Toshiyuki Aiyama

Earlier loss of hepatitis C virus RNA in interferon therapy can predict a long‐term response in chronic hepatitis C

To distinguish responders from non‐responders early in interferon (IFN) treatment would be beneficial in patients with chronic hepatitis C. Those patients unlikely to respond would be spared the cost and hazard of prolonged treatment. Forty‐three chronic hepatitis C patients who had received IFN‐α therapy (6–9 MU; six times weekly for 2 weeks followed by thrice weekly for 22 additional weeks) were randomly enrolled into the present study. Serially obtained sera were retrospectively tested for HCV‐RNA by reverse transcription‐polymerase chain reaction (AmplicorTM HCV) with a low limit detection of approximately 102 copies/mL. Genotypes were determined by type‐specific primers. Sixteen subjects were defined as sustained responders (SR), who showed sustained loss of viraemia with normalized alanine aminotransferase values for at least 6 months of follow‐up after completion of therapy. The other 27 subjects were non‐responders (NR), whose viraemia persisted during follow‐up. Pretreatment serum HCV‐RNA levels (P< 0.0001) and the genotype (P<0.01) were significant predictors for sustained response to IFN therapy. Hepatitis C virus RNA was detectable in only one (6%) SR and in 23 (85%) NR at the second week of therapy (P< 0.0001) and was detected in none of the SR subjects and in 18 (67%) NR at the fourth week of therapy (P< 0.0001). Pretreatment viral load was correlated with the time until loss of viraemia. Multivariate analysis revealed that loss of viraemia at the second week of therapy was the strongest predictor for a long‐term response, followed by the initial viral load and loss of viraemia at the fourth week of therapy. These findings suggest that it is possible to predict a long‐term response to IFN as early as at the second and fourth weeks after the start of therapy by identifying the presence or absence of HCV‐RNA with a sensitive assay.

Cyclosporine therapy affects aminotransferase activity but not hepatitis C virus RNA levels in chronic hepatitis C

Interferon (IFN) therapy is of proven efficacy in chronic hepatitis C, but it is not universally effective and is often limited by side effects. Cyclosporine A (CsA) is a potent immunosuppressant widely used in organ transplantation. We conducted a pilot study to determine whether CsA therapy could affect aminotransferase activity and hepatitis C virus RNA levels in patients with chronic hepatitis C. Cyclosporine A was administered to 10 patients (mean age of 59 years; male: female = 9:1) who did not respond to IFN therapy previously and who had elevated serum alanine aminotransferase (ALT) values for at least 6 months. All patients were positive for HCV‐RNA by RT‐PCR with genotype 1b. Their mean duration of hepatitis was 15 years. Oral CsA was given for 3 months in a dose that was increased at 1 month intervals from 1.5–2.0 to 2.0–3.0 and 3.0–4.0 mg/kg per day. All patients completed the treatment schedule, although two patients developed mild non‐symptomatic hypertension. Serum ALT levels gradually decreased in all but one patient. The mean percentage decrease was 59.5% at the end of therapy (from 153 ± 82 to 62 ± 48 IU/L; P < 0.02). The ALT levels fell to the normal range in five patients, although once therapy was discontinued the enzyme levels tended to return to pretreatment levels. Serum aspartate aminotransferase and g‐glutamyl transpeptidase levels similarly decreased. The serum HCV‐RNA titre, determined by competitive RT‐PCR, did not change in any patient throughout the study period. There were no appreciable alterations in other laboratory tests, such as serum creatinine levels and lymphocyte subsets, except for an increase in serum alkaline phosphatase levels. These findings suggest that CsA, even in a relatively low dose, reduces serum aminotransferase levels without serious side effects in patients with chronic‐hepatitis C, although an antiviral effect was not noted.

Serial analysis of hepatitis B virus core nucleotide sequence of patients with acute exacerbation during chronic infection

Recent studies suggest that hepatitis B virus (HBV) core region could be an immunological target and that amino acid (aa) substitutions are mostly restricted to a small segment located in the middle of the core region. We sequenced the middle portion of HBV core gene during the course of acute exacerbation of chronic hepatitis B, and compared aa variations between the region including ideal HLA‐A2 binding motifs and the nonbinding region. Five HBeAg+ chronic hepatitis patients with subtype adr (three with HLA‐A2 and two without HLA‐A2) were selected and using polymerase chain reaction (PCR) and cloning system, the central part of core region (nt 2063 to 2365, 303 bp) was sequenced in sera from each patient at three time points; before, at the peak of, and after exacerbation of hepatitis. The second set of sera showed higher aa substitution rates in five and in three out of five patients compared with those of the first and third sera, respectively. No significant difference was found in the aa substitution rates for the region with ideal HLA‐A2 binding motifs between patients with and without HLA‐A2. In asymptomatic HBV carriers with persistently normal aminotransferase values, alterations of the aa sequence were not observed within the same time frame. The results suggest that aa substitutions often occur at some particular positions in the middle of HBV core region during acute exacerbation of the disease under possible host immune pressures. Furthermore, unidentified epitopes appear to exist in the central part of HBV core region and HLA‐unrestricted lymphocytes may play a role in the immune response of chronic HBV carriers.

