Fabio Pavone

Therapy of chronic hepatitis C virus infection in the era of direct-acting and host-targeting antiviral agents

OBJECTIVES Chronic hepatitis C virus (HCV) infection represents a leading worldwide medical and social problem. The expanding knowledge of HCV lifecycle has led to the development of novel antiviral agents that: a) specifically target a viral function (direct-acting antivirals), or b) specifically inhibit viral replication. The present review describes the novel anti-HCV drugs that have been better studied at the time of this writing and the current two types of treatment, namely interferon-based and interferon-free regimens. In addition, predictive factors, virological responses, side-effects, and resistance mechanisms of the novel agents are summarized. CONCLUSIONS The introduction of novel antiviral agents is remarkably changing the therapeutic combinations aimed at improving virological responses both for easy-to-cure and difficult-to-treat patients. Since additional, effective drugs are under advanced development, it seems reasonable to expect that further therapeutic and prognostic improvements will be achieved in the near future.

Interleukin 28B gene polymorphisms in hepatitis C virus-related cryoglobulinemic vasculitis.

Objective. Single-nucleotide polymorphisms (SNP) in the interleukin 28B (IL-28B) gene region are strongly predictive of the response of infected patients to antiviral therapy for hepatitis C virus (HCV). We sought to determine the prevalence of SNP IL-28B rs12979860 C/C and non-C/C (C/T plus T/T) genotypes in HCV-related cryoglobulinemic vasculitis (CV), as compared with HCV-positive patients without CV. We also searched for their association with peculiar clinical manifestations of CV and potential influence on the complete response (virological, molecular, and immunological) to the therapy. Methods. The study cohort comprised 159 and 172 HCV-infected patients with and without CV, respectively, prospectively followed starting from 1990. SNP rs12979860 genotyping was performed by Taq-Man allelic discrimination. In 106 patients (66.6%) with CV, the profile of circulating B cell clonalities was determined as well. All patients with CV were treated with pegylated interferon-α/ribavirin-based antiviral therapy. Results. The T/T IL-28B genotype was more common in patients with CV than in those without (17% vs 8.1%, p = 0.02). In patients with CV, compared with non-C/C variants, the IL-28B C/C genotype was associated with a higher rate of complete response (52.6% vs 39.2%, p = 0.13), whereas a treatment response of 61.4% was demonstrated when solely virological response was considered (p = 0.008). A higher frequency of expanded B cell clonalities in the circulation (84.2% vs 55.9%; p = 0.005), kidney involvement (21% vs 2.9%; p = 0.003), and B cell non-Hodgkin lymphoma (17.5% vs 6.8%; p = 0.048), were also observed. Conclusion. In HCV-positive patients with CV, the IL-28B C/C genotype is distinguished biologically by a higher frequency of restriction of B cell response and clinically by a higher risk of cryoglobulinemic nephropathy and B cell malignancies, while acting as an independent predictor of a sustained virological response to antiviral therapy. In addition, we found that IL-28B T/T variant was more prevalent in patients with CV than in those without.

HCV-related liver and lymphoproliferative diseases: association with polymorphisms of IL28B and TLR2

To explore the relationship between innate immunity and hepatitis C Virus (HCV) in determining the risk of cirrhosis (CIR), hepatocellular carcinoma (HCC), mixed cryoglobulinemia syndrome (MCS) and non-Hodgkin lymphoma (NHL), we investigated the impact of the toll-like receptor-2 (TLR2) and interleukin-28B (IL28B) genetic variants. TLR2 −174 del variant was associated with TLR2 expression and with specific downstream molecules that drive the expression of different interleukins; rs12979860 Il28B was important in response to interferon-treatment and in spontaneous clearance of HCV. The risk for liver and lymphoproliferative diseases in HCV progression was clarified by stratifying 862 HCV-positive patients into groups based on liver (CIR, HCC) and lymphoproliferative HCV-related diseases (MCS, NHL) and compared with chronic HCV (CHC) infection. Analysis of TLR2-IL28B haplotypes showed an association of wild type haplotype with the lymphoproliferative diseases (OR 1.77, p = 0.029) and a slight increase in HCV viral load (HR 1.38, p = 0.054). Wild type haplotype (TLR2 ins/ins- IL28B C/C) was also found associated with older age in patients with an hepatic diseases (in CIR and in HCC p = 0.038 and p = 0.020, respectively) supporting an effect of innate immunity in the liver disease progression. TLR2 and IL28B polymorphisms in combination showed a role in the control of HCV viral load and different HCV disease progression.

