Akihito Tsubota

Disease progression and hepatocellular carcinogenesis in patients with chronic viral hepatitis: a prospective observation of 2215 patients

BACKGROUND/AIMS/METHODS The aim of this study was to elucidate the rate of development to cirrhosis and the rate of appearance of hepatocellular carcinoma in chronic viral hepatitis and to assess the risk factors for the development of disease in 2215 consecutive patients with viral hepatitis who were prospectively studied for a median observation period of 4.1 years. RESULTS The rates of development to cirrhosis were 7.6%, 21.7%, and 32.2%, at the 5th, 10th, and 15th year, respectively. The carcinogenesis rates were 3.4%, 10.5%, and 22.4% at the 5th, 10th, and 15th year, respectively. The appearance rates of cancer in 645 patients with only hepatitis B surface antigen and in 1500 patients with only anti-hepatitis C virus antibodies were 2.1% and 4.8% at the 5th year, 4.9% and 13.6% at the 10th year, and 18.8% and 26.0% at the 15th year, respectively. The proportional hazard model identified that the amount of alcohol intake (p= 0.0002) and the indocyanine green retention rate (p= 0.022) were independently associated with carcinogenesis in hepatitis type B; and stage of hepatitis (p<0.0001), gamma-glutamyl transpeptidase (p= 0.0046), history of blood transfusion (p=0.0093), albumin (p=0.012), and amount of alcohol intake (p= 0.031) were independently associated with the carcinogenesis rate in hepatitis type C. Although the severity of portal fibrosis was closely correlated with the future disease development and carcinogenesis in chronic hepatitis C, it was not a good predictor in chronic hepatitis B. CONCLUSION These epidemiological results suggest that there are some differences in the activity and modes of disease progression and cancer promotion between hepatitis B virus infection and hepatitis C virus infection.

A novel magnetic crystal-lipid nanostructure for magnetically guided in vivo gene delivery.

Cancer gene therapy requires a safe and effective gene delivery system. Polymer- and lipid-coated magnetic nanocrystals have been used to deliver silencing RNA, but synthesizing these magnetic vectors is difficult. Here, we show that a new nanoparticle formulation can be magnetically guided to deliver and silence genes in cells and tumours in mice. This formulation, termed LipoMag, consists of an oleic acid-coated magnetic nanocrystal core and a cationic lipid shell. When compared with the commercially available PolyMag formulation, LipoMag displayed more efficient gene silencing in 9 of 13 cell lines, and better anti-tumour effects when systemically administered to mice bearing gastric tumours. By delivering an optimized sequence of a silencing RNA that targets the epidermal growth factor receptor of tumour vessels, the intended therapeutic benefit was achieved with no evident adverse immune reaction or untoward side effects.

Genotypic subtyping of hepatitis C virus

Four subtypes of hepatitis C virus (HCV), Pt(I), K1(II), K2a(III) and K2b(IV), have been suggested based on the nucleotide sequences of the non‐structural (NS) 5 region. A fifth subtype from Japanese patients, Tr(V), which shows a less than 68% homology in nucleotide sequence when compared with other subtypes has been identified. A one‐step method which enables a quick determination of subtype using polymerase chain reaction with a mixed primer set deduced from the sequence of each subtype has been developed. Using this technique, the subtypes of 418 out of 478 Japanese patients (87.4%) were determined. The incidence of each subtype in Japan was as follows: K1(II), 307 (73.4%); K2a(III), 74 (17.7%); K2b(IV), 28 (6.7%); and Tr(V), 3 (0.7%). This one‐step subtyping technique should be useful for studying the epidemiology or biology of the HCV.

Interferon decreases hepatocellular carcinogenesis in patients with cirrhosis caused by the hepatitis B virus

To elucidate the influence of long term interferon administration on the rate of occurrence of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV)‐related cirrhosis, the authors analyzed 313 consecutive patients with cirrhosis.

Histological changes in liver biopsies after one year of lamivudine treatment in patients with chronic hepatitis B infection.

