B cells in chronically hepatitis C virus-infected individuals lack a virus-induced mutation signature in the TP53, CTNNB1, and BCL6 genes.

Hepatitis C virus (HCV) is considered to have a causative role in B-cell lymphoproliferative diseases, including B-cell lymphomas, in chronic virus carriers. Previous data from in vitro HCV-infected B-cell lines and peripheral blood mononuclear cells from HCV-positive individuals suggested that HCV might have a direct mutagenic effect on B cells, inducing mutations in the tumor suppressor gene TP53 and the proto-oncogenes BCL6 and CTNNB1 (ß-catenin). To clarify whether HCV indeed has a mutagenic effect on B cells in vivo, we analyzed naive and memory B cells from the peripheral blood of four chronic HCV carriers and intrahepatic B cells from the livers of two HCV-positive patients for mutations in the three reported target genes. However, no mutations were found in the TP53 and CTNNB1 genes. For BCL6, which is a physiological target of the somatic hypermutation process in germinal-center B cells, the mutation levels identified were not higher than those reported in the respective B-cell subsets in healthy individuals. Hence, we conclude that in chronic HCV carriers, the virus does not generally induce mutations in the cancer-related genes TP53, CTNNB1, and BCL6 in B cells. Based on these findings, new targets have to be investigated as potential mediators of HCV-associated B-cell lymphomagenesis.

Pegylated interferon-alpha, ribavirin, and rituximab combined therapy of hepatitis C virus-related mixed cryoglobulinemia: a long-term study.

This study illustrates the use and efficacy of a combination of pegylated interferon-alpha (Peg-IFN-alpha) and ribavirin (RBV), with or without rituximab (RTX), in hepatitis C virus (HCV)-related mixed cryoglobulinemia (MC). Twenty-two patients with HCV-related MC received Peg-IFN-alpha (2a: 180 mug or 2b: 1.5 mug/kg) weekly plus RBV (1000 or 1200 mg) daily for 48 weeks, and RTX (375 mg/m(2)) once a week for 1 month followed by two 5-monthly infusions (termed PIRR). Fifteen additional patients received Peg-IFN-alpha/RBV with the same modalities as the PIRR schedule. Complete response was achieved in 54.5% (12/22) and in 33.3% (5/15) of patients who received PIRR and Peg-IFN-alpha/RBV, respectively (P < .05). Clearance of HCV RNA and conversion of B-cell populations from oligoclonal to polyclonal in liver, bone marrow, and peripheral blood was maintained for up to 3 years in 10 of 12 (83.3%) and in 2 of 5 (40%) patients receiving PIRR and Peg-IFN-alpha/RBV, respectively (P < .01). Cryoproteins in 22.7% (5/22) of patients with PIRR and in 33.3% (5/15) with Peg-IFN-alpha/RBV persisted despite sustained HCV RNA clearance. No response occurred in remaining 5 patients of both groups. PIRR therapy is well tolerated and more effective than Peg-IFN-alpha/RBV combination in HCV-related MC. Its effect may last for more than 3 years.

Role of the receptor for the globular domain of C1q protein in the pathogenesis of hepatitis C virus-related cryoglobulin vascular damage.

Mixed cryoglobulinemia (MC) is a lymphoproliferative disorder observed in approximately 10 to 15% of hepatitis C virus (HCV)-infected patients. Circulating, nonenveloped HCV core protein, which has been detected in cryoprecipitable immune complexes, interacts with immunocytes through the receptor for the globular domain of C1q protein (gC1q-R). In this study, we have evaluated circulating gC1q-R levels in chronically HCV-infected patients, with and without MC. These levels were significantly higher in MC patients than in those without MC and in healthy controls and paralleled specific mRNA expression in PBL. Soluble gC1q-R circulates as a complexed form containing both C1q and HCV core proteins. Higher serum gC1q-R levels negatively correlated with circulating concentrations of the C4d fragment. The presence of sequestered C4d in the vascular bed of skin biopsies from MC patients was indicative of in situ complement activation. In vitro studies showed that release of soluble gC1q-R is regulated by HCV core-mediated inhibition of cell proliferation. Our results indicate that up-regulation of gC1q-R expression is a distinctive feature of MC, and that dysregulated shedding of C1q-R molecules contributes to vascular cryoglobulin-induced damage via the classic complement-mediated pathway.

