María P. Civeira

Pathological and virological findings in patients with persistent hypertransaminasaemia of unknown aetiology

BACKGROUND The histopathological spectrum and role of hepatitis viruses in cases of hypertransaminasaemia of unknown aetiology have not been correctly analysed in a sufficiently large number of patients. METHODS We studied 1075 consecutive patients referred for liver biopsy because of elevation of alanine aminotransferase (ALT) levels for more than six months. From this population we selected those cases in whom the aetiology could not be defined from clinical, biochemical, and serological data obtained before biopsy. In these patients liver biopsies were reviewed, and hepatitis B virus (HBV)-DNA and hepatitis C virus (HCV)-RNA were assayed in serum by polymerase chain reaction (PCR). Serum hepatitis G virus (HGV)-RNA was determined by PCR in 74 patients. RESULTS Of 1075 patients studied, the cause of the increased serum ALT levels remained elusive after appropriate testing in 109 patients (10.1%). Liver biopsies from these patients showed non-specific changes in 32.7% of cases, non-alcoholic steatohepatitis (NASH) in 15.8%, and chronic hepatitis or cirrhosis in 51.5%. HBV-DNA and/or HCV-RNA was detected more frequently in cryptogenic liver disease than in healthy blood donors (26.7% v 3.4%; p<0.001). HGV-RNA was found in only one patient. The proportion of cases with detectable HBV-DNA or HCV-RNA was 14.3% in patients with non-specific changes or NASH, 30.7% in patients with chronic hepatitis, and 61.5% in patients with cirrhosis. Cirrhosis was found more frequently in patients with positive HBV-DNA and/or HCV-RNA in serum than in those who tested negatively (p=0.005). CONCLUSIONS In our series, patients in whom biochemical and serological data did not determine the aetiology of the disease represented 10% of all cases referred for liver biopsy for persistent elevation of serum transaminases. Approximately 50% of patients had chronic hepatitis or cirrhosis and the remainder had NASH or non-specific changes. Occult viral infections were found in a high proportion of cases in the first group and in a low percentage of patients in the second.

Replication of hepatitis C virus in peripheral blood mononuclear cells. Effect of alpha-interferon therapy.

Hepatitis C virus (HCV) is a positive-stranded RNA virus which replicates through a negative-stranded RNA intermediate. Using a PCR procedure to detect positive and negative strands, we investigated the existence of HCV replication in lymphoid cells. Both positive and negative strands were found in the peripheral blood mononuclear cells (PBMC) of all patients (n = 10) with untreated chronic hepatitis C. No HCV sequences were detected in PBMC in any of the 8 healthy controls. Fifteen patients with chronic hepatitis C were studied at the end of a 12-month course of alpha-interferon therapy. The positive strand was detected in PBMC in all 9 non-responder patients, and the negative strand in 7. In contrast, in PBMC from responder patients (n = 6) the positive strand was found in 4 and the negative strand in only 2 cases. These results demonstrate that HCV can infect PBMC and replicate in these cells and that interferon seems to exert an inhibitory effect on this process. Persistence of HCV-RNA in PBMC may help explain disease relapse after successful interferon therapy.

Upregulation of Indoleamine 2,3-Dioxygenase in Hepatitis C Virus Infection

ABSTRACT Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.

Prediction of the response of chronic hepatitis C to interferon alfa: a statistical analysis of pretreatment variables.

Pretreatment variables that could predict the response of chronic hepatitis C to interferon alfa treatment have not been fully assessed. Eighteen baseline variables were evaluated in a series of 100 consecutive patients treated with a 12 month course of interferon alfa. For the purposes of this study, response was defined as the return to normal of aminotransferase activities before the third month of treatment. Seventy per cent of the patients responded to treatment. Six variables were associated with an increased likelihood of response assessed by univariate analysis. With stepwise multiple regression analysis assessment, however, only three variables remained independently predictive of response: low gamma glutamyltransferase (gamma GT) activities (p < 0.001), absence of obesity (p = 0.005), and absence of cirrhosis (p = 0.01). The response rate in patients with gamma GT activities < 0.66 mu kat/l (n = 55) was 78% and 60% in patients with values > 0.66 mu kat/l (n = 45) (p = 0.048). Response was attained in 75% of non-obese patients (n = 80), compared with only 50% of obese patients (n = 20) (p = 0.03). Finally, 80% of patients without cirrhosis (n = 76) responded, while among those with cirrhosis (n = 24) the response rate was only 37% (p < 0.001). All 23 patients without cirrhosis, <40 years old, and with gamma GT activities <0.66 mu kat/l responded to treatment, while only 28.5% of 14 patients with cirrhosis, >40 years old, and with gamma GT activities >0.66 mu kat/l responded to interferon alfa (p<0.001). Those findings may be useful when evaluating interferon alfa trials and it is suggested that this treatment should be applied early in the course of chronic hepatitis C.

