Eliana Persico

Natural history of hepatitis C virus carriers with persistently normal aminotransferase levels

BACKGROUND & AIMS Some patients with serum hepatitis C virus (HCV) have persistently normal aminotransferase (ALT) levels and are affected by cirrhosis. This study prospectively evaluated progression of the disease in a group of anti-HCV-positive patients with persistently normal ALT levels. METHODS Thirty-seven subjects were studied. Each subject underwent liver biopsy at baseline and after 5 years of follow-up. At baseline, serum samples were tested for genotypes and HCV RNA load. ALT levels and serum HCV RNA were tested every other month and every 6 months, respectively. Patients with increased ALT were discharged from the study and treated with IFN. Five years after the end of IFN therapy, a liver biopsy was performed. RESULTS Liver biopsy at baseline showed chronic hepatitis in 34 patients and normal histology in 3 patients, 2 of whom were negative for HCV RNA and 1 positive. HCV genotypes were distributed as follows: 2a, 56%; 1b, 41%; and 1a, 3%. At the end of 7-year follow-up, 73% of the patients still had normal ALT values. Liver histology after 5 years was comparable to that observed at entry to study. CONCLUSIONS Most patients with persistently normal ALT serum levels have very mild chronic hepatitis. However, healthy anti-HCV-positive subjects exist. In patients with HCV-related chronic hepatitis associated with persistently normal ALT levels, the grade of disease activity does not increase over years and progression to cirrhosis is slow or absent.

Suppressor of cytokine signaling 3 (SOCS3) expression and hepatitis C virus-related chronic hepatitis: Insulin resistance and response to antiviral therapy.

The response to antiviral therapy is lower in hepatitis C virus (HCV) patients with genotype 1 than in those with genotype 2. Overexpression of the suppressor of cytokine signaling 3 (SOCS3) gene in liver tissue is associated with a poorer treatment outcome in patients with chronic hepatitis C viral genotype 1. Also, insulin resistance has been implicated in nonresponse to an anti‐HCV treatment. To understand why HCV genotype 1 patients respond differently, we investigated SOCS3 gene expression, metabolic syndrome (MS), and the response to therapy in a cohort of patients with HCV‐related hepatitis. A total of 198 patients (108 with genotype 1 and 90 with genotype 2) treated with pegylated interferon plus ribavirin were consecutively enrolled in the study. We measured SOCS3 expression in Epstein‐Barr virus–transformed lymphoblastoid cell lines derived from peripheral lymphocytes of a subset of 130 patients. MS was more frequent in genotype 1 patients than in genotype 2 patients (P < 0.01). Nonresponders (P < 0.01), MS (P < 0.001), and genotype 1 (P < 0.001) were significantly related to SOCS3 overexpression. However, SOCS3 levels were higher in nonresponders also, regardless of the genotype (P < 0.01). In a univariate analysis, the genotype (P < 0.001), age (P < 0.001), SOCS3 (P < 0.001), and MS (P < 0.001) were significantly related to the response to therapy. However, in a multivariate analysis, SOCS3 was the only independent predictor of the response (odds ratio = 6.7; P < 0.005). Conclusion: We speculate that SOCS3 expression per se may influence the response to antiviral therapy and that the genotype 1b virus might induce its up‐regulation. This may account for the different responses to therapy between genotype 1–infected and genotype 2–infected patients. (HEPATOLOGY 2007.)

Elevated expression and polymorphisms of SOCS3 influence patient response to antiviral therapy in chronic hepatitis C

