Tadao Okuno

The virological and histological states of anti‐hepatitis C virus–positive subjects with normal liver biochemical values

We investigated anti‐hepatitis C virus (HCV) titers, HCV RNA levels in liver and serum, genetic variability in the hypervariable region of the genome, the form of the virus in the circulation, and liver histology in 21 anti‐HCV‐positive subjects with sustained normal liver biochemical values. Titer of anti‐HCV was determined by second generation anti‐HCV‐passive hemagglutination assay, and HCV RNA levels were semiquantitated by reverse transcriptase polymerase chain reaction (PCR). In 19 (90%) of the 21 subjects who had a higher titer of anti‐HCV (⩾214), HCV RNA was detected in both serum and liver, and histological examination showed minimal or mild chronic hepatitis in all. In the remaining 2 patients who had a lower titer of anti‐HCV, HCV RNA was not detected in serum and liver, and liver histology was normal. Anti‐HCV titers and HCV RNA levels in serum and liver in the 19 HCV RNA‐positive subjects were compared with those levels in the 41 patients with biopsy‐proven chronic hepatitis C and elevated serum aminotransferase levels as a control group. There were no significant differences in viral levels in serum and liver between the two groups. To further investigate virological differences between the two groups with regard to degree of genetic variability and the form in the circulation, we performed the PCR‐single strand conformation polymorphism (PCR‐SSCP) of the hypervariable region 1 and the immunoprecipitation analyses. PCR‐SSCP showed that the anti‐HCV‐positive subjects with normal liver biochemical values had quasispecies nature of the HCV genome similar to the patients with chronic hepatitis C, and the immunoprecipitation analysis showed that the virus circulated both in immune complexes and in the free form in both groups. These findings indicated that both groups had similar virological characteristics but showed different patterns of serum aminotransferase levels and histological findings, suggesting that the two groups may have different immune responses to the virus. (Hepatology 1995; 22: 418–425.)

Hepatic Hepatitis C Virus RNA as a Predictor of a Long-Term Response to Interferon-α Therapy