Double-jeopardy: scRNA-seq doublet/multiplet detection using multi-omic profiling.

The computational detection and exclusion of cellular doublets and/or multiplets is a cornerstone for the identification the true biological signals from single-cell RNA sequencing (scRNA-seq) data. Current methods do not sensitively identify both heterotypic and homotypic doublets and/or multiplets. Here, we describe a machine learning approach for doublet/multiplet detection utilizing VDJ-seq and/or CITE-seq data to predict their presence based on transcriptional features associated with identified hybrid droplets. This approach highlights the utility of leveraging multi-omic single-cell information for the generation of high-quality datasets. Our method has high sensitivity and specificity in inflammatory-cell-dominant scRNA-seq samples, thus presenting a powerful approach to ensuring high-quality scRNA-seq data.

Increased serum levels of the chemokine CXCL13 and up-regulation of its gene expression are distinctive features of HCV-related cryoglobulinemia and correlate with active cutaneous vasculitis.

Chemokine CXCL13, also known as BCA-1 (B cell-attracting chemokine-1) or BLC (B-lymphocyte chemoattractant), is a major regulator of B-cell trafficking. Hepatitis C virus (HCV) infection may be associated with B-cell dysfunction and lymphoproliferative disorders, including mixed cryoglobulinemia (MC). This study evaluates circulating levels of CXCL13 protein and specific mRNA expression in chronically HCV-infected patients with and without MC. Compared with healthy controls and HCV-infected patients without MC, CXCL13 serum levels were significantly higher in MC patients. The highest CXCL13 levels strongly correlated with active cutaneous vasculitis. CXCL13 gene expression in portal tracts, isolated from liver biopsy tissues with laser capture microdissection, showed enhanced levels of specific mRNA in MC patients with active cutaneous vasculitis. Specific CXCL13 gene mRNA expression was also up-regulated in skin tissue of these patients. These findings paralleled specific deposits of CXCL13 protein both in the liver and in the skin. Our results indicate that up-regulation of CXCL13 gene expression is a distinctive feature of HCV-infected patients. Higher levels of this chemokine in the liver as well as in the skin of patients with active MC vasculitis suggest a possible interrelation between these biologic compartments.

B cells and HCV: an infection model of autoimmunity.

In addition to cause acute and chronic liver disease, hepatitis C virus (HCV) infection is frequently associated to autoimmune disorders, such as mixed cryoglobulinemia, primary glomerulonephritis, monoclonal gammopathy of undetermined significance and post-transplant proliferative disorders. Progression to malignant phenotype of B cells may be the consequence of additional genetic events or abnormal conditions resulting from modification of host cell genes involved in the control of oncogenes and oncoproteins. In this review, we will address the potential immune disregulatory mechanism(s) underlying HCV persistence. In addition, HCV/B-cell interaction that might explain defects in humoral immunity in individuals who develop chronic virus carriage and lymphoproliferative disorders will be emphasized.

B-cell depletion in the treatment of mixed cryoglobulinemia.

A controlled study has been carried out to assess the efficacy of rituximab (RTX), a chimeric antibody that binds to the B-cell surface antigen CD20, in twenty patients with mixed cryoglobulinemia (MC) and HCV-positive chronic active liver disease, resistant to interferon-alpha (IFN-alpha) therapy. They received an intravenous infusion of 375 mg/m(2) RTX once a week for 4 consecutive weeks. Infusion of RTX had a good safety profile, and no severe side-effects were reported. Sixteen patients (80%) had a complete response (CR), characterized by rapid improvement of clinical signs (disappearance of purpura, weakness, arthralgias and improvement of peripheral neuropathy), and decreased cryocrit. CR was associated with a significant reduction in rheumatoid factor (RF) activity and anti-HCV antibody titers. Decline of IgG anti-HCV titers in the cryoprecipitates was usually associated with a favorable response (r= 0.81; p <0.005). No differences in the dynamics of B-cell depletion and recovery were found between responders and non-responders. Molecular monitoring of the B-cell response revealed disappearance/deletion of peripheral clones in the responders and great stability in the non-responders. RTX had a deep impact on hepatitis C viremia: HCV RNA increased to approximately twice the baseline level in the responders, whereas it remained much the same in the non-responders. Twelve out of 16 responders (75%) remained in remission throughout the follow-up. The results indicate that RTX has clinical and biological activity in HCV-positive MC patients. However, in view of the increased viremia in the responders, additional modes of application and combination of RTX with other agents need to be investigated.

