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Introduction {#sec1-1} ============ Infliximab (IFX), a chimeric anti-TNFα antibody, is effective in inducing and maintaining remission in a considerable proportion of IBD patients refractory to any other treatments \[[@ref1],[@ref2]\]. However, 8-12% of adult and/or pediatric patients fail to respond to the induction regimen (known as primary non responders) and approximately 40% of patients who respond initially and achieve clinical remission inevitably lose response over time\[[@ref3],[@ref7]\]. Lack of response to IFX is a stable trait and suggests that the differences in response might be in part genetically determined. Considering the high cost and safety profile of this drug, genetic targeting of patients responding to this therapy is certainly of great interest \[[@ref8]\]. So far, limited candidate gene association studies with response to IFX have been reported \[[@ref9]-[@ref11]\]. Recently, a genome-wide association study (GWAS) in paediatric IBD patients has revealed that the 21q22.2/BRWDI loci were associated with primary non response \[[@ref12]\]. Furthermore, although TNFa gene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date and some have led to contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\]. All anti-TNF agents share an IgG1 Fc fragment, but the contribution of the Fc portion to the response to treatment among currently used TNF blockers remains unknown. Receptors for IgG-Fc portion (FcR) are important regulatory molecules of inflammatory responses. FcR polymorphisms alter receptor function by enhancing or diminishing the affinity for immunoglobulins \[[@ref16]\]. Three major classes of FcR that are capable of binding IgG antibodies are recognised: FcγRΙ (CD64), FcγRΙΙ (CD32), and FcγRΙΙΙ (CD16). FcγRΙΙ and FcγRΙΙΙ have multiple isoforms (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. The most frequent polymorphism of *FcγRΙΙΙA* is a point mutation affecting amino acids in codon 158 in the extracellular domain. This results in either a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported that CD patients with *FcγRΙΙΙA* -158V/V genotype had a better biological and possibly better clinical response to IFX \[[@ref17]\]. However, further studies did not confirm this observation \[[@ref18]\]. The aim of this study was to assess whether the *TNF* and/ or *FcγRΙΙΙA* gene polymorphisms are genetic predictors of response to IFX, in a cohort of Greek patients with adult or paediatric onset of CD. Patients - Methods {#sec1-2} ================== Patients {#sec2-1} -------- We enrolled 106 consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at the 1^st^ Department of Gastroenterology, "Evangelismos" Hospital (79 adults) or the 1^st^ Department of Pediatrics, University Hospital of Athens "Aghia Sophia"(27 children). The diagnosis of CD was based on standard clinical, endoscopic, radiological, and histological criteria \[[@ref1],[@ref19]\]. Eligible patients should have inflammatory (luminal) disease and be naive to IFX. IFX was administered intravenously at a dose of 5mg/kg at weeks 0, 2, 6 and then every 8 weeks. Clinical and serological responses were assessed using the Harvey-Bradshaw Index (HBI) \[[@ref20]\] and the serum levels of C-reactive protein (CRP), respectively, at baseline (before the 1st infusion of IFX), the day before each subsequent IFX infusion and after 12 weeks of treatment. Ileocolonoscopy was performed by a single endoscopist (GJM) at baseline and after 12-20 weeks of therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \[[@ref21],[@ref22]\] \[[Table 1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapy as shown in [table 2](#T2){ref-type="table"}. The ethical committee of the participating hospitals approved the study. Research was carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient. ###### Grading of endoscopic mucosal lesions \[[@ref21],[@ref22]\]  ###### Classification of the study population due to response to infliximab therapy  Genotyping {#sec2-2} ---------- Genomic DNA from whole blood containing EDTA was extracted using standard techniques (NucleoSpin Blood kit, Macherey-Nagel, Germany). All polymerase chain reactions (PCRs) were run under conditions previously described \[[@ref23]\]. Primer sequences for the gene polymorphism at --308 were forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGG ACA CAC AAG CAT CAA GG-3′, for the polymorphism at −238 forward 5′-ATC TGG AGG AAG CGG TAG TG-3′ and reverse 5′-AGA AGA CCC CCC TCG GAA CC-3′. The PCR products were digested at 37 °C with NcoI to detect the SNP in the −308 gene allele and MspI to detect the polymorphism of the −238 nucleotide. The -857 C/T polymorphism was analyzed by allele-specific PCR method24 using the primers TNF857-C: 5′-aag gat aag ggc tca gag ag-3′, TNF857-N: 5′-cta cat ggc cct gtc ttc g-3′ and TNF857-M: 5′-t cta cat ggc cct gtc ttc a-3′. The --158V/F polymorphism of FcγRΙΙΙA gene was detected as described by Leppers-van de Straat et al \[[@ref25]\] using the primers 5′-CTG AAG ACA CAT TTT TACT CC CAA (A/C)-3′ and 5′-TCC AAA AGC CAC ACT CAA AGA C-3′. The PCR products were then subjected to 3% agarose-gel electrophoresis. "No target" controls were included in each PCR batch to ensure that reagents had not been contaminated. Statistical Analysis {#sec2-3} -------------------- Genotype frequencies were compared with the chi-square with Yate's correction using S-Plus (v. 6.2Insightful, Seattle, WA). Odds ratios (ORs) and 95 confidence intervals (CIs) were obtained with GraphPad (v. 3.00, GraphPad Software, San Diego, CA). The p values are all two-sided. Correction for multiple testing was not applied in this study. *P* values of \< 0.05 were considered to be significant. Results {#sec1-3} ======= Patient demographic and clinical characteristics are given in [Table 3](#T3){ref-type="table"}. There were 68 (64.15%) complete responders, 25 (23.58%) partial responders and 13 (12.26%) non responders to IFX in this study. There were no statistical differences in the mean age, gender, disease duration, location and behavior and smoking habits between complete or partial responders and primary non-responders. There was no disagreement between HBI scores and serum CRP levels. Although, the post-treatment CRP levels were significantly lower in complete responders compared to partial and non-responders, the decrease in CRP levels did not differ significantly between the three groups. Post-treatment CRP levels and mean HBI score were significantly lower in complete responders compared to pre-treatment values in contrast to partial and/or non-responders where the CRP levels and the mean HBI score did not differ significantly. ###### Demographic, clinical and biological characteristics of the study population  The -238 G/A, -308 G/A, and -857 C/T polymorphisms of the TNF gene and the -158 V/F polymorphism in the *FcγRΙΙΙA* gene were successfully determined in all subjects. The genotype distribution in complete, partial and non-responders were presented in [Table 4](#T4){ref-type="table"}. No significant difference was observed for the polymorphism tested. In addition, although there may be genetic differences in early (paediatric)-onset and late (adult)-onset CD we were unable to detect any such differences although the number of paediatric patients included in the current study did not allow firm conclusions. ###### Genotype frequency in complete responders, partial responders and non responders  In the present study, we could not correlate the decrease in serum CRP levels with the genotypes tested in any particular group of patients since in most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. However, no significant decrease in CRP was observed between the TNF genotypes tested. Regarding the -158 V/F polymorphism in the *FcγRΙΙΙA* gene, the relative decrease in serum CRP levels was greatest in VV homozygotes (78.15 ± 33.68%) and lowest in FF homozygotes (69.84 ± 28.7%) but this difference was not significant. Due to the small number of cases we did not stratify the genotype frequencies according to age. Discussion {#sec1-4} ========== The mechanism of IFX action in IBD seems to be multifactorial and the response to IFX is a complex phenomenon influenced by several parameters \[[@ref1]\]. Interestingly, a certain proportion of patients do not respond to IFX at all whereas a significant proportion will lose response over time \[[@ref3]-[@ref7]\]. This is the first Greek study aiming at identifying any significant associationbetween the -238 G/A, -308 G/A, and -857 C/T polymorphisms in the promoter region of the TNF gene and the -158V/F polymorphism in *FcγRΙΙΙA* gene and response to IFX in a cohort of adult and paediatric patients with CD and it was negative. Efficacy of IFX was assessed by clinical, serological and endoscopic parameters. Clinical response to IFX was evaluated using the HBI, which has been used in many clinical trials, is simple to use and has shown good correlation with the Crohn's Disease Activity Index (CDAI) \[[@ref26]\]. Serological evaluation of response to IFX was based on changes in serum levels of CRP, which has shown a good correlation with clinical activity and to a certain degree with endoscopic activity of CD \[[@ref27]\]. Finally, endoscopic activity of disease was assessed before and after IFX therapy using a simple description of healing of ulcerative and non ulcerative lesions \[[Table 1](#T1){ref-type="table"}\] as has been previously described \[[@ref21],[@ref22]\]. Endoscopic healing was assessed after 12-20 weeks of IFX treatment. It is conceivable that 12 weeks may be early to assess mucosal healing induced by biologic therapies \[[@ref27]\] but the vast majority of patients underwent endoscopy at least 16 weeks after initiation of IFX therapy (average time 17.6 weeks) and therefore it is unlikely that we have not obtained an objective view of the intestinal mucosal at follow up ileocolonoscopy. Regarding the *TNF* genotypes, our results are in agreement with Louis et al \[[@ref11]\] who did not find any significant difference between response groups when genotyped CD patients for the TNF -308G/A polymorphism and compared response rates after IFX treatment. The same results were reported by Mascheretti et al \[[@ref10]\] and Dideberg et al \[[@ref13]\]. Moreover, our results are in agreement with Tomita et al \[[@ref28]\] who reported no significant difference on *TNFa*, *FcgammaRIIA* and *FcgammaRIIIA* between responders and non responders 8 weeks after IFX treatment as well as with results of ACCENT I study where the relative decrease in serum CRP levels after IFX treatment was greatest in -158 VV homozygotes and lowest in FF homozygotes \[[@ref18]\]. In contrast, Louis et al \[[@ref17]\] observed a significant association between the -158V/F polymorphism in *FcγRΙΙΙA* and both the proportion of patients who had a drop in serum CRP levels after IFX treatment and the magnitude in decrease of serum CRP levels. This may account for the relatively small population of patients in our study, genetic differences in the studied populations and/or methodological differences between studies. Although it would be useful to genetically differentiate 'responders' from 'non-responders', there are not enough data on TNF polymorphisms in IBD and often only selected polymorphisms are genotyped. Small studies have shown possible associations between poor response to IFX and increasing mucosal levels of activated NF-kappaB, homozygosity for the polymorphism in exon 6 of TNFR2 (genotype Arg196Arg), positivity for perinuclear antineutrophil cytoplasmic antibodies and with the presence of increased numbers of activated lamina propia mononuclear cells producing interferon-gamma and TNFa \[[@ref29]\]. In conclusion, our study did not detect any associations between three TNFα gene polymorphisms or the -158 V/F polymorphism in the *FcγRΙΙΙA* gene and response to IFX in CD. However, in view of discrepant results in the literature large-scale pharmacogenetic studies in different populations, with similar baseline disease phenotypes and treatment protocols are needed to adequately estimate associations between genetic polymorphisms and treatment outcomes. Conflict of interest: None ^a^Evangelismos Hospital, ^b^Laboratory of Biology, School of Medicine, ^c^1^st^ Department of Pediatrics, School of Medicine, University of Athens, Greece

INTRODUCTION {#s1} ============ Hepatitis B virus (HBV) is still a major global health problem, with an estimated 257 million people worldwide that are chronically infected with HBV ([@B1]). HBV, together with duck hepatitis B virus (DHBV) and several other related animal viruses, belongs to the *Hepadnaviridae* family ([@B2]). The HBV virion is comprised of an outer envelope and an inner icosahedral nucleocapsid (NC) assembled by 240 copies of core protein (HBc) and packaged with a 3.2-kb partially double-stranded circular DNA genome ([@B3][@B4][@B8]). In addition to DNA-containing virions, a large amount of incomplete viral particles, such as hepatitis B surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be released from cells in the process of virus replication ([@B9]). Subviral HBsAg particles are spherical or rodlike and are present in vast excess over virions in sera of CHB patients ([@B2]). Empty virions share the same structure as DNA-containing virions but are devoid of nucleic acids ([@B10][@B11][@B14]). Naked capsids, which exit cells via a route different from that of virions ([@B15][@B16][@B17]), have the same structure as NCs but are either empty or filled with viral RNA and immature viral DNA ([@B7], [@B11], [@B18][@B19][@B20]). In NC, pgRNA undergoes reverse transcription into minus-strand DNA, followed by plus-strand DNA synthesis ([@B2], [@B21][@B22][@B24]). Intracellular NCs can be packaged with viral nucleic acids at all levels of maturation, including pgRNA, nascent minus-strand DNA, minus-strand DNA-RNA hybrids, and relaxed circular DNA (RC DNA) or double-stranded linear DNA (DSL DNA) ([@B5], [@B7]). Only the NCs with relatively mature viral DNA (RC or DSL DNA) are enveloped and secreted as virions. HBV replicating cells can release empty core particles assembled from HBc proteins and NCs that contain various species of replicative intermediate nucleic acids into the culture supernatant. However, while free naked capsids could be readily detected *in vitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are hardly found in the blood of HBV-infected patients ([@B17], [@B25], [@B26]). Although extracellular HBV RNA was detected in both *in vitro* cell culture systems and in clinical serum samples, its origin and composition remain controversial. It was proposed that extracellular HBV RNA represents pgRNA localized in virions ([@B27]). However, HBV spliced RNA and HBx RNA were also detected in culture supernatant of HBV stably replicating cells as well as in sera of CHB patients ([@B28], [@B29]). In addition, extracellular HBV RNA was also suggested to originate from damaged liver cells ([@B30]), naked capsids, or exosomes ([@B11], [@B29]). Hence, these extracellular RNA molecules have never been conclusively characterized. Here, we demonstrate that extracellular HBV RNAs are heterogeneous in length, ranging from full-length pgRNA (3.5 kilonucleotides \[knt\]) to RNA fragments with merely several hundred nucleotides. These RNA molecules represent 3′ receding pgRNA fragments that have not been completely reverse transcribed to DNA and pgRNA fragments hydrolyzed by the RNase H domain of polymerase in the process of viral replication. More importantly, extracellular HBV RNAs are localized in naked capsids and in virions in culture supernatants of HBV replicating cells and also circulate as CACs and virions in blood of hepatitis B patients. RESULTS {#s2} ======= Extracellular HBV RNAs are heterogeneous in length and predominantly integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {#s2.1} ------------------------------------------------------------------------------------------------------------------------------------------------------ To ascertain the origin of extracellular HBV RNA, we first examined viral particles prepared from culture medium of an *in vitro* HBV stably transduced cell line. A human hepatoma HepAD38 cell line was used in this study, as it sustains vigorous HBV replication under the control of a tetracycline-repressible cytomegalovirus (CMV) promoter ([@B31]). Total viral particles were concentrated and centrifuged over a 10% to 60% (wt/wt) sucrose gradient. Most of the subviral HBsAg particles, virions, and empty virions were detected between fractions 9 to 14 ([Fig. 1A](#F1){ref-type="fig"}, upper and middle). Naked capsids, detected only by anti-HBcAg and not by anti-HBsAg antibodies, settled in fractions 5 to 8 ([Fig. 1A](#F1){ref-type="fig"}, middle and lower). The majority of viral nucleic acids were detected in fractions between 4 and 11 ([Fig. 1B](#F1){ref-type="fig"}, upper), which coincided with the fractions containing virions (fractions 9 to 11), naked capsids (fractions 4 to 7), and the mixture of these particles (fraction 8). Consistent with previous observations, HBV virions are packed with mature viral DNA (RC or DSL DNA), while naked capsids contain both immature single-stranded DNA (SS DNA) and mature viral DNA ([Fig. 1B](#F1){ref-type="fig"}, upper). Moreover, Northern blot results showed that most of the HBV RNA was detected in the naked capsids ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 4 to 7), whereas only a very small amount was associated with virions ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). HBV RNA detected in naked capsids ranged from the full length of pgRNA down to a few hundred nucleotides (shorter than the HBx mRNA \[0.7 knt\]). Moreover, RNA molecules within virions were much shorter than those within naked capsids. We excluded the possibility of artifacts generated by the SDS-proteinase K extraction method, as a similar RNA blot pattern was obtained using a TRIzol