastic modulus of fiber posts and root dentin , distribution of loads in the root canal has an uniform pattern.11,12 so , less occurrence of root fracture has been reported for these restorations in comparison to metallic post retained restorations.7,8 the fiber post retention in root canal depends on the bond strength between different parts of the combined ' sandwich ' post - cement - dentin assembly.13 - 16 it has estimated that 60% of the fiber post failures occurred between the fiber post and resin cement.17,18 so , achieving effective bond strength between the frc post and resin cement is important.4,19 many post surface treatment protocols ( chemical and micro - mechanical modifications ) have been reported to improve surface energy characteristics of dental fiber reinforced posts.6,16,20 - 24 the bond strengths between post and resin cement can be improved chemically by post surface silanization.9,22,25,26 however , there is no definitive agreement on this subject.24,27 on the other hand , some studies concluded that roughening the post surface either with micro - mechanical methods like sandblasting20,24,28 - 34 or application of hydrogen peroxide35 - 37 can improve the retention of frc posts that are cemented with resin cements . however , the effectiveness of these roughening techniques is not completely confirmed.6,27,28 since chemical surface treatments alone may not create a strong bond between fiber posts and resin cements,21 some micro - mechanical treatment methods have also been performed to remove a surface layer of fiber posts and exposes the quartz fibers for silanization.38 this type of surface treatments that combine both a chemical and micromechanical method is called as alternative etching techniques.21 although , sandblasting and hydrofluoric acid etching are used to improve the bonding of fiber posts to resin cement,18,24,27 these techniques can damage the glass fibers and affect the post integrity.18 hydrogen peroxide ( h2o2 ) is one of the materials that can selectively dissolve the epoxy matrix without interfering with the glass fibers and can expose the fibers to be silanated.6,20,39,40 the purpose of this study was to investigate the effects of some post surface conditioning methods before silanization on the bond strength between fiber post and resin cement . the null hypothesis to be investigated was that the bond strength between fiber post and resin cement with the various surface conditioning methods are not significantly different . in this in vitro study , 40 human maxillary incisor single - rooted teeth free of caries , cracks , fractures , resorption , with fully developed roots and without previous root canal treatments or posts were selected . the teeth were cleaned of residues of soft tissue or calculus and disinfected by placing them in 2.5% naocl ( golrang , golrang co. , tehran , iran ) for two hours and then stored in 0.1% nan3 solution . diamond discs ( ref.070 , d&z , berlin , germany ) were mounted in a dental lathe machine ( kavo polishing unit . this job was done at low speed under water irrigation to achieve a 15 mm root length . the pulp tissues were removed with barbed broach ( dentsply / maillefer , ballaigues , switzerland ) . themaster apical file was the number 35 k file ( dentsply / maillefer , switzerland ) . canals were irrigated with 2 ml of 5.25% solution of sodium hypochlorite during the canal preparation stages ( golrang , iran ) . finally , paper points ( aria dent , asia chemiteb co , tehran , iran ) were used for drying the root canals . the obturation was performed with vertical condensation method , using ah26 ( dentsply caulk , milford , germany ) and gutta - percha ( aria dent , iran ) . a provisional restorative material ( gc caviton ; gc dental products corp . , tokyo , japan ) was used for filling the coronal root canal orifices , and then to ensuring the setting of the used sealer , the teeth were stored for one week in 100% humidity at 37. after one week , the gutta - percha in the coronal aspect of each root was removed with a gates glidden drill # 3 ( dentsply / maillefer , switzerland ) so that , 4 mm gutta - percha was preserved in the apex to maintain the apical seal . the all post spaces were prepared to a depth of 10 mm , measured from top of the sectioned root surface , with dedicated drills provided by a post manufacturer ( fibio , anthogyr , sallanches , france ) . to standardize the final shape of the post space , a no . 3 drill of this system was used as the last drill . finally , the canals were irrigated with distilled water and dried with paper points ( aria dent , iran ) . a glass reinforced fiber post ( hetco fiber post , hakim toos , mashhad , iran ) ( table 1 ) size # 3 was tried in the post spaces and then all posts were cut at a distance of 10 mm from the apical end with diamond discs ( ref . this procedure was done for post length standardization and creation diameter similarity among the used posts.19 in all groups , the shortened hetcoposts ( hakim toos , iran ) were cleaned with 70% ethanol for 60 seconds , rinsed with distilled water and air dried . the posts were randomly divided into four groups of 10 specimens each , according to surface treatments used : h2o2 + silane group . at the first , the 30% h2o2 were diluted to 20% h2o2 and then the posts in this group were immersed in diluted h2o2 for 20 minutes at room temperature . after 2 minutes rinsing with running water , they were air dried . then the posts surfaces were painted with a single layer of a silane coupling agent ( ultradent porcelain etch and silane , ultradent products inc . , ut , usa ) for 60 seconds and dried for 60 seconds with gentle air stream . the posts in this group were sandblasted with 50 m aluminum oxide particles using an oral microblaster ( dento - prep , ronviga , daugaord , denmark ) for 10 seconds at 0.25 mpa pressure from a 10 mm distance . after rinsing posts with 96% ethanol the posts surfaces were painted with a single layer of thesilane coupling agent ( ultradent porcelain etch and silane ) and were air dried for 60 seconds with gentle air stream . only a single layer of thesilane coupling agent ( ultradent porcelain etch and silane ) was applied on the posts surfaces for 60 seconds and then air dried for 60 seconds with gentle air stream . control group . the root canal dentin walls were conditioned with an autopolymerizing primer ( ed primer , kuraray medical inc . , one drop of each liquid ( a and b ) ( ed primer ) were mixed and applied to the root canal walls with a microbrush ( atlas microbrush ; atlas dandan co. , tehran , iran ) for 60 seconds . then , the post space was air dried and the excess primer was removed with paper points ( aria dent , iran ) . for fiber post cementation , equal amounts of base and catalyst pastes of an adhesive composite resin cement ( panavia f2.0 , kuraray medical inc . , japan ) were mixed and applied on the posts surface and into the root canals with a lentulo spiral instrument ( dentsply / maillefer , switzerland ) . using gentle finger pressure , the fiber posts were inserted into the prepared post spaces to full depth . finally , the remaining cement around the post was protected with oxygen inhibiting gel ( oxyguard ii , kuraray medical inc . ) . after the initial chemical polymerization , the resin cement was light cured for 60 seconds in such a way that the tip of the light unit ( coltolux50 , coltene , altstatten , switzweland ) was in close contact with the coronal end of the fiber post . accurate light intensity of the light output was monitored before each light exposure by coltolux light meter ( coltene , altstatten , switzerland ) . after the cementation procedures , all dental root specimens were placed in a light - proof box , containing 37 sterile saline for one week . all the specimens were subjected to thermocycling treatment for 5,000 cycles at temperatures alternating between 5 and 55 for 30 seconds each , with an intermediate pause of 15 seconds . the coronal part of each dental root was sectioned perpendicular toits long axis using a diamond disc ( ref.070 , d&z , germany ) mounted in a cutting machine ( tl-3000 , vafaeiindustrial , tehran , iran ) at low speed under constant distilled water irrigation to create 3 mm thick slices . in this manner , threepost / dentinsections ( coronal , middle and apical ) were obtained from each root . due to usingtaperedfiber posts , the post diameters were measuredusingadigital caliper ( mitutoyodigital caliper 500 - 714 - 10 , mitutoyo co. , tokyo , japan ) with 0.01 mm accuracy . a universal testing machine ( walt + bai ag testing machines industriestrass 4 , lhningen , switzerland ) was used for applying tensile force at acrosshead speed of 1 mm / min . the push - out pin was placed on the center of the apical end of the post surface and in an apico - coronal direction without inserting extra forces on the surrounding root canal walls . therefore , three push - out pins were constructed in three diameters ( 0.7 , 0.9 and 1.0 mm ) that were used for each three root sections respectively ( apical , middle and coronal parts ) . the peak force ( n ) required to extrude the fiber post from each root slice was recorded for all specimens . the bond strength in mpa was calculated by dividing the load at failure ( n ) by the total area of the cemented post . this calculation was done with the following formula : in this formula , is the constant 3.14 , r1 is the coronal post radius , r2 is the apical post radius and h is the slice thickness in mm . the collected data were analyzed ( spss / pc16.0 ; spssinc . , chicago , il , usa ) using two - way anova and post hoc tukeytest at p<.05 level of significance . table 2 shows the mean tensile bond strength ( tbs ) and standard deviation values for all experimental groups in different root canal regions . the two - way anova showed that different surface treatments ( p=.004 ) and root canal dentin regions ( p<.001 ) had significant effects on tbs , but interaction between surface treatments and root canal regions ( p=.068 ) had no significant effects on tbs ( table 3 ) . the h2o2 + silane group had the highest tbs mean value especially in the coronal region . the lowest tbs mean value was seen in the control group and in the apical region . post hoc tukeytest revealed that there was significant differences between h2o2 + silane group and other three groups ( p<.05 ) ( table 4 ) . based on the results of this investigation , the null hypothesis that the various surface treatments do not have any effects on the tbs between fiber post and resin cement was rejected . fiber posts are comprised of a matrix resin that surrounds different types of fibers . in the glass fiber posts , the fibers are made of glass and the matrix is made of epoxy resin.5 the epoxy polymers can not chemically bond with composite resin cements because of their highly cross - linked structure.41 therefore , some authors propose using silane coupling agent on the fiber post surface to increase the tbs between the fiber post and resin cement . however , it has been said that silane can not increase the bond strength of the fiber posts to resin cements or composite core materials.17,32,42 - 45 in fact this is due to this fact that the chemical bond is achieved mainly by creating covalent bonds between the silane coupling agent and the composite resin ; and between silane and the exposed glass fibers or filler particles of the post.46,47 in the present study , likes some other investigations , it was found that silane alone can not increase the tbs of the fiber posts to resin cements ( table 4 ) . roughening the fiber post surface with micro - mechanical procedures can bring the glass fibers in better contact with silane coupling agent.6,20,40 although , sandblasting is used to roughen the fiber posts surface for better bonding , 18,24,27 also it can damage the glass fibers.18 instead , hydrogen peroxide ( h2o2 ) can selectively dissolve the epoxy matrix without damaging the glass fibers.6,20,39,40 the present study showed that using h2o2 before silanization can significantly improve the tbs in comparison to sandblast + silane , silane and control groups ( p<.05 ) ( fig . 1 ) , which is in accordance with some other studies.18,24,27 yet , radovic et al.48 showed that even if sandblasting can improve the bond strength between fiber post and resin cement but water aging can reduce the bond strength of sandblasted specimens . it is said that by using h2o2 as a pretreatment for using silane coupling agent , a better chemical bonding between silane and fiber post was achieved.32,36,40,43,47 in the studies that bonding agent was applied immediately after using h2o2 ( without silane coupling agent as a mediator ) , the tbs mean value was reduced.1 however , some authors claimed that pre - treatments are not necessary before silane application.49 for root canal regions , our results are in accordance with other studies that reported higher bond strength for coronal region of the root than other tworegions.9,13,19 the tubular density and diameter of the coronal third of the root canal dentin are more than other regions . furthermore , the dentinal hybridization is not uniform and lateral branches of resin tags are not seen in the apical regions of root canal dentin.50 furthermore , more access to the coronal portion of the canal makes etching and applying the adhesive agents17,50 and light transmission to canal walls50 become more effective in this area . however , aksornmuang et al.9 found no significant differences in the bond strengths among different regions of the root canal walls . for example , the specimens had no coronal tooth structure , only one type of fiber post and adhesive were evaluated and the influences of fatigue loading on the push out tbs of specimens were not investigated . within the limitations of this in vitro study , the following conclusions were drawn : there were significant differences between the mean tensile bond strength values of different surface treatments and root canal regions . application of hydrogen peroxide before silanization increased the bond strength of resin cement to the fiber posts . the coronal region of the root canal showed significantly higher mean bond strength values than the other regions . | purposepost surface conditioning is necessary to expose the glass fibers to enable bonding between fiber post and resin cement .
the purpose of the present study was to evaluate the effect of different surface conditioning on tensile bond strength ( tbs ) of a glass fiber reinforced post to resin cement.materials and methodsin this in vitro study , 40 extracted single canal central incisors were endodontically treated and post spaces were prepared .
the teeth were divided into four groups according to the methods of post surface treatment ( n=10 ) : 1 ) silanization after etching with 20% h2o2 , 2 ) silanization after airborne - particle abrasion , 3 ) silanization , and 4 ) no conditioning ( control ) .
adhesive resin cement ( panavia f 2.0 ) was used for cementation of the fiber posts to the root canal dentin .
three slices of 3 mm thick were obtained from each root .
a universal testing machine was used with a cross - head speed of 1 mm / minute for performing the push - out tests .
two - way anova and tukey post hoc tests were used for analyzing data ( =0.05).resultsit is revealed that different surface treatments and root dentin regions had significant effects on tbs , but the interaction between surface treatments and root canal regions had no significant effect on tbs .
there was significant difference among h2o2 + silane group and other three groups.conclusionthere were significant differences among the mean tbs values of different surface treatments .
application of hydrogen peroxide before silanization increased the bond strength between resin cements and fiber posts .
the mean tbs mean values was significantly greater in the coronal region of root canal than the middle and apical thirds . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
adjustment and coping of children with pediatric illness has been extensively researched , with some emphasis placed on family adjustment and the reciprocal influence it has on ill children 's adjustment and coping [ 13 ] . the majority of studies conducted on family adjustment have focused on the adjustment of parents [ 46 ] . fewer studies have focused on the adjustment of siblings [ 710 ] , and most of these studies are specific to siblings of children with pediatric cancer . regardless of the severity of the pediatric chronic illness , siblings usually experience some emotional instability and disruption in their lives due to the diagnosis [ 1114 ] . breyer and colleagues found that 59% of children showed new externalizing symptoms and 26% showed new internalizing symptoms following their siblings ' cancer diagnosis . siblings are also exposed to unique situations that other children are not privy to , which may have positive or negative impacts on well siblings ' immediate and future well - being . specifically , siblings may experience positive behavior changes including greater maturity , empathy , and compassion . this new - found empathy and compassion may often lead to a desire to help and take care of others . siblings ' experiences with caring for and observing their ill siblings ' course of illness may also give them an appreciation for life and understanding of its fragility that siblings in well families may not be able to comprehend [ 16 , 17 ] . researchers disagree on the pervasiveness of the negative impact of the diagnosis on well siblings ' well - being . specifically , houtzager and colleagues conducted a longitudinal study on siblings ( 7 to 19 years ) of children diagnosed with cancer , following them up to 24 months after the cancer diagnosis , and found that siblings were most affected in the first few months following the diagnosis . conversely , alderfer et al . , in their study on adolescent siblings of childhood cancer survivors , found that siblings had elevated levels of posttraumatic stress when compared to nonaffected adolescents , suggesting that there may be some residual effects of the diagnosis on well siblings ' functioning . to date , no research has explored the continuing impact of growing up with an ill sibling , on well sibling 's late adolescent functioning . individuals between the ages of 17 and 24 years are typically categorized as late adolescents / early adults . these individuals face stressors and changes that could be challenging , including managing independence , leaving home for college and being separated from their immediate family , starting serious romantic relationships , and new responsibilities . siblings of children with chronic illnesses may have childhood experiences that could affect their adjustment to at least one of these stressors . siblings may have been separated from their families for extended periods of time due to their ill siblings ' hospitalizations . they may also have had to exercise some independence due to parents having to spend extended periods of time caring for their ill sibling or them taking on a caretaker role for younger well siblings at an early age . one can argue , therefore , that siblings of chronically ill children may have a unique advantage as they transition from adolescence to late adolescence , because of their childhood experiences . researchers have measured young adulthood adjustment of children with chronic illnesses and have found that these children tended to grow up to be more resilient adults , than those with a typical childhood experience , although others have found that some young adults display posttraumatic stress symptoms [ 2022 ] . the extent to which well siblings ' functioning mirror these findings are yet unknown . sibling relationships are believed to be one of the most powerful , extensive relationships in one 's lifetime and are distinguished by its love , jealousy , companionship , compassion , and rivalry [ 23 , 24 ] . this relationship is greatly affected when one sibling becomes seriously ill . for example , well children report feeling less close to their ill sibling . siblings of ill children have also reported high levels of anxiety and isolation [ 11 , 13 ] , envy , and contradictory feelings of guilt and resentment . some studies also found that well siblings have more difficulty adjusting and adapting to the illness than their ill siblings . the purpose of this study was to explore the continuing impact of growing up with an ill sibling on well siblings ' late adolescent functioning . by exploring well siblings ' psychological functioning in relation to community patterns and how they perceive their experience as influencing their lives currently , the researchers hope to provide a clearer picture on well siblings ' late adolescent functioning . additionally , by retrospectively exploring well siblings ' feelings and concerns about their ill siblings ' diagnosis , the researchers aim to provide insight on how these variables impact well siblings ' present functioning . participants ( n = 40 ) were randomly selected from enrollees in a university psychology subject pool who identified themselves as growing up with an ill sibling during prescreening . they were between the ages of 18 and 21 years ( mage = 18.78 , sd = 0.83 ) , and most were freshmen ( 67.5% ) at the university . the majority of participants reported living in nuclear ( 77.5% ) and step - parent ( 12.5% ) families at the time of their siblings ' diagnosis . place in the family in relation to ill sibling was evenly distributed with 50% reporting being older and 50% reporting being younger than their ill sibling . all fathers of participants worked full time , while they were growing up , while 52.5% of mothers worked full time . the majority of mothers ( 87.5% ) and fathers ( 82.5% ) of participants had at least some college education . specifically , questions were asked about family composition , sibling 's diagnosis , caretaker arrangements , and parent education and work status . the personality assessment screener ( pas ) was used to assess for behavioral and emotional problems of potential clinical significance . the pas is a 22-item measure that assesses a broad range of clinical problems and is comprised of the items proven to be maximally sensitive to clinical problems on the personality assessment inventory ( pai ) . the pas yields a total score and 10 domain scores including negative affect , acting out , health problems , psychotic features , social withdrawal , hostile control , suicidal thinking , alienation , alcohol problems , and anger control . the pas uses probability ( p ) scores , and the scores are interpreted as the probability that a person would obtain a problematic protocol if given the pai . therefore , any score above 50 p indicates that the person may be experiencing some clinical difficulty . this 27-item questionnaire was originally designed to assess perception and adaptation of well siblings to pediatric cancer of a sibling and included four factors . the first factor is interpersonal ( cronbach = 0.86 ) , including nine items assessing siblings ' perceptions of themselves in the experience . the second factor is intrapersonal ( cronbach = 0.80 ) , including seven items about how well siblings felt about their siblings ' diagnosis . factor 3 is communication ( cronbach = 0.67 ) , including four items that examined sibling 's perception of their ability to communicate with parents and adults about the diagnosis . the last factor is fear of disease ( cronbach = 0.65 ) , consisting of three items that assessed siblings ' fears and worries . because the measure was designed for use specifically with school - aged siblings of children with cancer , the wording ( replace cancer with illness ) and instructions ( think back to your childhood ) were modified . participants responded to all the statements on a 5-point likert scale ranging from 1 = never to 5 = always . questions were designed to elicit well siblings ' perceptions of retrospective and current adaptation and coping with having an ill sibling . responses to four of the questions were explored to meet the aims of this paper : sometimes good things come out of bad experiences . looking back , what would you want to happen differently about the experience ? what were your biggest fears then ? how has the disease affected your life ? , and how about now , do you have any fears or worries that stem from your sibling 's illness ? questions were developed based on information gathered from a review of the child literature on sibling coping and adaptation . informed consent was obtained from all participants and all participants agreed to have their interviews audio - recorded . participants were asked to complete the demographic questionnaire , mfcsq , and pas prior to the interview . the interviewer scanned the suicide items on the pas prior to participants leaving , and a suicide risk assessment was conducted on those endorsing at least slightly true on any of the items . participants were given credit for participation at the end of the interview and a debriefing form that included phone numbers through which they could seek psychological services if desired . confirmatory factor analyses ( cfa ) were performed to determine how well the items on the mcfsq yielded the same reliability coefficients reported by carpenter and sahler . to explore well siblings ' psychological functioning in relation to community norms , the pas was hand - scored , and the scores were converted to p scores . independent sample t tests were conducted to explore gender differences in pas scores . to examine the relationship between well siblings ' feelings and concerns about their siblings ' diagnosis during childhood and their current functioning , pearson correlations were computed between the pas domain and total scores and the mcfsq subscale scores . thematic analyses were used to elicit themes about how siblings perceived growing up with an ill sibling as influencing their lives , both in the past and currently . three of the four factors on the mcfsq produced similar reliability coefficients reported by carpenter and sahler in their study on siblings of children with cancer ( interpersonal = 0.89 , intrapersonal = 0.75 , and communication = 0.73 ) . the fear of disease subscale was excluded from the study because it produced a very low reliability coefficient ( = 0.15 ) . the low reliability of fear of disease may be attributed to this study 's focus on a variety of chronic and terminal illnesses rather than solely cancer . regarding well siblings ' psychological functioning , well siblings displayed psychological problems in the range described in the pas interpretive manual as being somewhat problematic but not necessarily of clinical significance for some domains ( > 60% of siblings had p scores in normal or mild range , see table 1 ) . for acting out , psychotic features , social withdrawal , and to a lesser extent hostile control , the distribution of p scores suggests that well siblings may be potentially experiencing clinically significant problems in these domains . the average total score , however , suggests that siblings are similar to well - adjusted community adults in their psychological functioning . when the scores were explored within the sibling sample , significant gender differences were found for acting out and alienation . male siblings were significantly more likely to report behavioral problems associated with impulsivity , sensation seeking , drug use , or a combination compared to female siblings . female siblings were significantly more likely to report failures of supportive relationships and a distrust or disinterest in such relationships . pearson correlations revealed a positive relationship between well siblings ' retrospective reporting of poor communication with parents during childhood and social withdrawal and alienation . given the exploratory nature of this study , we noted that the relationship between alcohol problem and communication also approached significance . the pas total score was positively related to retrospective reporting of interpersonal problems , that is , siblings ' perceptions of themselves in the experience . thematic analyses of siblings ' responses to interview questions are presented in tables 2 , 3 , and 4 . siblings identified greater awareness of illnesses ( 40% ) and family bonding and support ( 100% ) as good things that came from growing up with a sibling with a chronic illness . well siblings identified their ill siblings ' disease status , future , and their ability to help their siblings in the event of a crisis as fears they experienced while growing up . they also noted regretting not being more sensitive , jealous , and not spending enough time with family . the majority of siblings ( 62.5% ) reported that the experience continues to affect their lives positively with cautiousness about own health , maturity , and appreciation of life being the most common positive themes . approximately 17% of siblings noted that the experience continues to affect their lives negatively because they worry about the future and feel responsible for their siblings . twenty percent of siblings reported that growing up with an ill sibling had no effect on their lives . when asked if they had any fears or worries now that stem from their siblings ' illnesses , the majority of well siblings ( 60% ) reported that they had no fear or worries while others reported fears about ill - siblings ' health , siblings ' death , disease worry , and own future . results on well siblings ' current psychological well - being were mixed and were somewhat contradictory . examination of the mean scores in most domains suggested the possibility of siblings experiencing some problems , which may be expected given their developmental phase and the current stressors they may be experiencing in their lives such as adjusting to being freshmen in college . noteworthy is that all of the mean scores with the exception of anger problem are in the lower part of the range suggested by the interpretive manual as being potentially problematic . given the research on the psychological functioning of well siblings during childhood [ 7 , 29 ] , one would have expected these siblings to have much higher scores on the pas especially in the negative affect domain , which is a measure of personal distress , and experiences of apprehension and unhappiness . the results suggested that siblings may potentially experience clinically significant problems in acting out , which is indicative of behavioral problems . a significant gender difference emerged on this domain , with males having significantly higher scores than females . we speculate that these findings are due to males ' tendency to act more impulsively and late adolescents ' tendency to engage in more sensation - seeking behaviors and drug use on college campuses . also noteworthy the gender differences in acting out and alienation may be reflective of the different coping styles of males ( externalizing ) and females ( internalizing ) in general [ 31 , 32 ] . social withdrawal on the pas is characterized by social detachment and discomfort in close relationships , while hostile control is defined as an interpersonal style where there is a need for control and inflated self - image . the elevations on these two scales may be directly related to well - siblings ' childhood experiences . well siblings may have a history of being separated from their families for extended periods of time , and this may have contributed to the development of an avoidant attachment style . being forced to understand the fragility of life at an early age may also be responsible for them choosing to detach themselves from others . regarding hostile control , researchers have found that well siblings learn responsibility from an early age , and in some instances they may have to adopt the caretaker role to their other siblings as well as their parents . we hypothesize this early role reversal may potentially lead to a maladaptive manifestation of the behavior in late adolescence . additionally , competition for parents ' attention and affection during childhood may lead to siblings behaving in a manner to ensure they were attended to , and this too may be maladaptive in late adolescence . exploration of the relationship between retrospective accounts of feelings after the ill sibling was diagnosed and current psychological functioning resulted in some interesting findings with regard to communication . both communication and social withdrawal and communication and alienation were positively related . siblings who perceived their abilities to communicate with parents and adults about the diagnosis during childhood as poor were more likely to report social detachment and discomfort in social relationships and to report failure , distrust , or disinterest in supportive relationships . one explanation might be that siblings ' may be inclined toward withdrawal in their communication style and interaction with others due to their temperament . another potential explanation is that siblings may have learned to socially detach in response to not being able to communicate with parents and adults after the traumatic , life changing experience . a third explanation might be that parents may not have been forthright in their explanations about ill - siblings ' diagnosis when talking to well siblings , and as a result well siblings learn not to trust others . siblings who perceived their abilities to communicate with parents and adults about the diagnosis as poor were somewhat less likely to report negative consequences related to alcohol use and abuse . one possible explanation for this might be that well siblings ' tendency toward social withdrawal due to communication problems in childhood may mean that they do not engage in social activities that create opportunities for social drinking as frequently as others in their age . siblings who had a more negative perception of themselves during the experience were more likely to have emotional and behavioral problems of clinical significance . this is consistent with the childhood adjustment literature ; such that the more problematic and negative the experience was for the individual , the more the individual was at risk for emotional and behavioral problems [ 3335 ] . additionally , specific items on the interpersonal subscale assess parents ' relationship with the well sibling and parents ' and other adults ' preoccupations with the ill sibling . high scores on this subscale , therefore , are indicative of parents ' inability to meet the needs of the well sibling and failure to acknowledge their parental obligation to the well sibling despite having an ill sibling at home . these variables should be targets of intervention to reduce maladjustment and ineffective coping in well siblings . the qualitative data gathered from siblings provided some insight on well siblings ' adjustment in late adolescence . specifically , the data suggest a high level of positive outcomes for siblings who grew up with an ill sibling . the development of characteristics such as appreciation of life , cautiousness about health , and maturity may act as protective variables for late adolescents in college who may be exposed to several opportunities for engaging in health risk behaviors . empathy and compassion may also be helpful to well siblings as they engage in meaningful friendships and romantic relationships as well as when they transition to adulthood , in a number of important arenas . although participants reported negative experiences related to their siblings ' illness , very few stated that they felt any lasting effect that continued on to college . additionally , the majority of participants reported adapting to their family situation with no lasting hardship . this suggests that in spite of the potential for trauma from the experience , it may not necessarily result in posttraumatic stress . however , this is by no means uniform ; adjustment may vary greatly between families , siblings , or even the same sibling , depending on life experiences at that particular circumstance . participants who did report lasting effects were generally participants who lost a sibling to a terminal illness . while many participants stated that the change in their family dynamic was positive , others felt that the family became more distant or that the stress of medical visits and other illness - related appointments and events placed a large strain on the family system . the participants in this study already proved that they were somewhat resilient based on their enrollment in college . it is possible that if a community sample was used , different results would be obtained and should be considered in future studies . the retrospective measurement of siblings ' feelings and concerns regarding their siblings ' diagnosis was another limitation of the study ( i.e. , retrospective responding compromises the accuracy of the responses ) . however , future studies are likely to use retrospective measurement due to the challenges of longitudinal research . in addition , the research questions of interest include how an older adolescent / young adult looks back reflectively on how earlier experiences have shaped current experiences and perceptions . a third limitation of the study was the use of the pas as a measure of psychological functioning . the pas is very brief , focuses more on psychopathology , and only gives the probability of a specific domain being problematic . a measure of both positive and negative psychological functionings may have been more suitable for this study . future studies should measure positive and negative aspects of psychological functioning and also health - related quality of life . future studies should also include a larger sample size with control group of late adolescent participants with which to compare standard measures of psychological functioning . this study provides qualitative evidence that several positive outcomes are related to having a sibling with a chronic illness , including greater health literacy awareness and higher levels of family cohesion . however , for a subset of participants ( especially those whose sibling had a terminal illness ) , a number of negative outcomes emerged , including feelings of regret and fear and a breakdown in family communication and bonding . the present study also provides quantitative evidence for some lingering negative effects of growing up with an ill sibling on well siblings ' late adolescent functioning during transition to adulthood . specifically , well siblings ' inability to communicate with parents and adults about their ill - siblings ' diagnosis may have negative effects on their later social functioning , including their ability to be comfortable and trusting in social relationships . the results of the study also suggest that parents ' ability to manage the needs of their well children is crucial to their children 's resilience . specifically , if parents focus their attention on the ill sibling at the expense of the well sibling , a higher probability exists that the well sibling would be susceptible to behavioral and emotional problems in late adolescence . further research should explore how service providers may be able to increase protective factors and ameliorate risk factors , especially for families that may be at higher risk for negative adjustment outcomes . | the purpose of this study was to explore the continuing impact of growing up with an ill sibling on well siblings ' late adolescent functioning .
forty late adolescents ( mage = 18.78 , sd = 0.83 ) , who identified themselves as growing up with an ill sibling , completed a semistructured interview , demographic questionnaire , personality assessment screener , and my feelings and concerns sibling questionnaire .
participants reported clinically significant problems on some pas scales , and gender differences were found for acting out and alienation .
significant relationships were reported for communication and social withdrawal and alienation .
both positive and negative themes about the experience were elicited from the responses in the semistructured interview .
this study provides evidence for some lingering negative effects of growing up with an ill sibling on well siblings ' late adolescent functioning .
additionally , evidence for siblings ' development of positive characteristics that may act as protective variables as they face the stressors of late adolescence was also highlighted . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
euthanized mice by local iucac guidelines pin each limb on styrofoam surface and rinse abdominal surface with 70% ethanol . make one cut at the distal esophagus and a second cut at the distal large intestine and remove the gi tract from stomach to anus en bloc , carefully cutting ligaments at the duodenum and cecum using small scissors . to separate stomach , small intestine , and large intestine , cut at pylorus and ileocecal junction . rinse the lumen of each part of the gi tract with pbs using a 5ml syringe attached to a feeding needle ( older mice ) or blunt needle ( younger mice ) . cut two pieces for each part of the gi tract , one for whole mount visualization and one for cryosection . place the entire piece of each part of gi tract into a 1.5 cm microfuge tube filled with 1.0 ml of ice cold acetone and fix samples for at least 1 hour at 4c . carefully scrape off mucosal layer with a scalpel by holding one end with a pair of tweezers , leaving the muscularis . block and incubation steps can be done in one well of a 24-well plate or in a 1.5ml microfuge tube . block with 0.5 ml of blocking buffer ( 5% goat serum , 0.1% triton x-100 in pbs for 1 hour at 4 c ) . we have successfully used ack-2 rat anti - kit , rabbit anti - ano1 , and rabbit anti - pgp9.5 antibodies for whole mount immunostaining . incubate with secondary antibody diluted in blocking buffer for 2 hours at room temperature on rotator . we typically use alexa fluor 488 anti - rabbit and alexa fluor 594 anti - rat antibodies . mount on slide , serosal side on top to ensure the orientation of tissue during microscopy . this orientation will ensure the serosal side to face the coverslip and be encountered first as one focuses into the slide on a microscope . sometimes , we briefly examine the tissue under fluorescence microscopy to properly orient . under fluorescence microscopy to visualize kit or ano1 , one should encounter the icc - my network first from the side of the coverslip . add 50 l of hard set mounting media ( we use prolong gold ) and gently place cover slip on top of tissue . allow setting of mounting overnight at room temperature and store slides at -80 c . for microscopic examination , we used a wide - field microscope with a z - drive . we used either a 20x air objective ( na 0.75 , plan apochromat ) or 60x oil objective ( na 1.4 , plan apochromat ) and took 2 m or 1 m z - sections that span the entire muscularis . place each part of the gi tract , serosal side down , flat on filter paper . place tissue on filter paper in 4% paraformaldehyde ( pfa ) in pbs ( we dilute 32% paraformaldehyde in sealed vial into pbs right before use ) for 2 hours at room temperature . fixation can be done in a 6- or 12-well plate depending on size of tissue , or in a 1.5 ml microfuge tube . if done in plate , parafilm to minimize pfa exposure . longer fixation times may result in antigen masking , particularly for the ack-2 anti - kit antibody . when examining mice with different genotypes or with different treatments , it may be useful to mount tissue from different mice onto same cryomold to ensure parallel processing . freeze on dry ice and place in -80 c freezer for storage . cut 10 m cryosections onto slide and store at -80 c freezer . block with 150 l of blocking buffer ( same as for whole mount immunostaining ) for 1 hour . aspirate blocking buffer and add 150 l primary antibody diluted in blocking buffer and incubate overnight at 4 c in a humidified chamber . we typically use alexa 488 anti - rabbit and alexa 594 anti - rat antibodies . wash for 15 min 3x with pbs . add a drop of hard set mounting media with dapi ( we use prolong gold ) and place # 1.5 coverslip . acquire triple fluorescence images using dapi , fitc , and texas red filter sets . here stain of a sucrose protected fixed frozen section used for immunofluorescence shows some artifacts compared to a reference paraffin section ( figure 2 ) , where the yellow line demarks the myenteric plexus between the circular and longitudinal muscle layers and the green line demarks the border between the mucosa and muscularlis . examining the cryosection , from the serosa to mucosa , you first encounter the thinner longitudinal muscle ( lm ) layer followed by the thicker circular muscle ( cm ) layer . the section should be in parallel with the longitudinal muscle so the nuclei of lm should be somewhat elongated while the nuclei of the cm should be small and round . between the lm and cm the myenteric icc network ( icc - my ) surround the myenteric plexus and are made of multipolar cells . within the longitudinal muscle layer , there are rare intramuscular iccs ( icc - ims ) which are bipolar cells running in parallel with the muscle . within the circular muscle layer , there are abundant icc - ims which are also bipolar cells that run parallel with the circular muscle and are cross - sectioned with this cut . at the junction of the muscularis and submucosa , the submucosal icc network consisting of icc - smps is a flat network of multipolar iccs . examining the whole mount kit immunostaining of the large intestine , one should expect to encounter the icc - my network first , and then the icc - im of the cm , and icc - smp networks , as one focuses from the coverslip ( figure . 3 ) . volumetric measure of icc networks using 3d reconstruction of whole - mount images have been described . using wide - field microscope , there is significant out - of - plane fluorescence in the raw images which can be removed after deconvolution ( figure 3 ) . ano1 is another marker of icc s and co - immunostaining of kit and ano1 shows complete overlap of icc staining ( figure 4 ) . a ) representative h&e staining of the mouse large intestine of cryosections prepared as described and reference paraffin embedded section showing some shrinkage artifacts of cryosections . the yellow line demarks the myenteric plexus and the blue line separates the mucosa from the muscularis . b ) representative kit ( red , ack-2 ) and pgp9.5 ( green ) double immunofluorescence with dapi ( blue ) counterstaining of the mouse large intestine . representative kit ( red , ack-2 ) whole - mount immunofluorescence of the same field at three focal planes , the icc - my plane at the lm / cm border , the icc - im plane in the cm , and the icc - smp plane at the cm / submucosa border . representative double - immunofluorescence of ano1 ( green ) and kit ( red , ack-2 ) in large intestine . euthanized mice by local iucac guidelines pin each limb on styrofoam surface and rinse abdominal surface with 70% ethanol . make one cut at the distal esophagus and a second cut at the distal large intestine and remove the gi tract from stomach to anus en bloc , carefully cutting ligaments at the duodenum and cecum using small scissors . to separate stomach , small intestine , and large intestine , cut at pylorus and ileocecal junction . rinse the lumen of each part of the gi tract with pbs using a 5ml syringe attached to a feeding needle ( older mice ) or blunt needle ( younger mice ) . cut two pieces for each part of the gi tract , one for whole mount visualization and one for cryosection . place the entire piece of each part of gi tract into a 1.5 cm microfuge tube filled with 1.0 ml of ice cold acetone and fix samples for at least 1 hour at 4c . carefully scrape off mucosal layer with a scalpel by holding one end with a pair of tweezers , leaving the muscularis . block and incubation steps can be done in one well of a 24-well plate or in a 1.5ml microfuge tube . block with 0.5 ml of blocking buffer ( 5% goat serum , 0.1% triton x-100 in pbs for 1 hour at 4 c ) . we have successfully used ack-2 rat anti - kit , rabbit anti - ano1 , and rabbit anti - pgp9.5 antibodies for whole mount immunostaining . incubate with secondary antibody diluted in blocking buffer for 2 hours at room temperature on rotator . we typically use alexa fluor 488 anti - rabbit and alexa fluor 594 anti - rat antibodies . mount on slide , serosal side on top to ensure the orientation of tissue during microscopy . this orientation will ensure the serosal side to face the coverslip and be encountered first as one focuses into the slide on a microscope . sometimes , we briefly examine the tissue under fluorescence microscopy to properly orient . under fluorescence microscopy to visualize kit or ano1 , one should encounter the icc - my network first from the side of the coverslip . add 50 l of hard set mounting media ( we use prolong gold ) and gently place cover slip on top of tissue . for microscopic examination , we used a wide - field microscope with a z - drive . we used either a 20x air objective ( na 0.75 , plan apochromat ) or 60x oil objective ( na 1.4 , plan apochromat ) and took 2 m or 1 m z - sections that span the entire muscularis . place each part of the gi tract , serosal side down , flat on filter paper . cut filter paper around the tissue . place tissue on filter paper in 4% paraformaldehyde ( pfa ) in pbs ( we dilute 32% paraformaldehyde in sealed vial into pbs right before use ) for 2 hours at room temperature . fixation can be done in a 6- or 12-well plate depending on size of tissue , or in a 1.5 ml microfuge tube . if done in plate , parafilm to minimize pfa exposure . longer fixation times may result in antigen masking , particularly for the ack-2 anti - kit antibody . remove tissue from pfa and place in 30% sucrose in pbs . allow tissue to sink when examining mice with different genotypes or with different treatments , it may be useful to mount tissue from different mice onto same cryomold to ensure parallel processing . freeze on dry ice and place in -80 c freezer for storage . cut 10 m cryosections onto slide and store at -80 c freezer . first equilibrate slide at room temperature for 15 minutes . then draw circle around tissue using pap pen . block with 150 l of blocking buffer ( same as for whole mount immunostaining ) for 1 hour . aspirate blocking buffer and add 150 l primary antibody diluted in blocking buffer and incubate overnight at 4 c in a humidified chamber . we typically use alexa 488 anti - rabbit and alexa 594 anti - rat antibodies . add a drop of hard set mounting media with dapi ( we use prolong gold ) and place # 1.5 coverslip . place slide at room temperature overnight to set and store in -80 c freezer . h&e stain of a sucrose protected fixed frozen section used for immunofluorescence shows some artifacts compared to a reference paraffin section ( figure 2 ) , where the yellow line demarks the myenteric plexus between the circular and longitudinal muscle layers and the green line demarks the border between the mucosa and muscularlis . examining the cryosection , from the serosa to mucosa , you first encounter the thinner longitudinal muscle ( lm ) layer followed by the thicker circular muscle ( cm ) layer . the section should be in parallel with the longitudinal muscle so the nuclei of lm should be somewhat elongated while the nuclei of the cm should be small and round . between the lm and cm are myenteric plexi made of neurons that stain with pgp9.5 . the myenteric icc network ( icc - my ) surround the myenteric plexus and are made of multipolar cells . within the longitudinal muscle layer , there are rare intramuscular iccs ( icc - ims ) which are bipolar cells running in parallel with the muscle . within the circular muscle layer , there are abundant icc - ims which are also bipolar cells that run parallel with the circular muscle and are cross - sectioned with this cut . at the junction of the muscularis and submucosa , the submucosal icc network consisting of icc - smps is a flat network of multipolar iccs . examining the whole mount kit immunostaining of the large intestine , one should expect to encounter the icc - my network first , and then the icc - im of the cm , and icc - smp networks , as one focuses from the coverslip ( figure . 3 ) . volumetric measure of icc networks using 3d reconstruction of whole - mount images have been described . using wide - field microscope , there is significant out - of - plane fluorescence in the raw images which can be removed after deconvolution ( figure 3 ) . ano1 is another marker of icc s and co - immunostaining of kit and ano1 shows complete overlap of icc staining ( figure 4 ) . a ) representative h&e staining of the mouse large intestine of cryosections prepared as described and reference paraffin embedded section showing some shrinkage artifacts of cryosections . the yellow line demarks the myenteric plexus and the blue line separates the mucosa from the muscularis . b ) representative kit ( red , ack-2 ) and pgp9.5 ( green ) double immunofluorescence with dapi ( blue ) counterstaining of the mouse large intestine . ack-2 ) whole - mount immunofluorescence of the same field at three focal planes , the icc - my plane at the lm / cm border , the icc - im plane in the cm , and the icc - smp plane at the cm / submucosa border . representative double - immunofluorescence of ano1 ( green ) and kit ( red , ack-2 ) in large intestine . interstitial cells of cajal were initially characterized by santiago ramn y cajal exactly one century ago using methylene blue and silver chromate stains of gastrointestinal muscularis . cajal initially thought icc s were neurons based on their processes that are reminiscent of axons and dendrites . over many ensuing years , study of icc biology has been limited by lack of specific markers until the discovery of that kit not only is expressed in icc , but is also required for their development . since then , kit immunofluorescence has been widely used in the study of icc biology and has also led to the appreciation of icc in other contractile organs such as the urinary bladder . there have been numerous publications over the past 15 years using immunofluorescence to identify icc using various fixation and mounting techniques . in our hands " fixed - frozen " cryosections that have been prefixed with paraformaldehyde and sucrose protected works best compared to acetone or paraformaldehyde post - fixation after cryosectioning . for whole mounts , we have found that acetone fixation followed by mucosal dissection gives the most robust results . historically , the ack-2 has been the kit antibody of choice for mouse icc identification . it is a rat monoclonal that recognizes the extracellular domain and it is also a blocking antibody that causes ileus when given to live mice . however , the ack-2 epitope is slowly destroyed by paraformaldehyde fixation and is not rescued by standard antigen retrieval methods . compared to fixed frozen blocks and sections , paraffin embedded blocks and sections preserves morphology better and is more amenable to long term archiving ( figure 4 ) . we have characterized that d13a2 , a new rabbit monoclonal antibody , works exceptionally well in both fixed - frozen and paraffin sections of the gi tract . kit is expressed in several other cell types , including melanocytes , hematopoietic cells , germ cells , and in particular mast cells which are also found in the gi tract . luckily , most mast cells are found is the mucosa and not in the muscularis . double staining with rabbit anti - ano1 and rat anti - kit can eliminate non - specific staining of each antibody ( figure 4 ) . it is also worth noting that the relative expression of kit and ano1 between subclasses of icc seems different . for example , in the small intestine , kit staining is much more intense in myenteric icc compared to icc of the deep muscle plexus whereas ano1 staining is comparable . | the interstitial cells of cajal ( icc ) are mesenchymal derived " pacemaker cells " of the gastrointestinal ( gi ) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1 .
