Hiroshige Kojima

Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, trichostatin A, on human hepatoma cells.

Objective: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA. Methods: To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 antibody. Results: The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed the potential of TSA to induce apoptosis. The microarray analysis revealed that 8 genes including collagen type 1, α2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, α7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio >2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels of acetylated histones in these upregulated genes. Conclusions: This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in hepatoma cells.

Prevalence of diabetes mellitus and insulin resistance in patients with chronic hepatitis C: comparison with hepatitis B virus-infected and hepatitis C virus-cleared patients.

Background/Aims: Our aim was to evaluate the relationship between hepatitis C virus (HCV) infection and development of diabetes mellitus (DM) or insulin resistance (IR) in comparison with hepatitis B virus (HBV) infection and eradication of HCV infection by interferon treatment.

Fas polymorphisms influence susceptibility to autoimmune hepatitis.

BACKGROUND AND AIMS:Genetic factors associated with autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC), immune-mediated chronic inflammatory liver diseases of unknown etiology, remain to be elucidated. Polymorphisms of the gene encoding Fas have been linked to a variety of autoimmune diseases. We hypothesized that Fas gene polymorphisms might be genetic markers for AIH and PBC.METHODS:To determine the frequency and significance of Fas polymorphisms in patients with AIH and PBC, 74 Japanese AIH patients, 98 Japanese PBC patients, and 132 ethnically matched control subjects were investigated by the use of the Taqman assay.RESULTS:We found significant differences between AIH patients and controls in allele frequencies of Fas-670 (p= 0.009), Fas IVS (intervening sequence) 2nt176 (p= 0.018), Fas IVS3nt46 (p= 0.031), and Fas IVS5nt82 (p= 0.013) polymorphisms. Haplotype analysis revealed that one of the haplotypes, GATGC, was associated with increased AIH prevalence. On the other hand, we found no statistically significant differences between PBC patients and controls in allele frequencies of the Fas polymorphisms genotyped in this study.CONCLUSIONS:These results indicate a genetic link of Fas polymorphisms to the development of AIH. Further studies are needed to determine the genetic factors contributing to the development of AIH.

Spontaneous negativation of serum hepatitis C virus RNA is a rare event in type C chronic liver diseases: analysis of HCV RNA in 320 patients who were followed for more than 3 years

BACKGROUND/AIMS The natural course of hepatitis C virus (HCV) replication in type C liver diseases has not yet been elucidated. The aim of the study was to investigate the spontaneous outcome of the viremia by examining the changes in HCV RNA in patients with chronic type C liver diseases. METHODS Among patients who visited our liver clinic between June 1981 and December 1993, 320 patients with chronic type C liver diseases were followed for at least 3 years and had no history of interferon treatment. HCV RNA was examined by a highly specific reverse transcription-polymerase chain reaction method in paired serum samples obtained from these patients at the beginning and end of follow-up. RESULTS Among the 320 cases, HCV RNA was seropositive in 310 (97%) cases at the beginning of follow-up. Of these 310, HCV RNA remained seropositive in 304 (98%) and became seronegative in six (2%) cases by the end of follow-up. All of these six patients had liver cancer. HCV RNA became seronegative after the patients entered the state of liver failure because of the development of tumors or portal thrombosis. The remaining 10 cases who were seronegative for HCV RNA at the beginning were seropositive at the end of follow-up. Among the 320 cases, serum alanine aminotransferase normalized and remained normal for more than 12 months until the end of follow-up in 11 cases (0.6%/year/case), but none became negative for HCV RNA. CONCLUSIONS Thus, during the natural course of chronic HCV infection, spontaneous negativation of serum HCV RNA seems extremely rare, at least in patients with chronic active hepatitis or cirrhosis of the liver, and may occur primarily at the terminal stage when tumors cause liver failure.

Lower incidence of hepatic failure than hepatocellular carcinoma in Japanese patients with chronic hepatitis C.

Abstract: Background: Previous studies have shown that the development of hepatic failure was found more frequently than that of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C in the United States and European countries. We investigated the status in Japan in a retrospective cohort study.

Hepatitis A viral load in relation to severity of the infection

A correlation between hepatitis A virus (HAV) genomes and the clinical severity of hepatitis A has not been established. The viral load in sera of hepatitis A patients was examined to determine the possible association between hepatitis A severity and HAV replication. One hundred sixty‐four serum samples from 91 Japanese patients with sporadic hepatitis A, comprising 11 patients with fulminant hepatitis, 10 with severe acute hepatitis, and 70 with self‐limited acute hepatitis, were tested for HAV RNA. The sera included 83 serial samples from 20 patients. Viral load was measured by real‐time RT‐PCR. The detection rates of HAV RNA from fulminant, severe acute, and acute hepatitis were 10/11 (91%), 10/10 (100%), and 55/70 (79%), respectively. Mean values of HAV RNA at admission were 3.48 ± 1.30 logcopies/ml in fulminant, 4.19 ± 1.03 in severe acute, and 2.65 ± 1.64 in acute hepatitis. Patients with severe infection such as fulminant hepatitis and severe acute hepatitis had higher initial viral load than patients with less severe infection (P < 0.001). Viremia persisted for 14.2 ± 5.8 days in patients with severe infection and 21.4 ± 10.6 days in those with acute hepatitis after clinical onset (P = 0.19). HAV RNA was detectable quantitatively in the majority of the sera of hepatitis A cases during the early convalescent phase by real‐time PCR. Higher initial viral replication was found in severely infected patients. An excessive host immune response might follow, reducing the viral load rapidly as a result of the destruction of large numbers of HAV‐infected hepatocytes, and in turn severe disease might be induced. J. Med. Virol. 83:201–207, 2011.

