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Hepatitis C virus (HCV) genotype 1 infections are significantly more difficult to eradicate with PEG-IFN/ribavirin therapy, compared to HCV genotype 2. The aim of this work is to investigate the difference of immunological impairments underlying this phenomenon. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes and CD56+CD3- NK cells from cases of chronic hepatitis C were analyzed and assessed by treatment effect. Two strains of HCV were used to co-incubate with immune cells in vitro. NKG2D expression on peripheral CD56+CD3+ lymphocytes, but not NK cells, was significantly impaired in genotype 1 infection, compared to genotype 2. When peripheral blood mononuclear cells from healthy donors were co-incubated with TNS2J1, a genotype 1b/2a chimera strain, or with JFH1, a genotype 2a strain, genotype-specific decrease of NKG2D on CD56+CD3+ lymphocytes, but not NK cells, was observed.　Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes significantly correlated with reduction in serum HCV RNA levels from week 0 to week 4, and predicted treatment response. Ex vivo stimulation of peripheral CD56+CD3+ lymphocytes showed NKG2D expression-correlated IFN-γ production. In conclusion, Decreased NKG2D expression on CD56+CD3+ lymphocytes in chronic HCV genotype 1 infection predicts inferior treatment response to PEG-IFN/ribavirin therapy compared to genotype 2.

Treatment with pegylated interferon alpha-2b (PEGIFN) plus ribavirin (RBV) is standard therapy for patients with chronic hepatitis C. Although the effectiveness, patients with high titres of group Ib hepatitis C virus (HCV) respond poorly compared to other genotypes. At present, we cannot predict the effect in an individual. Previous studies have used traditional statistical analysis by assuming a linear relationship between clinical features, but most phenomena in the clinical situation are not linearly related. The aim of this study is to predict the effect of PEG IFN plus RBV therapy on an individual patient level using an artificial neural network system (ANN). 156 patients with HCV group 1b from multiple centres were treated with PEGIFN (1.5 µg/kg) plus RBV (400-1000 mg) for 48 weeks. Data on the patients' demographics, laboratory tests, PEGIFN, and RBV doses, early viral responses (EVR), and sustained viral responses were collected. Clinical data were randomly divided into training data set and validation data set and analyzed using multiple logistic regression analysis (MLRs) and ANN to predict individual outcomes. The sensitivities of predictive expression were 0.45 for the MLRs models and 0.82 for the ANNs and specificities were 0.55 for the MLR and 0.88 for the ANN. Non-linear relation analysis showed that EVR, serum creatinine, initial dose of Ribavirin, gender and age were important predictive factors, suggesting non-linearly related to outcome. In conclusion, ANN was more accurate than MLRs in predicting the outcome of PEGIFN plus RBV therapy in patients with group 1b HCV.

Aim: To predict treatment success using only simple clinical data from peg-interferon plus ribavirin therapy for chronic hepatitis C., Methods: We analyzed the clinical data of 176 patients with chronic hepatitis and hepatitis C virus genotype 1 who received 48 wk standard therapy, derived a predictive formula to assess a sustained virological response of the individual patient using a logistic regression model and confirmed the validity of this formula. The formula was constructed using data from the first 100 patients enrolled and validated using data from the remaining 76 patients., Results: Sustained virological response was obtained in 83 (47.2%) of the patients and we derived formulae to predict sustained virological response at pretreatment and weeks 4, 12 and 24. The likelihood of sustained virological response could be predicted effectively by the formulae at weeks 4, 12 and 24 (the area under the curve of the receiver operating characteristic: 0.821, 0.802, and 0.891, respectively), but not at baseline (0.570). The formula at week 48 was also constructed and validation by test data achieved good prediction with 0.871 of the area under the curve of the receiver operating characteristic. Prediction by this formula was always superior to that by viral kinetics., Conclusion: These results suggested that our formula combined with viral kinetics provides a clear direction of therapy for each patient and enables the best tailored treatment.