different subtypes of icc form distinct networks in the muscularis of the gi tract 2,3 . loss or injury to these networks
is associated with a number of motility disorders4 .
icc cells express the kit receptor tyrosine kinase on the plasma membrane and kit immunostaining has been used for the past 15 years to label the icc network5,6 .
importantly , normal kit activity is required for icc development5,6 .
neoplastic transformation of icc cells results in gastrointestinal stromal tumor ( gist ) , that frequently harbor gain - of - function kit mutations7,8 .
we recently showed that etv1 is a lineage - specific survival factor expressed in the icc / gist lineage and is a master transcriptional regulator required for both normal icc network formation and for of gist tumorigenesis9 .
we further demonstrate that it cooperates with activating kit mutations in tumorigenesis . here
, we describe methods for visualization of icc networks in mice , largely based on previously published protocols10,11 .
more recently , the chloride channel anoctamin 1 ( ano1 ) has also been characterized as a specific membrane marker of icc11,12 . because of their plasma membrane localization
, immunofluorescence of both proteins can be used to visualize the icc networks . here , we describe visualization of the icc networks by fixed - frozen cyrosections and whole mount preparations . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
exposure generally occurs through dental amalgams ,
vaccines , and/or fish consumption ( 1 ) ; this study will
focus on the latter . chronic mercury exposure in adults
has been linked to both neurologic damage and
cardiovascular disease ( 2 ) . mercury exposure is of
special concern in women of childbearing age as
mercury is a particularly potent neurotoxin during fetal
development ( 3 ) . in response to several studies showing these
adverse effects of mercury exposure , programs were
launched in both the united states and canada to
provide safe eating guidelines for locally caught and
commercial store - bought fish . vermont is a state that
has mobilized such educational efforts in the form of
informational brochures and posters , one of which is specifically targeted towards both pregnant and nonpregnant
women of childbearing age . while the content of vermont s fish advisory is
presented simply and clearly , there is little data to
determine whether the advisory is actually reaching
vermonters . here , we report the results of a crosssectional
analysis of 166 vermonters aimed to assess
fish consumption practices , estimate overall awareness
of vermont s safe - eating guidelines , and evaluate the
effectiveness of these educational efforts . surveying was conducted in person from september
through december 2006 at five different locations in
chittenden county , vermont ( table 1 ) . survey locations
were selected specifically to achieve a geographically
and socioeconomically diverse sample population . this
population is probably quite representative of
chittenden county as a whole , but perhaps less
representative of the entire state of vermont . in total ,
166 randomly selected men and women aged 18 and older were approached in person at the survey sites and
asked to complete this retrospective survey that was
developed by the authors specifically for this study . respondents completed the survey privately at a table
separate from the staff conducting the survey . the
survey gathered demographic information , detailed fish
consumption practices in an average month , evaluated
awareness of vermont s safe eating guidelines , and
assessed fish - consumption changes ( if any ) participants
may have enacted in response to these guidelines . one
meal of fish was defined as an uncooked 8 oz . fillet or a
6 oz . can of tuna . study methods were reviewed and
accepted by the university of vermont institutional
review board . surveys were excluded if the
demographic data was not completed or specific fish
consumption practice questions were not completed or
the answers were unclear . survey participants were stratified by gender , age ,
level of education , ethnic background , and whether or
not participants had children less than six years of age . women of childbearing age ( 18 44 years old ) were
also considered separately . chi - squared ( 2 ) analysis
was used to look for statistically significant differences
in fish consumption practices between different groups
of study subjects . data presented in this table has not been altered and includes
surveys with some responses left blank . a ) burlington public library ,
burlington , vermont ; b ) university pediatrics , williston , vermont ; c )
department of children and families , burlington , vermont ; d )
women , infants , and children ( wic ) , burlington , vermont ; e )
community health center , burlington , vermont ; f ) species high in
mercury defined as swordfish , shark , king mackerel , or tilefish . as shown in table 1 , 64% of respondents were female
( 75% of whom were of childbearing age ) . sixty - one
percent of respondents had at least some post - secondary
education and 85% of respondents were caucasian in
ethnicity . canned tuna was consumed most frequently ,
with 68% of respondents eating at least one meal per
month . nineteen percent reported eating vermont
caught fish at least once in an average month . furthermore , individuals high in one category of fish
consumption tended to be high in multiple categories . although 79% of respondents were aware of the
adverse health effects of mercury in fish prior to the
survey , only 48% were aware of specific vermont safeeating
guidelines . of note , in the subset of participants
that were both aware of the guidelines and ate fish , 67%
reported at least some change in their fish - consumption
practices in response to the guidelines . 2 analysis showed a statistically significant
association ( p<0.025 ) between high education level ( at
least some post - secondary education ) and reduced
monthly tuna consumption ( two or fewer meals per
month ) . a difference in awareness of safe - eating
guidelines was seen between subjects interviewed at the
burlington library compared to those interviewed at
the department of children and families ( p<0.05 ) . in
women of childbearing age , there was an association
between reduced level of canned tuna consumption and with both having seen a poster / brochure ( p<0.05 ) and
having specific knowledge of vermont s safe - eating
guidelines ( p<0.01 ) . there was no association between
fish consumption practices and whether or not the
respondent had children less than six years of age . previous studies assessing fish consumption patterns
in other states using a similar questionnaire have also
measured hair mercury levels in a subset of subjects to
estimate actual mercury exposure ( 3,4 ) . these studies
have found a highly significant correlation between
reported fish consumption and actual mercury levels in
the body . these considerations suggest that the results
of this study accurately reflect mercury exposure
through fish consumption in this study population . these data suggest that vermont is successfully
educating the public with regard to mercury exposure
through fish consumption ; 48% of those surveyed were
aware of specific safe - eating guidelines . compared to
other states this awareness is rather high , for example
similar studies in california , new mexico , and north carolina have reported awareness rates as low as 9% ,
9% , and 10% respectively ( 3 ) . a pooled analysis of
guideline awareness data in eight states bordering the
great lakes revealed ~50% awareness ( 5 ) , while
wisconsin reports a 77% awareness ( 4,5 ) . sixty - seven
percent of those who had seen advisory
posters / brochures reported changing their fish
consumption practices as a result . a last observation
was that vermonters of lower education / income may
not be as aware of the guidelines as vermonters as a
whole . this is evidenced , for example , by higher tuna
consumption in the lower education level subset
( p<0.05 ) , though other explanations for this trend a
certainly possible . study limitations included a large proportion of
respondents : ( a ) with post - secondary education level ,
( b ) reporting caucasian ethnicity , and ( c ) living in
vermont s largest county . additionally , the number of
respondents in this study ( 166 ) is rather low , and a more
extensive survey might reveal certain associations that
this study was not able to identify . it would also be
beneficial to assess advisory awareness throughout
vermont , focusing on more rural areas and populations
with less educational attainment to see if awareness
levels and fish consumption practices are similar . this study suggests that current efforts to educate
vermonters about adverse health effects of mercury in
fish are successful . additionally , they might be further
improved with additional emphasis on limits of fish
consumption across all types of mercury - containing
fish , not just those in individual categories . based on
these findings , the vermont department of health has modified its public education materials . although not
addressed by this study , a final recommendation , based
on recent work by mahafferty et al . ( 6 ) , would be to
include information regarding omega-3 fatty acid
content of different fish in addition to mercury levels . this will help the public make informed decisions by
weighing the health benefits ( omega-3 fatty acid
content ) versus the risks ( mercury levels ) in different
species of fish . | exposure to mercury from environmental sources , such as fish consumption , poses
potential health risks to the public .
the state of vermont has developed educational brochures and
posters displaying safe fish consumption guidelines in order to educate the public regarding mercury
exposure through fish . in this study ,
a group of medical students from the university of vermont
college of medicine , in partnership with the vermont department of health , conducted a study in
chittenden county , vermont in order to assess both fish consumption practices and overall awareness
of such safe eating guidelines and mercury advisories .
a total of 166 vermont residents were surveyed
during a six week period .
the results of this survey suggest that in chittenden county of vermont , these
educational efforts are markedly successful , with 48% of respondents being specifically aware of the
safe eating guidelines .
further , these results suggest that 61% of those respondents that reported low
monthly canned tuna consumption had a decreased their consumption in response to the safe eating
guidelines .
last , a series of specific , yet widely applicable recommendations are presented for future
public educational efforts regarding mercury exposure through fish consumption . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
robust data from many placebo - controlled trials have also demonstrated the efficacy of bisphosphonates in fracture risk reduction with a vertebral fracture risk reduction ranging from 40% to 70% and relative hip fracture reduction of 40% to 70% over 3 to 5 years of treatment . bisphosphonates have been the most widely used agent in south korea ( about 80% ) for the treatment of osteoporosis . however , some unexpected possible adverse effects have been reported , including osteonecrosis of the jaw and atypical femoral fractures . because bisphosphonates are incorporated into the skeleton and continue to exert an anti - resorptive effect for a period of time after the discontinuation of drugs , the concept of a drug holiday has emerged , whereby the risk of adverse effects might be decreased while the patient still benefits from anti - fracture efficacy . however , there is considerable controversy regarding the optimal selection of candidates for the drug holiday and monitoring during a drug holiday , both of which should be based on individual assessments of risks and benefits . in addition , responsiveness to anti - resorptive effects differs between the asian population and the white population , possibly owing to genetic , dietary , and environmental differences . hence , we propose a potential algorithm to guide physicians in south korea , despite no available clear recommendations regarding the length of use and drug holidays based on the current research . these statements can not and should not be used to govern health policy decisions about reimbursement or the availability of services . furthermore , a. a drug holiday should be viewed as a temporary , not permanent , suspension of active therapy.b . it should be remembered that discontinuing a bisphosphonate may not necessarily be a " holiday " from treatment because persistence of the anti - resorptive effect is expected for an undefined period of time.c . selection of candidates for the drug holiday and monitoring during a drug holiday needs to be tailored to the individual patients . a. a drug holiday should be viewed as a temporary , not permanent , suspension of active therapy . b. it should be remembered that discontinuing a bisphosphonate may not necessarily be a " holiday " from treatment because persistence of the anti - resorptive effect is expected for an undefined period of time . c. selection of candidates for the drug holiday and monitoring during a drug holiday it is unusual to contemplate a drug holiday in the treatment of most chronic diseases because with most therapies , beneficial drug effects rapidly diminish with discontinuation . however , the long skeletal residence time of bisphosphonates and concern about the risks of rare adverse events with long - term therapy raise the possibility that bisphosphonate therapy may be interrupted for a " drug holiday , " during which anti - fracture benefit might persist for a period of time while the potential risks are minimized . the first question is whether stopping bisphosphonates is safe in regard to maintaining adequate prevention against developing osteoporotic fractures . indeed , bisphosphonates are deposited in bones for at least 10 years , and when a bone containing bisphosphonate is resorbed , the bisphosphonate recirculates locally and systemically and is able to bind again to bone surfaces . bone resorption continues to be inhibited over time , and the anti - resorptive effect persists after the drug has been stopped . in assessing the maintenance of adequate prevention against developing osteoporotic fractures after stopping bisphosphonates , it would be ideal to have clinical trial data comparing fracture risk between patients who continue or stop therapy ; unfortunately , only three prospective studies have addressed this issue ( fig . long - term extension trial ( flex ) ( table 1 ) , subjects on alendronate for more than 5 years were randomized to either continue alendronate therapy for a total of 10 years or discontinue therapy after 5 years . although the subject number was small , those continuing alendronate for 10 years had fewer clinical vertebral fractures than the subjects receiving the drug for only 5 years ( 5.3% vs. 2.4% , respectively ) . there was no difference between the groups for morphometric vertebral or non - vertebral fractures . in the health outcomes and reduced incidence with zoledronic acid once yearly ( horizon)-pivotal fracture trial ( pft ; horizon - pft ) ( table 1 ) , subjects on zoledronic acid for 3 years were randomized to stop therapy or to continue on yearly zoledronic acid for 3 additional years . treatment for 3 additional years resulted in a 52% lower risk of morphometric vertebral fracture , compared with treatment for 3 years followed by placebo for the next 3 years ( fracture rates 3.0% vs. 6.2% , respectively ) . the risks of other fractures - including clinical or symptomatic vertebral fractures - did not differ between the groups . in the vertebral efficacy with risedronate therapy - multinational ( vert - mn ) , a small number of patients were given risedronate or placebo for 3 years and then followed for an additional one year after discontinuation ( table 1 ) ; the morphometric vertebral fracture incidence remained 46% lower in the former risedronate group , as compared with the former placebo group ( 6.5% vs. 11.6% , respectively ) . however , there was no group of patients continuing on risedronate , hence it was not possible to compare the fracture risk of discontinuing therapy with continuing therapy ( table 1 ) . the second question is whether stopping bisphosphonates reduce the risk of complications . in assessing the reduction of risk of complications after stopping bisphosphonates , it would be helpful to have clinical trials comparing the rates of adverse and serious adverse experiences in subjects randomized to continuing or discontinuing bisphosphonate therapy . it stands to reason that if rare undesirable medical occurrences are causally related to bisphosphonate use , then the risk should diminish over time as the bisphosphonate is eliminated from the bone . however , apart from the swedish study suggesting that the risk of atypical femoral fractures decreases following discontinuation of oral bisphosphonate , there are no data to answer this question . those data suggest the following after bisphosphonate exposure of 3 to 5 years in postmenopausal women with osteoporosis : the protection from fractures persists for an unknown interval of time in selected patients when therapy is withdrawn ; that the protection wanes within 3 to 5 years of discontinuation ; and that the risk of atypical femoral fractures increases with the duration of therapy but may decrease upon withdrawal of treatment . there are limited data to guide decision - making about the initiation and termination of " drug holidays . " currently , an observational study is in process to compare the effects on bone quantity and quality during a 2-year drug holiday after 2 years of bisphosphonate use ( nct01406613 ) . however , until these results are available and randomized controlled clinical trials are published , physicians are asked to use their best clinical judgment to determine who qualifies for a drug holiday . in the absence of clear evidence , any recommendations for the selection of candidates for the drug holiday and monitoring during a drug holiday can only be " expert opinion " and should be individualized according to a consideration of all available clinical information . furthermore , a drug holiday should be viewed as a temporary , not permanent , suspension of active therapy . it should be remembered that discontinuing a bisphosphonate might not necessarily be a " holiday " from treatment , because persistence of the anti - resorptive effect is expected for an undefined period of time . i. consider a drug holiday after 5 years of alendronate and risedronate treatment , and after 3 years of zoledronic acid , in individuals without high risk.ii . 1 ) t - score at any site still -2.5 after bisphosphonate therapy ( 5 years for alendronate and risedronate , and 3 years for zoledronic acid).2 ) previous fracture of the hip or spine.3 ) high risk of fracture because of secondary osteoporosis from chronic diseases or medication . factors guiding the determination of a drug holiday are the variable anti - resorptive potency and binding affinity of each bisphosphonate , as well as a demonstration of compliance with the therapy . i. consider a drug holiday after 5 years of alendronate and risedronate treatment , and after 3 years of zoledronic acid , in individuals without high risk.ii . 1 ) t - score at any site still -2.5 after bisphosphonate therapy ( 5 years for alendronate and risedronate , and 3 years for zoledronic acid).2 ) previous fracture of the hip or spine.3 ) high risk of fracture because of secondary osteoporosis from chronic diseases or medication . factors guiding the determination of a drug holiday are the variable anti - resorptive potency and binding affinity of each bisphosphonate , as well as a demonstration of compliance with the therapy . i. consider a drug holiday after 5 years of alendronate and risedronate treatment , and after 3 years of zoledronic acid , in individuals without high risk . 1 ) t - score at any site still -2.5 after bisphosphonate therapy ( 5 years for alendronate and risedronate , and 3 years for zoledronic acid).2 ) previous fracture of the hip or spine.3 ) high risk of fracture because of secondary osteoporosis from chronic diseases or medication . 1 ) t - score at any site still -2.5 after bisphosphonate therapy ( 5 years for alendronate and risedronate , and 3 years for zoledronic acid ) . 3 ) high risk of fracture because of secondary osteoporosis from chronic diseases or medication . factors guiding the determination of a drug holiday are the variable anti - resorptive potency and binding affinity of each bisphosphonate , as well as a demonstration of compliance with the therapy . in regard to determination of the optimal duration for bisphosphonate therapy , there are only 3 prospective studies containing information about fracture risk upon discontinuing bisphosphonate therapy , such as alendronate , risedronate , and zoledronic acid . hence , we suggest that the drug holiday may be considered after 5 years from alendronate and risedronate and after 3 years from zoledronic acid . in regard to selection of the appropriate candidates for drug holiday , there have been some suggestions ( table 2 ) . one study proposed to stratify patients based on their known or potential risk of fracture . high - risk patients include patients with prior history of fractures , low t - scores , and secondary osteoporosis because of the use of chronic glucocorticoids , or from diseases such as hyperthyroidism , hyperparathyroidism , rheumatoid arthritis , or other disease states than can cause severe immobility , for instance , multiple sclerosis . in those kinds of patients , it was recommended continuing bisphosphonate therapy for up to 10 years , and then a drug holiday could be considered ( table 2 ) . if a holiday from the bisphosphonate in high - risk patients is considered after 10 years , interval treatment with other agents , such as parathyroid hormone or selective estrogen receptor modulator , may be warranted . in moderate - risk patients , they suggested drug holiday after 5 to 10 years and in low - risk patients after 3 to 5 years of bisphosphonate therapy . bisphosphonates should then be resumed if bone mineral density ( bmd ) decreases or the patient has a fracture . the duration of a drug holiday is unknown , but the authors propose 1 to 2 years in high - risk patients , 3 to 5 years in moderate - risk , and indefinitely in low - risk patients . this proposal was also cited in the american association of clinical endocrinologists ( aace ) guideline . food and drug administration ( fda ) issued an advisory to provide additional guidance to physicians . the fda analyzed the 3 long - term extension trials : flex , horizon - pft , and vert - mn . although the primary endpoint in the extension trials was bmd , the fda analyses included both bmd and fracture outcomes ( vertebral and non - vertebral ) . in their review of flex , the panel reported that the rates of vertebral and non - vertebral osteoporotic fractures were similar whether participants continued to receive alendronate for up to 10 years or were switched to a placebo . also , the panel noted that fracture rates appear to be constant when all data on vertebral and non - vertebral osteoporotic fractures with long - term therapy are pooled from the 3 extension trials . for example , in patients who received continuous bisphosphonate treatment for at least 6 years , the fracture rates ranged from 9.3% to 10.6% , whereas the rate for patients switched to a placebo was 8.0% to 8.8% . these data question the benefit of extended therapy with bisphosphonates after 5 years of fracture prevention . therefore , the fda recommended periodic assessment of a patient 's need for continued therapy , taking into account their individual risks and benefits and patient preference to optimize the efficacy of bisphosphonates in reducing fracture risk . in this regard , patients at low risk for fracture ( e.g. , younger patients without a fracture history and with a bmd approaching normal ) may qualify for drug holiday after 3 to 5 years , whereas patients at higher risk of fracture ( e.g. , older patients with a history of fracture and a bmd remaining in the osteoporotic range ) may benefit from continued bisphosphonate therapy ( table 2 ) . these recommendations were also suggested by other groups . the response to the fda advisory commented that the fda analysis focused on the composite end point of all fractures , both vertebral and non - vertebral , when the original preplanned analyses of the extension studies separated vertebral and non - vertebral fractures because of " their distinct pathogenesis and different responses to treatment " . according to that analysis , the risk for vertebral fractures was shown to be reduced with continued bisphosphonate therapy beyond 3 to 5 years , whereas evidence was lacking for non - vertebral fractures . data from flex was used to estimate the number needed to treat ( for 5 additional years ) to prevent one clinical vertebral fracture ( in subgroups defined by bmd at the femoral neck and by prevalent vertebral fractures status at entry in flex ) ( table 3 ) . the risk of vertebral fracture is highest and the numbers needed to treat are lowest for patients with a femoral neck t - score -2.5 , which suggests that it may reasonably be expected that these patients would benefit by continuing bisphosphonate therapy . in addition , patients with a preexisting vertebral fracture with a femoral neck t - score -2.0 may also benefit from continuation ( table 2 ) . thus , in general , we consider that the following individuals are at a high risk for fractures and appear to benefit most from continuation of bisphosphonates : those with a t - score at any site still -2.5 after bisphosphonate therapy ( 5 years for alendronate and risedronate , and 3 years for zoledronic acid ) , previous fracture of the hip or spine , or secondary osteoporosis from chronic diseases or medication . alternative therapy ( e.g. , parathyroid hormone or selective estrogen receptor modulator ) may be used for individuals with a high risk . individuals without a high risk for fractures are unlikely to benefit from continued treatment after bisphosphonate therapy for 3 to 5 years . furthermore , there are some considerable factors guiding the determination of drug holiday , such as the variable anti - resorptive potency and binding affinity of each bisphosphonate , and a demonstration of compliance with the therapy . the variable anti - resorptive potency and binding affinity of each bisphosphonate are owing to their unique side chains . zoledronic acid has the highest potency , followed by risedronate , ibandronate , and alendronate . binding affinity is highest for zoledronic acid and decreases in order of magnitude for alendronate , ibandronate , and risedronate , respectively . this may be owing to a greater affinity of alendronate and zoledronic acid to hydroxyapatite , compared with risedronate and ibandronate . the skeletal binding sites for bisphosphonate are nearly unsaturable , thereby leading to a significant accumulation of bisphosphonates whereas release of bisphosphonates may be small , as it partly depends on bone turnover , which is reduced by the use of bisphosphonates . for example , after 10 years of alendronate use at a dose of 10 mg daily ( 70 mg weekly ) , the amount of alendronate released over several months or years would be equivalent to taking one - quarter of the usual dose . in general , zoledronic acid and alendronate maintain a prolonged effect after discontinuation , whereas others , such as risedronate , have a more rapid offset . another factor to consider in the drug holiday is a demonstration of compliance with the therapy . a recent retrospective register study about the residual treatment effect of alendronate and risedronate in swedish clinical practice suggests that the duration of bisphosphonate therapy is significantly inversely associated with the incidence of hospitalized fractures following discontinuation . specifically , during the first 6 months after terminating treatment , the adjusted fracture rates were considerably lower in patients who had been persistent with treatment for more than 12 months , compared with those who had stopped treatment within 1 month ( hazard ratio [ hr]=0.40 ) . it is known that 70% of bisphosphonate users discontinued their prescriptions after 1 year of use in south korea . therefore , the decision to go on drug holiday after 3 to 5 years should be after assurance of continuous use of bisphosphonates during the initial therapy period . a. parameter for monitoring during a drug holiday
i. consider the annual measurement of bmd using dual energy x - ray absorptiometry . b. restart therapy after a drug holiday
i. consider re - treatment if there is a significant decrease in bmd.ii . consider re - treatment if t - score reaches -2.5 or a new osteoporotic fracture occurs . a. parameter for monitoring during a drug holiday
i. consider the annual measurement of bmd using dual energy x - ray absorptiometry . b. restart therapy after a drug holiday
i. consider re - treatment if there is a significant decrease in bmd.ii . consider re - treatment if t - score reaches -2.5 or a new osteoporotic fracture occurs . ii . consider re - treatment if t - score reaches -2.5 or a new osteoporotic fracture occurs . there are no data providing information on how to monitor patients or when to restart therapy after a drug holiday . in the absence of guidance from clinical trials , empiric approaches although the approach has not been studied , bmd and biochemical markers of bone turnover ( btms ) measured after discontinuation may provide information about the persistence of the effect of the retained bisphosphonate . a significant decrease in bmd or a significant increase in btms suggests that the benefits of bisphosphonate therapy may be diminishing and that it may be time to return to active therapy . a recent study , a post hoc analysis of flex participants randomized to placebo after receiving 5 years of oral alendronate therapy , showed that older age and lower total hip bmd at the time of discontinuation were associated with higher rates of clinical fracture during the next 5 years , but neither 1-year changes in bmd nor 1- and 3-year changes in btms levels ( urinary type 1 collagen cross - linked n - telopeptide and bone - specific alkaline phosphatase ) were associated with fracture risk . similarly , most associations between 2- or 3-year changes in bmd and fracture risk were not statistically significant , although there were fewer fractures after the second ( n=70 ) and third ( n=57 ) annual visits included in the analysis . the risk of fracture was elevated among those with greater total hip bone loss after 2 or 3 years of follow - up , but after adjustment for age and baseline bmd at the time of discontinuation , only the 2-year total hip bone loss greater than 3% was significantly associated with fracture risk ( relative hr=1.68 , 95% confidence interval=1.05 - 2.72 ) . neither a 2- or 3-year change in bmd at the femoral neck nor a 3-year change in bmd at the lumbar spine was associated with fracture risk . therefore , more research is needed about the role of serial bone density testing and btms in monitoring fracture risk after therapy cessation , as well as the optimal therapies to use following a drug holiday . another untested approach is to reevaluate the patient after discontinuation , thereby making the decisions to restart therapy based on an updated assessment of fracture risk using algorithms initially developed for untreated individuals . for example , if the patient has a t - score -2.5 , or if the patient has a t - score between -1.0 and -2.5 and a world health organization 's fracture risk assessment estimate of fracture risk that meets treatment guidelines , then consider reinitiating therapy . if , at any time during the drug holiday there is a fracture , restarting therapy ( not necessarily a bisphosphonate ) is advised . monitoring with bmd and btms after discontinuation of alendronate therapy does not appear to improve fracture prediction , however , we propose the annual measurement of bmd and consideration of re - treatment if there is a significant decrease in bmd , a t - score reaches -2.5 , or new osteoporotic fracture occurs . randomized clinical trial evidence is not yet available on who may qualify for a drug holiday , thus many additional issues urgently need epidemiologic , clinical , and economic research in the korea . for example , the following several issues need to be addressed .
more research is needed about the length of time for which bisphosphonate treatments continue to work after they are stopped.does stopping bisphosphonates reduce the risk of complications such as osteonecrosis of the jaw and atypical femur fractures?how can the optimal duration of a drug holiday be determined for other bisphosphonates therapy such as ibandronate or pamidronate?how can we better identify the individuals at high risk for fractures who may benefit from continued bisphosphonate therapy?is there is a need to stratify patients based both on the femoral neck t - score , but not on the lumbar spine t - score and the preexisting vertebral fracture status?what approaches are most effective in treating osteoporosis in individuals with high risk?is there any difference in the optimal duration of a drug holiday according to each bisphosphonate with variable anti - resorptive potency and binding affinity?how can patients be better monitored after a drug holiday?when can therapy be restarted after a drug holiday?more research is needed about the role of serial bmd testing and btms in monitoring fracture risk after a drug holiday , as well as the optimal therapies to use following a drug holiday .
more research is needed about the length of time for which bisphosphonate treatments continue to work after they are stopped . does stopping bisphosphonates reduce the risk of complications such as osteonecrosis of the jaw and atypical femur fractures ? how can the optimal duration of a drug holiday be determined for other bisphosphonates therapy such as ibandronate or pamidronate ? how can we better identify the individuals at high risk for fractures who may benefit from continued bisphosphonate therapy ? is there is a need to stratify patients based both on the femoral neck t - score , but not on the lumbar spine t - score and the preexisting vertebral fracture status ? is there any difference in the optimal duration of a drug holiday according to each bisphosphonate with variable anti - resorptive potency and binding affinity ? how can patients be better monitored after a drug holiday ? when can therapy be restarted after a drug holiday ? more research is needed about the role of serial bmd testing and btms in monitoring fracture risk after a drug holiday , as well as the optimal therapies to use following a drug holiday . a. a drug holiday should be viewed as a temporary , not permanent , suspension of active therapy.b . it should be remembered that discontinuing a bisphosphonate may not necessarily be a " holiday " from treatment because persistence of the anti - resorptive effect is expected for an undefined period of time.c . selection of candidates for the drug holiday and monitoring during a drug holiday needs to be tailored to the individual patients . a. a drug holiday should be viewed as a temporary , not permanent , suspension of active therapy . b. it should be remembered that discontinuing a bisphosphonate may not necessarily be a " holiday " from treatment because persistence of the anti - resorptive effect is expected for an undefined period of time . c. selection of candidates for the drug holiday and monitoring during a drug holiday it is unusual to contemplate a drug holiday in the treatment of most chronic diseases because with most therapies , beneficial drug effects rapidly diminish with discontinuation . however , the long skeletal residence time of bisphosphonates and concern about the risks of rare adverse events with long - term therapy raise the possibility that bisphosphonate therapy may be interrupted for a " drug holiday , " during which anti - fracture benefit might persist for a period of time while the potential risks are minimized . the first question is whether stopping bisphosphonates is safe in regard to maintaining adequate prevention against developing osteoporotic fractures . indeed , bisphosphonates are deposited in bones for at least 10 years , and when a bone containing bisphosphonate is resorbed , the bisphosphonate recirculates locally and systemically and is able to bind again to bone surfaces . bone resorption continues to be inhibited over time , and the anti - resorptive effect persists after the drug has been stopped . in assessing the maintenance of adequate prevention against developing osteoporotic fractures after stopping bisphosphonates , it would be ideal to have clinical trial data comparing fracture risk between patients who continue or stop therapy ; unfortunately , only three prospective studies have addressed this issue ( fig . long - term extension trial ( flex ) ( table 1 ) , subjects on alendronate for more than 5 years were randomized to either continue alendronate therapy for a total of 10 years or discontinue therapy after 5 years . although the subject number was small , those continuing alendronate for 10 years had fewer clinical vertebral fractures than the subjects receiving the drug for only 5 years ( 5.3% vs. 2.4% , respectively ) . there was no difference between the groups for morphometric vertebral or non - vertebral fractures . in the health outcomes and reduced incidence with zoledronic acid once yearly ( horizon)-pivotal fracture trial ( pft ; horizon - pft ) ( table 1 ) , subjects on zoledronic acid for 3 years were randomized to stop therapy or to continue on yearly zoledronic acid for 3 additional years . treatment for 3 additional years resulted in a 52% lower risk of morphometric vertebral fracture , compared with treatment for 3 years followed by placebo for the next 3 years ( fracture rates 3.0% vs. 6.2% , respectively ) . the risks of other fractures - including clinical or symptomatic vertebral fractures - did not differ between the groups . in the vertebral efficacy with risedronate therapy - multinational ( vert - mn ) , a small number of patients were given risedronate or placebo for 3 years and then followed for an additional one year after discontinuation ( table 1 ) ; the morphometric vertebral fracture incidence remained 46% lower in the former risedronate group , as compared with the former placebo group ( 6.5% vs. 11.6% , respectively ) . however , there was no group of patients continuing on risedronate , hence it was not possible to compare the fracture risk of discontinuing therapy with continuing therapy ( table 1 ) . the second question is whether stopping bisphosphonates reduce the risk of complications . in assessing the reduction of risk of complications after stopping bisphosphonates , it would be helpful to have clinical trials comparing the rates of adverse and serious adverse experiences in subjects randomized to continuing or discontinuing bisphosphonate therapy . it stands to reason that if rare undesirable medical occurrences are causally related to bisphosphonate use , then the risk should diminish over time as the bisphosphonate is eliminated from the bone . however , apart from the swedish study suggesting that the risk of atypical femoral fractures decreases following discontinuation of oral bisphosphonate , there are no data to answer this question . those data suggest the following after bisphosphonate exposure of 3 to 5 years in postmenopausal women with osteoporosis : the protection from fractures persists for an unknown interval of time in selected patients when therapy is withdrawn ; that the protection wanes within 3 to 5 years of discontinuation ; and that the risk of atypical femoral fractures increases with the duration of therapy but may decrease upon withdrawal of treatment . there are limited data to guide decision - making about the initiation and termination of " drug holidays . " currently , an observational study is in process to compare the effects on bone quantity and quality during a 2-year drug holiday after 2 years of bisphosphonate use ( nct01406613 ) . however , until these results are available and randomized controlled clinical trials are published , physicians are asked to use their best clinical judgment to determine who qualifies for a drug holiday . in the absence of clear evidence , any recommendations for the selection of candidates for the drug holiday and monitoring during a drug holiday can only be " expert opinion " and should be individualized according to a consideration of all available clinical information . furthermore , a drug holiday should be viewed as a temporary , not permanent , suspension of active therapy . it should be remembered that discontinuing a bisphosphonate might not necessarily be a " holiday " from treatment , because persistence of the anti - resorptive effect is expected for an undefined period of time . i. consider a drug holiday after 5 years of alendronate and risedronate treatment , and after 3 years of zoledronic acid , in individuals without high risk.ii . 1 ) t - score at any site still -2.5 after bisphosphonate therapy ( 5 years for alendronate and risedronate , and 3 years for zoledronic acid).2 ) previous fracture of the hip or spine.3 ) high risk of fracture because of secondary osteoporosis from chronic diseases or medication . factors guiding the determination of a drug holiday are the variable anti - resorptive potency and binding affinity of each bisphosphonate , as well as a demonstration of compliance with the therapy .
a. determination of the duration for bisphosphonate therapy . i. consider a drug holiday after 5 years of alendronate and risedronate treatment , and after 3 years of zoledronic acid , in individuals without high risk.ii . 1 ) t - score at any site still -2.5 after bisphosphonate therapy ( 5 years for alendronate and risedronate , and 3 years for zoledronic acid).2 ) previous fracture of the hip or spine.3 ) high risk of fracture because of secondary osteoporosis from chronic diseases or medication . factors guiding the determination of a drug holiday are the variable anti - resorptive potency and binding affinity of each bisphosphonate , as well as a demonstration of compliance with the therapy . i. consider a drug holiday after 5 years of alendronate and risedronate treatment , and after 3 years of zoledronic acid , in individuals without high risk . 1 ) t - score at any site still -2.5 after bisphosphonate therapy ( 5 years for alendronate and risedronate , and 3 years for zoledronic acid).2 ) previous fracture of the hip or spine.3 ) high risk of fracture because of secondary osteoporosis from chronic diseases or medication . 1 ) t - score at any site still -2.5 after bisphosphonate therapy ( 5 years for alendronate and risedronate , and 3 years for zoledronic acid ) . 3 ) high risk of fracture because of secondary osteoporosis from chronic diseases or medication . factors guiding the determination of a drug holiday are the variable anti - resorptive potency and binding affinity of each bisphosphonate , as well as a demonstration of compliance with the therapy . in regard to determination of the optimal duration for bisphosphonate therapy , there are only 3 prospective studies containing information about fracture risk upon discontinuing bisphosphonate therapy , such as alendronate , risedronate , and zoledronic acid . there is no information about fracture risk upon discontinuing ibandronate therapy . hence , we suggest that the drug holiday may be considered after 5 years from alendronate and risedronate and after 3 years from zoledronic acid . in regard to selection of the appropriate candidates for drug holiday one study proposed to stratify patients based on their known or potential risk of fracture . high - risk patients include patients with prior history of fractures , low t - scores , and secondary osteoporosis because of the use of chronic glucocorticoids , or from diseases such as hyperthyroidism , hyperparathyroidism , rheumatoid arthritis , or other disease states than can cause severe immobility , for instance , multiple sclerosis . in those kinds of patients , it was recommended continuing bisphosphonate therapy for up to 10 years , and then a drug holiday could be considered ( table 2 ) . if a holiday from the bisphosphonate in high - risk patients is considered after 10 years , interval treatment with other agents , such as parathyroid hormone or selective estrogen receptor modulator , may be warranted . in moderate - risk patients , they suggested drug holiday after 5 to 10 years and in low - risk patients after 3 to 5 years of bisphosphonate therapy . bisphosphonates should then be resumed if bone mineral density ( bmd ) decreases or the patient has a fracture . the duration of a drug holiday is unknown , but the authors propose 1 to 2 years in high - risk patients , 3 to 5 years in moderate - risk , and indefinitely in low - risk patients . this proposal was also cited in the american association of clinical endocrinologists ( aace ) guideline . food and drug administration ( fda ) issued an advisory to provide additional guidance to physicians . the fda analyzed the 3 long - term extension trials : flex , horizon - pft , and vert - mn . although the primary endpoint in the extension trials was bmd , the fda analyses included both bmd and fracture outcomes ( vertebral and non - vertebral ) . in their review of flex , the panel reported that the rates of vertebral and non - vertebral osteoporotic fractures were similar whether participants continued to receive alendronate for up to 10 years or were switched to a placebo . also , the panel noted that fracture rates appear to be constant when all data on vertebral and non - vertebral osteoporotic fractures with long - term therapy are pooled from the 3 extension trials . for example , in patients who received continuous bisphosphonate treatment for at least 6 years , the fracture rates ranged from 9.3% to 10.6% , whereas the rate for patients switched to a placebo was 8.0% to 8.8% . these data question the benefit of extended therapy with bisphosphonates after 5 years of fracture prevention . therefore , the fda recommended periodic assessment of a patient 's need for continued therapy , taking into account their individual risks and benefits and patient preference to optimize the efficacy of bisphosphonates in reducing fracture risk . in this regard , patients at low risk for fracture ( e.g. , younger patients without a fracture history and with a bmd approaching normal ) may qualify for drug holiday after 3 to 5 years , whereas patients at higher risk of fracture ( e.g. , older patients with a history of fracture and a bmd remaining in the osteoporotic range ) may benefit from continued bisphosphonate therapy ( table 2 ) . the response to the fda advisory commented that the fda analysis focused on the composite end point of all fractures , both vertebral and non - vertebral , when the original preplanned analyses of the extension studies separated vertebral and non - vertebral fractures because of " their distinct pathogenesis and different responses to treatment " . according to that analysis , the risk for vertebral fractures was shown to be reduced with continued bisphosphonate therapy beyond 3 to 5 years , whereas evidence was lacking for non - vertebral fractures . data from flex was used to estimate the number needed to treat ( for 5 additional years ) to prevent one clinical vertebral fracture ( in subgroups defined by bmd at the femoral neck and by prevalent vertebral fractures status at entry in flex ) ( table 3 ) . the risk of vertebral fracture is highest and the numbers needed to treat are lowest for patients with a femoral neck t - score -2.5 , which suggests that it may reasonably be expected that these patients would benefit by continuing bisphosphonate therapy . in addition , patients with a preexisting vertebral fracture with a femoral neck t - score -2.0 may also benefit from continuation ( table 2 ) . thus , in general , we consider that the following individuals are at a high risk for fractures and appear to benefit most from continuation of bisphosphonates : those with a t - score at any site still -2.5 after bisphosphonate therapy ( 5 years for alendronate and risedronate , and 3 years for zoledronic acid ) , previous fracture of the hip or spine , or secondary osteoporosis from chronic diseases or medication . alternative therapy ( e.g. , parathyroid hormone or selective estrogen receptor modulator ) may be used for individuals with a high risk . individuals without a high risk for fractures are unlikely to benefit from continued treatment after bisphosphonate therapy for 3 to 5 years . furthermore , there are some considerable factors guiding the determination of drug holiday , such as the variable anti - resorptive potency and binding affinity of each bisphosphonate , and a demonstration of compliance with the therapy . the variable anti - resorptive potency and binding affinity of each bisphosphonate are owing to their unique side chains . zoledronic acid has the highest potency , followed by risedronate , ibandronate , and alendronate . binding affinity is highest for zoledronic acid and decreases in order of magnitude for alendronate , ibandronate , and risedronate , respectively . this may be owing to a greater affinity of alendronate and zoledronic acid to hydroxyapatite , compared with risedronate and ibandronate . the skeletal binding sites for bisphosphonate are nearly unsaturable , thereby leading to a significant accumulation of bisphosphonates whereas release of bisphosphonates may be small , as it partly depends on bone turnover , which is reduced by the use of bisphosphonates . for example , after 10 years of alendronate use at a dose of 10 mg daily ( 70 mg weekly ) , the amount of alendronate released over several months or years would be equivalent to taking one - quarter of the usual dose . in general , zoledronic acid and alendronate maintain a prolonged effect after discontinuation , whereas others , such as risedronate , have a more rapid offset . another factor to consider in the drug holiday is a demonstration of compliance with the therapy . a recent retrospective register study about the residual treatment effect of alendronate and risedronate in swedish clinical practice suggests that the duration of bisphosphonate therapy is significantly inversely associated with the incidence of hospitalized fractures following discontinuation . specifically , during the first 6 months after terminating treatment , the adjusted fracture rates were considerably lower in patients who had been persistent with treatment for more than 12 months , compared with those who had stopped treatment within 1 month ( hazard ratio [ hr]=0.40 ) . it is known that 70% of bisphosphonate users discontinued their prescriptions after 1 year of use in south korea . therefore , the decision to go on drug holiday after 3 to 5 years should be after assurance of continuous use of bisphosphonates during the initial therapy period . a. parameter for monitoring during a drug holiday
i. consider the annual measurement of bmd using dual energy x - ray absorptiometry . b. restart therapy after a drug holiday
i. consider re - treatment if there is a significant decrease in bmd.ii . consider re - treatment if t - score reaches -2.5 or a new osteoporotic fracture occurs . a. parameter for monitoring during a drug holiday
i. consider the annual measurement of bmd using dual energy x - ray absorptiometry . b. restart therapy after a drug holiday
i. consider re - treatment if there is a significant decrease in bmd.ii . consider re - treatment if t - score reaches -2.5 or a new osteoporotic fracture occurs . ii . consider re - treatment if t - score reaches -2.5 or a new osteoporotic fracture occurs . there are no data providing information on how to monitor patients or when to restart therapy after a drug holiday . in the absence of guidance from clinical trials , although the approach has not been studied , bmd and biochemical markers of bone turnover ( btms ) measured after discontinuation may provide information about the persistence of the effect of the retained bisphosphonate . a significant decrease in bmd or a significant increase in btms suggests that the benefits of bisphosphonate therapy may be diminishing and that it may be time to return to active therapy . a recent study , a post hoc analysis of flex participants randomized to placebo after receiving 5 years of oral alendronate therapy , showed that older age and lower total hip bmd at the time of discontinuation were associated with higher rates of clinical fracture during the next 5 years , but neither 1-year changes in bmd nor 1- and 3-year changes in btms levels ( urinary type 1 collagen cross - linked n - telopeptide and bone - specific alkaline phosphatase ) were associated with fracture risk . similarly , most associations between 2- or 3-year changes in bmd and fracture risk were not statistically significant , although there were fewer fractures after the second ( n=70 ) and third ( n=57 ) annual visits included in the analysis . the risk of fracture was elevated among those with greater total hip bone loss after 2 or 3 years of follow - up , but after adjustment for age and baseline bmd at the time of discontinuation , only the 2-year total hip bone loss greater than 3% was significantly associated with fracture risk ( relative hr=1.68 , 95% confidence interval=1.05 - 2.72 ) . neither a 2- or 3-year change in bmd at the femoral neck nor a 3-year change in bmd at the lumbar spine was associated with fracture risk . therefore , more research is needed about the role of serial bone density testing and btms in monitoring fracture risk after therapy cessation , as well as the optimal therapies to use following a drug holiday . another untested approach is to reevaluate the patient after discontinuation , thereby making the decisions to restart therapy based on an updated assessment of fracture risk using algorithms initially developed for untreated individuals . for example , if the patient has a t - score -2.5 , or if the patient has a t - score between -1.0 and -2.5 and a world health organization 's fracture risk assessment estimate of fracture risk that meets treatment guidelines , then consider reinitiating therapy . if , at any time during the drug holiday there is a fracture , restarting therapy ( not necessarily a bisphosphonate ) is advised . monitoring with bmd and btms after discontinuation of alendronate therapy does not appear to improve fracture prediction , however , we propose the annual measurement of bmd and consideration of re - treatment if there is a significant decrease in bmd , a t - score reaches -2.5 , or new osteoporotic fracture occurs . randomized clinical trial evidence is not yet available on who may qualify for a drug holiday , thus many additional issues urgently need epidemiologic , clinical , and economic research in the korea . for example , the following several issues need to be addressed . more research is needed about the length of time for which bisphosphonate treatments continue to work after they are stopped.does stopping bisphosphonates reduce the risk of complications such as osteonecrosis of the jaw and atypical femur fractures?how can the optimal duration of a drug holiday be determined for other bisphosphonates therapy such as ibandronate or pamidronate?how can we better identify the individuals at high risk for fractures who may benefit from continued bisphosphonate therapy?is there is a need to stratify patients based both on the femoral neck t - score , but not on the lumbar spine t - score and the preexisting vertebral fracture status?what approaches are most effective in treating osteoporosis in individuals with high risk?is there any difference in the optimal duration of a drug holiday according to each bisphosphonate with variable anti - resorptive potency and binding affinity?how can patients be better monitored after a drug holiday?when can therapy be restarted after a drug holiday?more research is needed about the role of serial bmd testing and btms in monitoring fracture risk after a drug holiday , as well as the optimal therapies to use following a drug holiday . more research is needed about the length of time for which bisphosphonate treatments continue to work after they are stopped . does stopping bisphosphonates reduce the risk of complications such as osteonecrosis of the jaw and atypical femur fractures ? how can the optimal duration of a drug holiday be determined for other bisphosphonates therapy such as ibandronate or pamidronate ? how can we better identify the individuals at high risk for fractures who may benefit from continued bisphosphonate therapy ? is there is a need to stratify patients based both on the femoral neck t - score , but not on the lumbar spine t - score and the preexisting vertebral fracture status ? is there any difference in the optimal duration of a drug holiday according to each bisphosphonate with variable anti - resorptive potency and binding affinity ? how can patients be better monitored after a drug holiday ? when can therapy be restarted after a drug holiday ? more research is needed about the role of serial bmd testing and btms in monitoring fracture risk after a drug holiday , as well as the optimal therapies to use following a drug holiday . though bisphosphonates are commonly used in the prevention of fracture and treatment of osteoporosis in south korea , there remains much controversy surrounding its potential side effects from prolonged use . until further evidence is published , there are no clear recommendations regarding the length of use and drug holidays . based on the current research , a potential algorithm to guide physicians in south korea if the patient does not have a high risk for fractures , then a drug holiday was considered after 3 to 5 years ( 5 years for alendronate and risedronate and 3 years for zoledronic acid ) . during the drug holiday , patients were re - evaluated for risk of fracture with an annual measurement of bmd . if patient meets the criteria for therapy ( such as a significant decrease in bmd , t - score -2.5 , or new osteoporotic fracture ) , then re - start a treatment . if the patient has a high risk of fractures ( t - score at any site still -2.5 after bisphosphonate therapy , previous fracture of the hip or spine , or high risk of fracture because of secondary osteoporosis from chronic diseases or medication ) , then can maintain a drug depending on their bmd and fracture status . | bisphosphonates have been widely used in the treatment of osteoporosis with robust data from many placebo - controlled trials demonstrating its efficacy in fracture risk reduction over 3 to 5 years of treatment .
