Akihiro Furusaka

Hepatitis C Virus JFH-1 Strain Infection in Chimpanzees Is Associated With Low Pathogenicity and Emergence of an Adaptive Mutation

The identification of the hepatitis C virus (HCV) strain JFH‐1 enabled the successful development of infectious cell culture systems. Although this strain replicates efficiently and produces infectious virus in cell culture, the replication capacity and pathogenesis in vivo are still undefined. To assess the in vivo phenotype of the JFH‐1 virus, cell culture–generated JFH‐1 virus (JFH‐1cc) and patient serum from which JFH‐1 was isolated were inoculated into chimpanzees. Both animals became HCV RNA‐positive 3 days after inoculation but showed low‐level viremia and no evidence of hepatitis. HCV viremia persisted 8 and 34 weeks in JFH‐1cc and patient serum–infected chimpanzees, respectively. Immunological analysis revealed that HCV‐specific immune responses were similarly induced in both animals. Sequencing of HCV at various times of infection indicated more substitutions in the patient serum–inoculated chimpanzee, and the higher level of sequence variations seemed to be associated with a prolonged infection in this animal. A common mutation G838R in the NS2 region emerged early in both chimpanzees. This mutation enhances viral assembly, leading to an increase in viral production in transfected or infected cells. Conclusion: Our study shows that the HCV JFH‐1 strain causes attenuated infection and low pathogenicity in chimpanzees and is capable of adapting in vivo with a unique mutation conferring an enhanced replicative phenotype. (HEPATOLOGY 2008.)

Expression of insulin receptor substrate-1 in hepatocytes : an investigation using monoclonal antibodies

To investigate the expression and subcellular distribution of insulin receptor substrate-1 in hepatocytes, which are major targets of insulin along with muscle and adipose tissue, we obtained monoclonal antibodies by immunizing mice with a fusion protein consisting of the C-terminal portion of the human insulin receptor substrate-1 and glutathione-S-transferase. Two of the monoclonal antibodies (designated as 7B3 and 6G5) were found to be useful for immunohistochemical studies. Using 6G5 we demonstrate a high level of expression of insulin receptor substrate-1 in liver cirrhosis hepatocytes and variable expression in hepatocellular carcinoma cells. These results suggest that insulin receptor substrate-1 may play a role in liver regeneration during cirrhosis and that an insulin signaling cascade may be involved in hepatocarcinogenesis.

Hepatocyte growth factor stimulates the growth of hepatocytes in altered foci and hyperplastic nodules induced by chemical hepatocarcinogens in rats

Abstract Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes. On the other hand, HGF also has cytostatic activity for several liver cancer cell lines. However, it is not known whether HGF can stimulate the growth of preneoplastic and neoplastic or precarcinomatous hepatocytes. Therefore, we investigated the ability of HGF to stimulate DNA synthesis by hepatocytes in altered foci (preneoplastic lesion) and hyperplastic nodules (neoplastic or precarcinomatous lesion) compared with those of transforming growth factor-alpha (TGF-α) and epidermal growth factor (EGF). Altered foci and hyperplastic nodules were induced in rats by the method of Solt and Farber which involved administration of diethylnitrosamine (DEN) and 2-acetylaminofluorene (2AAF), and partial hepatectomy. Hepatocytes were cultured in medium containing HGF (2, 5 or 10 ng/ml), TGF-α (20 ng/ml) or EGF (20 ng/ml), and autoradiography with 3H-thymidine incorporation was performed. Hepatocytes derived from these lesions were identified by positive immunostaining for glutathione S-transferase, placental form (GST-P). In altered foci, labeling indices (LI) in GST-P positive cells (altered focal cells) were 18.5% in the control (no growth factors added), and 76.8, 39.6 and 37.8%, in the hepatocytes treated with HGF (10 ng/ml), TGF-α and EGF, respectively. In hyperplastic nodules, LI in GST-P positive cells (hyperplastic nodular cells) were 10.8% in the control, and 66.2, 28.1 and 29.1% in hepatocytes treated with HGF (10 ng/ml), TGF-α and EGF, respectively. These data show that HGF and DNA-synthesizing activity in the preneoplastic and neoplastic or precarcinomatous hepatocytes induced by the chemical hepatocarcinogens.