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Aging is a risk factor for adipose tissue dysfunction, which is associated with inflammatory innate immune mechanisms. Since the adipose tissue/liver axis contributes to hepatosteatosis, we sought to determine age-related adipose tissue dysfunction in the context of the activation of the innate immune system fostering fatty liver phenotypes. Using wildtype and immune-deficient mice, we compared visceral adipose tissue and liver mass as well as hepatic lipid storage in young (ca. 14 weeks) and adult (ca. 30 weeks) mice. Adipocyte size was determined as an indicator of adipocyte function and liver steatosis was quantified by hepatic lipid content. Further, lipid storage was investigated under normal and steatosis-inducing culture conditions in isolated hepatocytes. The physiological age-related increase in body weight was associated with a disproportionate increase in adipose tissue mass in immune-deficient mice, which coincided with higher triglyceride storage in the liver. Lipid storage was similar in isolated hepatocytes from wildtype and immune-deficient mice under normal culture conditions but was significantly higher in immune-deficient than in wildtype hepatocytes under steatosis-inducing culture conditions. Immune-deficient mice also displayed increased inflammatory, adipogenic, and lipogenic markers in serum and adipose tissue. Thus, the age-related increase in body weight coincided with an increase in adipose tissue mass and hepatic steatosis. In association with a (pro-)inflammatory milieu, aging thus promotes hepatosteatosis, especially in immune-deficient mice.

Adult-derived human liver stem/progenitor cells (ADHLSCs) have the potential to alleviate liver injury. However, the optimal delivery route and long-term biodistribution of ADHLSCs remains unclear. In this paper, we used a triple fusion reporter system to determine the kinetic differences in the biodistribution of ADHLSCs following intrasplenic (IS) and intrahepatic (IH) administration in SCID-beige mice. ADHLSCs were transduced with a lentiviral vector expressing a triple-fusion reporter comprising renilla luciferase, monomeric red fluorescent protein and truncated HSV-1 thymidine kinase. The stability and duration of the transgenes, and the effects of transduction on the cell properties were evaluated in vitro. The acute retention and long-term engraftment in vivo were revealed by positron emission tomography (PET) and bioluminescence imaging (BLI), respectively, followed by histochemical analysis. We showed that ADHLSCs can be safely transduced with the triple fusion reporter. Radiolabeled ADHLSCs showed acute cell retention at the sites of injection. The IH group showed a confined BLI signal at the injection site, while the IS group displayed a dispersed distribution at the upper abdominal liver area, and a more intense signal. In conclusion, ADHLSCs could be monitored by BLI for up to 4 weeks with a spread out biodistribution following IS injection. .

Adult-derived human liver stem/progenitor cells (ADHLSCs) have the potential to alleviate liver injury. However, the optimal delivery route and long-term biodistribution of ADHLSCs remain unclear. In this article, we used a triple fusion reporter system to determine the kinetic differences in the biodistribution of ADHLSCs following intrasplenic (IS) and intrahepatic (IH) administration in severe combined immunodeficiency/beige mice. ADHLSCs were transduced with a lentiviral vector expressing a triple fusion reporter comprising renilla luciferase, monomeric red fluorescent protein, and truncated HSV-1 thymidine kinase. The stability and duration of the transgenes, and the effects of transduction on the cell properties were evaluated in vitro. The acute retention and long-term engraftment in vivo were revealed by positron emission tomography and bioluminescence imaging (BLI), respectively, followed by histochemical analysis. We showed that ADHLSCs can be safely transduced with the triple fusion reporter. Radiolabeled ADHLSCs showed acute cell retention at the sites of injection. The IH group showed a confined BLI signal at the injection site, while the IS group displayed a dispersed distribution at the upper abdominal liver area, and a more intense signal. In conclusion, ADHLSCs could be monitored by BLI for up to 4 weeks with a spread out biodistribution following IS injection., SCOPUS: ar.j, info:eu-repo/semantics/published

The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) can both mediate the entry of herpes simplex virus 1 (HSV-1). We have recently shown how these receptors contribute to infection of skin by investigating HSV-1 entry into murine epidermis. Ex vivo infection studies reveal nectin-1 as the primary receptor in epidermis, whereas HVEM has a more limited role. Although the epidermis represents the outermost layer of skin, the contribution of nectin-1 and HVEM in the underlying dermis is still open. Here, we analyzed the role of each receptor during HSV-1 entry in murine dermal fibroblasts that were deficient in expression of either nectin-1 or HVEM or both receptors. Because infection was not prevented by the absence of either nectin-1 or HVEM, we conclude that they can act as alternative receptors. Although HVEM was found to be highly expressed on fibroblasts, entry was delayed in nectin-1-deficient cells, suggesting that nectin-1 acts as the more efficient receptor. In the absence of both receptors, entry was strongly delayed leading to a much reduced viral spread and virus production. These results suggest an unidentified cellular component that acts as alternate but inefficient receptor for HSV-1 on dermal fibroblasts. Characterization of the cellular entry mechanism suggests that HSV-1 can enter dermal fibroblasts both by direct fusion with the plasma membrane and via endocytic vesicles and that this is not dependent on the presence or absence of nectin-1. Entry was also shown to require dynamin and cholesterol, suggesting comparable entry pathways in keratinocytes and dermal fibroblasts. IMPORTANCE Herpes simplex virus (HSV) is a human pathogen which infects its host via mucosal surfaces or abraded skin. To understand how HSV-1 overcomes the protective barrier of mucosa or skin and reaches its receptors in tissue, it is essential to know which receptors contribute to the entry into individual skin cells. Previously, we have explored the contributi

Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-beta-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol.