Hepatitis B virus infection of transplanted human hepatocytes causes a biochemical and histological hepatitis in immunocompetent rats

AIM: To characterize the host response to hepatitis B virus (HBV) infection in human hepatocytes transplanted into immunocompetent rodent rats tolerized by, and transplanted with primary human hepatocytes.METHODS: One week after the transplantation, rats were inoculated with HBV, and viral gene expression, replication,and host response was monitored.RESULTS: HBV DNA was detectable in serum for at least 60 days. HBsAg levels rose steadily for 3 weeks postinoculation and then plateaued at a level of about 0.6 pg/mi. HBV RNA was also found in liver at levels that remained constant through the time course. Immunofluorescence revealed clusters of hepatocytes that stained positive for HBcAg. The presence of HBV covalently closed circular DNA (cccDNA) in liver was demonstrated using nuclease digestion of single-stranded DNA followed by PCR. Serum ALT levels rose and reached a peak level of 180 IU/L on day 18, but remained elevated for 60 days. Histology revealed a progressive predominantly mononuclear lobular hepatitis.CONCLUSION: These data indicate that human hepatocytestransplanted into rats rendered tolerant to these cells, when infected by HBV, results in biochemical as well as histological evidence of hepatitis that accompanies viral gene expression,and DNA replication.

The Molecular and Structural Basis of HBV-resistance to Nucleos(t)ide Analogs

Infection with hepatitis B virus(HBV)is a worldwide health problem.Chronic hepatitis B can lead to fibrosis,liver cirrhosis,and hepatocellular carcinoma(HCC).Management of the latter two conditions often requires liver transplantation.Treatment with conventional interferon or pegylated interferon alpha can clear the virus,but the rates are very low.The likelihood,however,of viral resistance to interferon is minimal.The main problems with this therapy are the frequency and severity of side effects.In contrast,nucleos(t)ide analogs(NAs)have significantly lower side effects,but require long term treatment as sustained virological response rates are extremely low.However,long term treatment with NAs increases the risk for the development of anti-viral drug resistance.Only by understanding the molecular basis of resistance and using agents with multiple sites of action can drugs be designed to optimally prevent the occurrence of HBV antiviral resistance.

An Improved Method for Preparation of Uniform and Functional Mitochondria from Fresh Liver

Background and Aims:As the major energy source for mammalian cells,mitochondria have been the subject of numerous studies.However,the isolation and purification of healthy mitochondria,especially from fresh tissue,remains challenging.The most popular methods and kits involve various centrifugation steps which require substantial time and equipment but do not consistently provide pure preparations of functional mitochondria.The aim of this study was to determine whether methods could be devised to improve the purity and yield of functional mitochondria from fresh tissue.Methods:Fresh mouse liver was homogenized,and cells lysed.Particle size analysis,quantitation of mitochondrial DNA,mitochondrial oxygen consumption,and purity of mitochondria(by electron microscopy)were measured in samples after various purification steps and significant differences determined.Results:A two-step procedure consisting of centrifugation followed by filtration through 1.2μand 0.8μfilters resulted in uniform mitochondrial preparations with diameters between 520-540 nm,and approximately 5-times more pure samples.The mitochondria thus obtained had oxygen consumption and sensitivities to mitochondrial inhibitors that were indistinguishable from those purified by centrifugation alone.Electron microscopy confirmed the presence of more uniform and 4-5 times greater concentrations of mitochondria compared to centrifugation alone.Conclusions:A two-step procedure consisting of sequential centrifugation followed by filtration is a rapid method for the production of highly purified,uniform and functional mitochondria.

Secondary Structural Elements of the HCV X-region Involved in Viral Replication

Background and Aims:The noncoding regions in the 3'-untranslated region (UTR) of the hepatitis C virus (HCV)genome contain secondary structures that are important for replication.The aim of this study was to identify detailed conformational elements of the X-region involved in HCV replication.Methods:Ribonucleic acid (RNA) structural analogs X94,X12,and X12c were constructed to have identical conformation but 94%,12%,and 0% sequence identity,respectively,to the X region of HCV genotype 2a.Effects of structural analogs on replication of HCV genotypes 1b and 2a HCV RNA were studied by quantitative reverse transcriptase polymerase chain reaction.Results:In replicon BB7 cells,a constitutive replication model,HCV RNA levels decreased to 55%,52%,53%,and 54% after transfection with expression plasmids generating RNA structural analogs 5B-46,X-94,X-12,and X-12c,respectively (p<0.001 for all).In an HCV genotype 2a infection model,RNA analogs 5B-46,X-94,and X-12 in hepatic cells inhibited replication to 11%,9%,and 12%,respectively.Because the X-12 analog was only 12% identical to the corresponding sequence of HCV genotype 2a,the sequence per se,or antisense effects were unlikely to be involved.Conclusions:The data suggest that conformation of secondary structures in 3'-UTR of HCV RNA genome is required for HCV replication.Stable expression of RNA analogs predicted to have identical stem-loop structures might inhibit HCV infection of hepatocytes in liver and may represent a novel approach to design anti-HCV agents.