Y Okada

Suppression of Allogeneic Response by Viral IL-10 Gene Transfer:

Th1 cell activation and cytokine production shift the balance between Th1 and Th2, favoring the upregulation of proinflammatory activity that leads to destruction of allogeneic hepatocytes following transplantation. Th2-type cytokines, such as IL-10, have immune regulatory function. The aim of this study was to determine the antirejection efficacy of allogeneic hepatocytes with spheroidal shape (spheroids) genetically modified with viral IL-10 (vIL-10). Allogeneic hepatocyte spheroids, transferred vIL-10 gene by using adenovirus as the vector, were transplanted into the spleen of Nagases analbuminemic rats (NAR). NAR transplanted with vIL-10-transfected hepatocytes showed an abrupt rise in serum albumin levels that peaked on day 7 and remained at high levels up to day 21 after transplantation. The peak level of albumin on day 7 in vIL-10-transfected NAR was eminently higher than that in nontransfected NAR. Histopathological analysis revealed that in nontransfected NAR hepatocyte spheroids were more or less rejected on day 4, and, in contrast, vIL-10-transfected spheroids were still not rejected on day 14. This protective effect correlated with sustained high vIL-10 level in the splenic vein in NAR transplanted with vIL-10-transfected hepatocyte spheroids, suggesting that vIL-10 secreted from the transplanted hepatocytes induced an active suppression of allogeneic response. This study provides evidence to support the possibility of using vIL-10 gene therapy to prevent allogeneic response in hepatocyte transplantation.

Adenovirus-mediated viral IL-10 gene transfer prolongs survival of xenogeneic spheroidal aggregate-cultured hepatocytes

Abstract Xenotransplantation of hepatocytes appears to be a novel promising therapy for some forms of liver disease, and may well overcome the problem of donor shortage. We have previously reported that hepatocytes with a spheroidal shape (spheroids) are ideal for cell transplantation. The application of gene transfer techniques to this hepatocyte transplantation could possibly regulate the xenogeneic rejection reaction and, therefore, result in prolongation of the survival of the transplanted hepatocytes. In this study, we chose the adenovirus as a vector and an immunosuppressive cytokine named viral IL‐10 (vIL‐10) for transfection. A series of experiments was performed to elucidate the efficacy of transfection to the spheroids with adenovirus vectors and the effect of transfected vIL‐10 on the survival of xenogeneic hepatocytes. We examined the cell survival quantitatively by evaluating β‐galactosidase (β‐gal) activity, which was transfected into the hepatocytes in the xenogeneic spleen, and semi‐quantitatively by the histological findings. The results of in‐vitro studies identified an efficient expression of the β‐gal gene within the spheroids infected with Ad‐CMVLacZ (LacZ‐encoding adenovirus vector with CMV promotor) and the presence of BCRF1 mRNA within the spheroids transfected with AdCMVvIL‐10 (vIL‐10‐expressing adenovirus vector with CMV promotor) under the condition of 1 MOI, for 1 h. Xenogeneic hepatocytes with a spheroidal shape showed comparable survival to syngeneic hepatocytes for up to 4 days after transplantation with co‐transplantation of the vIL‐10‐transfected hepatocytes. From this study, we concluded that adenovirus‐mediated vIL‐10 gene transfer prolongs the survival of xenogeneic hepatocyte spheroids. Furthermore, spheroids possess ideal properties for gene transfection, as well as cell transplantation.

Hepatic energy booster effect and lipo-modulatory effect on postreperfusional endotoxemia by intraoperative lipid infusion in porcine liver transplantation

Abstract We investigated whether intraoperative infusion of fat emulsion could suppress postoperative endotoxemia and improve energy status in the transplanted liver graft using a swine orthotopic liver transplantation (OLT) model. Intraoperative free fatty acid (FFA) concentration, hepatic FFA clearance, serum hyaluronic acid levels, ATP content and hepatic energy charge (HEC) in liver grafts, postreperfusional endotoxin levels and recipient outcome were compared between a fat emulsion-treated group (LCT-treated group, n =6) and a saline infused-group (control group, n =7). Independent of massive surgical stress and administration of heparin, FFA concentration was significantly elevated in the LCT-treated group ( P =0.003). Since FFA clearance, ATP contents and HEC were also increased in the LCT-treated group ( P =0.024, 0.006, 0.005, respectively), intraoperative LCT-treatment was shown to increase FFA concentration and improve energy status of liver grafts. Because there were no significant differences in postoperative hyaluronic acid levels, infused fat emulsion (0.1 g/kg per hour) prevented endothelial cell injury. Significant improvement of postoperative endotoxemia was obtained at 1 and 2 h after reperfusion ( P =0.01 and 0.003, respectively). Energy booster effect and antiseptic effect led to prolonged survival of recipients in the LCT-treated group (28.5±13.3 days, P =0.013 vs. control). We concluded that the hepatic energy booster effect and the lipo-modulatory effect on postreperfusional endotoxemia caused by intraoperative infusion of fat emulsion may be of great use in human liver transplantation.

Adenovirus-mediated viral IL-10 gene transfer prolongs survival of allogeneic spheroidal aggregate-cultured hepatocytes

Xenotransplantation of hepatocytes appears to be a novel promising therapy for some forms of liver disease, and may well overcome the problem of donor shortage. We have previously reported that hepatocytes with a spheroidal shape (spheroids) are ideal for cell transplantation. The application of gene transfer techniques to this hepatocyte transplantation could possibly regulate the xenogeneic rejection reaction and, therefore, result in prolongation of the survival of the transplanted hepatocytes. In this study, we chose the adenovirus as a vector and an immunosuppressive cytokine named viral IL-10 (vIL-10) for transfection. A series of experiments was performed to elucidate the efficacy of transfection to the spheroids with adenovirus vectors and the effect of transfected vIL-10 on the survival of xenogeneic hepatocytes. We examined the cell survival quantitatively by evaluating β-galactosidase (β-gal) activity, which was transfected into the hepatocytes in the xenogeneic spleen, and semiquantitatively by the histological findings. The results of in-vitro studies identified an efficient expression of the β-gal gene within the spheroids infected with AdCMVLacZ (LacZ-encoding adenovirus vector with CMV promotor) and the presence of BCRF1 mRNA within the spheroids transfected with AdCMVvIL-10 (vIL-10-expressing adenovirus vector with CMV promotor) under the condition of 1 MOI, for 1 h. Xenogeneic hepatocytes with a spheroidal shape showed comparable survival to syngeneic hepatocytes for up to 4 days after transplantation with co-transplantation of the vIL-10-transfected hepatocytes. From this study, we concluded that adenovirus-mediated vIL-10 gene transfer prolongs the survival of xenogeneic hepatocyte spheroids. Furthermore, spheroids possess ideal properties for gene transfection, as well as cell transplantation.