Agostino Cividini

Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities

Hepatitis C virus (HCV) infection persists for an indefinite length of time in a major proportion of patients, inducing chronic liver lesions that evolve to cirrhosis and hepatocellular carcinoma (HCC) in approximately 20% of cases. We studied HCV viremia and genotypes by reverse transcription–polymerase chain reaction (RT‐PCR) in 341 consecutive anti‐HCV–positive patients. Of these, 167 patients had persistently normal or near normal alanine aminotransferase (ALT) levels (fluctuations ≤5 IU above the upper limit of normal); the remaining 174 patients presented with elevated ALT and histological evidence of chronic liver disease. Seventy percent of patients with normal ALT values had circulating HCV RNA despite the absence of biochemical indicators of liver damage and mild histological forms of chronic hepatitis were detected in most patients who underwent liver biopsy. Isolated genotype III infection was significantly more prevalent in this patient group with respect to control patients with abnormal ALT values (70% vs. 39%; P < .001). Conversely, isolated genotype II was more frequently found in patients with elevated ALT values and evidence of chronic liver disease (45% vs. 23%; P < .01) and it was progressively more represented in advanced liver disease, such as cirrhosis and HCC. Virological features of HCV infection might be associated with different clinical manifestations, suggesting a potential prognostic significance on disease outcome. (HEPATOLOGY 1995;21:285–290.)

Insulin resistance is associated with steatosis in nondiabetic patients with genotype 1 chronic hepatitis C

Conflicting data exist regarding the relationship between hepatitis C virus genotype 1 and hepatic steatosis as well as the latters role in the progression of fibrosis and treatment response. We assessed factors associated with hepatic steatosis in genotype 1 chronic hepatitis C and the impact of hepatic fat on fibrosis development and interferon responsiveness. Two hundred ninety‐one non‐diabetic patients with genotype 1 chronic hepatitis C were examined for the presence of steatosis and its correlation with clinical, virological, and biochemical data, including insulin resistance (IR), evaluated by the homeostasis model assessment (HOMA) score. Steatosis was graded as mild (1%‐20% of hepatocytes involved), moderate (21%‐40% of hepatocytes involved), and severe (>40% of hepatocytes involved). Steatosis was mild in 110 of 291 (37.8%) and moderate/severe in 55 of 291 (18.9%) subjects. By logistic regression, moderate/severe steatosis was independently associated with the female sex (odds ratio [OR] 2.74; 95% CI 1.40‐5.35), high γ‐glutamyltransferase levels (OR 1.52; 95% CI 1.22‐1.91), and HOMA‐score (OR 1.076; 95% CI 1.001‐1.26). By logistic regression, moderate/severe steatosis (OR 2.78; 95% CI 1.21‐6.4), and platelet counts (OR 0.97; 95% CI 0.96‐0.98) were independent predictors of advanced fibrosis. Patients with moderate/severe steatosis had an OR of 0.52 (95% CI 0.30‐0.90) for sustained virological response compared with patients with mild/absent steatosis. In conclusion, in nondiabetic European patients with genotype 1 hepatitis C at low risk for the metabolic syndrome, the prevalence of steatosis was nearly 60%. IR is a risk factor for moderate/severe steatosis, especially in men. Moderate/severe steatosis has clinical relevance, being associated with advanced fibrosis and hyporesponsiveness to antiviral therapy. (HEPATOLOGY 2006;43:64–71.)

Molecular epidemiology of hepatitis C virus infection among intravenous drug users

BACKGROUND/AIMS The clinico-pathological features of hepatitis C virus infection in intravenous drug users are different from those found in other hepatitis C virus-infected patients. Our aim was to test whether specific viral variants circulate within this particular patient population. METHODS We studied the distribution of hepatitis C virus genotypes in 90 drug addicts and 484 controls, according to the method described by Okamoto. RESULTS Hepatitis C virus type 1a and 3a infections were more frequent among intravenous drug users than in 125 age-matched controls (48.8% and 21.1% vs 17.6% and 11.2%), accounting for the majority of infections in intravenous drug users. Analysis of hepatitis C virus genotypes according to age showed that, in the general population, hepatitis C virus types 1a and 3a were more prevalent among patients younger than 40 years of age than in older individuals (17.6% and 11.2% vs 1.4% and 0.6%). CONCLUSIONS These findings suggest that hepatitis C virus types 1a and 3a were recently introduced in Italy, presumably via needle-sharing among intravenous drug users, and from this reservoir they are extending to the general population, particularly among younger subjects.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: exploring the immunological mechanisms

