Scott J. Cotler

Pretreatment symptoms and dosing regimen predict side-effects of interferon therapy for hepatitis C

We analysed data from a multicentre interferon (IFN) treatment trial to evaluate symptoms in patients with chronic hepatitis C and to identify factors that might predict development of debilitating IFN side‐effects. Two hundred and twenty‐two patients (120 US, 102 French) received 3 or 5 million units (MU) of IFN‐α three times weekly (t.i.w.) for 3 months. Those who had detectable hepatitis C virus (HCV) RNA, as detected by the branched DNA signal amplification (bDNA) assay, at 3 months were intensified to daily therapy, while patients who were bDNA negative continued t.i.w. dosing for the subsequent 3 months of treatment. Symptoms were assessed at baseline, and adverse effects were evaluated at 6 months of therapy. Prior to treatment, the most common symptom that interfered with daily functioning was fatigue, occurring in 25% of patients. The frequency of debilitating fatigue, myalgia, arthralgia, headache, the presence of dry eyes and dry mouth, and use of antidepressant medication increased significantly from baseline to 6 months of IFN therapy (all P < 0.01). In multivariate analysis, the development of a debilitating side‐effect at 6 months of treatment was associated with the presence of that symptom prior to therapy in all cases. Symptoms and adverse effects varied by gender and country. Compared with patients maintained on t.i.w. dosing, those who were dose intensified to daily IFN reported more debilitating fatigue, malaise, myalgia, arthralgia, fever, nausea, and headache, and the presence of dry mouth (all P < 0.05). In conclusion, patient characteristics, including pretreatment symptoms, gender and nationality, as well as daily IFN dosing are associated with the development of debilitating adverse effects on IFN therapy.

In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease

ABSTRACT Liver failure from chronic hepatitis C is the leading indication for liver transplantation in the United States. However, the pathogenesis of liver injury resulting from chronic hepatitis C virus (HCV) infection is not well understood. To examine the relationship between HCV replication in liver tissue and hepatocellular injury, a strand-specific in situ hybridization procedure was developed. The sensitivity and specificity of digoxigenin-labeled riboprobes were optimized by analyzing Northern blots and cell lines expressing HCV RNAs. For the current study, both genomic (sense) and replicative-intermediate (antisense) HCV RNAs were detected and quantified in 8 of 8 liver tissue specimens from infected patients versus 0 of 11 liver tissue specimens from noninfected controls. The distribution pattern for HCV replicative-intermediate RNA in liver was different from that for HCV genomic RNA. HCV genomic RNA was variably distributed throughout infected livers and was located primarily in the cytoplasm of hepatocytes, with some signal in fibroblasts and/or macrophages in the surrounding fibroconnective tissue. However, HCV replicative-intermediate RNA showed a more focal pattern of distribution and was exclusively localized in the cytoplasm of hepatocytes. There was no significant relationship between the distribution pattern for HCV genomic RNA and any indices of hepatocellular injury. However, a highly significant correlation was observed between the percentage of cells staining positive for replicative-intermediate RNA and the degree of hepatic inflammatory activity (P, < 0.0001). Furthermore, the ratio of cells staining positive for HCV replicative-intermediate versus genomic RNA correlated with the histological severity of liver injury (P, 0.0065), supporting the hypothesis that active replication of HCV in liver tissue may be a significant determinant of hepatocellular injury.

Daily interferon therapy for hepatitis C virus infection in liver transplant recipients.

