Overexpression of glutathione peroxidase with two isoforms of superoxide dismutase protects mouse islets from oxidative injury and improves islet graft function.

Primary nonfunction of transplanted islets results in part from their sensitivity to reactive oxygen species (ROS) generated during the isolation and transplantation process. Our aim was to examine whether coexpression of antioxidant enzymes to detoxify multiple ROS increased the resistance of mouse islets to oxidative stress and improved the initial function of islet grafts. Islets from transgenic mice expressing combinations of human copper/zinc superoxide dismutase (SOD), extracellular SOD, and cellular glutathione peroxidase (Gpx-1) were subjected to oxidative stress in vitro. Relative viability after hypoxanthine/xanthine oxidase treatment was as follows: extracellular SOD + Gpx-1 + Cu/Zn SOD > extracellular SOD + Gpx-1 > extracellular SOD > wild type. Expression of all three enzymes was the only combination protective against hypoxia/reoxygenation. Islets from transgenic or control wild-type mice were then transplanted into streptozotocin-induced diabetic recipients in a syngeneic marginal islet mass model, and blood glucose levels were monitored for 7 days. In contrast to single- and double-transgenic grafts, triple-transgenic grafts significantly improved control of blood glucose compared with wild type. Our results indicate that coexpression of antioxidant enzymes has a complementary beneficial effect and may be a useful approach to reduce primary nonfunction of islet grafts.

Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation.

Extracellular nucleotides play an important role in thrombosis and inflammation, triggering a range of effects such as platelet activation and recruitment, endothelial cell activation, and vasoconstriction. CD39, the major vascular nucleoside triphosphate diphosphohydrolase (NTPDase), converts ATP and ADP to AMP, which is further degraded to the antithrombotic and anti-inflammatory mediator adenosine. Deletion of CD39 renders mice exquisitely sensitive to vascular injury, and CD39-null cardiac xenografts show reduced survival. Conversely, upregulation of CD39 by somatic gene transfer or administration of soluble NTPDases has major benefits in models of transplantation and inflammation. In this study we examined the consequences of transgenic expression of human CD39 (hCD39) in mice. Importantly, these mice displayed no overt spontaneous bleeding tendency under normal circumstances. The hCD39 transgenic mice did, however, exhibit impaired platelet aggregation, prolonged bleeding times, and resistance to systemic thromboembolism. Donor hearts transgenic for hCD39 were substantially protected from thrombosis and survived longer in a mouse cardiac transplant model of vascular rejection. These thromboregulatory manifestations in hCD39 transgenic mice suggest important therapeutic potential in clinical vascular disease and in the control of serious thrombotic events that compromise the survival of porcine xenografts in primates.

Remission and pancreas isograft survival in recent onset diabetic NOD mice after treatment with low-dose anti-CD3 monoclonal antibodies.

Diabetes in NOD mice is an autoimmune disease similar to Type I diabetes in humans. Prior to hypoglycemia, changes in the islet infiltrate led to autoreactive T cell activation and destruction of the insulin-producing beta cells. If T cell activation can be inhibited before beta cell destruction is complete, islet cell rescue and regeneration can occur. Female NOD mice > 100 days old with blood glucose levels > 20 mM/l for less than 7 days were selected as 'recent onset' mice. Untreated, all of these animals would die of diabetes in < 40 days. Mice treated with anti-CD4 (GK1.5) achieved 14.3% permanent remission, while those treated with anti-CD8 (53.6.7) showed 33.3% permanent remission. Mice treated with anti-CD3 (145-2C1) also achieved 33.3% permanent remission, but 14% of these died of first dose syndrome. In mice treated with a low dose of anti-CD3 (10 microg KT3), which did not induce first dose syndrome, 50% remained in remission for > 100 days. This dose of mAb reduced insulitis but did not deplete splenic CD3 cells. When mice in remission were challenged with a vascularized pancreas isograft at 50 days, 9/22 remained normal and 13/22 had recurrent disease in both transplanted and native pancreas. Of the long-surviving isografts 7/9 were in KT3 treated recipients. Histology showed activated T cell infiltration in the native and transplanted pancreases of mice with transient remission. Benign insulitis with macrophages, B cells, CD4 > CD8 T cells and low levels of IL-2R, IL-2, IFN-gamma and IL-4 was seen in islets from the native pancreas and in long surviving pancreas isografts in mice that remained in remission. Thus, using low dose KT3, it was possible to halt the development of diabetes in 50% of animals treated soon after diagnosis, despite significant islet cell destruction at this stage. Of the KT3 treated mice in permanent remission, 70% had re-established tolerance to autoantigen and did not destroy vascularized pancreas isografts.