M.P. Civeira

Tumor necrosis factor α gene expression and the response to interferon in chronic hepatitis C

Tumor necrosis factor α (TNF‐α) is a cytokine with pleiotropic properties that is induced in a variety of pathological situations including viral infections. In this work, we analyzed the expression of TNF‐α gene in patients with chronic hepatitis C. Serum TNF‐α levels were found to be elevated in all chronic hepatitis C patients including those cases presenting sustained biochemical remission of the disease after interferon therapy. Untreated patients with chronic hepatitis C showed increased TNF‐α messenger RNA (mRNA) levels in the liver and mononuclear cells as compared with healthy controls. After completion of treatment with interferon, patients experiencing sustained complete response showed values of TNF‐α mRNA, both in the liver and in peripheral mononuclear cells, within the normal range, significantly lower than patients who did not respond to interferon and than those with complete response who relapsed after interferon withdrawal. Pretreatment values of TNF‐α mRNA were lower in long‐term responders to interferon than in cases who failed to respond to the treatment. Values of TNF‐α mRNA in the liver or in mononuclear cells were higher in specimens with positive hepatitis C virus (HCV) RNA than in those samples where the virus was undetectable. Neither the intensity of the liver damage nor the amount of HCV RNA in serum or in cells showed correlation with the levels of TNF‐α transcripts in peripheral mononuclear cells but it was found that high TNF‐α values were associated with genotype 1b. In conclusion, there is an enhanced expression of TNF‐α in HCV infection. High levels of this cytokine may play a role in the resistance to interferon therapy.

Superoxide Dismutase in Patients With Chronic Hepatitis C Virus Infection

It has been reported that hepatitis C virus (HCV) may cause oxidative stress in infected cells. Patients with chronic hepatitis C exhibit an increased production of tumor necrosis factor-alpha (TNF alpha), a cytokine that can produce oxidative stress by stimulating the generation of reactive oxygen species (ROS). Cell defense against ROS includes overexpression of Mn-superoxide dismutase (SOD), an inducible mitochondrial enzyme. To investigate cell defense against oxidative stress in HCV infection, we analyzed Mn-SOD mRNA in liver and in peripheral blood mononuclear cells (PBMC) from patients with chronic hepatitis C. Mn-SOD expression in PBMC was significantly increased in patients with HCV infection. Patients with sustained virological and biochemical response after therapy showed significantly lower Mn-SOD than patients with positive viremia. By contrast, Mn-SOD expression was not enhanced in the liver of patients with chronic hepatitis C. The values of Mn-SOD mRNA did not correlate with TNF alpha mRNA expression, viral load, or liver disease activity. Our results indicate that in HCV infection an induction of Mn-SOD was present in PBMC but absent in the liver, suggesting that this organ could be less protected against oxidative damage. Oxidative stress could participate in the pathogenesis of HCV infection.

Epidemiological, clinical and therapeutic associations of hepatitis C types in western European patients

BACKGROUND/AIMS Different variants of hepatitis C virus might show different susceptibility to interferon alpha treatment, but it is important to understand whether this difference in sensitivity reflects an association with other factors, such as cirrhosis or age. METHODS We have used an enzyme-linked immunosorbent hepatitis C virus typing assay based upon the detection of antibody in patients era to type-specific NS-4 antigens to investigate the effect of hepatitis C virus type in 610 patients with chronic hepatitis C virus infection. The influence of viral types and their interdependency with host factors were separately analyzed to establish which factors executed an independent effect on the probability of sustained response. RESULTS There was a marked difference in the distribution of hepatitis C virus types with age: infection with type 3 was more common in younger patients. The distribution of hepatitis C virus type with age is accounted for by differences in risk-factors for infection in different age groups. The frequency of cirrhosis increased markedly with age. Even after standardization for age, center and the presence of cirrhosis, viral type was strongly related to the outcome of infection. CONCLUSIONS Our data suggest that enzyme-linked immunosorbent hepatitis C virus typing could assist in patient selection for interferon treatment to improve sustained response rates. Together with measurement of viral load, hepatitis C virus typing may serve to indicate the probability of response in patients with chronic hepatitis C, and to elucidate antiviral mechanisms in the disease. The serotyping assay provides a relatively inexpensive screening method to determine the infecting hepatitis C virus type, which could facilitate therapeutic decisions and strategies in patients with chronic hepatitis C.

Early predictors of response to treatment in patients with chronic hepatitis C.