Background. Hepatitis C virus infection persists after liver transplantation and causes recurrent liver injury in the majority of patients. Standard dose interferon therapy has been largely unsuccessful for hepatitis C in transplant recipients. Methods. Twelve patients, at least 7 months posttransplant, with detectable hepatitis C virus RNA in serum and features of hepatitis C on liver biopsy were randomized to interferon-&agr;2a, 3 mU daily for 12 months (n=8) or no treatment (n=4). The tolerability of daily interferon dosing in liver transplant recipients was evaluated and effects on hepatitis C virus RNA level, quasispecies evolution, and liver histology were studied. Results. Treated patients had an improvement in histological activity index at the end of therapy relative to controls (median reduction of 2 versus median increase of 1.5) (P =0.04). Four treated patients had a virological response (all bDNA negative, one qualitative polymerase chain reaction negative) compared with none of the untreated patients. Only two of six treated patients tested had evidence of quasispecies diversification on therapy. Seven of eight patients in the treatment group required dose reduction for fatigue and/or depression. They tolerated 1.5 mU of interferon-&agr;2a daily. Two treated patients developed graft dysfunction, one of who had histological evidence of rejection and subsequent graft loss. Conclusions. Low daily doses of interferon were tolerated by liver transplant recipients and provided histological benefit without associated quasispecies diversification in most cases. These findings provide a rationale to study low dose daily or pegylated interferon maintenance therapy for the management of hepatitis C posttransplant.

Patients’ values for health states associated with hepatitis c and physicians’ estimates of those values

Patients’ values for health states associated with hepatitis c and physicians’ estimates of those values

Characterizing hepatitis B stigma in Chinese immigrants.

Summary.  Health‐related stigma is a cause of stress, alienation and discrimination that can serve as a barrier to prevention and care for infectious diseases such as HIV. Hepatitis B virus (HBV)‐related stigma is common in Asian immigrants, but has not been formally evaluated. The aim of this study was to develop and validate the first HBV stigma instrument and to begin to evaluate HBV stigma in Chinese immigrants. The HBV stigma instrument was developed based on constructs from validated HIV stigma scales and organized into five domains. A written survey was compiled to include demographic data, HBV knowledge questions and stigma items. The survey was pilot tested in English and Chinese and then finalized. Data were obtained from 201 patients seen in an urban Chinatown Internal Medicine practice. The stigma items showed a high degree of reliability when assessed in aggregate (α = 0.85) as well as within individual domains. Stigma was greatest in the Fear of Contagion domain. Knowledge questions showed a corresponding deficit in understanding of modes of HBV transmission. An inverse relationship between stigma scores and familiarity with HBV provided evidence of construct validity. In multivariable analysis, having a family member with HBV and higher HBV knowledge subset scores were associated with lower degrees of stigma. In conclusion, the hepatitis B stigma instrument showed reliability and construct validity. The relationship identified between familiarity and knowledge regarding HBV with lower stigma scores provides the basis for the development of interventions to reduce HBV stigma.

The Protein Kinase—Interacting Domain in the Hepatitis C Virus Envelope Glycoprotein—2 Gene Is Highly Conserved in Genotype 1—Infected Patients Treated with Interferon

The hepatitis C virus (HCV) envelope glycoprotein-2 inhibits the interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) via the PKR eukaryotic initiation factor-2alpha phosphorylation homology domain (PePHD). The present study examined the genetic variability of the PePHD in patients receiving IFN therapy. The PePHD from 12 HCV genotype 1 (HCV-1)-infected patients receiving daily IFN therapy was amplified by reverse-transcriptase polymerase chain reaction and analyzed by direct and clonal sequencing. The PePHD was highly conserved in 38 HCV GenBank isolates. There was no difference in pretreatment PePHD sequences isolated from IFN responders versus nonresponders. The major PePHD quasi-species variant did not change after 6 weeks of daily IFN therapy, and in 1 patient the major quasi-species variant did not change during 9 months of observation. Sequencing of 25 pretreatment PePHD clones from 3 patients confirmed that there was extremely low sequence variability surrounding the PePHD. The PePHD is highly conserved in HCV-1-infected IFN responders and nonresponders and does not appear to evolve in response to IFN therapy.