reagent to extract both intracellular nucleocapsid-associated and extracellular HBV RNA (not shown). Furthermore, quantification of viral RNA extracted by either the SDS-proteinase K method or TRIzol reagent produced a very similar copy number, except that the TRIzol reagent is known to preferentially extract RNA rather than DNA (not shown). Moreover, the RNA signal detected by Northern blotting could not be attributed to DNA fragments generated by DNase I treatment, which would reduce DNA to below the detection limit of the hybridization method (not shown). Furthermore, the RNA signal could be completely removed by an additional RNase A treatment (not shown). {#F1} To confirm the above-described results and to better separate naked capsids from HBV virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1.33 to 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}). The smearing bands of naked capsids were likely caused by high concentrations of CsCl salt, as fractionation of naked capsids in a 1.18-g/cm^3^ CsCl solution produced single bands. Virions, detected by both anti-HBcAg and anti-HBsAg antibodies ([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral DNA ([Fig. 2A](#F2){ref-type="fig"}, lower) and settled in fractions 13 to 15, with densities ranging from 1.23 to 1.25 g/cm^3^. In agreement with the results shown in [Fig. 1](#F1){ref-type="fig"}, HBV virions contained only the mature viral DNA (RC or DSL DNA), while naked capsids contained viral DNA replicative intermediates that ranged from the nascent minus-strand DNA to mature viral DNA ([Fig. 2B](#F2){ref-type="fig"} and [C](#F2){ref-type="fig"}). The lengths of viral minus- and plus-strand DNA in naked capsids and virions were determined by alkaline agarose gel electrophoresis analysis, a condition where denatured single-stranded DNA molecules migrate according to their lengths. In contrast to the complete minus- and mostly complete plus-strand DNA (closed to 3.2 knt) in virions, in naked capsids the minus-strand DNA and the plus-strand DNA can be both complete and incomplete (shorter than 3.2 knt) ([Fig. 2D](#F2){ref-type="fig"} and [E](#F2){ref-type="fig"}). Moreover, the length of HBV RNAs within naked capsids still ranged from 3.5 knt of pgRNA to shorter than the 0.7 knt of HBx mRNA. Full-length pgRNA accounted for only 10% of total RNA signal detected by Northern blotting (quantified from gray density of bands shown in [Fig. 2F](#F2){ref-type="fig"}). In contrast, HBV RNA species in virions are relatively shorter and barely detectable. In addition, we also determined viral DNA and RNA copy numbers in pooled naked capsids (fractions 3 to 7) and virions (fractions 10 to 21) by quantitative PCR. Quantification results showed that viral DNA in naked capsids and in virions accounted for about 60% and 40%, respectively, of total viral DNA signal in the HepAD38 cell culture supernatant ([Fig. 2G](#F2){ref-type="fig"}). More importantly, 84% of the HBV RNA was associated with naked capsids, while merely 16% was detected within virions ([Fig. 2G](#F2){ref-type="fig"}). Additionally, the DNA/RNA ratio was 11 in virions and 3 in naked capsids ([Fig. 2H](#F2){ref-type="fig"}), suggesting that more HBV RNA is present in naked capsids. {ref-type="fig"}).](zjv0241840640002){#F2} Extracellular HBV RNAs and immature viral DNA are detected in sera from CHB patients. {#s2.2} ------------------------------------------------------------------------------------- Employing the HepAD38 cell culture system, we demonstrated the presence of extracellular HBV RNAs and immature and mature viral DNA packaged in both the naked capsids and virions. Interestingly, Southern blot analyses showed that SS DNA could also be observed in serum samples from some CHB patients. We speculated that SS DNA in circulation would be carried by capsid particles that were released by HBV-infected hepatocytes into patients' bloodstreams. However, we reasoned that due to strong immunogenicity of naked capsids ([@B32], [@B33]), it would be difficult to detect them as free particles; rather, they would form complexes with specific anti-HBcAg antibodies and therefore circulate as antigen-antibody complexes ([@B25], [@B32][@B33][@B34]). To entertain this possibility, we then used protein A/G agarose beads to pull down the immune complexes. Forty-five serum samples obtained from CHB patients, with HBV DNA titers higher than 10^7^ IU per ml, were examined for the presence of particles containing SS DNA by a combination of protein A/G agarose bead pulldown assay and Southern blot analysis ([Fig. 3A](#F3){ref-type="fig"} and [B](#F3){ref-type="fig"}). SS DNA was detected, albeit to a different extent, in 34 serum samples ([Fig. 3A](#F3){ref-type="fig"} and [B](#F3){ref-type="fig"}, upper). The particles containing SS DNA were pulled down by protein A/G agarose beads from 11 out of the 34 samples ([Fig. 3A](#F3){ref-type="fig"} and [B](#F3){ref-type="fig"}, lower). Patient sera negative for SS DNA (patients 37, 38, 14, and 35) or positive for SS DNA (patients 17, 21, 42, and 44), as determined by the protein A/G agarose bead pulldown experiments, were selected for further studies ([Fig. 3C](#F3){ref-type="fig"}). {#F3} Northern blot analyses showed that HBV RNA was only detected in serum samples from patients 17, 21, and 42 ([Fig. 3D](#F3){ref-type="fig"}). Moreover, total viral DNA was analyzed by Southern blotting, and SS DNA was readily observed in serum samples from patients 17, 21, and 42 ([Fig. 3E](#F3){ref-type="fig"}). We also analyzed the lengths of DNA minus and plus strands in patients' sera. Despite the finding that most minus-strand DNA was complete, a small amount of viral DNA (that of patients 38, 35, 17, 21, and 42) was shorter than 3.2 knt ([Fig. 3F](#F3){ref-type="fig"}). Compared with viral minus-strand DNA, the length of plus-strand DNA, particularly in sera from patients 17, 21, and 42, was more variable, ranging from shorter than 2 knt to ∼3.2 knt ([Fig. 3G](#F3){ref-type="fig"}). Naked capsids form CACs with anti-HBcAg antibody in blood of CHB patients. {#s2.3} -------------------------------------------------------------------------- We showed that particles containing SS DNA were present in CHB patients' sera. To further examine these particles, we used CsCl density gradient centrifugation to fractionate a serum mixture from patients 37, 38, 14, and 35. In agreement with our earlier results ([Fig. 2A](#F2){ref-type="fig"}, lower, fractions 13 to 15, and B) and previous reports, HBV virions, with the characteristic mature viral DNA (RC or DSL DNA), were detected in fractions 12 to 14 with densities between 1.26 and 1.29 g/cm^3^ ([Fig. 4A](#F4){ref-type="fig"}) ([@B2]). Careful inspection of the blots revealed that SS DNA could be detected, albeit at very low level, in fractions 8 and 9, with densities from 1.33 to 1.34 g/cm^3^, and in fractions 18 to 21, with densities from 1.20 to 1.23 g/cm^3^ ([Fig. 4A](#F4){ref-type="fig"}). In contrast, CsCl density gradient separation of viral particles from serum of patient 17 showed a mixture of mature and immature viral DNA species. As SS DNA was detected at densities ranging from 1.37 to 1.20 g/cm^3^ ([Fig. 4B](#F4){ref-type="fig"}), no distinct viral DNA (mature RC or DSL DNA) specific to virions could be identified at densities between 1.27 and 1.29 g/cm^3^. Similar results were obtained using CsCl density gradient fractionation of sera from patient 21 (not shown) and patient 46 ([Fig. 4E](#F4){ref-type="fig"}). {#F4} We hypothesized that naked capsids could be released into blood circulation of CHB patients but were bound to specific antibodies. As SS DNA was detected in both high- and lower-density regions in CsCl gradient ([Fig. 4B](#F4){ref-type="fig"} and [E](#F4){ref-type="fig"}), we envisaged that the binding with specific antibodies led to a change of capsids' buoyant density. To test this, anti-HBcAg antibody was mixed with HepAD38 cell culture supernatant to mimic the postulated CACs in serum samples. The results demonstrated that in contrast to SS DNA from naked capsids, distributed to three fractions at densities between 1.33 and 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}, lower, and B), the mixture of naked capsids and CACs (SS DNA) was distributed more widely and could be detected in the lower density region (1.25 to 1.32 g/cm^3^) ([Fig. 4C](#F4){ref-type="fig"}, fractions 11 to 16). Similarly, intracellular capsids from HepAD38 cells were incubated with anti-HBcAg antibody, and a density shift of CACs to a lower-density region was also observed (not shown). To further confirm the lower density of CACs, NCs in virions secreted to HepAD38 cell culture supernatant were treated with NP-40 and mixed with anti-HBcAg antibody. CsCl fractionation showed that naked capsids and virion-derived NCs have become a homogenous mixture banding at densities from 1.37 to 1.27 g/cm^3^ ([Fig. 4D](#F4){ref-type="fig"}). Likewise, virion-derived NCs, obtained by treatment of serum sample from patient 46 with NP-40 bound with antibody, further formed new homogeneous CACs that settled at densities between 1.23 and 1.27 g/cm^3^ ([Fig. 4E](#F4){ref-type="fig"} versus F). However, NP-40 treatment alone did not produce a homogeneous mixture of naked capsids and virion-derived NCs, as these two particles still settled at distinct density regions with their characteristic viral DNA content ([Fig. 4G](#F4){ref-type="fig"}). On the other hand, DNA molecules in the two types of capsids still banded at densities between 1.38 and 1.31 g/cm^3^, further confirming that CACs have relatively lighter density ([Fig. 4G](#F4){ref-type="fig"}). Alternatively, the appearance of a homogenous mixture of virion-derived NCs and naked capsids ([Fig. 4D](#F4){ref-type="fig"} and [F](#F4){ref-type="fig"}) suggests the formation of higher-order antibody-mediated complexes of capsids. For instance, the complexes might not represent individual antibody-coated capsid particles but rather big CACs consisting of several capsid particles interconnected by antibodies. To verify whether intercapsid immune complexes exist, anti-HBcAg antibody was added to the purified HBV capsids expressed by Escherichia coli, and this mixture was examined by an electron microscope. E. coli-derived capsids were scattered as separate, distinct particles ([Fig. 5A](#F5){ref-type="fig"}). However, addition of antibody caused capsids to aggregate into clusters, making them too thick to be properly stained ([Fig. 5B](#F5){ref-type="fig"}). Despite this, a few capsids, which might not have been bound by antibodies or might have been associated with antibodies but did not form intercapsid antibody complexes, could be observed by electron microscopy (EM) ([Fig. 5B](#F5){ref-type="fig"}). {#F5} We then examined CACs in serum samples from CHB patients by EM. Sera from patients 11, 17, 21, 22, 23, 27, 28, 30, and 41, positive for SS DNA, were combined. Serum mixtures, with diminished HBsAg particles by centrifugation through a 20% and 45% (wt/wt) sucrose cushion, were examined by EM. The 27-nm capsid particles or CACs were visible ([Fig. 5C](#F5){ref-type="fig"}, arrow) along with the 42-nm HBV virions ([Fig. 5C](#F5){ref-type="fig"}, arrowheads) and the 22-nm spheres and rods of residual HBsAg particles (not indicated). However, the picture was not clear enough for us to conclusively determine if capsids were connected by or bound with antibodies, as described for unrelated virus in *in vitro* experiments ([@B35]). In addition, it is possible that some of the CACs are not visible by EM, as the complexes maybe too thick to gain clear contrast between lightly and heavily stained areas ([Fig. 5B](#F5){ref-type="fig"}). Lastly, CACs might be heterogeneous, having different molecular sizes and isoelectric points (pI) in hepatitis B patients' blood circulation. *In vitro* binding of naked capsids derived from HepAD38 cell culture supernatant with anti-HBcAg antibody changed their electrophoretic behavior and made them unable to enter the TAE-agarose gel ([Fig. 6A](#F6){ref-type="fig"}). Moreover, viral particles from sera of patients 0, 37, 38, 14, 35, 17, 21, 42, and 44 could not enter agarose gels prepared in TAE buffer. However, in buffer with higher pH value (10 mM NaCHO~3~, 3 mM Na~2~CO~3~, pH 9.4), they appeared as smearing bands on blots ([Fig. 6B](#F6){ref-type="fig"} and [C](#F6){ref-type="fig"}). Hence, the irregular electrophoretic behavior of these viral particles may result from changes in molecular size and/or pI value of capsid particles (pI  4.4) following their association with specific immunoglobulin G (or other types of antibodies) having different pI values (pI of human IgG may range from 6.5 to 9.5) ([@B36][@B37][@B39]). {#F6} Circulating HBV RNAs are of heterogeneous lengths and associated with CACs and virions in hepatitis B patient's plasma. {#s2.4} ----------------------------------------------------------------------------------------------------------------------- To characterize HBV RNAs circulating in CHB patients' sera, a plasma sample from patient 0 was studied. Similar to results obtained for patients 17, 21, and 46 ([Fig. 4B](#F4){ref-type="fig"} and [E](#F4){ref-type="fig"} and not shown), viral DNA in the plasma sample of patient 0 was detected in a broad density range in CsCl gradient and no distinct bands specific to HBV virions or naked capsids could be identified, indicating the presence of a mixture of virions and CACs ([Fig. 7A](#F7){ref-type="fig"}). {#F7} Furthermore, viral particles were pelleted through a 20% sucrose cushion and separated in a sucrose gradient. HBsAg was detected in fractions 5 to 14, peaking at fraction 11. The PreS1 antigen was found in fractions 5 to 12 with the peak at fractions 7 and 10, indicating its presence in HBsAg particles and HBV virions ([Fig. 7B](#F7){ref-type="fig"}, upper). Viral DNA, representing a combination of both mature and immature viral DNA, was detected in fractions 4 to 9 ([Fig. 7B](#F7){ref-type="fig"}, middle), suggesting the localization of CACs and virions in these fractions. HBV RNA was detected between fractions 5 and 7 and appeared in the same peak as viral DNA ([Fig. 7B](#F7){ref-type="fig"}, lower), indicating that HBV RNA is incorporated in the same viral particles as viral DNA. Therefore, circulating HBV RNA may be localized within CACs and/or virions. To better characterize HBV RNA in CACs and virions, plasma sample from patient 0 was centrifuged through a 20% sucrose cushion and pellets were fractionated in a homogenous CsCl solution (1.18 g/cm^3^) as previously described ([@B8]). However, possibly due to a tendency of capsid particles to aggregate and stick to the wall of the centrifugation tube and the low density of the initial CsCl solution ([@B8], [@B40]), only mature DNA species from virions were detected in densities ranging from 1.22 to 1.24 g/cm^3^ ([Fig. 7C](#F7){ref-type="fig"}, upper). Northern blot analyses demonstrated that the lengths of virion-associated HBV RNAs were approximately several hundred nucleotides ([Fig. 7C](#F7){ref-type="fig"}, lower). Virion-associated RNAs were unlikely to be contaminated by CAC-associated HBV RNAs, since the immature SS DNA could not be observed even after a long exposure of X ray film. Moreover, RNA molecules would have been longer if there were CAC contamination ([Fig. 7D](#F7){ref-type="fig"}, lower). Viral nucleic acids in pellets recovered from the centrifugation tube sidewalls could be readily detected on Northern ([Fig. 7C](#F7){ref-type="fig"}, lower, lane P) or Southern ([Fig. 7C](#F7){ref-type="fig"}, upper, lane P) blots using plus-strand-specific rather than minus-strand-specific riboprobe. To analyze viral nucleic acids in CACs, concentrated plasma sample was separated in a higher CsCl density gradient (1.18 g/cm^3^ and 1.25 g/cm^3^). Both mature and immature viral DNA species were only detected in fractions with densities from 1.21 to 1.26 g/cm^3^ ([Fig. 7D](#F7){ref-type="fig"}, upper), indicating the presence of a mixture of HBV virions and CACs. Viral RNAs were detected and ranged in length from a little shorter than the full-length pgRNA to a few hundred nucleotides ([Fig. 7D](#F7){ref-type="fig"}, lower). Compared to virion-associated RNAs ([Fig. 7C](#F7){ref-type="fig"}, lower), HBV RNA species detected in the mixture of CACs and virions were longer, with the longer RNA molecules possibly being associated with CACs. Extracellular HBV RNAs could serve as templates for synthesis of viral DNA. {#s2.5} --------------------------------------------------------------------------- Intracellular NCs are known to contain viral nucleic acids in all steps of DHBV DNA synthesis, including pgRNA, nascent minus-strand DNA, SS DNA, and RC DNA or DSL DNA ([@B5]). Our results showed that naked capsids contained almost the same DNA replicative intermediates as intracellular NCs ([Fig. 1B](#F1){ref-type="fig"} and [2B](#F2){ref-type="fig"}) ([@B7], [@B11]). We also demonstrated that extracellular HBV RNAs within the naked capsids, CACs, and virions were heterogeneous in length ([Fig. 1B](#F1){ref-type="fig"}, lower, [2F](#F2){ref-type="fig"}, and [7C](#F7){ref-type="fig"} and [D](#F7){ref-type="fig"}). In the presence of deoxynucleoside triphosphates (dNTPs), viral RNA could be degraded and reverse transcribed into minus-strand DNA by the endogenous polymerase *in vitro* ([@B5], [@B41], [@B42]). Also, incomplete plus-strand DNA with a gap of about 600 to 2,100 bases could be extended by endogenous polymerase ([@B43], [@B44]). Based on these results, we wished to examine whether extracellular HBV RNAs could serve as RNA templates for viral DNA synthesis and be degraded by polymerase in the process. As shown in [Fig. 8](#F8){ref-type="fig"}, endogenous polymerase assay (EPA) treatment of extracellular viral particles from either culture supernatant of HepAD38 cells or plasma sample from patients led to DNA minus ([Fig. 8A](#F8){ref-type="fig"} and [C](#F8){ref-type="fig"})- and plus ([Fig. 8B](#F8){ref-type="fig"} and [D](#F8){ref-type="fig"})-strand extension and, more importantly, HBV RNA signal reduction ([Fig. 8E](#F8){ref-type="fig"}, lane 4 versus 6 and lane 8 versus 10). The apparent low efficiency of EPA reaction might have been due to our hybridization method, which detected both extended and unextended DNA strands rather than detecting only newly extended DNA. {#F8} In the process of HBV DNA replication, prior to minus-strand DNA synthesis, capsid-associated RNA is the full-length pgRNA. Upon transfer of viral polymerase-DNA primer to the 3′ DR1 region of pgRNA and cleavage of the 3′ epsilon loop RNA (a 3.2-knt pgRNA fragment remained), minus-strand DNA synthesis initiates and the pgRNA template is continuously cleaved from 3′ to 5′ by RNase H activity of viral polymerase. Consequently, from the initiation to the completion of minus-strand DNA synthesis, there will be a series of pgRNA fragments with receding 3′ ends ranging from 3.2 knt to 18 nt of the 5′ cap RNA primer ([@B2], [@B21][@B22][@B24]), representing the RNA templates that have not yet been reverse transcribed into minus-strand DNA. In addition to pgRNA with receding 3′ ends, there are also short RNA fragments arising from intermittent nicks by the RNase H domain of polymerase. Therefore, we used RNA probes spanning the HBV genome to map whether these RNA molecules are present in extracellular naked capsids and virions. Five probes that spanned the HBV genome, except for the overlapping region between the 5′ end of pgRNA and the RNA cleavage site (nt 1818 to 1930), were prepared to map the extracellular HBV RNAs from HepAD38 cell culture supernatant ([Fig. 9A](#F9){ref-type="fig"}). Intracellular nucleocapsid-associated HBV RNA from HepAD38 cells was used as a reference. As the probes moved from the 5′ end to 3′ end of pgRNA, especially for probes 1 to 4, RNA bands shifted from a wider range, including both short and long RNA species, to a narrower range, close to full-length pgRNA, with fewer RNA species detected ([Fig. 9A](#F9){ref-type="fig"}, upper, lanes 2, 5, 8, 11, 14, and 17). Similarly, with the probes moving from the 5′ end to the 3′ end of pgRNA, a stronger intensity band representing extracellular HBV RNAs detected by each probe, especially for probes 1 to 4, was also shifting toward a longer RNA migration region ([Fig. 9A](#F9){ref-type="fig"}, upper, lanes 3, 6, 9, 12, 15, and 18). It should be noted that the shifting pattern was more apparent when RNAs were detected with probes 1 to 4 but not with probe 5. It is possible that the reverse transcription speed is relatively quicker in the initial step (from the 3′ end of pgRNA, which overlaps the probe 5 sequence), and as a result, fewer pgRNA fragments will harbor RNA sequence for probe 5. Also, a short RNA species from either intracellular nucelocapsids or naked capsids and virions migrated faster than 0.7 knt and could be detected by all probes ([Fig. 9A](#F9){ref-type="fig"}, upper, lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, and 18). These RNA molecules likely represent the pgRNA fragments that have been hydrolyzed by the RNase H domain of viral polymerase (including the 3′ epsilon loop RNA cleaved by polymerase in the reverse transcription step) ([@B24]). Collectively, as predicted, longer extracellular HBV RNA species that migrated slower and closer to the position of pgRNA had longer 3′ ends, the shorter viral RNA molecules that migrated faster had relatively shorter 3′ ends, and the RNA species detected by all probes may represent products of pgRNA hydrolysis. ). (B) Identification of 3′ ends of extracellular HBV RNAs. 3′ Ends of extracellular HBV RNAs were identified by the 3′ RACE method using different HBV-specific anchor primers (the same 5′ primers used for generating templates for producing riboprobes used in panel A, lower). Identified 3′ ends were numbered as described above, and numbers in parentheses indicate the amount of clones with the same 3′ ends. The asterisk indicates unknown nucleic acid copurified with intracellular capsid-associated viral RNA by TRIzol reagent. FL, full-length; Cap, 5′ cap of pregenomic RNA; pA, the polyadenylation site; An, poly(A) tail.](zjv0241840640009){#F9} These results were further confirmed by employing a 3′ rapid amplification of cDNA ends (RACE) method. Various 3′ ends spanning the HBV genome were identified ([Fig. 9B](#F9){ref-type="fig"}), validating the presence of 3′ receding RNA and the heterogeneous nature of extracellular HBV RNAs. EPA treatment clearly demonstrated that extracellular HBV RNAs could be used as templates for DNA synthesis, and the presence of 3′ receding-end pgRNA fragments further confirmed not only the existence but also the use of such molecules as templates for viral DNA synthesis. Therefore, just like the viral RNA counterpart within intracellular NCs, extracellular HBV RNA molecules represent the RNA molecules generated in the process of viral DNA replication. ETV reduces viral DNA level but increases extracellular HBV RNA level in naked capsids and virions *in vitro*. {#s2.6} -------------------------------------------------------------------------------------------------------------- Entecavir (ETV), widely used in anti-HBV therapy, is a deoxyguanosine analog that blocks the reverse transcription and plus-strand DNA synthesis steps in the HBV DNA replication process ([@B45][@B46][@B47]). Treatment of CHB patients with nucleos(t)ide analogs (NAs), including entecavir, efficiently reduces the level of serum viral DNA but at the same time increases circulating HBV RNA levels ([@B28], [@B48][@B49][@B52]). We examined the effect of entecavir on the levels of both intracellular and extracellular viral nucleic acids in HepAD38 cell culture. Total viral RNA level remained unchanged or marginally increased upon entecavir treatment ([Fig. 10A](#F10){ref-type="fig"}), and the intracellular capsid-associated viral RNA level was increased ([Fig. 10B](#F10){ref-type="fig"}, upper). In contrast and as expected, the intracellular capsid-associated viral DNA level was decreased ([Fig. 10B](#F10){ref-type="fig"}, lower). Similarly, extracellular viral DNA synthesis was significantly inhibited, while viral RNA was increased ([Fig. 10C](#F10){ref-type="fig"} and [D](#F10){ref-type="fig"}). Quantitative results showed that entecavir suppressed extracellular viral DNA to about one-tenth but at the same time increased viral RNA by about twofold the level for the untreated group ([Fig. 10E](#F10){ref-type="fig"}). {ref-type="fig"}. Viral particles in each fraction were separated by native agarose gel electrophoresis, followed by immunoblotting with anti-HBcAg antibody. Viral DNA and RNA were extracted and subjected to Southern or Northern blot analyses.](zjv0241840640010){#F10} Since viral DNA and RNA were enclosed in both naked capsids and virions, CsCl density gradient was used to separate these particles and to further study the antiviral effect of entecavir. As shown in [Fig. 10](#F10){ref-type="fig"}, DNA-containing naked capsids were detected in fractions 6 to 11 and virions in fractions 15 to 24 ([Fig. 10F](#F10){ref-type="fig"}). Entecavir effectively reduced viral DNA ([Fig. 10G](#F10){ref-type="fig"}, fractions 6 to 10 and 15 to 17; this was also seen in a longer exposure of [Fig. 10G](#F10){ref-type="fig"} \[not shown\]) but increased viral RNA content mainly in naked capsids ([Fig. 10H](#F10){ref-type="fig"}, fractions 6 to 9). Moreover, the increase in RNA content within naked capsids led to an increased density of naked capsids ([Fig. 10F](#F10){ref-type="fig"}, fractions 6 and 11, lower, versus fractions 6 and 11, upper). Interestingly, entecavir seemed to reduce HBcAg signal within virions (i.e., empty virions) ([Fig. 10F](#F10){ref-type="fig"}, fractions 15 to 21, upper, versus fractions 15 to 21, lower) while increasing the egress of naked capsids from HepAD38 cells (data not shown). DISCUSSION {#s3} ========== The RNA molecules in either intracellular NCs or extracellular virions were reported more than three decades ago ([@B5], [@B41], [@B42]), and naked capsids were shown to carry pgRNA *in vitro* ([@B9], [@B11]). Recently, it was suggested that the extracellular or circulating HBV RNA could serve as a surrogate marker to evaluate the endpoint of hepatitis B treatment ([@B27], [@B30], [@B48][@B49][@B53]). With this in mind and to facilitate its application as a novel biomarker for viral persistence, we studied the origin and characteristics of extracellular HBV RNA. In the present study, we extensively characterized extracellular HBV RNAs and demonstrated that extracellular HBV RNAs were mainly enclosed in naked capsids rather than complete virions in supernatant of HepAD38 cells ([Fig. 1B](#F1){ref-type="fig"} and [2F](#F2){ref-type="fig"}). These RNAs were of heterogeneous lengths, ranging from full-length pgRNA (3.5 knt) to a few hundred nucleotides. Furthermore, circulating HBV RNAs, also heterogeneous in length, were detected in blood of hepatitis B patients ([Fig. 3D](#F3){ref-type="fig"} and [7C](#F7){ref-type="fig"} and [D](#F7){ref-type="fig"}). Interestingly, the detection of HBV RNAs coincided with the presence of immature HBV DNA ([Fig. 3D](#F3){ref-type="fig"} and [E](#F3){ref-type="fig"}). Isopycnic CsCl gradient ultracentrifugation of RNA positive serum samples exhibited a broad range of distribution of immature HBV DNA, which contrasted with the results obtained in HepAD38 cells ([Fig. 2B](#F2){ref-type="fig"} versus [@B4]B and E, [@B7]A). For the first time, we provided convincing evidence that unenveloped capsids containing the full spectrum of HBV replication intermediates and RNA species that are heterogeneous in length could be detected in the circulation of chronic hepatitis B patients. In view of our results and literature reports ([@B2], [@B21][@B22][@B24]), the presence of extracellular HBV RNAs could easily be interpreted in the context of the HBV DNA replication model ([Fig. 11A](#F11){ref-type="fig"}). Since naked capsids contain viral DNA at all maturation levels, they will also carry HBV RNA molecules originating from pgRNA, including full-length pgRNA prior to minus-strand DNA synthesis, pgRNA with 3′ receding ends, and the pgRNA hydrolysis fragments. On the other hand, virions that contain only mature forms of viral DNA species would likely bear only the hydrolyzed short RNA fragments remaining in the nucleocapsid ([@B43]). Likewise, the HBV RNA species found in CACs are longer than those in virions in sera of hepatitis B patients ([Fig. 7D](#F7){ref-type="fig"}, lower, versus C, lower). In line with this reasoning, treatment of HepAD38 cells with entecavir reduced viral DNA in naked capsids and virions ([Fig. 10C](#F10){ref-type="fig"}, [E](#F10){ref-type="fig"}, and [G](#F10){ref-type="fig"}) but at the same time increased HBV RNA content within naked capsids ([Fig. 10H](#F10){ref-type="fig"}). This may be a result of the stalled activity of viral RT with concomitant shutdown of RNA hydrolysis ([@B46], [@B54]). {#F11} Contrary to a recent report claiming that the pgRNA-containing NCs can be enveloped and secreted as virions ([@B27]), we clearly demonstrated that secreted naked capsids carry the majority of HBV RNAs ([Fig. 1B](#F1){ref-type="fig"} and [2F](#F2){ref-type="fig"}) and that virion-associated RNAs are approximately several hundred nucleotides long ([Fig. 1B](#F1){ref-type="fig"} and [7C](#F7){ref-type="fig"}). Our results are consistent with earlier reports demonstrating that only mature nucleocapsids with RC/DSL DNA are enveloped and secreted as virions ([@B6][@B7][@B8], [@B11]), and under this condition, virions carry only short RNase H-cleaved pgRNA ([Fig. 11A](#F11){ref-type="fig"}, step 3). In this research, we were unable to separate hydrolyzed pgRNA fragments from the pgRNA and pgRNA with 3′ receding ends. Thus, the length of these RNA molecules could not be determined. The existence of hydrolyzed RNA products during reverse transcription is not without precedent. In some retroviruses, DNA polymerization speed of RT is greater than the RNA hydrolysis speed of RNase H, thus hydrolysis of RNA template is often incomplete ([@B55], [@B56]). For example, RT of avian myeloblastosis virus (AMV) hydrolyzed RNA template once for every 100 to 200 nt, while cleavage frequency of RTs of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) appeared to be around 100 to 120 nt ([@B57]). Moreover, RNA secondary structures, such as hairpins, may stall the RT activity promoting RNase H cleavage, producing shorter RNA fragments ([@B55], [@B56]). Furthermore, the cleaved RNA fragments may not disassociate but anneal to the nascent minus-strand DNA forming the DNA-RNA hybrids until they are displaced by plus-strand DNA synthesis ([@B55], [@B56]). Although similar studies on HBV replication were hampered by lack of fully functional viral polymerase *in vitro* ([@B58][@B59][@B61]), the reported presence of DNA-RNA hybrid molecules clearly indicated the existence of degraded pgRNA fragments that still annealed to the minus-strand DNA ([@B5], [@B41], [@B42], [@B62]). Consistent with a previous study, our results also showed that at least part of the SS DNA is associated with RNA molecules as the DNA-RNA hybrid molecules, as detected by either RNase H digestion or the cesium sulfate density gradient separation method ([@B5] and data not shown). Given the fact that HBV RNA and immature HBV DNA are packaged in naked capsids ([Fig. 1B](#F1){ref-type="fig"} and [2B](#F2){ref-type="fig"} and [F](#F2){ref-type="fig"}) ([@B11]), we postulated that, in CHB patients, unenveloped capsids are released into circulation, where they rapidly form CACs with anti-HBcAg antibodies ([Fig. 11B](#F11){ref-type="fig"}) ([@B25], [@B33], [@B34]). In support of this notion, we showed that protein A/G agarose beads could specifically pull down particles with mature and immature HBV DNA from sera of CHB patients, implying the involvement of antibody. Addition of anti-HBcAg antibody to HepAD38 cell culture supernatant led to a shift of naked capsids' buoyant density to lower-density regions ([Fig. 4C](#F4){ref-type="fig"} and [D](#F4){ref-type="fig"}), a pattern similar to that obtained in HBV RNA-positive serum samples ([Fig. 4B](#F4){ref-type="fig"} and [E](#F4){ref-type="fig"}, and [7A](#F7){ref-type="fig"}). These particles exhibited heterogeneous electrophoretic behavior that differed from that of particles in HepAD38 culture supernatant, suggesting that they are not individual naked capsid particles but are associated with antibodies and have nonuniform compositions ([Fig. 6](#F6){ref-type="fig"} and [11B](#F11){ref-type="fig"}) ([@B36][@B37][@B38]). In CHB patients, the high titers of anti-HBcAg antibodies, which exceed 10,000 IU/ml, preclude circulation of antibody-unbound naked capsids ([@B63]). Indeed, the excessive amounts of anti-HBcAg antibodies present in the plasma sample of patient 0 were able to pull down naked capsids from the culture supernatant of HepAD38 cells (not shown). We have demonstrated the presence of circulating CACs as the new form of naked capsids in CHB patients. It is known that naked capsid particles can be secreted either by the natural endosomal sorting complex required for transport (ESCRT) pathway ([@B15][@B16][@B17]) or possibly by cell lysis consequent to liver inflammation. Our preliminary clinical data (not shown) are in agreement with a recent study showing an association of circulating HBV RNA with serum ALT level ([@B64]). However, this connection can be interpreted in a different manner, as the capsid-antibody complexes might constitute a danger signal triggering inflammation. Interestingly, the release of naked capsids seems to be an intrinsic property of hepadnaviruses preserved through evolution. Recent studies by Lauber et al. provided evidence as to the ancient origin of HBV descending from nonenveloped progenitors in fish, with their envelope protein gene emerging *de novo* much later ([@B65]). Thus, it is reasonable to propose that the active release of HBV capsid particles should be deemed a natural course of viral egress. Apart from HBV particles, it was also reported that exosomes could serve as HBV DNA or RNA carriers ([@B29], [@B66], [@B67]). However, HBV DNA and RNA was detected in naked capsids or CACs and virion fractions rather than in lower-density regions where membrane vesicles like HBsAg particles (density of 1.18 g/cm^3^) and exosomes (density of 1.10 to 1.18 g/cm^3^) would likely settle ([@B2], [@B27], [@B48], [@B68], [@B69]) ([Fig. 1](#F1){ref-type="fig"} and [7B](#F7){ref-type="fig"}). As a result, it is not likely that exosomes serve as the main vehicles carrying HBV DNA or RNA molecules. Numerous pieces of data showed that HBV spliced RNAs also represent a species of extracellular HBV RNAs ([@B28], [@B70], [@B71]). However, in HepAD38 cells, as most of the RNAs are transcribed from the integrated HBV sequence other than the cccDNA template, pgRNA packaged into nucleocapsids is the predominant RNA molecule ([Fig. 9A](#F9){ref-type="fig"} and [10D](#F10){ref-type="fig"}), and viral DNA derived from pgRNA is the dominant DNA form ([Fig. 2D](#F2){ref-type="fig"} and [E](#F2){ref-type="fig"} and data not shown). For the same reason, it would be difficult for us to estimate the amount of spliced HBV RNAs in clinical samples. Although we could not completely rule out the possibility that HBV RNAs are released into blood circulation by association with other vehicles or other pathways, it is possible that the spliced HBV RNAs also egress out of cells in naked capsids and virions like the pgRNA. In summary, we demonstrated that extracellular HBV RNA molecules are pgRNA and degraded pgRNA fragments generated in the HBV replication process *in vitro*. Moreover, we provided evidence that HBV RNAs exist in the form of CACs in hepatitis B patients' blood circulation. More importantly, the association of circulating HBV RNAs with CACs or virions in hepatitis B patients suggests their pgRNA origin. Hence, our results here suggest the circulating HBV RNAs within CACs or virions in