Hallmark of acute hepatitis C virus (HCV) infection is a severe virus‐specific effector CD8+ T cell dysfunction that seems to be a critical factor in preventing the resolution of infection and in favoring the onset of chronic liver immunopathology. We suggest that this dysfunction is critical in the establishment of HCV persistence, unless it is compensated by multispecific responses, as found in individuals resolving infection. Analyses on purified populations indicate that central memory HCV‐specific CCR7+/CD8+ T cells efficiently proliferate and differentiate in vitro, although the large population of memory effector CCR7– cells found in the peripheral blood of acutely infected patients display poor effector functions ex vivo (semi‐effectors). However, we report strong evidence in support of IL‐2 being capable of pushing semi‐effector CTL to complete their effector cell program. Therefore, IL‐2 deficiency during T cell activation may be responsible for the dichotomy between memory CTL expansion and incomplete effector differentiation shown in patients with acute HCV infection. These data are consistent with the possible therapeutic treatment with IL‐2 to rebuild the effector T cell pool in these patients.

CYP ENZYME POLYMORPHISMS AND SUSCEPTIBILITY TO HCV-RELATED CHRONIC LIVER DISEASE AND LIVER CANCER

Cancer risk can be influenced by the exposure to endogenous or environmental toxins. Polymorphic enzymes involved in the metabolic activation/detoxification of carcinogens may account for individual variations of risk. We studied the polymorphisms of five enzymes of the P450 superfamily, CYP1A1, CYP1A2, CYP2D6, CYP2E1 and CY3A4, as risk factors for liver disease progression and cancer in hepatitis C virus‐infected patients. CYP genotyping was performed by polymerase chain reaction (PCR) restriction fragment length polymorphism or allele‐specific PCR. Different stages of disease were considered, as follows: 90 asymptomatic carriers and 87 chronic hepatitis, 92 cirrhosis and 91 hepatocellular carcinoma (HCC) cases. Reference allele frequencies were obtained from 99 blood donors. Allele distributions among categories were compared using the χ2 test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to express relative risks. Independent associations were modeled by correspondence analysis and logistic regression. Frequencies of the CYP1A1 highly inducible alleles, MspI m2 and Val, were increased in liver disease patients compared with carriers; no specific association with HCC was found. The high‐activity CYP2E1 c2 allele was underrepresented among HCC patients with respect to other HCV categories, including cirrhosis. CYP2D6 poor metabolizer (PM) genotypes were significantly more frequent in healthy subjects (7.1%) and carriers (11.1%) than in hepatitis/cirrhosis (4.6%) and HCC (1.2%) patients. This was confirmed by multivariable analysis. PM genotypes protected against progressive disease as ORs reduced proportionally to stage. The age at diagnosis for HCC was anticipated in non‐PM individuals. No differences were seen for CYP1A2 and CYP3A4 genes. Polymorphic variants of CYP genes may contribute to the progression of liver disease and HCC risk in HCV‐infected subjects.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: the role of the virus

In a companion study, we showed a dichotomy between the expansion of central memory (CCR7+) hepatitis C virus (HCV)‐specific CTL and the incomplete memory effector differentiation in patients with acute HCV infection. Indeed, effector cells were unable to perform immediate functions, despite expressing the tissue‐homing phenotype of effector memory cells (CCR7–; semi‐effectors). However, since they promptly differentiated into full‐effectors upon IL‐2 contact, we suggested that the inhibitory effect by environmental (possibly viral) factors on IL‐2 production may have a pivotal role in generating the large population of semi‐effector CCR7–/IFN‐γ– CTL. In accord with this view, we report here strong evidence in support of circulating HCVcore protein (HCVcore) playing a central role in inhibiting effector CTL differentiation, but not memory CTL expansion. The regulatory HCVcore effect is related to inhibition of the signal transduction pathway instrumental for IL‐2 production, supporting the evidence that IL‐2 was capable both of pushing semi‐effector CTL to complete their effector cell program and of restoring the HCVcore‐dependent inhibitory effect. Therefore, the strength of CTL activation is dependent on the balance between the threshold of stimulatory signals and the viral interference capacities provided during priming.

Hypervariable region 1 of hepatitis C virus: immunological decoy or biologically relevant domain?