Interferon- therapy has been reported [1, 2] to be beneficial in approximately 50% of patients with chronic hepatitis Cvirus (HCV) infection. However, approximately half of the patients who responded to therapy with a decrease of serum aminotransferase levels into the normal range had a relapse after therapy (short-term response), whereas the remaining patients showed sustained normal serum aminotransferase levels after therapy (long-term response). The reverse transcriptase-polymerase chain reaction (PCR) can now be used to test for the presence of HCV; therefore, we used PCR to investigate virologic events in patients during and after interferon- therapy [3-8]. Several groups [9-14] have investigated factors that might predict a sustained response to interferon therapy or a subsequent relapse after therapy in patients with chronic hepatitis C. However, such factors have not yet been clearly identified. Moreover, it is not well documented whether HCV can be completely eradicated in patients who had a long-term response to interferon therapy. To identify predictors of a long-term response and to determine whether HCV infection can be eradicated in patients with a long-term response, we investigated the clinical, virologic, and histologic effects of interferon- therapy during and 1 year after therapy in patients who had either a long-term or a short-term response to therapy. Methods Ninety patients with chronic hepatitis C were treated with human natural lymphoblastoid interferon- (Sumiferon, Sumitomo Pharmaceutical, Osaka, Japan) given intramuscularly at a dose of 6 MU three times a week for 6 months during 1991 and 1992. Written informed consent was obtained before the initiation of therapy. All patients had had increased serum aminotransferase levels (at least 1.5 times the upper limit of the normal range) for at least 6 months and had histologically proven chronic hepatitis. All patients had antibody to HCV (II) and detectable HCV RNA in serum, and all were negative for hepatitis B surface antigen. Patients with evidence of other forms of liver disease and with decompensated liver disease were excluded. Each month, all study patients were seen and tested for liver biochemical variables during and after therapy. During and at the end of interferon- therapy, serum alanine aminotransferase levels decreased into the normal range in 47 patients (52%, defined as responders); however, in the remaining 43 patients (48%, defined as nonresponders), the levels did not return to normal. The 47 responders were followed for more than 1 year after the cessation of therapy and were assigned to one of two groups according to the outcome after therapy: long-term responders and short-term responders. Long-term responders were defined as patients whose levels of serum alanine aminotransferase became normal during and at the end of therapy and who had sustained normal levels for more than 1 year after cessation of therapy. Short-term responders were defined as patients whose serum aminotransferase levels became normal during and at the end of therapy but whose levels increased again within 1 year after the cessation of therapy. Of the 47 responders, 22 were long-term responders and 25 were short-term responders. All patients gave written informed consent before they were enrolled in our study. Serum and liver biopsy samples (paired and 1-year follow-up) were tested for HCV RNA by reverse transcriptase-PCR using primers from the 5-noncoding region of the HCV genome. Portions of liver tissue weighing approximately 20 mg or more were immediately frozen in liquid nitrogen and were stored at 80C until used. Histologic diagnosis was made using both hematoxylin and eosin and reticulum-stained liver sections according to international standards, and these specimens were also graded using the numerical scoring system of Knodell and colleagues [15] by a pathologist who was blinded to the status of the patients. Nonspecific reactive hepatitis was defined as minimal inflammatory infiltration in portal tracts. Detection of Hepatitis C Virus RNA Hepatitis C virus RNA in the serum and the liver was assayed by the reverse transcriptase-double PCR technique using primers from the 5-noncoding region of the HCV genome, as previously described [5, 16, 17]. In brief, total RNA was extracted from frozen liver tissue by using guanidinium-phenol-chloroform methods [18]. The amount of nucleic acid extracted from the liver was determined by spectrophotometry, and 1.5 g of total cellular nucleic acid was subjected to reverse transcription and amplification with nested primers in a double-PCR technique. Nucleic acid extracted from 50 L of serum was dissolved in 50 L of RNase-free water, 5 L of which was used for reverse transcriptase-PCR. The oligonucleotide primers used were synthesized from the highly conserved 5-noncoding region of the HCV genome [19]: For the first cycle of the PCR assay, 5-GAGGAACTACTGTCTTCACGCAG-3 (sense; nucleotide numbers 37 to 59) and 5-CATGGTGCACGGTCTACGAGACC-3 (antisense; nucleotide numbers 310 to 332) primers were used. For the second cycle of the PCR assay, 5-GAGGAACTACTGTCTTCACGCAG-3 (sense; nucleotide numbers 37 to 59, the same sense primer used for the first cycle) and 5-GCACTCGCAAGCACCCTATCAGG-3 (antisense; nucleotide numbers 280 to 302) primers were used. The products of the PCR reaction were analyzed by electrophoresis in a 1% agarose gel containing ethidium bromide, 0.5 g/mL, and were visualized under ultraviolet light. The size of the expected HCV DNA product was 265 base pairs. The relative sensitivity of the PCR assay was determined by testing the synthetic HCV RNA; the assay detected 50 molecules of the synthetic HCV RNA. The end-point titer of HCV RNA was estimated in a semi-quantitative fashion by testing serial 10-fold dilutions of the nucleic acids extracted from 50 L of serum. Serial samples from individual patients were tested in the same run, and samples with known titers of HCV RNA were also tested as internal controls. Throughout our study, carryover PCR contamination was avoided by using techniques described by Kwok and Higuchi [20]. For each assay, known positive and negative serum samples were analyzed as controls in parallel with test samples. A PCR assay was also done using all the components except the reverse transcriptase enzyme, as well as the known negative and positive controls, to exclude the possibility of contamination by PCR products. In all the liver biopsy specimens, the integrity of the extracted RNA was shown by amplification of -actin messenger RNA as an internal control. Hepatitis C Virus Genotyping The HCV genotype was determined by reverse transcriptase-double PCR with type-specific primers in the core domain according to the methods of Okamoto and colleagues [21]. In brief, extracted RNA from 50 L of serum was reverse transcribed to complementary DNA with 10 pmol of the external antisense primer and with 100 units of Moloney murine leukemia virus-reverse transcriptase in a final volume of 10 L. Forty L of the first PCR solution containing 10 pmol of the external sense primer and 1 unit of Taq polymerase was added to the reverse transcriptase reaction solution in a final volume of 50 L. This mixture was amplified with 35 cycles of PCR (94 C for 1 minute, 55 C for 1 minute, and 72 C for 2 minutes). A 1-L aliquot of the amplified first PCR solution was added to the nested PCR solution with 6 pmol of the universal sense primer and with 3 pmol of each of the type-specific antisense primers, and 30 cycles of the nested PCR was done in the same program as the first PCR. A 20-L aliquot of the amplified nested PCR solution was analyzed by electrophoresis on 2.5% agarose gels containing ethidium bromide (0.5 g/mL) or on 4% agarose gels (NuSieve 3:1 Agarose, FMC Bioproducts, Rockland, Maine), followed by ethidium bromide staining. According to this method, type I specific DNA fragments were detected as a 49-base pair band; type II, type III, and type IV specific fragments were detected as 144-, 174-, and 123-base pair bands, respectively. Antibodies to Hepatitis C Virus Antibodies to HCV were assayed by HCV enzyme immunoassay (II) and by HCV passive hemagglutination second-generation kits (Dinabott, Tokyo, Japan) according to the manufacturers instructions. Statistical Analysis The chi-square test or the Fisher exact test was used for the statistical analysis of comparisons of two group frequencies. Where appropriate, laboratory features, titers of HCV RNA, and histologic activity index scores were compared by the Student t-test and by the paired t-test. Multivariate assessment by logistic regression was done to determine which variables were independently associated with a subsequent relapse after therapy. A multiple logistic regression model was built using 11 variables: 8 variables before therapy (age, sex, history of blood transfusion, HCV genotype, alanine aminotransferase value, serum HCV RNA levels, hepatic HCV RNA levels, and histologic activity index scores) and 3 variables at the end of therapy (the presence of serum HCV RNA, the presence of hepatic HCV RNA, and histologic activity index scores). Each variable was transformed into categorical data consisting of two or three simple ordinal numbers for univariate and multivariate analyses. All factors that were at least marginally associated with a future relapse (P < 0.05) were tested by multivariate logistic regression analysis. A P value of less than 0.05 was considered to be significant. The Statistical Package for the Social Sciences (SPS), version 4.0 (SPS, Chicago, Illinois), was used for all statistical computations. Results The characteristics of long-term and short-term responders are shown in Table 1. No significant differences were noted between the two groups in mean age, incidence of history of blood transfusion, or mean initial alanine aminotransferase levels. However, more women were in the long-term group than in the

