Xenotransplantation may be clinically feasible once the mechanisms of rejection are understood and graftsurvival can be achieved without compromising the recipient to the extent that systemic toxicity isencountered. Thrombotic and inflammatory reactions to porcine bone marrow (BM)-derived cells andvasculature are linked to difficulties in establishing mixed discordant chimerism in primates and thedevelopment of thrombotic microangiopathy in vascularized grafts. These responses may be associated withhumoral immunity to xenogeneic grafts and intrinsic molecular barriers between the discordant species. Thedevelopment of the GalT-KO pig and removal of the dominant xenoantigen has been a major advance in thisarea. However, there are still problems in inducing tolerance by generating mixed chimerism, either byvascularized thymic tissues or the bone marrow BM-derived cell approach; limited xenograft survival times andgraft injury are still a concern. The goals of this project are directed at delineating mechanisms of thethrombotic sequelae associated with the GalT-KO pig-to-baboon xenotransplant model. We will identify andcharacterize porcine antigenic targets of both natural and elicited xenoreactive antibodies directed againstGal negative xenografts in vivo by MALDI-TOF Mass Spectrometry. Vascular markers of thrombotic injurywill be also determined by porcine gene mini-arrays. Protein expression profiling will be undertaken tovalidate these vascular markers of xenograft rejection. The role of antithrombotic interventions will bedetermined addressing both pharmacological and genetic modalities. Antithrombin agents will beadministered to baboon recipients of porcine cell infusions and renal grafts, as dual anti-thrombotic and anti-inflammatory agents, at the time of graft implantation and with rejection episodes. We will also studytreatment with solCD39 (an ectonucleotidase) and ATL146e (an adenosine receptor A2a agonist), incombination with this antithrombin strategy. Gene therapeutic vectors will be employed to over-expressCD39, thrombomodulin and tissue factor pathway inhibitor in GalT-KO BM-derived cells, prior to theirinfusion into baboons. Outcomes with respect to inflammatory or thrombotic sequelae and engraftment willbe examined. We will finally evaluate transgenic approaches to over-express CD39 and the natural humananticoagulant factors in mice and pigs. Our studies will be judged successful if novel and clinically relevantantithrombotic strategies can be then developed and applied.
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