An analysis of acute changes in interleukin-6 levels after treatment of hepatitis C with consensus interferon

Cytokine production has been implicated in the antiviral response to interferon-alpha (IFN-alpha) in hepatitis C and in the development of IFN-alpha-related side effects. We characterized acute changes in serum cytokine levels following administration of a single dose of consensus IFN (IFN-con1) and during continuous treatment of chronic hepatitis C patients. Serum samples were collected at baseline, at multiple times early after IFN administration, and weekly thereafter. Viral RNA titers were assessed by RT-PCR, and viral kinetics were followed. ELISA assays were used to measure IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, and IL-16. Serum cytokine levels were low at baseline. IL-6 was detected in patients with hepatitis C but not in healthy control subjects by either ELISA or RT-PCR, indicating that low levels of circulating IL-6 were associated with hepatitis C infection. None of the cytokines measured increased significantly after IFN administration except for IL-6. IL-6 levels rose rapidly, peaked at 6-15 h in a dose-dependent manner, and returned to baseline by 48 h in both patients receiving a single dose of IFN and those receiving continuous treatment. This was confirmed by RT-PCR. Pretreatment IL-6 levels were directly correlated with area under the curve (AUC) for IL-6 during the 24 h after IFN dosing (r = 0.611, p = 0.007). Viral titers decreased within 24-48 h after a single dose of IFN-con1. Changes in hepatitis C RNA titers were not significantly associated with pretreatment IL-6 levels or with changes in IL-6 levels. In conclusion, (1) baseline serum cytokine levels, except for IL-6, were low or within the normal range in patients with hepatitis C, (2) IL-6 levels were detected in some patients with hepatitis C before treatment but not in healthy controls, (3) IL-6 levels increased acutely after a single dose of IFN-alpha, and IL-6 induction was related to baseline IL-6 level, and (4) changes in IL-6 levels did not correlate with the early virologic response to IFN.

A pilot study of the combination of cyclosporin A and interferon alfacon-1 for the treatment of hepatitis C in previous nonresponder patients

Goals To evaluate the safety and efficacy of the combination of interferon and cyclosporine for the treatment of hepatitis C in previous nonresponder patients. Background Preliminary data indicated that adding the immunosuppressive agent cyclosporin A to interferon might improve response rates in patients with hepatitis C. Study Ten previous virologic nonresponders with genotype 1 infection were included. Treatment consisted of interferon alfacon-1, 15 &mgr;g/d, and cyclosporine, 100 mg twice daily, for 4 weeks. The dose of interferon alfacon-1 was then decreased to 15 &mgr;g three times weekly, and cyclosporine was reduced to 50 mg twice daily. Therapy was continued for 48 weeks unless viremia persisted at week 24. Results Three of 10 subjects had an on-treatment virologic response, although one had a breakthrough with recurrent viremia during treatment and two relapsed after therapy was completed. On treatment responders had significantly higher trough cyclosporine levels at week 4 compared with nonresponders (P = 0.025). Serum creatinine levels remained stable, and no patient developed diabetes. Triglyceride levels increased during treatment. Cyclosporine was dose reduced in two patients for hypertension. Conclusions Selected patients with hepatitis C tolerated therapy, including cyclosporine without severe or irreversible toxicity. Despite an association between higher cyclosporine levels and on-treatment response, the combination of cyclosporine and interferon was ineffective in producing a sustained response in previous nonresponder patients.

A multicentre study of the usefulness of liver biopsy in hepatitis C.

