Mice containing livers repopulated with human hepatocytes would provide excellent in vivo models for studies on human liver diseases and hepatotropic viruses, for which no permissive cell lines exist. Here, we report partial repopulation of the liver of immunodeficient urokinase-type plasminogen activator (uPA)/recombinant activation gene-2 (RAG-2) mice with normal human hepatocytes isolated from the adult liver. In the transplanted mice, the production of human albumin was demonstrated, indicating that human hepatocytes remained functional in the mouse liver for at least 2 months after transplantation. Inoculation of transplanted mice with human hepatitis B virus (HBV) led to the establishment of productive HBV infection. According to human-specific genomic DNA analysis and immunostaining of cryostat liver sections, human hepatocytes were estimated to constitute up to 15% of the uPA/RAG-2 mouse liver. This is proof that normal human hepatocytes can integrate into the mouse hepatic parenchyma, undergo multiple cell divisions, and remain permissive for a human hepatotropic virus in a xenogenic liver. This system will provide new opportunities for studies on etiology and therapy of viral and nonviral human liver diseases, as well as on hepatocyte biology and hepatocellular transplantation. Persistent infection with hepatitis B virus (HBV) is a major worldwide health problem, and chronically infected individuals are at high risk for developing cirrhosis and hepatocellular carcinoma. 1,2 Despite the availability of an HBV vaccine, there are still more than 350 million chronically infected people worldwide, and the few antiviral treatments currently available have a limited rate of efficacy. The narrow host range of HBV and the lack of both in vitro systems and of convenient animal models have greatly hampered our understanding of the complete virus life cycle, as well as the development of more effective antiviral drugs aimed at eradicating the virus from chronic carriers. 3 Chimpanzees are the only animal species infectable with HBV, 4,5 but studies with these animals and evaluation of antiviral therapies are severely restricted because of their limited availability and high costs. Animal models based on HBV-related hepadnaviruses, such as woodchuck and Pekin duck hepatitis B viruses, are often used for assessment of antiviral drugs 6-8 and have provided important information about factors involved in establishment of virus infection, viral persistence, and hepatocarcinogenesis. 9-14 However, woodchucks are relatively large animals of outbred origins that are difficult to handle in many laboratories, and chronic hepadnavirus infection in birds does not lead to cancer. The development of HBV-expressing transgenic mice has also provided important insights regarding viral pathobiology and the role of HBV gene products in hepatocellular injury. 12,[15][16][17][18][19] Although infectious virus can be produced in transgenic mice, their hepatocytes are not permissive for infection. Therefore, the still-unknown early step...

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Chronic infection with hepatitis B virus (HBV) is associated with an increased risk for the development of cirrhosis and hepatocellular carcinoma (HCC).C hronic hepatitis B virus (HBV) infection is one of the major etiologic factors in the development of hepatocellular carcinoma (HCC). 1,2 Epidemiologic studies have shown that chronic HBV-infection is associated with a 100-fold increase in the risk for HCC development relative to noncarriers, placing HBV in the forefront among known human carcinogens. 3,4 Several lines of evidence suggest that HBV is tolerated by infected hepatocytes that do not show overt pathogenic reactions to virus replication. Therefore, liver damage observed in acute or chronic hepatitis B infection is mainly caused by cellular immune responses, which play a key role in the control of HBV infection. 5 Continuous necrosis and liver cell regeneration in the inflammatory context provide the driving force for carcinogenic progression in HBV carrier livers by allowing the selection of mutated hepatocytes. The long latency period for the development of HCC reveals a multistaged mechanism, in which selection of progressively mutated hepatocytes occurs. Moreover, virus-host interactions have been related to the oncogenic properties of the virus. Tumor progression may also be promoted by the presence of viral gene products, such as the X protein, 6-8 or by overexpression and accumulation of the large envelope protein. 9 The inappropriate expression of a truncated form of the middle envelope protein produced from integrated viral sequences has also been reported. 10 A direct role for integration of a mammalian hepadnavirus in the development of HCC has been identified for the woodchuck hepatitis virus (WHV). Integrations of WHV DNA into the N-myc and c-myc protooncogenes have shown that WHV integration can activate cellular protooncogenes by enhancer or promoter insertion. 11,12 Molecular analysis of genomic DNA from human HCCs revealed the presence of clonally propagated viral DNA integrations and the majority of tumors from chronic HBV carriers harbor integrated viral DNA, often multiple copies per cell. [13][14][15][16] Although a cellular protooncogene that is commonly activated by HBV has not been identified, the high prevalence of HBV integrations at sites of chromosomal abnormalities, such as deletions, duplications, and chromosomal translocations, has suggested that the integration process may occur in a manner that promotes mutagenesis. 1,17,18 Studies on the natural history of integrations of duck hepatitis B virus (DHBV) have directly shown that hepadnavirus integrations