We investigated the relationship between serum ribavirin concentrations and clearance, as well as therapeutic efficacy and adverse reactions, in 97 Japanese patients with chronic hepatitis C virus infections treated with a 6-month course of high-dose alpha2b interferon (6 million units/day) plus ribavirin (600 to 800 mg/day) combination therapy. This randomized trial showed that the saturation of ribavirin uptake after taking ribavirin capsules does not occur within a dose range of 600 to 800 mg/day, which is a standard dosage used clinically in Japan. Serum ribavirin concentrations and clearance did not correlate with sustained virological response rates. Fourteen patients discontinued therapy because of adverse reactions, and sustained virological response rates were significantly reduced by discontinuation of therapy, while dose reduction of ribavirin did not alter the therapeutic effects. Ribavirin concentrations after 1 week and ribavirin clearance were significantly correlated with discontinuation of ribavirin; however, a multiple-regression analysis revealed that only hemoglobin concentration, but not ribavirin clearance, was a significant factor for discontinuation of therapy (odds ratio, 0.514; 95% confidence interval, 0.311 to 0.85; P = 0.0095). It appears that peripheral erythrocytes may act as a reservoir for ribavirin and regulate serum ribavirin levels in the very early phase of treatment.

Background/aims: Two distinct natural interferon-alpha (BALL-1 and Namalwa) are available for patients with chronic hepatitis C in Japan, but the efficacy has not been well documented. We investigated two studies using a natural BALL-1 interferon-alpha treatment for chronic hepatitis C and assessed its efficacy., Methodology: In interferon-alpha monotherapy (Study I), 42 patients with chronic hepatitis C received 10 mega units of BALL-1 interferon-alpha intramuscularly consecutively for an initial 2 weeks followed by three times a week for 6 months totally. In a combination therapy of natural interferon-alpha and interferon-beta (Study II), 24 patients received intravenous 3 mega units of interferon-beta twice daily for the initial 2 weeks followed by 10 mega units of natural BALL-1 interferon-alpha consecutively for 2 weeks and three times a week for 6 months totally. Efficacy and predictive factors for sustained viral response was investigated., Results: Study II included significant younger patients than study I. Sustained virological response was obtained in 31.0% in Study I and 56.5% in Study II by intention-to-treat analysis. Sustained viral response in the group of genotype 1b and viral load more than 100 KIU/mL was 3/23 (13.0%) and 8/18 (44.4%) in Study I and II, respectively. The response rate in Study II was higher than that of Study I especially among the patients with high pretreatment viral load or genotype 1b (p<0.05). Multivariate analysis showed that pre-treatment HCV-RNA levels, HCV-genotype, and histological staging before the interferon treatment were significant predictive factors of sustained viral response., Conclusions: These studies suggest that natural BALL-1 interferon-alpha is useful for inducing sustained viral response in patients with chronic hepatitis C, even in those possessing genotype 1b and high viral load. In addition, the combination therapy with a starting regimen with twice-daily interferon-beta administration for 2 weeks may be more effective than monotherapy.

Background/aims: The development of retinal lesions induced by a uniform regimen of interferon-alpha therapy for chronic hepatitis C was prospectively investigated., Methods: Eighty-one patients received 6 mega units of natural interferon-alpha intramuscularly daily for 2 weeks and then 3 times a week for 22 weeks. The total dose of interferon-alpha administered was uniformly 478 mega units per patient. Two expert ophthalmologists prospectively examined the patients for retinal lesions before, during and after the therapy., Results: Retinopathy was not found in comparison groups or any of the patients before treatment. In total 34.6% (28/81) of the patients showed cotton-wool spots or minor retinal hemorrhage, or both lesions, during therapy, but these lesions were reversed during or after interferon therapy. The occurrence rates of cotton-wool spots alone, retinal hemorrhage alone and both lesions were 13.6% (11/81), 6.2% (5/81), and 14.8% (12/81), respectively. The appearance of retinopathy did not correlate with patients' background including viral loads and response to the therapy, but was more frequently found in older patients and patients with hypertension and/or diabetes mellitus; disappearance of retinopathy was more prolonged than in patients without these complications. Almost all the lesions appeared 2-4 months after the start of the therapy, and the severity of the lesions did not differ between patients with and without hypertension and/or diabetes mellitus., Conclusion: Although it is not clear if interferon-associated retinopathy occurs in a dose-dependent manner, the present study shows a standard pattern of the occurrence of retinopathy in patients with chronic hepatitis C receiving a uniform dosage of natural interferon-alpha.