HCV-associated B cell clonalities in the liver do not carry the t(14;18) chromosomal translocation.

Infection with HCV can be associated with B-cell non-Hodgkin lymphoma. Polymerase chain reaction (PCR) amplification assays for Bcl-2/IgH rearrangement were performed on nucleic acids extracted from portal tract inflammatory infiltrates, isolated with laser capture microdissection (LCM), from liver biopsy sections of 16 hepatitis C virus (HCV)-infected patients with and without extrahepatic B cell-related disorders. Results were compared with total DNA extracted from core liver biopsy specimens and from peripheral blood mononuclear cells (PBMCs). We failed to demonstrate specific Bcl-2/IgH amplicons either in liver tissue or in PBMCs in all patients of the current series. Multiple PCR assays for variable diversity joining (VDJ) IgH gene rearrangements were also performed in the liver compartment. Selective amplification compatible with mono or oligoclonal B cell clonotypes was demonstrated in 80% (6/8) and 25% (2/8) of patients with and without clinical evidence of B-cell disorders. V(H)1 and V(H)3 were the most represented V(H) families. In situ expression of Bcl-2 protein was carried out by immunohistochemistry on liver biopsy sections. Bcl-2 protein was detected in 2 (12.5%) patients who did not associate extrahepatic disorders. In conclusion, current data support the concept that production of IgH gene rearrangements is not associated with Bcl-2/IgH chromosomal translocation in hepatic compartment. Liver overexpression of Bcl-2 protein may occur in at least a minor proportion of HCV-infected patients.

Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20.

A controlled study has been carried out to assess the efficacy of rituximab, a chimeric antibody that binds to the B-cell surface antigen CD20, in 20 patients with mixed cryoglobulinemia (MC) and hepatitis C virus (HCV)-positive chronic active liver disease, resistant to interferon alpha (IFN-alpha) therapy. They received an intravenous infusion of 375 mg/m(2) rituximab once a week for 4 consecutive weeks. Infusion of rituximab had a good safety profile and no severe side effects were reported. Sixteen patients (80%) showed a complete response (CR), characterized by rapid improvement of clinical signs (disappearance of purpura and weakness arthralgia and improvement of peripheral neuropathy), and decline of cryocrit. CR was associated with a significant reduction of rheumatoid factor (RF) activity and anti-HCV antibody titers. Decline of IgG anti-HCV titers in the cryoprecipitates was usually associated with a favorable response (r = 0.81; P <.005). No differences in the dynamics of B-cell depletion and recovery were found between responders and nonresponders. Molecular monitoring of the B-cell response revealed disappearance/deletion of peripheral clones in the responders and great stability in the nonresponders. Rituximab had a deep impact on hepatitis C viremia; HCV RNA increased approximately twice the baseline levels in the responders, whereas it remained much the same in the nonresponders. Twelve (75%) of 16 responders remained in remission throughout the follow-up. The results indicate that rituximab has clinical and biologic activity in patients with HCV(+) MC. However, in view of the increased viremia in the responders, additional modes of application and combination of rituximab with other agents need to be investigated.

Intrahepatic B cell clonal expansions and extrahepatic manifestations of chronic HCV infection.

B cell repertoire in three biological compartments (liver, bone marrow and peripheral blood) of 30 unselected patients chronically infected with HCV has been characterized. Restriction of humoral immune response defined by enrichment of B cell clonal expansions occurred in the liver of 15 patients (50%), in the bone marrow and peripheral blood of 2 (6.7%) and 8 (26.7%) patients, respectively. An in situ hybridization technique was developed for the detection of dominant B cell clones in patients with monoclonal expansions. It was shown that morphologically distinct B cell expansion contributes to the formation of intraportal follicle-like structures. Sequence analyses of CDRH3 gene segments revealed a wide range of variations. Clones derived from the same founder were demonstrated simultaneously in the three compartments explored. The occurrence of B cell clonal expansions profoundly influenced the clinical expression of HCV infection, since it was associated with extrahepatic manifestations. In sharp contrast, no extrahepatic signs or disease occurred in patients without evidence of intrahepatic B cell clonalities. These findings emphasize the profound B cell function derangement in at least half of HCV-infected patients. Thus, the restriction of V gene usage has a direct impact on the clinical spectrum of HCV infection.