BACKGROUND/AIMS The aim of this study was to examine the histological changes in liver biopsies induced by 52 weeks of lamivudine therapy in patients with e-antigen positive and e-antigen negative chronic hepatitis B infection. METHODS Twenty patients were enrolled into this open-label study. All patients had a liver biopsy within the 4 weeks before starting lamivudine therapy. Lamivudine was given orally at a dose of 100 mg OD for 52 weeks. A second liver biopsy was taken for comparison at the end of week 52. Blinded biopsies were evaluated by a histopathologist and scored according to Knodells histology activity index (HAI). RESULTS Ninety-five percent (19/20) patients had a reduction of their hepatic necroinflammatory HAI score (components 1 through 3) by > or =2 points at the end of 52 weeks of lamivudine therapy compared to their pretreatment values. Not only were improvements in necroinflammatory activity observed, but 7/20 (35%) of patients had improvement in fibrosis. This histologic improvement was independent of the presence or absence of e-antigen. CONCLUSIONS Significant improvements in liver histology can be obtained in the majority of patients when they are treated with lamivudine for 1 year.

Factors contributing to ribavirin dose reduction due to anemia during interferon alfa2b and ribavirin combination therapy for chronic hepatitis C.

BackgroundRecent studies indicate that combination therapy with ribavirin and interferon alfa2b (IFNΑ2b) is effective for chronic hepatitis C virus (HCV) infection. However, reversible hemolytic anemia is a common side effect of this therapy.MethodsWe determined those factors that contribute to ribavirin dose reduction due to anemia during this treatment by using multiple logistic regression analysis in Japanese patients. The study included 123 patients with chronic hepatitis C (85 male, 38 female; mean age, 50 years; range, 20–70 years), who received 24-week combination therapy. All patients were treated with IFNΑ2b daily for 2 weeks, followed by three times weekly dosing for 22 weeks, with oral ribavirin twice daily, at a total daily dose of 600 or 800 mg.ResultsOf the 123 patients, 34 patients required dose reduction of ribavirin, and 78 patients required no dose reduction. Overall, 20 patients discontinued. On univariate analysis, reduction of the ribavirin dose correlated significantly with pretreatment hemoglobin (Hb) levels of less than 14 g/dl, female sex, and patient age 55 years or older. On multivariate analysis, pretreatment Hb of less than 14 g/dl level and age 55 years or older were significantly associated with ribavirin dose reduction. The hazard ratios were 3.56 (95% confidence interval [CI], 1.48–8.53) for pretreatment Hb levels of less than 14 g/dl, and 2.50 (95% CI, 1.05–5.94) for age 55 years or more.ConclusionsBecause patient age of 55 years or more, and Hb levels of less than 14 g/dl are significant factors that influence ribavirin-induced hemolytic anemia, more careful monitoring is necessary during combination therapy for patients with these risk factors.

Molecular analysis of intraspousal transmission of hepatitis C virus

BACKGROUND/AIMS Although intraspousal transmission of hepatitis C virus (HCV) has been speculated, there is no direct evidence. METHODS To investigate whether transmission of HCV occurs by this route, 295 spouses of persons diagnosed with HCV were studied. Of these, 25 (8.8%) tested positive for anti-HCV. Next, the HCV genotype was determined by polymerase chain reaction using a mixed primer set, and cDNA was obtained in 17 of the 25 couples for comparison of the genotypes. RESULTS Of these 17, 14 (82.4%) spouses were shown to be infected with HCV of the same genotype. Analysis of the nucleotide sequences of putative E1 gene of eight couples having the same HCV genotype revealed that five couples had remarkably high nucleotide sequence homologies (> 97%), whereas samples obtained from the remaining three couples showed relatively low homology (91-92%). Nucleotide sequence homologies were significantly higher between spouses than between non-spouse pairs of isolates. Phylogenetic analysis using the neighbor-joining method suggests that infection in these five couples probably occurred after marriage. Furthermore, none of the five couples had shared other possible transmission routes such as intravenous drug use, dental treatment or acupuncture. CONCLUSION These data strongly suggest the occurrence of intraspousal transmission of HCV.

Efficacy of lamivudine therapy and factors associated with emergence of resistance in chronic hepatitis B virus infection in Japan.