Background/aims: Immunological status has been considered to correlate to the response to interferon therapy for chronic hepatitis C. The aim of this study was to evaluate the correlation between humoral immunity and long-term response to interferon treatment for chronic hepatitis C., Methodology: Seventy-one patients with chronic hepatitis C received 10 million units of interferon-alpha 2b three times a week for 24 weeks. Peripheral blood mononuclear cells were obtained before interferon-alpha 2b was administered and were cultured for 7 days. Immunoglobulin concentration in the culture supernatants was measured by enzyme-linked immunosorbent assay and correlation with the response to the therapy was evaluated., Results: Serum ALT levels normalized in 51.4% and hepatitis C virus RNA disappeared in 35.7% six months after the end of therapy. Immunoglobulin production was significantly lower in the patients in whom serum ALT levels normalized than those in whom serum ALT levels remained elevated. The similar result was obtained when efficacy was evaluated on the basis of hepatitis C virus RNA disappearance., Conclusions: These results suggest that the less humoral immunity, the better response to interferon will be obtained in patients with chronic hepatitis C, meaning that the balance in T-helper function is one of key factors in the response to interferon treatment.

Results from a multicentre, clinical trial of interferon-alpha2a (IFN-alpha2a) for the treatment of chronic hepatitis C are reported. Serum hepatitis C virus (HCV) RNA levels were monitored as follows: before, and 2 days after, the first administration of IFN-alpha2a; during and at the end of treatment; and 6 months after completion of therapy. Peripheral blood lymphocyte subpopulations were measured, by two-colour flow cytometry, before and 3 h after the first intramuscular (i.m.) administration of 9 mega units (MU) of IFN-alpha2a. Virological responders had a significantly lower pretreatment level of CD11+ CD8- lymphocytes. Biochemical responders had significantly lower pretreatment levels of CD11- CD8+, human leucocyte antigen (HLA) DR- CD4- and HLA DR- CD8+ populations, and a higher pretreatment HLA DR+ CD4- population. These pretreatment differences disappeared 3 h after the first i.m. administration of IFN-alpha2a. CD11- CD8+ and HLA DR+ CD8+ cell populations became significantly lower in virological responders 3 h after the first i. m. administration of IFN-alpha2a. HLA DR+ CD4+ cell populations were increased less in biochemical responders. Thus, T-lymphocyte subpopulations were different between responders and non-responders to IFN therapy and IFN-modulated host immunity. Multivariate analysis showed that the pretreatment CD11+ CD8- cell population was an independent predictive factor of response to therapy. On the other hand, patients whose serum HCV RNA cleared or decreased within the first 2 days of IFN-alpha2a therapy were more likely to achieve a virological response. This predictive factor, however, was not an independent factor by multivariate analysis. These results suggest that host immunity is an important factor in response to IFN therapy, and HCV clearance within the first 2 days is a good predictive factor of response.

The effect of IFN-alpha on the growth of 5 hepatoma cell lines and a normal liver-derived cell line were examined. IFN dose-dependently inhibited the growth of cell lines except for HLE and PLC/PRF/5 in which the inhibition only occurred at a high concentration over 10,000 IU/ml. IFN-alpha induced the G1 arrest of these cells according to upregulation of p21WAF-1 expression, which is induced in PLC/PRF/5 and HLE only at a high IFN concentration. The receptor for IFN-alpha was well expressed in all the cell lines. DNA rearrangement of IRF-1 was detected in HLE and PLC/PRF/5 by Southern blotting, while IRF-2 gene was preserved. IFN-induced gene expressions were compared between HCC-T, HCC-M and PLC/PRF/5. RT-PCR demonstrated that the full-length IRF-1 and -2 mRNA was transcribed in all cell lines, but the mRNA amount of former gene in PLC/PRF/5 was less than that in HCC-T and HCC-M, about 1/10 by competitive RT-PCR. The sequence analysis of IRF-1 cDNA was performed and the full-length mRNA transcription was reconfirmed in PLC/PRF/5, but no significant point mutation was detected. These results suggest that IFN-alpha inhibits hepatoma growth by increasing p21WAF-1 expression only when the IRF-1 gene is preserved normally.