Summary.  The role of liver biopsy in the assessment of chronic hepatitis C is generally accepted yet there is no prospective data available to quantify its contribution. A previous single centre pilot study suggested that the clinician could predict the amount of fibrosis and to a lesser extent, inflammation with moderate accuracy. The 2002 National Institute of Health Hepatitis C Consensus Conference recommended further study of the role of liver biopsy. Our objective was to compare a prediction of biopsy findings by expert clinicians using usually available clinical and laboratory data to actual biopsy results in order to determine whether biopsy is required routinely. This was a prospective observational study conducted at seven university centres in which the accuracy of clinicians predictions of the degree of inflammation and fibrosis were compared with the actual liver biopsy using an adaptation of a standard histological scoring system. We studied 81 adults with previously untreated chronic hepatitis C, raised serum transaminases and positive HCV‐RNA in serum. Clinicians predicted the inflammatory grade in 44 of 80 cases (55%) and the fibrosis stage in 46 of 81 cases (57%). Nine of 17 cirrhotic cases were predicted (sensitivity 53%, specificity 56%). No unexpected additional diagnoses were made on the biopsies. Thus despite knowledge of the clinical and laboratory investigations of patients with hepatitis C, clinicians are unable to accurately predict the hepatic inflammatory grade and fibrotic stage. Liver biopsy is an essential investigation to accurately evaluate the grade and stage of liver disease patients with hepatitis C.

Neutralizing antibodies in hepatitis c virus infection: a review of immunological and clinical characteristics

Chronic hepatitis C virus (HCV) infection is the most common cause of chronic liver disease in the United States, affecting more than 2.7 million people.1 Most cases of acute HCV result in chronic infection despite the appearance of antibodies to both structural and nonstructural HCV antigens. Hepatitis C replicates rapidly, at an estimated 1012 virions per day,2 and has a high mutation rate of approximately 10 3 nucleotide substitutions per genome site per year.3 The host humoral immune response contributes to the generation of viral diversity. Although HCV infection frequently becomes chronic, neutralizing antibodies to viral epitopes have been identified. Neutralizing antibodies interfere with viral binding to cellular receptors and entry into hepatocytes through antibody coating of the virus or by inducing conformational changes in viral structural proteins. Neutralizing antibodies differ from binding antibodies by their ability to prevent viral infectivity. The lack of a cell-culture system supporting efficient HCV replication and particle assembly has made it difficult to characterize the envelope proteins present on the virion and to elucidate the process of viral binding and entry into hepatocytes.4 Two structural glycoproteins that form the viral envelope, named E1 and E2, are a target for the host immune response and also represent the putative viral attachment proteins. A recent study that used a cell fusion assay to examine the initial steps of HCV infection showed that the presence of both chimeric E1 and E2 proteins were necessary for cell fusion and that low-pH treatment enhanced the fusion activity of the HCV envelope proteins.5 The experimental findings suggested that HCV enters target cells through an endosomal pathway and that a low-pH– dependent conformational change of the E1 and/or E2 proteins occurs in the endosome that leads to membrane fusion and nucleocapsid entry into the cytoplasm. The N-terminus of the E2 protein contains a specific region approximately 27–31 amino acids in length, termed hypervariable region (HVR) 1, that appears to lack structural constraints and allows extensive amino acid variation. This region likely forms part of the E2 protein located outside the HCV envelope, where it is a prime target of neutralizing antibodies.6 Variability in the amino acid sequence of HVR1 among different isolates results from humoral immune pressure leading to the selection of escape mutants.6,7 Studies showing conservation of HVR1 in an agammaglobulinemic patient,8 a reduced rate of HVR1 variability in patients with common variable immunodeficiency compared to patients with normal immune responses,9 and a marked decrease in nucleotide diversity of HVR1 in human immunodeficiency virus–infected patients with CD4 counts 50/ L10 support the hypothesis that the immune response is important in driving generation of viral diversity. The development of mutations in HVR1 may contribute to viral persistence. Other factors that may play a role in persistent infection include the relatively low immune reactivity of HCV proteins, the high specificity of antibodies to HVR1 that do not cross-neutralize emerging quasispecies (multiple isolates in the same individual), and an inadequate cytotoxic T-lymphocyte response to viral epitopes.11–14 This review analyzes the characteristics of naturally occurring and vaccine-induced hepatitis C antibodies, their role in the course of infection, and possible clinical uses of these antibodies.