Objective: Several reports have examined the efficacy of long-term lamivudine therapy and the risk factors involved in emergence of viral resistance in Japanese patients with hepatitis B virus (HBV) infection. However, the patient cohorts in such studies are relatively small. Methods: We analyzed 234 chronically HBV-infected Japanese patients who were treated with lamivudine for more than 12 months. They comprised patients with HBV genotype A (n = 8), genotype B (n = 21), genotype C (n = 203) and other HBV genotypes (n = 2). Results: In most patients, lamivudine resulted in normalization of alanine transaminase (ALT) levels at 6 and 12 months, and suppression of serum HBV DNA to undetectable levels by the branched chain DNA probe assay (bDNA). Rates of ALT normalization and non-detection of HBV DNA were higher among patients with genotype B than genotype C disease. The proportions of patients who achieved HBeAg loss were 27, 42 and 45% after 6 months, 1 year and 2 years, respectively. The emergence of mutations was not different among genotypes A, B and C by the Kaplan-Meier method. Multivariate analyses identified high HBV DNA level (bDNA ≧100 MEq/ml) as an independent factor associated with emergence of the YMDD motif mutation in all patients. Among patients with genotype C disease, which is the predominant HBV genotype in Japan, multivariate analysis also identified high HBV DNA level and HBeAg positivity as factors associated with emergence of resistance. Conclusion: Patients exhibiting these factors at the commencement of lamivudine treatment must be monitored carefully at regular intervals for emergence of viral resistance.

Diagnostic Problems Caused by HBsAg Mutants – A Consensus Report of an Expert Meeting

A panel of 16 experts from 9 countries convened on April 14 at Schloss Reinhartshausen near Wiesbaden in Germany, to discuss the diagnostic significance of mutants, variants and genotypes of hepatitis B virus (HBV). Since the description of Australia antigen in 1965 and the subsequent observation that it was the envelope of the HBV and now designated hepatitis B surface antigen (HBsAg), this lipoprotein has been a mainstay in the diagnosis of HBV infections. HBsAg tests are used routinely in the diagnosis of acute and chronic liver disease, the screening of blood or organ donors and the surveillance of persons at risk to acquire or to transmit HBV. Current immunoassays for HBsAg are very specific and sensitive (both 199%) and are usually able to detect !0.5 ng HBsAg/ml serum. Their performance is validated in extensive trials before licensing and their detection limit is assayed with an International Standard for HBsAg. An immanent problem of virology is the variability of viruses. Due to the low fidelity of the viral nucleic acid polymerases and the high replication rates, virtually all nucleotide positions of a viral genome can be mutated within a relatively short time. However, the viability of the virus and its adaptation to the host allow the selection and outgrowth of only a limited number of mutants. The survival strategy of HBV in the population is mainly based on induction of immune tolerance and persistence of high viremia. In this state of infection the existing viral genome is favored whereas mutants are less fit and selected against or may be subject to an immune reaction and preferably eliminated. In fact, most of the HBsAg carriers in Germany have a very similar S gene sequence. However, under selective pressure the virus can express many different viable HBsAg mutants. Summary of the Presentations

Single nucleotide polymorphism of the MxA gene promoter influences the response to interferon monotherapy in patients with hepatitis C viral infection

Summary. The biological activity of interferon (IFN) is mediated by the induction of intracellular antiviral proteins, such as 2′–5′ oligoadenylate synthetase, dsRNA‐activated protein kinase and MxA protein. Among these, MxA protein is assumed to be the most specific surrogate parameter for IFN action. This study was performed to elucidate whether a single nucleotide polymorphism (SNP) (G/T at nt‐88) in the promoter region of the MxA gene influences the response to IFN therapy in patients with chronic hepatitis C virus (HCV) infection. Polymorphisms of the MxA gene in 235 HCV patients were determined by polymerase chain reaction‐restriction fragment length polymorphism. The frequency of SNP was compared between sustained‐responders (n = 78) and nonresponders (n = 157), as determined by biochemical and virological responses to IFN. Multivariate analysis showed that among all patients, HCV genotype, HCV RNA level and the SNP of the MxA gene were independent and significant determinants of the outcome of IFN therapy [odds ratio 3.8 (95% confidence interval 2.0–7.0), P < 0.0001; 0.27 (0.15–0.50), P < 0.0001; 1.8 (1.0–3.4), P = 0.0464, respectively]. Furthermore, among patients with a low viral load (≤2.0 Meq/mL), MxA‐T‐positive patients were more likely to show a sustained response compared with MxA‐T‐negative patients [2.87 (1.3–6.3); 62%vs 36%; P = 0.0075]. Our findings suggested that the SNP of the MxA gene is one of the important host factors that independently influences the response to IFN in patients with chronic HCV infection, especially those with a low viral load.