Autoimmune Hepatitis: Factors Involved in Initiation and Methods of Diagnosis and Treatment

Autoimmune hepatitis is an acute or mostly chronic liver disease that can affect both adults and children and has a clear prevalence for the female sex. A definite etiology has not been established, but it is known that genetic predisposing profiles and exogenous trigger factors are involved. The main diagnostic criteria include typical histological features, the occurrence of serum auto-antibodies, and increased levels of transaminases and gamma-globulins. Instances of autoimmune hepatitis sharing features with other autoimmune liver diseases have also been observed. An imbalance of the immune system with persistent activation of effector T cells has been emphasized to account for the sustained liver injury. Clinical manifestations are variable both at presentation and throughout the course of the disease, ranging from an asymptomatic state or the occurrence of non-specific symptoms to the features of end-stage liver disease such as jaundice, ascites, and gastrointestinal bleeding. A clinical and biochemical remission is achieved in at least 80% of patients receiving corticosteroids with or without the addition of azathioprine. Alternative therapeutic schedules have been proposed for unresponsive and intolerant patients. Given that relapse often occurs after therapy withdrawal, maintenance treatment is usually required.

Thymic stromal lymphopoietin in hepatitis C virus-related cryoglobulinemic vasculitis: gene expression level and protein distribution

IntroductionHepatitis C virus (HCV) infection can be detected in virtually all patients with cryoglobulinemic vasculitis (CV). Among its many effects, the virus is able to stimulate the production of thymic stromal lymphopoietin (TSLP) by infected hepatocytes. In this study, we assessed the systemic levels and tissue distribution of TSLP in 60 chronically HCV-infected patients, 36 with and 24 without CV.MethodsSerum TSLP levels were measured by an enzyme-linked immunosorbent assay (ELISA) method. TSLP mRNA was assessed in patient samples by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). TSLP protein in liver and skin biopsy samples was revealed by indirect immunofluorescence. All other methods were carried out according to standardized procedures.ResultsSerum TSLP levels were significantly higher in patients with than in those without CV and in healthy individuals. Higher TSLP levels paralleled specific mRNA expression and the up-regulation of TSLP protein in liver tissue. Compared with non-CV patients, higher TSLP levels in CV were accompanied by a higher frequency of circulating mono/oligoclonal B-cell expansions (8% vs. 92%, p < 0.0001) and a higher number of peripheral CD20+ B-cells (10.3% vs. 15.5% p = 0.04). In addition, TSLP mRNA expression in the liver of CV patients was lower than in their correspondent skin tissue and paralleled specific immune deposits of TSLP protein in keratinocytes.ConclusionOverall, this study shows that TSLP secreted by hepatocytes and keratinocytes of HCV-infected patients with CV is involved in the pathogenesis of vasculitis and may possibly support the therapeutic use of TSLP-targeted monoclonal antibodies.

Activation‐induced cytidine deaminase in b cells of HCV‐related cryoglobulinemic vasculitis

Immunoglobulin variable region heavy chain (IgVH) somatic gene diversification is instrumental in the transformation process that characterizes hepatitis C virus (HCV)‐related B cell lymphoproliferative disorders. However, the extent to which activation‐induced cytidine deaminase (AID), an enzyme essential for IgV gene somatic hypermutation (SHM), is active in cryoglobulinaemic vasculitis (CV) remains unclear. AID mRNA expression in the peripheral blood of 102 chronically hepatitis C virus (HCV)‐infected patients (58 with and 44 without CV) and 26 healthy subjects was investigated using real‐time reverse transcription–polymerase chain reaction (RT–PCR). The features of activation‐induced cytidine deaminase (AID) protein and mRNA transcripts were explored in liver tissue biopsies and portal tracts isolated using laser capture microdissection. In chronically HCV‐infected patients, AID mRNA expression was almost threefold higher in those with than in those without CV and sevenfold higher than in healthy subjects (median‐fold: 6·68 versus 2·54, P = 0·03 and versus 0·95, P = 0·0003). AID transcript levels were significantly higher in polyclonal than in clonally restricted B cell preparations in either CV or non‐CV patients (median‐fold, 15·0 versus 2·70, P = 0·009 and 3·46 versus 1·58, P = 0·02, respectively). AID gene expression was found to be related negatively to age and virological parameters. AID protein was found in portal tracts containing inflammatory cells that, in several instances, expressed AID mRNA transcripts. Our data indicate that the aberrant expression of AID may reflect continuous B cell activation and sustained survival signals in HCV‐related CV patients.