hepatitis B patients could serve as novel biomarkers to assess efficacy of treatment. MATERIALS AND METHODS {#s4} ===================== Cell culture. {#s4.1} ------------- HepAD38 cells that replicate HBV in a tetracycline-repressible manner were maintained in Dulbecco's modified Eagle's medium (DMEM)-F12 medium supplemented with 10% fetal bovine serum, and doxycycline was withdrawn to allow virus replication ([@B31]). Patients and samples. {#s4.2} --------------------- Serum samples from 45 chronic hepatitis B patients with HBV DNA titer higher than 10^7^ IU per ml were randomly selected. Detailed medical records of these patients are included in [Table 1](#T1){ref-type="table"}. ###### Medical records of hepatitis B patients used in this research[^*a*^](#T1F1){ref-type="table-fn"} Patient no. Sex Age (yr) HBV DNA titer (IU/ml) HBeAg (IU/ml) HBsAg (IU/ml) ALT (IU/liter) SS DNA result ------------- ----- ---------- ----------------------- --------------- --------------- ---------------- --------------- 0 NA NA 2.67E + 06 4,932 396 \+ 1 M 54 1.24E + 07 25 \>250 69 \+ 2 F 32 1.20E + 07 1,067 69,384 38 \+ 3 F 21 1.36E + 07 1,712 200 149 \+ 4 M 33 \>5.00E + 07 4,812 113,933 133 \+ 5 NA NA 1.25E + 07 3,423 33 − 6 M 26 1.17E + 07 545 2,759 22 − 7 M 36 1.77E + 07 4,332 19,541 136 **+** 8 M 35 \>5.00E + 07 1,199 \>250 104 **+** 9 M 26 2.20E + 07 \>250 143 − 10 M 30 \>5.00E + 07 2 4,265 123 − 11 F 23 \>5.00E + 07 20 5,757 120 **+** 12 M 37 2.07E + 07 2,315 16,128 177 **+** 13 M 28 \>5.00E + 07 3,495 60,676 58 NA 14 F 28 \>5.00E + 07 16,515 89,575 78 \+ 15 M 37 1.62E + 07 574 +, ND 112 \+ 16 M NA \>5.00E + 07 1,601 \>250 22 NA 17 M 15 2.28E + 07 2,038 32,739 180 \+ 18 M 41 2.71E + 07 694 \>250 313 \+ 19 M 34 2.35E + 07 80 32,514 148 \+ 20 F 44 \>5.00E + 07 1,596 4,306 172 − 21 M NA 3.48E + 07 107 \>250 103 \+ 22 NA NA \>5.00E + 07 2024 45,873 147 \+ 23 M 20 1.32E + 07 13,411 12,387 344 \+ 24 M 48 \>5.00E + 07 5,511 76,914 33 − 25 M NA 3.15E + 07 15,984 366 − 26 M 31 4.16E + 07 10,251 50,469 442 \+ 27 M 60 1.35E + 07 749 \>250 105 \+ 28 F 41 \>5.00E + 07 4,173 \>52,000 194 \+ 29 NA NA \>5.00E + 07 4,233 49,125 39 \+ 30 M 29 1.42E + 07 25 5,800 940 \+ 31 M 27 2.34E + 07 1,117 22,412 129 \+ 32 M 37 2.65E + 07 70 109 NA 33 NA NA 2.03E + 07 4,902 111 \+ 34 M 32 \>5.00E + 07 993 43,582 249 \+ 35 NA NA 2.94E + 07 4,641 93,336 12 \+ 36 NA NA \>5.00E + 07 10,956 2,496 108 \+ 37 F 43 \>5.00E + 07 1,021 \>250 74 \+ 38 F 28 \>5.00E + 07 215 446 26 \+ 39 M 31 \>5.00E + 07 +, ND 38,165 194 \+ 40 NA NA \>5.00E + 07 25 \>250 69 \+ 41 M 26 1.52E + 07 +, ND +, ND 95 \+ 42 M 25 \>5.00E + 07 6,300 43,151 373 \+ 43 M 22 \>5.00E + 07 3,844 23,620 329 \+ 44 M 27 1.36E + 07 1,185 11,106 149 \+ 45 M 44 1.28E + 07 663 23,330 425 − 46 F 29 \>5.00E + 07 +, ND +, ND 667 \+ NA, not available; ND, not determined; M, male; F, female; sera from patients 0 and 46 were not included with sera from other patients for SS DNA screening. Plasma sample was the plasma exchange product obtained from an HBeAg-negative hepatitis B patient (patient 0) (HBV genotype B with A1762T, G1764A, and G1869A mutation) who died of fulminant hepatitis as a consequence of reactivation of hepatitis B ([Table 1](#T1){ref-type="table"}). Ethics statement. {#s4.3} ----------------- All samples from HBV-infected patients used in this study were from an already-existing collection supported by the National Science and Technology Major Project of China (grant no. 2012ZX10002007-001). Written informed consent was received from participants prior to collection of clinical samples ([@B72]). Samples used in this study were anonymized before analysis. This study was conducted in compliance with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the ethics committee of the Shanghai Public Health Clinical Center. Preparation of viral particles. {#s4.4} ------------------------------- HepAD38 cell culture supernatant was mixed with polyethylene glycol 8000 (PEG 8000) to a final concentration of 10% (wt/vol) and incubated on ice for at least 1 h, followed by centrifugation at 925 × *g* for 20 min. Pellets were suspended in TNE buffer (10 mM Tris-Cl \[pH 7.5\], 100 mM NaCl, and 1 mM EDTA) containing 0.05% β-mercaptoethanol to 1/150 of the original volume, followed by a brief sonication ([@B73], [@B74]). Alternatively, viral particles in HepAD38 cell culture supernatant were concentrated 50- to 100-fold by ultrafiltration using a filter unit (Amicon Ultra-15, 100 kDa). Plasma samples from patient 0 were centrifuged through a 20% (wt/vol) sucrose cushion at 26,000 rpm for 16 h in an SW 32 Ti rotor (Beckman), and pellets were resuspended in 1/200 the original volume of TNE buffer and sonicated briefly ([@B75]). Samples prepared using methods described above were either used immediately or aliquoted and stored at −80°C for later use. Sucrose density gradient centrifugation. {#s4.5} ---------------------------------------- HepAD38 cells culture supernatant concentrated by PEG 8000 was centrifugation at 500 × *g* for 5 min to remove aggregates. Ten percent, 20%, 30%, 40%, 50%, and 60% (wt/wt) sucrose gradients were prepared by underlayering and incubated for 4 h in a water bath at room temperature to allow gradient to become continuous. Five hundred microliters of concentrated sample was layered over the gradient and centrifuged at 34,100 rpm for 14 h at 4°C in a Beckman SW 41 Ti rotor. Fractions were collected from top to bottom, and the density of each fraction was determined by refractometry ([@B10]). Fractions containing viral particles were subjected to native agarose gel analysis, and HBsAg level was determined by enzyme-linked immunosorbent assay (ELISA) (Shanghai Kehua). Cesium chloride density gradient centrifugation. {#s4.6} ------------------------------------------------ HepAD38 cell culture supernatant (1.5 ml), concentrated by ultrafiltration, or serum samples from chronic hepatitis patients diluted with TNE buffer to 1.5 ml were mixed with equal volumes of 37% (wt/wt) CsCl-TNE buffer (1.377 g/cm^3^) and underlayered with 1.9 ml 34% (wt/wt) CsCl-TNE buffer (1.336 g/cm^3^), followed by centrifugation at 90,000 rpm at 4°C for 12 h (Beckman VTi 90 rotor) ([@B8]). The tube was punctured from the bottom, and every six to seven drops were collected as one fraction. Densities of separated fractions were determined by weighing. Each fraction was then desalted against TNE buffer by ultrafiltration, followed by native agarose gel separation or nucleic acid extraction. All of the CsCl density gradient centrifugation experiments were carried out at 90,000 rpm at 4°C for 12 h in a Beckman VTi 90 rotor. Native agarose gel analysis of viral particles and capsid-associated DNA. {#s4.7} ------------------------------------------------------------------------- Viral particles were resolved by native agarose gel (0.8% agarose gel prepared in Tris-acetate-EDTA \[TAE\] buffer) electrophoresis and transferred in TNE buffer to either a nitrocellulose membrane (0.45 μM) for detection of viral antigens with specific antibodies or a nylon membrane for Southern blot analysis of viral DNA. For viral antigens detection, the membrane was first fixed as previously described ([@B74]), and HBV core antigen was detected by anti-HBcAg antibody (Dako) (1:5,000). The same membrane then was soaked in stripping buffer (200 mM glycine, 0.1% SDS, 1% Tween 20, pH 2.2) and reprobed with anti-HBsAg antibody (Shanghai Kehua) (1:5,000). For Southern blot analysis of viral DNA, the membrane was dipped in denaturing buffer (0.5 N NaOH, 1.5 M NaCl) for 10 s and immediately neutralized in 1 M Tris-Cl (pH 7.0)--1.5 M NaCl for 1 min, followed by hybridization with minus-strand-specific riboprobe ([@B76]). Viral nucleic acid extraction, separation, and detection. {#s4.8} --------------------------------------------------------- **(I) Nucleic acid extraction.** To extract total viral nucleic acids (DNA and RNA), the SDS-proteinase K method was used ([@B77]). Samples were digested in solution containing 1% SDS, 15 mM EDTA, and 0.5 mg/ml proteinase K at 37°C for 15 min. The digestion mixture was extracted twice with phenol and once with chloroform. Aqueous supernatant were added with 1/9 volume of 3 M sodium acetate (pH 5.2) and 40 μg of glycogen and precipitated with 2.5 volumes of ethanol. In addition to the SDS-proteinase K method, viral RNA was also extracted with TRIzol LS reagent according to the manufacturer's instructions (Thermo Fisher Scientific). To isolate intracellular capsid-associated viral RNA, HepAD38 cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl \[pH 7.8\], 1 mM EDTA, 1% NP-40), and cytoplasmic lysates were incubated with CaCl~2~ (final concentration, 5 mM) and micrococcal nuclease (MNase) (Roche) (final concentration, 15 U/ml) at 37°C for 1 h to remove nucleic acids outside nucleocapsids. The reaction was terminated by addition of EDTA (final concentration, 15 mM), and then proteinase K (0.5 mg/ml without SDS) was added to the mixture, followed by incubation at 37°C for 30 min to inactivate MNase. Viral nucleic acids were released by addition of SDS to a final concentration of 1% and extracted as described above. **II. Separation. (i) TAE agarose gel.** Viral DNA was resolved by electrophoresis through a 1.5% agarose gel in 1× TAE buffer, followed by denaturation in 0.5 M NaOH--1.5 M NaCl for 30 min and neutralization with 1 M Tris-Cl (pH 7.0)--1.5 M NaCl for 30 min. **(ii) Alkaline agarose gel.** Viral DNA was denatured with a 0.1 volume of solution containing 0.5 M NaOH and 10 mM EDTA and resolved overnight at 1.5 V/cm in a 1.5% agarose gel with 50 mM NaOH and 1 mM EDTA. After electrophoresis, the gel was neutralized with 1 M Tris-Cl (pH 7.0)--1.5 M NaCl for 45 min ([@B78]). **(iii) Formaldehyde-MOPS agarose gel.** Viral RNA was obtained by treatment of total nucleic acids extracted using the above-described SDS-proteinase K method with RNase free DNase I (Roche) for 15 min at 37°C. The reaction was stopped by addition of equal amounts of 2× RNA loading buffer (95% formamide, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol FF, and 1 mM EDTA) supplemented with extra EDTA (20 mM), followed by denaturing at 65°C for 10 min. Viral RNA extracted by TRIzol LS reagent was mixed with 2× RNA loading buffer and denatured. Denatured mixtures were separated by electrophoresis through a 1.5% agarose gel containing 2% (vol/vol) formaldehyde solution (37%) and 1× MOPS (3-\[N-morpholino\]propanesulfonic acid) buffer. The gels described above were balanced in 20× SSC solution (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) for 20 min, and viral nucleic acids were transferred onto nylon membranes overnight with 20× SSC buffer. III. Detection. {#s4.10} --------------- Digoxigenin-labeled riboprobes used for detection of HBV DNA and RNA were prepared by *in vitro* transcription of a pcDNA3 plasmid that harbors 3,215 bp of HBV DNA (nt 1814 to 1813) by following the vendor's suggestions (12039672910; Roche). Riboprobes used for HBV RNA mapping were transcribed from DNA templates generated by PCR by incorporating T7 promoter into the 5′ end of reversed primers ([Fig. 9A](#F9){ref-type="fig"}). Hybridization was carried out at 50°C overnight, followed by two 5-min washes in 2× SSC--0.1% SDS at room temperature and two additional 15-min washes in 0.1× SSC--0.1% SDS at 50°C. The membrane was sequentially incubated with blocking buffer and anti-digoxigenin-AP Fab fragment (Roche) at 20°C for 30 min. Subsequently, the membrane was washed twice with washing buffer (100 mM maleic acid, 150 mM NaCl, and 0.3% Tween 20, pH 7.5) for 15 min, followed by detection with diluted CDP-Star substrate (ABI) and exposure to X-ray film. Protein A/G agarose bead pulldown of antibody-antigen complexes. {#s4.11} ---------------------------------------------------------------- Two hundred microliters of serum sample was first mixed with 300 μl of TNE buffer, and then 15 μl of protein A/G agarose bead slurry (Santa Cruz) was added to the mixture, followed by incubation overnight at 4°C in a sample mixer. Subsequently, protein A/G agarose beads were washed three times with TNE buffer, and viral DNA in input serum samples (40 μl) and agarose bead pulldown mixtures were extracted and subjected to Southern blot analysis. EM. {#s4.12} --- Serum samples from patients 11, 17, 21 22, 23, 27, 28, 30, and 41 were pooled (200 μl each) and mixed with 200 μl of 20% (wt/wt) sucrose. Serum mixtures were centrifuged through 2 ml of 20% (wt/wt) and 2 ml of 45% (wt/wt) (1.203 g/cm^3^) sucrose cushions at 34,100 rpm for 8 h at 4°C in an SW 41 Ti rotor (Beckman) to remove HBsAg particles. Supernatants were decanted and the centrifugation tube was placed upside down for 20 s, and residue sucrose was wiped out. One milliliter of phosphate buffer (10 mM Na~2~HPO~4~, 1.8 mM KH~2~PO~4~, and no NaCl) (pH 7.4) was added, and the bottom of the tube was gently washed without disturbing the pellet. A volume of 11.5 ml of phosphate buffer then was added into the tube and centrifuged again at 34,100 rpm for 3 h at 4°C. The pellet was resuspended in a drop of distilled water and dropped onto a carbon-coated copper grid, followed by staining with 2% phosphotungstic acid (pH 6.1) and examining in an electron microscope (Philip CM120) ([@B13], [@B79]). Viral DNA and RNA quantification. {#s4.13} --------------------------------- Viral DNA used for quantification was extracted using the SDS-proteinase K method as described above. Viral RNAs were extracted by TRIzol LS reagent, and DNase I was used to remove the remaining DNA, followed by phenol and chloroform extraction and ethanol precipitation. Reverse transcription was carried out using Maxima H minus reverse transcriptase (Thermo Fisher Scientific) with a specific primer (AGATCTTCKGCGACGCGG \[nt 2428 to 2411\]) according to the manufacturer's guidelines, except the 65°C incubation step was skipped to avoid RNA degradation. To ensure removal of viral DNA signal (below 1,000 copies per reaction), a mock reverse transcription, without addition of reverse transcriptase, was carried out. Quantitative real-time PCR (qPCR) was carried out using Thunderbird SYBR qPCR mix (Toyobo) in a StepOnePlus real-time PCR system (ABI). Primer pairs (F, GGRGTGTGGATTCGCAC \[nt 2267 to 2283\]; R, AGATCTTCKGCGACGCGG \[nt 2428 to 2411\]) conserved among all HBV genotypes and close to the 5′ end but not in the overlap region between the start codon and the poly(A) cleavage site of pgRNA were chosen. The cycling conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 5 s, 57°C for 20 s, and 72°C for 30 s. DNA fragment containing 3,215 bp of full-length HBV DNA was released from plasmid by restriction enzymes, and DNA standards were prepared according to a formula in which 1 pg of DNA equals 3 × 10^5^ copies of viral DNA. EPA. {#s4.14} ---- HepAD38 cell culture supernatant or plasma from patient 0 were concentrated as described above and mixed with equal volumes of 2× EPA buffer (100 mM Tris-Cl, pH 7.5, 80 mM NH~4~Cl, 40 mM MgCl~2~, 2% NP-40, and 0.6% β-mercaptoethanol) with or without dNTPs (dATP, dCTP, dGTP, and dTTP, each at a final concentration of 100 μM) ([@B80]). The reaction mixtures were incubated at 37°C for 2 h and stopped by addition of EDTA to a final concentration of 15 mM. 3′ RACE. {#s4.15} -------- Concentrated HepAD38 cell culture supernatant (by ultrafiltration) was digested with MNase in the presence of NP-40 (final concentration, 1%) for 30 min at 37°C. EDTA (final concentration, 15 mM) and proteinase K (final concentration, 0.5 mg/ml) were then added and incubated for another 30 min at 37°C. Viral nucleic acids were extracted with TRIzol LS reagent followed by DNase I treatment to remove residue viral DNA. Poly(A) tails were added to the 3′ end of HBV RNA by E. coli poly(A) polymerase (NEB). The preincubation step at 65°C for 5 min was omitted to reduce potential RNA degradation, and reverse transcription was carried out with Maxima H minus reverse transcriptase (Thermo Scientific) using an oligo-dT(29)-SfiI(A)-adaptor primer (5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′) in reverse transcription buffer \[1× RT buffer, RNase inhibitor, 1 M betanine, 0.5 mM each dNTP, and 5 μM of oligo-dT(29)-SfiI(A)-adaptor primer\] at 50°C for 90 min, followed by heating at 85°C for 5 min and treatment with RNase H at 37°C for 15 min. PCR amplification of cDNA fragments was then performed with 5′ HBV-specific primers \[the same sequences of forward primers used for riboprobe preparation ([Fig. 9A](#F9){ref-type="fig"}), except each primer containing a flanking sequence plus a SfiI(B) site (5′-AGTGATGGCCGAGGCGGCC-3′)\] and 3′ adaptor primer (5′-AAGCAGTGGTATCAACGCAGAGTG-3′). The reaction was carried out with PrimeSTAR HS DNA polymerase (TaKaRa) at 95°C for 5 min, followed by 5 cycles of 98°C for 5 s, 50°C for 10 s, and 72°C for 210 s, 35 cycles of 98°C for 5 s, 55°C for 10 s, and 72°C for 210 s, and a final extension step at 72°C for 10 min. PCR amplicons were digested with SfiI enzyme and cloned into pV1-Blasticidin vector (kind gift from Zhigang Yi, Shanghai Medical College, Fudan University). Positive clones were identified by sequencing, and only clones with 3′ poly(dA) sequence were considered authentic viral RNA 3′ ends. We thank Zhuying Chen and Xiurong Peng for handling serum samples and compiling the clinical data used in this research. This research was supported by the National Natural Science Foundation of China (NSFC) (81671998, 91542207), National Key Research and Development Program (2016YFC0100604), National Science and Technology Major Project of China (2017ZX10302201001005), Shanghai Science and Technology Commission (16411960100), and Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-07-E00057). [^1]: **Citation** Bai L, Zhang X, Kozlowski M, Li W, Wu M, Liu J, Chen L, Zhang J, Huang Y, Yuan Z. 2018. Extracellular hepatitis B virus RNAs are heterogeneous in length and circulate as capsid-antibody complexes in addition to virions in chronic hepatitis B patients. J Virol 92:e00798-18. <https://doi.org/10.1128/JVI.00798-18>.