Background: The response to antiviral therapy of chronic hepatitis C virus (HCV) infection is determined by virological, environmental and genetic factors. Objective: The hypothesis was tested that the expression of specific genes and their haplotype frequencies can differentiate between non-responders (NRs) and sustained virological responders (SVRs) to antiviral treatment. Methods: A methodological approach based on molecular marker discovery and validation was used to study the genes influencing the antiviral treatment in lymphoblastoid cell lines from 74 genotype 1b HCV patients (44 from Southern Italy and 30 from Northern Italy) treated with pegylated interferon (IFN) α and ribavirin. Furthermore, an association study was performed, testing three single nucleotide polymorphisms (SNPs) of suppressor of cytokine signalling 3 (SOCS3) in 162 NR and 184 SVR subjects (SOCS3 −8464 A/C (rs12952093), −4874 A/G (rs4969170) and 1383 A/G, (rs4969168)). Results: SOCS3 basal expression levels were significantly increased in two independent sets of NR groups (p<0.05). A highly significant association was found between NRs and both the positively associated haplotype (OR = 2.01, 95% CI 1.45 to 2.79, p = 0.0002) and the negatively associated haplotype (OR = 0.56, 95% CI 0.42 to 0.76, p = 0.0014). In particular, the SOCS3 −4874 AA genotype was strongly associated with failure of antiviral therapy (OR = 4.00, 95% CI 2.09 to 7.66, p = 0.0003) and the AA genotype carriers had significantly higher SOCS3 mRNA and protein levels (p<0.05). Conclusions: Basal levels of SOCS3, an inhibitor of the IFNα-induced Janus kinase–signal transducer and activator of transcription pathways, and its genetic polymorphisms influence the outcome of antiviral treatment. SOCS3 thus represents a novel blood biomarker for the a priori prediction of treatment response.

Hepatitis C virus carriers with persistently normal ALT levels: biological peculiarities and update of the natural history of liver disease at 10 years.

Summary.  Some chronic hepatitis C (CHC) patients exhibit persistently normal alanine aminotransferase (ALT) levels (PNAL). Patients with PNAL experience significantly milder disease. In order to understand the differences between CHC patients with elevated ALT levels compared with those with PNAL better, we compared epidemiological, immunological and histological findings, in particular, the value of proliferating hepatocyte activity (PCNA) between the two groups of patients. We studied 40 chronic hepatitis C virus (HCV) carriers with increased ALT who underwent liver biopsy for histological diagnosis and determination of clinical prognosis, and 24 PNAL patients under follow‐up for 10 years. Immunological response to different HCV genomic epitopes was tested in both the control group and in PNAL subjects. PCNA values from liver specimens of all patients as well as liver biopsies of PNAL patients at time points 0 and 5 years were calculated according to Hall et al.Age, sex and body mass index (BMI) were not significantly different between the two groups. The median liver histology stage was significantly higher in HCV carriers vs the PNAL group (2.5, range = 2–6 vs 1.5, range = 1–2; P < 0.01). Among PNAL patients, histological stage was not statistically different at the three time points considered. Interferon (IFN)‐gamma production was comparable in the two groups. PCNA was significantly higher in the group with elevated ALT levels vs the PNAL group (8%, range = 4–15%vs 5% range = 3–8%; P < 0.05) and no statistically significant differences were found in PNAL patients at time points 0, 5 and 10 years. This study confirms that progression to cirrhosis is slow or absent in PNAL patients after 10 years of follow‐up. Accordingly, the hepatic proliferative activity index is low and seems to be stable over time.

Prevalence and incidence of cryoglobulins in hepatitis C virus-related chronic hepatitis patients: a prospective study.

OBJECTIVES:A high prevalence of cryoglobulins has been reported in patients with hepatitis C virus (HCV)-related liver disease. The aim of this study was to evaluate the prevalence and the incidence of cryoglobulins and their association with clinical symptoms in chronic hepatitis and cirrhosis patients.METHODS:The prevalence of cryoglobulins and cryoglobulinemic syndrome was investigated at enrollment in 237 patients (213 with chronic hepatitis and 24 with cirrhosis). A 7-yr follow-up was conducted evaluating the occurrence of cryoglobulins and/or cryoglobulinemic syndrome every 6 months. Rheumatoid factor was also tested in all patients.RESULTS:Prevalence of rheumatoid factor, cryoglobulins, and cryoglobulinemic syndrome in chronic hepatitis patients were 2%, 0.8%, and 0%, respectively. In cirrhosis patients the prevalence was 4%, 8%, and 0%, respectively. No statistically significant differences were found between the two groups. During the follow-up only one patient for each group abruptly developed cryoglobulinemic syndrome, and none of the patients who showed signs of cryoglobulinemia developed the syndrome or showed signs of evolution of the disease.CONCLUSIONS:Our data demonstrate that the presence of cryoglobulins and/or cryoglobulinemic syndrome in HCV-related liver disease is unusual, as is the occurrence of cryoglobulinemia over time in these patients. This leads us to think that HCV-related cryoglobulinemic syndrome and HCV-related liver disease are independent diseases. This supports new and indirect evidence for an independent and direct role of HCV in liver and blood disorders.

