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Background: The infusion of pig growth factor-mobilized peripheral blood leukocytes (containing 1 to 2% progenitor cells) (pPBPC) into baboons is associated with a thrombotic microangiopathy, which results from a direct effect of these pig cells on platelet aggregation. Ajoene is a synthetic derivative of garlic that inhibits aggregation of human platelets induced by all known agents. To assess its potential use in models of xenotransplantation, this agent was tested for its effect on baboon platelet aggregation in vitro and in vivo., In Vitro Studies: Baboon platelet aggregation assays, using adenosine diphosphate (ADP) (20 or 40 microm) or collagen (12.5 microg/ml), were performed after incubation with ajoene (0 to 150 microg/ml) or dipyridamole (0 to 200 microg/ml). Platelets were also incubated with pPBPC (5 x 10(6) cells) without or with ajoene in the absence of a known agonist. In vivo studies: Baboons received either a single intravenous dose of ajoene (10 to 25 mg/kg) or dipyridamole (0.8 mg/kg), or repeated doses of both agents at 2 to 3 h intervals. Platelet-rich plasma was obtained for platelet aggregation assays at time points up to 4 h post-drug administration., Results: In vitro, platelet aggregation was inhibited by 95% (ADP assay) and 89% (collagen assay) by ajoene at concentrations of > or =75 microg/ml. Dipyridamole had no effect at concentrations of <100 microg/ml, but inhibited aggregation almost completely at higher concentrations. Ajoene inhibited the aggregation caused by pPBPC by 33 to 50%. In vivo, platelet aggregation was completely inhibited for 2 h by ajoene at 25 mg/kg. Dipyridamole at 0.8 mg/kg reduced aggregation by 20% for 15 min, but the effect was lost by 60 min. In combination, the two agents prolonged inhibition marginally. Repeated doses of both agents at 2 h intervals maintained complete inhibition of aggregation, but did not do so when the interval between doses was extended to 2.5 or 3 h. Combined therapy was not associated with any bleeding complications., Conclusions: Although ajoene is a powerful inhibitor of platelet aggregation, the need for repeated administration and its partial effect on pPBPC-induced platelet aggregation would suggest that it may be of only limited value in preventing the thrombotic microangiopathy that develops when pPBPC are infused into baboons. However, it would seem worthy of further investigation when used in combination with other agents.

Background: Natural anti-Gal antibodies (NAb) to Gal epitopes play a key role in the rejection of pig cells or organs transplanted into primates. We have investigated the effect on NAb return after extracorporeal immunoadsorption (EIA) of the continuous intravenous (i.v.) infusion of (i) bovine serum albumin conjugated to Gal type 6 oligosaccharides (BSA-Gal) or (ii) a poly l-lysine backbone conjugated to Gal type 2 or 6 oligosaccharides (PLL-Gal)., Methods: Porcine mobilized peripheral blood progenitor cells (PBPC) obtained by leukapheresis from MHC-inbred miniature swine (n = 9) were infused intravenously (i.v.) into baboons: Group 1 baboons (n = 4) received whole body and thymic irradiation, splenectomy, antithymocyte globulin, cobra venom factor, cyclosporine, mycophenolate mofetil, anti-CD154mAb, porcine hematopoietic growth factors, and EIA before transplantation of high doses (2 to 4 x 1010 cells/kg) of PBPC; Group 2 baboons (n = 3) received the Group 1 regimen plus a continuous i.v. infusion of BSA-Gal for up to 30 days; Group 3 baboons (n = 5) received the Group 1 regimen plus a continuous i.v. infusion of PLL-Gal type 2 (n = 2) or both PLL-Gal types 2 and 6 (n = 3) for up to 30 days., Results: Group 1: NAb returned to pre-PBPC levels within 20-30 days, but there was no induction of antibody to Gal or non-Gal determinants; Group 2: NAb was undetectable or at very low level during BSA-Gal therapy. In one baboon, however, IgG to Gal type 2, but not to type 6, returned during BSA-Gal therapy; Group 3: NAb was undetectable or at very low level during PLL-Gal therapy. In two baboons that received PLL-Gal type 2, NAb to Gal type 6, but not to type 2, returned during PLL-Gal treatment. Two of five baboons, however, developed systemic infection. Four of five baboons died within 14 days; autopsy revealed focal hemorrhagic injury to their hearts, lungs, and small intestines, with histologic abnormalities that varied between animals from hemorrhage and/or thrombosis in some organs (heart, lungs, or intestine) to signs of infections (bacteria in intestine, cytomegalovirus in liver)., Conclusions: (i) BSA-Gal and PLL-Gal therapy maintained depletion of NAb. (ii) Some heterogeneity in specificity of NAb was identified, indicating that the infusion of a combination of Gal type 2 and 6 glycoconjugates may be required. (iii) The addition of PLL-Gal to the immunosuppressive regimen was associated with a high incidence of morbidity and mortality without a clear histopathologic entity underlying the cause of death.

Organs transplanted from pig to primate are rejected within minutes or hours by an antibody-dependent, complement-mediated mechanism [hyperacute rejection (HAR)]. Even after depletion of anti-Gal alpha 1-3Gal (Gal) antibody (Ab), for example by extracorporeal immunoadsorption, return of natural Ab is believed to be a major factor in the initiation of acute humoral xenograft rejection. Various non-human primates are used as recipients of pig organs in experimental discordant xenotransplantation (XTx) models. However, anti-Gal IgM and IgG levels in non-human primates may differ from those in humans. Serum levels of anti-Gal IgM and IgG were measured by enzyme-linked immunosorbent assay (ELISA) in humans (n=14), chimpanzees (n=8), baboons (n=214), cynomolgus monkeys (n=29), rhesus monkeys (n=23) and Japanese monkeys (n=6). The mean level of anti-Gal IgM was significantly higher in chimpanzees than in other groups, while in rhesus monkeys it was significantly lower than in other groups, except baboons and Japanese monkeys. The mean human anti-Gal IgG level was higher than in other groups and this difference reached statistical significance except with regard to chimpanzees. The mean anti-Gal IgG level in baboons was significantly lower than that in humans, chimpanzees and cynomolgus monkeys. The measured differences in anti-Gal IgM and IgG levels may affect the kinetics of Ab removal and rate of return in different species, and thus may have relevance for translating work in non-human primate models to the clinical setting.