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Background: Xenotransplantation using pigs as source species carries a risk for the activation of latent herpesviruses from the porcine donor and potential transmission to the recipient. In pig-to-baboon xenotransplantation, activation of porcine cytomegalovirus (PCMV) has been associated with xenograft injury and an increased incidence of consumptive coagulopathy and graft loss. Activation of porcine lymphotropic herpesvirus (PLHV)-1 was not observed in pig-to-baboon solid organ xenotransplantation, but was associated with a syndrome of post-transplantation lymphoproliferative disorder (PTLD) after allogeneic stem cell transplantation in pigs., Material and Methods: Early weaning of piglets was used to try to reduce the viral burden of xenograft donors. This consisted of separating the piglets of a litter from the sow within the first 2 weeks after birth and raising them in isolation from the remaining herd., Results: We have previously demonstrated that PCMV could be excluded from source animals by early weaning of piglets. However, early weaning failed to exclude PLHV-1 from source pigs., Conclusions: This disparity between PCMV and PLHV-1 reflects differing pathogenesis of infection of these herpesviruses. New approaches will be needed to exclude PLHV-1 from pig colonies.

Background: Acute humoral xenograft rejection (AHXR) is an immunologic barrier in pig-to-baboon organ transplantation (Tx). We report microvascular thrombosis and myocardial necrosis in a series of cardiac xenografts., Methods: Ten baboons underwent heterotopic heart Tx from pigs transgenic for human decay-accelerating factor. Recipients were treated with soluble Gal glycoconjugates and multiple immunosuppressive agents. Grafts were removed when palpable contractions stopped. Stained tissue sections from harvested grafts were analyzed by light and fluorescence microscopy., Results: Xenograft survival ranged from 4 to 139 (mean 37, median 27) days. Some histology was typical for AHXR (n = 4; median survival 22 days). Hemorrhage and edema were only focal in the longer-surviving grafts (n = 4, median survival 54 days). All grafts had multiple platelet-rich fibrin thrombi occluding myocardial vessels. Ischemic damage was manifested by contraction band necrosis in four grafts, myocytolysis in eight, coagulative necrosis in nine, and patchy myocyte dropout in all grafts. A notable paucity of interstitial mononuclear cells was observed in all grafts. Marked intimal thickening resembling that of allograft vasculopathy was observed in one graft. Immunofluorescence showed immunoglobulin (Ig)G and/or IgM deposition in five grafts. Multivessel C4d deposition appeared in seven grafts. Significant C3 deposition was absent., Conclusions: Cardiac xenograft survival in the pig-to-baboon model can be significantly prolonged by vigorous immunosuppressive treatment of recipient animals. Additional efforts to block humoral activation of graft endothelial cells and/or to overcome species-specific molecular coagulation pathway incompatibilities may prevent the development of microvascular thrombosis and myocardial infarction. Cardiac xenograft vasculopathy (chronic rejection) can occur with prolonged graft survival., (Copyright 2004 Blackwell Munksgaard)

Background: Successful hematopoietic cell allotransplantation results in donor-specific tolerance, but this approach has been unsuccessful in the wild-type pig-to-baboon xenotransplantation model, as pig cells were lost from the circulation within 5 days. However, after cessation of immunosuppressive therapy on day 28, all baboons demonstrated non-specific unresponsiveness on mixed leukocyte reaction (MLR) for at least 30 days. We have now investigated the transplantation of bone marrow (BM) cells from miniature swine homozygous for alpha1,3-galactosyltransferase gene-knockout (GalT-KO)., Methods: Baboons (n = 3) were pre-treated with whole body and thymic irradiation, anti-thymocyte globulin, and splenectomy, and received immunosuppressive and supportive therapy for 28 days. BM was harvested from GalT-KO swine (n = 3). The baboons were monitored for the presence of pig cells by flow cytometry and colony-forming units (CFUs), and for cellular reactivity by MLR., Results: A mean of 11 x 10(8) BM cells/kg was infused into each baboon. The mean absolute numbers and percentages of pig cells detected in the blood at 2 h and on days 1, 2 and 4, respectively, were 641/microl (9.5%), 132/microl (3.4%), 242/microl (3.9%), and 156/microl (2.9%). One baboon died (from accidental hemorrhage) on day 6, at which time chimerism was present in the blood (2.0%) and BM (6.4%); pig cell engraftment in the BM was confirmed by polymerase chain reaction (PCR) of CFUs. In the two other baboons, blood chimerism was lost after day 5 but returned at low levels (<1%) between days 9 to 16 and 7 to 17, respectively, indicating transient BM engraftment. Both surviving baboons showed non-specific unresponsiveness on MLR until they were euthanized on days 85 and 110, respectively., Conclusions: By using BM cells from GalT-KO pigs, chimerism was detected at levels comparable with previous studies when 30-fold more growth factor-mobilized peripheral blood progenitor cells had been transplanted. In addition, cellular hyporesponsiveness was prolonged. However, long-term engraftment and chimerism were not achieved.