although bisphosphonates are generally safe and well tolerated , concerns have emerged about the adverse effects related to its long - term use , including osteonecrosis of the jaw and atypical femur fractures . because bisphosphonates are incorporated into the skeleton and continue to exert an anti - resorptive effect for a period of time after the discontinuation of drugs , the concept of a " drug holiday " has emerged , whereby the risk of adverse effects might be decreased while the patient still benefits from anti - fracture efficacy . as randomized clinical trial evidence is not yet available on who may qualify for a drug holiday ,
there is considerable controversy regarding the selection of candidates for the drug holiday and monitoring during a drug holiday , both of which should be based on individual assessments of risk and benefit .
this statement will provide suggestions for clinicians in south korea on the identification of possible candidates and monitoring during a bisphosphonate drug holiday . |
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systemic lupus erythematosus ( sle ) is a heterogeneous , multisystem autoimmune disease associated with the production of antibodies against self - antigens . its pathologic basis is vasculitis induced by immune complex , which suggests that blood vessels might be an important target of autoimmunity overactivity . as a result , there is a wide variation in vessel involvement in sle , including arteriostenosis and deep venous thrombosis , while glomerulonephritis and defused alveolar hemorrhage can also occur to a certain extent . cerebral venous sinus thrombosis ( cvst ) is an uncommon disorder in sle that is a manifestation of overlapping neuropsychiatric lupus ( nple ) and vessel disease . cvst has been rarely reported and is potentially fatal to the patient due to the acute neurological deterioration that can occur . as such , practitioners should be familiar with this condition , so the correct diagnosis can be made as soon as possible to improve patient prognosis . the etiology of cvst is complicated . in sle , the main culprits are vascular injuries caused by vasculitis . antiphospholipid antibodies ( apls ) and prothrombotic tendencies might also be responsible for cvst . by investigating the clinical features of cvst in sle and analyzing the differences between sle patients with and without cvst , this study seeks to explore the characteristics and risk factors for cvst in sle patients . 4747 sle patients admitted to the peking union medical college hospital from january 2000 to december 2013 were reviewed . all patients were diagnosed as having sle according to 1997 american college of rheumatology ( acr ) revised classification criteria . in the sle cohort , after exclusion of patients with infectious and traumatic cvst or incomplete data , we identified 17 cases ( 0.36% ) complicated with cvst as indicated by clinical features , lumbar puncture , imaging materials ( magnetic resonance venography ( mrv ) ) , and at least one neurologist 's confirmation . for each case ( n = 17 ) , three age- and sex - matched controls ( n = 51 ) were randomly selected from the contemporaneously admitted sle patients without cvst . the local institutional review board approved the study . because the study was based on a review of medical records that had been obtained for clinical purposes , the requirement for written informed consent was waived . numerical data and categorical data were expressed as mean sd ( range ) and percentage , respectively . the significance was estimated by student 's t - test , pearson 's chi - square , or fisher 's exact test ( when expected frequencies were < 5 ) . the median age was 30 years old ( range 1252 ) and the sle disease duration was 12 months ( range 2120 ) . the mode of onset was acute ( < 48 h ) in 12 patients ( 70.6% ) and progressive ( subacute or chronic ) for the other 5 patients . headache was the most common neurological manifestation ( 15/17 , 88.2% ) , and among these cases , 8 patients suffered from nausea / vomiting ( 47.1% ) . seven ( 41.2% ) cases had conscious disturbance , 6 ( 35.3% ) had edema of eyelids or conjunctiva , and 6 ( 35.3% ) had blurred vision or diplopia . hearing loss occurred in 2 cases ( 11.8% ) while 6 cases had seizures ( 35.3% ) . as complications , subarachnoid hemorrhage ( sah , 4/17 , 23.5% ) , sinusitis ( 3/17 , 17.6% ) , and cerebral ischemia and infarction ( 2/17 , 11.8% ) were also found in sle patients with cvst ( table 1 ) . all patients underwent lumbar puncture , which showed that the intracranial pressure ( icp ) was higher than 330 mm h2o in 7 patients ( 41.2% ) and 6 patients ' icp ranged from 180 to 320 mm h2o , while the remaining 4 were lower than 180 mm h2o . biochemical analysis of cerebrospinal fluid ( csf ) indicated that the protein level was higher than normal in 9 patients ( 52.9% ) and glucose and chloride levels were normal . myelin basic protein ( mbp ) was elevated in 5 patients . from examination of mrv images , we found that thrombosis in the transverse sinus ( ts ) was the most common site ( 14/17 , 82.4% ) , followed by sigmoid sinus ( ss , 9/17 , 52.9% ) . thrombosis in the superior sagittal sinus ( sss , 6/17 , 35.3% ) and inferior sagittal sinus ( iss , 1/17 , 5.9% ) was also seen . twelve patients suffered from more than one sinus thrombosis ( 12/17 , 70.6% ) ( figure 1(a ) ) . most sle patients with cvst ( 16/17 , 94.1% ) also had involvement of other organs , with lupus nephritis ( 12/17 , 70.6% ) , hemocytopenia ( 10/17 , 58.8% ) , arthritis ( 6/17 , 35.3% ) , fever ( 6/17 , 35.3% ) , rash ( 5/17 , 29.4% ) , and serositis ( 5/17 , 29.4% ) being the most common conditions . the mean sle disease activity index ( sledai ) score ( 8 points of cvst excluded ) of these patients was 12.1 3.7 . twelve cases had hypoalbuminemia ( 12/17 , 70.6% ) and the mean serum albumin level of all 17 cases was ( 30.4 7.5 ) g / l . the prothrombin time and activated partial thromboplastin time were normal in most cases . apls , including anticardiolipin ( 5/17 , 29.4% ) antibodies , anti-2gp1 antibodies ( 4/17 , 23.5% ) , and lupus anticoagulants ( 6/17 , 35.3% ) , were detected in 7 cases ( 7/17 , 41.2% ) in this cohort . thrombophilia factors ( protein s and protein c ) were detected in 12 patients , and no protein c or s abnormality was found . only one patient accompanied other thromboses in the veins ( table 2 ) . compared with sle patients without cvst , the incidence of thrombocytopenia ( 58.8% versus 23.5% , p = 0.007 ) and prevalence of apls ( 41.2% versus 15.7% , p = 0.043 ) were significantly higher in sle patients with cvst . sle patients with cvst also had much higher disease activity ( sledai scores ) ( 20.1 3.7 ) when compared with sle patients without cvst ( 12.8 5.3 , p = 0.001 ) ( table 3 ) . thirteen patients were treated by pulse therapy with gc ( methylprednisolone 0.5~1 g / d 3~5 day ) . cyclophosphamide ( ctx ) was selected as the preferred immunosuppressant for 17 patients , and fk506 or mycophenolate mofetil was added for 2 cases . moreover , anticoagulation was utilized in 11 patients , with heparin or low molecular weight heparin ( lmwh ) being used with preference . seven patients had been intrathecally injected about 3 to 5 times with dexamethasone ( 10 mg ) , after which their csf pressure decreased from over 330 mm h2o to 115~250 mm h2o . specifically , one patient died of cerebral hernia , the other one died of pneumonia , and the third one committed suicide . during the follow - up period ( mean 36.8 21.7 months ) , repeat of cerebral mrv was performed in 8 patients and 5 of them had partial or total recanalization of cvst ( figure 1(b ) ) . nple is one of the most important manifestations of sle , which occurs in 14%~80% of adult and 22%~95% of pediatric [ 6 , 7 ] sle patients . in 1999 , acr named nple into 19 categories . cvst is one of the rare and severe cerebrovascular diseases , and its clinical features lack specificity . thus , rheumatologists should be aware of this rare but potentially fatal complication of sle . as an uncommon disorder that accounts for about 0.5% of all strokes , cvst presents as a variety of neurologic symptoms and can lead to severe morbidity and mortality . the causes of cvst include infection , dehydration , local trauma , rheumatologic diseases such as sle and behet 's disease ( bd ) as well as thrombophilia including nephritic syndrome , antithrombin iii deficiency , pregnancy , cancer , and use of oral contraceptives . the current study showed that the incidence of cvst was about 0.36% in sle , which is much lower than that of cvst in bd ( 7.8% ) , making this condition quite rare . it is not clear when and why cvst could happen and whether this condition is due to high sle activity . endothelial cell injury caused by immune complex - induced vasculitis was thought to play an important role in nple , which includes cvst . thrombophilia is related to some sle complications , such as nephritic syndrome and hyperfibrinogenemia caused by chronic inflammatory status and could represent another etiologic factor . infections of the middle ear , facial skin , or intracalvarium , which were known as the most common causes of cvst , were secondary consequences of gc and immunosuppressant treatment and are sometimes minor factors associated with cvst in sle . based on our data , no related craniofacial infections were found in the sle patients complicated with cvst . renal involvement was documented in 12 cases , but few of them fulfilled the criteria of nephritic syndrome . apls were found much more frequently ( 41.2% ) in this group compared to sle patients without cvst ( 15.7% ) , and it hints that these molecules are involved in cvst etiology in sle . moreover , most cases had high disease activities and good responses to gc ( systemic administration or intrathecal injection ) and immunosuppressants , which supports that cerebral vasculitis might be another possible cvst mechanism . headaches , which can be persistent and severe and the only neurological sign , are the most frequent symptom of cvst and occur in about 90 percent of cvst patients . in the current study , 88.2% patients suffered from headache , most of which increased gradually over days or weeks , while some had acute - onset mimicking or were accompanied by or even initially presenting as a sah [ 16 , 17 ] . the cause of headaches is likely intracranial hypertension ( ich ) , which can also cause vomiting , papilledema , and diplopia due to involvement of the cranial nerves . thirteen patients ( 76.5% ) were confirmed as ich and the pressures in 7 cases were higher than 330 mm h2o in the current study . most lupus headaches are chronic and are not accompanied by ich or other neurological signs , while headaches in the majority of cvst cases are acute or subacute . besides headaches , about half of patients develop other neurological signs to indicate their location to practitioners . sss involvement frequently leads to paralysis and seizure , while thrombosis of the ts can be an underlying cause of acute aphasia . isolated or multiple cavernous sinus syndrome , manifesting as oculomotor nerve palsies , facial pain , sensory loss in the distribution of the trigeminal nerve , or proptosis and chemosis , is often caused by infections and is very rare in sle . analysis of the cerebrospinal fluid through lumbar puncture is almost always nonspecific but is necessary to differentiate infections . in this study , slightly elevated csf protein was found in more than half of patients ( 9/17 , 52.9% ) . mrv is now the most sensitive tool for detecting cvst , but its specificity is based on meticulous knowledge of the anatomy and common variations of the cerebral sinus such as left transverse sinus hypoplasia or atresia could be seen in some patients . therefore , the diagnosis of cvst should be determined not only by imaging but also by clinical manifestations and csf measures . sss was reported to be the most frequently involved site ( 62.5% ) and ts was the second ( 41.2%~44.7% ) . infections always cause thrombosis of the cavernous sinus , ts , or ss . previously , there were no data concerning cvst associated with sle . in the current study , ts was the most common site of thrombosis occurrence ( 14/17 , 82.4% ) followed by ss ( 9/17 , 52.9% ) , sss ( 6/17 , 35.3% ) , and the inferior sagittal sinus ( iss , 1/17 , 5.9% ) . the data represented that most cases were accompanied by renal and hematological involvement or arthritis , fever , and rash . the higher prevalence of thrombocytopenia in sle patients with cvst might partly result from damage caused by autoantibodies and depletion of platelets when thrombosis occurs . the meaning of apls was apparent and confirmed that these antibodies were involved in cvst in sle . concerning sle activity , the sledai of the cvst group was higher than the controls , indicating that the occurrence of cvst might parallel the systemic active situation of sle . the treatment of patients with cvst should include four aspects : removal of precipitating factors , administration of antithrombotic therapy , lowering of ich , and relieving of neurological symptoms [ 15 , 21 , 22 ] . the current study showed that these cases had high sle activity and rapid disease progression so that aggressive treatments to control sle are necessary and important . in order to achieve clinical remission as soon as possible , most cases needed gc pulse therapy combined with ctx , which is one of the most powerful immunosuppressants . heparin or lmwh was utilized by 11 cases and followed by warfarin in the current study , while sah and severe thrombocytopenia were contraindications to anticoagulation in other patients . patients had also been treated with dehydration drugs and intrathecal injections of dexamethasone ( 10 mg ) to normalize their icp . in the past , the mortality rate of cvst in sle reached 30%~50% . in part due to modern neuroimaging and lmwh administration , the mortality rate was reduced to 8%~14% and the outcomes continued to improve [ 26 , 27 ] . in the current study , the mortality was 11.8% ( 2/17 , 1 case died as the result of suicide rather than cvst ) . complete or partial recovery was observed in most patients who survived ( 13/17 , 76.5% ) , while no patient experienced a relapse of cvst or had a poor outcome with permanent neurological deficits during the mean 3-year follow - up . as one of the rare and severe complications of sle , cvst usually occurs in active sle patients and is accompanied by systemic involvement ( especially thrombocytopenia ) . cvst in sle may be caused by a variety of pathogenic factors , such as vasculitis and apls . the clinical picture of cvst is nonspecific and may vary significantly due to different venous sinus involvements , which can make the diagnosis quite difficult . early diagnosis and prompt management of cvst and the underlying disease could significantly improve the prognosis of lupus with cvst . | clinical characteristics of systemic lupus erythematosus ( sle ) patients complicated with cerebral venous sinus thrombosis ( cvst ) between 2000 and 2013 were analyzed through this retrospective , single - centre study .
of 4747 hospitalized sle patients , 17 ( 0.36% , 12 females , average age 30 ) had cvst .
headache ( 88.2% ) was the most common neurological symptom followed by nausea or vomiting ( 47.1% ) , conscious disturbance ( 41.2% ) , edema of eyelids or conjunctiva ( 35.3% ) , blurred vision or diplopia ( 35.3% ) , and seizure ( 35.3% ) . increased intracranial pressure ( icp ) occurred in 13 cases ( 76.5% ) .
magnetic resonance venography ( mrv ) detected thrombosis in the transverse ( 82.4% ) , sigmoid ( 52.9% ) , and sagittal ( 35.3% ) sinuses , with frequent ( 70.6% ) multiple sinus occlusions .
compared to sle patients without cvst , sle patients with cvst had a higher prevalence of thrombocytopenia and positive antiphospholipid antibodies and a higher sle disease activity index ( sledai ) score .
13 patients achieved improvement following glucocorticoids and immunosuppressants treatment , as well as anticoagulants , while 3 patients died at the hospital .
cvst is relatively rare in sle and tends to occur in active lupus patients .
intracranial hypertension is common but nonspecific clinical feature , so mrv evaluation is necessary to establish a diagnosis .
aggressive treatment for the rapid control of sle activity combined with anticoagulants can improve the prognosis . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
non - melanomatous skin cancer ( nmsc ) affects over 3.5 million patients per year in the united states . there are several available treatment options including surgery , topical chemotherapies , external beam radiation therapy , and brachytherapy [ 2 , 3 , 4 , 5 , 6 ] . although effective , surgery can be disfiguring and morbid for lesions located on cosmetically sensitive areas of the body such as the face . for these lesions and for medically inoperable patients , radiotherapy can offer an equally effective alternative to surgery . when using conventional megavoltage radiation , treatments can last between 3 - 6 weeks depending on size and location of the lesion [ 6 , 7 ] . in addition , to ensure adequate margins and dose to the skin surface , water - equivalent bolus and clinical evaluation are commonly utilized [ 6 , 7 ] . superficial / orthovoltage radiation ( 40 - 300 kvp ) has also been used to treat thin superficial nmsc [ 8 , 9 ] . finally , for large tumors with irregular shapes , high - dose - rate ( hdr ) brachytherapy molds , flaps , interstitial catheters , or radionuclide applicators can be used with an ir source [ 3 , 5 , 10 , 11 , 12 , 13 , 14 , 15 ] . since 2009 , electronic brachytherapy ( ebt ) , a 50 kvp miniaturized x - ray source with specialized cone - shaped applicators has been used to treat superficial nmsc . xoft ebt ( axxent ebx ; xoft a subsidiary of icad , inc . , sunnyvale , ca , usa ) does not require a radioactive isotope and involves minimal shielding [ 16 , 17 , 18 ] . it also does not require the same regulations of a radioactive source such as ir . xoft ebt uses a hypofractionated regimen as do other orthovoltage or hdr brachytherapy techniques [ 13 , 17 , 18 , 20 ] . another system similar to xoft ebt is esteya ( elekta ab , stockholm , sweden ) but uses a 69.5 kv x - ray source [ 21 , 22 , 23 ] . because of ebt low energy x - rays , the maximum dose is at the surface , and the dose at 5 mm depth is 50 - 60% of the surface dose depending upon the applicator , e.g. , if the prescription depth is 5 mm then ~200% of dose is at the skin surface [ 24 , 25 ] . in a previously published study using xoft ebt for lesions 1 - 7 mm thick treated with 40 gy in 8 fractions , treatment resulted in a local control of 100% and an excellent cosmesis in 93% of patients with no toxicity at a follow - up of 1 year . in this particular study , in many clinics , however , a default depth is selected for ebt calculations and is used universally for all patients . the lateral extension is usually determined with visual inspection on the skin surface and a varying margin is applied based on the physician 's discretion [ 6 , 7 ] . in our initial ebt experience , using clinical estimates of depth and lateral extent , led to complications such as grade 4 skin necrosis and prolonged wound healing , especially with thicker lesions and lower extremity lesions ( figure 1 ) . this motivated us to investigate a more accurate approach in choosing an applicator size and dose depth prescription by utilizing clinically available ultrasound ( us ) to measure the depth and lateral extent in order to prevent overdosing the skin . two lesions on the lower extremity were treated to 40 gy in 8 fractions ( 5 gy per fraction ) twice per week using a 35 mm cone based on clinical exam and default depth of 3 mm in our early electronic brachytherapy ( ebt ) experiences . the patient developed a non - healing grade 4 ulceration that persisted even after 4 months post - ebt . although this toxicity could be due to a number of contributing factors , it illustrates the need for a more precise and accurate depth of prescription . a ) pre - treatment , b ) 4 months post - ebt in the past , nmsc lateral extension and depth measurements have commonly been determined by visual estimation during clinical examination [ 6 , 7 ] but this method can be quite subjective and physician - dependent . measurements from punch biopsies have also been utilized but have led to questions regarding its accuracy . this study reports on the feasibility of using standard us technology as a measuring tool to determine the diameter and the depth of a lesion in order to adequately prescribe radiation dose in ebt treatment planning . from december 2013 to april 2015 , 24 patients with 28 biopsy confirmed nmscs were referred for definitive xoft ebt . the depth and lateral extension for all lesions five lesions , 4 basal cell carcinomas ( bcc ) , and 1 squamous cell carcinoma ( scc ) , could not be visualized on us and therefore they were not included in this study . these were treated at a default depth of 1 mm and with a radial margin based on the biopsy scar . due to limitation of the ebt technique ( low energy and high surface dose ) , in addition , the largest available xoft applicator size is 50 mm in diameter , so the largest lateral dimension of any treated lesion was limited to 36 mm due to inclusion of a 7 mm radial margin to cover microscopic disease and setup errors . lesions were ineligible if evidence of clinical or pathologic positive lymph nodes , metastatic disease , or melanoma histology . radiation oncology and radiology records , referring physician documentation , and hospital records were examined . data was gathered on : patient demographics , pathology , physical examination , photographs , threedimensional ( 3d ) tumor measurements with us , and radiation treatment planning sheets , calculations , and quality assurance ( qa ) . lesions were staged based on the american joint committee on cancer staging manual 7 edition . toxic effects were graded according to the national cancer institute common toxicity criteria for adverse events , version 4 . this retrospective study was approved by our institutional review board . written consent from each patient was received to publish their data and photographs . a patient with a suspicious skin lesion first underwent a shave biopsy by a dermatologist . after a diagnosis of nmsc was rendered , the patient was referred for radiotherapy and underwent a consultation with a board - certified radiation oncologist . the patient was then sent for us imaging of the lesion for lateral extent and depth measurements , which were documented in a report within an accessible electronic medical record . focused us of the lesions was performed by a boardcertified radiologist using either : the logiq e9 ( ge healthcare , milwaukee , wi , usa ) or s-3000 us units ( acuson ; siemens medical solutions , mountain view , ca , usa ) . linear array transducers with upper frequencies of 14 and 18 mhz were utilized to obtain sonograms . imaging was performed using either a custom made gel standoff pad or copious amounts of cold us gel acting as a standoff , especially on body parts where the gel pad could potentially cause image degradation due to poor apposition . two - dimensional ( 2d ) b - mode images were acquired with transverse and longitudinal sweeps across the lesion . the us probe was oriented along the longest dimension of the lesion to obtain sagittal and transverse measurements . measurements of depth were made at the most vertical extension of the nmsc in either plane . in order to cover potential subclinical disease , a margin of 5 mm was added radially to the gross tumor volume ( gtv ) diameter measurements to create the clinical target volume ( ctv ) , even though dermato - pathologic data has shown that the lateral extent estimate derived from us measurements tends to overestimate the extent of the lesion . since ebt is a contact therapy with the applicator applied directly and firmly to the skin , there was no expected movement during treatment but an inter - fractional error in positioning the applicator was possible . therefore , a 2 mm margin was added to the ctv to create a planning target volume ( ptv ) . however , when treating difficult anatomical sites ( e.g. eyelid , lip ) , ctv and ptv were reduced based on practicality and clinical judgment . with 4 ebt applicator sizes available ( 10 , 20 , 35 , and 50 mm ) , we used the cone size that adequately covered the ptv . no extra margin was added to depth based on us measurements and mohs pathology data showing that us may overcompensate for depth by about 27% , and there was no expected setup error in the vertical dimension . the median dose used was 40 gy in 10 fractions delivered every other day ; median fraction size was 4 gy . two lesions on the upper lip and tip of the nose received a more protracted course of 50 gy in 20 fractions of 2.5 gy per fraction due to central anatomical locations and greater depths . one patient stopped her treatment after 32 gy due to grade 3 erythema . during simulation , patients with lesions on the face were immobilized using an aquaplast facemask ( civco medical solutions , coralville , ia , usa ) and the area to be treated was cut out from the facemask . when using ebt , the maximum dose occurs at the skin surface due to the use of low energy x - rays , therefore output of the source was defined as the dose rate ( gy / minute ) at the skin surface for each applicator . this x - ray source calibration for each applicator followed the american association of physicists in medicine ( aapm ) task group ( tg ) 61 protocol [ 24 , 25 ] . the beam - on time was computed by the ratio of prescribed dose over the product of dose rate and vendor provided percentdepth - dose ( pdd ) values . therefore , for a given prescription dose , 2 parameters were essential in treatment planning calculations : prescription depth determined the pdd value while the lateral extension determined the applicator size . at the time of treatment , after the patient was setup on the treatment couch , the lesion was demarcated with a fine tip marker by the physician and compared to photographs taken during simulation the patient was monitored by 2 therapists and a physicist who remained in the room to ensure the patient kept the same position as planned . , mundelein , il , usa ) was provided for the treated area during the ebt course and as long as the radiation - induced erythema persisted ( usually a month ) . the percent of prescription dose at the surface is considered for clinical purposes ( table 1 ) . cone applicator size with a constant depth also shows small variation in percent of prescription dose at the surface . a patient with a suspicious skin lesion first underwent a shave biopsy by a dermatologist . after a diagnosis of nmsc was rendered , the patient was referred for radiotherapy and underwent a consultation with a board - certified radiation oncologist . the patient was then sent for us imaging of the lesion for lateral extent and depth measurements , which were documented in a report within an accessible electronic medical record . focused us of the lesions was performed by a boardcertified radiologist using either : the logiq e9 ( ge healthcare , milwaukee , wi , usa ) or s-3000 us units ( acuson ; siemens medical solutions , mountain view , ca , usa ) . linear array transducers with upper frequencies of 14 and 18 mhz were utilized to obtain sonograms . imaging was performed using either a custom made gel standoff pad or copious amounts of cold us gel acting as a standoff , especially on body parts where the gel pad could potentially cause image degradation due to poor apposition . two - dimensional ( 2d ) b - mode images were acquired with transverse and longitudinal sweeps across the lesion . the us probe was oriented along the longest dimension of the lesion to obtain sagittal and transverse measurements . measurements of depth were made at the most vertical extension of the nmsc in either plane . in order to cover potential subclinical disease , a margin of 5 mm was added radially to the gross tumor volume ( gtv ) diameter measurements to create the clinical target volume ( ctv ) , even though dermato - pathologic data has shown that the lateral extent estimate derived from us measurements tends to overestimate the extent of the lesion . since ebt is a contact therapy with the applicator applied directly and firmly to the skin , there was no expected movement during treatment but an inter - fractional error in positioning the applicator was possible . therefore , a 2 mm margin was added to the ctv to create a planning target volume ( ptv ) . however , when treating difficult anatomical sites ( e.g. eyelid , lip ) , ctv and ptv were reduced based on practicality and clinical judgment . with 4 ebt applicator sizes available ( 10 , 20 , 35 , and 50 mm ) no extra margin was added to depth based on us measurements and mohs pathology data showing that us may overcompensate for depth by about 27% , and there was no expected setup error in the vertical dimension . the median dose used was 40 gy in 10 fractions delivered every other day ; median fraction size was 4 gy . two lesions on the upper lip and tip of the nose received a more protracted course of 50 gy in 20 fractions of 2.5 gy per fraction due to central anatomical locations and greater depths . during simulation , an immobilization device was fabricated depending upon the location of the nmsc . patients with lesions on the face were immobilized using an aquaplast facemask ( civco medical solutions , coralville , ia , usa ) and the area to be treated was cut out from the facemask . when using ebt , the maximum dose occurs at the skin surface due to the use of low energy x - rays , therefore output of the source was defined as the dose rate ( gy / minute ) at the skin surface for each applicator . this x - ray source calibration for each applicator followed the american association of physicists in medicine ( aapm ) task group ( tg ) 61 protocol [ 24 , 25 ] . the beam - on time was computed by the ratio of prescribed dose over the product of dose rate and vendor provided percentdepth - dose ( pdd ) values . therefore , for a given prescription dose , 2 parameters were essential in treatment planning calculations : prescription depth determined the pdd value while the lateral extension determined the applicator size . at the time of treatment , after the patient was setup on the treatment couch , the lesion was demarcated with a fine tip marker by the physician and compared to photographs taken during simulation . then the applicator was placed firmly against the skin and treatment initiated . the patient was monitored by 2 therapists and a physicist who remained in the room to ensure the patient kept the same position as planned . , mundelein , il , usa ) was provided for the treated area during the ebt course and as long as the radiation - induced erythema persisted ( usually a month ) . the percent of prescription dose at the surface is considered for clinical purposes ( table 1 ) . surface dose increases with increasing depth . cone applicator size with a constant depth also shows small variation in percent of prescription dose at the surface . characteristics of non - melanomatous skin cancer lesions bcc basal cell carcinoma , scc squamous cell carcinoma normal anatomy : the layers of skin : epidermis ( thickness 0.06 - 0.6 mm ) , dermis ( thickness 1 - 4 mm ) , and hypodermis ( subcutaneous tissues ; thickness 5 - 20 mm ) can be visualized on us . the epidermis appears as the most superficial , well - defined , hyperechoic , linear band producing the entry echo between the us gel and skin ( epidermal entry echo [ eee ] ) . the dermis below the epidermis is also hyperechoic , usually less echogenic than epidermis , and with hypoechoic hair follicles , vessels , and sebaceous glands . the hypodermis is hypoechoic with intervening hyperechoic connective tissue septa separating fat lobules . underneath the skin , superficial fascia covering muscle figure 2b illustrates each layer described on us . clinical setup and ultrasound image . a right forehead basal cell carcinoma ( a ) delineated with a pen ( dotted line ) and ( b ) on ultrasound found to have a hypoechoic lesion that measures 7.5 mm in the transverse dimension , 3 mm in depth and 5 mm in the sagittal dimension ( not shown ) . a 20 mm applicator was chosen to treat to a depth of 3 mm . a hyperechoic epidermis ( thin arrow ) , a hypoechoic dermal layer with a more echoic signal from subcutaneous fat beneath ( arrowhead ) , and the strongly echoic layer below subcutaneous fat is bone that creates shadowing so nothing deeper is anatomically distinguishable ( thick arrows ) us provides 2d or 3d images of deep dermal or subdermal layers by measuring differences in sound impedance . on high resolution us , bcc is a solid , heterogeneous and hypoechoic mass that shows irregular margins containing echogenic foci of keratin nests . scc also appears hypoechoic but may be more aggressive at the time of presentation with infiltration of deeper tissues [ 29 , 30 ] . figure 2a and b shows a nmsc on the forehead and us image depicting the clarity and capability of us in providing measurements for ebt prescriptions . the mean largest diameter of all lesions was 8 mm ( range 2.6 - 20 mm ) . the mean depth of all lesions was 2.1 mm ( range 1 - 3.4 mm ) . for bcc , the largest lateral extent ranged from 2.6 - 12 mm and depth ranged from 1 - 3.4 mm . for scc , the largest lateral extent ranged from 8 - 20 mm and depth ranged from 2.5 - 3.1 mm . the only upper extremity lesion had the largest lateral extent ( 20 mm ) . with us depth measurements , prescription depth could be rounded or prescribed to the fractional depth ( e.g. 1.5 mm rather than every millimeter ) . figure 3a and b shows the largest lateral extent and the second largest lateral extent , and also depicts the largest diameter and depth for each lesion using us - determined measurements for ebt . no correlation between location , lateral size , and depth was seen in our study . ultrasound ( us ) measurements of lateral extensions and depth . the largest lateral extent ( mm ) and second largest lateral extent ( mm ) of each non - melanomatous skin cancer measured with us and used for electronic brachytherapy planning are shown ( a ) . the largest lateral extension ( mm ) and depth ( mm ) of each non - melanomatous skin cancer measured with us and used for electronic brachytherapy planning are shown ( b )
all patients have had a complete response , and with a median follow - up of 12 months ( range 6 - 22 months ) , there have been no failures . more importantly , there have been no cases of grade 4 skin necrosis or delayed healing . figure 4 shows progressively worsening radiation - induced erythema during ebt for a patient with bcc . a scalp lesion measured to be 6.3 mm 4 mm 2 mm with ultrasound was treated with a 35 mm applicator to a 2 mm depth . the dose regimen was 40 gy in 10 fractions every other day and the lesion is shown : ( a ) prior to initial treatment , ( b ) after 5 fractions , ( c ) after completion of 10 fractions , and ( d ) after 1 month follow - up . there was progressively worsening erythema over the course of electronic brachytherapy that resolved after 1 month characteristics of non - melanomatous skin cancer lesions bcc basal cell carcinoma , scc squamous cell carcinoma normal anatomy : the layers of skin : epidermis ( thickness 0.06 - 0.6 mm ) , dermis ( thickness 1 - 4 mm ) , and hypodermis ( subcutaneous tissues ; thickness 5 - 20 mm ) can be visualized on us . the epidermis appears as the most superficial , well - defined , hyperechoic , linear band producing the entry echo between the us gel and skin ( epidermal entry echo [ eee ] ) . the dermis below the epidermis is also hyperechoic , usually less echogenic than epidermis , and with hypoechoic hair follicles , vessels , and sebaceous glands . the hypodermis is hypoechoic with intervening hyperechoic connective tissue septa separating fat lobules . underneath the skin , superficial fascia covering muscle figure 2b illustrates each layer described on us . clinical setup and ultrasound image . a right forehead basal cell carcinoma ( a ) delineated with a pen ( dotted line ) and ( b ) on ultrasound found to have a hypoechoic lesion that measures 7.5 mm in the transverse dimension , 3 mm in depth and 5 mm in the sagittal dimension ( not shown ) . a 20 mm applicator was chosen to treat to a depth of 3 mm . a hyperechoic epidermis ( thin arrow ) , a hypoechoic dermal layer with a more echoic signal from subcutaneous fat beneath ( arrowhead ) , and the strongly echoic layer below subcutaneous fat is bone that creates shadowing so nothing deeper is anatomically distinguishable ( thick arrows ) us provides 2d or 3d images of deep dermal or subdermal layers by measuring differences in sound impedance . on high resolution us , bcc is a solid , heterogeneous and hypoechoic mass that shows irregular margins containing echogenic foci of keratin nests . scc also appears hypoechoic but may be more aggressive at the time of presentation with infiltration of deeper tissues [ 29 , 30 ] . figure 2a and b shows a nmsc on the forehead and us image depicting the clarity and capability of us in providing measurements for ebt prescriptions . the mean largest diameter of all lesions was 8 mm ( range 2.6 - 20 mm ) . the mean depth of all lesions was 2.1 mm ( range 1 - 3.4 mm ) . for bcc , the largest lateral extent ranged from 2.6 - 12 mm and depth ranged from 1 - 3.4 mm . for scc , the largest lateral extent ranged from 8 - 20 mm and depth ranged from 2.5 - 3.1 mm . the only upper extremity lesion had the largest lateral extent ( 20 mm ) . with us depth measurements , prescription depth could be rounded or prescribed to the fractional depth ( e.g. 1.5 mm rather than every millimeter ) . figure 3a and b shows the largest lateral extent and the second largest lateral extent , and also depicts the largest diameter and depth for each lesion using us - determined measurements for ebt . no correlation between location , lateral size , and depth was seen in our study . ultrasound ( us ) measurements of lateral extensions and depth . the largest lateral extent ( mm ) and second largest lateral extent ( mm ) of each non - melanomatous skin cancer measured with us and used for electronic brachytherapy planning are shown ( a ) . the largest lateral extension ( mm ) and depth ( mm ) of each non - melanomatous skin cancer measured with us and used for electronic brachytherapy planning all patients have had a complete response , and with a median follow - up of 12 months ( range 6 - 22 months ) , there have been no failures . more importantly , there have been no cases of grade 4 skin necrosis or delayed healing . figure 4 shows progressively worsening radiation - induced erythema during ebt for a patient with bcc . this grade 2 erythema usually resolves by the 1 month follow - up . non - melanomatous skin cancer treated with electronic brachytherapy . a scalp lesion measured to be 6.3 mm 4 mm 2 mm with ultrasound was treated with a 35 mm applicator to a 2 mm depth . the dose regimen was 40 gy in 10 fractions every other day and the lesion is shown : ( a ) prior to initial treatment , ( b ) after 5 fractions , ( c ) after completion of 10 fractions , and ( d ) after 1 month follow - up . there was progressively worsening erythema over the course of electronic brachytherapy that resolved after 1 month in this methodology paper , we describe the use of commonly available us for determining the lateral extent and depth of a pathologically proven nmsc , and the clinical application of such measurements to guide ebt planning . without a standard objective mode of measuring nmscs , many clinicians determine prescription depth and applicator size based on clinical judgment [ 6 , 7 ] or biopsy information . when using small contact applicators , this clinical approach bears potential issues such as inadequate lateral coverage and/or under - treating the nmsc or the alternative of over - treatment , which may lead to unnecessary toxicity ( skin necrosis or delayed healing ) . , this leads to questions about whether the punch biopsy removes the deepest portion of the lesion prior to us measurement for ebt . in our study , shave biopsies were used for pathologic verification allowing a greater chance that the remaining nmsc depth and margins were adequately assessable by us . unfortunately , there are few available modalities that can be used for non - invasive and accurate measurement of a superficial lesion 's lateral extent and depth . a prior study has discussed us use for nmsc treated with hdr - bt . there has been report of using ct for depth assessment in prior ebt studies [ 17 , 18 ] but in our experience , ct does not have enough discrimination for accurate depth determination when dealing with superficial thin lesions . we conducted this pilot study to provide an element of qa and guidelines for accurate treatment planning that is currently not performed routinely rather than proving clinical outcomes . we have found that us evaluation can visualize and measure thin nmscs in the range of 1 - 5 mm . thus , we advocate that rather than using clinical exam measurements ( the current standard of care ) , which can be subjective based on practitioner , objective depth , and lateral extent us measurements that should be used as guidance when prescribing radiotherapy for these superficial cancers . the detailed use of us for nmsc ebt planning has not been described before and is an important consideration for future ebt skin cancer guidelines . our intent for this conceptual paper is to describe a more standard technique for prescribing ebt for nmsc ; however , we do feel it is important to provide our preliminary clinical outcomes data in order to show that the use of us does not worsen local control or cosmesis compared to previously published studies [ 17 , 18 ] . compared to previously published data with median follow - ups ranging from 4.1 to 10 months , the 1 year patient outcomes data for our study shows 100% local control and no significant toxicity , essentially similar to reported data by other studies [ 17 , 18 , 22 , 26 ] . only long - term follow - up will determine whether us - guided prescriptions will result in better cosmetic outcomes due to the precision of depth prescriptions . a novel point from our study is that with the use of us , physicians are not limited to depth prescriptions of every millimeter ( i.e. 3 mm or 4 mm ) but can prescribe ebt to tenths of millimeters ( i.e. 2.7 mm or 3.5 mm ) in order to spare superficial layers from receiving higher doses with unnecessary deeper prescriptions . a limitation of current commercially available us is its capacity to detect nmscs that are < 1 mm deep . five biopsy - proven nmscs could not be detected by our us modality . a high frequency ultrasound unit ( hfus ) such as 50 mhz may have better resolution to detect previously undetectable lesions , and improve the quality and detail of these radiologic images [ 30 , 32 ] . hfus could also be a useful tool for defining diameter and characterizing changes over time . various other clinical imaging modalities for evaluating nmsc have been described such as dermoscopy , optical coherence tomography , and reflectance confocal microscopy [ 33 , 34 , 35 , 36 , 37 , 38 , 39 ] but each has its own caveats . standard imaging with a commonly available us unit eliminates guesswork at the time of prescribing and planning ebt . there are no studies to our knowledge that have reported on the systematic use of us measurements of depth and lateral extension for ebt treatment planning . this study may bode importance in further clinical applications for adequately treating and following nmsc patients . we feel that as the use of ebt in the treatment of nmsc continues to grow , it becomes more important to have standard measurement techniques for prescribing treatment and reporting outcome data . ultrasound technology provides an easy and objective modality to determine the diameter and depth of a lesion when treating thin nmsc lesions with xoft ebt . the suggested guidelines provide a set of simple parameters to follow when treating superficial skin cancers over cosmetically sensitive parts of the body where skin toxicity could jeopardize the anticipated good cosmetic results . | purposeelectronic brachytherapy ( ebt ) has gained acceptance over the past 5 years for the treatment of non - melanomatous skin cancer ( nmsc ) .
although the prescription depth and radial margins can be chosen using clinical judgment based on visual and biopsy - derived information , we sought a more objective modality of measurement for ebt planning by using ultrasound ( us ) to measure superficial ( < 5 mm depth ) lesions.material and methodsfrom december 2013 to april 2015 , 19 patients with 23 pathologically proven nmscs underwent a clinical examination and us evaluation of the lesions prior to initiating a course of ebt .
twenty lesions were basal cell carcinoma and 3 lesions were squamous cell carcinoma .