The efficacy of different doses and administration periods of interferon (IFN)-alpha treatment for patients with chronic hepatitis C was retrospectively evaluated by using the return of serum alanine aminotransferase (ALT) to normal levels 12 months after therapy as an index. Two hundred forty-one patients with chronic hepatitis C received 6 to 9 mega units of IFN-alpha once, twice, or three times per week for a different number of weeks. ALT levels generally returned to normal in 77 of the 241 patients (32.0%). The efficacy of IFN-alpha therapy with a total dose of 350 mega units or more was significantly superior to that with 350 mega units or less (30 to 45%, p < 0.05). However, the efficacy was the same if a total dose was more than 350 mega units, and more than 700 mega units did not improve the response rate. Administration periods of more than 6 months and less than 1 year appeared to be the most effective in this study. To achieve a complete response, administration three times a week was better than once a week. The higher the dose administered within a certain period, the higher was the efficacy of the IFN-alpha treatment. However, the factors affecting response must be further studied, because there are patients who respond to therapy with a total of only about 200 mega units, and patients who do not respond even to 800 mega units given for more than 1 year.

The liver extract preparation Adelavin9 (referred to as EXT-FAD hereinafter) is a combination product that consists of hog liver extract and flavin-adenine dinucleotide (FAD). EXT-FAD has been reported to improve the histology of chronic liver injury and is widely used in Japan. Interferon (IFN) has been used to treat viral hepatitis. In this study, the action of EXT-FAD on the antiviral activity of IFN was examined using experimental models of viral infection in vitro and in vivo. EXT-FAD significantly enhanced the 2',5'-oligoadenylate synthetase (2-5AS) activity in human peripheral blood mononuclear cells (PBMC) treated with human IFN-alpha-2a. EXT-FAD is likely to affect the late phase of IFN action to produce the antiviral activity. Combination of IFN-alpha-2a with EXT-FAD produced a synergistic inhibitory effect against herpes simplex virus type-1 (HSV-1) in vitro. The effect of EXT-FAD in combination with IFN-alpha-2a against influenza virus type A (Fluv A) was also examined and a similar effect to that found in the anti-HSV-1 assay was observed. In addition, administration of EXT-FAD to mice infected with HSV-1 significantly prolonged the mean survival periods. The intracellular redox state plays a role in inhibiting virus replication. EXT-FAD was found to increase the intracellular glutathione (GSH) level. EXT-FAD also enhanced the nitric oxide synthase (NOS) activity, which plays a role in antiviral effects. This result indicate that the effect of EXT-FAD on the antiviral activity of IFN is partially related to increased intracellular GSH levels and NOS activation.

Correlation between the major histocompatibility complex class I antigens (HLA-A, -B and -C) and the elimination from serum of hepatitis C virus in patients with chronic hepatitis C has not been understood. We analyzed HLA phenotypes and their relationship to the efficacy of interferon treatment. Of the 172 patients who were treated with 9 million units of interferon-alpha 2a three times a week for 6 months, 54 patients were responders and 118 patients were non-responders. No significant difference was observed between the 172 patients and 199 healthy subjects with regard to the frequencies of HLA-A, -B and -C antigen phenotypes. However, HLA-B55, B62, CW3 and CW4 frequencies were significantly higher in responders than in non-responders to the interferon treatment. CW4 was found to link with B62, but other phenotypes were independent each other. Patients with HLA B55, B62 and CW3 had a significantly lower viral load, and showed a better response to interferon. These results suggest that HLA system does not have an influence on the evolution towards chronicity of the disease due to hepatitis C virus, but HLA B55, B62 or CW4, and CW3 may be a virus quantity-regulating factors which then affect to response to the interferon treatment, indicating that these HLA antigens in conjunction with a viral peptide is a key target antigen for cytotoxic T lymphocytes in patients with chronic hepatitis C.

We have reported the Keio multicenter randomized trial of interferon-alpha 2b treatment for chronic hepatitis C, hypothesizing that disappearance of serum hepatitis C virus (HCV) ribonucleic acid (RNA) during the first 2 days by one does administration of interferon is a predictive factor of final response to the high-dose interferon treatment. In this study we quantified HCV RNA by multicyclic reverse transcription-polymerase chain reaction in the stored sera of the same patients with our previous study. The multivariate analysis confirmed that the pretreatment HCV RNA levels and HCV genotype were significantly correlated with the response to the 6-month course interferon treatment. Although the relationship between decreased HCV RNA titers within the first 2 days after one dose administration of interferon and the efficacy of the therapy was not obtained, the cases in which HCV RNA disappeared within 2 days significantly responded to the 6-month course treatment (73.7% vs 12.5% in other cases, p < 0.01). The present study has confirmed the hypothesis suggested in the previous study that clearance of HCV RNA during the first 2 days has a predictive value for the final outcome.