Changes in serum hepatitis C virus RNA titer and response to interferon therapy in patients with chronic hepatitis C

Response to interferon (IFN) therapy for chronic hepatitis C has been determined by the alteration of serum alanine aminotransferase (ALT) values. However, eradication of hepatitis C virus (HCV) could be another aim of the therapy. Thus, we serially measured serum HCV RNA levels during therapy and for at least 12 months after cessation of therapy in 65 patients with chronic hepatitis C who received IFN-α (49 cases) or -β (16 cases). The presence of HCV and its amount were measured by the combination of nested and competitive PCR. Twenty-seven patients, who were categorized as complete responders, showed sustained normalization of ALT values for more than six months posttreatment. The viral RNA titers at pretreatment were significantly lower in complete responders (logarithmic copy numbers/ml: 5.4±1.3,P<0.001) than in partial and nonresponders. Complete response rate was significantly higher in patients with HCV genotype III (68.4%,P<0.01) than those with type II (23.6%). Among 27 complete responders, HCV RNA became undetectable in only 13 patients six months after completion of therapy, and 11 still had low levels of viremia; however, none experienced relapse of the disease during follow-up of 12–24 months. Three complete responders showed lasting high-titered viremia, and their ALT values rose again during follow-up. Our data suggest that IFN treatment of chronic hepatitis C is often ineffective in eradicating HCV infection even in responders, and long-term follow-up study is necessary to determine the sustained beneficial effect of IFN.

Deficiency of antibody response to hypervariable region of hepatitis C virus in patients with chronic hepatitis C

BACKGROUND/AIMS Frequent mutations in the hypervariable region of hepatitis C virus have been suggested to be a cause of persistent infection by providing a way for the virus to escape host immunity. However, the variation rate in the hypervariable region is often low in patients with chronic hepatitis. The aim of this study was to elucidate the mechanism of persistent infection in patients with chronic hepatitis by investigating the relationship between the antibody response to and the variation in hypervariable region. METHODS The hypervariable regions of 26 clones of six patients with chronic hepatitis C were expressed as proteins fused with glutathione S-transferase, and sera of the patients were serially tested for antibody to these proteins. RESULTS The extent of antibody response to the hypervariable region differed considerably among the patients. Three patients showed no or only scanty antibody response. These had a lower variation rate in the hypervariable region (0-1.3/year) than in the others with frequent or persistent antibody response (2.1-14.6/year). In two patients, serum samples were found to be reactive with the clones even before the appearance of the clones. In three patients, coexistence of the antibodies and corresponding clones were noted. An augmentation of antibody response always followed rises in serum alanine aminotransferase levels. CONCLUSION Deficiency of antibody response to the hypervariable region may be one of the causes of persistency in hepatitis C virus infection.

Serum HCV RNA titer at the end of interferon therapy predicts the long-term outcome of treatment

BACKGROUND/AIMS Serum HCV RNA was quantitated by the competitive polymerase chain reaction before, at the end of, and after interferon therapy. We assessed whether serum HCV RNA titer at the end of interferon therapy predicts the long-term outcome of treatment. METHODS/RESULTS Of 71 patients treated with various doses of interferons, 21 became negative for HCV RNA persistently during follow up of 2 years, and they were considered as complete responders. The serial determinations of HCV RNA titer for each individual showed that in patients with HCV RNA negative at the end of therapy, the complete response rate was quite high (78.6%), while in patients with HCV RNA titer > or = 10(4) copies/ml at the end of therapy, none became complete responders in long-term follow up. The percentage decreasing to < or = 10(2) copies/ml of HCV RNA at termination of interferon tended to be higher in patients with genotype 2a (14/21, 66.7%) than in those with genotype 1b (18/42, 42.9%). The complete response rate of patients whose viral load was < or = 10(2) copies/ml at termination of interferon was significantly higher in genotype 2a (11/14, 78.6%) than in genotype 1b (5/18, 27.8%) (p < 0.01). Pretreatment HCV RNA titer appeared to correlate to the titer at the end of therapy (r = 0.596, p < 0.001); even when HCV RNA decreased to < or = 10(2) copies/ml, the higher pretreatment titer indicated a lower likelihood of complete response (p < 0.05). CONCLUSIONS These results indicate that HCV genotype and pretreatment viral titer are important factors in the response to interferon therapy. In addition, our study suggests that it is possible to stop interferon therapy at an appropriate time by monitoring HCV RNA titer.

Different Constitution of Hepatitis C Virus Population in Peripheral Blood Mononuclear Cells and Plasma in Patients with Type C Chronic Liver Disease

We searched for the presence of the plus orminus strand of hepatitis C virus (HCV) RNA inperipheral blood mononuclear cells (PBMCs) from threepatients with chronic HCV infection using thestrand-specific reverse transcription and polymerase chainreaction (RT-PCR) method. To examine whether the HCVpopulation of PBMCs differs from that of plasma, asequence of the hypervariable region (HVR) in the E2/NS1 region was analyzed. All three patients hadboth plus and minus strands of HCV RNA in their PBMCs.Sequence study revealed that the HCV population in PBMCswas homogenous in all patients, while that in plasma was composed of two main clones. One ofthese had the same sequence as the clones seen in PBMCs,except for one patient. Our results suggest that PBMCsrepresent one of the extrahepatic replication sites of HCV and that tissue tropism isexpressed by some of the HCV population.