Activation-induced cytidine deaminase in B cells of hepatits C virus-related cryoglobulinaemic vasculitis

Immunoglobulin variable region heavy chain (IgVH) somatic gene diversification is instrumental in the transformation process that characterizes hepatitis C virus (HCV)‐related B cell lymphoproliferative disorders. However, the extent to which activation‐induced cytidine deaminase (AID), an enzyme essential for IgV gene somatic hypermutation (SHM), is active in cryoglobulinaemic vasculitis (CV) remains unclear. AID mRNA expression in the peripheral blood of 102 chronically hepatitis C virus (HCV)‐infected patients (58 with and 44 without CV) and 26 healthy subjects was investigated using real‐time reverse transcription–polymerase chain reaction (RT–PCR). The features of activation‐induced cytidine deaminase (AID) protein and mRNA transcripts were explored in liver tissue biopsies and portal tracts isolated using laser capture microdissection. In chronically HCV‐infected patients, AID mRNA expression was almost threefold higher in those with than in those without CV and sevenfold higher than in healthy subjects (median‐fold: 6·68 versus 2·54, P = 0·03 and versus 0·95, P = 0·0003). AID transcript levels were significantly higher in polyclonal than in clonally restricted B cell preparations in either CV or non‐CV patients (median‐fold, 15·0 versus 2·70, P = 0·009 and 3·46 versus 1·58, P = 0·02, respectively). AID gene expression was found to be related negatively to age and virological parameters. AID protein was found in portal tracts containing inflammatory cells that, in several instances, expressed AID mRNA transcripts. Our data indicate that the aberrant expression of AID may reflect continuous B cell activation and sustained survival signals in HCV‐related CV patients.

HCV RNA genomic sequences and HCV-E2 glycoprotein in sural nerve biopsies from hcv-infected patients with peripheral neuropathy

Peripheral neuropathy (PN), the major neurological complication of chronic HCV infection, is frequently associated with mixed cryoglobulinaemia (MC) and small‐vessel systemic vasculitis. While humoral and cell‐mediated immune mechanisms are suspected to act together in an aberrant immune response that results in peripheral nerve damage, the role of HCV remains largely speculative. The possible demonstration of HCV in peripheral nerve tissue would obviously assume important pathogenic implications.

Features of peripheral CD8+CD57+ lymphocytes in patients with autoimmune hemolytic anemia

Abstract Autoimmune hemolytic anemia (AIHA) is an acquired condition characterized by the presence of autoantibodies recognizing erythrocyte-related antigens. Several components of the immune system are involved in disease pathogenesis. Among them, as for other autoimmune disorders, a role for specific CD8+CD57+ regulatory cells subset could be hypothesized. We evaluated this lymphocyte subset by flow cytometry in 18 AIHA patients randomly selected in a retrospective population of 29 cases. Secondary forms were observed in 65.5% of cases, whereas frequencies of warm, cold, mixed, and atypical forms were similar. Cold agglutinins and cryoglobulins tested positive in 44.8% and 10.3% of cases, respectively. These patients exhibited a higher frequency of peripheral vascular symptoms (odds ratio = 8.2, p = .04) and complement consumption (odds ratio = 7.2, p = .02). Frequency of CD8+CD57+ cells resulted significantly higher in AIHA patients than in control group (17.0 ± 15.8% vs 8.2 ± 5.0%, p = .04). Regardless of therapeutic schedule, patients with partial or no response to therapy (8/18) showed higher frequencies of CD8+CD57+ cells as compared with controls (23.6 ± 21.3% vs 8.9 ± 4.9%, p = .01), whereas 10/18 complete responders (CR) showed lower levels of CD8+CD57+ cells (11.7 ± 6.9%, p = .11). CR and controls showed similar values (p = .24). This study suggests that monitoring this lymphocyte subset before and after treatment administration might have a prognostic value. Moreover, CD8+CD57+ cells may represent a possible therapeutic target to restore the normal balance between lymphocyte populations.