The term sustained response should be applied to patients with negative serum HCV-RNA and normal values of serum transaminases 6 months after interferon withdrawal. To investigate which factors identify sustained responders early during treatment we analysed 18 reports which used the definition sustained response. Eight reports, comprising 988 patients, have studied the value of early clearance of viraemia as a predictor of sustained response using multivariate analysis and all of them found that this was the strongest predictor of a sustained response. Determination of HCV-RNA 4 or 12 weeks after initiation of IFN therapy predicts treatment outcome more accurately than baseline viral load (the best pre-treatment predictor). ALT levels during the first 12 weeks of treatment have lower predictive value than early viral clearance. The sensitivity of a negative HCV-RNA test is similar at week 4 and at week 12 of therapy while the specificity and the accuracy is higher at week 4. The value of persistent viraemia for early prediction of no response is higher than 97%, with similar values at weeks 4 and 12. Persistence of HCV-RNA in serum at week 4 strongly indicates that the patient will not respond to treatment and in these cases interruption of treatment or other therapeutic options could be considered.

Nuclear factor‐κB in the liver of patients with chronic hepatitis C: Decreased RelA expression is associated with enhanced fibrosis progression

The mechanisms of liver damage in chronic hepatitis C virus (HCV) infection are poorly understood. The transcription factor, nuclear factor‐κB (NF‐κB), regulates the expression of genes involved in apoptosis, inflammation, and antiviral response. It plays a protective role in several forms of liver damage. In this study, we analyzed NF‐κB by gel mobility shift assay and immunohistochemistry in liver biopsies from HCV‐infected patients, and we have determined the hepatic levels of the components of the NF‐κB system by semiquantitative polymerase chain reaction (PCR). We found that NF‐κB was activated in the liver of patients with chronic hepatitis C. Neither NF‐κB activity nor the RNA levels of NF‐κB subunits showed correlation with liver inflammatory activity, viral load, or HCV genotype. By contrast, hepatic mRNA values of RelA, the main element of active NF‐κB, correlated inversely with apoptosis (r = −.68; P < .05) and with the rate of fibrosis progression (r = −.51; P < .04). In intermediate/rapid fibrosers, RelA mRNA levels were significantly decreased as compared with slow fibrosers (P < .003) and with normal livers (P < .03). In conclusion, we found that NF‐κB is activated in chronic HCV‐infected livers, and that the expression of RelA is inversely correlated with liver cell apoptosis and with the rate of fibrosis progression. Our data thus suggest that RelA expression may protect against liver fibrosis and hepatocellular damage.

Production of interleukin-2 in response to synthetic peptides from hepatitis C virus E1 protein in patients with chronic hepatitis C: relationship with the response to interferon treatment

BACKGROUND/AIMS The role of cellular immunity in the clearance of hepatitis C virus after interferon therapy has not yet been elucidated. Here, we analyzed the T cell response to peptides from hepatitis C virus E1 protein in untreated and interferon-treated patients with chronic hepatitis C virus infection. METHODS We used thirty-six 15-mer synthetic peptides from hepatitis C virus E1 protein (genotype 1a) in a sensitive interleukin-2 production assay in two groups of controls (healthy seronegative individuals and patients with liver diseases unrelated to hepatitis C virus), and three groups of patients with chronic hepatitis C: nine patients who cleared the virus after interferon treatment (group 1), nine patients who failed to respond to the therapy (group 2) and nine previously untreated patients (group 3). RESULTS None of the controls responded to any of the peptides tested, whereas 8/9 (88%) of patients from group 1 responded positively. In contrast, only 2/9 (22%) of patients from group 2 showed peptide recognition. In group 3, 5/9 patients (55%) displayed positive response against E1 peptides. When E1 peptides from the sequence corresponding to genotype 1b (the commonest in patients who were non-responders to interferon) were tested in nine additional interferon-resistant patients (group 2*) a positive response was detected in only three of them (33%). CONCLUSIONS T cell recognition of hepatitis C virus E1 peptides in patients with chronic hepatitis C who exhibit sustained response to interferon therapy is increased as compared with interferon-resistant cases, suggesting that T cell immunity to hepatitis C virus structural proteins may play a role in the clearance of this viral infection.