SOCS3 and IRS-1 gene expression differs between genotype 1 and genotype 2 hepatitis C virus-infected HepG2 cells

Abstract Background: The poor response to antiviral treatment of hepatitis C virus (HCV)-infected patients with genotype 1b has been associated with a higher prevalence of metabolic syndrome. However, the molecular link between these clinical entities is not clear. The goal of this study was to clarify the role of genotype 1b and 2 in the genetic expression of suppressor of cytokine signaling 3 (SOCS3) and insulin receptor substrate 1 (IRS-1). Methods: We infected human hepatocellular carcinoma cell line (HepG2) cells with human HCV genotype 1b or 2 and measured the gene and protein expression of SOCS3 at various times. We also evaluated impairment in the insulin pathway by analysis of IRS-1 and phospho-AKT. For the control, we used HepG2 cell cultures treated with non-infectious serum. We also demonstrated the occurrence of HCV infection by the detection of both positive and negative strands in the cells and culture medium. To test infection of the HepG2 cells, we performed quantitative real-time polymerase chain reaction (qRT-PCR) of viral load at different time points. We analyzed the viral genotype in the pellet and supernatant. Results: At each time point, we found positive and negative strands in the infected cells, while in the medium we found positive, but no negative strands. We also detected the presence of the correct genotype in the medium. Two weeks following infection when the viral load was higher, we tested genotype 1b and 2 infected cells. SOCS3 gene expression was significantly higher in genotype 1b-infected cells (median 2.56; mean 2.82±0.59) compared with genotype 2 (median 1.34; mean 1.46±0.31) (p=0.04) and control cells (median 1.09; mean 1.02±0.11, p=0.02). There was no difference between cells exposed to genotype 2 and control cells. Conversely, IRS-1 was significantly lower in genotype 1b-infected cells (median 15.97; mean 15.45±0.67) compared with genotype 2-infected cells (median 16.45; mean 16.44±0.01, p=0.04). Statistically significant differences were seen when comparing the pAKT/AKT ratio in genotype 1b-infected cells (0.19±0.034) and not genotype 1b-infected (genotype 2-infected and non-infected) cells (0.253±0.004, p=0.03). This inverse regulation is compatible with interactions between the molecular expression of SOCS3, IRS-1 and phospho-AKT mediated by the genotype 1b virus. Conclusions: Up-regulation of the SOCS3 gene might be one of the mechanisms governing non-response to therapy and expression of insulin resistance mediated via a direct mechanism at this level of genotype 1b HCV. Clin Chem Lab Med 2009;47:1217–25.

Occult hepatitis B virus infection in patients with non‐Hodgkin lymphoma: the need for early diagnosis in anti‐Hbc positive patients

Occult hepatitis B virus (HBV) infection can be defined as the long lasting persistence of viral genome in the liver tissue of patients without HBV surface antigen (HBsAG), with or without antibodies to hepatitis B core antigen (anti-HBc) or hepatitis B surface antigen (anti-HBs).1 The clinical relevance of occult B infection is well documented.2–6 Immunosuppressive therapy can promote viral replication and disease progression. Discontinuation of immunosuppressive drugs may lead to the reconstitution of the immune response to the virus and hence to immune mediated destruction of infected hepatocytes. This is a well recognised occurrence in patients with hepatitis B infection or non-Hodgkin lymphoma (NHL).7,8 We studied the prevalence of occult HBV infection in 58 consecutive NHL patients, six of whom were HBsAG positive and 52 were …