Randomised trial of lymphoblastoid α-interferon in chronic hepatitis C: Effects on inflammation, fibrogenesis and viremia

Seventy-two patients with chronic hepatitis C were included in a randomised trial of lymphoblastoid interferon versus no treatment. Thirty-six patients entered each group. Interferon was given in a step-down schedule for 12 months. Aminotransferase activities became normal during treatment in 30 of 36 (83.3%) treated patients, but in only 1 out of 36 (2.7%) non-treated cases (p < 0.001). However, a reactivation of the disease during the interferon course was observed in 12 patients after a mean of 5.58 +/- 1.55 months of therapy, and a post-treatment relapse was observed in 5 additional cases. The remaining 13 patients (36%) had sustained normalization of the aminotransferase levels for 15.27 +/- 10.34 months (range 3-30) after discontinuation of interferon, thus representing a long-term sustained remission of the disease. Knodells histological activity index decreased in all treatment patients, except for 3 non-responders (91.5%), but in only 9 of 36 non-treated cases (25%) (p < 0.001). Procollagen type III peptide levels normalized in most cases (83%) with a sustained response. A significant decrease in the detection of hepatitis C virus RNA was observed in patients with a sustained response (p < 0.05). Anti-interferon antibodies were only detected in one non-responder. Thus, interferon diminishes inflammatory and fibrogenic activity in the majority of patients with chronic hepatitis C and abolishes viremia in most of the patients with a sustained response.

Altered expression and activation of signal transducers and activators of transcription (STATs) in hepatitis C virus infection: in vivo and in vitro studies

Background: Signal transducers and activators of transcription (STATs) play a critical role in antiviral defence. STAT3 is also important in cell protection against inflammatory damage. STAT proteins are activated by interferons and by hepatoprotective cytokines of the interleukin 6 superfamily, including cardiotrophin 1. Methods: We analysed the status of STATs in hepatitis C virus (HCV) infected livers and the relationship between expression and activation of STATs and HCV replication in Huh7 cells transfected with HCV genomic replicon. Results: STAT3α expression was reduced in HCV infected livers showing an inverse correlation with serum alanine aminotransferase. In patients with HCV infection, nuclear staining for phosphorylated STAT3 was faint in parenchymal cells (although conspicuous in infiltrating leucocytes), in contrast with strong nuclear staining in hepatocytes from control livers. Expression and activation of STAT1 (a factor activated by both interferon (IFN)-α and IFN-γ) were increased in HCV infected livers, particularly in those with high inflammatory activity. Conversely, phosphorylated STAT2 (a factor selectively activated by IFN-α) was undetectable in livers with HCV infection, a finding that was associated with marked downregulation of the two functional subunits of the IFN-α receptor. HCV replication in Huh7 cells caused STAT3α downregulation and blocked STAT3 phosphorylation by either IFN-α or cardiotrophin 1. HCV replication in Huh7 cells also inhibited STAT1 and STAT2 activation by IFN-α while there was no impairment of STAT1 phosphorylation by the proinflammatory cytokine IFN-γ. Conclusions: STAT3 is downregulated in HCV infected livers and in Huh7 cells bearing the full length HCV replicon. HCV replication is associated with impaired Jak-STAT signalling by antiviral and cytoprotective cytokines. These effects may favour viral replication while facilitating the progression of liver disease.

Ribavirin in the treatment of chronic hepatitis C unresponsive to alfa interferon

For the 30-50% of patients with chronic hepatitis C who do not respond to alpha-interferon therapy there is no alternative treatment. Some previously untreated patients have shown a biochemical response to ribavirin, but the antiviral effects of this substance on alpha-interferon-resistant cases is largely unknown. Twelve patients with chronic hepatitis C who had not responded to a 6-12 month course of alpha-interferon were included in this study. Oral ribavirin was administered at a dose of 16 mg/kg per day for 6 or 9 months. Aminotransferase levels had not significantly changed during interferon therapy but decreased significantly during ribavirin treatment (mean alanine aminotransferase at baseline, 102 +/- 18 IU/l vs. 55 +/- 14 IU/l at 6 months; P = 0.0001). Aminotransferase levels became normal in 6 cases (50%), significantly decreased in 3 patients (25%), and did not significantly change in the remaining 3 cases (25%). All patients with normalized aminotransferase values relapsed after ribavirin was discontinued and aminotransferase activity returned to pretreatment levels. Before therapy serum hepatitis C virus RNA was detected by polymerase chain reaction in 10 cases. None of them had cleared viral RNA when tested following 3, 6 and 9 months of ribavirin therapy. Side-effects were mild and reversible. In conclusion, about half of the patients with chronic hepatitis C who are unresponsive to alpha-interferon show a clear-cut biochemical response after 6-9 months of ribavirin administration. However, ribavirin does not clear circulating hepatitis C virus RNA and relapses occur after withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)