The aim of this study was to evaluate the inhibitory effect of the nucleotide analogue adefovir on woodchuck hepatitis B virus (WHV) replication and, in particular, to determine whether the pool of covalently closed circular DNA (cccDNA) could be reduced by adefovir treatment in primary cultures of woodchuck hepatocytes isolated from a chronic carrier. Strong reduction of WHV-DNA synthesis (90%) and secretion (up to 98%) was observed with all 3 doses of adefovir used (1, 10, and 100 mol/L), whereas in the absence of the drug, high amounts of viral particles were continuously secreted in the culture medium until the end of the study (27 days). Secretion of envelope proteins and viral RNA levels remained constant both in the adefovirtreated and -untreated cultures for the entire course of the study. Intracellular core protein levels declined by approximately 50% in all the cultures, independent of adefovir treatment. There was no indication of cccDNA loss in the adefovir-treated hepatocyte cultures even when cell turnover was induced for 14 days by the addition of epidermal growth factor (EGF) to the culture medium. Our data show that adefovir has a very strong inhibitory effect on WHV-DNA synthesis in chronically infected primary hepatocyte cultures and indicate that cccDNA is a very stable molecule that appears to be efficiently transmitted to the dividing hepatocytes. (HEPATOLOGY 2000;32:139-146.)Persistent infection with hepatitis B virus (HBV) continues to be a major health problem as it is associated with various degrees of liver damage, cirrhosis, and hepatocellular carcinoma. Despite the existence of an effective vaccine, more than 350 million people are infected worldwide. 1,2 Current treatment regimens primarily rely on interferon alfa therapy. However, interferon treatment is associated with a variety of side effects and has been successful in only approximately one third of the patients, being very poorly effective in individuals with perinatally acquired HBV infection. In recent years, a variety of new anti-HBV agents have been developed, and these are currently evaluated in clinical trials. 3,4 Because HBV replication proceeds through reverse transcription, treatment with inhibitors of HBV reverse transcriptase is a very attractive option.During hepadnavirus infection, the relaxed circular partially double stranded DNA genome is transported to the nucleus of the hepatocyte, where it is converted into a covalently closed circular molecule, named cccDNA, which serves as template for the transcription of all viral RNAs. 5 The synthesis of the new relaxed circular viral DNA genome is mediated by the viral P-protein, which has reverse transcriptase and DNA-dependent DNA polymerase activity. Synthesis of the viral DNA takes place in the cytoplasm of the hepatocytes within immature nucleocapsids. 6 To establish a sufficient pool of nuclear cccDNA, some of the newly synthesized viral DNAs need to be retransported to the nucleus of the infected hepatocyte. 7 Therefore, the replicative forms of viral DNA are a...