HEPATITIS G VIRUS IN PATIENTS WITH HODGKIN'S LYMPHOMA

The responsibility of hepatitis C virus (HCV) in causing mixed cryoglobulinaemia was documented several years ago (Ferri et al, 1991). More recently, a significantly high prevalence of HCV infection has been described in patients with B-cell lymphoproliferative disorders (B-LPD) in Italy as well as in other countries. In these studies the prevalence of HCV in patients with Hodgkin’s disease (HD) was low and did not differ significantly from that of the general population (De Rosa et al, 1997). Hepatitis G virus (HGV) is a new member of the Flaviviridae family sharing large structural and biological similarities with HCV. HGV role in causing chronic viral hepatitis is still open to debate; its replication site has not been clearly located, but lymphotropism has been reported (Thomas et al, 1996). Recently, its potential oncogenic role in non-Hodgkin’s lymphoma (NHL) has been suspected (Keenan et al, 1997). We screened for the presence of HGV-RNA the sera of 71 patients with HD (31 males and 40 females) consecutively recruited at diagnosis, and of 71 ageand sex-matched healthy subjects. All patients and controls were HIV negative and had never been transfused. No HD patient showed signs of chronic hepatitis. Since it has been reported that about 10% of patients infected with HCV are co-infected with HGV, the prevalence of HCV infection was simultaneously tested in the same groups of patients and controls. Sera from patients and controls were rapidly frozen and stored at 1 208C, until required for parallel analysis. HGV-RNA was tested by using a nested RT-PCR with primers amplifying the non-structural regions 3 (NS3) and 5 (NS5) and the 5 non-coding region (Schlueter et al, 1996) in patient and control sera and in patient’s circulating lymphocytes. The test was considered positive when results were concordant. Every PCR was carried out with a positive and a negative control. RT-PCR was repeated for all positive and indeterminate specimens. HCV-RNA was searched for using primers expanding the highly conserved 5 non-coding region of the HCV genome. Carryover PCR contamination was avoided by applying the measures suggested by Kwok & Higuchi (1989). HCV antibodies were detected by third-generation recombinant immunoblot assay (RIBA, Ortho Diagnostic Systems). The prevalence of HGV viraemia in HD patients was significantly higher than in healthy controls (15% v 1·4%; x P<0·007) (Table I). The odds ratio (OR) for HD in subjects with HGV viraemia relative to those without HGV viraemia was 12·8 (95% confidence limits 1·63–81), indicating a 12fold increased association between HD and the presence of HGV viraemia. No differences of staging and grading were found between HGV positive and negative patients. Only one subject in the control group showed positivity for HCV and none had double infection. In HD patients the prevalence of HCV was similar to that observed in the general population, confirming our previous report; only two patients with HD were HCV-RNA positive and one had HGV and HCV coinfection. The rarity of co-infection with HCV/HGV eliminates that the possibility of correlation between HGVand HD is mediated by HCV. In a smaller series, Keenan et al (1997) found HGV-RNA in one out of 17 HD patients. In long-term survivors of haematological malignancies, HGV viraemia has been attributed to immunosuppression caused by the cytotoxic treatments administered; by analogy, one may think that the higher prevalence of HGV viraemia in HD is secondary to the immunodeficiency often associated with this disease. However, since we studied HD patients at diagnosis it is unlikely that a substantial proportion of them were infected in the relatively short period between onset of the disease and diagnosis. HGV-RNA may be detected in lymphocytes of infected patients (Thomas et al, 1996); we did not find viral RNA in the circulating lymphocytes of our HD patients with HGV viraemia. This can be explained either by a low undetectable viral load in lymphocytes, or because in HD the site of viral replication may occur in cells other than circulating lymphocytes. By analogy, reports in the literature suggest that HHV-8 may play a pathogenic role in myeloma by infecting dendritic cells and not plasma cells (Said et al, 1997). A better assessment of the association between HGV and HD may come from larger epidemiological surveys. Biological studies testing the presence of HGV in neoplastic and/or accessory cells in HD patients are also necessary to clarify the role, if any, that HGV plays in the pathogenesis of HD.