Koolstra K, Beenakker J‐WM, Koken P, Webb A, Börnert P. Cartesian MR fingerprinting in the eye at 7T using compressed sensing and matrix completion‐based reconstructions. Magn Reson Med. 2019;81:2551--2565. 10.1002/mrm.27594 30421448 **Funding information** This project was partially funded by the European Research Council Advanced Grant 670629 NOMA MRI. 1. INTRODUCTION {#mrm27594-sec-0005} =============== Ophthalmologic disease diagnosis conventionally relies mainly on ultrasound and optical imaging techniques such as fundus photography and fluorescent angiography (FAG), MRI is increasingly being used in the radiological community.[1](#mrm27594-bib-0001){ref-type="ref"}, [2](#mrm27594-bib-0002){ref-type="ref"}, [3](#mrm27594-bib-0003){ref-type="ref"} One of the main advantages of MRI is its capability to assess nontransparent tissues such as ocular tumors or structures behind the globe such as the eye muscles. Currently, however, these applications are mainly based on qualitative MRI methods using the large number of tissue contrasts addressable by MR. As an example, in Graves' ophthalmopathy fat‐suppressed T~2~‐weighted MRI is the standard to detect inflammation in the eye muscles,[4](#mrm27594-bib-0004){ref-type="ref"}, [5](#mrm27594-bib-0005){ref-type="ref"} whereas in the diagnosis of retinoblastoma, a rare intraocular cancer in children, standard T~1~‐ and T~2~‐weighted MRI is often performed to confirm the presence of the tumor and to screen for potential optic nerve involvement.[2](#mrm27594-bib-0002){ref-type="ref"} In more recent ophthalmologic applications of MRI, such as uveal melanoma (the most common primary intraocular tumor), quantitative MRI techniques including DWI[6](#mrm27594-bib-0006){ref-type="ref"} and DCE imaging[7](#mrm27594-bib-0007){ref-type="ref"} have been shown, but currently diagnosis is still based on qualitative methods.[3](#mrm27594-bib-0003){ref-type="ref"} To personalize treatment plans quantitative parameters of the tissues involved, as can be acquired invasively for example by performing biopsies,[8](#mrm27594-bib-0008){ref-type="ref"} are highly desirable. However, quantitative parameter mapping by means of MRI requires long examination times, which would result in significant eye‐motion artifacts, as well as patient discomfort.[9](#mrm27594-bib-0009){ref-type="ref"} MR fingerprinting (MRF) is a recently introduced method for rapid quantitation of tissue relaxation times and other MR‐related parameters.[10](#mrm27594-bib-0010){ref-type="ref"} It uses a flip angle sweep to induce a unique signal evolution for each tissue type. Incoherent undersampling can be applied during sampling of the MRF train, enabling acceleration of the MRF scans.[10](#mrm27594-bib-0010){ref-type="ref"} Together with its ability to measure simultaneously T~1~ and T~2~, MRF offers a solution to the problem of obtaining quantitative measures in an efficient manner and in relatively short scanning times. One of the main challenges in ocular imaging is in‐plane and through‐plane eye motion, often associated with eye blinking.[11](#mrm27594-bib-0011){ref-type="ref"}, [12](#mrm27594-bib-0012){ref-type="ref"}, [13](#mrm27594-bib-0013){ref-type="ref"} The motion results in corrupted k‐space data that introduces artifacts and blurring throughout the entire image. Shortening the scans would reduce motion‐related artifacts, but standard acceleration techniques are not optimal for the current eye application due to the following 3 reasons. First, a cued‐blinking protocol is typically used to control and reduce the eye motion.[3](#mrm27594-bib-0003){ref-type="ref"}, [11](#mrm27594-bib-0011){ref-type="ref"} This requires an instruction screen placed at the end of the MR tunnel to be visible to the patient which complicates the use of small phased array receive coils in front of the eye, blocking the view. Instead, a custom‐built single‐element eye loop coil is used, which provides a high local SNR[3](#mrm27594-bib-0003){ref-type="ref"} and screen visibility, but which clearly excludes the possibility of scan acceleration by means of parallel imaging.[14](#mrm27594-bib-0014){ref-type="ref"} Second, the gel‐like vitreous body has an extremely long T~1~, particularly at high field.[15](#mrm27594-bib-0015){ref-type="ref"} Its value of 3 to 5 s requires a long duration of the MRF sequence to encode the MR parameters (T~1~,T~2~) sufficiently. Thus, using a flip angle train with a small number of RF pulses is not feasible, hindering scan time reduction. Finally, a time‐efficient spiral sampling scheme, usually applied in MRF,[10](#mrm27594-bib-0010){ref-type="ref"}, [16](#mrm27594-bib-0016){ref-type="ref"}, [17](#mrm27594-bib-0017){ref-type="ref"}, [18](#mrm27594-bib-0018){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} introduces off‐resonance effects in each of the individual MRF images.[20](#mrm27594-bib-0020){ref-type="ref"} This occurs even when combined with unbalanced sequences such as fast imaging with steady state precession,[16](#mrm27594-bib-0016){ref-type="ref"} which are in themselves robust to off‐resonance effects.[21](#mrm27594-bib-0021){ref-type="ref"} The off‐resonance effects present in spiral sampling schemes are much stronger at high field, where they result in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} caused by strong main field inhomogeneities (particularly in the eye region due to many air‐tissue‐bone interfaces), as well as the presence of significant amounts of off‐resonant orbital fat around the eye. In this work, a Cartesian sampling scheme is used, which is more robust than spiral sampling to off‐resonance effects, but which is significantly less time‐efficient.[23](#mrm27594-bib-0023){ref-type="ref"} With such a Cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the MRF image series.[10](#mrm27594-bib-0010){ref-type="ref"}, [20](#mrm27594-bib-0020){ref-type="ref"} In this case, direct matching of the measured MRF signal reconstructed by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurate for high undersampling factors.[24](#mrm27594-bib-0024){ref-type="ref"}, [25](#mrm27594-bib-0025){ref-type="ref"} Therefore, the quality of the reconstructed MRF data has to be improved before the matching process. Compressed sensing (CS) has been introduced as a technique to reconstruct images from randomly undersampled data by enforcing signal sparsity (in the spatial dimension only or both in spatial and temporal dimensions),[26](#mrm27594-bib-0026){ref-type="ref"}, [27](#mrm27594-bib-0027){ref-type="ref"} allowing a scan time reduction in many applications.[28](#mrm27594-bib-0028){ref-type="ref"}, [29](#mrm27594-bib-0029){ref-type="ref"}, [30](#mrm27594-bib-0030){ref-type="ref"} The flexibility of MRF toward different sampling schemes and undersampling factors makes it possible to reconstruct the source images by means of CS.[27](#mrm27594-bib-0027){ref-type="ref"}, [31](#mrm27594-bib-0031){ref-type="ref"}, [32](#mrm27594-bib-0032){ref-type="ref"} Higher acceleration factors might be feasible if the correlation in the temporal dimension is better used.[33](#mrm27594-bib-0033){ref-type="ref"} Examples of such reconstructions specifically tailored to MRF are given in Davies et al, Pierre et al, and Zhao et al[34](#mrm27594-bib-0034){ref-type="ref"}, [35](#mrm27594-bib-0035){ref-type="ref"}, [36](#mrm27594-bib-0036){ref-type="ref"} which take into account the simulated dictionary atoms in the image reconstruction process. Recent work has shown that the temporal correlation in the MRF data can be exploited even further by incorporating the low rank structure of the data into the cost function,[37](#mrm27594-bib-0037){ref-type="ref"} a technique which was introduced into MR in Liang[38](#mrm27594-bib-0038){ref-type="ref"} and in MRF in Zhao[39](#mrm27594-bib-0039){ref-type="ref"} and used by many others[40](#mrm27594-bib-0040){ref-type="ref"}, [41](#mrm27594-bib-0041){ref-type="ref"}, [42](#mrm27594-bib-0042){ref-type="ref"}: these techniques can also be combined with sparsity constraints.[43](#mrm27594-bib-0043){ref-type="ref"}, [44](#mrm27594-bib-0044){ref-type="ref"} Most of the aforementioned techniques involve Fourier transformations in each iteration, making the reconstruction process time‐consuming. In this application, the single‐element receive coil allows us to perform the reconstruction process entirely in k‐space when exploiting the low rank structure of the MRF data as is performed in matrix completion (MC)‐based reconstructions.[42](#mrm27594-bib-0042){ref-type="ref"}, [45](#mrm27594-bib-0045){ref-type="ref"} In this work, undersampled Cartesian ocular MRF is investigated using CS and MC‐based reconstructions. Simulations and experiments performed in 6 healthy volunteers for confirmation are compared with fully sampled MRF in terms of the quality of the parameter maps, and mean relaxation times were derived for different ocular structures at 7T. Finally, parameter maps after an MC‐based reconstruction are included for a uveal melanoma patient, showing the feasibility of ocular MRF in eye tumor patients. 2. METHODS {#mrm27594-sec-0006} ========== 2.1. Fingerprinting definition {#mrm27594-sec-0007} ------------------------------ The MRF encoding principle is based on a variable flip angle train with relatively short TRs, so that the magnetization after each RF pulse is influenced by the spin history. Following closely the implementation of the sinusoidal MRF pattern described in Jiang et al,[16](#mrm27594-bib-0016){ref-type="ref"} a flip angle pattern of 240 RF excitation pulses ranging from 0° to 60° (see Figure [1](#mrm27594-fig-0001){ref-type="fig"}A) was defined by the function$$FA\left( x \right) = \left\{ \begin{matrix} {20\,\text{sin}(\frac{\pi}{110}x)\,\text{for}\, 1 \leq x \leq 110} \\ {60\,\text{sin}(\frac{\pi}{130}\left( {x - 110} \right))\,\text{for}\, 110 < x \leq 240} \\ \end{matrix} \right.$$ {#mrm27594-fig-0001} preceded by an inversion pulse (16). A fast imaging with steady state precession sequence was used,[16](#mrm27594-bib-0016){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} in which the TE was chosen as 3.5 ms and 4.0 ms for low resolution scans and high resolution scans, respectively. The selected excitation RF pulse had a time‐bandwidth product of 10, resulting in a reasonably sharp slice profile. The RF pulse phase was fixed to 0°. To simplify dictionary calculations, because of the simplification of the magnetization coherence pathways,[46](#mrm27594-bib-0046){ref-type="ref"} the TR was set to a constant value of 11 ms. A 3D dictionary was calculated following the extended phase graph formalism,[21](#mrm27594-bib-0021){ref-type="ref"}, [46](#mrm27594-bib-0046){ref-type="ref"} based on the Bloch equations,[47](#mrm27594-bib-0047){ref-type="ref"}, [48](#mrm27594-bib-0048){ref-type="ref"} incorporating 27,885 signal evolutions.[46](#mrm27594-bib-0046){ref-type="ref"} T~1~ values ranged from 10 to 1000 ms in steps of 10 ms, and from 1000 to 5000 ms in steps of 100 ms. T~2~ values ranged from 10 to 100 ms in steps of 10 ms and from 100 to 300 ms in steps of 20 ms. A B~1~ ^+^ fraction ranging from 0.5 to 1.0 in steps of 0.05 was incorporated into the dictionary calculation. To shorten the scan time, we used a short waiting time between repetitions of the MRF train (called the repetition delay) of 2.5 s. Therefore, each MRF scan was preceded by 3 dummy trains to establish steady state magnetization,[19](#mrm27594-bib-0019){ref-type="ref"} which was considered in the dictionary calculation. The longitudinal magnetization after the 3 dummy trains, required for correction of the M~0~ maps, was calculated for each T~1~/T~2~ combination. The repetition delay of 2.5 s was efficiently used as the blink time.[3](#mrm27594-bib-0003){ref-type="ref"}, [11](#mrm27594-bib-0011){ref-type="ref"} 2.2. Experimental setup {#mrm27594-sec-0008} ----------------------- All experiments were approved by the local medical ethics committee, and all volunteers and patients signed an appropriate informed consent form. The experiments in this study were performed on 6 healthy volunteers and 1 uveal melanoma patient using a 7T MR system (Philips Healthcare) equipped with a quadrature head volume coil (Nova Medical) for transmission and a custom‐built single‐element eye coil for reception, with a diameter of approximately 4 cm.[3](#mrm27594-bib-0003){ref-type="ref"}, [49](#mrm27594-bib-0049){ref-type="ref"} A cued‐blinking protocol was followed, which means that all subjects were instructed to focus on a fixation target shown on a screen during data acquisition and to blink in the 2.5 s repetition delay. This was performed using a small mirror integrated into the eye coil, allowing visualization of a screen placed outside the magnet through 1 eye, while the eye to be imaged was closed and covered by a wet gauze to reduce susceptibility artifacts in the eye lid.[50](#mrm27594-bib-0050){ref-type="ref"} This setup is shown schematically in Figure [1](#mrm27594-fig-0001){ref-type="fig"}B. 2.3. MR data acquisition {#mrm27594-sec-0009} ------------------------ Because of the presence of significant orbital fat around the eye, and the sensitivity of the spiral to off‐resonance resulting in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} a Cartesian sampling scheme was used to acquire all data. The fingerprinting scans were acquired as a single slice at 2 different spatial resolutions: 1.0 × 1.0 × 5.0 mm^3^ and 0.5 × 0.5 × 5.0 mm^3^. The lower resolution scan was performed twice, the first fully sampled to serve as a reference, and the second one undersampled. The scan time of the fully sampled scan was 7:02 min, while the scan time of the undersampled scan, in which 15% of the data was acquired, was 1:16 min. The high resolution scan was only acquired as an undersampled data set, in which 12.5% of the data was acquired, resulting in a scan time of 1:57 min. In the undersampled scans a simple variable density k‐space sampling was applied, schematically shown in Figure [1](#mrm27594-fig-0001){ref-type="fig"}C, supporting both CS and MC‐based reconstructions. A fully sampled center of k‐space was acquired for each time point consisting of 6/8 k‐space lines for the low resolution/high resolution scans, respectively. For all scans, the FOV was set to 80 × 80 mm^2^, resulting in an acquisition matrix of 80 × 80 and 160 × 160 for the low and the high resolution scans, respectively. The phase encoding direction was set from left‐to‐right to minimize contamination by any residual motion artifacts in the eye lens, and the read out direction was set to the anterior‐posterior direction. B~1~ ^+^ maps were acquired using the dual refocusing echo acquisition mode method[51](#mrm27594-bib-0051){ref-type="ref"} with the following scan parameters: FOV = 80 × 80 mm^2^, in‐plane resolution 1 mm^2^, slice thickness 5 mm, 1 slice, TE~1~/TE~2~ = 2.38/1.54 ms, TR = 3.7 ms, FA = α:60°/ß:10°: the scan time for a single slice was less than 1 s. 2.4. Reconstruction {#mrm27594-sec-0010} ------------------- For each time point, the corresponding images were reconstructed from the available data, using custom software written in MATLAB (Mathworks, Inc) and run on a Windows 64‐bit machine with an Intel i3‐4160 CPI @ 3.6 GHz and 16 GB internal memory. Different reconstructions were performed: (i) a fast Fourier transform (FFT) of the fully sampled data and of the zero‐filled undersampled data; (ii) a CS reconstruction with total variation regularization in the spatial dimension (2D CS), and with total variation in both spatial and temporal dimensions (3D CS) of the undersampled data; (iii) an MC‐based reconstruction of the undersampled data. ### 2.4.1. CS reconstruction {#mrm27594-sec-0011} In this reconstruction, the complete image series is reconstructed by iteratively solving the nonlinear problem$$\hat{\mathbf{x}} = \text{argmin}_{\mathbf{x}}TV\left( \mathbf{x} \right)s.t.\, RF\mathbf{x} = \mathbf{y}_{u}$$ through the unconstrained version$$\hat{\mathbf{x}} = \text{argmin}_{\mathbf{x}}{\frac{\mu}{2}{|RF\mathbf{x} - \mathbf{y}_{u}|}}_{2}^{2} + \frac{\lambda}{2}TV{(\mathbf{x})}$$ In this formulation, $F \in \mathbb{C}^{Nt \times Nt}$ is a block diagonal matrix with the 2D Fourier transform matrix in each diagonal block, $R \in \mathbb{C}^{Nt \times Nt}$ is a diagonal matrix incorporating the sampling locations, $\mathbf{y}_{u} \in \mathbb{C}^{Nt \times 1}$ is the undersampled k‐t space data, $\hat{\mathbf{x}} \in \mathbb{C}^{Nt \times 1}$ is an estimate of the true image series and $\mathit{TV}$ is a total variation operator which is used to enforce sparsity in the reconstruction.[52](#mrm27594-bib-0052){ref-type="ref"}, [53](#mrm27594-bib-0053){ref-type="ref"} Here, $N$ is the number of k‐space locations per image frame and $t$ is the number of measured time points (or flip angles in the MRF train). The regularization parameters $\mu$ and $\lambda$ in Equation [\[Link\]](#mrm27594-disp-0001){ref-type="disp-formula"} were determined empirically and set to $\mu = 0.1\, and\,\lambda = 0.2.$ Two basic versions of the total variation operator,$$TV\left( \mathbf{x} \right) = {|\nabla_{x}{\mathbf{x}|}}_{1} + {|\nabla_{y}{\mathbf{x}|}}_{1}$$ $$TV\left( \mathbf{x} \right) = {|\nabla_{\mathbf{x}}{\mathbf{x}|}}_{1} + {|\nabla_{\mathbf{y}}{\mathbf{x}|}}_{1} + {|\nabla_{\mathbf{t}}{\mathbf{x}|}}_{1}$$ were implemented to investigate the effect of promoting sparsity either only in the spatial dimension (2D CS) or in both the spatial and temporal dimensions (3D CS). In these expressions, $\nabla_{x},\nabla_{y}$ and $\nabla_{t}$ are the first derivative operators acting on the spatial $x$ and $y$ dimensions and the time dimension, respectively. Solving the problem given in Equation [\[Link\]](#mrm27594-disp-0001){ref-type="disp-formula"} is done in this work using Split Bregman. For details on this algorithm the reader is referred to Goldstein and Osher.[54](#mrm27594-bib-0054){ref-type="ref"} ### 2.4.2. MC reconstruction {#mrm27594-sec-0012} Similar to CS with the TV operator acting in 3 dimensions (see Equation [(1)](#mrm27594-disp-0003){ref-type="disp-formula"}), MC uses the information from the temporal dimension.[45](#mrm27594-bib-0045){ref-type="ref"}, [55](#mrm27594-bib-0055){ref-type="ref"} A main difference between CS and MC, however, is that sparsity of singular values, which is a priori information in the MC reconstruction, can be observed both in image space and in k‐space. This allows one to complete the entire reconstruction in k‐space, which is computationally efficient, especially if only a single receiver coil is used.[42](#mrm27594-bib-0042){ref-type="ref"} The MC‐based reconstruction iteratively solves$$\hat{M} = \mathit{argmin}_{M}{|M|}_{\ast}\, s.t.\,\mathcal{P}_{\Omega}M = M_{u}$$ with ${| \bullet |}_{\ast}$ being the nuclear norm, $\mathcal{P}_{\Omega}$ the sampling operator selecting the measured k‐t space locations, $M_{u} \in {}^{t \times N}$ the undersampled k‐t space data and $\hat{M} \in \mathbb{C}^{t \times N}$ an estimate of the true k‐t space. The nuclear norm of *M* sums the singular values of *M*, and can thus be written as ${|\sigma(M)|}_{1}$, where $\sigma$ transforms $M$ into a vector containing the singular values of $M$. The central k‐t space is used as calibration data, of which the rank can be used as a priori information in the reconstruction of undersampled data. In this process, a projection matrix $\mathcal{P}_{U_{n}} \in \mathbb{C}^{t \times t}$ projects in each iteration $i$ the undersampled data matrix $M^{i}$ onto a low‐rank subspace spanned by the columns of $U_{n} \in \mathbb{C}^{t \times n}$, such that$$\overset{\mspace{600mu}}{M^{i}} = \mathcal{P}_{U_{n}}M^{i}$$ with$$\mathcal{P}_{U_{n}} = U_{n}U_{n}^{H}.$$ Here, $U_{n}$ contains the $n$ most significant left singular vectors of the calibration matrix $M_{c} \in \mathbb{C}^{t \times p}$ and is constructed from the full singular value decomposition $M_{c} = U\Sigma V^{H}$, $U \in \mathbb{C}^{t \times t}$, $\Sigma \in \mathbb{R}^{t \times p}$, $V \in \mathbb{C}^{p \times p}$, which is performed once at the beginning of the algorithm. In the second step of each iteration, the data are updated according to$$M^{i + 1} = M_{u} + {(I - \mathcal{P}_{\Omega})}{\overset{\sim}{M}}^{i}.$$ The value $n$ was determined empirically from the singular value plots (shown in Figure [1](#mrm27594-fig-0001){ref-type="fig"}D for 1 volunteer) and set to 4 for all MC‐based reconstructions. Further details of the adopted algorithm to solve Equation [(2)](#mrm27594-disp-0004){ref-type="disp-formula"}, and its implementation can be found in Doneva et al.[42](#mrm27594-bib-0042){ref-type="ref"} To ensure convergence of the iterative CS and MC‐based reconstructions, 40 Split Bregman iterations (1 inner loop) were used for the CS reconstructions and 100 iterations were used for all MC‐based reconstructions. To judge the performance of the reconstruction methods, relative error measures are defined throughout the manuscript as$$\mathit{RelativeError}\left( \mathbf{u} \right) = \frac{{{|\mathbf{u} -}\mathbf{u}_{\mathbf{r}\mathbf{e}\mathbf{f}}|}_{2}}{{|\mathbf{u}_{\mathbf{r}\mathbf{e}\mathbf{f}}|}_{2}},$$ where $\mathbf{u}_{\mathit{ref}}$ is the fully sampled image series and both $\mathbf{u}$ and $\mathbf{u}_{\mathit{ref}}$ are vectorized. 2.5. Dictionary matching process {#mrm27594-sec-0013} -------------------------------- For each subject, the measured B~1~ ^+^ map was used to calculate an average B~1~ ^+^ value in the eye. Based on this value, a 2D subdictionary was chosen that matches the drop in B~1~ ^+^ for each volunteer. Each voxel signal in the reconstructed MRF image series was then matched to an element of the subdictionary. In this process, the best match between the measured signal and the dictionary elements was found for each voxel by solving$$m = \mathit{argmax}_{\mathbf{i} \in {\{{1,..,\mathbf{M}}\}}}\left\{ {\mathbf{d}_{\mathbf{i}} \bullet \mathbf{s}} \right\}$$ where $\mathbf{d}_{i} \in \mathbb{C}^{t \times 1}$ is the ith normalized dictionary element and $\mathbf{s} \in \mathbb{C}^{t \times 1}$ is the normalized measured signal. The index $m$ that maximizes the inner product describes the dictionary element $\mathbf{d}_{m}$ (with corresponding T~1~ and T~2~ values) that gives the best match with the measured signal. Finally, the scalar proton density per voxel was determined from the model$$\mathbf{S}{= rM}_{0}\mathbf{D}_{\mathbf{m}},$$ where $\mathbf{S} \in \mathbb{C}^{t \times 1}$ is the nonnormalized signal per voxel and $\mathbf{D}_{m} \in {}^{t \times 1}$ the nonnormalized dictionary element corresponding to the best match $\mathbf{d}_{m}$, such that$$M_{0} = \frac{1}{r}\frac{(\mathbf{D}_{m} \bullet \mathbf{S})}{(\mathbf{D}_{m} \bullet \mathbf{D}_{m})}$$ *r* is a value between 0 and 1, describing the fraction of the initial longitudinal magnetization that is left after the dummy trains, also depending on T~1~ and T~2~, which takes into account the short repetition delay in between the MRF trains. M~0~ maps are all shown on a log‐scale due to the high dynamic range of the respective proton densities, with that of the vitreous body being more than an order of magnitude larger than other structures. The processed T~1~, T~2~, and M~0~ maps were compared for different reconstruction methods (FFT, 2D CS, 3D CS, and MC) and for different acquisitions (low spatial resolution, high spatial resolution). T~1~ and T~2~ values were averaged in different regions of interest, annotated in Figure [1](#mrm27594-fig-0001){ref-type="fig"}E for each volunteer. These values were used to determine mean ± SD values over all volunteers for the different reconstructions. 