the most common location was the nose ( 10 lesions ) .
a 14 or 18 mhz us unit was used by an experienced radiologist to determine depth and lateral extension of lesions .
the us - measured depth was then used to define prescription depth for ebt planning without an added margin .
a margin of 7 mm was added radially to the us lateral extent measurements , and an appropriate cone applicator size was chosen to cover the target volume.resultsthe mean depth of the lesions was 2.1 mm with a range of 1 - 3.4 mm , and the mean largest diameter of the lesions was 8 mm with a range of 2.6 - 20 mm .
dose ranged from 32 - 50 gy in 8 - 20 fractions with a median dose of 40 gy in 10 fractions .
all patients had a complete response and no failures have occurred with a median follow - up of 12 months ( range of 6 - 22 months ) . also , no prolonged skin toxicities have occurred.conclusionsa routinely available radiological us unit can objectively determine depth and lateral extension of nmsc lesions for more accurate ebt treatment planning , and should be considered in future ebt treatment guidelines . |
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the history of facial reconstructive surgery has progressed slowly and naturally and is punctuated by highlights from sudden profound changes . facial transplantation has only recently become possible through the discovery of innovative drugs and the courage of innovative surgeons . facial allotransplantation is a new surgical technique that could be considered a new paradigm in facial reconstruction . facial transplantation is among the most prominent areas of composite tissue allotransplantation ( cta ) or vascular composite allograft ( vca ) , along with hand transplantation . since the first facial allograft transplantation was reported in france in 2005 , 25 cases have been performed worldwide with encouraging results ( 1 ) . as with any novel and previously developed procedure , many questions have been posed and many concerns have emerged . for example , what is an acceptable risk - benefit ratio , and what are the indications for transplantation ? today , the number of facial transplantations remains small ; however , the total number of allograft recipients , including those receiving hands , faces , bones , joints , and abdominal walls , has surpassed 150 , and more than 10 yr of follow - up data are available for some of these patients ( 2 ) . the indications for facial transplantation require considerable discussion . although many superb techniques and solutions exist , all reconstructive surgeons agree that specific unmet needs and limitations also exist . bullet injuries and severe burn trauma seem to be definite and primary indications for transplantation , as these injuries include severe skin damage , tissue loss , and disfigurement ( 3 ) . however , reconstructive options , multiple conventional surgical reconstructive procedures , and even very sophisticated supermicrosurgical techniques can not facilitate recovery and thus reveal the limits of autologous tissue . full or partial facial transplantation can be selected as the surgical strategy according to the anatomic deficiencies . inequitable patient characteristics such as immunosensitization or mental illness can present hurdles to such surgeries ( 4 ) . the transplantation team should thoroughly examine the previous medical histories of blood transfusions or cadaveric skin grafts . psychological issues such as major depressive , psychotic , cognitive , and behavioral disorders represent another contraindication because of problems with a postoperative loss of control and cooperation . postoperative immune therapy is associated with many harmful complications such as renal toxicity , malignancy , diabetes , and metabolic disorders . ultimately , patients should decide whether the benefits of facial transplantation outweigh the risks of surgery and immunosuppression . each practicing physician has the duty and responsibility to provide his / her patients with the best treatment options available , and patients should be allowed to decide for themselves whether to accept these risks and proceed with cta . in most asian countries , patients and their families may experience feelings of guilt regarding the procedure of facial harvesting from the donor patient . this guilt acts as another major hurdle that is not as prominent in the usa and european countries . after donor facial harvesting is completed , a prefabricated facial mask should be applied to the facial deficit area ( 5 ) . the recipient might experience problems with identity because of the expected transformation resulting from the transplantation surgery . anesthesia for the long facial transplantation procedure requires advanced planning for airway management , vascular access , anesthetic technique , and fluid management . the preparation and grafting phases are highly hemorrhagic ( > 1 blood volume ) and therefore massive transfusion is required ( 6 ) . during facial allograft transplantation , the anesthesiologist must be prepared for a long period of anesthesia with rapid blood loss following graft reperfusion . severe postoperative graft edema is present in most patients . in the intensive care unit ( icu ) , most patients encounter complications such as renal insufficiency , acute respiratory distress syndrome , and jugular thrombosis . opportunistic bacterial infections are also an issue in these highly immunosuppressed patients during the postoperative period . the anesthesia team should be involved in the early planning phase and should have access to the donor 's detailed medical history ; alternative anticoagulation treatments for the donor and recipient have been suggested to avoid possible complications . craniofacial and orthognathic considerations should be emphasized with respect to functional effects when planning and executing facial transplants that include both bone and soft tissue elements . these steps are taken to restore the normal anatomy by fixing the face in a proper relationship with the skull base . traditional orthognathic planning via cephalometric parameters yields the most anatomical restoration of occlusion , speech , and airway function ( 7 ) . typical candidates have extremely complex vascular anatomies due to severe injury and/or multiple prior reconstructive attempts ; therefore , each procedure is uniquely determined according to the candidate 's defects and vascular anatomy . ct angiography vascular mapping has demonstrated the clinical relevance of imaging , the angiosome concept , and noninvasive key vessel delineation as well as the current controversies related to the vascular anastomoses . the facial artery is the main arterial supply to the facial skin envelope and serves as the main pedicle for a number of facial flaps , including the facial transplant graft ( 8) . a careful vascular workup prior to facial transplantation and a unipedicled flap procedure are therefore essential . previously , surgical planning software was used to create specific facial defects ( mandibular , midface , or large ) in recipient models , followed by restoration using allografts extracted from the donor models . most immunosuppression protocols are triple - therapy regimens , which comprise tacrolimus ( fk-506 ) , mycophenolate mofetil , and prednisolone . the initial functional and aesthetic outcomes have been very encouraging , and good motor and sensory recovery and improved important facial functions have been observed . phonation recovery has been impressive and has allowed patients to talk , smile , chew , swallow , and blow normally ( 9 ) . as predicted , episodes of acute rejection have been common but are easily controlled with increased systemic immunosuppression treatments . all candidates are fully informed with regard to the risks of cytomegalovirus / infection transmission and the institution of aggressive anti - viral , bacterial , and fungal prophylaxis . despite some long - term complications , which are similar to the complications reported after solid organ transplantation , the patients have been generally satisfied with their new faces , and the results have led to the recovery of some social interactions . the psychological improvements have been remarkable and have resulted in the reintegration of patients into the outside world , social networks , and even the workplace ( 10 ) . cellular therapy in the context of transplantation can significantly benefit the allograft survival and reduce the healing time . the molecular characterizations of these cells and the mechanisms associated with their participation in allograft acceptance and rejection will contribute to the future of modern transplantology . anatomical research , immunosuppression , and immune tolerance are important topics in cta research . before initiating human facial transplantation in any country , the preclinical laboratory results should be determined and animal surgeries performed for 2 reasons : first , to develop an immune tolerance strategy and second , to provide surgical simulation opportunities . because these fields do not target life - saving organs , however , without this presurgical preparation , it is difficult to obtain appreciation and compassion from other scientists and the population . review articles about transplantation immunology as well as a few research papers about topical immunosuppressive agents , immune tolerance strategies that incorporate cell culture , and pretreatments have been published ( 11,12,13 ) additionally , a few preclinical animal allotransplantation studies using rat , rabbit , dog , and cadaver models have been published ( 14,15,16,17,18,19 ) . surgical simulation opportunities in medium to large - sized animals are very important for transplantation surgeons . although the facial transplantation technique has been described as similar to the currently used surgical methods , this is not true in reality . experience with large animal transplantation research can lead to an understanding of the considerable existing differences . in any country , it is very difficult and challenging to establish a facial transplantation program . the development of such a program in korea would involve a successful collaboration between a strong project leader with a vested clinical research interest , a multidisciplinary team of investigators , an institutional review board ( irb ) , the korean network for organ sharing ( konos ) , and a special fundraiser ( 20 ) as this program far exceeds the presently occurring tasks in plastic surgery departments . there had been intense debate regarding the risks and benefits of this type of experimental surgery prior to the start of the facial transplant program . a sound research protocol , solid infrastructure , expert personnel , and adequate funding will be required before launching human applications . to date , only a few active facial transplantation programs have been implemented worldwide in areas with growing interests . the members of the facial transplantation team responsible for carrying out the protocol will include a team leader , a program manager / coordinator , clinical and rehabilitation specialists , social workers , bioethicists , nurses , and administrative staff . the demonstrated experience of this team indicates that patients will receive optimal care both before and after facial transplantation via the collaborations , creativity , and unique multidisciplinary approaches of the members . the establishment of this process can be slow and lengthy ; therefore , the project leader must strive to maintain the enthusiasm and continue to drive the project forward ( 21 ) . many korean surgeons expected that this program would begin at an earlier time point ; however , there remain a few hurdles to overcome . first , facial transplantation should be categorized as a new medical technology and protected under the organ transplantation law with respect to facial harvesting from a brain - dead patient . second , during the donor harvest , close and intimate coordination is necessary between the teams harvesting the face and those harvesting other organs such as the heart , liver , kidney , and cornea . the related situations and circumstances differ between countries ; therefore , each program must find its own way . the risk / benefit balance must be weighed carefully by each program and individually for each patient . our duty as doctors and scientists is to collect and report the informative data and knowledge needed to develop a social consensus regarding these important issues . this will only be possible through candid discussions and presentations of the strategic protocols among many interested stakeholders . the costs associated with facial transplantation are higher than those associated with heart or any other solid organ transplantation and are 2-fold higher than the costs associated with liver transplantation ( 22 ) . this cost might decrease as the surgical teams gain experience , which might also reduce the requirement for postoperative intensive care and the overall hospital stay durations . the 2 largest areas of cost utilization are surgery and nursing , followed by anesthesia and pharmacy . the cost of facial transplantation is similar to that of multiple conventional reconstructions ( 23 ) . although the cost of facial transplantation is considerable , the alleviation of psychological and physiological suffering , exceptional functional recovery , and fulfillment of long - lasting hopes for social reintegration may be priceless . the overall early outcomes of the facial transplant program have been generally more positive than many had predicted . dissemination of the outcomes and ongoing refinement of the process might eventually allow facial transplantation to become a first - line reconstructive option for patients with extensive facial disfigurements . facial transplantation has the unique potential to restore the facial form and function in patients with severe facial defects . this procedure represents the most comprehensive type of transplantation performed to date . to effectively address this challenging surgery , the establishment of a facial transplantation program is complicated by multiple problems concerning the various involved medical fields , administration , society , ethics , and laws ; however , if we cooperate , this paradigm change in facial reconstruction can deliver new hope to the few patients with no other alternatives . the establishment strategy involves the foundation of a basic science laboratory , the cultivation of a supportive institutional clinical environment , and the innovative application of technologies . patients should be extensively educated about the risks and benefits of facial transplantation and then allowed to act as the primary decision - makers . improving patients ' quality of life is a major goal of plastic surgery , and facial transplantation can help us to accomplish this goal . | severely disfiguring facial injuries can have a devastating impact on the patient 's quality of life . during the past decade , vascularized facial allotransplantation has progressed from an experimental possibility to a clinical reality in the fields of disease , trauma , and congenital malformations .
this technique may now be considered a viable option for repairing complex craniofacial defects for which the results of autologous reconstruction remain suboptimal . vascularized facial
allotransplantation permits optimal anatomical reconstruction and provides desired functional , esthetic , and psychosocial benefits that are far superior to those achieved with conventional methods . along with dramatic improvements in their functional statuses ,
patients regain the ability to make facial expressions such as smiling and to perform various functions such as smelling , eating , drinking , and speaking .
the ideas in the 1997 movie " face / off " have now been realized in the clinical field .
the objective of this article is to introduce this new surgical field , provide a basis for examining the status of the field of face transplantation , and stimulate and enhance facial transplantation studies in korea . |
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cystic fibrosis ( cf ) , caused by mutations in the cystic fibrosis transmembrane conductance regulator ( cftr ) , is characterized by a multiorgan pathology affecting the upper and lower airway , gastrointestinal and reproductive tracts , and endocrine system ( riordan et al . , 1989 ; collins , 1992 ; rowe et al . , 2005 ; cutting , 2015 ) . cf is one of the most common lethal autosomal - recessive diseases , with a prevalence of one in 3500 in the united states and one in 2500 in the european union ( farrell , 2008 ; pettit and fellner , 2014 ) . lack of functional cftr expression at the apical membrane of secretory epithelia results in defective cl and bicarbonate secretion , coupled to enhanced na absorption and mucus secretion , which in airway epithelia leads to dehydration and acidification of the airway surface liquid ( tarran et al . , 2001 ; chen et al . , 2010 ; as a consequence , impaired mucociliary clearance provokes recurrent infection and uncontrolled inflammation culminating in lung damage , which is the primary cause of morbidity and mortality in cf ( ratjen and doring , 2003 ; boucher , 2007 ; stoltz et al . , 2015 ) . cftr is member of the atp - binding cassette ( abc ) subfamily c ( abcc7 ) ( kerr , 2002 ) . it consists of two homologous halves , each containing a hexa - helical membrane - spanning domain ( msd1 and msd2 ) and a nucleotide - binding domain ( nbd1 and nbd2 ) that are connected by an unstructured regulatory domain ( riordan , 1993 ; riordan et al . , cf is caused by 2000 mutations in the cftr gene with a wide range of disease severity ( www.genet.sickkids.on.ca/home.html ; www.cftr2.org ; sosnay et al . , 2013 ) , which is further influenced by modifier genes ( collaco and cutting , 2008 ; cutting , 2010 ) and by the environmental and socioeconomic status of patients ( schechter et al . taylor - robinson et al . , 2014 ; kopp et al . , the first classification of cf mutations into four classes according to their primary biological defect was proposed by welsh and smith in a landmark paper ( welsh and smith , 1993 ) . currently , six major classes are distinguished ( rowe et al . , 2005 ; zielenski and tsui , 1995 ) ( figure 1 ) . traditional classification of cf mutations based on their cellular phenotype . class i : protein synthesis defect ; class ii : maturation defect ; class iii : gating defect ; class iv : conductance defect ; class v : reduced quantity ; and class vi : reduced stability . er , endoplasmic reticulum ; tgn , trans - golgi network . class i encompasses frameshift , splicing , or nonsense mutations that introduce premature termination codons ( ptc ) , resulting in severely reduced or absent cftr expression . class ii mutations lead to misfolding , premature degradation by the endoplasmic reticulum ( er ) quality - control system , and impaired protein biogenesis , severely reducing the number of cftr molecules that reach the cell surface . class iii mutations impair the regulation of the cftr channel , resulting in abnormal gating characterized by a reduced open probability . class iv mutations alter the channel conductance by impeding the ion conduction pore , leading to a reduced unitary conductance ( sheppard et al . class v mutations do not change the conformation of the protein but alter its abundance by introducing promoter or splicing abnormalities ( highsmith et al . class vi mutations destabilize the channel in post - er compartments and/or at the plasma membrane ( pm ) , by reducing its conformational stability ( haardt et al . , 1999 ) and/or generating additional internalization signals ( silvis et al . , 2003 ) . this results in accelerated pm turnover and reduced apical pm expression ( haardt et al . , 1999 ; silvis et al . , the disease liability is unknown , but efforts are under way to assess their functional consequence and clinical severity ( www.cftr2.org ; sosnay et al . , 2013 ) . defining the cellular and molecular pathology of cftr mutations proved to be invaluable for development of small - molecule compounds targeting the underlying defect(s ) in cf . the fact that some cftr variants carrying class iii or iv mutations can be expressed at the apical membrane of secretory epithelia at a density similar to that of the wild - type protein , although they are functionally impaired ( e.g. , g551d ) , led to the development of gating potentiators that increase the open probability and thereby the pm chloride conductance ( yang et al . , 2003 ) . food and drug administration approved for cf treatment ; it directly targets the gating defect of the class iii mutation g551d - cftr ( van goor et al . , 2009 ) . this compound was developed by vertex pharmaceuticals in conjunction with cystic fibrosis foundation therapeutics , inc . ( cfft ) , and shows remarkable clinical benefit in patients carrying the mutation in either one or two alleles ( van goor et al . the approval of vx-770 was extended to eight additional class iii mutations ( g178r , s549n , s549r , g551s , g1244e , s1251n , s1255p , and g1349d ) ( yu et al . , 2012 ; vertex , 2014a ) and recently to the class iv mutation r117h ( vertex , 2014b ) . the prototypical class ii mutation , f508-cftr ( phe508del ) , elicits a complex folding defect that compromises both nbd1 stability and the channel s cooperative domain assembly ( du and lukacs , 2009 ; du et al . , 2005 ; mendoza et al . , 2012 ; rabeh et al . , for many years , large - scale efforts have been under way to isolate correctors that act as pharmacological chaperones by directly binding to and promoting the biogenesis of class ii cftr mutations . the most promising corrector compound at present , vx-809 ( lumacaftor ) , partially reverts the f508-cftr functional expression defect by stabilizing the nbd1-msd1/2 interface ( farinha et al . okiyoneda et al . , 2013 ; ren et al . , 2013 ) , leading to a marked correction from 3 to 15% of wild - type channel activity in vitro ( van goor et al . , 2011 ) . a clinical trial , however , failed to observe significant clinical benefit in homozygous f508-cftr patients ( clancy et al . , 2012 ) . acute addition of vx-770 to vx-809-corrected f508-cftr doubled the pm activity in vitro ( van goor et al . , 2011 ) , and the combination therapy showed modest but significant clinical improvement ( boyle et al . , 2014 ; wainwright et al . , the combination treatment has been approved for cf patients 12 years and older with two copies of the f508 mutation ( vertex , 2015 ) . other class ii mutations that can be corrected by vx-809 in vitro include e56k , p67l , e92k , r170 g , l206w , v232d , f508 g , and a561e ( caldwell et al . , 2011 ; okiyoneda et al . , 2013 ; ren et al . , 2013 ; veit et al ribosomal read - through allows synthesis of full - length cftr carrying class i mutations . to this end , ataluren ( ptc124 ) was developed as a drug that promotes near - cognate aminoacyl - trna incorporation at ptcs ( lentini et al . ataluren partially restores g542x - cftr ( class i ) expression in a mouse model and modestly corrects cftr function in nasal epithelia in patients with class i mutations ( du et al . , 2008 ; sermet - gaudelus et al . , 2010 ; 2011 ) . in a recent phase 3 clinical trial , however , ataluren treatment failed to produce significant clinical benefit , perhaps due to an adverse drug drug interaction with tobramycin , which is a commonly administered , inhaled antibiotic used to treat lung infections in cf patients ( kerem et al . , the efficacy of available monotherapies for some mutant alleles , which have been designated as class i , class ii , or class iii / iv mutations , is currently limited . thus comprehensive mapping of the multiple molecular defects caused by a single or combination of mutant alleles could offer considerable advantage for improving therapeutic interventions and for future development of drug combinations . in the following list , we present a subset of mutations that display combinatorial molecular defects . f508 : the most prevalent class ii mutation impairs cftr conformational maturation and leads to its targeting for premature er - associated degradation ( cheng et al . , 1990 ; cyr , 2005 ; kim and skach , 2012 ; lukacs et al . , 1994 ) . however , f508-cftr molecules that either constitutively or following rescue procedures escape the er quality control and accumulate at the pm of airway epithelia exhibit a channel - gating defect , which is a hallmark of class iii mutations ( dalemans et al . , 1991 ) , as well as accelerated turnover in post er compartments and at the pm , a class vi mutation characteristic ( lukacs et al . , 1993 ) . unless the folding and conformational dynamics of the rescued f508-cftr are fully restored to that of the wild - type protein by pharmacological treatment , this mutation remains partially defective and requires correction of its gating and/or peripheral stability defect . rescue of the gating defect can be achieved with potentiators ( e.g. , vx-770 ) ( van goor et al . , 2009 ) . peripheral stabilization of the f508-cftr could be attained by 1 ) the peptide inhibitor ical36 ( cushing et al . , 2010 ) , 2 ) preventing post - golgi ubiquitination ( fu et al . , 2015 ; okiyoneda et al . , 2010 ) , 3 ) restoring autophagosome formation ( luciani et al . , 2012 ) , or 4 ) modulating cellular protein homeostasis ( hutt et al . , 2010 ) . thus the most common mutant has multiple defects that extend beyond the features of a class ii mutation.w1282x : this ptc represents a class i mutation , though recent studies suggest a more complex phenotype . first , the level of the w1282x transcript is reduced by nonsense - mediated rna decay ( hamosh et al . , 1992 ; second , the ptc deletes part of the nbd2 , which likely compromises nbd1-nbd2 dimerization and w1282x - cftr folding and activity . moreover , if the primary defect is corrected either with spontaneous or drug - induced read - through , some of the fully translated channel will contain nonconservative amino acid substitutions . these missense mutations may cause structural defects ( class ii characteristic ) , as suggested by the phenotype of cf patients with a missense mutation at the w1282 residue ( faucz et al . , 2007 ; ivaschenko et al . , 1993 ; visca et al . , 2008 ) , as well as a gating defect ( class iii characteristic ) , which can be inferred based on w1282x - cftr channel activation after exposure to vx-770 ( xue et al . , 2014).p67l : p67l is a mild class ii mutation that results in attenuated cftr biogenesis , as indicated by the reduced ratio between post - er complex glycosylated ( band c ) and er - resident core - glycosylated protein ( band b ) ( ren et al . , 2013 ; sosnay et al . , 2013 ; van goor et al . , 2014 ) . treatment with the corrector vx-809 increases the abundance of the complex - glycosylated form and pm density to nearly the level of wt - cftr ( ren et al . , 2013 ; veit et al . , 2014 ) . however , the mutant channel is also sensitive in vitro to potentiator treatment ( a class iii characteristic ) , both in the presence and absence of corrector ( van goor et al . accordingly , treatment with vx-770 ameliorated the cf lung disease in a heterozygous p67l/f508 patient ( yousef et al . , 2015).r117h : this mutation in conjunction with the 5 t variant in the polythymidine tract in intron 8 was originally categorized as a class iv mutation , but it also exhibits a gating defect ( class iii trait ) that , at least in part , can be rectified by vx-770 treatment ( sheppard et al . , 1993 ; van goor et al . , 2014 ) . the r117h mutation also results in reduced complex - glycosylated cftr expression , which is a class ii characteristic ( fanen et al . , 1997 ; this potentially explains the limited success of vx-770 treatment in patients carrying this mutation ( char et al . , 2014 ; moss et al . , f508 : the most prevalent class ii mutation impairs cftr conformational maturation and leads to its targeting for premature er - associated degradation ( cheng et al . , 1990 ; cyr , 2005 ; kim and skach , 2012 ; lukacs et al . , 1994 ) . however , f508-cftr molecules that either constitutively or following rescue procedures escape the er quality control and accumulate at the pm of airway epithelia exhibit a channel - gating defect , which is a hallmark of class iii mutations ( dalemans et al . , 1991 ) , as well as accelerated turnover in post er compartments and at the pm , a class vi mutation characteristic ( lukacs et al . , 1993 ) . unless the folding and conformational dynamics of the rescued f508-cftr are fully restored to that of the wild - type protein by pharmacological treatment , this mutation remains partially defective and requires correction of its gating and/or peripheral stability defect . rescue of the gating defect can be achieved with potentiators ( e.g. , vx-770 ) ( van goor et al . , 2009 ) . peripheral stabilization of the f508-cftr could be attained by 1 ) the peptide inhibitor ical36 ( cushing et al . , 2010 ) , 2 ) preventing post - golgi ubiquitination ( fu et al . , 2015 ; okiyoneda et al . , 2010 ) , 3 ) restoring autophagosome formation ( luciani et al . , 2012 ) , or 4 ) modulating cellular protein homeostasis ( hutt et al . , 2010 ) . thus the most common mutant has multiple defects that extend beyond the features of a class ii mutation . w1282x : this ptc represents a class i mutation , though recent studies suggest a more complex phenotype . first , the level of the w1282x transcript is reduced by nonsense - mediated rna decay ( hamosh et al . , 1992 ; second , the ptc deletes part of the nbd2 , which likely compromises nbd1-nbd2 dimerization and w1282x - cftr folding and activity . moreover , if the primary defect is corrected either with spontaneous or drug - induced read - through , some of the fully translated channel will contain nonconservative amino acid substitutions . these missense mutations may cause structural defects ( class ii characteristic ) , as suggested by the phenotype of cf patients with a missense mutation at the w1282 residue ( faucz et al . , 2007 ; ivaschenko et al . , 1993 ; visca et al . , 2008 ) , as well as a gating defect ( class iii characteristic ) , which can be inferred based on w1282x - cftr channel activation after exposure to vx-770 ( xue et al . , 2014 ) . p67l : p67l is a mild class ii mutation that results in attenuated cftr biogenesis , as indicated by the reduced ratio between post - er complex glycosylated ( band c ) and er - resident core - glycosylated protein ( band b ) ( ren et al . , 2013 ; sosnay et al . , 2013 ; treatment with the corrector vx-809 increases the abundance of the complex - glycosylated form and pm density to nearly the level of wt - cftr ( ren et al . , 2013 ; veit et al . , 2014 ) . however , the mutant channel is also sensitive in vitro to potentiator treatment ( a class iii characteristic ) , both in the presence and absence of corrector ( van goor et al . , 2014 ; veit et al . , 2014 ) . accordingly , treatment with vx-770 ameliorated the cf lung disease in a heterozygous p67l/f508 patient ( yousef et al . , 2015 ) . r117h : this mutation in conjunction with the 5 t variant in the polythymidine tract in intron 8 was originally categorized as a class iv mutation , but it also exhibits a gating defect ( class iii trait ) that , at least in part , can be rectified by vx-770 treatment ( sheppard et al . the r117h mutation also results in reduced complex - glycosylated cftr expression , which is a class ii characteristic ( fanen et al . , 1997 ; this potentially explains the limited success of vx-770 treatment in patients carrying this mutation ( char et al . , 2014 ; moss et al . , 2015 ) . we propose a modification of the current classification scheme , which would entail permutations of the traditional class i vi cf mutations . this expanded classification of the major mechanistic categories ( welsh and smith , 1993 ; zielenski , 2000 ; rowe et al . , 2005 ) accommodates the unusually complex , combinatorial molecular / cellular phenotypes of cf alleles . it consists of 31 possible classes of mutations , including the original classes i , ii , iii / iv , v , and vi , as well as their 26 combinations , as depicted in the venn diagram shown in figure 2 . for the sake of simplicity , class iii and iv mutations , representing functional ( gating and conductance , respectively ) defects , are combined . for example , according to the expanded classification , g551d will be designated as a class iii mutation as before ( welsh and smith , 1993 ) , while f508 will be classified as class ii vi , w1282x as class i ii iii vi , p67l as class ii iii / iv , reflecting the composite defects in mutant cftr biology ( figure 2 and table 1 ) . a recent study by vertex pharmaceuticals successfully demonstrated that 24 of 54 tested missensse mutations display both a processing ( class ii ) and gating ( class iii ) defect in the fischer rat thyroid epithelial expression system ( van goor et al . , 2014 ) . characterization of several rare cf mutations is ongoing in laboratories of the cftr2 consortium , the cftr folding consortium , cfft , vertex pharmaceuticals , and many others ( caldwell et al . , 2011 ; yu et al . , 2012 ; sosnay et al . , 2013 ; harness - brumley et al . , 2014 ; hong et al . , 2014 ; van goor et al . , 2014 ; . this work will likely provide further examples of combinatorial mechanistic defects exhibited by cf mutants . in - depth analysis of the biology of cf mutants distinguishes them according to their complex molecular pathology and suggests drug combinations for treatment of different patient populations . ( cutting , 2015 ) , will pave the way to personalized medicine in cf . however , reliable prediction of the responsiveness of a mutant phenotype to pharmacotherapy could be challenging and is dependent on the cellular model system ( pedemonte et al . , 2010 ) . emerging evidence also suggests that the efficacy of approved and preclinical drugs may vary with different mutations within the same class . for example , while nearly complete processing correction of p67l- and r170g - cftr ( class ii ) was achieved with vx-809 treatment ( okiyoneda et al . 2014 ) , vx-809 only partially reversed the folding defect of some other class ii mutants ; for example , n1303k and f508 ( okiyoneda et al . , 2013 ; awatade et al . , this differential susceptibility to correction is attributed to the nature of the primary folding / structural defect . according to one hypothesis , robust folding correction of f508-cftr requires corrector combinations to avert its nbd1-msd1/2 interface and nbd1 stability defects ( mendoza et al . , 2012 ; rabeh et al . , 2012 ; he et al . , 2013 ; okiyoneda et al . , 2013 ) . the n1303k mutation in nbd2 was not rescued by vx-809 , and only modest processing was observed by targeting both the nbd1/msds and nbd2 interfaces with c4 and c18 ( a vx-809 analogue ) ( okiyoneda et al . , 2013 ; rapino et al . , although r347h- and t338i - cftr cause severe functional defects with no or modest loss of protein expression , only r347h - cftr is potentiated by vx-770 to near wild type like conductance ( van goor et al . , 2014 ) . likewise , the p5 potentiator activates f508-cftr , but it has no effect on g551d - cftr chloride permeation ( yang et al . , 2003 ) . thus identification of mutation - specific novel potentiators or their combinations may further optimize channel rescue for specific class iii / iv mutations . additive enhancement of g551d - cftr activity by the combination of the potentiators genistein and curcumin supports the feasibility of combining potentiators ( yu et al . , 2011 ) . likewise , we envision that mutation - specific read - through drugs will ultimately need to be combined with other correctors and potentiators , based on the pleiotropic defects associated with this class of mutations ( as illustrated for w1282x above ) . the ultimate goal of theratyping is to achieve optimal correction of a specific mutant defect by selecting the most efficacious cftr modulator(s ) , including correctors(s ) , potentiator(s ) , and/or read - through drugs , or a combination of these drugs . based on accumulating observations , however , mechanistic subdivisions of some of the major classes of mutations ( classes i , ii , and iii ) may be necessary to further improve the success of drug - selection strategies . this will facilitate the theratyping of cf alleles and their combinations and expedite the identification and approval process for combination therapies . theratyping has already proven successful in identifying class iii mutations that are responsive to vx-770 ( yu et al . , 2012 ) , leading to the approval of this drug for eight rare mutations besides g551d ( vertex , 2014a ) . in fact , the results of large - scale theratyping could be overlaid as a third dimension on the venn diagram presented in figure 2 . thus , during the 22 years following the initial classification of cf mutations ( welsh and smith , 1993 ) , our understanding of the molecular complexity of cf alleles has evolved remarkably , establishing the need for an advanced mutation classification scheme in conjunction with personalized cf therapy . | more than 2000 mutations in the cystic fibrosis transmembrane conductance regulator ( cftr ) have been described that confer a range of molecular cell biological and functional phenotypes .
most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis ( cf ) with variable disease severity . based on the molecular phenotypic complexity of cftr mutants and their susceptibility to pharmacotherapy
, it has been recognized that mutations may impose combinatorial defects in cftr channel biology .
this notion led to the conclusion that the combination of pharmacotherapies addressing single defects ( e.g. , transcription , translation , folding , and/or gating ) may show improved clinical benefit over available low - efficacy monotherapies .
indeed , recent phase 3 clinical trials combining ivacaftor ( a gating potentiator ) and lumacaftor ( a folding corrector ) have proven efficacious in cf patients harboring the most common mutation ( deletion of residue f508 , f508 , or phe508del ) .
this drug combination was recently approved by the u.s .
food and drug administration for patients homozygous for f508 .
emerging studies of the structural , cell biological , and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different cftr alleles .
establishment of a set of combinatorial categories of the previously defined basic defects in cf alleles will aid the design of even more efficacious therapeutic interventions for cf patients . |
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beginning 50 years ago , the framingham heart study forever changed our approach to predict and prevent coronary heart diseases by identifying major risk factors for myocardial infarction ( mi ) [ 14 ] and developing risk assessment function based on the major risk factors [ 5 , 6 ] . since then many other cardiovascular diseases ( cvds ) risk prediction functions or scores have been developed [ 712 ] . all these previous prediction models require laboratory and clinical examinations and target at high - risk individuals who visit a clinic . a simplified risk assessment tool is , however , required for cvd prevention through population approach that aims to reduce the risk factors at population levels through lifestyle and environment changes . such a simplified tool is also useful in less developed countries where the health care resources are limited but the largest increase in cvd will occur . recently , an american study showed that a model that uses non - laboratory - based risk factors predicted cvd events as accurately as one that relied on laboratory - based values . this has , however , not been confirmed yet by other studies . based on data of the national finrisk study , we developed a model which does not include laboratory variables and blood pressure measurement and compared it to the models which includes these measurements with regard to their predictive ability of first events of coronary heart disease ( chd ) . the national finrisk study has been carried out every 5th year since 1972 to monitor cvd risk factor levels and evaluate health strategies to lower cvd events in finland based on random population representative samples . cohorts first examined in 1982 , 1987 , 1992 , 1997 , and 2002 , including about 14815 finnish men and 16617 finnish women aged 2464 years , were followed up using national registers until 31 december of 2007 . the maximum follow - up length was 5.9 years for the 2002 cohort and 25.9 years for the 1982 cohort . the survey methods followed the standard protocol ( http://www.ktl.fi/ehrm ) , which have been described in details elsewhere . first ever chd events were ascertained through computerized record linkage of the unique national i d numbers of the survey participants to the national causes of death registry and the national hospital discharge registry . international classification of disease ( icd ) , 9th revision ( 10th revision ) , was used for the classification of either the fatal or the nonfatal events . icd codes of 410414 ( i20i25 ) for fatal chd and 410 - 411 ( i21-i22 , i24 ) for nonfatal acute mi were applied . the first nonfatal mi and the first fatal chd events constitute the incident chd events . subjects with a self - reported history of ischemic heart diseases or ascertained from the registers as having ischemic heart diseases before the baseline survey of each study cohort were excluded from the data analysis . cox proportional hazard model analyses were performed to relate factors to the first ever chd events using stata 11.0 . the assumption of the proportionality was tested based on schoenfeld residuals ( phtest ) method for each explanatory variable for each cohort and for men and for women . the results showed the assumption of the proportionality of hazards holds ; p values of the global tests varied from 0.20 to 0.93 . 50 candidate variables , including age , marital status , spouse and children , self - evaluation of one ' personal or family life , education and occupation , stress from work and family , family income , dietary components , physical activities at work and leisure time , tobacco and alcohol consumption , self - consideration of one 's health status and functional capacity , history of diabetes or hypertension or elevated total cholesterol , parents ' history of cardiovascular diseases , body mass index ( bmi ) , and laboratory measurements of total cholesterol and hdl - cholesterol , ratio of total cholesterol to hdl cholesterol ( cho / hdl ) , were examined in either univariable or stepwise multivariable models . a variable list with detailed variable description is presented in the supplemental digital table 1 in supplementary material available online at doi : 11.55/2011/823782 . age , residential areas , survey years , and years of education were fitted in all models . cohort specific baseline survival function ( or rate ) at the mean values of risk factors of the whole study population was estimated . all the continuous variables were logarithmically transferred to improve discrimination and calibration of the models and to minimize the influence of extreme observations . three different cox models were finally constructed : one laboratory based and two non laboratory based . both the chol / hdl and the systolic blood pressure entered into the laboratory based model . bmi did not enter into the laboratory based model , but into the non - laboratory - based models at the absence of the chol / hdl . chol / hdl was removed from the laboratory - based model to form the non - laboratory - based models , and systolic blood pressure was further taken out from the non - laboratory - based model to form the layperson - oriented models . the regression coefficient of each factor was estimated based on pooled data of all five cohorts , but the cohort - specific baseline survival rates at 10 years derived from the cox models were used to calculate the 10-year absolute risk for each cohort using the formula described in the framingham heart study . discriminationreceiver operating characteristic ( roc ) curves of the 10-years absolute chd risk calculated based on the estimates of the three different cox models were plotted . areas under the curves ( aucs ) were calculated to evaluate discrimination of the models , and the differences in aucs were tested based on c statistic . receiver operating characteristic ( roc ) curves of the 10-years absolute chd risk calculated based on the estimates of the three different cox models were plotted . areas under the curves ( aucs ) were calculated to evaluate discrimination of the models , and the differences in aucs were tested based on c statistic . calibrationcalibration , a measure of agreement between observed and predicted chd events within 10 years , was made for the layperson - oriented model . the 10-year absolute chd risk was used to divide individuals into deciles of the predicted risk . the mean of the predicted probability of having the chd in each decile and the observed probability obtained using kaplan - meier method was plotted . calibration , a measure of agreement between observed and predicted chd events within 10 years , was made for the layperson - oriented model . the 10-year absolute chd risk was used to divide individuals into deciles of the predicted risk . the mean of the predicted probability of having the chd in each decile and the observed probability obtained using kaplan - meier method was plotted . the net reclassification improvement ( nri)nri was calculated according to pencina et al . to quantify the improvement in the right reclassification of the layperson - oriented model against the other two models in either event or non - event groups . according to the guidelines for cvd prevention from different major organizations or study groups based on 10-year absolute risk assessment [ 1820 ] , 10-year chd risk categories of < 5% , 5%9% , 10%19% , and 20% were made . if a model moves a chd event from a low risk category to a high one , the reclassification was considered right and the mode prediction was improved compared with the reference model . a downgrade for the non - events nri was calculated according to pencina et al . to quantify the improvement in the right reclassification of the layperson - oriented model against the other two models in either event or non - event groups . according to the guidelines for cvd prevention from different major organizations or study groups based on 10-year absolute risk assessment [ 1820 ] , 10-year chd risk categories of < 5% if a model moves a chd event from a low risk category to a high one , the reclassification was considered right and the mode prediction was improved compared with the reference model . a downgrade for the non - events bmi and the prevalence of diabetes have increased while blood pressure and unfavourable lipid profiles decreased during the last 35 years in finland as reported . altogether 1131 ( 7.6% ) first ever chd events in men and 561 ( 3.4% ) in women were observed during the followup . individuals who developed chd events were older and had higher bmi , blood pressure and worse lipid profiles than those who were free of chd at the end of the followup ( table 2 ) . the proportion of people with prior history of diabetes or hypertension , parent(s ) suffering from mi , and self - perceived worse health conditions were also higher in the former than in the latter . current smoking in men and lower education level in both genders were related to incidence of chd . the regression coefficients of the predictors and the baseline survival function for each cohort are shown in table 3 . the estimated 10-year mean absolute chd risk were 3.76% , 3.85% , and 3.76% in men for the laboratory based model , the non - laboratory based models with and without blood pressure ; they was 1.25% , 1.37% , and 1.33% , respectively , in women . given a positive test at the risk of 20% , the positive predictive values for 10-years chd incidence were similar between the models ( 22.4% in men and 25.0% in women by the layperson - oriented model , 20.2% and 25.6% by the laboratory - based model and 21.2% and 21.8% by the non - laboratory - based model with systolic blood pressure ) . however , the number of individuals referred to further tests or intervention was largest for the laboratory - based model ( n = 286 ) than for the non - laboratory based models with ( n = 258 ) and without ( n = 209 ) blood pressure measurement . all three predictive models adequately discriminated people with chd events from those without as shown in figure 1 ( the roc curves ) . the auc ( 95% ci ) was 0.823 ( 0.8070.839 ) for the laboratory - based model , 0.808 ( 0.7910.824 ) and 0.803 ( 0.7870.820 ) for the non - laboratory - based models with and without systolic blood pressure in men , and 0.878 ( 0.8560.901 ) , 0.871 ( 0.8480.894 ) , and 0.864 ( 0.8400.887 ) , respectively , in women . the layperson - oriented model gave slightly smaller auc than others ( p < 0.001 for overall comparisons in both genders ) . calibration of the layperson - oriented model showed that the predicted 10-year event rate matched well with the observed event rate across deciles of the predicted risk in men ( = 14.15 , 9df , p = 0.117 ) and in women ( = 12.36 , 9df , p = 0.194 ) ( figure 2 ) . additional calibration was made for each of the five study cohorts at 5 , 10 , 15 , 20 , and 25 years , respectively , for cohorts 2002 , 1997 , 1992 , 1987 , and 1982 . the predicted probability of having chd events at 5 , 10 , 15 , 20 , and 25 years was close to those observed at each of the 6 risk categories as shown in supplemental digital figure s1 . table 4 shows the net reclassifications of individuals with and without events across 10-year risk categories based on different models . the laboratory - based model correctly reclassified individuals in both event and non - event groups , but the overall nri was significant only in men ( 8.1% , p = 0.001 ) not in women ( 2.9% , p = 0.432 ) as compared with the layperson - oriented model . addition of the systolic blood pressure to the layperson - oriented model upgraded both events and non - events , with an overall nri of 3.8% ( p = 0.053 ) in men and 7.1% ( p = 0.017 ) in women . the roc curves and the nri for two additional models that do not contain either the history of diabetes or hypertension are shown in supplemental digital figure s2 and table 2 in supplementary material available online at doi:11.55/2011/823782 . the predictive ability of the models without either of the two was not impaired substantially in terms of discrimination and reclassification of individuals . the performance of the two non - laboratory - based chd risk assessment models is good in terms of discrimination and calibration . addition of the lipid measurements to the non - laboratory - based model slightly improved the reclassification of individuals in men but not in women , whereas addition of the systolic blood pressure into the layperson model improved the reclassification of individuals with the events but overestimated the risk in individuals without events . laboratory tests and blood pressure measurement have been required in most previous prediction models [ 512 , 16 ] . a recent study based on the national health and nutrition examination survey has , however , challenged the traditional way of building the models by showing that laboratory based model including total cholesterol performed no better than the non - laboratory based model requiring bmi rather than total cholesterol for predicting cvd outcomes . our current study lent further support to the approach to simplify the risk prediction model . moreover , we show that a non - laboratory - based model not requiring measurement of the systolic blood pressure performed no worse than that requiring regarding to the prediction of the 10-year chd incidence . the advantage of the simplified model over the traditional ones is that it does not require a clinic visit , and therefore , is both time and cost saving . this is important for cvd prevention using population approach and in the countries where the medical care resources are short and blood pressure measurement is not widely available . in spite of that the guidelines for cvd prevention have been produced by different organizations and study groups [ 1820 ] , most developing countries do not have a systematic screening program for cvd . a doctor has no obligation to estimate a patient 's future cvd risk according to the patient 's current health status . thus there is no additional information rather than the complaint disorders will be discussed and handled during a hospital visiting . the layperson - oriented model as a supplemental tool may promote cvd risk screening and prevention by raising the public awareness of the disease through population approach , which is beyond the original intention to apply a cvd risk prediction score in a clinic setting targeting at high - risk individuals . to include bmi in the model is particularly important in public health considering the recent increase in obesity and diabetes worldwide , and can inform lifestyle changes . incapable to walk 500 meters without rest appeared to be a strong early sign of the future chd in spite of the causes of the inability . thus , either a single component of the model or the integrated risk score estimated based on the model may serve as a trigger leading to early diagnosis and treatment of the chd as well as other comorbid conditions such as hypertension , dyslipidemia and hyperglycemia . it needs , however , to be borne in mind that the layperson - oriented model derived from the finnish population has not been externally validated and might need calibration before it is applied to other populations considering differences in cvd risk among countries . in spite of the differences in absolute risks , relative risks of certain risk factors such as smoking are relatively less variable across countries and age groups . a risk score based on estimate of relative risk has , thus , been suggested but its predictive property needs to be further investigated against the one based on estimate of the absolute risk . strengths of the current study include its large sample size , population - based study design , standard survey protocols and questionnaire , reasonable participation rates and complete followup with first ever hard chd events , including both fatal and nonfatal events . the finnish causes of death register and hospital discharge register , the source of the data on chd events in this study , have high coverage and diagnostic accuracy as reported previously . the weakness of the finrisk study is that the individual 's risk factors were measured only at the baseline examinations and the changes in individual 's risk factor levels during the followup are not known . considering the decline in chd risk and changes in risk factors in the population level in finland during the last 35 years , the birth cohort - specific baseline survival function calculated at the mean values of covariates of the whole study population of all cohorts was used to calculate the absolute chd risk of an individual person . the risk prediction function developed in this way has been shown to fit the data of individual cohort well even though of the cohorts were recruited over a 25-year period . in conclusion , the predictive ability of a layperson - oriented model is similar to the models including laboratory and systolic blood pressure measurements , but it is simple , noninvasive , time and cost saving , easy to use and has great potential to be applied in cvd prevention campaign through population approach . | one laboratory - based and two non - laboratory - based models with and without blood pressure measures are developed based on data of 14815 men and 16617 women aged 2564 years . during the followup 1134 men and 566 women developed coronary heart disease ( chd ) .
