Orthotopic heart transplantation in a transgenic pig-to-primate model

BACKGROUND: Previous studies demonstrated that hearts from transgenic pigs expressing human decay-accelerating factor (hDAF) were not hyperacutely rejected when transplanted heterotopically into the abdomen of cynomolgus monkeys. This study examines orthotopic transplantation of hDAF transgenic pig hearts into baboon recipients. METHODS: Orthotopic xenogeneic heart transplantation was performed using piglets, transgenic for hDAF, as donors. Ten baboons were used as recipients and were immunosuppressed with a combination of cyclophosphamide, cyclosporine, and steroids. RESULTS: Five grafts failed within 18 hr without any histological signs of hyperacute rejection. Pulmonary artery thrombosis induced by a size mismatch was observed in two of these animals. The other three recipients died because of failure to produce even a low cardiac output and/or dysrhythmia. The remaining five animals survived between four and nine days. One animal died of bronchopneumonia on day 4. Three xenografts stopped beating on day 5 due to acute vascular rejection. The longest survivor was killed on day 9 with a beating, histologically normal xenograft, because of pancytopenia. CONCLUSIONS: The results reported here demonstrate that hDAF transgenic pig hearts are not hyperacutely rejected when transplanted into baboon recipients. Orthotopically transplanted transgenic pig hearts are capable of maintaining cardiac output in baboons. An optimum immunosuppressive regimen is the subject of ongoing research.     

Comparative study of target antigens for primate xenoreactive natural antibodies in pig and rat endothelial cells

BACKGROUND: A rat-to-primate cardiac xenograft model has been proposed as an alternative to the clinically relevant but more cumbersome pig-to-primate model for assessing the efficacy of strategies aimed at preventing xenograft hyperacute rejection. As in pig xenografts, the rejection of rat hearts was mediated by the binding of xenoreactive natural antibodies (XNA) and complement activation. The present study was conducted to identify target antigens recognized by cynomolgus and rhesus monkey IgM XNA on rat tissues and cells in comparison with pig cells. METHODS: The reactivity of rhesus or cynomolgus serum on pig and rat endothelial cells (ECs) was studied by flow cytometry, ELISA, and complement-dependent cytotoxicity, after removal of primate XNA by perfusion of pig livers, immunoadsorption on a Gal alpha(1,3)Gal affinity column, and enzymatic removal of alpha-galactosyl epitopes from the cell surface. Rat and pig EC extracts were also immunoprecipitated with primate serum and resolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The expression of the Gal alpha(1,3)Gal epitope was analyzed on rat tissues and ECs by immunohistochemistry, flow cytometry, and Western blot, using the isolectin B4 from Griffonia simplicifolia. RESULTS: Removal of primate XNA or of alphaGal epitopes resulted in a decrease in XNA binding to pig and rat cells, leaving a similar degree of residual reactivity in the two species. At least five proteins of 260, 210, 110, 56, and 50 kDa were immunoprecipitated on rat ECs, with molecular weight similar to several proteins identified on pig ECs. These results suggest that primate XNA recognize similar antigens on rat and pig ECs. Rat cells expressed lower levels of the Gal alpha(1,3)Gal epitope than pig cells. A large proportion, but not all, of primate XNA react with this epitope on pig and rat ECs. CONCLUSION: This study suggests that the rat is a valuable species for the evaluation of genetic engineering strategies on the vascular endothelium aimed at preventing hyperacute xenograft rejection.     

Effects of DL-penicillamine on cytotoxic reaction between baboon performed xenoantibodies and pig endothelial cells

The purpose of this study was to evaluate effects of DL-Penicillamine (DLP), a compound interrupting S-S bonds (IgM pentamers) on binding and cytotoxicity of adult baboon performed xenoantibodies to pig endothelial cells. Pooled baboon serum was treated with different concentrations of DLP during various periods of time. Complement-mediated cytotoxicity assay was used to determine the reactivity of baboon xenoantibodies to pig aortic endothelial cells (PAEC). To assess IgM and IgG binding to PAEC, ELISA method was applied. Serum treated with DLP revealed significant reduction of cytotoxicity in a dose dependent manner. Cytotoxicity was also reduced during time prolongation of DLP exposure to PAEC. Results indicate that baboon performed IgM and IgG xenoantibodies bind to pig endothelial cells, but only IgM is able to cause degradation of the complement. DLP significantly reduces cytotoxicity and eliminates binding of IgMs to PAEC in spite of continued binding of IgG xenoantibodies to the surface of endothelium.     

Life-supporting pig-to-primate renal xenotransplantation using genetically modified donors

BACKGROUND: In order to circumvent the complement-mediated hyperacute rejection of discordant xenografts, a colony of pigs transgenic for the human regulator of complement activity, human decay-accelerating factor (hDAF), has been produced. METHODS: Seven kidneys from hDAF transgenic pigs and six kidneys from nontransgenic control pigs were transplanted into cynomolgus monkeys; both native kidneys were removed during the same operation. The recipient animals were immunosuppressed with cyclosporine, steroids, and cyclophosphamide. RESULTS: In the transgenic group, the median survival time was 13 days (range, 6-35 days); the median survival time in the control group was 6.5 days (range, 0.3-30 days). There were no cases of hyperacute rejection in the transgenic group, and the two longest-surviving kidneys in this group showed no evidence of rejection on histological examination. In contrast, all control kidneys underwent antibody-mediated rejection, one demonstrating hyperacute rejection and the others acute vascular rejection. CONCLUSION: This study demonstrates that (i) a kidney from an hDAF transgenic pig can support the life of a primate for up to 35 days (and also shows the basic physiological compatibility between the pig and nonhuman primate); (ii) nontransgenic kidneys are not routinely hyperacutely rejected; and (iii) the presence of hDAF on the kidney confers some protection against acute vascular rejection. Improved immunosuppression and immunological monitoring may enable extended survival.     