3. RESULTS {#mrm27594-sec-0014} ========== 3.1. Simulation results {#mrm27594-sec-0015} ----------------------- Figure [2](#mrm27594-fig-0002){ref-type="fig"} shows the parameter maps (T~1~, T~2~, and M~0~) obtained for different reconstruction methods, after subsampling the fully sampled k‐space data of 1 healthy volunteer. Even though an incoherent sampling scheme was used, a zero‐filled FFT reconstruction does not lead to accurate parameter maps. The CS reconstruction with total variation regularization in the spatial domain leads to only minor improvement for the high undersampling factor that was chosen. The results show that including the sparsity constraint in the temporal dimension on top of the spatial dimension improves the CS reconstruction, with the largest improvement in the optic nerve and the lens nucleus, indicated by the white arrows. The total undersampling factor of 6.7, however, in combination with the low resolution reconstruction matrix and the single channel signal, results in loss of detail in the CS approach. {#mrm27594-fig-0002} This is not the case for the MC‐based reconstructions. The parameter maps resulting from the MC‐based approach are very close to the parameter maps obtained from the fully sampled scan, enabling visualization of the extraocular muscles and the orbital fat, indicated by the white circles. The error maps in Figure [2](#mrm27594-fig-0002){ref-type="fig"}, defined as the relative difference with the parameter maps from the fully sampled scan, given in percentages, confirm these findings. The error has a more noise‐like behavior for the MC‐based reconstruction compared with the CS reconstruction, and is much lower in the sensitive region of the eye coil. The error maps for T~1~ show larger percentage improvements compared with T~2~. These general trends were also true for different undersampling factors (see Supporting Information Figure [S1](#mrm27594-sup-0001){ref-type="supplementary-material"}, which is available online). 3.2. Experimental results {#mrm27594-sec-0016} ------------------------- Parameter maps obtained in an undersampled experiment are shown in Figure [3](#mrm27594-fig-0003){ref-type="fig"} for low spatial resolution images. The experimental results confirm the findings from the simulation study. The parameter maps obtained from the undersampled MRF scan with a 3D CS reconstruction show loss of detail compared with the parameter maps obtained with an MC‐based reconstruction. This is especially visible in the M~0~ maps. For the MC‐based reconstruction, the parameter maps are similar quality to those obtained from the fully sampled scans, showing the feasibility of accelerating MRF in the eye using a Cartesian sampling scheme. It should be noted that the full k‐space data and the undersampled k‐space data originate from different scans, which is why residual motion artifacts are different between the resulting parameter maps. The parameter maps at high resolution in Figure [4](#mrm27594-fig-0004){ref-type="fig"} show more detail compared with the parameter maps at low resolution in Figure [3](#mrm27594-fig-0003){ref-type="fig"}, indicated by the white circle. For the high resolution case, however, the 3D CS reconstruction gives larger improvements compared with the low resolution case. {ref-type="fig"}). The parameter maps obtained from a CS reconstruction show loss of detail. The quality of the maps obtained from the undersampled scan after an MC‐based reconstruction is comparable to the quality of the maps from a fully sampled scan. Inhomogeneities are visible in the vitreous body, which is very hard to accurately encode due to the low sensitivity of the MRF train for very long T~1~ values](MRM-81-2551-g003){#mrm27594-fig-0003} {ref-type="fig"} show more structural detail, indicated by the white circle. Note that Figure [3](#mrm27594-fig-0003){ref-type="fig"} and Figure [4](#mrm27594-fig-0004){ref-type="fig"} were different scans, in which motion artifacts are also different. Fully sampled data sets were not acquired for the high resolution case due to the prohibitively long scanning times required](MRM-81-2551-g004){#mrm27594-fig-0004} Parameter maps obtained in the 6 different volunteers for the low resolution scans are shown in Figure [5](#mrm27594-fig-0005){ref-type="fig"}. In all volunteers, some inhomogeneities are visible in the vitreous body, which is a region that is very sensitive to any type of motion or system imperfections because of the low sensitivity of the MRF sequence for very long T~1~ compared with short T~1~. This effect is illustrated in Figure [6](#mrm27594-fig-0006){ref-type="fig"}, where differences in short T~1~ values (500‐1000 ms) result in more distinguishable dictionary elements compared with the same absolute differences in long T~1~ values, (3500‐4000 ms) especially in the first half of the MRF train. These inhomogeneities differ slightly between successive scans in the same volunteer, and are more visible in the scans of volunteer 3 (Figure [5](#mrm27594-fig-0005){ref-type="fig"}C) and volunteer 5 (Figure [5](#mrm27594-fig-0005){ref-type="fig"}E). Overall, the shortened scan time reduces the risk of motion artifacts, which is clearly visible in volunteers 5 and 6 (Figure [5](#mrm27594-fig-0005){ref-type="fig"}E,F). The high resolution parameter maps for the same volunteers are shown in Supporting Information Figure [S2](#mrm27594-sup-0001){ref-type="supplementary-material"}A‐F, with several regions of improved structural detail indicated by the white circles. {ref-type="table"}. In some volunteers the inhomogeneities in the vitreous body appear stronger than in others, which probably correspond with cases of more motion. This can also be seen in (E,F), where the quality of the maps is better for the shorter scans (MC) compared with the fully sampled ones](MRM-81-2551-g005){#mrm27594-fig-0005} {#mrm27594-fig-0006} Average T~1~ and T~2~ values in the lens nucleus, the vitreous body, the orbital fat, and the extraocular muscles are reported in Table [1](#mrm27594-tbl-0001){ref-type="table"} for the different low resolution scans and reconstruction methods. The relaxation times obtained with a CS reconstruction are relatively close to those of the MC‐based reconstruction, but differences are observed in small anatomical structures such as the extraocular muscles and the eye lens. Differences between the relaxation times from the MC‐based reconstructions and the FFT of the fully sampled data can in part be explained by the fact that motion artifacts differ from scan to scan. Average relaxation times obtained from high resolution scans (not reported) follow the results for the low resolution scans. Reference T~1~ values at 7T reported in Richdale et al[15](#mrm27594-bib-0015){ref-type="ref"} are included in Table [1](#mrm27594-tbl-0001){ref-type="table"}; it should be noted that these reported values show large differences in relaxation times between different measurement techniques. ###### T~1~ and T~2~ values for different ocular structures (annotated in Figure [1](#mrm27594-fig-0001){ref-type="fig"}C), averaged within the structure and over 6 volunteers[a](#mrm27594-note-0002){ref-type="fn"} CS 3D MC Full 7T Richdale et al. -------------------- --------------- ---------- ---------- -------------------- Lens nucleus 1403±178 1037±220 996±248 1520/1020 Vitreous body 3632±375 3614±444 3599±334 5000/4250 Orbital fat 93±23 100±29 95±26 -- Extraocular muscle 731±342 1736±346 1545±191 -- **T~2~ (ms)** Lens nucleus 29±9 29±12 21±10 -- Vitreous body 139±14 147±20 145±12 -- Orbital fat 55±12 51±16 51±19 -- Extraocular muscle 67±26 50±12 55±25 -- Values, given in milliseconds, were averaged in different regions of interest (lens nucleus, vitreous body, orbital fat, and extraocular muscle) from the different scans at low resolution, using different reconstruction methods, for each of the 6 healthy volunteers. The resulting values were used to determine mean ± SD values over all volunteers. The CS reconstruction produced different relaxation times in small anatomical regions such as the lens nucleus and the extraocular muscles. The TRs for the MC‐based reconstructions are close to the values for the fully sampled scans. Remaining differences can be explained by motion artifacts that differ from scan to scan. Reference values at 7T (variable flip angle gradient echo/inversion recovery) from previous literature were reported in the last 2 columns, showing large differences in T~1~ values between different techniques. John Wiley & Sons, Ltd Parameter maps in a uveal melanoma patient are shown in Figure [7](#mrm27594-fig-0007){ref-type="fig"}, together with a T~2~‐weighted, fat‐suppressed, TSE image for anatomical reference. The tumor and the detached retina are characterized in the MRF maps by much lower T~1~, T~2~, and M~0~ values compared with the vitreous body, which allows for clear discrimination between tumor and healthy tissue. Dictionary matches and measured signals (both normalized) in the detached retina, the lens nucleus, the eye tumor, and the fat are also shown. The average values in regions of interest are reported in Table [2](#mrm27594-tbl-0002){ref-type="table"}. {#mrm27594-fig-0007} ###### T~1~ and T~2~ values for different ocular structures in a uveal melanoma patient[a](#mrm27594-note-0003){ref-type="fn"} T~1~(ms) T~2~(ms) ------------------------------- ---------- ---------- Lens nucleus 916 24 Vitreous body 4218 209 Orbital fat 112 84 Extraocular muscle 1282 56 Eye tumor 883 36 Liquid behind detached retina 1814 64 T~1~ and T~2~ values in milliseconds were averaged over drawn regions of interest. The eye tumor shows different relaxation times (both T~1~ and T~2~) compared with the vitreous body and with the liquid behind the detached retina, which allows for discrimination between tumor and healthy tissue. John Wiley & Sons, Ltd Reconstruction times for the different reconstruction methods were averaged over 6 healthy volunteers and reported in Table [3](#mrm27594-tbl-0003){ref-type="table"}. The iterative nature of CS and MC increases the reconstruction times compared with the direct FFT reconstruction, but the MC‐based reconstruction is much more time‐efficient because it is performed entirely in k‐space, and uses only fast matrix vector multiplications.[42](#mrm27594-bib-0042){ref-type="ref"} ###### Reconstruction times[a](#mrm27594-note-0004){ref-type="fn"} Computation time (s) -------------------------- ---------------------- ------ CS 3D (40 SB iterations) 584 2734 MC (100 iterations) 12 44 FFT 0.1 0.5 Mean values of reconstruction times in seconds calculated over 6 healthy volunteers for CS 3D, MC, and the direct FFT. The reconstruction times for both CS and MC take longer compared to the direct FFT due to the iterative process, but the MC‐based reconstruction is much more time‐efficient than the CS reconstruction because it is performed entirely in k‐space. John Wiley & Sons, Ltd 4. DISCUSSION {#mrm27594-sec-0017} ============= The results in the simulation study clearly show the benefit of using the temporal dimension in the reconstruction of MRF data, as is performed using MC. The low rank property of the signal evolutions allows higher undersampling factors than in a CS reconstruction, in which the TV operator was used to enforce sparsity in the temporal as well as in the spatial dimensions. The experimental results confirmed these findings, and showed the feasibility of reducing the MRF scan time with the proposed MC‐based reconstruction from 7:02 min to 1:16 min. Using MC, high resolution parameter maps can be obtained, which was out of practical reach for full sampling due to the long scan time. The technique was also demonstrated in a uveal melanoma patient, in which relaxation times showed a clear difference between tumor and healthy tissue. The CS reconstruction resulted in smoothed parameter maps, which averages out motion artifacts, but also reduces the amount of visible detail. One reason why the CS reconstruction did not perform as well as the MC‐based reconstruction might be that the TV operator is not the optimal sparsifying transform for transforming the measured data along the temporal domain. Other sparsifying transforms, such as the Wavelet transform or even learned transforms or dictionaries,[56](#mrm27594-bib-0056){ref-type="ref"}, [57](#mrm27594-bib-0057){ref-type="ref"} might result in improvements of the parameter maps after a CS reconstruction. For the high resolution data, however, the 3D CS reconstruction seemed to perform better compared with the low resolution case, while the MC‐based reconstruction performed well in both the low and the high resolution cases. This suggests that the CS reconstruction is more dependent on the resolution of the acquired data than MC, which might be explained by the fact that MC, as implemented here, does not incorporate any spatial correlation into the reconstruction process. Furthermore, reducing the resolution might reduce the sparsity of the images in appropriate transform domains, while this is one of the key ingredients for CS to work. Images from undersampled scans were reconstructed with MC, in which the chosen rank of the projection matrix influences the error. Here, the number of incorporated singular values was determined empirically in a simulation study: 4 singular values resulted in the smallest error after 100 iterations of the algorithm. Other sampling patterns, flip angle trains or anatomies will likely require new optimization of the projection matrix. In the current acquisition, 15% or 12.5% of the data was acquired with 6 or 8 fully sampled central k‐space lines for each image frame. Further tuning of the sampling pattern might improve the accuracy of the reconstructions or allow even shorter scan times. One should keep in mind, however, that the sampled k‐t lines are used to reconstruct the missing k‐t lines. Because higher undersampling factors result in shorter scan times, this reduces the risk of motion‐corrupted k‐space lines, but if there is still significant motion, this affects a larger percent of the acquired data. Therefore, care should be taken to find a balance between the scan time and the robustness of the reconstruction algorithm to motion. In this work, the projection matrix was constructed from the central k‐t lines of the measurement data. In Doneva et al,[42](#mrm27594-bib-0042){ref-type="ref"} it was shown that this type of projection matrix results in a more accurate reconstruction compared with a projection matrix constructed from randomly selected k‐t lines due to the lower SNR in the latter case. Other works have used the simulated MRF dictionary as calibration data, which would eliminate the need to fully sample the centers of k‐space.[41](#mrm27594-bib-0041){ref-type="ref"} Such an approach will probably show a steeper decay in normalized singular values due to the absence of noise and motion in the simulations (see Supporting Information Figure [S3](#mrm27594-sup-0001){ref-type="supplementary-material"}). The central k‐space based projection matrix, however, results in a smaller reconstruction error, indicating that the central k‐space approximates the rank of the measurement data better. Further work should investigate whether this approach could be advantageous in terms of mitigating motion artifacts. As an alternative approach to the method used in our work, in which a low‐rank constraint is added as a penalty term to the cost function, the low‐rank property of the unknown image series can be incorporated directly in the data fidelity term, transforming the minimization problem into a linear one, which may be beneficial in terms of computational costs.[41](#mrm27594-bib-0041){ref-type="ref"} It would be interesting to compare the accuracy of the 2 methods in future work. Although this study has shown the feasibility of using MR fingerprinting to characterize the relaxation times of different anatomical structures in the eye, eye motion can still be a limiting factor. The parameter maps presented in the results section show inhomogeneities in the vitreous body, which can be a result of different types of motion in the eye (see Supporting Information Figure [S4](#mrm27594-sup-0001){ref-type="supplementary-material"}). The presence of motion in combination with the long T~1~ of the vitreous body and the low sensitivity of the MRF train to these long values, make it challenging to accurately map the relaxation times in the vitreous body itself, as was shown in Figure [6](#mrm27594-fig-0006){ref-type="fig"}. Adopting a longer MRF train, as well as pattern optimization of the MRF train, might help to increase the encoding capability, but a longer time between the cued‐blinks will strongly increase the chance of blink‐induced artifacts. However, one should recognize from a clinical point‐of‐view that for almost all ocular conditions the vitreous body is not affected and, therefore, an accurate quantification of its T~1~ is clinically not relevant. Outer volume suppression pulses, applied immediately before the inversion pulse or during 0 flip angle phases in the MRF train, might offer a way to reduce the flow of fresh magnetization (caused by motion) coming from slices above and below the imaging slice or from the left and the right of the imaging field of view, during repetitions of the flip angle train. However, such an approach and its effect on the quality of the parameter maps has to be investigated further. The parameter maps corresponding to patient data showed a very large difference between tumor tissue and healthy vitreous body, suggesting that fully homogeneous regions of T~1~ in the vitreous body are not necessary for disease quantification and classification. Future work should investigate the extension of the current single slice approach to a 3D approach, such that the entire eye can be efficiently quantified from 1 scan. The measured relaxation times are different between volunteers, potentially explained by anatomical or other volunteer‐specific differences. Small differences in relaxation times were observed for different scans in the same volunteer, caused by motion artifacts that change from scan to scan, but overall they are consistent within each volunteer, which is important for the use of this technique in practice. Considering the large deviations in measured relaxation times between different studies, it will be interesting to compare the MRF technique to standard T~1~ and T~2~ mapping techniques on a patient‐specific basis, and in this way investigate the origin of deviations from mean values as well as compare the robustness to motion for the different techniques. It should be noted, however, that in Ma et al,[58](#mrm27594-bib-0058){ref-type="ref"} it was already observed that MRF values do not always agree perfectly with reference values from other techniques, and potential reasons for this need to be investigated. Parameter maps in the current study were not corrected for slice profile effects, but all experiments were performed using an RF pulse with a very high time‐bandwidth product, minimizing the effects as demonstrated in Ma et al.[58](#mrm27594-bib-0058){ref-type="ref"} The flip angle map, which is used as an input in the matching process, was produced with DREAM, in which the B~1~ ^+^ encoding slice thickness was set to be double the acquisition slice thickness to eliminate the slice profile effect.[51](#mrm27594-bib-0051){ref-type="ref"} Values for the optic nerve were not reported in this study because the optic nerve was not visible in all scans due to small differences in planning and anatomy, and the slice thickness of 5 mm makes the measured values in the optic nerve very sensitive to partial volume effects. These partial volume effects also complicate quantification of heterogeneous tumors. In particular, tumor relaxation values could become inaccurate due to averaging with the strong signal coming from the surrounding vitreous body. Planning the imaging slice through the tumor as well as through the center of the vitreous body, such that the imaging plane is perpendicular to the tangent along the retina, would help to reduce these effects. One limitation of the current study is the rather high slice thickness used (which is limited by the gradient strengths). With small changes in the sequence such as using a slightly longer echo time, acquisition and reconstruction of a 2‐mm‐thick slice is feasible (see Supporting Information Figure [S5](#mrm27594-sup-0001){ref-type="supplementary-material"}). The in‐plane resolution of 0.5 mm is satisfactory for tumor quantification and classification, as well as visualizing small structures such as the sclera and the ciliary body. The results in this study show the potential to perform ocular MRF in tumor patients. To adopt ocular MRF in clinics, the technique could be further tailored to quantify specifically the relevant T~1~ and T~2~ values of tumors. Extensions to multislice or 3D acquisitions could be developed such that the whole tumor volume can be covered and quantified. Further studies should investigate which clinical applications will benefit from ocular MRF and in that way explore the clinical relevance of the technique. In conclusion, the high undersampling factors used for this Cartesian, nonparallel imaging‐based approach shorten scan time and in this way reduce the risk of motion artifacts, which is most relevant for elderly patients, who typically experience difficulties focusing on a fixation target. Supporting information ====================== ###### **FIGURE S1** The effect of the undersampling factor on the performance of different reconstruction methods. Undersampled data sets were obtained by subsampling a fully sampled data set, while fixing the number of central k‐space lines to six for all undersampling factors. For larger undersampling factors, MC outperforms 2D and 3D CS. For undersampling factors smaller than three, MC has a slightly higher error compared to 3D CS. Overall, the error appears to be less affected by the undersampling factor for MC compared to the other reconstruction methods. Error measures are defined according to Equation 5 **FIGURE S2** The parameter maps in all healthy volunteers for high resolution scans. Parameter maps obtained in six healthy volunteers are shown in (a)‐(f), respectively. The CS 3D reconstruction performs better for the high resolution scans than for the low resolution scans, but the parameter maps still show loss of detail compared to the maps obtained from the undersampled scan after an MC‐based reconstruction, with examples indicated by the white circles. Fully sampled reference scans were not obtained due to the long scan time required. A zoomed‐in version of the MC result in volunteer 1 is shown in (g), and repeated in (h) with a different color scale **FIGURE S3** Comparison of 2 different projection matrices. (a) The normalized singular value vector of the simulated MRF dictionary shows a steeper decay compared to the normalized singular vector of the central k‐space data. (b) The reconstruction error (defined as in Equation 5) as a function of the n most significant left singular values, is smaller when using the central k‐space as calibration data. A rank 3‐4 projection matrix results in the smallest reconstruction error when using the central k‐space data **FIGURE S4** The effect of motion on the parameter maps. (a) Motion was simulated by randomly replacing 1 of the 12 acquired k‐space lines in each MRF frame by (type 1) its phase‐modulated version with a random phase shift between 0 and 2π, mimicking in‐plane rigid body motion and (type 2) white gaussian noise (matching the maximum intensity of the replaced k‐space line), representing the worst case scenario of a completely corrupted signal. For motion type 1 larger differences are visible in the vitreous body. Motion type 2 results in noise break‐through in the parameter maps. For both types of motion, less than 6% change in T~1~ was observed in the vitreous body, while the T~2~ of the eye lens was changed by more than 20%, underlining the nonlinear effect of motion on the parameter maps. (b) The singular values of the calibration data show a less steep decay when k‐space lines are corrupted by motion **FIGURE S5** Parameter maps obtained from a thinner slice. By increasing the echo time from 3.5 ms to 4.6 ms, a slice of 2 mm can be acquired, spatial resolution 1×1×2 mm^3^. With this slice thickness the resulting parameter maps are less susceptible to partial volume effects, but slightly more noise is present in the maps due to the reduced SNR in the MRF images ###### Click here for additional data file. The authors thank Mariya Doneva for helpful discussions on reconstruction, and Thomas O'Reilly and Luc van Vught for useful insights during data acquisition.