the area under the receiver operating characteristic curve ( 95% ci ) for prediction of chd incidence was 0.823 ( 0.8070.839 ) for the laboratory - based model , 0.808 ( 0.7910.824 ) and 0.803 ( 0.7870.820 ) for the non - laboratory - based models with and without systolic blood pressure in men ( p < 0.01 for overall comparison ) , and 0.878 ( 0.8560.901 ) , 0.871 ( 0.8480.894 ) , and 0.864 ( 0.8400.887 ) , respectively , in women ( p < 0.01 ) .
the predicted rates matched well with the observed ones ( p > 0.10 ) . compared with the model without blood pressure ,
the non - laboratory - based model with blood pressure tended to reclassify individuals into the higher risk categories for both event and nonevent groups in both genders .
the study concludes the predictive property of the non - laboratory - based models are good . |
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it has been shown to affect 18% of women and 6.5% of men in united states . worldwide approximately 240 millions of people have an estimated 1.4 billion attacks of migraine each year . severe migraine has been included in the list of global burden of diseases by world health organization ( who ) and it represents a serious health problem both for individuals and society as this disabling disorder has a direct impact on quality of life . effective management of migraine depends on accurate diagnosis ruling out other causes for headache . in 1988 the international headache society published criteria for the diagnosis of a number of different headache types . this is diagnosed when there are at least five headache attacks lasting 4 - 72 hours ( untreated or unsuccessfully treated ) , which has at least two of the four following characteristics : unilateral location , pulsating quality , moderate or severe intensity ( inhibits or prohibits daily activities ) , aggravated by walking stairs or similar routine physical activity during headache at least one of the two following symptoms occur : phonophobia and photophobia , nausea and/or vomiting migraine with aura ( ma ) is diagnosed when : at least two attacks occur fulfilling with at least three of the following : one or more fully reversible aura symptoms indicating focal cerebral cortical and/or brain stem functions , at least one aura symptom develops gradually over more than four minutes , or two or more symptoms occur in succession . no aura symptom lasts more than 60 minutes ; if more than one aura symptom is present , accepted duration is proportionally increased . headache follows aura with free interval of at least 60 minutes ( it may also simultaneously begin with the aura . migraine with typical aura is diagnosed when there is homonymous visual disturbance , unilateral paresthesias and/or numbness , unilateral weakness and aphasia or unclassifiable speech difficulty . pharmacological treatment of migraine has equally important therapeutic and prophylactic components . besides nsaids , opioids , and ergot alkaloids , triptans are the major contributor to the management of acute attack of migraine but producing partial relief or providing consistent response in 50 - 60% of patients . prophylactic treatment is indicated if acute treatment alone is inadequate , patient is experiencing more than two to three attacks per month , when acute therapy is contraindicated , use of abortive medicine more than twice a month , presence of migraine with prolonged aura , hemiplegic migraine , migranous infarction . a variety of diverse pharmacological classes of drugs are in use for prevention of attack . the goal of preventive therapy is to improve patients quality of life by reducing migraine frequency , severity , and duration , and by increasing the responsiveness of acute migraine to treatment . the mechanism of action of most of prophylactic drugs is poorly understood as they have multiple mechanisms . it is postulated that their mode of action converge on twotargets that is inhibition of cortical excitation and resting nociceptive dysmodulation . trials have favoured the use of drugs like -blockers ( propranolol , timolol , metoprolol ) , antidepressant ( amitriptyline ) , anticonvulsants ( divalproex , topiramate , tiagabin , levetiracetam , zonisamide ) as the first - line drugs for prevention but none is effective in all cases and none abolishes the attacks totally . few clinical trials done so far have failed to prove any superiority of one prophylactic agent over the other . despite the availability of a number of drugs for aborting / preventing the migraine , a significant numbers of migraineurs are unable to achieve satisfactory results . most of the drugs are associated with adverse drug reactions ( adrs ) which are common and severe too . this has necessitated the need for searching altogether newer classes of drugs with different mechanisms which may be more efficacious and better tolerated than the already existing ones . some clinical trials and case reports have justified the benefits of ssris , high - dose riboflavin , monteleukast sodium , magnesium , botulinum toxin and coumarin derivatives in migraine.[279 ] this is diagnosed when there are at least five headache attacks lasting 4 - 72 hours ( untreated or unsuccessfully treated ) , which has at least two of the four following characteristics : unilateral location , pulsating quality , moderate or severe intensity ( inhibits or prohibits daily activities ) , aggravated by walking stairs or similar routine physical activity during headache at least one of the two following symptoms occur : phonophobia and photophobia , nausea and/or vomiting migraine with aura ( ma ) is diagnosed when : at least two attacks occur fulfilling with at least three of the following : one or more fully reversible aura symptoms indicating focal cerebral cortical and/or brain stem functions , at least one aura symptom develops gradually over more than four minutes , or two or more symptoms occur in succession . no aura symptom lasts more than 60 minutes ; if more than one aura symptom is present , accepted duration is proportionally increased . headache follows aura with free interval of at least 60 minutes ( it may also simultaneously begin with the aura . migraine with typical aura is diagnosed when there is homonymous visual disturbance , unilateral paresthesias and/or numbness , unilateral weakness and aphasia or unclassifiable speech difficulty . pharmacological treatment of migraine has equally important therapeutic and prophylactic components . besides nsaids , opioids , and ergot alkaloids , triptans are the major contributor to the management of acute attack of migraine but producing partial relief or providing consistent response in 50 - 60% of patients . prophylactic treatment is indicated if acute treatment alone is inadequate , patient is experiencing more than two to three attacks per month , when acute therapy is contraindicated , use of abortive medicine more than twice a month , presence of migraine with prolonged aura , hemiplegic migraine , migranous infarction . a variety of diverse pharmacological classes of drugs are in use for prevention of attack . the goal of preventive therapy is to improve patients quality of life by reducing migraine frequency , severity , and duration , and by increasing the responsiveness of acute migraine to treatment . the mechanism of action of most of prophylactic drugs is poorly understood as they have multiple mechanisms . it is postulated that their mode of action converge on twotargets that is inhibition of cortical excitation and resting nociceptive dysmodulation . trials have favoured the use of drugs like -blockers ( propranolol , timolol , metoprolol ) , antidepressant ( amitriptyline ) , anticonvulsants ( divalproex , topiramate , tiagabin , levetiracetam , zonisamide ) as the first - line drugs for prevention but none is effective in all cases and none abolishes the attacks totally . few clinical trials done so far have failed to prove any superiority of one prophylactic agent over the other . despite the availability of a number of drugs for aborting / preventing the migraine , a significant numbers of migraineurs are unable to achieve satisfactory results . most of the drugs are associated with adverse drug reactions ( adrs ) which are common and severe too . this has necessitated the need for searching altogether newer classes of drugs with different mechanisms which may be more efficacious and better tolerated than the already existing ones . some clinical trials and case reports have justified the benefits of ssris , high - dose riboflavin , monteleukast sodium , magnesium , botulinum toxin and coumarin derivatives in migraine.[279 ] this is diagnosed when there are at least five headache attacks lasting 4 - 72 hours ( untreated or unsuccessfully treated ) , which has at least two of the four following characteristics : unilateral location , pulsating quality , moderate or severe intensity ( inhibits or prohibits daily activities ) , aggravated by walking stairs or similar routine physical activity during headache at least one of the two following symptoms occur : phonophobia and photophobia , nausea and/or vomiting migraine with aura ( ma ) is diagnosed when : at least two attacks occur fulfilling with at least three of the following : one or more fully reversible aura symptoms indicating focal cerebral cortical and/or brain stem functions , at least one aura symptom develops gradually over more than four minutes , or two or more symptoms occur in succession . no aura symptom lasts more than 60 minutes ; if more than one aura symptom is present , accepted duration is proportionally increased . headache follows aura with free interval of at least 60 minutes ( it may also simultaneously begin with the aura . migraine with typical aura is diagnosed when there is homonymous visual disturbance , unilateral paresthesias and/or numbness , unilateral weakness and aphasia or unclassifiable speech difficulty . pharmacological treatment of migraine has equally important therapeutic and prophylactic components . besides nsaids , opioids , and ergot alkaloids , triptans are the major contributor to the management of acute attack of migraine but producing partial relief or providing consistent response in 50 - 60% of patients . prophylactic treatment is indicated if acute treatment alone is inadequate , patient is experiencing more than two to three attacks per month , when acute therapy is contraindicated , use of abortive medicine more than twice a month , presence of migraine with prolonged aura , hemiplegic migraine , migranous infarction . a variety of diverse pharmacological classes of drugs are in use for prevention of attack . the goal of preventive therapy is to improve patients quality of life by reducing migraine frequency , severity , and duration , and by increasing the responsiveness of acute migraine to treatment . the mechanism of action of most of prophylactic drugs is poorly understood as they have multiple mechanisms . it is postulated that their mode of action converge on twotargets that is inhibition of cortical excitation and resting nociceptive dysmodulation . trials have favoured the use of drugs like -blockers ( propranolol , timolol , metoprolol ) , antidepressant ( amitriptyline ) , anticonvulsants ( divalproex , topiramate , tiagabin , levetiracetam , zonisamide ) as the first - line drugs for prevention but none is effective in all cases and none abolishes the attacks totally . few clinical trials done so far have failed to prove any superiority of one prophylactic agent over the other . despite the availability of a number of drugs for aborting / preventing the migraine , a significant numbers of migraineurs are unable to achieve satisfactory results . most of the drugs are associated with adverse drug reactions ( adrs ) which are common and severe too . this has necessitated the need for searching altogether newer classes of drugs with different mechanisms which may be more efficacious and better tolerated than the already existing ones . some clinical trials and case reports have justified the benefits of ssris , high - dose riboflavin , monteleukast sodium , magnesium , botulinum toxin and coumarin derivatives in migraine.[279 ] a new target which has recently caught the attention of researchers in migraine is renin angiotensin system ( ras). ras has neurophysiological , chemical and immunological effects that are relevant to pathophysiology of migraine . this fact directed the scientists to explore the usefulness of angiotensin - converting enzymes inhibitors ( ace inhibitors)/angiotensin ii receptor antagonists in the management of migraine . the use of ace inhibitors as prophylactic agents in headache was first proposed by siceturi in 1981 . dramatic improvement was seen in 50% patients experiencing improvement in headache and 31% had partial relief . , in 1986 . in 1992 , research by paterna et al . , found the utility of captopril in preventing migraine . these initial steps strongly led to further research on the use of these drugs in migraine . there is no clear underlying mechanism of action relating , as the effect produced by them is probably not due to their effects on blood pressure . it has been suggested that the effect of ace inhibitors can be related to their ability to increase norepinephrine and serotonin action on vascular tone . the postulated pharmacological effects which can be relevant in migraine are inhibition of conversion of angiotensin i to angiotensin ii , inhibition of free radicals , alteration of sympathetic activity , increase in prostacyclin synthesis , inhibition of degradation of bradykinin , enkephalin and substance p. acting through angiotensin receptors , angiotensin ii also modulates cerebrovascular flow and has effect on homeostasis , autonomic pathways and neuroendocrine system . angiotensin ii has been found experimentally to influence the tonic modulation of pineal melatonin synthesis . angiotensin ii has been shown to activate neurokin in factor kappa b through at1 and at2 ( angiotensin receptors ) receptors which is associated with increased expression of no synthase . thus angiotensin , by its multifaceted actions , is involved in pathogenesis of migraine providing one additional mechanism by virtue of which ace inhibitors / angiotensin ii receptor antagonists possess prophylactic role in migraine . recently it has been found that genetic basis of migraine contributes a lot to define the role of ace inhibitors / angiotensin ii receptor antagonist in migraine prophylaxis . in human serum , ace levels individuals who are homozygous for dd allele ( deletion allele ) have increased ace activity . migraine is more common in persons with dd allele correlating higher ace activity to more migraine attacks . in a study , it has been seen that incidence of dd genotype in migraine with aura was significantly higher than controls ( 25.9% versus 12.5% , p value < 0.01 ) . studies have shown that ace inhibitors ( enalapril , lisinopril ) as well as angiotensin ii receptor antagonists ( candesartan , telmisartan ) have proved to be effective in reducing frequency as well as severity of migraine attacks with minimal side effects . outcome measures studied in most of trials showed decrease in number days of headache , number of days with migraine , hours with migraine , headache severity index , level of disability , improved quality of life and decrease in consumption of specific or nonspecific analgesics . case series , open label studies , randomized controlled clinical trials and meta - analysis have been done so far evaluating the role of ace inhibitors / angiotensin ii receptor antagonists for prevention of migraine . in a meta - analysis done by etminanet al . the risk of headache was about one - third lower in patients taking an angiotensin ii receptor antagonist than in those taking placebo ( rr 0.69 ; 95% confidence interval [ ci ] : 0.62 to 0.76 ; the test of heterogeneity was negative , p value 0.2 ) . the odds ratio for having a headache per unit dose of the reference drug losartan was 0.81 ( 95% ci : 0.68 - 0.93 ) . relevant studies predicting the clinical efficacy and tolerability of ace inhibitors / angiotensin ii receptor antagonists are summarized in table 1 . clinical studies of ace inhibitors / angiotensin ii receptor antagonists in prophylaxis of migraine results of the above - mentioned studies clearly indicate the effectiveness and safety of ace inhibitors / angiotensin ii receptor antagonists , providing a new hope for chronic migraineurs . a special indication for the use of ace inhibitors and angiotensin ii receptor antagonists is migraineurs with bronchial asthma , intermittent claudication and conduction defects . pregnancy is a known contraindication to the use of these drugs because of their ability to produce teratogenic effects in second and third trimester . ace inhibitors and angiotensin ii receptor antagonists show a potential in prophylactic management of migraine . patients with frequent headaches who do not respond to conventional prophylactic agents or in whom these drugs are contraindicated , trial of ace inhibitors / angiotensin ii receptor antagonists can be useful . their use should be considered as a long - term therapeutic approach to migraine prophylaxis . further assessment by larger studies is warranted in future to evaluate whether the positive effects are shared by all ace inhibitors / angiotensin ii receptor antagonists . | migraine constitutes 16% of primary headaches affecting 10 - 20% of general population according to international headache society . till now
nonsteroidalanti - inflammatory drugs ( nsaids ) , opioids and triptans are the drugs being used for acute attack of migraine .
substances with proven efficacy for prevention include -blockers , calcium channel blockers , antiepileptic drugs and antidepressants .
all the already available drugs have certain limitations .
either they are unable to produce complete relief or 30 - 40% patients are no responders or drugs produce adverse effects .
this necessitates the search for more efficacious and well - tolerated drugs . a new class of drugs like angiotensin - converting enzyme inhibitors ( ace inhibitors ) and angiotensin ii receptor antagonists
have recently been studied for their off label use in prophylaxis of migraine .
studies , done so far , have shown results in favour of their clinical use because of the ability to reduce number of days with headache , number of days with migraine , hours with migraine , headache severity index , level of disability , improved quality of life and decrease in consumption of specific or nonspecific analgesics .
this article reviews the available evidence on the efficacy and safety of these drugs in prophylaxis of migraine and can give physician a direction to use these drugs for chronic migraineurs .
searches of pubmed , cochrane database , medscape , google and clinicaltrial.org were made using terms like ace inhibitors , angiotensin ii receptor antagonists and migraine .
relevant journal articles were chosen to provide necessary information . |
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the silkworm ( bombyx mori ) , domesticated over the last 5000 years from a wild progenitor bombyx mandarina ( 1 ) , is an important source of livelihood for subsistence farmers engaged in silk production in many countries . it is believed to be a central model for lepidopteran genomics and genetics , and second only to fruitfly ( drosophila melanogaster ) ( 2 ) as an insect model for genetic studies ( 3 ) . as many basic physiological processes of insects are conserved through evolution , study of silkworm will help further elucidate the function of gene homologs and facilitate studies of insect domestication , morphogenesis , endocrinology , reproduction , behavior and immunity . bombyx mori has an estimated haploid nuclear genome size of 530 mb ( 4 ) broken into 28 chromosomes . a 3x coverage draft sequence was reported previously ( 5 ) and many expressed sequence tag ( est ) sequences have been released ( 6 ) . many other resources will be generated by the international lepidopteran genome project ( http://www.ab.a.u-tokyo.ac.jp/lep-genome ) . at the beijing genomics institute ( bgi ) , the major genome sequencing center in china the silkworm genome sequence is an important contribution to functional genomics for the silkworm and comparative and functional genomics for lepidopteran species , and will provide a solid foundation for integrating biological information for lepidopteran and even insects in general . in order to facilitate the usage of most up - to - date knowledge about the silkworm genome , we developed the silkworm knowledgebase ( silkdb ) as a highly integrated information system for silkworm data storage , retrieval , visualization and analysis . the current version of silkdb is focused on assembling contigs and anchoring contigs onto scaffolds based on mapped genetic markers and bac - based physical maps . in the process , we have developed software packages for sequence assembly , identification and annotation of genes and transposable elements ( tes ) . we utilize silkworm as a framework genome to organize information for other lepidoptera , which represent a diverse and important group of insect pests in agriculture , so as to bridge the model insect and its family members . silkdb , together with its database , search engine and genome - oriented mapview provides both an information resource and a comparative analysis workbench for genomic research of silkworm and other insects . owing to the complexity and the large - scale nature of the genomic data , the strategy of comprehensive organization and effective management are of essence for successive analyses . in silkdb , we organize the genomic data in three different modules of scaffold / contig , gene / cdna and te classes , and link the data of different modules through genome - oriented mapview . in scaffold module , silkdb contains the 428.7 mb b.mori genomic sequences covering 90.9% of all known silkworm genes . the raw sequences were produced by using a whole genome shotgun ( wgs ) ( 7 ) technique and sequence reads were assembled by using an updated version of our reps software ( 8) . there are 23 156 scaffolds for the 28 chromosomes . the average contig and scaffold sizes , by using n50 statistics , are 12.5 and 26.9 kb , respectively . genomic sequences were annotated for gene content by using bgf ( bgi gene finder ) and database searches against public resources . bgf is a self - developed ab initio program based on genscan ( 9 ) and fgenesh ( 10 ) , and was successfully utilized for our rice genome annotation ( 11 ) . after correction of partial and erroneous predictions , the estimated gene count is 18 510 , which exceeds the 13 379 genes reported for d.melanogaster ( 12 ) . interpro domains ( 13 ) were annotated by interproscan release 7.0 and functional assignments were mapped onto gene ontology ( go ) ( 14 ) . to further the study of silkworm genome biology , we investigated the biologically important genes in comparison with spider and butterfly , such as silk gland , wing patterning , development , immunity and defense , hormones and receptors , etc . besides the 18 510 annotated genes , the gene module hosts a collection of 212 known silkworm genes ( with full - length cdna sequences ) from genbank ( 15 ) , 16 425 est clusters based on our sequencing of 80 470 ests from different b.mori tissues , 554 genbank genes of other lepidoptera and 521 b.mori homologs of other lepidopteran genes . snps mined from the b.mori est sequences ( 16 ) were collected and mapped onto the genome . a set of b.mandarina ests was also produced , clustered and compared with the b.mori dataset for domestication study . genome expansion is believed to be due to te insertions . to explore the increase in genome size from fruitfly ( 116.8 mb ) ( 17 ) to silkworm ( 428.7 mb ) , we applied repeatmasker ( http://www.repeatmasker.org/ ) for identifying tes and tagging te classes . a total of 601 225 tes were identified , most of which are from a single gypsy - ty3-like retrotransposon ( 18 ) . classes of tes and their detailed information are stored in the third module , the te_class module . a simple way for users to access data stored in the silkdb database is through the data module , where users can get an overview of the data content , data statistics and the correlations between each data type . mapview and search engine are two self - developed tools built on top of the database for rapid visualization and querying of the data at many levels . as an efficient visualization tool , mapview currently displays the b.mori genome assembly on the scaffold scale with sequence contigs aligned to , and allows users to browse a series of tracks aligned with the genomic sequence ( figure 1 ) . users may center the map upon a point on the scaffold of interest and expand to obtain a more detailed view of genetic markers , predicted genes , cdnas , est clusters of both the domestic and wild silkworm , and classes of tes . b.mori gene homologs of other lepidoptera are also marked out with distinct color - coding . every sequence record is linked to several display options in mapview system . a text - based tabular report for each element contained in the visualization system cross - referenced links to related database entries , such as interpro , go and genbank , are also provided if available . the silkdb search engine is the entry point for searching the major data types stored in the silkdb . it provides two kinds of searches for users : keyword - based subject search and blast - based homology search , including searches for scaffolds , contigs , genes , cdnas , classes of tes , etc . users can define concrete limitations to extract records that are best suited to their research needs . the front end consists of a set of jsp scripts running on tomcat web server . a large set of java servlets and javabeans mediate the user 's interaction with the database . to handle the large amount of yet complex silkworm genome data , we developed a standard set of genome - based bio - xml format that lays the foundation for our research work and allows silkdb to accommodate the fast - accumulating data and to integrate new data types when encountered . we are aiming at building a genomic information resource and comparative analysis workbench for silkworm with an intention to expand to other lepidoptera and model insects . continued efforts will be made for the improvement of data quality , including anchoring scaffolds onto b.mori chromosomes , improving functional annotations based on phenotypically identified mutants and gene expression at the transcription and translation levels , updating est clusters as more data are generated and annotating the clusters with respect to potential encoded protein products . besides the timely updated silkworm genome information , silkdb has been constantly incorporating more data , as they become available , from other lepidoptera genomes , and different types of biological data , such as phenotype and gene expression data . refinement of the system and addition of new applications are continuous efforts for the silkdb project . we will introduce into silkdb a versioning system and references around different versions . in the near future , it will be possible for users to retrieve the data of different versions , trace up and locate changes of a given entity between different versions . a key enhancement to comparative analysis will be the development of a comparative map viewer , allowing users to evaluate the alignment of conserved regions with alternative views of genome evolution . based on the comparative map viewer , further comparative studies on genomic sequences between lepidoptera , d.melanogaster , anopheles gambiae ( 19 ) , caenorhabditis elegans ( 20 ) , and other invertebrates will be conducted for the study of lepidopteran - specific genes , many of which are potential candidates for targets of lepidopteran - selective insecticides , and will help further our understanding of the molecular mechanism of genetic diversity among insects . this project was funded by chinese academy of sciences ( kscx2-sw-223 ) , national development and reform commission , ministry of science and technology ( 2002aa104250 ; 2002aa234011 ; 2001aa231061 ; 2001aa231011 ; 2001aa231101 ; 2004aa231050 ; 30200163 ; 90208019 ) , china national grid ( 2002aa104250 ) and 863 high technology foundation of china ( 2004aa2z1020 ) . | the silkworm knowledgebase ( silkdb ) is a web - based repository for the curation , integration and study of silkworm genetic and genomic data . with the recent accomplishment of a 6x draft genome sequence of the domestic silkworm ( bombyx mori )
, silkdb provides an integrated representation of the large - scale , genome - wide sequence assembly , cdnas , clusters of expressed sequence tags ( ests ) , transposable elements ( tes ) , mutants , single nucleotide polymorphisms ( snps ) and functional annotations of genes with assignments to interpro domains and gene ontology ( go ) terms .
silkdb also hosts a set of ests from bombyx mandarina , a wild progenitor of b.mori , and a collection of genes from other lepidoptera .
comparative analysis results between the domestic and wild silkworm , between b.mori and other lepidoptera , and between b.mori and the two sequenced insects , fruitfly and mosquito , are displayed by using b.mori genome sequence as a reference framework .
designed as a basic platform , silkdb strives to provide a comprehensive knowledgebase about the silkworm and present the silkworm genome and related information in systematic and graphical ways for the convenience of in - depth comparative studies .
silkdb is publicly accessible at http://silkworm.genomics.org.cn . |
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diabetic nephropathy ( dn ) is the most common complication and leading cause of mortality associated with diabetes . dn is a leading contributor to cases of kidney failure in developed countries , and both type i and type ii forms of diabetes can result in dn . while the global pattern of dn incidence is not fully surveyed , it is nonetheless a widespread occurrence presently that is expected to increase in prevalence . numerous factors , both environmental and genetic , have been reported to influence dn onset , severity , and the rate of progression . dn is assessed clinically through a five - stage system of criteria , with each stage featuring a distinct set of functional and structural changes and reflected alterations in benchmark renal function markers . the structural changes begin with glomerular and tubular hypertrophy and the thickening of the basement membrane and mesangium expansion , leading to end - stage glomerular closure and tubulointerstitial fibrosis [ 5 , 6 ] . these changes are driven primarily by the dysregulation of typical glucose metabolism pathways , leading to the characteristic loss of blood glucose control and aberrant adipose function [ 79 ] . this loss of glucose control can lead to a number of other changes , including inflammation and cellular stress . while perhaps not as dramatically observed as in some other conditions , inflammatory processes are heavily involved in the structural deterioration that occurs in dn . this involvement is only natural given that inflammation is known to be involved in the pathogenesis of diabetes , with elevation of serum inflammation markers in long term diabetes patients . the cause(s ) of inflammation in dn are not clear , but some combination of pathogen insult and/or tissue damage may be responsible . the former is not likely to be the strongest contributor however , as the kidney is not typically exposed to pathogens and has not been shown to be more vulnerable during dn . since it is very likely that the inflammation occurring in dn is sterile and chronic , intrinsic kidney cell injury , namely , injury to glomerular , tubular , and vascular cells , may be the main cause . damage or danger signals released by injured renal cells can trigger remodeling processes by stimulating renal cells and activating immune cells of both the innate and adaptive systems . other metabolic signals may also contribute . in this review , the authors will review the roles played by , and crosstalk between , several t cells , macrophages , dendritic cells , and renal tubular cells , which are among the most important cell types contributing to the inflammation mediated acceleration of dn progression . the authors will consider the impact glucose metabolism and other mechanisms of metabolic control may have on these different cell types as a key explanatory factor for dn pathogenesis . dendritic cells are the professional apcs in the body , and their function is intimately tied to the progression of diabetic nephropathy . dcs exist primarily in two types : the more common myeloid - derived type ( mdc ) and the less common , but more highly pathogenic , plasmacytotic ( pdc ) variety . mdcs and pdcs differ primarily in terms of tnf production , with the high levels of tnf secreted by pdcs having been shown to result in significant morphological changes in dn as well as other autoimmune and inflammatory disorders . more importantly however , dcs can act as keen sensors of damage , being armed with a complete repertoire of receptors for both extrinsic pathogens and intrinsic factors . these include most classes of pattern - recognition receptors , including toll - like receptors , nod - like receptors , and c - type lectin receptors , which may also coligate . following activation by these stimuli , dcs can begin initiating an inflammatory response to counter a perceived threat , potentially via nlrp3 or alternative licensing [ 13 , 14 ] . dc linkage with t cells is sufficient to activate the t cell receptor ( tcr ) and drive adaptive responses . signaling along these complex pathways generally ends in the activation of transcription factors such as irf7 , nf-b , and irak , which can then coordinate the secretion of varied chemokines to attract pmns and alter the behavior of other nearby cells [ 15 , 16 ] . dcs are able to sense extracellularly through cell surface receptors and intracellularly via endosome - surface receptors . these latter receptors , such as tlr8 , may also permit dcs to directly sense micrornas and other molecules taken up as exosomes . overall , dc phagocytosis and the ensuing processing mechanisms are critical to the initiation of inflammation . dcs are widely present in the renal tubulointerstitium in normal mice , and immunohistochemistry has also revealed the significant numbers of both mdcs and pdcs in normal human kidneys . as such , resident dcs in kidney may be readily capable of processing damaged cells and signals and consequently initiate the cycle of tissue damage and repair present in dn . dcs have been shown to be critically involved in tubulointerstitial inflammation in various progressive nephritic diseases and in the remnant kidney model . unfortunately however , the role for dcs in dn inflammation has not been well studied , in part due to difficulties associated with plasticity and the relative instability of cultured dc cell lines . similar to dcs , macrophages also function as apcs , but few resident macrophages are normally present in the kidneys . curiously however , a heavily increased macrophage presence is observed in both the glomeruli and tubulointerstitium in human t2d , and the prevention of such macrophage infiltration has been the focus of some recent work [ 1921 ] . such an increase in the macrophage population has been correlated with the degree of glomerulosclerosis , increased proteinuria , increased serum creatinine levels , and the presence of interstitial fibrosis . importantly , deletion or inhibition of macrophage accumulation by decreasing intracellular adhesion molecule 1 , monocyte chemoattractant protein 1 , or colony - stimulating factor receptor 1 ameliorated dn in both db / db and stz - induced diabetic mice . given that these knockouts primarily affect chemotaxis and late polarization , it stands to reason that the increase in macrophages around the kidney in dn is primarily a result of outside recruitment , and not simply a local population expansion . similarly , smad7 deletion , which impacts the tgf signaling pathway , has been shown to enhance macrophage recruitment to the kidneys . since outside recruitment typically only occurs in response to damage sensing , it can thus be expected that these macrophages only come into play some time after the initial damage event(s ) . two main types of macrophages , the classically activated and proinflammatory m1 and the alternatively activated and anti - inflammatory m2 , are commonly recognized , with the m2 type being further divided into three subtypes . macrophage populations are generally highly plastic however , such that rigid / exclusive production and expression patterns are rarely observed . several authors have also suggested various other unique macrophage populations , based on factors such as the differential expression of lyc6 . tissue - resident macrophages may also generally express other markers that are tissue - specific , unlike classical bone - marrow derived macrophages [ 2628 ] . nonetheless , the simple classification scheme is useful for understanding many basic aspects of macrophage function . transitions along the spectrum from the two extremes have been an area of particular focus and are also highly relevant to the progression of dn . many of the infiltrating macrophages in the middle stages of dn are likely to adopt the m1 type and cause significant damage that accelerates the condition , although the precise origin of these macrophages is not fully clear . many inducers of m1 activity and m1 signature products have been implicated with dn , with tnf being perhaps the most notable . tnf signaling induces production of nf-b along a jak / stat pathway , leading to inflammation . the cytosolic adaptor protein myd88 conducts the classical pathway for m1 function downstream of tlr signaling . myd88 may activate various members of the traf family , leading to further activation of irak as well as irf7 . m1 may also be stimulated via induction of other pattern - recognizing receptors such as nlrs . regardless of the precise channel used , m1 can be stimulated by both classical pathogen - associated molecular patterns ( pamps ) , such as lipopolysaccharide ( lps ) , and damage - associated molecular patterns ( damps ) such as extracellular dna . the relevance of pamp - driven signaling to dn mainly lies in possible changes in response to secondary infection , a situation that is not wholly clear . given the known damage and ecm changes that occur in dn however , it seems that damps are equally important to consider , if not more so . the transcription factor irf5 is intimately involved in m1 processes , such that irf5 is also considered to be one of the defining characteristics of the m1 phenotype . irf5 is also critical for m1 production of type one interferons and also influences the activity of inos , a primary means by which m1 can generate ros . these signaling molecules work in concert to prevent wound healing but may also prevent hyperresponse via inhibition of other proinflammatory cell types . after all , ros production has been shown to lead to the inhibition of several t effector subtypes , among others . despite being proinflammatory , m1 are also sensitive to oxidative stress . m1 are suppressed by an overabundance of no via the nitration and subsequent loss of function of irf5 and are actually more abundant in inos deficient mice as a consequence . similarly , hypoxia has been shown to inhibit m1 polarization via altered expression of hif-1 . oxidative stress has been shown to lead to changes in autophagy and other macrophage activities as well . interestingly however , high levels of oxidative stress observed in dn have been previously shown to result in tubular cell and podocyte injury , and the debris from cells killed in such manner may also serve to further stimulate m1 and overcome the inhibitory pressure m1 may experience otherwise . m1 may also produce sizeable amounts of il-6 , but the precise importance of this production is unclear given the fact that many other cell types also secrete the cytokine during inflammation . m1 macrophages can also form inflammasomes to further their function , with the nlrp3 and aim2 varieties being the ones best characterized thus far . these inflammasomes may form under a wide variety of stimuli and gather together large amounts of caspase 1 , which can then cleave and activate interleukins 1beta , 18 , and 33 . card and pyr domains on these inflammasomes may recruit and involve a broad assortment of different proteins to further m1 function . damage sensing by m1 macrophages has been demonstrated to contribute to such inflammasome activation , although the exact molecular machinery may be varied and has not been completely characterized . metabolic control of m1 has become subject of increasing interest and may be particularly relevant to dn given the metabolic disruption that is implicit in the condition . recent work has shown that m1 macrophages may have an altered expression of the krebs cycle enzyme isocitrate dehydrogenase , making it a possible target . this altered expression may have important ramifications given the change in glucose availability present in dn . the glucose and salt sensor aldose reductase has also been shown to play a key role in m1 metabolic control , although it is not fully clear if its control is mostly mediated by its production of sorbitol or through other protein interactions . high salt has been demonstrated to result in increased inflammasome activity , but it is not clear if the response is to the salt itself or just to osmolality increase . advanced glycation end products , commonly observed in diabetes , can also strongly influence m1 macrophage function . it is clear , however , that the provision of high amounts of glucose is a facilitator for m1 stimulation , partly mediated through the increased expression of srebp1 . interestingly , additional stimulation using insulin may significantly reverse these additions , such that they remain responsive along the standard pathways of metabolism . arginine metabolism has also been suggested to be involved in m1 control , while other metabolites are probably also relevant to uncertain capacities . in addition , as part of their phagocytotic capacity , macrophages may also uptake large amounts of cholesterol to become foam cells . foam cells have been identified to occur in a number of renal conditions , and some recent work has focused on promoting cholesterol efflux to prevent foam cell buildup , but it remains to be seen if foam cells should truly be targeted or simply regarded as a marker . in addition , the nuclear receptors fxr , lxr , and pxr have been identified to have important function in regulation of m1 , particularly in the kupffer cells of the liver and in resident macrophage populations in the kidneys . activation of the bile acid receptor fxr has been shown to repress macrophage function and infiltration capability in dn , partly by suppressing the ifn mediated activation of stat1 . however , fxr activation may not be wholly anti - inflammatory , with its mechanistic control still not fully understood . lxr function in macrophages has largely been explored in the context of atherosclerosis , in which lxr activation has been reported to induce efflux of cholesterol from macrophages . these effects have been confirmed in dn , with lxr activation leading to the amelioration of the condition . this amelioration might be highly dependent on macrophage function , as the ap-1 and nf-b signaling pathways have been shown to be important in dn , since those pathways feature prominently in macrophages . pxr may also influence macrophage sensitivity to cholesterol , although it may play a more important role in regulating the metabolism of the drugs being used to treat dn given its general role in controlling drug responses . at the same time , activation of these nuclear receptors may also lead to the induction of ppar family members such as ppar , which can then serve to negatively regulate inflammation . the balancing of the proinflammatory effects metabolites may have and the anti - inflammatory effects of their activated receptors remains to be clarified . overall , macrophages tend to trend towards m1 at the onset of inflammation and then switch to m2 to promote healing some time thereafter . the precise mechanisms that induce such a switch are not understood , although damage sensing , further cytokine stimulation , and autocrine regulation have been proposed as possible reasons [ 5355 ] . changes in the expression of surface markers such as from cd11b to cd163 may be used to monitor these changes in part but are not wholly sufficient given the persistent expression of perceived m1 markers well after a change in cytokine production has been observed . a large and varied assortment of compounds have attempted to target this transdifferentiation process as a means for alleviating the inflammation in dn and other autoimmune disorders , but none have been extremely successful thus far , despite revealing some interesting target options . many of these compounds have focused on repressing the activity of m1 transcription factors through selective inhibition , but it is not clear if the transition is actually controlled along a specific pathway . m2 macrophage differentiation has been one of the key goals for immunology - based treatments for dn , due to the protective effects m2 may induce . in particular , m2 are responsible for the secretion of il-10 , a potent cytokine that can act to suppress the activity of most proinflammatory cell types . il-10 function runs counter to that of tnf , helping to shut down its efficacy , partly by disrupting the p38/mapk pathway . il-10 may also lead to the suppression of inos activity via the induction of ho-1 and consequent co salvage . in addition , il-10 mediated induction of a pi3k / akt pathway for cell survival may directly improve wound healing despite otherwise hindering proliferation . polyamine secretion from m2 may also encourage the resolution of inflammation by influencing transcription programs in other cells . curiously , m2 function is coordinated to a large extent by irf4 , which is typically understood to be a proliferative and proinflammatory transcription factor across a wide range of different cell types . the exact signal transduction network in play within m2 macrophages that reverses that typical functional pathway is unknown . t helper cells become relevant at the site of inflammation later than apcs but still have an important functional role in regulating inflammation resolution or lack thereof . t helper cells do not typically have as strong of a residency status as other immune cells , but some tissue - specific markers have been identified . once activated , cd4 + t cells can organize many of the hallmark signs of inflammation , such as widespread recruitment and chemokine release . of particular interest in dn is the fact that cd4 + t helpers have been shown to engage in significant crosstalk with fibroblasts to influence fibrosis . as such , targeting against several of these subsets has been shown to be useful in treating dn , particularly th17 and th1 . t helper cells can be conventionally split into t effector or t regulatory phenotypes , with the proinflammatory th1 , th2 , th17 , th9 , and th22 subsets forming the cd4 + subset of the former . each of these subtypes has the capability of producing signature cytokines , although such production is not necessarily exclusive . lineage - defining transcription factors may be highly expressed and critically regulate the function of these subtypes , with dynamic interplay between them potentially contributing to the nature of their polarization . the precise strength of tcr signaling at the beginning of their activation has been shown to bias future differentiation towards certain phenotypes [ 64 , 65 ] . different metabolites have been demonstrated to also have variable impact across these different phenotypes , although many of those differences remain undiscovered . cd4 + t cells can also be highly plastic , adopting multiple phenotypes either over time or even simultaneously . as such , it is useful to first consider all of the cd4 + cells in context before examining the particular factors that a particular phenotype may hold somewhat exclusively . cd4 + t cell activation occurs along a multistep pathway , with bona fide activation being somewhat ambiguous . the energy need during this differentiation process , while not well studied , is likely to be quite high , given the clear disparity in metabolic intake between differentiated t effector types and nave th0 cells . during this differentiation process , cells may be directly activated and subsequently polarized or potentially undergo a failed activation and instead enter into a state of anergy . the nature of cd4 + anergy is not well known , with much of the current information on this phenomenon having been gleaned from analogous study on cd8 + t cells . anergetic t cells are understood to have very weak , if any , capability of sending further stimulation to other cells , especially via cytokine production . several types of viral infections are known to induce t cell anergy as a means of escaping immune suppression , and tgf signaling is thought to be involved , but much remains to be clarified . intriguingly , tlr7 activation following damage sensing has been suggested to induce this state of anergy , implying a potential increase in the population of anergetic t cells in the context of dn . following activation , t helpers can then pass through a functional stage of variable duration before eventually making a choice to become memory cells or otherwise undergo exhaustion or apoptosis . cd4 + t helper memory is thought to be critically regulated by tslp , which may induce the cells to adopt a lower metabolic profile . more recently , a newer exhaustion phenomenon has been uncovered , which has been treated as being distinct from simple plastic transitions across different phenotypes . cd4 + t cells are known to require significant autocrine secretion of il-2 ( as well as lesser amounts of il-5 and il-7 ) in order to maintain their viability and continue functioning . in some viral diseases however , t cell function has been observed to fade over time , with il-2 autocrine signaling being suppressed [ 72 , 73 ] . this suppression has been correlated with the increased expression of the cell surface marker pd-1 and the transcription factor bcl6 , among others . while some early speculation held that these cells were in fact merely becoming tfh - like , the observation of other markers such as tim-3 suggests otherwise . once cd4 + cells begin to undergo exhaustion , they gradually lose functions one at a time . recently , several authors have proposed that this type of exhaustion may also occur in cases of chronic inflammation , where the continued activation and function of cd4 + cells may eventually be suppressed through some sort of internal transition . this type of exhaustion - mediated transition has thus far been identified to occur between th17 and treg phenotypes but may also occur between other t effector types and t regulatory types . this type of exhaustion is likely caused by some kind of damage sensing mechanism or perhaps the extreme buildup of stress that may occur in chronic inflammation . cd4 + t helper tolerance of glucose has been shown to be an important factor in the pathogenesis of diabetes and as such is also highly relevant to dn . th17 cells are a highly active t cell population classically activated through the cytokines il-6 and tgf- or alternatively activated into a pathogenic form via il-1 , il-6 , and il-23 . as noted previously , these cytokines are often present in significant concentrations in the local environment during inflammation , with significant amounts of them being released by epithelial cells , particularly by those under stress . once th17 cells receive signaling along either tract , a jak / stat pathway is activated , culminating in stat3 activation of rort and ensuing production of il-17 . changes in the production of il-17 and in the expression of rort are understood to be the most conclusive indicators of change in th17 function , but it is also possible that other changes may emerge even earlier . th17 function has been explored as a possible contributor to worsening dn , but the actual strength of the connection is unclear . some studies have suggested that th17 cells might be uniquely upregulated in dn , although it is also possible that the increase is simply a part of a general increase in proinflammatory cytokines . th1 cells are a similar proinflammatory subset that often responds in a similar fashion to th17 cells in the face of stimulus . by contrast , th1 cells produce large amounts of ifn and express the transcription factor t - bet ( tbx21 ) . stimulated by the common apc product il-12p40 , th1 cells rely on stat4 activation at the end of a jak / stat pathway to function . like other t helper subtypes , th1 are also influenced by batf - driven and irf4-driven transcription networks . cd4 + t helpers may also respond differently to common forms of oxidative stress common in dn . th17 cells may also be preferentially activated by the hypoxia environment that can form in dn , although they are also susceptible to inhibition by nitric oxide and other ros stress . in this manner , they differ from other cd4 + t helpers which are less responsive to no or otherwise feed off of it to increase inflammation , such as th9 . unlike apcs , t helper cells have significantly muted expression of classical damp and pamp receptors , such that they are not as likely to be directly influenced by danger signals such as extracellular dna . recent work has shown that human t helpers may be inhibited via activation of tlr7 however , raising hope that other direct influences may also be uncovered . apc transduction of such signaling might occur at a rapid pace in dn however , especially given the broad scale damage that is present during accelerated remodeling . in addition , t cells can directly receive signaling via other extracellular metabolites , such as atp . extracellular atp may then activate the ampk pathway to increase inflammation by upregulating metabolism . while it is not intuitively obvious that increases in metabolism must correlate with an increase in inflammation , it is almost always the case , as basic increases of metabolism can strongly impact cellular activation . metabolic control of cd4 + t helpers has been the focus of increased study in recent years , with many metabolic products having been identified to bias cellular polarization . for instance , succinate , an intermediate in the krebs cycle , has been shown to play an important role in modulating th17 activity . provision of excess amino acids has also been shown to somewhat preferentially induce th17 activation , while its opposite starvation selectively inhibits the phenotype [ 84 , 85 ] . other metabolites , such as salt , have also been shown to induce t effector activity , although it is also possible that the change is primarily a result of an increase in osmolality [ 86 , 87 ] . interestingly , in many of these cases , the th2 and treg phenotypes are not affected , for reasons not fully clear . one particularly striking example of this phenomenon is the fact that brd4 inhibition seems to sharply limit th1 and th17 activity while leaving th2 and treg populations at original levels , despite the fact that brd4 is broadly expressed across cd4 + lineages and is a very general factor critical for the assembly of the superelongation complex for replication and transcription [ 89 , 90 ] . it may also be possible that cd4 + t helpers can respond differently to the provision of nucleotides or other metabolites of different classes . regardless , this differing need for various metabolites leads to a useful potential source for t helper targeting or at the very least cautions against the use of some methods for treatment . for instance , the inhibition of th17 cells has been a longstanding goal in many conditions besides dn , and one of the prime means for achieving this has been through the treatment of broad - acting corticosteroids . unfortunately however , recent research suggests that th17 cells may actually be able to more effectively respond to corticosteroid stress than some other subsets and actually survive such inhibition over the short term . as such , it is important to observe that these metabolic differences can only be targeted in cases where one cell type expresses and requires a metabolite that other polarization states do not also need . while not immune cells , tubular cells in the kidney may serve to propagate many of the immune signals present in dn , both through direct cytokines and through metabolites . after all , tubular cells handle filtered glucose and other metabolites , while also transporting electrolytes and water . although currently there are debates about whether or not the primary role of tubular cells is acting in parallel with glomerular cells in the pathogenesis of dn , it is a well received fact that functional dysregulation in the tubules and cell injury are main contributors to dn . as noted previously , one of the early pathologic changes in dn is tubular cell hypertrophy , followed by increased thickness of tubular basement membranes . these changes affect the composition of the extracellular matrix and drive feedback for further changes . subsequently , tubular atrophy , atubular glomeruli , extensive immune cell infiltration , and fibrosis develop and progress . multiple factors including hypermetabolism , excessive free radicals , and chronic inflammation have been implicated in dn tubulointerstitial lesions . naturally , work load in tubular cells in diabetes is greatly elevated as a result of hyperfiltration , along with accumulated high glucose and related metabolites . accordingly , energy generation and consumption are increased to meet the functional need , leading to a state of hypermetabolism that provokes cell stress and free radical overgeneration . moreover , nonenzymatic interactions between high glucose pressure and proteins promote the generation of carbonyls in the process , as well as advanced glycation end products which provoke free radical synthesis . this cycle may also cause positive feedback in the absence of a functional response to insulin . tubular cells may also directly produce tgf in response to glucose to directly modulate immune function . free radicals may directly damage cell structures and modify cellular proteins and dna , resulting in cell stress and injury , which may subsequently activate dcs and macrophages to initiate a pattern of damage associated sterile inflammation . additionally , free radicals may oxidize nitric oxide ( no ) to decrease no availability and cause vasoconstriction , leading to decreased tubular blood supply and hypoxia . hypoxia suppresses prolyl hydroxylase domain proteins and activates oxygen sensing transcription factor hypoxia inducible factor ( hif-1 ) . it is noted that free radicals can also increase hif-1 transcription and function by directly activating the hif-1 promoter and regulating hydroxylase function of the asparaginyl hydroxylase . indeed , the levels of hif have been noted to increase in the tubular and interstitial cells of dn , and the suppression of hif has been shown to ameliorate dn . one of important functions of hif is to switch cell metabolism from oxidative phosphorylation to glycolysis to increase cell survival under hypoxic condition . however , this metabolic shift also causes the changes in immune cell phenotypes that favor inflammation . hif - family proteins increase macrophage aggregation , invasion , and mobility and are intimately involved in macrophage polarization . for instance , macrophages lacking hif-2 can not be stimulated via lps to generate m1 inflammatory responses . hypoxia induces the production of proinflammatory cytokines tnf and il-1 by dcs . on the other hand , dcs cultured under hypoxia have less costimulatory molecules and maturation markers and show decreased migration in response to c - c chemokine receptor type 7 ligands . since the process of t cell activation and differentiation is tightly associated with metabolic switch , it is not unexpected to find a role for hif in t cell activation and differentiation . th1 , th2 , and th17 cells exhibited increased glycolysis and less oxidative phosphorylation compared to t regulatory cells , which show a greater reliance on lipid oxidation and oxidative phosphorylation . th17 has high levels of hif-1 and deletion of hif-1 in t cells inhibits th17 differentiation in vitro and in experimental autoimmune encephalomyelitis . in contrast , hypoxia and deletion of hif ubiquitination ligase vhl both promote stabilization of hif-1 and enhance generation of th17 cd4 + cells . the generation of treg is usually opposite to the induction of th17 cd4 + cells . thus , enhanced treg cell differentiation was observed in the case of hif-1 deletion , a condition not favoring th17 cells lineage differentiation . one possible molecular mechanism for hif attenuation of treg cell development is direct binding and targeting of the treg lineage - defining transcription factor foxp3 for degradation . hif-1 can also affect metabolic adaptations in cd8 + t cells and control expression of many molecules associated with effector function and migration . overall , hif activation by hypoxic microenvironment as well as free radicals in tubulointerstitial area favors inflammation via modulation of immune cells . however , since the preferred metabolic fuels for tubular cells are not glucose , the act of hif on these cells may differ . both high glucose and advanced glycation end products can activate nf-b and inflammasome and upregulate mcp-1 and vcam-1 in tubular cells , while also promoting further ecm modifications . large amounts of albuminuria have also been implicated in the proinflammatory phenotype of tubular cells . finally , apart from genetic susceptibility , dn takes about 1020 years to develop as the result of multiple vicious interplays among dysregulated metabolism , cell stress and injury , chronic inflammation , and epigenetic changes . although we have gained some knowledge about the pathogenesis of dn and have developed some treatment in recent years , at present we are still unable to prevent disease progression to diabetic renal failure . as such , it is critical to understand the details of metabolic abnormalities , the pros and cons of hif activation , and the keys to block chronic inflammation in order to enable more effective treatment of dn . | diabetic nephropathy ( dn ) is a serious complication of diabetes . at its core , dn is a metabolic disorder which can also manifest itself in terms of local inflammation in the kidneys .
