Lorenz Theilmann

An estimated 170 million persons worldwide are infected with hepatitis C virus (HCV), a major cause of chronic liver disease. Despite increasing knowledge of genome structure and individual viral proteins, studies on virus replication and pathogenesis have been hampered by the lack of reliable and efficient cell culture systems. A full-length consensus genome was cloned from viral RNA isolated from an infected human liver and used to construct subgenomic selectable replicons. Upon transfection into a human hepatoma cell line, these RNAs were found to replicate to high levels, permitting metabolic radiolabeling of viral RNA and proteins. This work defines the structure of HCV replicons functional in cell culture and provides the basis for a long-sought cellular system that should allow detailed molecular studies of HCV and the development of antiviral drugs.

Primary human hepatocytes were used to study bile salt hepatotoxicity and the hepatoprotective potential of ursodeoxycholate in vitro. Hepatocytes were obtained by collagenase perfusion of healthy human liver tissue and were treated with glycochenodeoxycholate for 24 hr 1 day after plating. Clear signs of cytotoxicity were observed at concentrations of about 100 mumol/L glycochenodeoxycholate. Toxicity was determined by release of alkaline phosphatase, gamma-glutamyl transferase, AST, ALT or lactate dehydrogenase into the culture medium, by measuring DNA synthesis of the cultured liver cells and by testing the viability of the hepatocytes using trypan-blue dye exclusion. Addition of ursodeoxycholate, which by itself proved to be of little toxicity, significantly reduced the hepatotoxic effects of glycochenodeoxycholate: 72% +/- 6% of the cells survived treatment with 500 mumol/L glycochenodeoxycholate alone, but addition of 100 mumol/L ursodeoxycholate increased the survival rate to 87% +/- 4% (p less than 0.05). Moreover, all enzymes tested were secreted at a significantly lower level when ursodeoxycholate was present. Similarly, the cellular DNA synthesis was maintained at significantly higher levels as a result of ursodeoxycholate treatment. We conclude that (a) primary human hepatocytes are a suitable model for studying hepatotoxicity of bile salts in vitro, (b) ursodeoxycholate reduces hepatotoxicity of other bile salts and (c) ursodeoxycholate can act hepatoprotectively by itself (i.e., alteration of the metabolism of other bile salts is not necessarily required).

To study possible extrahepatic sites for the replication of hepatitis C virus (HCV), we examined fresh and cultured peripheral blood mononuclear leukocytes (PBML), as well as different subpopulations of PBML of HCV-infected patients, for the presence of viral genomic and antigenomic RNA. Sense and antisense oligonucleotide primers derived from HCV sequences were used for reverse transcription (RT) followed by an amplification with the polymerase chain reaction assay (PCR). Using antisense primers for RT, genomic viral RNA could be detected in serum, liver, total PBML and B lymphocytes of chronically infected patients. However, only liver tissue and PBML specimens were positive when a sense primer was used. To demonstrate further the specificity of these findings, total PBML were stimulated using pokeweed mitogen and synthesis of HCV RNA was determined by incorporation of [3H]uridine into nascent viral RNA molecules using a hybrid release assay. Additionally, total PBML from an uninfected person could be infected in vitro using an HCV RNA-positive serum. The PCR products obtained from serum, liver and PBML specimens of an HCV-positive individual were found to have nearly identical sequences. Our findings suggest that PBML could be a site for viral replication of HCV during the natural course of infection and may represent a reservoir for hepatitis C virions.