Historical conceptualizations of depression =========================================== There is a long tradition in phenomenologlcal psychopathology that stresses basic bodily alterations as core features of depressive states. Thus, Wernicke used the term "vital feelings" to describe certain somatic symptoms occurring in affective psychoses.^[@ref1]^ Vital feelings refer to the close relationship of the body to the awareness of self. They determine the way we experience our body and the impression we assume our physical presence makes on other people. Vital feelings are somatic affects localized In different parts of the body. Whereas vital feelings constitute the bodily background of our normal experiences, they may move to the fore In a depressive mood. For example, depressed patients very often complain of a headache which is described not exactly as an ordinary pain, but more as an unbearable pressure "like a band around the head." Other disturbed vital feelings affect the chest or the abdomen, and mediate unpleasant sensations of weight, tension, heaviness, or Inhibition, totally absorbing the focus of attention. In quite a similar way Dupré speaks of "coenestopathic states" which mean a distressing, qualitative change of normal physical feeling In certain areas of the body during an episode of depressive mood. It Is a global loss of vitality In which all bodily parts and functions may be altered, and all their performances depressed.^[@ref2]^ Kurt Schneider considered these disturbances of vital feelings to be the core of cyclothymic depression. In his psychopathologlcal assessment they were of paramount diagnostic significance In depressive Illness, more or less equivalent to the first-rank symptoms In schizophrenia.^[@ref3]^ Huber discriminated between vital disturbances on the one hand and vegetative symptoms In depression on the other.^[@ref4]^ Vital disturbances refer to the vital feelings just mentioned. They comprise a loss of general vital tone of the body, a prevailing fatigue or exhaustibility, and various forms of somatic dysesthesia, typically of a static, more localized character affecting head, chest, heart region, or abdomen. All-pervasive sensations of anesthesia, stiffness, and alienation of the total body may characterize a somatopsychic depersonalization in depression which may appear as a Cotard\'s syndrome in its extreme form. If the vital disturbances take on a peculiar form that is difficult to describe in ordinary everyday words, Huber speaks of a "coenesthetic" depression which must be typologically differentiated from the bizarre states of coenesthetic schizophrenia. Vegetative symptoms are closely associated with these vital disturbances and coenesthesias in depression. Disturbances of sleep, appetite, and digestion are most frequent. However, there may be many other vegetative symptoms in depression such as disordered salivation, transpiration and lacrimation, cardiac arrhythmias and dyspnea, loss of libido and various sexual dysfunctions, dys- or amen? orrhea, loss of or increase in body weight, decreased turgor of the skin, loss of hair, decrease in body temperature, nausea, vomiting, meteorism, dizziness, sweating, or sensations of coldness. Both vital disturbances, coenesthesias and vegetative symptoms, are typically coexistent with the well-known affective, behavioral, and cognitive symptoms of depression. With respect to the different settings of medical care, however, these psychological symptoms of depression may be masked by a dominant reporting of somatic symptoms. M. Bleuler addressed the point in his book *Depressions in Primary Care,* in 1943: *"It is a common and frequent observation that depressive patients with single somatic complaints come to the consulting room of the general practitioner, internal specialist, and even the surgeon, gynecologist, ophthalmologist, urologist and other medical specialists, and spontaneously, they only speak of somatic phenomena while concealing their state of depressive mood. They report palpitations, tightness of the chest, loss of appetite, obstipation, pollakiuria, amenorrhea and many others. Only when one looks at their psychic state does one discover that they report numerous hypochondriac ideas also in other areas, that in addition they produce depressive ideas of impoverishment and sin, that beyond that their whole stream of thoughts is inhibited, that the depression manifests itself not only in the somatic complaints reported, but in various other bodily expressions."^[@ref5]^* In spite of this long-standing psychopathological view on the somatic foundation of depressive mood, at least in moderate and severe clinical states, it is bewildering that the official psychiatric classification systems of the *Diagnostic and Statistical Manual of Mental Disorders,* 4th edition *(DSM-IV)* and the *ICD-10* *Classification of Mental and Behavioral Disorders. Clinical Descriptions and Diagnostic guidelines (ICD-10)* only marginally appreciate somatic symptoms as diagnostic criteria for depressive disorders while focussing on the psychological symptoms of affect and cognition. So, *DSM-IV* lists only three criteria of somatic symptoms for major depressive disorder: sleep disturbance, appetite disturbance, and fatigue or loss of energy. And correspondingly, in *ICD-10,* disturbances of sleep and appetite, loss of libido, and amenorrhea are the only somatic symptoms considered to be of diagnostic significance for major depression. Beyond this short list of predominantly vegetative symptoms, no painful physical symptoms are mentioned in either the *DSM-IV* or *ICD-10.* There seems to be a major shift In diagnostic practice, however; the second version of the *Diagnostic and Statistical Manual of Mental Disorders,* 4th edition, Text Revision *(DSM-IV TR)* now Includes new criteria referring to "excessive worry over physical health and complaints of pain (eg, headaches or joint, abdominal, or other pains)."^[@ref6]^ This supplement of diagnostic criteria Is Indicative of an againIncreasing awareness of the importance of somatic symptoms in depression. What is meant by "somatic" in somatic symptoms of depression? ============================================================= In the literature there are many terms used to describe somatic symptoms in depression: somatic, somatlzed, physical, bodily, somatoform, painful, psychosomatic, vegetative, medically unexplained, masked, etc.^[@ref7]^ These diverse terms refer to different theoretical or diagnostic concepts. For states of depressive mood the neutral term "somatic" is preferred, comprising various bodily sensations that a depressed individual perceives as unpleasant or worrisome. These dysesthesias are very often localized In certain body parts or organs, or may affect the whole body In Its vital condition, as In the case of fatigue or loss of energy. Several basic physical dysfunctions, such as those of sleep, appetite, or digestion, are also to be included in the term "somatic." In addition, It may be clinically relevant to differentiate between painful and nonpalnful somatic symptoms of depression. From a diagnostic perspective one has to keep in mind that somatic symptoms play a significant role both in primary psychiatric disorders, first and foremost depressive and anxiety disorders, and in somatoform disorders. And In differential diagnosis, somatic symptoms must be considered as possibly even Indicative of underlying somatic diseases. A diagnostic challenge may be seen In the well-known fact that depressive, anxiety, somatoform disorders, and medical conditions are frequently coexistent, or Interact In the Individual patient.^[@ref8]-[@ref10]^ Regarding the assessment of somatic symptoms, Kroenke correctly points out that diagnosis very often is more approximative than precise. Presented somatic symptoms may be either clearly attributed to a distinct medical disorder or be placed into one of the following heuristic categories: somatoform disorder, another primary psychiatric disorder (often depression and/or anxiety), functional somatic syndrome (eg, irritable bowel syndrome, fibromyalgia, chronic fatigue syndrome), "symptom-only" diagnosis (eg, low back pain, idiopathic dizziness) or only partially explained by a defined medical disorder (eg, many states of chronic pain).^[@ref11]^ Epidemiological studies may provide an illuminating survey of the prevalence of somatic symptoms in depressive disorders, especially those encountered in primary care, and the prognostic value of somatic symptoms regarding their development in the further course of illness. Somatic symptoms of depressive disorders in inpatient care and primary care =========================================================================== In a clinical study, Hamilton reported that somatic symptoms prevailed in a great majority of depressed patients.^[@ref12]^ Somatic symptoms, particularly somatic anxiety and fatigue, were documented in up to 80% of a sample of 260 women and 239 men suffering from major depression. These somatic symptoms very frequently had an underlying psychopathologically relevant hypochondriasis, both in women and men. This study confirmed earlier studies showing that depressive disorders with predominantly somatic presentation were likely to be the most common form of depression, both in inpatient and outpatient care.^[@ref13],[@ref14]^ Hagnell and Rorsman stressed the Indicative significance of somatic symptoms in depressed primary care patients regarding their risk of suicide.^[@ref15]^ Epidemiological studies designed to establish prevalence figures for depressive disorders In primary care during recent years have uniformly demonstrated that depressive disorders are highly prevalent at this level of medical care.^[@ref16]-[@ref19]^ For the great majority of depressed patients seeking professional help in the official health care system, general practitioners and internists are the decisive interface for diagnosis and treatment of depression.^[@ref20]^ Primary-care patients with depression very often present with somatic complaints. This seems to be more the rule than the exception worldwide.^[@ref21],[@ref22]^ Two of the three most common symptoms reported during a current depressive episode were somatic (tlred/no energy/listless: 73%, broken sleep/decreased sleep: 63%) as shown by the European Study Society study (DEPRES II).^[@ref23]^ This study, however, also underlined that 65% of the depressed primary care patients suffered from a concomitant medical condition pointing to some likely difficulties In differential diagnosis. The multlcenter International study (n =1146) conducted by the World Health Organization (WHO) confirmed that two thirds of the patients presented their depressive mood with somatic symptoms exclusively, and more than half complained of multiple medically unexplained somatic symptoms.^[@ref24]^ In another primary care study, Kirmayer et al arrived at a similar finding of patients presenting their depressive or anxiety disorders exclusively with somatic symptoms in an overwhelming majority (73%). The identified somatic symptoms were the main reason for the initial visit to the primary care physician.^[@ref25]^ In a US study in 573 patients with the diagnosis of major depression, two thirds (69%) complained of general aches and pains, hinting at a close relationship between pain symptoms and depression.^[@ref26]^ The diagnostic situation In primary care frequently manifests Itself, however, as somewhat more complicated. Many patients present only with a single or a few somatic symptoms which remain medically unexplained and do not fulfill the affective and cognitive criteria for a discrete depressive or anxiety disorder at the end of the clinical interview. Single somatic symptoms are the primary reason for more than 50% of patients visiting a general practitioner or an outpatient clinic. In some 20% to 25%, these somatic symptoms are recurrent or chronic. Somatic symptoms that remain unexplained after a careful medical assessment generally bear a high risk for psychiatric morbidity, regardless of the type of symptoms.^[@ref27]-[@ref29]^ Up to two thirds of these patients develop a depressive disorder in the medium term, and between 40% to 50% fulfill the criteria for an anxiety disorder.^[@ref30]-[@ref33]^ In a cross-sectional study in 1042 primary care patients, Gerber et al investigated the differential relationship between specific somatic complaints and underlying depressive symptoms. Some somatic symptoms showed a high positive predictive value (PPV) for depression: Sleep disturbances (PPV: 61%), fatigue (PPV: 60%), three or more complaints (PPV: 56%), nonspecific musculoskeletal complaints (PPV: 43%), back pain (PPV: 39%), amplified complaints (PPV: 39%), vaguely stated complaints (PPV: 37 %).^[@ref34]^ Some somatic symptoms are typically covarlant In the patients\' complaints without having received the nosological status of a discrete medical condition. These clusters of symptoms are instead considered as functional somatic syndromes and termed according to the diagnostic standards of the various medical disciplines, eg, fibromyalgia, chronic fatigue syndrome, and irritable bowel syndrome, etc. For some authors in psychiatry these functional somatic syndromes represent typical variants of somatoform disorders. There is still a controversial dispute in the medical literature, however, as to whether to assemble all these functional somatic syndromes within one general category of somatization,^[@ref35],[@ref36]^ or to split them up into separate clinical entities.^[@ref37]^ From an empirical standpoint, it is remarkable that among these syndromes there is a significant overlap on the level of symptoms and a strong association with depressive and anxiety disorders.^[@ref38]-[@ref41]^ A close relationship between states of depressive mood and symptoms of pain, especially of chronic pain, has been impressively established in many empirical studies.^[@ref26],[@ref42]-[@ref44]^ Depression and painful symptoms commonly occur together. As both conditions are highly prevalent in the general population, their frequent co-occurrence might be due to mere statistical coincidence.^[@ref45],[@ref46]^ From an empirical standpoint, however, the prevalence figures of coexistence are far beyond statistical expectation. In a meta-analytical survey, Bair et al demonstrated that around two thirds of all depressed patients treated in primary, secondary, and tertiary centers, both in outpatient and inpatient settings, report distressing painful somatic symptoms.^[@ref26]^ Conversely, the prevalence rate of major depression in patients with various pain syndromes is about 50%. There seem to be higher rates in clinical states characterized by multiple diffuse pain symptoms than by more defined types of pain. The risk of major depression is considered to be dependent on the severity, frequency, persistence, and number of pain symptoms.^[@ref47],[@ref48]^ From the perspective of primary care an epidemiological study assessing the predictive power of chronic pain for depressive morbidity showed that the prevalence rate of at least one chronic painful physical condition (CPPC) in the general population was 17.1%. At least one depressive symptom was present in 16.5% of subjects; 27.6% of these subjects had at least one CPPC. Major depression was diagnosed in 4% of subjects, and 43.4% of these subjects had at least one CPPC, which was 4 times more often than in subjects without depressive disorder.^[@ref49]^ This significant Interrelationship of CPPC and depression confirmed the earlier clinical advice of Katon, suggesting that if all patients with painful physical conditions were systematically assessed regarding a possible underlying depression, some 60% of all states of depression could be detected in primary care.^[@ref50]^ Generally, one has to keep in mind that, both from a cross-sectional and a longitudinal perspective, there is a relevant overlap of depressive, anxiety, and somatoform disorders, especially chronic painful physical conditions, among primary care patients presenting with medically unexplained symptoms.^[@ref51]-[@ref58]^ It is an important clinical finding that, with an increasing number of medically unexplained symptoms, the risk of an underlying depressive disorder increases in an impressive dose-response relationship. In a study which included 1000 adults and another study comprising 500 patients with a chief complaint of somatic symptoms, the presence of any somatic symptom increased the likelihood of a mood or anxiety disorder by two- or threefold. Only 2% of patients with no or only one somatic symptom had a mood disorder, but 60% of those patients presented nine or more somatic symptoms.^[@ref31],[@ref59]^ Patients with multiple medically unexplained somatic symptoms also show a greater amount of associated other psychiatric comorbidity.^[@ref60],[@ref61]^ Somatic symptoms in depression and rates of diagnostic recognition within primary care ====================================================================================== The typical form of presenting a depression In primary care Is via somatization. This form of somatic presentation, however, is considered to be one of the main reasons for low rates of recognition of depression In this sector of the medical care system.^[@ref20],[@ref62]^ It must be acknowledged that the alarmingly low figures of diagnosed and consecutively treated depressive disorders in only 25% to 33% of affected patients found in epidemiological studies during the early 1990s have increased up to some 60%. ^[@ref17],[@ref19]^ From a perspective of primary care, general practitioners are consulted by two groups of depressed patients who may pose a diagnostic challenge. Patients suffering from a medical condition have a frequent depressive comorbidity^[@ref23],[@ref63]^ These associated depressions often remain undetected, as the diagnostic focus of the primary care physicians is led by a dominant model of somatic disease.^[@ref64]^ Indeed, certain somatic symptoms such as sleep disturbances, diffuse bodily pains and aches, fatigue, changes of appetite, etc, may characterize both the pathophysiological process of a discrete medical condition and a depressive disorder as well. The differential diagnosis may be difficult. The role and significance of somatic symptoms for the diagnosis of depression in medically ill patients have been a controversial issue in the scientific literature. Meanwhile, a clinically reasonable consensus has been arrived at that the *DSM-IV* criteria for major depression do not require significant modification for patients with medical comorbidities.^[@ref65]-[@ref67]^ Somatic symptoms can positively contribute to a diagnosis if they are assessed in line with typical concomitant affective, behavioral, and cognitive symptoms of depression.^[@ref9]^ For a primary care physician It Is Important to know that at least 20% to 30% of patients with chronic medical conditions suffer from a coexisting depression.^[@ref68]^ It must be assumed that, even In those patients being diagnosed with an acute somatic disease for the first time, depression coexists In a significant percentage.^[@ref69]^ All In all, patients with medical conditions are to be considered as a risk group for nonrecognitlon of concomitant depression.