Aim: to study whether sensitization to pig antigens results in humoral and/or cellular sensitization to alloantigens in baboons, and thus increases the risks of organ allotransplantation after xenotransplantation. Serum from baboons that were naive (n = 4), sensitized to Gal alpha 1,3Gal (Gal) antigens (n = 2), or sensitized to Gal + non-Gal pig antigens (n = 2) were tested by flow cytometry for the presence of immunoglobulin G (IgG) and IgM antibodies that bind to pig or baboon peripheral blood mononuclear cells (PBMC). Two allosensitized baboons were used as positive controls. The same 10 sera were tested in a complement-mediated cytotoxicity assay to detect cytotoxic antibodies against pig, allo and self-PBMC. The T-cell responses of the same baboons to allogeneic and pig PBMC stimulators in mixed lymphocyte reaction (MLR) were studied. All baboon sera contained cytotoxic antibodies that bound to pig PBMC. Binding and cytotoxicity were higher in xenosensitized baboons, particularly in those sensitized to Gal + non-Gal antigens (P < 0.001). None of the naive or xenosensitized baboon sera bound to baboon PBMC. Serum from allosensitized baboons showed anti-baboon IgG and IgM binding, but there was no increase in binding to pig PBMC or in cytotoxicity to pig cells. The MLR response to pig stimulators in baboons sensitized to non-Gal pig antigens was greater than that of naive or Gal-sensitized baboons (P < 0.001), but there was no increase in the response to baboon cells. In baboons, no in vitro evidence that a previous pig xenograft might endanger the outcome of a subsequent allograft was documented.

There is a shortage of human blood for transfusion. The possibility of using alpha-galactosidase-treated pig red blood cells (pRBCs) for transfusion into humans has been investigated. pRBCs were treated in vitro with alpha-galactosidase. In vitro binding of antibodies (Abs) in baboon or human sera to untreated/treated pRBCs was assessed by flow cytometry and serum cytotoxicity. In vivo clearance rates of (1) autologous baboon red blood cells (RBCs), (2) unmodified pRBCs, and (3) alpha-galactosidase-treated pRBCs were measured after transfusion into baboons receiving either no treatment or depletion of complement +/- depletion of anti-Gal alpha 1-3Gal (Gal) Ab or of macrophage phagocytes. In vitro binding of baboon or human Abs to treated pRBCs was absent or minimal compared with untreated pRBCs, and serum cytotoxicity was completely inhibited. In vivo autologous baboon RBCs survived for >16 days and unmodified pRBCs for <15 min in an untreated baboon. Treated pRBCs survived for 2 h in an untreated baboon, for 24 h in a complement-depleted baboon, and for 72 h when the baboon was depleted of both complement and anti-Gal Ab, or of complement and macrophage phagocytes. All baboons, however, became sensitized to Gal antigens. Failure to prolong the in vivo survival of treated pRBCs could be due to inadequate removal of Gal epitopes because sensitization to Gal developed, or could imply other, as yet unidentified, causes for RBC destruction. To fully assess the potential of pRBC transfusion in humans, more complete alpha-galactosidase treatment of pRBCs will be required.

Gal alpha 1,3Gal (Gal) is the first target in antibody-mediated rejection of pig-to-non-human primate xenograft. Its expression may vary between organs and constituents of organs. Gal expression was studied in pancreas, testis, spleen and thymus of 22 pigs, with ages ranging from 1 to 22 months. The immunoperoxidase technique using the biotinylated lectin, Griffonia simplicifolia (IB4), was used. In the pancreas, neither endocrine (islet cells) nor exocrine cells expressed Gal. The Sertoli cells in the testis were negative. The spleen capsule and trabeculae did not stain for Gal, although both splenic T and B lymphocytes expressed Gal (B > T). Thymocytes were weakly positive, whereas thymic epithelial cells were negative for Gal. No age-related differences were seen in any tissues. Porcine islets of Langerhans, Sertoli cells, and the splenic and thymic structural frameworks did not express Gal, and therefore, should be relatively resistant to anti-Gal antibody-mediated rejection. The availability of pigs deficient in Gal as a source of islets may therefore not be beneficial in extending islet graft survival in non-human primate models.