Peginterferon α‐2a (40 KD) plus ribavirin for the treatment of chronic hepatitis C in Japanese patients

Background:  The efficacy and safety of peginterferon alpha‐2a (40 KD) (peg‐IFNα‐2a) plus ribavirin has not been reported for Japanese patients with chronic hepatitis C. The aim of this study was to evaluate this combination in treatment‐naïve patients and in non‐responders or relapsers to interferon monotherapy.

Varying incidence of cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis C responding differently to interferon therapy

To determine whether interferon (IFN) therapy can reduce incidence of the development of cirrhosis and hepatocellular carcinoma equally in patients with chronic hepatitis C virus (HCV) who responded differently to therapy, a retrospective analysis of 250 patients treated with IFN was conducted.

Virological Response in Patients with Hepatitis C Virus Genotype 1b and a High Viral Load

Background and objective:In Japan the prevalence of the hepatitis C virus (HCV) antibody is highest in the elderly population. Therefore, it is important for elderly patients to undergo interferon (IFN) therapy. In patients with HCV genotype 1b and a high viral load, the sustained virological response (SVR) rate is lower in older compared with younger patients receiving combination antiviral therapy. In addition, inadequate adherence to combination therapy is often seen in elderly patients, and is associated with reduced response rates. The aim of this retrospective analysis was to evaluate the effects of host-related factors (i.e. sex, age, baseline HCV RNA level, bodyweight and fibrosis stage) and peginterferon (PEG IFN)-α-2a plus ribavirin dose reductions on SVR rates.Methods:A total of 192 treatment-naive patients with a HCV genotype 1b infection and a high viral load were included in the analysis. Patients had been enrolled into a phase III trial of 48 weeks of treatment with PEG IFN-α-2a plus ribavirin or PEG IFN-α-2a plus placebo. All patients were evaluated for effect of drug exposure on SVR. In addition, the impact of host-related factors or dose reductions on SVR was assessed.Results:Approximately 30% of patients were considered elderly (≥60 years of age). The overall SVR rate was significantly higher in patients treated with combination therapy versus monotherapy (59.4% vs 24.0%, p < 0.001). Attainment of an SVR following combination therapy was not influenced by any factor evaluated in the analysis, although elderly males were associated with decreased SVR rates. Younger age (odds ratio [OR] 1.081; 95% CI 1.125, 1.034; p = 0.0009), lower baseline HCV RNA levels (OR 1.003; 95% CI 1.006, 1.001; p = 0.006) and a severe fibrosis stage (F3/4) [OR 6.194; 95% CI 1.037, 37.000; p = 0.0455] significantly increased the likelihood of achieving an SVR with monotherapy. In the combination therapy group, patients maintaining a full dosage schedule of PEG IFN-α-2a and ribavirin and those requiring dose reductions of either study drug had similar SVR rates (64.5% vs 61.9%). However, the SVR rate was reduced to 33.3% among patients who discontinued combination therapy. Three out of the 31 patients who received the full dosage schedule were elderly patients. In addition, of the 15 patients who discontinued combination therapy, three were <50 years of age and six were ≥60 years of age. The SVR rate was reduced in patients with cumulative PEG IFN-α-2a and ribavirin doses of <60%; the majority of these patients were elderly.Conclusion:The attainment of an SVR following PEG IFN-α-2a plus ribavirin combination therapy was not influenced by any of the host-related factors evaluated in this analysis, although elderly males were associated with a decreased SVR rate. Younger age, male sex and lower baseline HCV RNA levels significantly increased the likelihood of achieving an SVR with monotherapy. In addition, dose reductions appeared to have a negative impact on SVR in elderly patients. Therefore, it is important to minimize PEG IFN-α-2a and ribavirin dose reductions by effectively managing treatment-related adverse events in elderly patients.