Phylogenetic analysis of hepatitis A virus in sera from patients with hepatitis A of various severities

Background: We analysed the association of the 5′ nontranslated region (5′NTR), nonstructural proteins 2B and 2C of the hepatitis A virus (HAV) genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems, in order to align all our data and examine whether genomic differences in HAV are responsible for the range of clinical severities.

Quantitative evaluation of telomerase activity in small liver tumors: analysis of ultrasonography-guided liver biopsy specimens

BACKGROUND/AIMS Telomerase activity which restores the length of telomere repeat arrays is frequently detectable in various malignancies, including hepatocellular carcinoma. The aim of this study was to determine the diagnostic usefulness of the quantitative measurement of telomerase activity in small liver tumors, which has not yet been established. METHODS Fifty-eight liver specimens from tumorous and non-tumorous portions of 29 small liver tumors equal to or less than 3.0 cm were obtained by ultrasonography-guided liver biopsy, and of these, 25 were diagnosed as hepatocellular carcinoma and four as adenomatous hyperplasia. The telomerase activities in these specimens and control specimens were examined quantitatively by telomeric repeat amplification protocol with standard control. RESULTS The mean telomerase activity in cirrhosis without liver tumor was 0.4+/-0.6 (+/-S.D.) arbitrary units (AU) and that in 29 non-tumorous parts of the tumors was 4.5+/-7.4 AU. The mean telomerase activity in 13 tumors equal to or less than 2 cm in diameter was 77.1+/-133.7 AU and that in 16 tumors more than 2 cm was 152.7+/-215.2 AU. The mean telomerase activity in the 4 adenomatous hyperplasias was 5.5+/-4.5 AU; those of hepatocellular carcinoma with Edmondson-Steiner classification I, II, III and IV were 49.6+/-47.4 (n = 10), 240.1+/-273.6 (n = 9), 119.2+/-174.6 (n = 4) and 144.6+/-80.2 (n = 2) AU, respectively. CONCLUSIONS The telomerase activity was significantly higher in hepatocellular carcinoma compared to adenomatous hyperplasia and non-neoplastic tissue, indicating that the quantitation of telomerase activity would be useful for the diagnosis of small hepatocellular carcinoma.

Lamivudine treatment in a patient with hepatitis B virus reactivation after allogenic peripheral bone marrow transplantation

We report the case of a 52-year-old woman with hepatitis B virus (HBV) reactivation during treatment for chronic graft vs. host disease (GVHD) after peripheral bone marrow transplantation (PBSCT) to treat chronic myelocytic leukemia. She was given cyclosporine and prednisolone orally to treat chronic GVHD after PBSCT. Liver dysfunction first developed 25 months after transplantation with the appearance of hepatitis B s antigen (HBsAg), hepatitis B e antigen (HBeAg), and elevation of HBV-DNA up to 4.5 log copies/ml. Retrospective examination of her serum before PBSCT proved negative for HBsAg and HBeAg, and positive for anti-HBsAg, anti-HBeAg, anti-hepatitis B core antigen, and HBV-DNA (2.7 log copies/ml), showing that she was in a state of occult HBV infection. Nucleotide sequences of the HBV genome obtained from her serum showed no core promoter mutations at nt 1762 and 1764 and no pre-core mutation at nt 1896. Polymerase chain reaction-restriction fragment length polymorphism showed that she was infected with HBV genotype B. The administration of lamivudine, a nucleoside analog, improved her liver function and reduced HBV-DNA replication. We conclude that antiviral agents, such as lamivudine, are effective for treating hepatitis B reactivation during immunosuppressive treatment, such as for GVHD. The administration of a nucleoside analog before transplantation should also be considered in the light of HBV genotypes and mutations, even if HBsAg was negative and the viral load was low before transplantation.

Chronic graft-versus-host disease complicated by acute hepatitis B.

We observed a 45-year-old man with acute hepatitis B while receiving treatment of chronic graft-versus-host disease (GVHD) of the liver. When he developed a sudden elevation of serum aminotransferases 17 months after bone marrow transplantation, he was under immunosuppressive therapy consisting of cyclosporin A against chronic GVHD of the liver. Serologic tests for hepatitis B virus (HBV) showed no reactivation but de novo acute infection. The serum levels of aminotransferases after elevation of biliary tract enzymes increased mildly. However, icterus was not observed in his sequential course. A liver biopsy specimen revealed mild acute liver injury accompanied by slight degeneration of bile ducts. It is presumed that owing to immunosuppressive therapy, his liver dysfunction was relatively mild, and the hepatitis became quiescent without becoming serious. On the other hand, the serum of the patient remained hepatitis B surface antigen positive for more than 1 year after the onset of the hepatitis, which suggested chronicity of HBV infection.