Subsequently, a non-myeloablative conditioning regimen employing cyclosporine and/or anti-CD154, mycophenolate mofetil (MMF) and cobra venom factor (CVF) was found to be effective in inducing multilineage peripheral blood chimerism for up to 5 days following transfusion of high dose porcine PBMCs (2.7 to 4.6 1010 cells/kg) into baboons that experienced undergone extracorporeal immunoadsorption of anti-Gal antibodies [16]. therapy for 28 days. All animals received GalT-KO bone marrow (1 to 2 2 109 cells/kg) in two fractions on days 0 and 2, and were thereafter monitored for the presence of pig cells by circulation cytometry, for porcine progenitor cells by PCR of BM colony-forming models, and for cellular reactivity to pig cells by combined lymphocyte reaction (MLR). In vitro antibody formation to LoCD2b and rATG was tested by ELISA; antibody reactivity to GalT-KO pig cells was tested by circulation cytometry and cytotoxicity assays. Additionally, Baboons 3 and 4 received orthotopic kidney transplants on days 17 and 2, respectively, to test the potential effect of the protocol on renal transplantation. Results None of the animals showed detectable pig cells by circulation cytometry for 7-Chlorokynurenic acid sodium salt more than 12 h post-BM infusion. However, porcine progenitor cell engraftment, as evidenced by pig-derived colony forming units in the BM, as well as peripheral microchimerism in the thymus, lymph node, and peripheral blood was recognized by PCR in baboons 1 and 2 for at least 28 days post-transplant. ELISA results confirmed humoral immunocompetence at time of 7-Chlorokynurenic acid sodium salt transplantation as antibody titers to rat (LoCD2b) and rabbit (ATG) improved within 2 weeks. However, no induced antibodies to GalT-KO pig cells or improved donor specific cytotoxicity was detectable by circulation cytometry. In contrast, baboons 3 and 4 designed serum antibodies to pig cells as well as to rat and rabbit immunoglobulin by day time 14. Retrospective analysis exposed that although all four baboons possessed low levels of antibody-mediated cytotoxicity to GalT-KO cells prior to transplantation, the two baboons (3 and 4) that became sensitized to pig cells (and declined pig kidneys) experienced relatively high pre-transplantation titers of antiCnon-Gal IgG detectable by circulation cytometry, whereas baboons 1 and 2 experienced undetectable titers. Conclusions Engraftment and specific non-responsiveness to pig cells has been accomplished in two of four baboons following GalT-KO pig-to-baboon BMT. Engraftment correlated with absence of preformed antiCnon-Gal IgG serum antibodies. These results are encouraging with regard to the possibility of achieving transplantation tolerance across this xenogeneic barrier. Keywords: bone marrow, chimerism, GalT-Ko, tolerance, xenotransplantation Intro Organ transplantation remains the only definitive remedy for end-stage organ failure, yet there currently is present a critical shortage of human being organs. Xenotransplantation of organs from pigs could provide a potential answer to this shortage. Pig organs are attractive because of the unlimited availability, physiologic compatibility with humans, and relative lack of ethical issues surrounding their use, as compared to nonhuman primates. However, interspecies transplantation poses additional immune barriers to the people seen with human-to-human allogeneic transplantation [1]. Reliance on high-dose, non-specific pharmacologic immunosuppression for pig xenograft acceptance in primates results in serious and often fatal side effects, including fulminate illness and malignancy [2]. Thus, overcoming the immune difficulties posed by medical xenografts may depend on inducing immunological tolerance [3]. We have previously reported success in the induction of allogeneic tolerance through administration of donor bone marrow to produce combined lymphohe-matopoietic chimerism in rodent [4,5], pig [6], and non-human primate models [7], as well as in recent clinical tests [8,9]. Tolerance has also been achieved by 7-Chlorokynurenic acid sodium salt this modality for concordant xenografts, including rat-to-mouse [10] and baboon-to-cynomolgus monkey [11]. However, successful induction of tolerance through combined chimerism across the discordant pig-to-non-human primate barrier has not yet been achieved. Earlier attempts using bone marrow KIAA0849 (BM) [12,13] or mobilized peripheral blood progenitor cells (PBPC) [14C16] following ex lover vivo immunoadsorption of natural anti-Gal antibodies have demonstrated only transient chimerism. In these studies, the quick reappearance of anti-Gal antibodies was thought to preclude long-term hematopoietic engraftment [3,16]. The recent production of l-3-Gal transferase 7-Chlorokynurenic acid sodium salt gene knock-out (GalT-KO) pigs through nuclear transfer offers circumvented the problems posed by anti-Gal antibodies [17,18]. Using these animals as donors inside a earlier study, we have demonstrated successful multilineage peripheral blood microchimerism for 9 to 16 days in three animals. However, one recipient died on post-transplant day time 6 and the remaining two recipients by no means recovered baseline immune function [19]. In the current study, we have used an attenuated non-myeloablative conditioning regimen to improve survival and immunocompetence in order to determine the effect of the Gal knock-out on engraftment. Materials and methods Animals Recipients were baboons (n = 4 = B156, B158, B167, B175) of known ABO blood type and body 7-Chlorokynurenic acid sodium salt weight 8 to 15 kg.