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is highly heterogeneous and is responsible for significant inter- and intra-individual variation of the infecting virus, which may represent an important pathogenetic mechanism leading to escape and persistent infection. Moreover, a binding site for neutralizing antibodies (Ab) has been allegedly identified in this region. Prospective studies of serological responses to synthetic oligopeptides derived from HVR1 sequences of patients with acute and chronic HCV infection showed extensive serological cross-reactivity for unrelated HVR1 peptides in the majority of the patients. A significant correlation was found between HVR1 sequence variation, and intensity, and cross-reactivity of humoral immune responses providing strong evidence in support of the contention that HCV variant selection is driven by the host immune pressure. Monoclonal Ab (mAb) generated following immunization of mice with peptides derived from natural HVR1 sequences also showed cross-reactivity for several HVR1 sequences attesting to the existence of conserved amino acid motifs among different variants. These findings suggest that it is possible to induce a broadly cross-reactive immune response to HVR1 and that this mechanism can be used to generate protective immunity for a large repertoire of HCV variants.

Antibody responses to the hepatitis C virus E2 protein: relationship to viraemia and prevalence in anti-HCV seronegative subjects.

A small proportion of patients with chronic hepatitis C virus (HCV) infection show no serological responses to the HCV polypeptides incorporated in commercial III generation immunoassays. To determine whether sera from these subjects contain antibodies to the highly immunoreactive second envelope polypeptide E2, which is not included in current anti‐HCV assays, we studied 59 anti‐HCV negative subjects who were found consistently to be HCV RNA positive by polymerase chain reaction (PCR). Controls included 167 anti‐HCV seropositive patients with or without serum HCV RNA and normal subjects. Antibodies to the E2 region were sought for by ELISA using the following antigens: a full length E2 protein expressed in insect cells using a baculovirus vector and extracted under denaturing conditions (dE2), and a C‐terminal truncated soluble E2 (sE2) protein (a.a. 390–683), also expressed with a baculovirus vector, containing a signal peptide of rabies virus G protein which allows its secretion into the culture supernatant. Sera from only two (3.4%) of the 59 anti‐HCV negative, HCV RNA positive patients recognised sE2 and none dE2. In sharp contrast, 82% of seropositive, viraemic patients recognised sE2 and 60% dE2, the difference in immunoreactivity being statistically significant (P < 0.0003). A significantly lower proportion of sera from anti‐HCV positive, HCV RNA negative subjects recognised either sE2 or dE2 (16% and 13%, respectively, P < 0.000001). Healthy controls were consistently negative. These results indicate that antibody responses to predominantly conformational epitopes on the HCV E2 protein are common in patients with chronic HCV infection and are strictly related to the presence of circulating viral genomes. In contrast, only a minor proportion of HCV RNA positive patients, but anti‐HCV seronegative by commercial immunoassays, have humoral immune responses to the HCV E2 region. J Med Virol 51:1–5, 1997.

Hepatitis C virus infection among institutionalised psychiatric patients: a regression analysis of indicators of risk

BACKGROUND/AIMS Institutionalised psychiatric patients are at increased risk of developing chronic infection with hepatitis B virus (HBV). However, little information is available on transmission and epidemiology of hepatitis C virus (HCV) in this setting. The aim of this study was to identify potential risk factors of acquiring HCV infection in two large psychiatric institutions in northern Italy. METHODS We designed a case-control study using randomly selected controls from the same study database, consisting of a total of 1180 patients, in order to satisfy the principle that both cases and controls should be representative of the same base experience. A multiple regression logistic analysis was used to identify features that could predict exposure to HCV as evidenced by the presence of circulating anti-HCV antibodies. RESULTS Anti-HCV was detected in 79 patients (6.7%). The prevalence of viraemia and the distribution of genotypes were very similar to those found in subjects with chronic HCV infection drawn from the same geographical area. Multivariate analysis indicated that a diagnosis of psychosis and a history of trauma were statistically significant independent risk factors associated with a positive anti-HCV result (OR 2.615, 1.273-5.373 95% CI and OR 2.096, 1.133-3.877 95% CI, respectively). CONCLUSIONS The findings of this large epidemiological study show for the first time that prolonged residence in psychiatric institutions does not entail per se a significant risk of acquiring HCV infection. Since transmission of HCV in this setting appears to occur predominantly via classical parenteral routes, simple prophylactic measures appear to be adequate to prevent infection.

Kinetics and Significance of Serum Hepatitis C Virus Core Antigen in Patients with Acute Hepatitis C

ABSTRACT An immunoassay detecting hepatitis C virus core antigen was evaluated for its ability to predict clinical outcome in a series of patients with acute hepatitis C. In subjects who cleared the virus, core antigen was no longer detectable within 16 weeks of onset, whereas considerable fluctuations were noted among patients progressing to chronic hepatitis, one of whom showed consistently negative values despite the intermittent presence of viral RNA.