Rejection was correlated with a drop in left ventricular pressure demonstrated by telemetry. this study, we are reporting the survival of cardiac xenotransplantation recipients (n=3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported Mutated EGFR-IN-2 successful immunosuppression regimen and effective control of non-Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co-stimulation blockade based immunomodulatory regimen. Introduction Recent technological advancements in the field of genetic engineering may facilitate the development of genetically modified pigs with either deletion of certain molecules immunogenic to humans or with expression of human transgenes to overcome various incompatibilities between humans /non-human primates and pigs. We believe that organs from these genetically engineered (GE) animals may overcome the unmet demand of solid organs for patients with end-stage organ failure (1). However, identifying the ideal genetic construct of a pig donor for clinical xenotransplantation remains a challenge. Recently, transplantation of cells and solid organs from GE pig donors has resulted in significant improvement in xenograft survival (2C10). Thrombomodulin (TBM) is usually a coagulation regulatory protein that binds thrombin and enhances its activity by converting protein C to activated protein C (aPC). The aPC then inhibits activated factors V and VIII, leading to decreased thrombin formation and prevention of a hypercoagulable state (11). Pig TBM does not demonstrate analogous efficacy within human or non-human primate systems. The lack of a functional TBM is usually hypothesized to lead to microthrombosis and premature graft loss through a mechanism impartial of Mutated EGFR-IN-2 immunologic graft rejection and is unmitigated by immunosuppressive regimens. The ability to express hTBM in Rabbit polyclonal to FBXW8 donor pigs has provided a mechanism to overcome this graft loss secondary to microthrombosis. Recently, we have exhibited long-term cardiac xenotransplantation survival from a GE donor pig, expressing human thrombomodulin (GTKO.CD46. hTBM) using immunosuppression including co-stimulation blockade by anti-CD40 (2C10R4) mAb in pig-to-baboon cardiac xenotransplantation model (4). One question raised from this work is the necessity of human hTBM expression in the presence of anti-CD40 mAb based co-stimulation blockade; it is unclear whether it confers additional graft protection from rejection over co-stimulation blockade alone. In this study, using a comparable cardiac xenotransplantation model described by us earlier (3, 4, 12) we substituted GTKO.CD46.hTBM pig hearts with hearts from donor pigs which do not express hTBM (i.e. GTKO.CD46 only) along with our already tested modified immunosuppression regimen which include costimulation blockade by anti-CD40 mAb. Materials and Methods Animals Specific pathogen free (SPF) baboons of either sex weighing 7C15 kg (2C3 years of age) from University of Oklahoma (Norman, OK) were housed in a clean pathogen free facility and were used as recipients. Six to eight week old genetically modified alpha 1,?3-Galactosyltransferase gene knockout (GTKO) pigs of either sex with an overexpression of human CD46 without hTBM (GTKO.hCD46) (n=3) were used as donors (Revivicor Inc., Blacksburg, VA). These GTKO.CD46 pigs were produced by breeding over 6 generations and were homozygous for human CD46 and homozygous Gal knockout. The method of generation of these pigs has been described previously (13). The transgenes of hCD46 were stable at the genomic and protein level (3, 4). The weights of donor pigs were matched with the baboon recipient to accommodate the heart in the abdomen. All animals were used in compliance with guidelines provided by the National Heart, Lung and Blood Mutated EGFR-IN-2 Institute (NHLBI) Animal Care and Use Committee (IACUC). Immunosuppression Immunosuppressive regimen for all recipient baboons included induction therapy comprised of anti-thymocyte globulin (ATG), Mutated EGFR-IN-2 anti-CD20 antibody (Rituximab) and co-stimulation blockade with anti-CD40 (Clone 2C10R4) mAb, Cobra venom factor (CVF; Quidel, San Diego, CA) which was used to inhibit complement activation. High dose anti-CD40 mAb, Mycophenolate Mofetil (MMF), and tapered dose of steroids were also administered throughout the course as maintenance therapy. Details of the immunosuppressive regimen are.