^[@ref70]^ This especially applies to elderly medically ill patients.^[@ref71]^ In the other major group of depressed primary care patients, the somatic symptoms complained of very often remain medically unexplained. If one focuses on the mode of presentation, about 50% of the patients report somatic symptoms exclusively, and a minor percentage of some 20% present their depressive disorder with prevailing psychological, ie, affective and cognitive symptoms.^[@ref7],[@ref21],[@ref72],[@ref73]^ There is not, however, a categorical split between a somatic mode of presentation on the one hand and a psychological mode on the other. Rather, a broad spectrum of transition must be assumed, and the grading of somatization has an impact on the probability of recognition of an underlying depression.^[@ref25]^ As a rule, primary care physicians do not recognize a depression with an individual patient better when he or she is complaining of many actual medically unexplained somatic symptoms (here they rather prefer a diagnostic standpoint of wait and see), but when the patient returns again and again to consult because of these symptoms.^[@ref74]^ In addition, the extent of hypochondriacal worries and health anxieties facilitate, a correct diagnosis of depression.^[@ref75],[@ref76]^ Patients with somatic complaints that are not explained medically in an adequate way, however, do not represent a uniform group regarding diagnostic categorization. Besides depressive disorders, which in primary care manifest themselves according to the traditional concept of an endogenous type only in minority but instead show many atypical features,^[@ref77]-[@ref79]^ one must consider various anxiety and somatoform disorders in differential diagnosis.^[@ref60],[@ref61],[@ref80]-[@ref82]^ Again as a rule, there exists an Impressive overlap on the level of symptoms among all these diagnostic categories.^[@ref10]^ Aspects facilitating somatic symptoms in depression =================================================== Many factors may contribute to the form and extent to which a depression is presented in somatic symptoms. Female gender has been confirmed to be closely associated with somatization in many studies covering differential aspects on various theoretical levels.^[@ref83]^ In a gender differential analysis, Sllversteln draws some Interesting conclusions from the epidemiological data of the National Comorblty Survey.^[@ref84],[@ref85]^ By dividing respondents Into those who met overall criteria for major depression and exhibited fatigue, appetite, and sleep disturbances ("somatic depression") and those who met overall criteria without these somatic symptoms ("pure depression") she demonstrated gender differences only for "somatic depression" but not for "pure depression." The higher prevalence of "somatic depression" In females was strongly associated with a high frequency of anxiety disorders. Interestingly, this type of "somatic depression" among female patients already had Its onset during early adolescent years with predominantly bodily pains and aches. Wenzel et al attributed the higher prevalence of "somatic depression" in women largely to changes in appetite.^[@ref86]^ Gender differences can also be found in primary care. Women consistently reported most typical somatic symptoms at least 50% more often than men. Although mental disorders, above all depressive and anxiety disorders, were found to be correlated with this mode of somatic presentation, there was also an independent female gender effect on somatic symptom reporting.^[@ref87]^ In a later study Jackson et al found that among primary care patients with somatic symptoms, on the whole, women were younger, more likely to report stress, endorsed more "other, currently bothersome" symptoms, were more likely to have a mental disorder, and were less likely to be satisfied with the care.^[@ref88]^ A greater susceptibility of women, both to psychosocial stress and somatic illness stress, was held responsible for this higher prevalence of depressive and anxiety disorders in female patients.^[@ref89]^ A greater vulnerability to depressive and anxiety disorders on the one hand, and a strong neurobiological association to defined functional somatic syndromes (eg, fibromyalgia, irritable bowel syndrome, chronic fatigue syndrome) on the other may further increase the extent of this gender difference.^[@ref40],[@ref90]^ The disposition both to somatization and to depressive and anxiety disorder may be intermingled in various ways. Thus, a depressive mood may trigger the immediate illness behavior to enter the medical care system and to report somatized problems caused otherwise.^[@ref91]^ The very high frequency of somatic anxiety symptoms in patients with major depression may be interpreted by the idea that anxiety appears to be a major source of bodily distress and consecutive hypochondriasis, thus fostering somatization behavior.^[@ref12]^ Indeed, specific effects of depression, panic, and somatic symptoms on illness behavior must be considered.^[@ref92]^ Various causal illness interpretations, a tendency to amplify somatic distress, and difficulties In Identifying and communicating emotional distress, all have an impact on the form and extent of a somatic mode of presentation.^[@ref93]-[@ref95]^ Again, regarding the course of Illness, depressive and anxiety disorders following somatoform disorders may significantly contribute to the chronlflcatlon and complication of the latter.^[@ref39],[@ref96]^ From a perspective of etiologically relevant risk factors It Is a well-established epidemiological finding that the extent and severity of early adverse events, especially manifold traumatic experiences, are tightly connected with the mental and somatic state of adults. This general disposition may be detected In a series of psychiatric disorders, as In conversion and somatization syndromes,^[@ref97]-[@ref103]^ several chronic pain conditions,^[@ref104]-[@ref106]^ hypochondriacal attitudes,^[@ref107]^ factitious disorders,^[@ref98]^ and depressive, anxiety, and substance disorders.^[@ref108]-[@ref110]^ One can draw a basic conclusion from many epldemiologlcally designed longitudinal studies that the more a person has been exposed to severe and early trauma, the higher the risk will be that she/he will suffering from recurrent or chronic depression with pronounced suicidality, multiple medically unexplained somatic symptoms, especially chronic physical pain conditions with an onset already during adolescence or young adulthood, the more her/his psychic and somatic state as a whole will be negatively affected, and the more she/he will demonstrate abnormal illness behavior.^[@ref61],[@ref111]^ Culture and society are other factors that may have an important impact on the way a depressive mood is presented in a predominantly somatic way.^[@ref25]^ Interestingly, the comprehensive international WHO study on depression in primary care, conducted in 12 countries on different continents, was not able to identify clear cultural influences on the somatic mode of presenting a depression. A somatic presentation was much more common at centers where patients lacked an ongoing relationship with a primary care physician than at centers where most patients had a personal physician. This variable had a robustly differentiating effect beyond the various cultural settings.^[@ref24]^ Besides gender, culture, and type of patient-physician relationship, there may be many other factors influencing a more somatic mode of presentation, such as different ages in life cycle, association with medical conditions, earning a lower income, and imprisonment.^[@ref7],[@ref112]^ Burden of somatic symptoms in depression ======================================== Most patients who are psychopharmacologically treated for depression fail to reach full remission.^[@ref113]-[@ref114]^ A majority of patients may respond to antidepressants (by definition a reduction of symptoms by some 50% or more), but still suffer from residual symptoms. These residual symptoms are often somatic in nature. Symptoms of somatic anxiety and various painful conditions seem be especially common in states of incomplete remission.^[@ref115]^ Residual symptoms which are not treated must effectively be considered as a negative risk factor with respect to earlier relapse, and a more severe and chronic future course of illness.^[@ref116]-[@ref119]^ The clinical significance of somatic symptoms in depression may best be illustrated with the relationship between depression and painful physical conditions. In general, the worse the painful somatic symptoms, the more severe and the longer a depressive episode persists. In their general population-based study, Ohayon and Schatzberg found that depressed patients with chronic pain symptoms reported a longer duration of depressive mood (19.0 months) than those without chronic pain (13.3 months). In addition, a chronic physical pain condition in persons with at least one key symptom of depression was associated with an elevated rate of suicidal thoughts.^[@ref49]^ Fishbain considered chronic pain as a major suicide risk factor in depression.^[@ref120]^ Von Korff and Simon demonstrated a significant correlation between the intensity of pain symptoms and a worse outcome of depressive disorders. This worse outcome included more pain-related functional impairments, a worse state of general health, higher rates of unemployment, use of more opiates, more frequent polypharmacy, and more intensive utilization of medical services due to pain complaints. ^[@ref121]^ Although both painful and nonpainful somatic symptoms improve with antidepressant treatment, It Is the Intensity and extent of pain symptoms at baseline that significantly contribute to a less favorable response to medication, and to a longer duration of treatment necessary for a satisfying result, if at all.^[@ref122]-[@ref124]^ If one asssembles painful and nonpainful somatic symptoms of depression into a single dimension of somatization, It is this factor that must be correlated with an impressively increased overall use of health care services,^[@ref125]-[@ref127]^ to significant treatment nonadherence and a resulting higher risk of relapse and more chronic course of illness.^[@ref128]^ Again, a recurrent or chronic depression includes a higher risk of suicide^[@ref129]^ and an increased morbidity and mortality due to Illness-inherent factors or associated natural causes.^[@ref130]-[@ref132]^ All in all, it must be concluded that: when somatic symptoms, above all painful physical conditions, accompany the already debilitating psychiatric and behavioral symptoms of depression, the economic burden that ensues for patients and their employers increases considerably,^[@ref133]-[@ref134]^ the functional status may be hampered signifiacantly,^[@ref135]^ and the health-related quality of life is lowered dramatically^[@ref136]^ Neurobiological underpinnings of somatic symptoms in depression =============================================================== Various psychosocial and biological stressors may trigger a depression. Neurobiological processes underlying any depressive illness are manifold; this applies to the different somatic symptoms in particular. A strong heritable disposition, polygenetic in nature, seems to be established, but maladaptive neurobiological stress response systems already acquired by stressful and traumatic experiences during early development may play a major role in the pathophysiology of depression as well.^[@ref137]^ Dysfunctions in the serotonergic, noradrenergic, and dopaminergic neurotransmitter systems have been considered as relevant for quite a long time. Drawing from the neuroanatomical serotonergic tracts, starting in the midbrain raphe cell bodies and projecting to the frontal cortex, basal ganglia, limbic system, and hypothalamus on the one hand, of noradrenergic pathways originating in the locus ceruleus of the brain stem and projecting again to the same regions of the frontal cortex, limbic areas, and hypothalamus, but also uniquely to other parts of the frontal cortex and to the cerebellum on the other, Stahl stressed that deficiencies in the activity of specific pathways of serotonin and norepinephrine might account for the differential clinical phenomenology in depression. This seems to be true both for the typical psychological and somatic symptoms. Regarding somatic symptoms, especially vegetative symptoms such as changes in appetite or weight, lack of pleasure and sexual appetence, and sleep abnormalities, dysfunctional hypothalamic and sleep centers may be of paramount importance, all influenced by both serotonin and norepinephrine.^[@ref138]^ Fatigue, exhaustibility, or loss of energy, common distressing symptoms during a depressive episode, but also obstinate residual symptoms, may be mediated by different malfunctioning neuronal circuits that are regulated by multiple neurotransmitters.^[@ref139]^ Fatigue can be experienced as reduction in either mental or more physical vital feeling. Likely candidates for the neuronal structures that may mediate physical fatigue refer to brain areas regulating motor functions, such as striatum or cerebellum, but also to certain spinal pathways transferring sensory input from the body and thus modulating the perception of physical tiredness. In addition to serotonin and norepinephrine, dopamine may be involved in this process. Mental tiredness, on the other hand, may be mediated by diffuse cortical circuits and be influenced by cholinergic, histaminergic, noradrenergic, and dopaminergic neurotransmitters. The various painful somatic symptoms in depression may essentially be associated with serotonergic and noradrenergic pathways descending from brain stem centers to the spinal cord. An imbalance in these neurotransmitters, normally serving to inhibit the sensory input from the intestines, musculoskeletal system, and other body regions, may accentuate pain sensitivity.^[@ref26],[@ref140]^ As a matter of course, neither psychological nor somatic symptoms in depression can be explained by dysfunctional neurotransmitters exclusively. Many other neurobiological processes are involved in the pathophysiology of depression, such as an abnormal HPA axis with a disordered feedback mechanism of the corticotropin-releasing factor (CRF) -adrenocorticotropic hormone (ACTH) - Cortisol stress response, a reduced secretion of the neuropeptide hypocretin thus contributing to a desynchronization of the sleep-wake cycle, various abnormalities in the inflammatory system with an increased production of certain proinflammatory cytokines, a resulting depletion of the serotonin system, sickness behavior and depressive mood, reduced concentrations of various neurotrophlns such as brain-derived neurotropic factor (BDNF) causing Impaired neuroplastlcity, cell resistance, and neurogenesis.^[@ref137],[@ref141]-[@ref147]^ The intricate pathophysiological interplay of neuroendocrine stress response, inflammation, and neurotransmitter systems, both centrally and peripherally, may perhaps best be illustrated by the relationship between chronic pain conditions and depressive mood states (succinctly summarized in refs 148-150). In short, chronic stress evoked by chronic pain leads to a loss of negative glucocorticoid feedback in the (hypothalamic-pituitary-adrenocortical (HPA) axis and downregulation of the glucocorticoid receptors within the brain and the body periphery. Inflammation and nerve injury stimulate nociresponsive neurons within the dorsal horn of the spinal cord, and the relay of the nociceptive information ascends to the brain stem to be gated within the thalamus, prior to its cognitive appraisal within the somatosensory cortex. Monoamlnergic neurons In the brain stem normally descend to the spinal cord to act as a "brake" on nociceptive transmission. During chronic pain, loss of serotonergic and noradrenergic tone In response to glucocortlcold-lnduced monoamlnergic depletion may lead to descending Inhibitory Impulses to the spinal cord to effect an enhancement of pain sensation. Loss of glucocorticoid Inhibition of proinflammatory cytokines leads to proliferation of peripheral inflammatory events, contributing to pain sensitization. Although acute stress may be analgesic, implying an inhibitory circuitry between the limbic and somatosensory cortices, chronic stress evoked by chronic pain, leads to downregulation of glucocorticoid-mediated activity of this inhibitory connection, causing enhanced pain perception. Similarly, although acute pain may be mood-enhancing via both sympathetic and glucocorticoid routes (implying an excitatory reciprocal link between the somatosensory and limbic cortices), chronic pain-Induced downregulation of glucocorticoid modulation of this link may lead to depressed mood. Psychopharmacological implications for the treatment of somatic symptoms in depression ====================================================================================== Numerous trials with antidepressants have demonstrated that full remission of the psychological, and especially of the somatic, symptoms in depression can be achieved only by a minority group of depressed patients within a usual 6- to 8-week treatment period.^[@ref62],[@ref151],[@ref152]^ These sobering facts are reflected by a higher risk of relapse, a worse course of illness with many associated psychosocial disabilities, and a hampered health-related quality of life. Therefore, achieving a state of symptomatic remission must be a treatment goal of utmost clinical importance. Targeting both serotonin and norepinephrine in those neuronal circuits that mediate somatic symptoms is the most widely employed strategy to reduce painful and nonpainful somatic symptoms in depression.^[@ref90]^ In comparison with selective serotonin reuptake inhibitors, antidepressants with a dual action on both the serotonin and norepinephrine system were significantly superior in alleviating these somatic symptoms and achieving full symptomatic remission of depression. This may be a promising approach, even to treating chronic pain conditions, eg, fibromyalgia, without prevailing depressive symptoms.^[@ref153],[@ref154]^ This seems to have been well established In clinical trials with venlafaxlne,^[@ref155]-[@ref159]^ duloxetlne,^[@ref160]-[@ref163]^ mllnaclpran,^[@ref164]^ or mlrtazaplne.^[@ref165]^ In order to Improve distressing symptoms of fatigue, the use of psychostimulants, modafinil, bupropion, or selective norepinephrine reuptake inhibitors such as reboxetine or atomoxetine may be recommended.^[@ref166]^ As a rule, psychopharmacological efforts to treat severe states of depression or states of depression with prominent somatic symptoms effectively must be guided by a perspective of a longer duration than usual. Higher dosages of a selected antidepressant have to be used very often. Sometimes shifts within or between pharmacological classes of antidepressants or an augmentation with, eg, lithium or tri-iodthyronine, are necessary to arrive at the desired aim. From a pragmatic standpoint, clinically rational algorithms may favorably guide this endeavor.^[@ref167]^ Finally, it must be stressed that a reasonable combination of pharmacological and psychotherapeutic approaches can improve the treatment results in many depressed patients.^[@ref168],[@ref169]^

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