Chronic active hepatitis with histological features of primary biliary cirrhosis

SummaryA 51-year-old woman was evaluated because of Raynauds phenomenon, Sjögrens syndrome, and general malaise. There was neither skin itching nor jaundice. Endoscopic retrograde cholangiopancreatography showed a normal extrahepatic as well as intrahepatic biliary tree. Serum GOT and GPT fluctuated with episodes of marked increases. The alkaline phosphatase was slightly increased and total cholesterol was normal. There were marked increases of IgG and IgM. AMA was positive at a titer of 1:320, which was measured by an indirect immunofluorescence method. PBC-specific AMA (anti-M2) was positive, but mixed-form AMA (anti-M4) negative. An LE-cell test, ASMA, ANA, and anti-DNA antibody were all positive on several repeated tests. A wedge liver biopsy tissue showed mixed features of PBC and CAH. A diagnosis of CAH was made on the basis of the clinical, serological, and morphologic findings. The patient responded well to prednisolone treatments with prominent improvement of her symptoms and liver function tests. Subsequently, AMA fell to undetectable levels by indirect immunofluorescence method.

Polymorphisms of the core, NS3, and NS5A proteins of hepatitis C virus genotype 1b associate With development of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the common sequelae of hepatitis C virus (HCV) infection. It remains controversial, however, whether HCV itself plays a direct role in the development of HCC. Although HCV core, NS3, and NS5A proteins were reported to display tumorigenic activities in cell culture and experimental animal systems, their clinical impact on HCC development in humans is still unclear. In this study we investigated sequence polymorphisms in the core protein, NS3, and NS5A of HCV genotype 1b (HCV‐1b) in 49 patients who later developed HCC during a follow‐up of an average of 6.5 years and in 100 patients who did not develop HCC after a 15‐year follow‐up. Sequence analysis revealed that Gln at position 70 of the core protein (core‐Gln70), Tyr at position 1082 plus Gln at 1112 of NS3 (NS3‐Tyr1082/Gln1112), and six or more mutations in the interferon/ribavirin resistance‐determining region of NS5A (NS5A‐IRRDR≥6) were significantly associated with development of HCC. Multivariate analysis identified core‐Gln70, NS3‐Tyr1082/Gln1112, and α‐fetoprotein (AFP) levels (>20 ng/L) as independent factors associated with HCC. Kaplan‐Meier analysis revealed a higher cumulative incidence of HCC for patients infected with HCV isolates with core‐Gln70, NS3‐Tyr1082/Gln1112 or both than for those with non‐(Gln70 plus NS3‐Tyr1082/Gln1112). In most cases, neither the residues at position 70 of the core protein nor positions 1082 and 1112 of the NS3 protein changed during the observation period. Conclusion: HCV isolates with core‐Gln70 and/or NS3‐Tyr1082/Gln1112 are more closely associated with HCC development compared to those with non‐(Gln70 plus NS3‐Tyr1082/Gln1112). (HEPATOLOGY 2013;58:555‐563)