Expression of interferon-α subtypes in peripheral mononuclear cells from patients with chronic hepatitis C: a role for interferon-α5

Interferon (IFN)‐α is a family of antiviral proteins encoded by different genes. The biological significance of the existence of various IFN‐α subtypes is not clear. We have investigated the interferon system in chronic hepatitis C virus (HCV) infection, a disease that responds to interferon‐α2 therapy in only a limited proportion of cases. We analysed the expression of interferon regulatory factor (IRF)‐1, IRF‐2, and IFN‐α subtypes in nonstimulated and Sendai virus‐stimulated peripheral blood mononuclear cells (PBMC) from HCV infected patients and healthy controls. We observed that the IRF‐1 mRNA and IRF‐1/IRF‐2 ratios were increased in PBMC from hepatitis C patients with respect to normal subjects. Sendai virus stimulation of PBMC led to a significant increase in the levels of IRF‐1, IRF‐2 and IFN‐α mRNAs and in the production of IFN‐α protein with respect to basal values in healthy controls as well as in patients with HCV infection. In addition, we found that while natural HCV infection induced increased IFN‐α5 expression in PBMC, in vitro infection of these cells with Sendai virus caused a raise in the expression of IFN‐α8 in both patients and normal controls. In summary, our results indicate that virus‐induced activation of the IFN system in human PBMC is associated with selective expression of individual IFN‐α subtypes, IFN‐α5 being the specific subtype induced in PBMC from patients with chronic HCV infection.

Characterization of T‐cell responses against immunodominant epitopes from hepatitis C virus E2 and NS4a proteins

Summary.  Successful clearance of hepatitis C virus (HCV) infection has been associated with strong cellular immune responses against viral antigens. However, although the magnitude of these responses is clearly important for viral eradication, more studies are needed to unravel the fine specificity of the protective anti‐HCV immunity in infected patients. This was the aim of the present study. Overlapping peptides spanning the sequence of HCV E2 and NS4a proteins were used to stimulate T cells from patients with chronic hepatitis C divided into three groups: naïve patients, patients who exhibited sustained response to interferon (IFN)‐α therapy and patients who failed to respond to the treatment. Interleukin‐2 production by stimulated cells was measured in each case. Patients with sustained response to therapy had stronger responses to E2 peptides than nonresponders, whereas naïve patients demonstrated intermediate reactivity. In the case of NS4a, responses against peptides where similar in all groups of patients. Analysis of the peptides recognized by T cells showed that responses were broad and heterogeneous, and some immunodominant epitopes, preferentially recognized by patients exhibiting sustained response to treatment, were found. These results confirm the role of cellular immune responses in viral clearance, and stress the importance of immunodominant regions within HCV antigens. These viral sequences may represent valuable immunogens for preparation of therapeutic or prophylactic vaccines.

Interleukins in chronic active hepatitis B. Relationship with viral markers.

Interleukin-2, a product of helper T cells, is essentially involved in the regulation of cell-mediated immunity. Two monocyte-derived factors, interleukin-1 and prostaglandin E2, influence interleukin-2 synthesis with opposite actions. To analyse immunoregulatory function in HBsAg-positive chronic active hepatitis, T cell subsets in peripheral blood and the levels of interleukin-2, interleukin-1 and prostaglandin E2 in supernatants from lectin- or lipopolysaccharide-activated peripheral mononuclear cell cultures were determined in 16 healthy controls and 33 patients with chronic active hepatitis B. Interleukin-2 activity was comparable to the controls in patients without delta infection who had seroconverted to anti-HBe (group 1), but it was significantly reduced in both HBeAg-positive subjects (group 2) (P less than 0.05 vs. controls and group 1) and those cases with positive delta markers (group 3) (P less than 0.01 and less than 0.05 vs. controls and group 1, respectively). In group 3, interleukin-2 was similarly diminished in both anti-HTLV-III-positive and -negative cases as well as in HBeAg- and anti-HBe-positive subjects. Notwithstanding the changes in interleukin-2 activity, no significant differences in the number of T4 cells, or in the levels of either interleukin-1 or prostaglandin E2, were found among the various groups of subjects studied. However, in those groups with reduced interleukin-2 activity an increased number of T8 cells was observed. It is suggested that the low levels of interleukin-2 found in the replicative phase of chronic active hepatitis B and in delta superinfection reflect a disturbed immunoregulation that may contribute to persistent viral replication in these two conditions.

Detection of hepatitis C virus antibodies with new recombinant antigens: assessment in chronic liver diseases

A new serological assay to detect antibodies against hepatitis C, based on a recombinant protein (BHC10) which incorporates structural and non-structural viral antigens, was tested in 67 healthy subjects and 409 patients with various forms of liver disease. Results were compared with the current assay based on the recombinant non-structural viral antigen c100 and with the recently introduced second-generation assay, Ortho2. None of the healthy subjects was positive by any of the assays. In patients with chronic non-A, non-B hepatitis the prevalence of anti-BHC10 was 96.8%, higher than anti-c100 (83.3%, p less than 0.001) and similar to Ortho2 (94.3%). False-positive results were less frequently found when BHC10 was used. These findings show that assays incorporating structural and non-structural antigens provide higher sensitivity to detect hepatitis C virus infection and they define an almost exclusive role of hepatitis C virus in the genesis of chronic non-A, non-B hepatitis.