Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer

Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo

A global scientific strategy to cure hepatitis B

Chronic hepatitis B virus (HBV) infection is a global public health challenge on the same scale as tuberculosis, HIV, and malaria. The International Coalition to Eliminate HBV (ICE-HBV) is a coalition of experts dedicated to accelerating the discovery of a cure for chronic hepatitis B. Following extensive consultation with more than 50 scientists from across the globe, as well as key stakeholders including people affected by HBV, we have identified gaps in our current knowledge and new strategies and tools that are required to achieve HBV cure. We believe that research must focus on the discovery of interventional strategies that will permanently reduce the number of productively infected cells or permanently silence the covalently closed circular DNA in those cells, and that will stimulate HBV-specific host immune responses which mimic spontaneous resolution of HBV infection. There is also a pressing need for the establishment of repositories of standardised HBV reagents and protocols that can be accessed by all HBV researchers throughout the world. The HBV cure research agenda outlined in this position paper will contribute markedly to the goal of eliminating HBV infection worldwide

The utility of the new generation of humanized mice to study HIV-1 infection: transmission, prevention, pathogenesis, and treatment

Substantial improvements have been made in recent years in the ability to engraft human cells and tissues into immunodeficient mice. The use of human hematopoietic stem cells (HSCs) leads to multi-lineage human hematopoiesis accompanied by production of a variety of human immune cell types. Population of murine primary and secondary lymphoid organs with human cells occurs, and long-term engraftment has been achieved. Engrafted cells are capable of producing human innate and adaptive immune responses, making these models the most physiologically relevant humanized animal models to date. New models have been successfully infected by a variety of strains of Human Immunodeficiency Virus Type 1 (HIV-1), accompanied by virus replication in lymphoid and non-lymphoid organs, including the gut-associated lymphoid tissue, the male and female reproductive tracts, and the brain. Multiple forms of virus-induced pathogenesis are present, and human T cell and antibody responses to HIV-1 are detected. These humanized mice are susceptible to a high rate of rectal and vaginal transmission of HIV-1 across an intact epithelium, indicating the potential to study vaccines and microbicides. Antiviral drugs, siRNAs, and hematopoietic stem cell gene therapy strategies have all been shown to be effective at reducing viral load and preventing or reversing helper T cell loss in humanized mice, indicating that they will serve as an important preclinical model to study new therapeutic modalities. HIV-1 has also been shown to evolve in response to selective pressures in humanized mice, thus showing that the model will be useful to study and/or predict viral evolution in response to drug or immune pressures. The purpose of this review is to summarize the findings reported to date on all new humanized mouse models (those transplanted with human HSCs) in regards to HIV-1 sexual transmission, pathogenesis, anti-HIV-1 immune responses, viral evolution, pre- and post-exposure prophylaxis, and gene therapeutic strategies

Woodchuck hepatitis virus X protein is present in chronically infected woodchuck liver and woodchuck hepatocellular carcinomas which are permissive for viral replication.

The woodchuck hepatitis virus (WHV) X gene (WHx) is required for infectivity of WHV in woodchucks, and the gene encodes a broadly acting transcription factor. Several lines of evidence from cell culture and transgenic mice suggest that X proteins can promote hepatocarcinogenesis. To determine whether WHx-encoded proteins are present during persistent infection and hepatocellular carcinoma (HCC) in woodchucks, we surveyed livers and HCCs from a panel of WHV carrier woodchucks for the presence of WHx by utilizing an immunoprecipitation-Western blot (immunoblot) procedure. We detected a single 15.5-kDa WHx gene product in 100% of the persistently infected livers but not in livers from animals which had recovered from acute infection or in those of uninfected woodchucks. Analysis of HCCs revealed that all of the tumors which contained WHV replication intermediates were also positive for WHx. In contrast, WHx was undetectable in HCCs which did not contain replicative intermediates. Subcellular localization studies detected WHx in the cytoplasm but not in the nuclei of primary woodchuck hepatocytes. Comparative immunoprecipitation experiments revealed that there were 4 X 10(4) to 8 X 10(4) molecules of WHx per primary woodchuck hepatocyte. Four lines of WHx transgenic mice did not develop HCC spontaneously. However, when one line was treated with diethylnitrosamine, the occurrence of precancerous lesions was enhanced compared with that in diethylnitrosamine-treated nontransgenic controls. The apparent absence of WHx in some woodchuck HCCs indicates that WHx may not be required to maintain the tumor phenotype, whereas its presence in all persistently infected livers leaves open the possibility that it plays a role in hepatocarcinogenesis

Impact of Refinements to Handling and Restraint Methods in Mice

There is increasing evidence that, compared to non-aversive handling methods (i.e., tunnel and cupping), tail handling has a negative impact on mouse welfare. Despite this evidence, there are still research organisations that continue to use tail handling. Here, we investigated handling for routine husbandry by three different methods: tail, cupping and tube in a relevant real-world scenario involving mice bred off-site. After transfer to the destination unit, mice were assessed for overt behaviours associated with anxiety and fear. Mice that experienced tail handling were less easy to handle, were more responsive to the box opening, and scored lower in a hand approach test. One barrier to non-tail handling methods is the current practice of restraining mice by the tail for procedures. We therefore next assessed whether a modified method for restraint that takes the animal from cupping to restraint without the use of the tail was associated with better welfare. This refined restraint method reduced overt signs of distress although we did not find any differences in corticosterone levels or anxiety-related behaviours. These findings suggest that avoiding tail handling throughout the animal’s laboratory experience, including during restraint, benefits their welfare