Role of the thymus in transplantation tolerance in miniature swine: V. Deficiency of the graft-to-thymus pathway of tolerance induction in recipients of cardiac transplants.

BACKGROUND: We have previously shown that both thymic immigrants (graft to thymus pathway) and thymic emigrants (thymus to graft pathway) are involved in tolerance to renal allografts in miniature swine treated with a short course of calcineurin inhibitors. This study investigates the role of these pathways in cardiac transplant survival in recipients treated with a short course of tacrolimus. METHODS: Eleven animals received two-haplotype fully MHC-mismatched cardiac grafts with a 12-day course of tacrolimus. Recipients were thymectomized on day -21 (n=5) or day 0 (n=3), or were left euthymic (n=3). Two of the day -21 thymectomized animals received a day 0 host-MHC matched thymocyte infusion. RESULTS: Euthymic recipients of cardiac grafts treated with an immunosuppressive regimen identical to that previously shown to induce tolerance in euthymic recipients of renal allografts all rejected their grafts. Although no animal became tolerant, animals that were euthymic or thymectomized on day 0, as well as recipients of day 0 host-type thymocyte infusions following thymectomy on day -21, developed donor-specific hyporesponsiveness and maintained their cardiac grafts for markedly prolonged periods. In contrast, all animals thymectomized on day -21 that did not receive thymocyte infusions developed strong antidonor CTL responses and rejected their grafts by day 35. CONCLUSIONS: The graft-to-thymus pathway that plays an important role in tolerance induction to renal allografts appears to be relatively deficient in recipients of cardiac grafts. Strategies to increase donor antigen migration to the host thymus might therefore assist in tolerance induction to cardiac allografts.

Long-term acceptance of fully allogeneic cardiac grafts by cotransplantation of vascularized thymus in miniature swine.

BACKGROUND: We have previously reported the ability of both thymokidney and vascularized thymic lobe (VTL) allografts to induce transplantation tolerance to renal allografts across a full major histocompatibility complex (MHC) mismatch in thymectomized miniature swine. However, whether vascularized thymus is capable of inducing tolerance to less tolerogeneic organs when it is transplanted simultaneously is not yet known. The present study investigates cardiac allograft survival and the mechanism of long-term acceptance in recipient swine following cotransplantation of VTL and cardiac grafts from fully MHC-mismatched donors. METHODS: Animals received a heart graft, a heart graft and a VTL, or a heart graft and a donor thymocyte infusion. Immunosuppressive regimens consisted of 12 or 28 days of tacrolimus. RESULTS: All animals that received a VTL maintained their grafts significantly longer than their counterparts that received only a heart graft, and those receiving 28 days of tacrolimus maintained their heart grafts long-term. Recipients of a donor thymocyte infusion demonstrated slightly prolonged cardiac graft survival but all rejected their grafts, highlighting the importance of thymic stroma. Cytotoxic T-lymphocyte responses against third-party antigens by cells from tolerant animals showed restriction by both self and donor MHC, whereas responses of controls were restricted to self MHC only. The presence of donor dendritic cells in the VTL grafts and results of co-culture assays suggest that both central and regulatory mechanisms were involved in achieving long-term acceptance. CONCLUSION: This is the first demonstration of the long-term acceptance of fully MHC-mismatched cardiac allografts in large animals.

Vascularized thymic lobe transplantation in a pig-to-baboon model: a novel strategy for xenogeneic tolerance induction and T-cell reconstitution.

BACKGROUND: This laboratory has previously demonstrated the induction of allogeneic tolerance by vascularized thymic lobe (VTL) transplantation in miniature swine. We report here our initial attempt to induce tolerance by VTL transplantation in the clinically relevant, discordant, pig-to-baboon model of xenotransplantation. METHODS: Six baboons received xenografts of hDAF VTLs. Four of these baboons also received omental thymic tissue implants. All recipients were treated with an immunosuppressive conditioning regimen that included thymectomy, splenectomy, extracorporeal immunoadsorption of anti-alpha Gal antibodies, and T-cell depletion. Two control baboons received sham operations, of which one also received 5x10 hDAF porcine thymocytes/kg intravenously. RESULTS: Transplanted VTL grafts supported early thymopoiesis of recipient-type immature thymocytes, and facilitated engraftment of nonvascularized thymic omental implants. Recipients of the VTL grafts demonstrated donor-specific unresponsiveness in MLR assays, development of peripheral CD45RAhigh/CD4 double positive (DP) cells, and positive cytokeratin staining of thymic stroma in the grafts for 2 months following xenotransplantation. The control baboons did not show these markers of thymic reconstitution. The eventual return of Gal natural antibodies led to the destruction of graft epithelial cells and the rejection of all VTL grafts by 3 months posttransplantation. CONCLUSIONS: VTL transplantation from hDAF swine to baboons induced early thymopoiesis in the recipients and donor-specific cellular unresponsiveness in vitro. When coupled with additional strategies aimed at silencing humoral rejection, VTL transplantation may significantly prolong xenograft survival and result in long-term tolerance.

Pneumopericardial tamponade in a patient with partial sternal dehiscence.

We present a rare case of sudden pneumopericardial tamponade in a patient with partial sternal dehiscence after cardiac surgery. Urgent decompression was needed in the management of the condition. Vacuum-assisted closure therapy was also used to prevent the problem from recurring. When there is acute hemodynamic deterioration in a patient with sternal wound dehiscence, pneumopericardial tamponade should be considered as a possible complication.

Anti-Gal alpha 1-3Gal IgM and IgG antibody levels in sera of humans and old world non-human primates.

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.

Inhibition of baboon platelet aggregation in vitro and in vivo by the garlic derivative, ajoene.

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.

Depletion of anti-Gal antibodies by the intravenous infusion of Gal type 2 and 6 glycoconjugates in baboons.

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.