The clinical significance of core promoter and precore mutations during the natural course and interferon therapy in patients with chronic hepatitis B

Abstract OBJECTIVE: We aimed to determine the clinical significance of mutations in core promoter and precore regions in chronic hepatitis B. We investigated changes in these mutations during the natural course and interferon therapy in patients with chronic hepatitis B. METHODS: A total of 93 patients with hepatitis B virus surface antigen were divided into four groups according to hepatitis B e antigen (HBeAg)/anti-HBe status and serum aminotransferase levels. Group I (n = 16) comprised HBeAg-positive patients with normal aminotransferase levels, group II (n = 31) HBeAg-positive patients with elevated aminotransferase levels, group III (n = 30) anti-HBe–positive patients with normal aminotransferase levels, and group IV (n = 16) anti-HBe–positive patients with elevated aminotransferase levels. All patients of group II and seven of group IV were treated with interferon. Three serial serum samples per untreated patient and eight samples per treated patient were tested for HBV DNA levels and core promoter and precore mutations by polymerase chain reaction combined with restriction fragment length polymorphism, and some were cloned and sequenced. RESULTS: Core promoter mutation was found in 38% of group I, 74% of group II, 97% of group III, and 100% of group IV. Precore mutation was found in 6% of group I, 90% of group II, and 100% of groups III and IV. The HBV DNA levels were significantly higher in groups I, II, IV, and III, in that order. Serial determination of these two mutations and viral levels showed that the core promoter mutation appeared to occur first, followed by a completion of the precore mutation along with a decrease in viral levels in patients who seroconverted to anti-HBe after interferon therapy. Interferon therapy suppressed both precore wild- and mutated-type viral levels equally. However, it did not induce any specific mutations. CONCLUSIONS: Core promoter mutation appeared to develop or complete first, followed by completion of the precore mutation, and the virus with these two mutations seemed to be the form to persist in the natural course of chronic hepatitis B. The clinical significance of these mutations appeared to be profoundly associated with the viral levels.

The clinical value of grading and staging scores for predicting a long-term response and evaluating the efficacy of interferon therapy in chronic hepatitis C

BACKGROUND/AIMS To determine the clinical usefulness of a new histological scoring system (grading and staging scores) for predicting a long-term response to interferon therapy and evaluating the efficacy of therapy, we examined biochemical, virological and histological findings during and 1 year after interferon therapy in 109 patients with chronic hepatitis C. METHODS Hepatitis C virus RNA was assayed by reverse transcriptase polymerase chain reaction, hepatitis C virus genotype was determined by reverse transcriptase polymerase chain reaction using type-specific primers, and histological grading and staging scores were determined according to a newer scoring system. RESULTS The patients were divided into two groups according to the outcome of serum alanine aminotransferase levels and HCV RNA level during and after therapy: 31 long-term responders whose serum aminotransferase level became and remained normal for 1 year after therapy with undetectable HCV RNA in serum and liver and 78 non-responders whose aminotransferase levels did not normalize during therapy or rose again after therapy. Before therapy, the long-term responders had significantly lower viral levels, lower incidence of genotype 1b, and lower staging scores than those of the non-responders. There was no significant difference in grading score between the long-term and non-responders. Multivariate analysis showed that the viral level and genotype are more important predictors of a long-term response than the staging score. Both grading and staging scores decreased significantly at the end of therapy in both the long-term and non-responders. The 1-year follow-up liver biopsy examination in the long-term responders showed that the grading score, but not the staging score, continued to decrease significantly. CONCLUSIONS These findings suggest that: (1) the staging score, but not the grading score, appears to be associated with a long-term response, but the viral level and genotype are more important predictors than the staging score; and (2) both the grading and staging scores decreased significantly with interferon therapy, but the staging score appeared to take longer to improve than the grading score.

In vivo interferon system assessed by 2′‐5′ oligoadenylate synthetase activity in chronic hepatitis C virus patients treated with pegylated interferon and ribavirin

Aim:  2′,5′ oligoadenylate synthetase (2‐5AS), an enzyme induced by interferon, is an accurate indicator of the antiviral effect of interferon. We measured it during pegylated interferon based therapies in patients with chronic hepatitis C virus (HCV) in order to determine the dynamics of antiviral status in vivo and the relationship between the response to exogenous interferon and the outcome of therapy.