such inflammation can then drive the classical markers of fibrosis and structural remodeling . as
a result , resolution of immune - mediated inflammation is critical towards achieving a cure for dn .
many immune cells play a part in dn , including key members of both the innate and adaptive immune systems .
while these cells were classically understood to primarily function against pathogen insult , it has also become increasingly clear that they also serve a major role as internal sensors of damage .
in fact , damage sensing may serve as the impetus for much of the inflammation that occurs in dn , in a vicious positive feedback cycle .
although direct targeting of these proinflammatory cells may be difficult , new approaches that focus on their metabolic profiles may be able to alleviate dn significantly , especially since dysregulation of the local metabolic environment may well be responsible for triggering inflammation to begin with . in this review ,
the authors consider the metabolic profile of several relevant immune types and discuss their respective roles . |
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in recent years , there has been a shift from the traditionally secret experimental data kept by the pharmaceutical industry to a more open - access culture in relation to data sharing ( 1 ) . for this reason , we have been witnessing a steady increase in public repositories of bioactive small molecules such as chembl ( 2 ) and pubchem ( 3 ) . however , as public repositories of bioactive small molecules have only just recently been made available , the problem of how to handle chemical entities is still largely unsolved . even though standards have been widely adopted to describe genes and proteins ( e.g. ensembl i d , entrez i d for genes , and uniprot i d for proteins ) , small molecule identifiers , as well as measures for properties such as biological activities , are not necessarily standardized across different resources ( 4 ) . one could claim that the bottleneck in understanding how small molecules perturb biological systems is no longer in the generation , gathering and availability of experimental data but in their organization , presentation and visualization ; in other words , in the development of centralized systems that would better enable their exploitation . the problem is not only how to extract data from different ( federated ) resources , it is also important to provide solutions that facilitate provenance tracking , visualization , uniform and systematic description of data and their integration in ways that can preserve the semantic relationships between the different entities . furthermore , the number of failures of drug candidates in advanced stages of clinical trials has increased and the number of submissions for us food and drug administration ( fda ) approval has decreased in the last decade . one of the reasons may be our reductionist approach to discovery , whereby a complex system , namely a drug and its metabolites interacting with many proteins across multiple cellular compartments and tissues over time , is reduced to a simplistic ligand target interaction model . this is probably too crude and emphasizes the need to look at the effects of compounds on global systems aided by the integration of multiple biological and temporal data sources . with the emerging fields of chemogenomics ( 5 ) , systems pharmacology ( 6 ) and systems chemical biology ( 7,8 ) , it becomes feasible to investigate the drug action at different levels from molecular to pathway , cellular , tissues and clinical outcomes ( 9 ) . for example , it has become apparent that many common diseases such as cancer , cardiovascular diseases and mental disorders are much more complex than initially anticipated , as they are caused by multiple molecular and cellular dysfunctions rather than being the result of a single defect . therefore , network - centric therapeutic approaches that consider entire pathways rather than single proteins must be investigated ( 10 ) . among the recent advances in the field of systems chemical biology , servers supporting drug profiling such as stitch ( 11 ) , disgenet ( 12 ) or the new database promiscuous ( 13 ) should be mentioned . stitch3 provides confidence scores that reflect the level of confidence and significance of compound protein interactions . promiscuous is a resource focused on drug compounds , including withdrawn and experimental , containing drug disease association database focused on the current knowledge of human genetic diseases including mendelian , complex and environmental diseases . we have previously reported the development of chemprot , a disease chemical biology database ( 14 ) . compared with other approaches , chemprot-1.0 offered a high level of integration of chemical and biological data , including internally curated disease - associated protein protein interactions ( ppis ) ( 15 ) . here , we present the second release of chemprot , a resource of annotated and predicted disease chemical biology interactions . chemprot-2.0 can be accessed at http://www.cbs.dtu.dk / services / chemprot-2.0/. the present release contains a compilation of over 1 100 000 unique chemicals with biological activity for > 15 000 proteins . we have added a visual interface that supports user - friendly navigation through the data , biological activities and disease associations . chemprot-2.0 now enables the user to query the database not solely by chemicals or proteins but also through therapeutic effects , adverse drug reactions and diseases . the similarity ensemble approach ( sea ) developed by keiser et al . ( 16 ) has also been implemented , so that protein sequence similarity can be used when examining chemical promiscuity . with these updates , chemprot-2.0 offers an integrative approach to understand the impact of small molecules on biological systems and contributes to the investigation of molecular mechanisms related to diseases and clinical outcomes . user can query chemprot-2.0 using chemical , protein , disease , atc code and ses . user can query chemprot-2.0 using chemical , protein , disease , atc code and ses . chemical protein interactions data were gathered in june 2012 from updated open - source databases chembl ( version 14 ) , bindingdb ( 17 ) , pdsp ki database ( 18 ) , drugbank ( version 3.0 ) ( 19 ) , pharmgkb ( 20 ) , active compounds from the pubchem bioassay ( 2012 ) targeting human proteins and the two commercial databases : wombat ( version 2011 ) and wombat - pk ( version 2011 ) ( 21 ) . the iuphar - db database ( 22 ) was also integrated in the new version of chemprot-2.0 . protein annotations that lack explicit bioactivity data might be of interest in the mining of a large and diverse integrated database . therefore , we included also data from ctd ( 23 ) and stitch ( 11 ) . ctd extracts literature data about environmental chemicals and how they modulate gene expression , whereas stitch provides chemical protein relationships from text mining the co - occurrence of a chemical term and a protein ( gene ) term in medline abstracts . clinical outcomes were of special interest in this version and we decided to include information from the anatomical therapeutic chemical ( atc ) classification system ( 24 ) developed by the world health organization , as well as se data from dailymed ( http://dailymed.nlm.nih.gov/dailymed/ ) . from a biological perspective , we updated our internal human interactome platform to reach 14 421 genes interacting through 507 142 unique ppis . the updated version of omim ( 25 ) , genecards ( 26 ) , kegg ( 27 ) , reactome ( 28 ) and gene ontology ( 29 ) databases was also downloaded ( june 2012 ) , curated and integrated in chemprot-2.0 . based on the assumption that compounds sharing similar structure have potential similar bioactivities , we encoded the chemical structure with two different types of fingerprints : the 166 maccs key which encode the presence or absence of some predefined substructural or functional groups ( 31 ) and the fp2 fingerprints computed with openbabel ( 32 ) . chemical similarity between two compounds is quantitatively assessed using the tanimoto coefficient . by including the sea method ( 16 ) , one can also predict potential new targets for a compound . for the internal development of sea , compounds with an activity value < 100 m were considered ( only ic50 , ec50 , potency , ac50 , ki values were used ) . furthermore , to complete the set of active protein ligands , annotated compound protein interactions from ctd , drugbank and pharmgkb were also included , together with annotated protein compound in the stitch database . for this dataset , the raw similarity score , i.e. the sum of ligand pair wise tanimoto coefficients based on the fp2 fingerprint , in addition , for all protein targets , we operated under the assumption of promiscuity , i.e. proteins with high - sequence similarity may share similar functions and may be targeted by the same compound ( likely with different bioactivities ) . protein sequences were obtained from uniprot ( 33 ) , and sequence comparisons were computed using blastp ( 34 ) . the similarity of two sequences was assessed using an e - score , an expectation value related to the probability that sequence similarity between two proteins is not achieved by random chance ( 34 ) . we filtered the output and proteins with an e - value < 10 ( as default ) are depicted . with respect to ses , 988 small molecule drugs were matched against 174 se as described ( 35 ) . term frequency vectors compiled from dailymed were integrated in chemprot-2.0 and proteins associated to each drug are then depicted . in chemprot-2.0 , a visual interface was implemented to facilitate the visualization of the results using html 5 and javascript . protein associations are depicted in a pie - chart heatmap where each pie corresponds to the database from which we gathered the information . hovering over the pie - charts with the pointer , activity values are then displayed . a valuable feature is the handling of multiple activities that have been gathered for a given compound target pair by selecting all values . a color spectrum from blue ( low activity ) to red ( strong activity ) it is also possible to select a specific database or / and a specific activity type and define a range of activities ( threshold ) of interest in order to optimize the query . results from the sea approach are also integrated in the activity type.
figure 2.example of the graphical interface output based on a compound query . on the top the heatmap on the left represents the bioactivities gathered for the input compound ( in blue ) and structurally similar compounds ( in pink ) in the x - axis and the proteins in the y - axis . a color spectrum from blue ( low ) to red ( high ) is used to represent the activity . if several binding data have been measured for the same chemical protein interaction , intensity of the colors is represented inside the circle . the heatmap on the left represents the bioactivities gathered for the input compound ( in blue ) and structurally similar compounds ( in pink ) in the x - axis and the proteins in the y - axis . a color spectrum from blue ( low ) to red ( high ) is used to represent the activity . if several binding data have been measured for the same chemical protein interaction , intensity of the colors is represented inside the circle . the compound query is always shown in the first column followed by similar compounds ( sorted in descending order of similarity ) whereas the protein queried is depicted in the first row . to optimize the display , the heatmap is limited to a section of 100 rows 100 columns . if the chemical protein matrix is larger , we have included an arrow feature ( ) that allows the user to upload the next 100 data items for both axes . the user has still the possibility to view the data in a table format and to download the results in a flat - file format . in the table format , display mode the user can dynamically sort and group the activities according to compound , target , species , activity type , etc . a second heatmap that depicts protein disease categories next to it , the diseases link redirects the user to the disease - associated proteins complex around the selected protein . a new , dynamic interface has been implemented , where the proteins associated to a biological term are shown when highlighting the term of interest ( figure 3 ) . figure 3.example of the disease complexes network representation for the dopamine receptor d2 ( drd2 ) . twenty - five proteins interact directly to the protein drd2 and pointing the cursor to schizophrenia , seven genes are associated to this disease . example of the disease complexes network representation for the dopamine receptor d2 ( drd2 ) . twenty - five proteins interact directly to the protein drd2 and pointing the cursor to schizophrenia , seven genes are associated to this disease . the chemprot-2.0 database interface is accessible freely online . in addition to the chemical and protein search that was previously implemented , the user can search by diseases , atc codes and ses . for example , the query epilepsy returns 2662 compounds active on 13 proteins associated to this disease . similarly , looking for the se hallucinations , 15 drugs ( with the term frequency associated to it ) active on 470 proteins are displayed . some of these drugs ( ropinirole , pergolide , amantadine and pramipexole ) are used for the treatment of parkinson diseases , by affecting the dopaminergic and serotonergic systems . interestingly , visual hallucinations are symptoms of the parkinson s disease and perturbing the serotonergic system could help to alleviate these symptoms ( 36 ) . for example , there is a possible association between the polymorphism of the serotonin transporter ( htt ) and the development of osteoporosis ( 37 ) . some of these drugs bind to htt and could thus be potentially investigated for drug repurposing . many diseases seem not to be the result of a single defect but are rather caused by multiple molecular and cellular abnormalities . therefore , observations of a drug effect not only at the molecular level but also at cellular and systems levels should guide therapeutic strategies for the development of better and safer drugs . chemprot-2.0 offers the possibility of interrogating multiple layers of information by linking chemically induced biological perturbations to disease and phenotype . we believe with the advances in proteomics , metabolomics and other omics sciences , combined with next - generation sequencing technologies , we will no longer evaluate the bioactivity profile of a chemical solely at the molecular level , but rather we will investigate biomedical knowledge with the integration of genetic polymorphisms and clinical effects ( 38 ) . the innovative medicines initiative joint undertaking , openphacts ( to l.w . , j.k . ) ; innovative medicines initiative joint undertaking , etox ( to u.p.j . , o.t . ) ; gendinob project supported by the danish council for strategic research ( to k.a . ) ; lundbeck foundation ( to s.k.k . ) ; national institutes of health [ r21 gm095952 to t.i.o . ] . | chemprot-2.0 ( http://www.cbs.dtu.dk/services/chemprot-2.0 ) is a public available compilation of multiple chemical
protein annotation resources integrated with diseases and clinical outcomes information .
the database has been updated to > 1.15 million compounds with 5.32 millions bioactivity measurements for 15 290 proteins .
each protein is linked to quality - scored human protein
protein interactions data based on more than half a million interactions , for studying diseases and biological outcomes ( diseases , pathways and go terms ) through protein complexes . in chemprot-2.0 , therapeutic effects as well as adverse drug reactions have been integrated allowing for suggesting proteins associated to clinical outcomes .
new chemical structure fingerprints were computed based on the similarity ensemble approach .
protein sequence similarity search was also integrated to evaluate the promiscuity of proteins , which can help in the prediction of off - target effects .
finally , the database was integrated into a visual interface that enables navigation of the pharmacological space for small molecules .
filtering options were included in order to facilitate and to guide dynamic search of specific queries . |
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mutations in the lecithin : cholesterol acyltransferase ( lcat ) gene cause either familial lecithin : cholesterol acyltransferase deficiency ( fld ) ( omim # 245900 ) or fish - eye disease ( fed , omim # 136120 ) . fld patients are characterized by a significant reduction in serum high - density lipoprotein cholesterol ( hdl - c ) level , corneal opacity , normochromic anemia and proteinuria with progression to end - stage renal disease [ 1 , 2 ] . plasma low - density lipoprotein cholesterol ( ldl - c ) levels are widely variable among fld patients although plasma lcat activity catalyzing hdl ( lcat activity ) and catalyzing apolipoprotein b containing lipoproteins ( lcat activity ) is absent or nearly absent . the loss of lcat activity leads to increased levels of phosphatidylcholine and free cholesterol ( fc ) in the blood and to the formation of an abnormal lipoprotein called lipoprotein - x ( lp - x ) . as a result , accumulation of fc occurs in various tissues , including the cornea , erythrocyte membrane and the kidney . recent report suggests an etiological role for lp - x in the development of glomerulosclerosis . here , we describe two novel mutations of lcat gene , a c to t transition in exon 2 converting proline 69 to leucine ( pro69leu ) and a t to c transition in exon 6 converting methionine 293 to threonine ( met293thr ) . since baass et al . hypothesized that the presence of the apoe 2 allele contributed to the accumulation of intermediate - density lipoprotein ( idl ) in the patients with lcat deficiency , we investigated their apoe genotypes and phenotypic manifestations of the patients with fld . a 27-year - old japanese man was referred to okayama university hospital because he had extremely low hdl - c and ldl - c concentrations . on physical examination laboratory findings demonstrated a mild degree of normochoromic - normocytotic anemia ( 11.3 g / dl ) . although his renal function was normal , ( + 1 ) proteinuria was revealed via the dipstick method ( 0.21 g / day ) . the consanguinity was found in his parents as shown in figure 1a . presented cases with fld . squares are male , circles are female . filled symbols indicate homozygous carriers and left filled symbols indicate heterozygous carriers . ( c , d ) determination of the lcat gene sequences of proband and their families . heterozygous mutation of mother and elder sister and homozygous mutation of proband are shown in cases 1 ( c ) and 2 ( d ) . electron micrograph shows lipid deposits with a vacuolar lucent appearance in the gbm ( bar = 2 m ) . ( g i ) high - performance liquid chromatography patterns of cases 1 ( g ) , 2 ( h ) and normal healthy control ( i ) . a 30-year - old japanese woman was referred because of very low hdl - c and ldl - c associated with ( 1 + ) proteinuria via dipstick ( 0.08 g / day ) . she also showed bilateral corneal opacity and mild hemolytic anemia ( 11.2 g / dl ) , demonstrating target cells on her peripheral blood smear . direct sequencing of lcat gene and comparison with reference sequence ( nm_000229 ) revealed that case 1 had a c to t nucleotide substitution in exon 2 resulting in pro69leu [ c.278 c > t ( p.pro69leu ) ] ( figure 1c ) and case 2 had a t to c substitution in exon 6 resulting in met293thr mutations [ c.950 t > c ( p.met293thr ) ] ( figure 1d ) . case 1 carried apoe 4/3 genotype , whereas case 2 carried apoe 2/2 genotype ( table 1 ) . the mother of case 1 carried apoe 3/3 genotype and genomic dnas were not available from the deceased father . renal biopsy in both cases revealed similar histological findings ; diffuse vacuolization of the glomerular basement membranes ( gbm ) by periodic acid - methenamine silver stain and lipid deposits with a vacuolar lucent appearance in the mesangial matrix and the gbm by electron microscopy ( figure 1e and f ) . in both cases , half normal lcat activity was observed by using exogenous substrate , suggesting the diagnosis of fed . however , clinical manifestations with hemolytic anemia and renal involvement coincided with the features of fld . characteristics and lipoprotein profiles of presented casesa
reml - c , remnant - like particles cholesterol measured with metabolead reml - c ; nd , not determined . both cases 1 and 2 revealed similar lipid profile ; ldl - c and hdl - c were extremely low ( table 1 ) . furthermore , fc / total cholesterol ( tc ) ratio was much higher than normal value , suggesting reduced activity of esterification of plasma cholesterol due to lcat deficiency . in both cases , the lipoproteins in fraction 710 , corresponding to idl particle size , were characterized by triglycerides ( tg)- and phospholipid ( pl)-rich . since lp - x is fc- and pl - rich lipoprotein particle , the peaks appeared in fraction 710 seem to be remnant lipoproteins containing idl remnant ( figure 1 g and h ; arrows ) . in addition , lp - x was not detected by agarose gel electrophoresis in both cases . the remnant - like particles cholesterol was measured with metabolead reml - c well - reflects idl remnant compared with immunoseparation assay , the high values of reml - c in both cases also support the elevation of idl remnant . a 27-year - old japanese man was referred to okayama university hospital because he had extremely low hdl - c and ldl - c concentrations . on physical examination laboratory findings demonstrated a mild degree of normochoromic - normocytotic anemia ( 11.3 g / dl ) . although his renal function was normal , ( + 1 ) proteinuria was revealed via the dipstick method ( 0.21 g / day ) . the consanguinity was found in his parents as shown in figure 1a . presented cases with fld . squares are male , circles are female . filled symbols indicate homozygous carriers and left filled symbols indicate heterozygous carriers . ( c , d ) determination of the lcat gene sequences of proband and their families . heterozygous mutation of mother and elder sister and homozygous mutation of proband are shown in cases 1 ( c ) and 2 ( d ) . electron micrograph shows lipid deposits with a vacuolar lucent appearance in the gbm ( bar = 2 m ) . ( g i ) high - performance liquid chromatography patterns of cases 1 ( g ) , 2 ( h ) and normal healthy control ( i ) . a 30-year - old japanese woman was referred because of very low hdl - c and ldl - c associated with ( 1 + ) proteinuria via dipstick ( 0.08 g / day ) . she also showed bilateral corneal opacity and mild hemolytic anemia ( 11.2 g / dl ) , demonstrating target cells on her peripheral blood smear . a 27-year - old japanese man was referred to okayama university hospital because he had extremely low hdl - c and ldl - c concentrations . on physical examination laboratory findings demonstrated a mild degree of normochoromic - normocytotic anemia ( 11.3 g / dl ) . although his renal function was normal , ( + 1 ) proteinuria was revealed via the dipstick method ( 0.21 g / day ) . the consanguinity was found in his parents as shown in figure 1a . presented cases with fld . squares are male , circles are female . filled symbols indicate homozygous carriers and left filled symbols indicate heterozygous carriers . ( c , d ) determination of the lcat gene sequences of proband and their families . heterozygous mutation of mother and elder sister and homozygous mutation of proband are shown in cases 1 ( c ) and 2 ( d ) . electron micrograph shows lipid deposits with a vacuolar lucent appearance in the gbm ( bar = 2 m ) . ( g i ) high - performance liquid chromatography patterns of cases 1 ( g ) , 2 ( h ) and normal healthy control ( i ) . a 30-year - old japanese woman was referred because of very low hdl - c and ldl - c associated with ( 1 + ) proteinuria via dipstick ( 0.08 g / day ) . she also showed bilateral corneal opacity and mild hemolytic anemia ( 11.2 g / dl ) , demonstrating target cells on her peripheral blood smear . direct sequencing of lcat gene and comparison with reference sequence ( nm_000229 ) revealed that case 1 had a c to t nucleotide substitution in exon 2 resulting in pro69leu [ c.278 c > t ( p.pro69leu ) ] ( figure 1c ) and case 2 had a t to c substitution in exon 6 resulting in met293thr mutations [ c.950 t > c ( p.met293thr ) ] ( figure 1d ) . case 1 carried apoe 4/3 genotype , whereas case 2 carried apoe 2/2 genotype ( table 1 ) . the mother of case 1 carried apoe 3/3 genotype and genomic dnas were not available from the deceased father . renal biopsy in both cases revealed similar histological findings ; diffuse vacuolization of the glomerular basement membranes ( gbm ) by periodic acid - methenamine silver stain and lipid deposits with a vacuolar lucent appearance in the mesangial matrix and the gbm by electron microscopy ( figure 1e and f ) . in both cases , half normal lcat activity was observed by using exogenous substrate , suggesting the diagnosis of fed . however , clinical manifestations with hemolytic anemia and renal involvement coincided with the features of fld . characteristics and lipoprotein profiles of presented casesa
reml - c , remnant - like particles cholesterol measured with metabolead reml - c ; nd , not determined . both cases 1 and 2 revealed similar lipid profile ; ldl - c and hdl - c were extremely low ( table 1 ) . furthermore , fc / total cholesterol ( tc ) ratio was much higher than normal value , suggesting reduced activity of esterification of plasma cholesterol due to lcat deficiency . in both cases , the lipoproteins in fraction 710 , corresponding to idl particle size , were characterized by triglycerides ( tg)- and phospholipid ( pl)-rich . since lp - x is fc- and pl - rich lipoprotein particle , the peaks appeared in fraction 710 seem to be remnant lipoproteins containing idl remnant ( figure 1 g and h ; arrows ) . in addition , lp - x was not detected by agarose gel electrophoresis in both cases . the remnant - like particles cholesterol was measured with metabolead reml - c well - reflects idl remnant compared with immunoseparation assay , the high values of reml - c in both cases also support the elevation of idl remnant . lynn et al . recently reported that accumulation of lp - x in renal lesion can stimulate monocyte chemoattractant protein-1 expression in mesangial cells via nuclear factor-b . reported lp - x is larger than normal ldl - c in size , and the percentages for tg , cholesteryl ester , fc and pl were 9.5 , 6.2 , 27.6 and 56.8% in lp - x , respectively . in our cases , we found tg- and pl - rich remnant idl particles , where tc and fc contents are very low . previous research has observed that apoe 2 allele carriers reveal decreased ldl - c and increased tg and lipoprotein remnants , whereas apoe 4 carriers have increased ldl - c and tg in comparison to 3/3 subjects [ 8 , 9 ] . it has been suggested that lipoproteins of 2 carriers have a low affinity for lipoprotein receptors , which induces decreased cholesterol delivery to the hepatocytes and a following upregulation of hepatic sterol synthesis and ldl receptors . therefore , a decreased conversion of very low - density lipoprotein into ldl and appearance of idl is observed in 2 carriers . baass et al . hypothesized that the presence of the apoe 2 allele contributed to the accumulation of idl instead of lp - x in the patients with lcat deficiency , but idl remnant is even higher in case 1 carrying apoe 4/3 genotype compared with case 2 with apoe 2/2 genotype ( figure 1 g and h ) . the discrepancy of these observations may attribute to the difference in mutations of lcat gene rather than apoe genotype and further characterization of enzymatic activities of mutated gene products is required in future investigations . in conclusion , we describe two unrelated fld patients with corneal opacity , hemolytic anemia , abnormalities in serum lipoprotein profile and lipid accumulation in gbm . instead of lp - x , we detected idl remnant in both cases and similar pathological findings in renal biopsy . genotyping apoe gene revealed apoe 4/3 in case 1 and a rare association of apoe 2/2 in case 2 , thus apoe 2/2 may not be the major modifier gene for the appearance of idl in fld patients . more cases need to be investigated to draw the definitive conclusions on the relation among lcat mutation , apoe genotype and lipoprotein profile in fld patients . | familial lecithin : cholesterol acyltransferase deficiency ( fld ) is an autosomal recessive disorder characterized by corneal opacity , hemolytic anemia , low high - density lipoprotein cholesterol ( hdl - c ) and proteinuria .
two novel lecithin : cholesterol acyltransferase ( lcat ) mutations[c.278 c > t ( p.pro69leu ) ; c.950 t > c ( p.met293thr ) ] were identified in a 27-year - old man and in a 30-year - old woman , respectively .
both patients manifested corneal opacity , hemolytic anemia , low low - density lipoprotein cholesterol and hdl - c and proteinuria . lipid deposits with vacuolar lucent appearance in glomerular basement membranes were observed in both cases .
apoe genotype was also investigated : the first case results 4/3 , the second 2/2 ; however , they shared a similar phenotype characterized by the presence of intermediate - density lipoproteins ( idl ) remnant and the absence of lipoprotein - x . in conclusion , our findings suggest that apoe 2/2 may not be the major determinant gene for the appearance of idl in fld patients . |
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yawning is a physiological response defined by a sequence of involuntary mouth opening , deep inspiration , brief apnea , and slow expiration . the james lange theory suggests that feedback of afferent signals from muscles and viscera integrates at the brain to create arousal , with yawning as a manifestation of bodily consciousness . certain emotional states and drowsiness are physiological events that have been shown to be associated with excessive yawning , as have pathological conditions such as stroke and frontal lobe / brain stem tumors , , , . there are only a few cases in the literature where yawning has been described as a symptom of epilepsy . while such cases are rare , they describe yawning as a periictal phenomenon or as an ictal manifestation of epilepsy , most commonly temporal lobe epilepsy ( tle ) , , , , , . based on the current review of the literature , we present the first case of controllable yawning as a primary seizure semiology due to dominant frontal lobe involvement . a 20-year - old right - handed man presented with brief episodes of strange symptoms with yawning noted since age 18 years . he described these as brief episodes of yawning lasting a few seconds , with associated lightheadedness , at times with muscle spasms over his arms or neck without loss of awareness . these usually occurred upon waking up in the morning or when sitting at a computer , also when tired or sleep - deprived . he added that he could restrain his urge to yawn . to characterize his spells , an 8-hour video electroencephalography ( veeg ) however , 8 typical yawning episodes were recorded , which at times were accompanied by right arm movement and showed rhythmic diffuse theta range activity with left - hemispheric predominance lasting for up to 7 s on eeg ( fig . a.1 ) . a follow - up study performed after 6 months recorded episodes when he restrained his yawn with no ictal correlates on eeg . magnetoencephalography ( meg ) was also completed with a 148-channel neuromagnetometer system ( 4d neuroimaging ) with 32 channels of simultaneous digital eeg . overall functional brain connectivity was imaged with meg coherence source imaging ( meg - csi ) . results were encoded as a color spectrum for values between 1 ( entirely coherent ) and 0 ( no coherence ) and overlaid on the patient 's mri with the solutions restricted to the gray matter . the meg - csi exclusively lateralized high coherence to the left hemisphere , and persistent neuronal networks were most reliably present in the left lateral orbitofrontal gyrus ( fig . cortical thickness of the brain was evaluated based on the t1-weighted magnetic resonance images ( mri ) , acquired on a 1.5 t philips healthcare scanner and processed using the freesurfer software ( version 5.3.0 ; http://www.surfer.nmr.mgh.harvard.edu ) . the intensities of the t1-weighted images were bias - corrected using the nonparametric nonuniform intensity normalization implemented in the n3 v1.10 package along with their default parameters . then , skull stripping was performed using a hybrid watershed algorithm . the cortical regions of the brain were extracted , and the surface of each hemisphere was registered to the surface of the freesurfer 's atlas . finally , the cortical thickness of the left hemisphere ( lh ) was subtracted from the cortical thickness of the right hemisphere ( rh ) point - by - point . a smoothed version of the lh rh map , spatially smoothed using a 5-mm fhwm gaussian kernel , is also shown on the patient 's lh surface , using the same thresholding and color - coding scheme . left lateral orbitofrontal and medial orbitofrontal ( marked in red color ) regions showed thicker cortex ( fig . the patient was started on lacosamide , and seizures were better controlled with dose optimization to 200 mg twice daily . in addition , he could control the progression of his seizures by consciously restraining his yawn . our case demonstrates controllable yawning as a novel primary seizure semiology in dominant frontal lobe epilepsy as diagnosed with veeg and meg and supported by mri - based cortical thickness analysis . this suggests that epileptogenic areas could also be intricate with widespread networks involving the dominant frontal lobe symptomatogenic areas . physiologic yawning is thought to be primarily controlled by the paraventricular nucleus of the hypothalamus , the locus coeruleus , and reticular activating system . one theory proposes that nerve endings of the incertohypothalamic dopaminergic system project into oxytocinergic neurons that , in turn , connect to the hippocampus , pons , and medulla oblongata , stimulating yawning . yawning is still seldom reported in connection with epilepsy , since first described by penfield and jasper . found 12 patients , with associated periictal or ictal yawing : in 2 patients as preictal , in 4 patients as exclusively ictal , and in 6 patients as postictal manifestation of the seizures . mechanisms have been proposed to explain periictal yawning , namely brainstem activation due to changes in alertness , activation due to epileptic discharge , or seizure - mediated release of neurohormonal substances . yawning as an ictal phenomenon has been reported , although rarely , as a pathological condition in the elderly originating from diencephalic / brain stem structures . three other case reports reveal ictal yawning as a manifestation of tle , , , while others displayed either focal spike - and - waves or diffuse attenuation on eeg . as a postictal phenomenon , yawning occurred in 5 of 6 patients with tle reviewed by specchio et al . , of which 4 had nondominant tle with histopathology showing hippocampal sclerosis , ganglioglioma , cavernous angioma , and dsyembryoplastic neuroepithelial tumor . another patient with postictal irresistible , forceful yawning had nondominant hemisphere epilepsy with right centroparietal discharges on eeg . in contrast , our patient appears to be a rare case with yawning occurring as an ictal manifestation of dominant frontal lobe epilepsy . occasionally associated symptom of right arm jerk could explain contralateral frontal lobe involvement as well . a study involving slow event - related fmri experiments in volunteers suggested that ventromedial prefrontal cortex ( vmpfc ) activation is associated with the urge to yawn by contagion , independently of mirror neuron network . similarly , the urge to control yawning in our patient could possibly indicate executive function being accessed from the involved frontal lobe . in addition , it is not uncommon to encounter a paucity of interictal epileptiform discharges on eeg in frontal lobe epilepsy . after obtaining veeg findings that correlated yawning with ictal left - hemispheric predominant slow activity , further investigation with meg - csi confirmed an area of high coherence within the epileptiform network over the left lateral orbitofrontal regions . although the routine mri of the brain was unremarkable , advanced analysis examining the cortical thickness showed increased gray matter in the same area as meg - csi . this is the first report showing left frontal lobe involvement in ictal yawning diagnosed with functional testing by veeg and meg and structural brain mapping by mri cortical thickness analysis . although the exact mechanism for our patient 's seizure semiology is unclear , it is possible that neural networks within vmpfc and the lateral orbitofrontal region activated by epileptic discharges somehow trigger the reflex in brainstem leading to yawning . although our patient appeared to be a rare case , it is possible that many cases go unreported , especially if seizures are controllable . in conclusion , the case we described expands our view on the possibilities of localization for ictal yawning that could also be present with dominant frontal lobe epilepsy . our understanding in such cases is still inadequate , and further studies with advanced diagnostic investigative modalities will likely help better define such epilepsies with intricate neuronal networks . | excessive yawning was described in some neurological conditions as part of periictal or ictal manifestations of epilepsy , most commonly temporal lobe .
we present the first case of controllable yawning as a primary seizure semiology with dominant frontal lobe involvement in a 20-year - old man .
video electroencephalography recorded 8 yawning episodes accompanied with right arm movement correlating with rhythmic diffuse theta range activity with left hemispheric predominance .
magnetoencephalography coherence source imaging was consistent with persistent neuronal networks with areas of high coherence reliably present over the left lateral orbitofrontal region .
epileptogenic areas may have widespread networks involving the dominant frontal lobe in unique symptomatogenic areas . |
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bile duct injuries remain one of the most devastating complications of both open and laparoscopic cholecystectomy ( lc ) . heuristic errors of perception in which surgeons fix anatomical reference points incorrectly and commence dissection in an area of danger is perhaps the most common cause of bile duct injuries . injury prevention theories have suggested that strategies to prevent injuries would be most effective at the time of anatomical identification and orientation , and prior to dissection . we present three cases of right sectoral duct injuries , two of which had right posterior sectoral duct ( rpsd ) injuries noted on intraoperative cholangiogram ( ioc ) ( strasberg type c injuries ) ( figures 1 , 2 ) , while the third had a right sectoral duct injury noted on postoperative endoscopic retrograde cholangiopancreatography ( ercp ) ( strasberg type a injuries ) ( figure 3 ) ( table 1 ) . biliary injuries were primarily repaired and stented over 14 french ( fr . ) caliber t - tubes brought through separate choledochotomies in the first two cases , while an ercp was utilized for postoperative evaluation and therapy in the third . although most leaks originate from the cystic duct ( cd ) stump and common bile duct ( cbd ) , those from aberrant sectoral bile ducts are also common but rarely discussed . biliary tree anomalies are present in up to 25% of patients , with aberrant right hepatic ducts being the most common . aberrant sectoral duct arise most commonly from the right liver and drain into the common hepatic duct ( chd ) or cd ( figure 1(b ) ) . an aberrant right sectoral hepatic duct usually represents the only route of biliary drainage for the portion of the right hepatic lobe it drains . the most dangerous sectoral variant is when the cd runs along side of a low - lying aberrant right sectoral duct . most commonly this is the rpsd , which drains segments 6 and 7 and is present in 4.88.4% of the population [ 4 , 5 ] . injuries to these ducts are likely underreported since they may be asymptomatic and often unrecognized as the injured area atrophies over time . three patients underwent lc and sustained iatrogenic rpsd injuries requiring operative or endoscopic management . in the first two cases , a review of iocs ( figures 1 , 2 ) demonstrated aberrant biliary anatomy and injuries to low - lying rpsds . a tangential injury to the rpsd in both cases was successfully recognized and repaired primarily by duct - to - duct anastomosis . t - tube ( 14 fr . ) stenting of the repair was performed through a separate choledochotomy , with the upper arm of the t - tube crossing the injured rpsd . post - repair cholangiogram obtained through the t - tube revealed opacification of all segments of the right liver without extravasation or stricture ( figure 2 ) . these injuries may have escaped detection and opacification at ercp due to the lack of communication of the injured ducts with main biliary channels , thereby rendering endoscopic management impossible . such injuries should be suspected when a bile leak persists despite normal cholangiography and there is a presumed failure of a cd stump leak to close after biliary stent placement . in the third case , an ercp ( figure 3 ) revealed the bile leak to arise from an injured aberrant right sectoral duct with anomalous communication to the left biliary system . pilots and astronauts need to make accurate judgments of motion to perform critical aerospace tasks safely and effectively to avoid disaster . although laparoscopic biliary surgery is over 15 years old , biliary injuries continue to be a major source of morbidity and concern for surgeons . despite continued efforts , the rate of injury has leveled off at 0.5% for lc . typically , biliary injuries occur either due to technical problems or anatomical misidentification of the cd , latter being the most common cause . two structures most often misidentified as the cd are the cbd or an aberrant right hepatic duct ( rhd ) . the resultant injuries are of two main types cbd mistaken for cd is clipped and divided ; and the segment of an aberrant rhd ( between entry of the cd and junction of the chd ) is misidentified and either ligated , transected or injured during dissection in calot 's triangle . way et al . analyzed 252 patients with laparoscopic bile duct injuries and concluded that these injuries stem principally from visual perceptual illusion , and not from errors of skill , knowledge , and judgment . this misperception theory is compelling in that even on identification of irregularities corrective feedback did not occur , thus supporting the notion that the native human mindset to hold firm to our assumptions is strong . in spite of excellent laparoscopic visualization , misidentification of extrahepatic accessory bile ducts during lc is a frequent cause of complications . ioc is good at avoiding misidentification of the cbd , but poor at detecting an aberrant rhd depending on the location of the cd puncture site . nevertheless , performing complete circumferential dissection of the gallbladder neck and cd before cholangiography or clip placement should enable visualization of any variant anatomy . this recommendation was not followed in two of our cases , hence non - circumferential dissection and early clipping was performed . a choledochotomy for the cholangiogram was mistakenly made into an aberrant rhd instead of the cd ( figure 1 ) . this type of injury has been described as a class iv lawrence way injury due to misidentification of the rpsd as the cd resulting in a lateral injury , transection or ligation of an unseen low - lying rhd . when correctly interpreted , routine ioc increases the chances of recognizing bile duct injuries , but it may not lower their incidence . this prompts us to conclude that more than a routine ioc may be essential to prevent biliary injuries . magnetic resonance cholangiopancreatography ( mrcp ) is an excellent noninvasive imaging modality that is capable of demonstrating aberrant biliary anatomy . analyzed anatomical variations of the biliary tree with multislice - computed tomography ( mct ) cholangiography in 113 patients . mct cholangiography provided clear images of aberrant bile ducts in 18 patients major type ( draining liver segment ) in 9 ( 8% ) and minor type ( draining liver subsegment ) in 9 ( 8% ) . alibrahim et al . demonstrated the efficacy of spiral ct - iv cholangiography with 3d reconstructions for imaging the biliary tree . using this technique excellent visualization of the biliary anatomy was achieved with good opacification of at least first - order or third - order bile ducts in 91% or 84% of patients , respectively . despite their efficacy , routine ercp , mrcp , and contrast - enhanced mct cholangiography are expensive tools , and should be reserved for select cases . irrespective of information derived on imaging , improvisation of surgical technique is necessary to avoid biliary injuries . careful dissection , conclusive identification of anatomy with clearing of the hepatocystic triangle of all fat , and determination of endpoint in the dissection is pivotal in prevention of intraoperative complications . in 1995 strasberg et al . introduced a technique for conclusive identification of the cystic structures at lc based on a other important strategies include the use of a 30-degree telescope which provides en face view of the calot 's triangle and its intermittent withdrawal gives the surgeon an overall perspective and spatial orientation . simulation - based surgical training and the availability of biologically - based models of human body architecture will permit virtual reality to serve as quantitative models of human self - motion perception . performance analysis in heading estimation and related motion perception tasks will aid in the design of training regimes for surgeons in the near future . robotic instruments to simulate the motions of the surgeon 's wrist and the use of 3d imaging to immerse the surgeon in a 3d video operating field may also offer potential solutions to the inherent problems of traditional laparoscopic surgery . though exciting , these technologies are still in an early stage of development , and each device entails its own set of challenges and limitations . despite all the emerging advances bile duct injuries will still occur , and if they do conversion to open procedure is usually indicated . repair should be attempted by general surgeons only if the techniques of dissection and reconstruction are common and familiar , and every effort should be made to conserve bile duct length . repair techniques for minor injuries to the lateral wall of the chd or cbd are familiar to most general surgeons , but injuries to aberrant ducts and those at or above the confluence of hepatic ducts require operative skills more likely to be available at specialized hepatobiliary units . when such expertise is unavailable at the time of injury , closed suction drains should be placed in the region of hepatoduodenal ligament and referral made expeditiously . | although laparoscopic cholecystectomy ( lc ) has been widely accepted as the standard of care , it continues to have a higher complication rate than open cholecystectomy .
