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

High-Resolution Electron Microscopy of Semiconductor Heterostructures and Nanostructures

This chapter briefly describes the fundamentals of high-resolution electron microscopy techniques. In particular, the Peak Pairs approach for strain mapping with atomic column resolution, and a quantitative procedure to extract atomic column compositional information from Z-contrast high-resolution images are presented. It also reviews the structural, compositional, and strain results obtained by conventional and advanced transmission electron microscopy methods on a number of III–V semiconductor nanostructures and heterostructures

Sleep and immune function

Sleep and the circadian system exert a strong regulatory influence on immune functions. Investigations of the normal sleep–wake cycle showed that immune parameters like numbers of undifferentiated naïve T cells and the production of pro-inflammatory cytokines exhibit peaks during early nocturnal sleep whereas circulating numbers of immune cells with immediate effector functions, like cytotoxic natural killer cells, as well as anti-inflammatory cytokine activity peak during daytime wakefulness. Although it is difficult to entirely dissect the influence of sleep from that of the circadian rhythm, comparisons of the effects of nocturnal sleep with those of 24-h periods of wakefulness suggest that sleep facilitates the extravasation of T cells and their possible redistribution to lymph nodes. Moreover, such studies revealed a selectively enhancing influence of sleep on cytokines promoting the interaction between antigen presenting cells and T helper cells, like interleukin-12. Sleep on the night after experimental vaccinations against hepatitis A produced a strong and persistent increase in the number of antigen-specific Th cells and antibody titres. Together these findings indicate a specific role of sleep in the formation of immunological memory. This role appears to be associated in particular with the stage of slow wave sleep and the accompanying pro-inflammatory endocrine milieu that is hallmarked by high growth hormone and prolactin levels and low cortisol and catecholamine concentrations

Strong antiviral activity of the new l-hydroxycytidine derivative, l-Hyd4FC, in HBV-infected human chimeric uPA/SCID mice

BACKGROUND: Suppression of viral replication with nucleoside/nucleotide inhibitors has been shown to greatly improve the outcome of chronic HBV infection. beta-l-nucleoside analogues, especially beta-l-deoxycytidine derivatives represent one of the most efficient groups of antiretroviral compounds. We recently described that hydroxylation of the amino group of these beta-l-deoxycytidine derivatives preserved their strong HBV inhibitory activity in vitro, but strongly reduced their cytotoxicity. From this new group of compounds we selected beta-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxy-5-fluorocytidine (l-Hyd4FC) for a first in vivo investigation. The aim of this study was to determine the antiviral activity of l-Hyd4FC in HBV-infected human liver chimeric urokinase plasminogen activator (uPA)/SCID mice. METHODS: Stably infected animals (median 6x10(7) HBV DNA/ml) were injected daily with either l-Hyd4FC (50 mg/kg) or saline as controls. Mice treated with lamivudine served to compare the in vivo antiviral potency of l-Hyd4FC. Virological changes were determined by quantitative PCR. RESULTS: Treatment with l-Hyd4FC for 4 weeks induced a 2-log reduction of viraemia, while a median 1.5-log decline was achieved with lamivudine. Intrahepatically, l-Hyd4FC induced a median eightfold decline of viral activity (relaxed circular DNA/covalently closed circular DNA), and threefold reduction of pregenomic RNA/GAPDH levels. No significant decline of subgenomic HBV transcripts, as well as of circulating hepatitis B e antigen and hepatitis B surface antigen was detected. Maintenance of human serum albumin concentrations throughout the study, negative TUNEL staining and occurrence of viral rebound after drug withdrawal indicated that l-Hyd4FC was not toxic in human hepatocytes. CONCLUSIONS: Administration of l-Hyd4FC in uPA/SCID mice harbouring HBV-infected human hepatocytes demonstrated the high antiviral potency of this drug in vivo. Such characteristics make l-Hyd4FC a good candidate for further investigations a as potential HBV therapeutic agent

Measurement of the displacement field of dislocations to 0.03 Å by electron microscopy

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