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Erythrocytes bind circulating immune complexes (ICs) and facilitate IC clearance from the circulation. Chronic hepatitis C virus (HCV) infection is associated with IC-related disorders. In this study, we investigated the kinetics and mechanism of HCV and HCV-IC binding to and dissociation from erythrocytes. Cell culture-produced HCV was mixed with erythrocytes from healthy blood donors, and erythrocyte-associated virus particles were quantified. Purified complement proteins, complement-depleted serum, and complement receptor antibodies were used to investigate complement-mediated HCV-erythrocyte binding. Purified HCV-specific immunoglobulin G (IgG) from a chronic HCV-infected patient was used to study complement-mediated HCV-IC/erythrocyte binding. Binding of HCV to erythrocytes increased 200- to 1,000-fold after adding complement active human serum in the absence of antibody. Opsonization of free HCV occurred within 10 minutes, and peak binding to erythrocytes was observed at 20-30 minutes. Complement protein C1 was required for binding, whereas C2, C3, and C4 significantly enhanced binding. Complement receptor 1 (CR1, CD35) antibodies blocked the binding of HCV to erythrocytes isolated from chronically infected HCV patients and healthy blood donors. HCV-ICs significantly enhanced complement-mediated binding to erythrocytes compared to unbound HCV. Dissociation of complement-opsonized HCV from erythrocytes depended on the presence of Factor I. HCV released by Factor I bound preferentially to CD19+ B cells compared to other leukocytes. Conclusion: These results demonstrate that complement mediates the binding of free and IC-associated HCV to CR1 on erythrocytes and provide a mechanistic rationale for investigating the differential phenotypic expression of HCV-IC-related disease.

The goal of this study was to determine whether an association exists between circulating microRNA (miRNA) levels and disease progression in chronic hepatitis C (CHC), whether plasma or extracellular vesicles (EVs) were optimal for miRNA measurement and their correlation with hepatic miRNA expression, and the mechanistic plausibility of this association. We studied 130 CHC patients prospectively followed over several decades. A comprehensive miRNA profile in plasma using microarray with 2578 probe sets showed 323 miRNAs differentially expressed between healthy individuals and CHC patients, but only six that distinguished patients with mild versus severe chronic hepatitis. Eventually, let-7a/7c/7d-5p and miR-122-5p were identified as candidate predictors of disease progression. Cross-sectional analyses at the time of initial liver biopsy showed that reduced levels of let-7a/7c/7d-5p (let-7s) in plasma were correlated with advanced histological hepatic fibrosis stage and other fibrotic markers, whereas miR-122-5p levels in plasma were positively correlated with inflammatory activity, but not fibrosis. Measuring let-7s levels in EVs was not superior to intact plasma for discriminating significant hepatic fibrosis. Longitudinal analyses in 60 patients with paired liver biopsies showed that let-7s levels in plasma markedly declined over time in parallel with fibrosis progression. However, circulating let-7s levels did not parallel those in the liver. Conclusion: Of all miRNAs screened, the let-7 family showed the best correlation with hepatic fibrosis in CHC. A single determination of let-7s levels in plasma did not have superior predictive value for significant hepatic fibrosis compared with that of fibrosis-4 index, but the rate of let-7s decline in paired longitudinal samples correlated well with fibrosis progression. Pathway analysis suggested that low levels of let-7 may influence hepatic fibrogenesis through activation of transforming growth factor β signaling in hepatic stellate cells. (Hepatology 2016;64:732-745)

To investigate the quantitative characteristics of humoral immunity in patients with hepatitis C, we established an enzyme-linked immunosorbent spot (ELISpot) assay for detection of anti-hepatitis C virus (HCV)-secreting B cells. Receiver operating characteristic curve analysis demonstrated 100% specificity and 58% to 92% sensitivity for detecting B-cell responses to NS5b, NS3, E2, and core antigens. The median sum of anti-HCV-secreting B cells to all HCV antigens tested was significantly higher in 39 patients with chronic hepatitis C (47.3 spot forming cells [SFCs]/10(6) peripheral blood mononuclear cells [PBMCs]) than in 9 recovered subjects (15.3 SFCs/10(6) PBMCs; P = .05) or 11 uninfected controls (5.3 SFCs/10(6) PBMCs; P < .001); the significant difference (P = .018) in chronic versus recovered patients was in reactivity to nonstructural antigens NS3 and NS5b. Anti-HCV immunoglubulin M (IgM)-secreting B cells were also readily detected and persisted decades into HCV infection; there was no difference in IgM-positive cells between chronic and recovered patients. ELISpot reactivity to genotype 1-derived antigens was equivalent in patients of genotypes 1, 2, and 3. There was significant correlation between the numbers of anti-HCV IgG-secreting B cells and serum aminotransferase and to the level of circulating antibody. In conclusion, ELISpot assays can be adapted to study B-cell as well as T-cell responses to HCV. Measurement at the single-cell level suggests that humoral immunity plays a minor role in recovery from HCV infection and that B-cell immunity is strongest in those with persistent infection.

SEN virus (SEN-V) is a recently identified single-stranded, circular DNA virus. Two SEN-V variants (SENV-D and SENV-H) were assayed by polymerase chain reaction (PCR) to investigate their role in the causation of transfusion-associated non–A to E hepatitis. The incidence of SEN-V infection after transfusion was 30% (86 of 286) compared with 3% (3 of 97) among nontransfused controls (P < .001). Transfusion risk increased with the number of units transfused (P < .0001) and donor-recipient linkage for SEN-V was shown by sequence homology. The prevalence of SEN-V in 436 volunteer donors was 1.8%. Among patients with transfusion-associated non–A to E hepatitis, 11 of 12 (92%) were infected with SEN-V at the time of transfusion compared with 55 of 225 (24%) identically followed recipients who did not develop hepatitis (P < .001). No effect of SEN-V on the severity or persistence of coexistent hepatitis C virus (HCV) infection was observed. In 31 infected recipients, SEN-V persisted for greater than 1 year in 45% and for up to 12 years in 13%. SEN-V–specific RNA (a possible replicative intermediate) was recovered from liver tissue. In summary, SENV-D and -H were present in nearly 2% of US donors, and were unequivocally transmitted by transfusion and frequently persisted. The strong association of SEN-V with transfusion-associated non–A to E hepatitis compared with controls raises the possibility, but does not establish that SEN-V might be a causative agent of posttransfusion hepatitis. The vast majority of SEN-V–infected recipients did not develop hepatitis. (HEPATOLOGY 2001;33:1303-1311.)