bile duct injury with lc has often been attributed to surgical inexperience , but it is also clear that aberrant bile ducts are present in a significant number of patients who sustain biliary injuries during these procedures .
we present three cases of right sectoral hepatic duct injuries which occurred during lc and provide a discussion of the conditions which are likely to lead to these injuries , as part of a strategy to prevent them . |
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the incidence of obesity increased rapidly among children and adolescents in the united states over the last few decades . in 2007 - 08 , obesity prevalence ( with body mass index ( bmi ) greater than or equal to the 95th percentile of the cdc growth chart ) was 19.6% for children 6 to 11 years of age and 18.1% for adolescents aged 12 to 19 years . parallel to the rising prevalence of obesity among children and adolescents , there was a rapid rise in the consumption of , and total energy intake derived from , food away from home , particularly at fast food restaurants [ 2 , 3 ] . consumption of at least one serving of fast food per day was reported by 30.3% of children and adolescents in 19941998 . fast food restaurants were found to be an important source of food away from home among children aged 2 through 17 years ; the percentage of total calories derived from food consumed at fast food restaurants rose from 2% in the 1977 - 78 period to 9% during the 199496 period . the increased proportion of total caloric intake derived from fast food has been variously attributed to bigger portion sizes , increased convenience , taste , accessibility , and affordability [ 3 , 57 ] . furthermore , the frequency of fast food consumption was reported to be rising with age among children ages 9 through 19 as their consumption decisions become more independent of parental influences [ 6 , 8 ] . fast food consumption has been associated with higher total caloric intake , higher total fat and sodium intake , poorer nutrient and vitamin intake , higher bmi , and increased likelihood of obesity [ 4 , 913 ] . an increase in fast food intake during the transition period from adolescence to adulthood was linked to greater weight gain . potentially modifiable determinants of childhood eating behaviors and food choices include environmental and contextual factors , such as the availability of healthy food , promotion of appealing and convenient foods , and variations in food prices [ 16 , 17 ] . food prices , in particular , have been shown to be key determinants of consumption [ 1823 ] and therefore may be good candidates for effective policy intervention aimed at improving dietary patterns among children and adolescents . a systematic review of time series and household survey studies on price elasticity of demand for food found that consumption of food away from home was more responsive to price changes than any other food category with a 10% increase in price associated with a 8.1% reduction in consumption . a limited number of studies on price sensitivity among children and adolescents found mixed evidence on the association between prices and intake of high - fat energy - dense foods . one such study using data from the 19941998 continuing survey of food intakes by individuals ( csfii ) found that higher fast food prices were associated with lower fast food consumption for children aged 29 years and adolescents aged 1018 years although the association was not statistically significant for the adolescent sample . using data from a high school cafeteria , another study found that a 10% increase in the price of higher - fat foods ( french fries , cookies , and cheese sauce ) and a 25% reduction in the price of lower - fat foods ( fresh fruit , low - fat cookies , low - fat chips , and cereal bars ) did not substantially alter revenue , suggesting that consumption patterns shifted towards healthier foods . a vending machine price study found low - fat snack price reductions of 10 , 25 , and 50 percent corresponded to increased sales of 9 , 39 , and 93 percent , respectively , among both adolescents and adults . a recent study by sturm and datar using a single cross - section of 5th graders found a positive relationship between the price of fast food and intake . however , the authors indicated that this result was not robust and disappeared in alternative specifications potentially due to the correlation between fast food and meat prices in the data . linking food price to fast food consumption is important given that a number of recent studies have found statistically significant negative associations between fast food prices and the prevalence of overweight among children and adolescents [ 2833 ] . the link between fast food prices and consumption helps to establish that the observed negative association between fast food prices and obesity operates through fast food consumption and is not due to unobserved confounding factors . therefore , we expanded on prior studies by using a longitudinal individual - level random effects estimation model , which controls for unobserved individual factors , to examine the relationship between children 's fast food consumption and prices of fast food and food at home . economic contextual factors including median household income and availability of fast food restaurants at the zip code level were included in the analysis to further control for confounding factors . this study drew on two waves of individual - level data ( 5th graders in 2004 and 8th graders in 2007 ) from the early childhood longitudinal study , kindergarten class of 1998 - 99 ( ecls - k ) . trained evaluators assessed children in their schools , collected information from their parents over the telephone , and contacted teachersand school administrators to complete the questionnaires . information on the frequency of fast food consumption was self - reported by the children , anthropometric variables such as height and weight were measured by the evaluators , home environment and school characteristics were self - reported by parents , teachers , and school officials . the outcome of interest was child fast food consumption , which was comprised of how many times in the past 7 days the child ate a meal or snack from a fast food restaurant such as mcdonald 's , pizza hut , burger king , kentucky fried chicken , taco bell , wendy 's , and other similar establishments . the responses given by the children were converted from a categorical scale to a numerical scale using midpoints to facilitate analysis . the contextual data consisted of food prices from the american chambers of commerce researchers association ( accra ) , fast food outlet density data from dun and bradstreet , and median household income from the census 2000 . food price indices were computed from the accra cost of living index reports , which contain quarterly information on prices in the united states , and which were matched to each child for each year based on the closest zip code match available using the child 's home zip code identifier . an index of fast food price was computed using three food items in the accra data , which includes a mcdonald 's quarter - pounder with cheese , a thin - crust regular cheese pizza at pizza hut and/or pizza inn , and fried chicken ( thigh and drumstick ) at kentucky fried chicken and/or church 's fried chicken . in order to compute the price index of food at home , a basket of grocery items was utilized , which included meats ( steak , ground beef , fried chicken , and tuna ) , fruits and vegetables ( potatoes , bananas , lettuce , sweet peas , peaches , and frozen corn ) , dairy products ( half gallon of whole milk , a dozen eggs , margarine , and parmesan cheese ) , and soft drinks . both price indices were weighted based on expenditure shares provided by accra derived from the bureau of labor statistics ( bls ) consumer expenditure survey . all prices were deflated by bls consumer price index ( cpi ; 19821984 = 1 ) . in addition to the price data , a number of economic contextual variables , including the census median household income and fast food restaurant density were merged to the restricted geocoded version of ecls data at the zip code level to account for differences in neighborhood characteristics . the median household income data for each zip code was obtained from the census 2000 . information on fast food restaurant outlet density was obtained by year and zip code from the dun and bradstreet ( d&b ) business list data at the primary 8-digit sic code level for categories of fast food restaurants and stands and chain and independent pizzerias but not coffee or ice cream shops . the basic controls consisted of standard individual and household characteristics including age , gender , race ( white , african american , hispanic , other race , and more than one race ) , mother 's highest level of education completed ( less than high school , high school , some college , bachelor 's degree or more ) , family income ( categories consisting of income below $ 20,000 , $ 20,001$35,000 , $ 35,001$50,000 , $ 50,001$75,000 , $ 75,001$100,000 , and $ 100,001 and above ) , degree of urbanicity ( urban , suburban , and rural ) , and an indicator for the year . two variables indicating the number of times per week a child ate breakfast and dinner with parents were included to account of unobservable characteristics such as the importance the parents place on children 's food consumption patterns . number of hours of television watched per week was included to control for lifestyle behaviors . all data analysis for this project this study was approved by the institutional review board at the university of illinois at chicago . children 's food consumption may be influenced by a number of economic contextual factors , including food prices . changes in relative prices of different food products are expected to influence the relative demand for these products . consumption of fast food will have own - price effects from fast food prices , and cross - price substitution effects from prices of other foods ( such as food at home ) . a model of children 's fast food consumption of the following form was estimated :
( 1)ffconsist=0+1ffpricest+2fhpricest+3ffrestst + 4mhincs+5xit+6dt+vi+wist ,
where ffpricest and fhpricest were indices of fast food and food at home prices for geographic area s at time t , respectively , for which we also controlled for the average distance of the price match . ffrestst measured the per capita availability of fast food restaurants for geographic area s at time t. mhincs , was the median household income in geographic areas . xit was a vector of individual and household characteristics for individual i at time t. the characteristics in the vector xit included gender , race / ethnicity , highest level of schooling completed by mother , parental income , urbanicity indicators , number of days per week child ate breakfast with parents , number of days per week child ate dinner with parents , and number of hours per week child watched television . were the parameters to be estimated , vi was the constant individual - specific error , and wist was a standard error term . to take advantage of the longitudinal nature of data , an individual - level random effects model was estimated to provide a weighted average of the between and within estimates where vit and the independent variables were assumed to be uncorrelated . we tested this assumption based on a hausman test which indicated that a random effects model was appropriate . further , we conducted functional form comparisons and determined that a linear form of individual - level random effects model was more appropriate than count data forms . the poisson goodness - of - fit and the likelihood ratio tests indicated that the poisson distribution was not suitable for this sample . the point estimates from the linear random effects models were comparable to binomial count models ; however , the latter models did not permit the use of clustering and reporting of robust standard error due to the limitation in the statistical software . further , previous work also examined the distribution of these outcomes and estimated them using linear ols . sensitivity analysis was conducted to assess the robustness of the fast food price estimates to exclusions of additional contextual variables . the data showed ( not shown in tables ) that the frequency of fast food consumption was significantly higher ( p 0.01 ) for children who were male , african american , low - income , and frequent television viewers compared to their counterparts . it is likely that these individuals with greater frequency may be more price sensitive , given that their overall expenditure on fast food would be greater than those with less frequency . thus , we expanded our analysis to estimate models by various subgroups across gender , race , parental income , and child television usage . further , given that previous research found that adolescents with body mass index ( bmi ) in the upper tail of the distribution was more responsive to fast food prices , we also estimated models separately by overweight status . the average weekly frequency of fast food consumption by the 5th and 8th graders was roughly 2.5 times in the past week . among the household characteristics , about half ( 50.2% ) of the sample was female . additionally , about 66.5% were white , 9.2% were african american , 15.2% were hispanic , 2.2% defined themselves as multiple races , and 6.9% were of another race . on average , more than one - half of the children ( 63.6% ) had mothers who completed some college or more ; about 23.7% of children had mothers whose highest level of education completed was high school . approximately , 10.6% of children lived in families with parental income below $ 20,000 , and 38.4% lived in families with income above $ 75,000 . nearly 67.3% of the households were from urban residential areas , while 13.4% were from suburban areas and 19.3% were from rural areas . among the economic contextual factors , the average fast food price was $ 2.66 while the average price of food at home was $ 1.19 ( based on the 19821984 deflated price indices ) . on average , finally , there were approximately 3.6 fast food restaurants per 10,000 capita per zip code . table 2 presents the regression estimates of childhood fast food consumption using an individual - level random effects model . a one - standard deviation increase in the price of fast food ( a $ 0.17 increase ) was associated with a reduction in fast food consumption of approximately 0.09 times per week based on the coefficient estimate that a one - dollar increase in the price was associated with an approximate decline of 0.53 in the weekly frequency of fast food consumption . measured as a price elasticity ( the percentage change in consumption associated with a one - percent change in price ) , the estimates corresponded to a price elasticity of 0.57 , suggesting that a 10% increase in price of fast food was associated with a reduction in weekly consumption by 5.7% , as shown in table 3 . the results from sensitivity analyses presented in table 3 show that the relationship between fast food prices and consumption was generally robust to the exclusion of the various other contextual factors . the association of the price of fast food increased slightly by about 20% ( elasticity of 0.69 versus 0.57 ) when all other contextual factors were excluded from the model . higher median household income was associated with lower fast food consumption : a $ 10,000 rise in median household income lowered fast food consumption by 0.13 times per week . greater availability of fast food outlets measured as outlets per capita was associated with higher frequency of fast food consumption . higher prices of food at home were associated with higher frequency of fast food consumption although the estimate was not statistically significant . table 4 shows that consumption among males was more responsive to increases in the price of fast food , which was expected given that they were more frequent fast food consumers than were females . we also found greater fast food price sensitivity among children from lower - income households , but the estimates were not statistically significant . the estimations across ethnicities show that the price of fast food was important and statistically significant for white children but not for african american or hispanic children despite the fact that minority children consumed fast food more frequently . this suggests minority children 's preferences for fast food may be influenced by factors other than food prices . children who frequently watched television ( nine or more hours per week ) were found to be more price sensitive than their counterparts . this suggests that higher fast food prices might reduce their consumption to a greater extent , which is important given television watching in general is associated with childhood obesity [ 38 , 39 ] and fast food ads are the most prevalent food ads seen on television by children and adolescents . finally , we also found stronger price sensitivity among children who were already overweight ( bmi greater than or equal to 85th percentile of the cdc growth chart ) , perhaps because when they consumed from fast food restaurants they purchased more calories . additionally , there were also significant differences across the subgroups in their response to the other contextual factors . fast food consumption by children from low - income households was more strongly influenced by zip code level median household income and fast food restaurant outlet density compared to children from high - income households . furthermore , the protective ( reducing ) effect of the higher median household income on fast food consumption was 3-times - greater for african american and hispanic minority children than for white children and also greater for more frequent television viewers . policymakers continue to consider a number of potential public health policy interventions aimed at reducing the negative health implications from the increasing rates of obesity prevalence among children and adolescents in the united states . public health policy may help to reduce obesity prevalence in children and adolescents by utilizing mechanisms that improve diets through the influence of food prices . children and adolescents often have disposable income and are able to make independent meal selections when food is not purchased for them directly by their parents . more specifically , the third highest spending category for this age group after beauty / apparel and videogames / electronics was restaurants [ 42 , 43 ] . since children are less likely to understand the nutritional and health implications of their high caloric consumption patterns , this age group is more likely to be potentially influenced by prices as opposed to health consequences as they may have high rates of time preference . thus , this study aimed to describe the relationship between fast food and food at home prices and fast food consumption among children in 5th and 8th grade using a nationally representative panel dataset . the results based on longitudinal estimation methods suggested that pricing policies in the form of taxes may be an effective tool for reducing fast food consumption . the fast food price elasticity of consumption of 0.57 indicated that this age group was sensitive to price variations such that a ten percent increase in price was associated with a 5.7% reduction in weekly fast food consumption . this estimate is in the mid- to lower - range of estimates of price elasticity of demand for food away from home . earlier studies suggested that pricing mechanisms may have the strongest impact on those individuals who were in the upper tail of the bmi distribution . consistent with these previous findings , this study found that that price of fast food had a stronger association with consumption among overweight children . we also found a number of subpopulations who were more frequent fast food consumers to be relatively more price sensitive . this suggests that pricing policy instruments such as fast food taxes might be particularly effective in reducing fast food consumption among frequent consumers and those at risk for overweight . this is important given that previous research had showed that adolescents who were overweight were less likely to compensate for their fast food meals by properly adjusting their energy intake downward throughout the remainder of the day . first , the fast food consumption data were self - reported total number of days per week consumed , not actual amounts of consumption ( e.g. , caloric intake ) . second , the accra price data had a number of limitations , which included that the data were only collected in a limited number of cities and metropolitan statistical areas , the data were based on establishment samples that reflect a higher standard of living , and they did not always sample the same cities continuously and hence the data were not fully comparable over time . third , the outlet density count measures were subject to count error and we were limited to using sic codes which may have classification errors . fourth , this study was only able to assess the contextual variables at the zip code level due to data limitations in the availability of more proximate geographic identifiers in the esls - k data . despite these limitations , the results from the individual - level random effects model in this paper suggested that higher fast food prices were associated with lower frequency of children 's fast food consumption . therefore , public health policy interventions such as taxes may be effective in reducing consumption of fast food and possibly reducing the prevalence of overweight and obesity among children . given that the price effects were stronger among children who were male , white , overweight , and frequent television viewers , additional studies based on longitudinal data are needed to develop the evidence base with regard to the potential effectiveness of pricing interventions among various groups of children to help improve food consumption patterns , overall diet , and health outcomes that may possibly translate into healthier outcomes later in adulthood . | fast food consumption is a dietary factor associated with higher prevalence of childhood obesity in the united states .
the association between food prices and consumption of fast food among 5th and 8th graders was examined using individual - level random effects models utilizing consumption data from the early childhood longitudinal study , kindergarten class of 1998 - 99 ( ecls - k ) , price data from american chamber of commerce researchers association ( accra ) , and contextual outlet density data from dun and bradstreet ( d&b ) .
the results found that contextual factors including the price of fast food , median household income , and fast food restaurant outlet densities were significantly associated with fast food consumption patterns among this age group .
overall , a 10% increase in the price of fast food was associated with 5.7% lower frequency of weekly fast food consumption .
these results suggest that public health policy pricing instruments such as taxes may be effective in reducing consumption of energy - dense foods and possibly reducing the prevalence of overweight and obesity among us children and young adolescents . |
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nevus sebaceus ( ns ) is a common congenital birthmark that occurs most frequently on the head and face . removal is preferable because various tumors have been reported to develop in ns [ 1 , 2 ] . basal cell carcinoma ( bcc ) seldom occurs in ns , and it is very important to be able to clinicopathologically distinguish bcc from trichoblastoma . herein , we describe a case of bcc and trichoblastoma occurring simultaneously in the same ns , including the differential dermoscopic features . a 77-year - old woman had been diagnosed at the age of 15 years with ns which had started from the left side of her ear and extended to her chest . she was referred to our hospital with a 3-year history of black macules on the left ear . a physical examination showed yellowish plaques around the left side of the neck and small , numerous yellow papules in the ns . a black macule of 2 mm in diameter was observed on the left auricle ( fig . a dermoscopic examination revealed multiple blue - gray ovoid nests and whitish veils , but arborizing vessels were not found ( fig . a surgical specimen showed that tumor cells were proliferating from the epidermis to the upper dermis and were arranged in a palisading pattern at the periphery with basal melanosis . deposits of mucin and clefts were observed in the surrounding area with dilated vessels in the papillary dermis ( fig . three months after the surgery , a small black macule appeared on the left earlobe which slowly enlarged . a physical examination indicated the presence of a small black macule with a diameter of 1 mm on the left earlobe ( fig . a dermoscopic examination revealed a round , homogeneous , brownish structure without arborizing vessels ( fig histopathologically , the surgical specimen showed that the tumor cells were proliferating in the dermis , presented as multiple different shapes , were either clear or basophilic , and were arranged in a partial palisading pattern . the tumor cells also resembled follicular germinative cells without keratinizing cysts , and islands of tumor cells were present as hair bulbs surrounding fibrous stroma ( fig . a study on tumors occurring in ns found that trichoblastoma was observed in 4.7% of cases ( 28 of 596 cases ) and bcc in 0.8% of cases ( 5 of 596 cases ) . in a dermoscopic examination of bcc , arborizing vessels were observed in 84.1% of cases and multiple blue - gray ovoid nests in 36% of cases . multiple black structures , and the trichoblastoma showed a single black structure without arborizing vessels . we theorize that this difference is a specific clue when distinguishing between bcc and trichoblastoma in ns . in a dermoscopic examination of ns , it is difficult to precisely evaluate a macule with vessels directly under the epidermis due to histopathological modifications of the background , particularly under the influence of epidermal proliferation . this is why our bcc did not reveal arborizing vessels . in conclusion , most ns are removed to avoid secondary tumors , including bcc ; thus , there have been no reports on their dermoscopic findings . | nevus sebaceus ( ns ) is a common congenital birthmark , and various tumors have been reported to develop in ns .
basal cell carcinoma ( bcc ) seldom occurs in ns , and it is very important to be able to clinicopathologically distinguish bcc from trichoblastoma .
herein , we describe a case of bcc and trichoblastoma occurring simultaneously in the same ns , including the differential dermoscopic features .
bcc is clinically difficult to distinguish from trichoblastoma because the clinical manifestations are similar . in a dermoscopic examination of bcc ,
arborizing vessels are one of the diagnostically significant features . in our case
, the bcc showed
multiple black structures , and the trichoblastoma showed a single black structure without arborizing vessels .
to the best of our knowledge , there have been no reports on the dermoscopic findings of secondary tumors on ns . |
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the ability and efficiency of mastication is the first step in the digestion of food , and this ability declines with age ( locker , 2002 ; morley , 2001 ; takata et al . , 2006 ) . masticatory ability is closely associated with maintaining adequate nutrition in older populations ( de morais et al . , 2013 ; kwon , suzuki , kumagai , shinkai , & yukawa , 2006 ; walls , steele , sheiham , marcenes , & moynihan , 2000 ) . lower masticatory ability reduces the variety in a person s diet , leading to malnutrition , weight loss , and a low body mass index ( bmi ) among the older adults ( nordenram , ljunggren , & cederholm , 2001 ; tamura , bell , masaki , & amella , 2013 ) . it is also reported that impairment of mastication is associated with general health status ( miura et al . , 1998 ; miura , araki , & umenai , 1997 ; miura , kariyasu , yamasaki , arai , & sumi , 2005 ; sun et al . , 2007 ) , quality of life ( qol ; brennan , spencer , & roberts - thomson , 2008 ; kimura et al . , 2009 ; takata et al . , 2006 ) , and cognitive function ( elsig et al . , 2015 ; mummolo , ortu , necozione , monaco , & marzo , 2014 ; teixeira et al . , active life expectancy also related chewing ability ( nasu & saito , 2006 ) . impaired masticatory ability often leads to frailty and institutionalization . maintenance or recovery of sufficient chewing ability for older adults is related to a longer active life expectancy . this study investigated whether self - rated masticatory ability is associated with a self - reliant life with adequate subjective health conditions in a community - dwelling older population . the older population in japan is expanding over time and accounts for more than 25% of the population ( i.e. , 32 million ) . the percentage of long - term care recipients above 65 years of age was 17.9% in may 2014 . the gap between life expectancy and active life expectancy is 9.13 years in females and 12.68 years in males , respectively , and this is a pressing issue in japan with increasing older populations , despite the fact that society has achieved the highest level of life expectancy . the extension of active life expectancy constitutes a pivotal issue for not only the well - being of the older population but also the demographics and socioeconomic of the society . this study population consists of participants in survey , which is a prospective and population - based study , and is designed to identify factors for self - reliant life in community - dwelling older adults in japan ( kayama , hironaka , & tani , 2014 ) . this study recruited 1,706 individuals who were 65 years and above , living in kumamoto city , and obtained informed consent from 1,554 of those subjects . those subjects had involvement with regional welfare commission volunteers and community comprehensive care center staffs . the study excluded subjects who died , moved , or were hospitalized during the study , and thus , 1,377 individuals participated ( 459 men and 910 women , mean age 79.8 6.6 years ) . this structured questionnaire survey was conducted in conjunction with regional welfare commission volunteers and community comprehensive care center staffs . the methods for data collection were described previously ( kayama et al . , 2014 ) . subjective health status was established by questionnaire with four levels of choice ; queried items included very healthy , moderately healthy , not very healthy , and not healthy . instrumental activity of daily living ( iadl ) was evaluated using an eight - item questionnaire that included walking for 15 min with or without a cane ; going out at least once a week , answering the telephone ; and using home electronic appliances , daily shopping activity , taking trash out , managing money , and ventilating rooms . forgetfulness was queried by the question item : have you been horribly forgetful lately . daily shopping activity was compared among those living with their family / caregiver and those who did not . daily shopping activity was categorized as supermarket store , convenience store , home delivery , and no shopping . the level of social activity evaluation included attending culture lessons or pursuing a hobby and interacting with neighborhoods . the level of neighborly ties as social capital were evaluated using a questionnaire that asked whether subjects shared any gifts ; had frequent conversations ; usually exchanged greetings ; and rarely interacted with their neighbors . family composition was characterized as living alone , living with spouses , living with family , and spending alone in the daytime . self - rated masticatory ability was measured on a 5-point scale from being able to eat whatever subjects wanted to eat , able to eat most foods except some hard ones , limited foods because of inadequate chewing , unable to eat few foods because of insufficient chewing , and a fluid diet . in this survey , this study was approved by the ethical committee of kumamoto health science university , kumamoto , japan ( no . 23 - 12 for epidemiology ) , and informed content was obtained from all participants . a logistic regression analysis was used to determine which categorical factors were independent predictors of having self - perceived health without requiring long - term care services . a multivariate logistic regression analysis was used to calculate the odds ratios ( ors ) and 95% confidence intervals ( cis ) after controlling simultaneously for potential confounders . all statistical analyses were performed using ibm spss statics for japan version 19.0 for windows ( ibm , japan ) . a value of p < .05 was considered to be statistically significant . this study population consists of participants in survey , which is a prospective and population - based study , and is designed to identify factors for self - reliant life in community - dwelling older adults in japan ( kayama , hironaka , & tani , 2014 ) . this study recruited 1,706 individuals who were 65 years and above , living in kumamoto city , and obtained informed consent from 1,554 of those subjects . those subjects had involvement with regional welfare commission volunteers and community comprehensive care center staffs . the study excluded subjects who died , moved , or were hospitalized during the study , and thus , 1,377 individuals participated ( 459 men and 910 women , mean age 79.8 6.6 years ) . this structured questionnaire survey was conducted in conjunction with regional welfare commission volunteers and community comprehensive care center staffs . the methods for data collection were described previously ( kayama et al . , 2014 ) . subjective health status was established by questionnaire with four levels of choice ; queried items included very healthy , moderately healthy , not very healthy , and not healthy . instrumental activity of daily living ( iadl ) was evaluated using an eight - item questionnaire that included walking for 15 min with or without a cane ; going out at least once a week , answering the telephone ; and using home electronic appliances , daily shopping activity , taking trash out , managing money , and ventilating rooms . forgetfulness was queried by the question item : have you been horribly forgetful lately . daily shopping activity was compared among those living with their family / caregiver and those who did not . daily shopping activity was categorized as supermarket store , convenience store , home delivery , and no shopping . the level of social activity evaluation included attending culture lessons or pursuing a hobby and interacting with neighborhoods . the level of neighborly ties as social capital were evaluated using a questionnaire that asked whether subjects shared any gifts ; had frequent conversations ; usually exchanged greetings ; and rarely interacted with their neighbors . family composition was characterized as living alone , living with spouses , living with family , and spending alone in the daytime . self - rated masticatory ability was measured on a 5-point scale from being able to eat whatever subjects wanted to eat , able to eat most foods except some hard ones , limited foods because of inadequate chewing , unable to eat few foods because of insufficient chewing , and a fluid diet . in this survey , this study was approved by the ethical committee of kumamoto health science university , kumamoto , japan ( no . 23 - 12 for epidemiology ) , and informed content was obtained from all participants . a logistic regression analysis was used to determine which categorical factors were independent predictors of having self - perceived health without requiring long - term care services . a multivariate logistic regression analysis was used to calculate the odds ratios ( ors ) and 95% confidence intervals ( cis ) after controlling simultaneously for potential confounders . all statistical analyses were performed using ibm spss statics for japan version 19.0 for windows ( ibm , japan ) . a value of p < .05 was considered to be statistically significant . the initial evaluation of the participants ( 1,377 individuals ) in the present study showed that 578 subjects could eat whatever they wanted to eat , 686 could eat most foods except a few that were difficult , 83 could eat a limited number of foods because of insufficient chewing ability , 6 could eat few foods because most were too difficult to chew , and 3 were on a fluid diet . participants who answered unknown ( 21 persons ) were excluded from the current statistical analysis . the association between the subjects self - rated masticatory ability with having good self - perceived health without requiring long - term care as a self - reliant life among the older adults is shown in table 1 . the population that could chew whatever they wanted to eat included the highest proportion ( 68.6% ) of those having good self - rated health without requiring long - term care ( p < .001 , test ) , and only 6.2% of those with poor self - rated health and requiring long - term care insurance were found in this population . in contrast , the population that could eat a limited number of foods because of insufficient chewing ability accounted for 56.6% of those with poor self - rated health and requiring long - term care . association of self - rated masticatory ability with self - reliant life in older adults . note . category numbers of mastication are as follows : 1 = whatever wanting to eat , 2 = most foods expect some hard ones , 3 = limited foods because of chewing not well , 4 = few foods because of the difficulty of chewing or fluid diet . the association between self - rated masticatory ability with other characteristics of the participants is shown in table 2 . the group with adequate self - rated masticatory ability included a younger population ( 65 - 74 , 53.2% ) than the other groups ( 75 - 84 , 43.0% ; above 85 , 32.7% ) . the group having a bmi < 18.5 included a significantly lower proportion of those with good mastication . the characteristics associated with mastication were walking for 15 min ; anxiety about falling ; going out once or more per week ; becoming forgetful ; using the telephone availing electronic appliance ; daily shopping ; taking the trash out ; managing money ; having a hobby ; interacting with neighbors ; and sharing with neighbors . in addition , having good self - rated health and not requiring long - term care with good subjective health was also significantly associated with the subjects self - rated masticatory ability ( p < .001 , test ) . association between self - rated masticatory ability with demographic , nutritional , status , iadl items , and self - reliant life in elderly . note . data was analyzed by chi - square test ( having good self - rated masticatory ability vs. rest of all ) . self - reliant life with good subjective health : having good self - perceived health without receiving long - term care insurance . the ors and 95% cis for having good self - rated health without receiving long - term care insurance were calculated using a logistic regression analysis adjusted for multiple confounding factors , such as demographics , iadl , social capital , cognitive function , and self - rated masticatory ability ( table 3 ) . an unadjusted univariate analysis revealed that better mastication , younger age , better iadl ( walking for 15 min , going out once and over a week , using the telephone , using home electronic appliances , going to shopping daily , taking the trash out , managing money , and ventilating rooms ) , good social capital ( having a hobby , interaction with neighbors ) , and better cognitive function ( not forgetful ) were associated with good self - rated health without receiving long - term care . the crude or for mastication was 3.47 ( 95% ci = [ 2.76 , 4.38 ] ) . model 1 adjusted for age and gender attenuated the or for mastication but remained statistically significant ( or = 3.26 , 95% ci = [ 2.58 , 4.13 ] ) . model 2 , adjusted for iadl , social capital , and cognitive function , and the or for mastication , was still statistically significant ( or = 2.16 , 95% ci = [ 1.64 , 2.83 ] ) . association of self - reliant later life with self - rated masticatory ability determined by multilevel logistic regression . note . or = odds ratio ; ci = confidence interval . the participants were divided into four groups based on their self - rated masticatory ability and self - reliant life , and the number of iadl items ( eight items maximum ) in each group were analyzed ( figure 1 ) . a multiple anova adjusted for gender and age revealed that there were significantly more iadl items in individuals having good mastication ( 7.64 0.84 ) than those without good mastication ( 7.32 1.28 ) even among those with self - rated good health who were not receiving long - term care . similarly , there were significantly more iadl items in the individuals having good mastication ( 6.55 1.71 ) than in those who did not ( 5.44 2.19 ) , even in the group that did not have good self - rated health and were not receiving long - term care . comparison of number of iadl items for the four groups by self - perceived masticatory ability and health . the word , in figure , health indicates self - perceived health without receiving long - term care insurance , and mastication the current study found that self - rated masticatory ability was significantly associated with the self - perceived good health in those not receiving long - term care insurance in community - dwelling age of 65 and above . the self - perceived good health in subjects who were not receiving long - term care insurance defined those with a self - reliant later life . the present study is the first report on the relationship between a self - reliant later life with positive self - perceived health and self - rated masticatory ability . the significant association between the subjects self - rated masticatory ability and their self - perceived good health without receiving long - term care insurance indicated that satisfactory masticatory ability could be an independent predictor of a self - reliant later life . rising a rate of long - term institutionalization of older adults damages both the sustainability of social security and the aged own qol . health , labour , and welfare ministry in japan estimated a reduction in health care cost would be 8.9 billion dollar per year in 2020 , if increase in active life expectancy would exceed increase in total life expectancy . to maintain their active life and to live in their familiar environment until the end of the life without institutionalization or hospitalization , it is critical to expand active life expectancy . in addition , multiple lines of evidences demonstrated that life expectancy crucially related with the self - perceived health ( kaplan & camacho , 1983 ; larue , bank , jarvik , & hetland , 1979 ; rakowski , mor , & hiris , 1994 ) . taken together , we defined the aged self - reliant status as self - perceived adequate health without long - term care - required certification . the current findings are consistent with those of previous studies reporting the relationship between patients qol and their masticatory ability . a previous study found that poor chewing ability based on the number of chewable foods was significantly associated with poor qol status in 80-year - old subjects ( takata et al . , 2006 ) . miura et al . ( 2000 ) reported that satisfactory chewing status is correlated with qol evaluated by the total score of the philadelphia geriatric center ( pcg ) morale . a report on older brazilians showed that those satisfied with their ability to chew food was closely correlated with the score of each domain ( physical , psychological , environment , and social relations ) on the world health organization quality of life questionnaire - brief version ( hugo , hilgert , de sousa mda , & cury , 2009 ) . another recent study reported a relationship between certification of long - term care insurance and self - rated chewing ability . the result of the kaplan meier analysis for individuals aged 65 to 79 years clearly demonstrated that the proportion of individuals not certified for long - term care insurance significantly declines in those with self - rated fair or poor masticatory ability compared with those having good mastication ( moriya , notani , murata , inoue , & miura , 2014 ) . a cox proportional hazard analysis also shows that the certification for long - term care insurance was 1.87-fold ( ci = [ 1.07 , 3.27 ] ) higher in the individuals with fair or poor masticatory ability in comparison with those with adequate ability ( moriya et al . , 2014 ) . a 6-year follow - up study on rural community dwelling older adults in japan by shinkai et al . ( 2003 ) demonstrated that depression of basic activity of daily living ( adl ) and iadl were 1.88-fold ( ci = [ 1.26 , 2.82 ] ) and 2.22-fold ( ci = [ 1.50 , 3.27 ] ) , respectively , higher in individuals with poor chewing ability in comparison with those with better ability . taken together , maintaining the chewing ability in later life would advantage self - reliant life through keeping well both mentally and physically . these evidences suggest that masticatory ability should be the concern in gerontological research . to ensure sustainability of social security system , it is important to close the gap between life expectancy and active life expectancy . ( 2011 ) conducted a study of 882 individuals aged 65 and above , and a multivariate logistic regression demonstrated that severely impaired masticatory ability is significantly related to higher hospitalization costs . kimura et al . ( 2013 ) assessed masticatory efficiency using color - changing chewing gum in 269 community - dwelling japanese older adults above the age of 75 , and concluded that lower masticatory efficiency is significantly associated with lower adl , lower cognitive function , and depression . the result of panel interview surveys for age of 65 and above in japan indicated that active life expectancy of individuals who could chew relatively hard foods was 2 to 3 years longer than people who could chew only relatively soft foods in daily diet ( nasu & saito , 2006 ) . these evidences , including our findings , strongly suggested that maintenance of adequate chewing ability in older adults is closely related to expansion of the active life expectancy , leading to the sustainability of social security system . the participants of the current study were armed with a regional welfare commission volunteer or community comprehensive care center . therefore , the proportion of participants receiving long - term care insurance in the present study was 33.8% , higher than the average proportion in japan ( 17.9% in aged 65 and above ) . the proportion of participants whose health was perceived to be well was 67.2% , slightly lower than a population based on a recent survey on health consciousness by the ministry of health , labour , and welfare in japan ( 77.9% in aged 65 years and above in 2014 ) . this study conducted a structured - questionnaire interview - based investigation in conjunction with the regional commission volunteers or public nurses on the staffs of community comprehensive care center , closely interacting with community - dwelling older adults through regular home visits . therefore , the volunteers or the public nurses commissioned this investigation had built a good rapport with the participants in the study , allowing them to collect true - to - life data . the self - rated masticatory ability was closely correlated with neighborhood social capital , such as interacting with neighbors and sharing something with neighbors , as shown in table 2 . results from a prospective cohort study based on the aichi gerontological evaluation study indicated that both community and individual levels of horizontal social capital ( such as volunteer group , sports clubs , and hobby clubs ) are significantly associated with retaining 20 or more teeth in individuals aged 65 and above ( aida et al . , 2009 ) . conducted another study based on a survey in ohsaki city and demonstrated that neighborhood social capital , accomplished by four kinds of networks ( civic , sports and hobby , volunteer , and friendship networks ) , is closely correlated with dentate status in older adults ( aida et al . , in contrast , social capital is not correlated with the use of oral prosthesis , such as a denture or dental bridge ( yamamoto et al . , 2014 ) . there were few questionnaire items to measure social capital in the current study ; thus , conclusions based on the relationship between social capital and masticatory ability were limited . further study on the association between social capital and masticatory ability is needed . first , self - rated masticatory ability has not correlated with any objective masticatory function such as bite force . this may weaken our argument , although there are multiple evidences on good correlation between subjective and objective chewing ability . second , participants of this study were selected by regional welfare commission volunteers and community comprehensive care center staffs , suggesting to have bias . further study is needed to assess the community - dwelling elderly people who have no relation with regional welfare commission volunteers or community comprehensive care center staffs . third , we did not have any socioeconomic status , such as income and length of educations , of participants . although society in kumamoto area narrowed the gap between the rich and the poor in older adults , subjective masticatory ability may be related with socioeconomic status in japanese older adults . fourth , there is a geographic limitation , as the participants were all from one city . some clinical data , such as serum albumin level for nutrition condition or blood pressure for general condition , might strengthen our findings . sixth , to ensure this mechanism , effect of clinical intervention on masticatory ability , such as denture wearing , dental implant , and muscle training , would be a considerable research task . in conclusion , there was a significant association between self - rated masticatory ability and having self - perceived good health in older adults who were not receiving long - term care insurance , suggesting that a satisfactory level of chewing could be an independent predictor for self - reliant living in the community - dwelling older population . the result of our study demonstrated that asking the self - rated masticatory ability could be an easier and a useful tool for non - healthcare professionals to assess the health status of regional older adults . first , self - rated masticatory ability has not correlated with any objective masticatory function such as bite force . this may weaken our argument , although there are multiple evidences on good correlation between subjective and objective chewing ability . second , participants of this study were selected by regional welfare commission volunteers and community comprehensive care center staffs , suggesting to have bias . further study is needed to assess the community - dwelling elderly people who have no relation with regional welfare commission volunteers or community comprehensive care center staffs . third , we did not have any socioeconomic status , such as income and length of educations , of participants . although society in kumamoto area narrowed the gap between the rich and the poor in older adults , subjective masticatory ability may be related with socioeconomic status in japanese older adults . fourth , there is a geographic limitation , as the participants were all from one city . some clinical data , such as serum albumin level for nutrition condition or blood pressure for general condition , might strengthen our findings . sixth , to ensure this mechanism , effect of clinical intervention on masticatory ability , such as denture wearing , dental implant , and muscle training , would be a considerable research task . in conclusion , there was a significant association between self - rated masticatory ability and having self - perceived good health in older adults who were not receiving long - term care insurance , suggesting that a satisfactory level of chewing could be an independent predictor for self - reliant living in the community - dwelling older population . the result of our study demonstrated that asking the self - rated masticatory ability could be an easier and a useful tool for non - healthcare professionals to assess the health status of regional older adults . | objective : the aim of the present study was to elucidate the influence of self - rated masticatory ability on independent living in community - dwelling older adults . method : a total of 1,377 subjects aged 65 and over who lived in kumamoto city , japan were participated in a survey to investigate critical factors for self - reliance in older adults . in this study , we defined independent life in older adults as self - perceived adequate health without long - term care certification .
logistic regression analysis was used to assess self - perceived masticatory ability in relation to the independent life .
results : the population with adequate self - rated masticatory ability included a significantly higher proportion of subjects with good self - perceived health without long - term care ( 72.7% ) than the remaining subjects ( 27.3% ) .
a logistic regression analysis revealed that there was significant relationship between subjective adequate mastication and living a self - reliant healthy life ( p < .001 ) .