Tracking and tracing of transplanted cells in mice is required in many fields of research. Examples are transplantation of stem cells into organs of mice to study their differentiation capacity and injection of tumor cells to examine metastatic behavior. In the present study we tested the lipid dye CM-DiI and red fluorescent nanoparticles Qdot655 for their applicability in tagging and tracing of human cells in mice. Labeling of different cell types, including MCF-7 human breast cancer cells, human cord blood derived cells, human NeoHep cells and human hepatopancreatic precursor cells, is technically easy and did not compromise further cell culture. After transplantation of CM-DiI or Qdot655 marked cells, red fluorescent structures could be detected already in unprocessed paraffin slices of the studied organs, namely liver, lung, pancreas, kidney, spleen and bone marrow. Next, we examined whether the red fluorescent structures represent the transplanted human cells. For this purpose, we established an in situ hybridization (ISH) technique that allows clear-cut differentiation between human and murine nuclei, based on simultaneous hybridization with human alu and mouse major satellite (mms) probes. We observed a high degree of coincidence between CM-DiI-marked cells and alu positive nuclei. However, also some mms positive cells contained CM-DiI, suggesting phagocytosis of the transplanted CM-DiI-marked cells. The degree of such CM-DiI-positive mouse cells depended on the cell type and route of administration. From a technical point of view it was important that CM-DiI-positive structures in paraffin slices remained fluorescent also after ISH. In contrast, Qdot655 positive structures faded during further staining procedures. In conclusion, marking of cells with CM-DiI or Qdot655 prior to transplantation facilitates recovery of human cells, since a high fraction of positive structures in the host's tissue originate from the transplanted cells. However, CM-DiI or Qdot655 positive staining of individual cells in transplanted tissues is not sufficient to prove their human origin. Additional procedures, such as ISH with alu-probes, are essential, when characterizing individual cells.

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Isolated hepatocytes represent a relevant model of the liver and are highly required both for research and therapeutic applications. However, sources of primary liver cells from human beings and from some animal species are limited. Therefore, cryopreservation of hepatocytes could greatly facilitate advances in various research areas. The aim of this study was to evaluate whether cryopreserved primary woodchuck hepatocytes could be used for woodchuck hepatitis B virus (WHV) infection studies, and whether they could maintain their regenerative potential in vivo after thawing. Critical steps for good quality of cryopreserved hepatocytes included the use of University of Wisconsin (UW) solution as a main component of the freezing medium, stepwise reduction of dimethylsulfoxide (DMSO) to avoid osmotic shock, and maintenance of low concentrations of DMSO in the culture medium. After cryopreservation, cell viability was still high (70% to 80%), and 50% to Liver transplantation is a successful and well-established treatment for end-stage liver disease and liver failure. However, donor-organ scarcity is a fundamental limitation of this therapy. The availability of highly differentiated primary liver cells to be used for cell-based therapies, such as hepatocyte transplantation, tissue-engineered organs, or for extracorporeal liver support systems, represents an attractive alternative to whole-organ transplantation. 1,2 Freshly isolated normal adult hepatocytes are already widely used in various research areas of hepatology, pharmacology, and toxicology, and initial clinical trials have also shown their potential for therapeutic applications. 3 Essential prerequisites for therapeutic use of hepatocyte transplantation in humans is that primary liver cells must be promptly available, remain highly differentiated, and maintain their proliferative capabilities within the host liver, because only a limited number of cells can be infused into a patient. 4 In past years, numerous research laboratories established procedures for the isolation of primary hepatocytes from commonly used laboratory animals, rat hepatocytes being the most studied. However, only a few laboratories have the possibility to procure livers and perform the isolation of highly viable hepatocytes from humans and animal species that are scarcely available. Therefore, efficient cryopreservation and banking of hepatocytes would greatly expand and facilitate the use of primary liver cells both for research and therapeutic applications, while decreasing the need of freshly procured livers for the preparation of hepatocytes both from animal species and humans.Persistent infection with hepatitis B virus (HBV) is one of the major causes of liver disease in humans, being associated with various degrees of chronic hepatitis, cirrhosis, and the development of hepatocellular carcinoma. 5,6 Replication of HBV can be successfully achieved by transfecting hepatoma cell lines with cloned HBV-DNA genomes. These systems have significantly contributed to elucidating various asp...

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