Prediction of sustained remission of chronic hepatitis C after a 12-month course of alfa interferon

alpha-Interferon therapy normalizes aminotransferase levels in approximately 50% of the patients with chronic hepatitis C, but post-therapy relapses are common and predictive factors of sustained response remain largely unknown. We retrospectively assessed several parameters as predictors of sustained remission after a 12-month course of lymphoblastoid alpha-interferon: the Knodell histological activity index, serum levels of procollagen type III peptide, serum HCV-RNA, anti-alpha-interferon antibodies, and anti-HCV antibodies (C-100-3), all at month 12. Thirty-seven patients were studied. Fourteen patients were non-responders (38%), 15 patients experienced a sustained response (40.5%) and eight patients responded similarly but relapsed after alpha-interferon withdrawal (21.5%). A decrease in the histological activity index above 5, normalization of procollagen type III peptide levels (< 12 ng/ml) and the absence of viremia after treatment were all significantly associated with a sustained response (p = 0.008, p = 0.007 and p = 0.037, respectively). Anti-interferon antibodies were detected in only one non-responder patient. Anti-C-100-3 antibodies became undetectable at month 12 in 5 of the 15 sustained responders. The best prediction of sustained response was obtained from the three variables independent of multivariate analysis according to the following equation: F = 0.872 + 0.067 x K (decrease of histological index) -0.052 x P (procollagen type III peptide levels at month 12) -0.28 x R (HCV-RNA at month 12; R = 2 when present and R = 1 when absent). A score higher than 0 predicted sustained remission with a 100% sensitivity and specificity in this series of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenicity of variable regions of hepatitis C virus proteins: selection and modification of peptide epitopes to assess hepatitis C virus genotypes by ELISA

The immunogenicity of variable regions of hepatitis C virus (HCV) proteins was studied by ELISA by using 543 synthetic peptides from 120 variable regions and 90 sera from HCV-infected patients. Some regions from certain genotypes were less immunogenic, or even non-immunogenic, compared with their equivalents in other genotypes. However, the mean recognition of all peptides from genotypes 1a, 1b and 3 by sera infected with genotypes 1a, 1b and 3, respectively, showed no significant differences, suggesting a similar overall immunogenicity of variable regions from these genotypes. Proteins NS4a, NS4b and NS5a were found to be the most immunogenic. Recognition of individual peptides by the sera of infected patients showed that the humoral response against HCV is patient-dependent. The work shows that 15-mer peptides may encompass several B-cell epitopes. These epitopes may lie in slightly different positions in different genotypes. Thirty-one percent of the 543 peptides were recognized by some of the 35 healthy donors. This may be a reflection of the large number of antigens to which they had been exposed, but it may also reflect a strategy of HCV to respond to immune pressure. After selection and modification, a set of 40 peptides was used to assess genotypes 1a, 1b, 1, 2 and 3 in the sera of HCV-infected patients, with sensitivities of 34.1, 48.5, 68.8, 58.3 and 48.9% and specificities of 100, 99.1, 97.1, 99.5 and 99%, respectively. The overall sensitivity and specificity for the assessment of genotypes 1, 2 and 3 were 64 and 98%, respectively.

Dysregulation of interferon regulatory factors impairs the expression of immunostimulatory molecules in hepatitis C virus genotype 1-infected hepatocytes

Background IL-7 and IL-15 are produced by hepatocytes and are critical for the expansion and function of CD8 T cells. IL-15 needs to be presented by IL-15Rα for efficient stimulation of CD8 T cells. Methods We analysed the hepatic levels of IL-7, IL-15, IL-15Rα and interferon regulatory factors (IRF) in patients with chronic hepatitis C (CHC) (78% genotype 1) and the role of IRF1 and IRF2 on IL-7 and IL-15Rα expression in Huh7 cells with or without hepatitis C virus (HCV) replicon. Results Hepatic expression of both IL-7 and IL-15Rα, but not of IL-15, was reduced in CHC. These patients exhibited decreased hepatic IRF2 messenger RNA levels and diminished IRF2 staining in hepatocyte nuclei. We found that IRF2 controls basal expression of both IL-7 and IL-15Rα in Huh7 cells. IRF2, but not IRF1, is downregulated in cells with HCV genotype 1b replicon and this was accompanied by decreased expression of IL-7 and IL-15Rα, a defect reversed by overexpressing IRF2. Treating Huh7 cells with IFNα plus oncostatin M increased IL-7 and IL-15Rα mRNA more intensely than either cytokine alone. This effect was mediated by strong upregulation of IRF1 triggered by the combined treatment. Induction of IRF1, IL-7 and IL-15Rα by IFNα plus oncostatin M was dampened in replicon cells but the combination was more effective than either cytokine alone. Conclusions HCV genotype 1 infection downregulates IRF2 in hepatocytes attenuating hepatocellular expression of IL-7 and IL-15Rα. Our data reveal a new mechanism by which HCV abrogates specific T-cell responses and point to a novel therapeutic approach to stimulate anti-HCV immunity.