conclusion : our results showed that satisfactory masticatory function was positively related to a self - reliant life with subjective healthy conditions in community - dwelling older adults , which was associated with an extended active life expectancy . |
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gestational trophoblastic neoplasias ( gtns ) are rare malignant tumors arising from placental trophoblastic tissues . approximately 50% of gtns occur after molar pregnancies , 25% after normal pregnancies and 25% after ectopic pregnancies or abortions . they are classified into four groups , namely , invasive mole , choriocarcinoma , placental site trophoblastic tumor and epithelioid trophoblastic tumor . approximately 1520% of complete hydatidiform moles and 1% of partial moles develop into one of the gtns . penetrating deep into the myometrium , sometimes with involvement of the peritoneum , adjacent parametrium or vaginal vault . in the past , hysterectomy was a standard treatment for women diagnosed with gtns of poor prognosis . however , this has changed since the introduction of highly effective chemotherapy , and gtn has become a disease that can be treated with chemotherapy alone in most cases . although the role of hysterectomy has become limited in the modern treatment of gtn , the procedure may be necessary in certain cases . a 50-year - old woman , gravida 3 , para 1 , presented to our department with irregular genital bleeding for 2 months . her obstetrical history included a normal vaginal delivery 13 years before and two spontaneous abortions 8 and 11 years previously . transvaginal ultrasonography showed an enlarged uterus with endometrial thickening with an echogenic pattern ( fig . as endometrial cancer was suspected first , an endometrial biopsy and magnetic resonance imaging ( mri ) were performed . the endometrial biopsy revealed decidual tissues , and the mri revealed invasion of a uterine corpus tumor into the myometrium ( fig . a week later , the patient admitted to the hospital with continuation of irregular vaginal bleeding . second endometrial biopsy was done and serum -human chorionic gonadotropin ( hcg ) level was also measured owing to suspicion of a hydatidiform mole . the serum hcg level was over 225 000 miu / ml , and the endometrial biopsy revealed a complete hydatidiform mole . because the patient 's hemoglobin level was 7.0 g / dl , owing to the continuation of uterine bleeding , we transfused 800 ml of red cell concentrate and discharged the patient . a lung metastasis , 1 cm in diameter , was observed on a contrast computer tomography scan ( fig . 3 ) , and an invasive mole with lung metastasis was diagnosed . because the patient did not desire preservation of her uterus or adnexa , we offered her a total abdominal hysterectomy ( tah ) and bilateral salpingo - oophorectomy ( bso ) . at her next consultation for the continuous genital bleeding , a tumor of the right labium minus was recognized and histologically confirmed as a metastatic lesion of the mole ( fig . unfortunately , a tumor of right labium minus had spontaneously fallen off during surgery . in a gross specimen , molar vesicles were recognized at the uterine corpus with the right ovary a little swollen ( fig . the invasions into myometrium by both villi and the trophoblasts were histopathologically confirmed ( fig . in addition to the labium minus tumor , a metastatic lesion of the right ovary was diagnosed . the patient 's serum hcg level fell to 10 779 miu / ml after her operation . the patient was then started on administered methotrexate 20 mg / body intramuscularly , 5 days a week at 2 weeks intervals . her serum hcg level rapidly fell to 1.2 miu / ml after four courses of methotrexate ( fig . two additional courses of methotrexate were completed , and there has been no evidence of recurrent disease for 6 months . figure 2:mri showing invasion into the uterine cervix ( a ) and myometrium invasion in the posterior wall of the uterus ( b ) . figure 5:gross specimen obtained after the tah and bilateral salpingo - oophorectomy . in the close - up on the right , molar vesicles can be seen at the uterine corpus . figure 6:histopathological staining shows the invasions into the myometrium by both the villi and trophoblasts . figure 7:graph showing the change in serum hcg levels from the first consultation to the end of treatment . the green triangle indicates when the patient underwent surgery ( tah and bso ) , and the yellow triangle indicates methotrexate treatments . mri showing invasion into the uterine cervix ( a ) and myometrium invasion in the posterior wall of the uterus ( b ) . gross specimen obtained after the tah and bilateral salpingo - oophorectomy . in the close - up on the right , molar vesicles can be seen at the uterine corpus . graph showing the change in serum hcg levels from the first consultation to the end of treatment . the green triangle indicates when the patient underwent surgery ( tah and bso ) , and the yellow triangle indicates methotrexate treatments . gtns in postmenopausal women are rare and although , they can be benign or malignant in the perimenopausal and postmenopausal age group , they are usually malignant in the postmenopausal age group . over 20 years ago , tsukamoto et al . reported 20 cases of trophoblastic disease in women aged 50 or older with 25% of the patients diagnosed with a choriocarcinoma , 35% with a hydatidiform mole and 40% with an invasive mole . the majority of women with gtns are of reproductive ages and desire to preserve their fertility . most gtns can be treated with chemotherapy alone without hysterectomy . in cases with metastatic gtn indications for hysterectomy in the gestational trophoblastic disease cases , include women with no need to preserve fertility with a suspicious lesion confined to the uterus , a chemoresistant lesion , hemoperitoneum or coexistence of other uterine diseases . because gtns are more likely to occur in older age groups37.5% of patients are 50 years old versus 27.5% of patients 4049 years old versus 13.9% of patients 15 years after molar evacuation , hysterectomy plays an essential role in the management of these patients . hysterectomy decreases the overall risk for postmolar gtn to ~3.5% from 20% following suction dilation and evacuation . because most women with hydatidiform mole are of reproductive age < 40 years old , the adnexa should not be removed unless the patient has an obvious adnexal metastasis , is perimenopausal or postmenopausal , or has complications related to theca lutein cysts . serum hcg levels must be monitored in all patients even after the hysterectomy to assure that no postmolar gtns are arising . reported that when comparing similar patients at low - risk disease , patients undergoing primary hysterectomy followed by adjuvant chemotherapy had a shorter duration and lower total dose of chemotherapy than patients receiving chemotherapy alone . therefore , primary hysterectomy followed by chemotherapy is a reasonable solution for patients at low - risk gtn who do not desire to preserve fertility , especially for those of postmenopausal age . when perimenopausal patients presented with irregular genital bleeding , we tend to think first of uterine cancer , but we must recognize gestational trophoblastic disease as one of the differential diagnosis . | gestational trophoblastic neoplasias ( gtns ) are rare tumors that constitute < 1% of all gynecological malignancies .
gtns in postmenopausal women are rare and usually malignant .
we present a rare case of an invasive mole of the uterus with metastasis to the right ovary and labium minus treated by total abdominal hysterectomy followed by chemotherapy . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
summer 2009 , adult individuals of culex pipiens were caught by using different models of co2 and light traps in rural habitats at 10 different european locations ( fig . mosquitoes were preserved in 90% ethanol and sent to uppsala by mail by the local mosquito researchers . pipiens population was represented by 30 adult individuals , all from wild , over - ground populations . dna from individual mosquitoes was extracted by a chelex procedure proteinase - k - based extraction ( 16 ) using the protocol provided by the manufacturer . pipiens
( 4 , 1719 ) ( cq13 , cq29 , cxpgt9 , cxpgt20 , cxpgt40 , cxpgt46 , cxpgt51 , and cxpgt53 ) and followed the pcr - protocols given in the original paper . we used the software microchecker ( 20 ) to check for null alleles and other technical problems . we detected a significant number of null alleles in several loci , apparent as an uncommonly large proportion of homozygotes for a few alleles , which created large deviations from hardy weinberg equilibrium . the repeatability of the amplifications was 95% ; so , there are no reasons to believe that technical problems caused this pattern . since several samples did not amplify at all for a given locus , there are reasons to suspect that there are alleles present that did not amplify . null alleles are commonly found in lepidoptera ( 21 ) and seem to be present also in this dipterian sample . the main effect of null alleles seems to be an overestimate of f
st
( 22 ) , but no major distortions of genetic structure . we used the method by chapuis and estoup ( 23 ) to correct for the presence for null alleles since this method performed best in a comparative study . the method is implemented in the software freena ( 23 ) . to perform basic statistics , such as the number of alleles , allelic richness , and expected heterozygosity , we used the softwares arlequin 3.5.1.2 ( 24 ) and fstat 2.9.3.2 ( 25 ) . to calculate pairwise f
st , we used freena using both uncorrected data and data corrected for null alleles ; 95% confidence intervals were obtained through 1000 bootstrap replicates . since f
st is dependent on the level of heterozygosity , a low estimate can be due to a high within population variation rather than lack of differentiation . we therefore also calculated gst
( 26 ) , which rescales f
st to have bounds between zero and one to get an intuitive picture of the degree of differentiation . to tests for possible isolation by distance patterns , we used the program mantel 2.0 ( 27 ) . finally , to make neighbor - joining ( nj ) trees based on the distances obtained , we used mega5 ( 28 ) . to assess if the sampling and the number of loci used was enough to detect a structure if there is one ( i.e. power of the tests ) , we used the program powsim ( 29 ) . we used all loci and simulated f
st - values for two populations being separated for 250 generations , each with an effective population size ( n
e ) of 5000 and with a sampling effort of 30 individuals ( as in this study ) . the values were used to simulate a case where the split of the populations are very recent , and the populations are very large . hence , this is a very conservative test of power , as if time is longer and n
e is smaller , it would be much easier to find significant results . nevertheless , even with these very conservative start values , we found that the expected f
st is 0.025 ( 95% range , 0.01390.0392 ) and the power 0.99 . thus , we are confident that , given the number of loci and sampling effort , we can find differences if they exist . consequently , we were not able to estimate genetic differentiation , as our data adjustments can not cope with functions such as , for example , the estimation of gene flow . when the data are adjusted for null alleles ( alleles that do not amplify ) , multilocus estimates of gene flow instead we used so - called private alleles ( i.e. alleles only found in one population ; 30 ) assuming that there were a number of private alleles that did not amplify ( null alleles ) . with help of genepop and by using private alleles , we estimated the number of migrants per generation across all populations . the aim of estimating the gene flow with this method was to test if the observed amount of divergence was affected by gene flow or if it was what we could expect when no gene flow has occurred since separation . to implement this , we calculated expected f
st by simulations using powsim with n
e values ranging from 100 to 10,000 and time since separation ( t ) ranging from 100 to 5000 generations and allele frequencies taken from the observed data . this will give us a range of expected f
st - values for different combinations of n
e and t ( for results , see fig . 4 ) . populations from romania , turkey , and france were excluded from this calculation since they probably belong to another group of culex . to estimate n e , we used the method of xu and fu 2004 ( 31 ) designed for highly variable markers such as microsatellites , where = 4 n
e , and is the mutation rate . if the parameter values needed to get the observed f
st - values are outside what is realistic in this system , we can reject the hypothesis of no gene flow between them . summer 2009 , adult individuals of culex pipiens were caught by using different models of co2 and light traps in rural habitats at 10 different european locations ( fig . mosquitoes were preserved in 90% ethanol and sent to uppsala by mail by the local mosquito researchers . pipiens population was represented by 30 adult individuals , all from wild , over - ground populations . dna from individual mosquitoes was extracted by a chelex procedure proteinase - k - based extraction ( 16 ) using the protocol provided by the manufacturer . pipiens
( 4 , 1719 ) ( cq13 , cq29 , cxpgt9 , cxpgt20 , cxpgt40 , cxpgt46 , cxpgt51 , and cxpgt53 ) and followed the pcr - protocols given in the original paper . we used the software microchecker ( 20 ) to check for null alleles and other technical problems . we detected a significant number of null alleles in several loci , apparent as an uncommonly large proportion of homozygotes for a few alleles , which created large deviations from hardy weinberg equilibrium . the repeatability of the amplifications was 95% ; so , there are no reasons to believe that technical problems caused this pattern . since several samples did not amplify at all for a given locus , there are reasons to suspect that there are alleles present that did not amplify . null alleles are commonly found in lepidoptera ( 21 ) and seem to be present also in this dipterian sample . the main effect of null alleles seems to be an overestimate of f
st
( 22 ) , but no major distortions of genetic structure . we used the method by chapuis and estoup ( 23 ) to correct for the presence for null alleles since this method performed best in a comparative study . the method is implemented in the software freena ( 23 ) . to perform basic statistics , such as the number of alleles , allelic richness , and expected heterozygosity , we used the softwares arlequin 3.5.1.2 ( 24 ) and fstat 2.9.3.2 ( 25 ) . to calculate pairwise f
st , we used freena using both uncorrected data and data corrected for null alleles ; 95% confidence intervals were obtained through 1000 bootstrap replicates . since f
st is dependent on the level of heterozygosity , a low estimate can be due to a high within population variation rather than lack of differentiation . we therefore also calculated gst
( 26 ) , which rescales f
st to have bounds between zero and one to get an intuitive picture of the degree of differentiation . to tests for possible isolation by distance patterns , we used the program mantel 2.0 ( 27 ) . finally , to make neighbor - joining ( nj ) trees based on the distances obtained , we used mega5 ( 28 ) . to assess if the sampling and the number of loci used was enough to detect a structure if there is one ( i.e. power of the tests ) , we used the program powsim ( 29 ) . we used all loci and simulated f
st - values for two populations being separated for 250 generations , each with an effective population size ( n
e ) of 5000 and with a sampling effort of 30 individuals ( as in this study ) . the values were used to simulate a case where the split of the populations are very recent , and the populations are very large . hence , this is a very conservative test of power , as if time is longer and n
e is smaller , it would be much easier to find significant results . nevertheless , even with these very conservative start values , we found that the expected f
st is 0.025 ( 95% range , 0.01390.0392 ) and the power 0.99 . thus , we are confident that , given the number of loci and sampling effort , we can find differences if they exist . consequently , we were not able to estimate genetic differentiation , as our data adjustments can not cope with functions such as , for example , the estimation of gene flow . when the data are adjusted for null alleles ( alleles that do not amplify ) , multilocus estimates of gene flow instead we used so - called private alleles ( i.e. alleles only found in one population ; 30 ) assuming that there were a number of private alleles that did not amplify ( null alleles ) . with help of genepop and by using private alleles , we estimated the number of migrants per generation across all populations . the aim of estimating the gene flow with this method was to test if the observed amount of divergence was affected by gene flow or if it was what we could expect when no gene flow has occurred since separation . to implement this , we calculated expected f
st by simulations using powsim with n
e values ranging from 100 to 10,000 and time since separation ( t ) ranging from 100 to 5000 generations and allele frequencies taken from the observed data . this will give us a range of expected f
st - values for different combinations of n
e and t ( for results , see fig . populations from romania , turkey , and france were excluded from this calculation since they probably belong to another group of culex . to estimate n e , we used the method of xu and fu 2004 ( 31 ) designed for highly variable markers such as microsatellites , where = 4 n
e , and is the mutation rate . if the parameter values needed to get the observed f
st - values are outside what is realistic in this system , we can reject the hypothesis of no gene flow between them . the number of alleles , allelic richness , and expected heterozygosity did not differ much between the populations ( table 1 ) . the global f
st was 0.090 ( 95% ci : 0.0490.14 ) using the uncorrected data set and 0.054 ( 95% ci : 0.0320.80 ) . the pairwise f
st - values were low but were not different from zero , except in one case ; between denmark and serbia ( table 2 ) . however , the comparisons that involved the samples from france , rumania , and turkey were highly differentiated from the rest of the populations . 2 , the distance between populations was on average lower using the corrected data set . if we exclude the three deviating populations , global f
st was now only 0.029 ( 0.0180.041 ) and 0.022 ( 0.0150.028 ) , respectively , which corresponds to gst - values of 0.20 and 0.15 , respectively . the two estimates of f
st was strongly correlated ( r=0.99 , p<0.001 ; mantel test ) . there was a significant correlation between geographic and genetic distances [ log ( distance ) versus f
st/(1 f
st ) , r=0.31 , p=0.0.032 for the corrected data set , mantel test ] . neighbor - joining ( nj ) trees illustrating the genetic distance between populations ( a ) without correction for null alleles , ( b ) with correction , ( c ) using the lower 2.5 interval , and ( d ) using the upper 2.5 interval . number of individuals sampled , number of alleles ( se ) , allelic richness , ar ( se ) , and expected heterozygosity ( se ) for the different populations pairwise distances with ( a ) uncorrected f
st and ( b ) corrected values two of the markers used here were the same that were used in fonseca et al . ( 4 ) , where two distinct lineages were found . in one of the loci cxpgt9 , the allele frequency spectrum of the three outlying populations ( france , romania , and turkey ) was similar to the underground populations discovered in ref . ( 4 ) and the allele frequency spectrum for the rest was close to the one found in the above - ground populations found in ref . 3a , where the allele size 116 [ corresponding to allele size 114 in ref . ( 4 ) ] is shown to be very frequent in france , romania , and turkey . in ref . ( 4 ) , the same allele reached a frequency of around 0.5 , while in the other populations , the frequencies were much lower . allele size 124 , on the other hand , was not found in romania , turkey , or france , but in all other populations , at about the same frequency . it is also worth noting that allele size 116 , being the most frequent one in the putative underground populations , was found in almost all of the other populations , albeit at a much lower frequency . this suggests a gene flow between the underground populations and the above - ground populations . the same pattern can be seen in locus cq29 where allele size 178 reaches a high frequency in the above - ground populations , but not in the below - ground populations ( fig . 3b ) , while allele size 184 is very common in france , being almost the only allele found , and relatively common in romania , but having a low frequency in turkey . both alleles are found in both groups , but at a much lower frequency , again indicating some gene flow between the groups . frequencies of two alleles in ( a ) locus cxpgt9 and ( b ) qt29 in the different populations . the mean frequency of private alleles was 0.026 , which when corrected for sample size yields an estimate of the number of migrants of 5.74 . the number of migrants between malta and sweden ( the most distant populations ) was as high as 2.04.we found that the estimate of effective population , = 2.85 , which assuming a mutation rate for microsatellites ( ) of 510 , gives an estimate of n
e1500 , and with a more conservative estimate of 10 , gives n
e7100 . to obtain the range of observed f
st - values by drift alone , we would expect n
e in the range of at least 6000 , which is possible given the conservative estimate of mutation , but only at short times since separation ( fig . even if the observed f
st - values are not unattainable , the conditions under which this might happen are quite restricted . expected f
st values ( shaded areas ) in relation to time to separation in generations ( x - axis ) and effective population size ( n
e ) based on simulations in powsim . the line indicates the upper limit of f
st - levels observed in the current study , and thus the observed values are only possible under a model of pure genetic drift for parameter values above the line . the dotted line indicates the average of the estimated n
e over all populations . the study showed two major results : first , three of the analyzed populations ( france , romania , and turkey ) were very different from the rest of the populations ( sweden , denmark , alsace , croatia , serbia , spain , and malta ) and they also greatly differed from each other ( table 2 , fig . 2 ) . in fact , they were so different that there are reasons to believe that they belong to differentiated lineages . furthermore , the allele frequency distribution in these populations match the one that has been earlier found in the underground populations ( 4 ) . second , most pairwise differences were low and showed a shallow structure among the populations apart from the three deviating ones ( fig . the low f
st - values between populations suggest that there is an extensive gene flow between the populations , and this was confirmed by the simulations where the conditions for differentiation by drift alone was far too stringent given what is known about these populations . a low f
st by itself does not mean that there is extensive gene flow ( 32 ) since f
st is dependent on the level of heterozygosity . however , even if we rescale the values to account for within - population heterozygosity , the overall level of differentiation is still low in the above - ground populations . thus , we are confident that basically all european populations of the above - ground cx . the estimate of gene flow was high , which together with the simulations , suggests that the genetic similarity among populations is due to ongoing gene flow rather than a result of a recent shared history and expansion . an alternative explanation is that the species only very recently spread throughout europe , and thus due to incomplete lineage sorting and subsequent local stochastic effects , the populations have not diverged more , which is reflected in the unclear relationships between the populations . however , this species were described by linnaeus in 1758 ( 33 ) , and there are no reasons to believe that it was an uncommon species at that time . even if the species had very recently arrived to northern europe when linnaeus described it , 250 years is ample time to develop a population structure if gene flow is restricted or absent . the results from the power analysis confirm this ; the expected f
st - values are in our simulation higher than observed here , even though we used very conservative input - values . this strengthens the support for a wide - ranging dispersal of mosquito - individuals across large distances in europe , and hence the possibility of a spread of vector - borne pathogens . vector competence of mosquitoes describes their ability to transmit pathogens after taking an infected blood meal . the ability of vectors transmitting a pathogen is known to vary between populations of mosquito species . the exact relationship between vector competence and environmental factors is however not clear and can be influenced by many external factors ( 8) . according to our study , an extensive amount of gene exchange , and hence exchange of individuals , occurs between populations of cx . , infectious diseases spread by this species may not be associated with a certain vector genotype , but rather with suitable environmental conditions . the distribution and intensity of mosquito - borne infections is strongly influenced by the interactions between temperature , vectors , hosts , and pathogen genetics . temperature often determines both the latitudinal boundary and upper elevation limit of pathogen transmission when the extrinsic incubation period becomes greater than the life span of the vector ( 8) . temperature is also linked to the intensity and transmission of pathogens . in temperate regions , seasonality is one of the key components of the climate . in many temperate regions , however therefore , tropical mosquito - borne diseases can be introduced and transmitted during the right season in the temperate regions , but in most cases , they will be eliminated during the winter ( 1 ) . some viruses , however , such as the usutu virus in austria , have been shown to be able to maintain in a natural cycle with a minimum of at least 10 days / year with a temperature > 30c ( 10 ) . it is also predicted that usutu virus will become endemic in large parts of central europe before the end of the century and that optimal environmental conditions for outbreaks of this virus will occur in about 10 years from now ( 10 , 34 ) . pipiens and molestus . because pipiens is strictly ornithophilic while molestus has a more catholic taste and readily bites humans and other mammals , these hybrid populations are thought to be the most high - capacity vectors in southern europe and north america ( 35 ) . this gives rise to concern , should those hybrids spread to northern europe or if another hybridization event should occur . pipiens in sweden and the rest of northern europe are less likely to be involved in zoonotic transmission of , for example , wnf since they are not constituted of hybrids . however , in a recent paper , reusken et al . ( 35 ) showed the presence of hybrid pipiensmolestus mosquitoes in the netherlands . since our results imply an extensive gene flow between populations of cx . this could potentially alter the epidemiology of many arthropod - borne viruses in northern europe giving rise to epidemics / epizootics similar to the wnf outbreak in north america from 1999 and onward . consequently , up - to - date surveillance systems are called for all over the europe as , without them , detection and response to emerging vector - borne diseases can be severely impaired ( 36 ) . the emergence of wnf in 1999 in new york is a good example of the consequences that a delayed response can have on the outcome of novel pathogen introduction . studies show that impaired mosquito surveillance can make the management costs of epidemics several times higher than applying a strategy with sustained surveillance and early case detection ( 36 ) . the authors have not received any funding or benefits from industry or elsewhere to conduct this study . | backgroundspecies in the culex pipiens complex are common almost all over the world and represent important vectors for many serious zoonotic diseases . even if , at the moment , many of the pathogens potentially transmitted by cx .
pipiens are not a problem in northern europe , they may , with increasing temperatures and changing ecosystems caused by climate change , move northward in the future .
therefore , the question whether or not the cx .
pipiens populations in northern europe will be competent vectors for them is of high importance .
one way to estimate the similarity and the rate of contact between european cx .
pipiens populations is to look at the gene exchange between these populations.methodsto test the genetic diversity and degree of differentiation between european cx .
pipiens populations , we used eight microsatellite markers in 10 mosquito populations originating from northern , central , and southern europe.resultswe found that three of the analyzed populations were very different from the rest of the populations and they also greatly differed from each other . when these three populations were removed , the variance among the rest of the populations was low , suggesting an extensive historic gene flow between many european cx .
pipiens populations.conclusionsthis suggests that infectious diseases spread by this species may not be associated with a certain vector genotype but rather with suitable environmental conditions .
consequently , we would expect these pathogens to disperse northward with favorable climatic parameters . |
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k channels play an important role in regulating the membrane potential of smooth muscles and their excitability . generally , outward k current oppose membrane excitability via k channels . in a variety of cells including gastrointestinal ( gi ) tract , several types of k channels which are activated by diverse intracellular factors , such as ca and atp have been reported ( 1 - 7 ) . the best known among them is ca - activated k channel ( k(ca ) channel ) whose gating is regulated by concentration of intracellular free ca ( [ ca]i ) ( 3 - 6 ) . k(ca ) channel participates principally in the rapid repolarization of ca - dependent action potentials ( 8) . voltage - dependent and ca - independent k channels ( k(v ) channel ) and atp - sensitive k channel ( katp channel ) in smooth muscles are also known well ( 1 , 7 , 9 ) . in gi tract , basal activation of katp channel has been shown to contribute to control of resting membrane potential ( rmp ) ( 9 ) . recently , ph - sensitive two - pore k channels of the task family have also been reported in murine gi tract ( 10 ) . therefore , it seems that more types of k channels still exist in gi smooth muscles . in fact , there is another class of k channel not reported in gi smooth muscle cells . this is known as k(na ) channel , which is activated by intracellular na ( [ na]i ) . it was reported in guinea pig ventricular myocytes ( 11 ) . since its isolation from cardiac myocytes , the gating of k(na ) channel has been known to be activated by intracellular na , but not by ca and atp or other nucleotides ( 12 ) . depending on cell types and recording conditions , reported unitary conductance of k(na ) channel ranges from 105 to 200 ps and half - maximal activation by na occurs between 7 and 80 mm ( 13 ) . k(na ) channel of the heart was suggested to be activated under pathophysiological conditions such as during failure of the na - k pump ( 11 ) . also k(na ) channel has been proposed to be activated by a single action potential and could be involved in setting rmp in neuronal cell ( 14 - 16 ) . to date , however , the existence of k(na ) channel in smooth muscles has not been reported yet . in gi tract , spontaneous contraction and slow wave are dependent on extracellular na concentration : in the duodenum and ileum , low na decreased amplitude , frequency , and rate of rise of the upstroke potentials ( 17 , 18 ) . acetylcholine ( ach)-induced inward current ( iach ) in gi smooth muscles is known as na permeable channel ( 19 ) . in addition , na current has been reported in human jejunal circular smooth muscle ( 20 ) . therefore , it is highly likely that pathways for na influx apparently exist in gi smooth muscles . it is known that intracellular na concentration in resting state reaches less than 10 mm in cardiac and smooth muscle cells ( 21 , 22 ) , and [ na]i in smooth muscles is increased or expected to increase by the inhibition of na - k pump ( 18 , 21 ) . these earlier observations suggest a possibility that increased level of intracellular na might play an important role in gi smooth muscle . and therefore , this study was designed to prove the existence of ik(na ) in guinea pig gastric myocytes . guinea pigs of both gender , weighing 300 - 350 g , were exsanguinated after stunning . all experiments were performed in accordance with the guidelines for the animal care and use approved by the chungbuk national university . the antral portion of stomach was cut , and the mucosal layer was separated from the muscle layers in ca - free physiological salt solution ( ca - free pss ) . the circular muscle layer was dissected from the longitudinal layer using fine scissors and made into small segments ( 23 mm ) . these segments were incubated in ca - free pss for 30 min at 4. then , they were incubated for 15 - 25 min at 35 in the digestion medium containing 0.1% collagenase ( wako pure chemicals , osaka , japan ) , 0.05% dithiothreitol , 0.1% trypsin inhibitor and 0.2% bovine serum albumin . following incubation in the enzyme solution , the softened muscle segments were transferred into ca - free pss medium , and tissues were washed repeatedly ( 4 - 5 times ) with ca - free pss medium . tissues were then gently agitated with a wide - bore glass pipette to prepare a cell suspension . isolated cells were transferred to a small chamber on the stage of an inverted microscope ( ix-71 , olympus , tokyo , japan ) . glass pipettes with a resistance of 2 - 5 m were used to make a giga seal of 5 - 10 g , by using standard patch clamp techniques ( 23 ) . membrane currents were amplified with an axopatch-1c or 200b patch - clamp amplifier ( axon instruments , california , u.s.a . ) , and a data were digitized with digidata 1,220 or digidata 1,322 and stored directly and digitized on - line using pclamp software ( version 5.5.1 or 9.2 ) . data were displayed on a digital oscilloscope , pen recorder ( gould , cleveland , oh , u.s.a . ) and a computer monitor , and data were analyzed using pclamp 6.0 ( pclamp 9.2 ) and origin software 6.1 . ca - pss , containing ( in mm ) nacl 140 , kcl 5 , cacl2 2 , mgcl2 1 , glucose 5 , and hepes ( n-[2-hydroxyethyl ] piperazine - n'-[2-ethanesulphonic acid ] ) 10 , and equimolar concentrations of external na ( [ na]o ) were replaced by k for various extracellular k ( [ k]o ) . in some experiments , [ na]o and [ k]o were replaced by 145 mm n - methyl - d - glucamate chloride ( nmdg ) or cscl2 . pipette solution , containing ( mm ) kcl 145 , gtp ( tris form ) 0.1 , egta ( ethylene glycol - bis ( 2-aminoethyl ether- n , n , n',n'-tetraacetic acid ) 10 , creatine phosphate ( crp , tris form ) 2.5 , hepes 10 , atp ( tris form ) 4 , mgcl2 5 , and ph was adjusted to 7.3 with trizma . equimolar concentration of k in k - rich pipette solution was replaced by li or na for making li- and na - rich pipette solution , respectively . for low cl pipette solution , containing na - gluconate 100 , kcl 13 , na2gtp 0.1 , na2atp 2.5 , na2crp 2.5 , egta 10 , hepes 10 , mg2atp 1.5 , mgcl2 3.5 , k -gluconate 22 , ph r56865 ( n-[1-(4-(4-fluorophenoxy ) butyl)-4-piperidinyl]-n - methyl-2-benzothiazolamine was a kind gift from janssen research foundation , beerse , belgium ) . all drugs used in this study were purchased from sigma ( st . louis , mo , u.s.a . ) . the student 's t - test was used wherever appropriate to evaluate differences in data . guinea pigs of both gender , weighing 300 - 350 g , were exsanguinated after stunning . all experiments were performed in accordance with the guidelines for the animal care and use approved by the chungbuk national university . the antral portion of stomach was cut , and the mucosal layer was separated from the muscle layers in ca - free physiological salt solution ( ca - free pss ) . the circular muscle layer was dissected from the longitudinal layer using fine scissors and made into small segments ( 23 mm ) . these segments were incubated in ca - free pss for 30 min at 4. then , they were incubated for 15 - 25 min at 35 in the digestion medium containing 0.1% collagenase ( wako pure chemicals , osaka , japan ) , 0.05% dithiothreitol , 0.1% trypsin inhibitor and 0.2% bovine serum albumin . following incubation in the enzyme solution , the softened muscle segments were transferred into ca - free pss medium , and tissues were washed repeatedly ( 4 - 5 times ) with ca - free pss medium . tissues were then gently agitated with a wide - bore glass pipette to prepare a cell suspension . isolated cells were transferred to a small chamber on the stage of an inverted microscope ( ix-71 , olympus , tokyo , japan ) . glass pipettes with a resistance of 2 - 5 m were used to make a giga seal of 5 - 10 g , by using standard patch clamp techniques ( 23 ) . membrane currents were amplified with an axopatch-1c or 200b patch - clamp amplifier ( axon instruments , california , u.s.a . ) , and a data were digitized with digidata 1,220 or digidata 1,322 and stored directly and digitized on - line using pclamp software ( version 5.5.1 or 9.2 ) . data were displayed on a digital oscilloscope , pen recorder ( gould , cleveland , oh , u.s.a . ) and a computer monitor , and data were analyzed using pclamp 6.0 ( pclamp 9.2 ) and origin software 6.1 . ca - pss , containing ( in mm ) nacl 140 , kcl 5 , cacl2 2 , mgcl2 1 , glucose 5 , and hepes ( n-[2-hydroxyethyl ] piperazine - n'-[2-ethanesulphonic acid ] ) 10 , and ph was adjusted to 7.4 with naoh . equimolar concentrations of external na ( [ na]o ) were replaced by k for various extracellular k ( [ k]o ) . in some experiments , [ na]o and [ k]o were replaced by 145 mm n - methyl - d - glucamate chloride ( nmdg ) or cscl2 . pipette solution , containing ( mm ) kcl 145 , gtp ( tris form ) 0.1 , egta ( ethylene glycol - bis ( 2-aminoethyl ether- n , n , n',n'-tetraacetic acid ) 10 , creatine phosphate ( crp , tris form ) 2.5 , hepes 10 , atp ( tris form ) 4 , mgcl2 5 , and ph was adjusted to 7.3 with trizma . equimolar concentration of k in k - rich pipette solution was replaced by li or na for making li- and na - rich pipette solution , respectively . for low cl pipette solution , containing na - gluconate 100 , kcl 13 , na2gtp 0.1 , na2atp 2.5 , na2crp 2.5 , egta 10 , hepes 10 , mg2atp 1.5 , mgcl2 3.5 , k -gluconate 22 , ph was adjusted to 7.3 with trizma . r56865 ( n-[1-(4-(4-fluorophenoxy ) butyl)-4-piperidinyl]-n - methyl-2-benzothiazolamine was a kind gift from janssen research foundation , beerse , belgium ) . the student 's t - test was used wherever appropriate to evaluate differences in data . membrane potential was held at -60 mv , and hyperpolarizing - ramp pulse , ranging from 80 to -120 mv ( -0.05 v / sec ) , was applied to cells with 15 sec interval . after the whole - cell configuration was established , inward current was produced by increasing the external k from 4.5 mm to 140 mm . the amplitude of the steady - state inward current was -4713.1 pa under k rich pipette solution ( n=4 , fig . however , significantly large amplitude of the steady - state inward current was produced by increasing [ k]o ( 60 mm ) under 110 mm of intracellular na concentration ( [ na]i ) ( -1,052145.7 pa , n=5 , fig . 1ab shows the current / voltage ( i / v ) relationship of na - induced inward current by increasing [ k]o ( 60 mm ) under 110 mm [ na]i ( [ k]i=35 mm ) . reversal potential ( erev ) of this current in i / v relationship was 12.5 mv and it was close to expected k equilibrium potentials ( 13.6 mv ) . 2a shows i / v relationship of na - induced inward current by increasing [ k]o ( 60 and 140 mm ) under 80 mm [ na]i . erev of each current in i / v relationship was 1 and 19 mv , respectively ( fig . each potential was shifted to rightwards , following expected potentials by increasing [ k]o ( 60 and 140 mm ) . these inward currents which was activated by increasing [ k]o was blocked by total replacement of external solution with [ nmdg]o and [ cs]o , respectively ( fig . effects of k channel blockers , such as ba , glibenclamide , tetraetylammonium ( tea ) , 4-aminopyridine ( 4-ap ) , apamine , and quinidine , on the steady - state of na - induced current were studied . either of ba ( 100 m ) , glibenclamide ( 10 m ) , 4-ap ( 10 mm ) , or quinidine ( 3 m ) did not block na - induced inward current ( p>0.05 ; data not shown ) . however , tea ( 5 mm ) decreased the steady - state current of na - induced current to 682.7% of the control ( n=5 , p<0.05 , fig . 2b ) . since k(ca ) and katp channels are present in gi tract , na - induced inward current was studied in the presence of diverse k channel blockers to eliminate the possibility of contamination with other types of k channels . in the presence of k channel blocker cocktail ( mixture of k channel blockers using ba [ 100 m ] , glibenclamide [ 10 m ] , tea [ 10 mm ] , 4-ap [ 10 mm ] , apamine [ 300 nm ] , and quinidine [ 3 m ] ; ph was adjusted to 7.4 before use ) , na - dependent current was still recorded . the amplitude of inward current with 140 mm [ k]o was -1,063182.1 pa ( fig . 2c , 3a ) . however , small steady - state inward current ( -6038.1 pa ) was shown under li - rich pipette solution ( n=4 , p<0.05 ; fig . it has recently been reported that certain types of kna channels ( slick and slack channels ) have a cl dependence ( 24 ) . to evaluate the cl dependence of na - induced inward current in guinea pig gastric myocytes , amplitude of the na - induced inward current the amplitude of na - induced inward current under 20 mm cl and 110 mm [ na]i in the presence of k channel blocker cocktail was -47186.0 ( n=5 , fig . 2c ) . to evaluate the ionic selectivity of inward current obtained in the presence of k channel blocker cocktail , erev was also determined from the i / v relationship and plotted against logarithmic scale of [ k]o ( fig . 3bb ) . when the line - fitting was applied to the data points , a slope of recorded data under k channel blocker cocktail and low cl pipette solution was obtained . in 3 tested cells , these results suggest that na - induced inward current in guinea pig gastric myocytes is selective to k ions . finally , to confirm that na - induced inward current is ik(na ) , we studied the effect of r-56865 which is known to block ik(na ) . ten and twenty m r-56865 inhibited na - induced inward current to 115.7 and 42.9% of the control ( n=3 and 5 ) , respectively ( p<0.05 , fig . intracellular ca is regarded as one of main regulatory factor in many biological responses including activation of ion channels ( 6 ) . in contrast to ca , na has not generally been considered as an important intracellular messenger to say nothing of muscular cells . however , since k(na ) channel was first identified in guinea pig cardiac myocytes , its existence and importance of [ na]i have also been reported in various neuronal cells ( 11 , 12 , 15 , 16 , 25 ) . these facts naturally raise a question of whether k(na ) channel exists in gi smooth muscle . as can be seen in fig . 1aa , increasing [ k]o ( 140 mm ) failed to produce inward current without na in pipette solution . this is also true when na in pipette solution was replaced with li in the presence of k channel blocker cocktail ( fig . 2c ) . however , addition of na into pipette solution produced inward current by stimulation of increasing [ k]o . 1ab ( 110 mm [ na]i ) and 2a ( 80 mm [ k]o ) , increasing [ k]o produced larger inward currents , and the erev was moved to rightward direction following the values expected . however , this current was not affected by pretreatment with ouabain ( 10 m ) known to blocker of na - k pump ( data not shown ) . even though k(na ) channel has been reported to exist in the heart and neurons , its electrophysiological and molecular properties furthermore , characteristics of k(na ) channel , have not yet been reported in any smooth muscle including gi tract . to date , two representative molecular identities known as kna channel subunits have been reported : slack and slick gene families ( 24 , 26 ) . in both type of kna channels , tea ( 20 mm ) was reported to inhibit kna channels significantly ( 24 ) . as seen in fig . 2b , tea ( 5 mm ) significantly inhibited na - induced current in guinea pig gastric myocytes . since k(ca ) and katp channels are present in gi tract , we tried also to exclude activation of k(ca ) and katp channels by high ca buffering capacity , using 10 mm egta and 4 mm atp , at holding potential of -60 mv . in addition , to prevent possible activation of other types of k channels , we studied [ na]-induced current in the presence of k channel blocker cocktail which contained ba ( 100 m ) , glibenclamide ( 10 m ) , tea ( 10 mm ) , 4-ap ( 10 mm ) , apamine ( 300 nm ) , and quinidine ( 3 m ) ( fig . 2c , 3a ) . in the presence of k channel blocker cocktail , na - induced inward current was activated , although the amplitude of the steady state current was reduced . as shown in fig . 2c , ik(na ) was further reduced under low cl in the presence of k channel blocker cocktail . it has recently been reported that certain types of kna channels ( slick and slack channels ) are cl dependent and slick is highly sensitive to cl ( 27 ) . 2c , 3ba and 4a , the amplitude of na - induced current in the presence of k channel blocker cocktail was significantly reduced by low cl pipette solution . however , as shown in fig . 1b , na - induced inward current was blocked by 140 mm [ nmdg]o and [ cs]o . therefore , it seemed that ik(na ) in guinea pig gastric myocytes might also be cl - dependent for its activation . ionic selectivity of na - induced current was studied under low cl pipette solution in the presence of k channel blocker cocktail to eliminate all possible contaminations . as shown in fig . 3bb , the slope of reversal potential obtained versus [ k]o in the presence of k channel blocker cocktail by linear - fitting was 522.5 mv per 10-fold change in [ k]o ( 28 ) . finally , fig . 4 shows reversible inhibitory effect of r-56865 ( 10 and 20 m ) on na - induced inward current . these results are very similar to the results observed in cardiac cells ( 29 , 30 ) . all the above findings suggest that na - induced inward current recorded in our experiment is ik(na ) found in guinea pig gastric myocytes . for activation of k(na ) channel of cardiac myocytes in single channel level , it required at least 10 - 20 mm [ na]i ( 11 , 12 , 16 ) . a recent report also suggests that the half - maximal activation of k(na ) channel by na occurs between 7 and 80 mm , depending on cell types and recording conditions in single channel levels ( 13 ) . therefore , single channel study appears to be required in the future to determine the threshold of [ na]i for activation of ik(na ) in guinea pig gastric myocytes . in cardiac myocytes , it was reported that normal level of [ na]i is less than 10 mm ( 22 ) . therefore , it is unlikely that k(na ) channel would ever be activated under normal conditions . instead , a considerable increase of [ na]i is possible in case of a prolonged failure of na pumping after the onset of acute ischemia or during anoxia . increase of [ na]i would cause reversal of na - ca exchange due to a reduced driving force for na ions ( 11 , 31 , 32 ) . however , the activation of ik(na ) following intracellular na accumulation may set membrane potential to more negative ranges so that na - ca exchanger may maintain the driving force for na influx , thereby improving ca transport ( 33 ) . in the case of neurons , [ na]i in resting state also ranges between ~4 and 15 mm ( 27 ) , however , [ na]i in neurons can also reach as high as ~100 mm by stimuli in dendrites ( 27 , 34 ) . therefore , it has earlier been suggested that the activation of ik(na ) could be involved in setting rmp and generating the spindle waves of neurons ( 15 , 16 , 35 ) . as known well , there are several na influx pathways in gi tract such as na channel , iach and na - k pump ( 18 - 21 ) . therefore , ik(na ) may play an important physiological role in the regulation of membrane potential , hence the motility of gi tract . since there has been no report until now on the identification of k(na ) channel in either gi tract or whole smooth muscle research field , the present study is the first report to prove the existence of ik(na ) in guinea pig gastric myocytes . | this study was designed to identify and characterize na+-activated k+ current ( ik(na ) ) in guinea pig gastric myocytes under whole - cell patch clamp . after whole - cell configuration
was established under 110 mm intracellular na+ concentration ( [ na+]i ) at holding potential of -60 mv , a large inward current was produced by external 60 mm k+ ( [ k+]o ) .
this inward current was not affected by removal of external ca2 + .
k+ channel blockers had little effects on the current ( p>0.05 ) .
only tea ( 5 mm ) inhibited steady - state current to 682.7% of the control ( p<0.05 ) . in the presence of k+ channel blocker cocktail ( mixture of ba2 + , glibenclamide , 4-ap , apamin , quinidine and tea ) ,
a large inward current was activated . however , the amplitude of the steadystate current produced under [ k+]o ( 140 mm ) was significantly smaller when na+ in pipette solution was replaced with k+- and li+ in the presence of k+ channel blocker cocktail than under 110 mm [ na+]i . in the presence of k+ channel blocker cocktail under low cl- pipette solution , this current was still activated and seemed k+-selective , since reversal potentials ( erev ) of various concentrations of [ k+]o - induced current in current / voltage ( i / v ) relationship were nearly identical to expected values .
r-56865 ( 10 - 20 m ) , a blocker of ik(na ) , completely and reversibly inhibited this current .
the characteristics of the current coincide with those of ik(na ) of other cells .
our results indicate the presence of ik(na ) in guinea pig gastric myocytes . |
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