Evidence of human non-alpha-galactosyl antibodies involved in the hyperacute rejection of pig lungs and their removal by pig organ perfusion

BACKGROUND: Human natural xenoantibodies represent a major hurdle to the clinical application of pig lungs in transplantation by initiating hyperacute rejection within minutes to hours. OBJECTIVE: The object was to compare pig organ perfusion and specific depletion of anti-alpha-galactosyl xenoantibodies for prevention of hyperacute rejection in the pig to human lung combination. METHODS: Large White pig (20-25 kg) left lungs were removed and continuously ventilated and reperfused ex vivo either with (1) whole human blood previously perfused in situ through pig right lung (group I), liver (group II), or spleen (group III) or with (2) human plasma in vitro immunoabsorbed on columns containing alpha-galactosyl disaccharide (Gal-alpha-(1-3)Gal-beta-(CH2)3NH2; B disaccharide) (group IV). Each study group included 6 animals. RESULTS: The in situ and in vitro preperfusions depleted anti-alpha-galactosyl xenoantibodies and all in situ perfused pig organs showed histologic signs of hyperacute rejection. After the ex vivo reperfusion, group I xenografts had a significantly (P < .001) longer functional and histologic survival than did xenografts in groups II, III, and IV. Human blood reperfusing group I xenografts had a significantly (P < 0.05) lower (1) decline of clotting factors and total circulating immunoglobulins, (2) total and membrane attack complex (C5b,6,7,8,9) complement activation, and (3) hemolysis. By Western blot analysis, the in situ lung preperfusion removed antibodies against non-alpha-galactosyl proteins of low molecular weight that were not eliminated by the alpha-galactosyl column. CONCLUSIONS: Results demonstrate that specific depletion of anti-alpha-galactosyl antibodies alone incompletely protects pig lungs from hyperacute rejection. It is speculated that the more complete prevention of this rejection afforded by pig lung preperfusion relates to the removal of other, non-alpha-galactosyl antibodies.     

Serial sodium Amytal interviews in the clinical setting

A prospective final crossmatch with patient serum and donor lymphocytes using the complement-dependent cytotoxicity assay to identify any performed anti-donor antibody is required for kidney transplantation. The presence of pre-existing antibody may lead to hyperacute rejection of the transplanted kidney. Certain anti-donor antibodies have previously been shown to be ineffective in promoting hyperacute rejection, such as IgM autoantibodies and non-specific IgM lymphocytotoxic antibodies. In this report, we present evidence that IgM HLA alloantibody specific to the donor does not lead to hyperacute rejection and produces graft survival results equivalent to transplants with negative pre-transplant final crossmatches. Forty-eight (48) of 402 patients transplanted over and 8 yr period were transplanted across a positive final crossmatch due to IgM antibodies alone. Three patients exhibited IgM autoantibodies and 26 patients demonstrated non-specific IgM antibodies to lymphocytes. In 15 patients, following a detailed serum screening analysis, a significant correlation (r > 0.9, p < 0.001) was observed between HLA Class I antigens and the presence of corresponding IgM alloantibodies. Five of these patients were subsequently transplanted despite a positive final crossmatch that was clearly demonstrated to be the result of IgM alloantibody to donor HLA Class I specificities. All of these patients continue to have graft function. These results suggest that hyperacute rejection is not mediated by any type of IgM antibody to donor lymphocytes and that kidney transplantation when only IgM antibody is present against donor lymphocytes represents a reasonable opportunity for a safe transplant and successful long-term graft survival.     

Intracellular expression in pig cells of anti-alpha1,3galactosyltransferase single-chain FV antibodies reduces Gal alpha1,3Gal expression and inhibits cytotoxicity mediated by anti-Gal xenoantibodies

BACKGROUND: The carbohydrate structure Gal alpha1,3Gal expressed on pig cells is the major antigen recognized by xenoreactive natural antibodies in the higher primates. In xenotransplantation, natural antibodies binding to that structure initiate hyperacute rejection, and the anti-Gal alpha1,3Gal antibodies that are elicited probably take part in later phases of vascularized graft rejection. This epitope also appears to be involved in innate cellular responses. Inactivation of alpha1,3 galactosyltransferase in transgenic pigs would certainly lead to the success of xenotransplantation, but gene knockout in pigs is not feasible yet. METHODS: As a novel strategy to inhibit alpha1,3 galactosylation, we generated recombinant single-chain Fv (ScFv) antibodies directed against pig alpha1,3-galactosyltransferase and evaluated the effect of their intracellular expression on enzyme activity and Gal alpha1,3Gal expression. RESULTS: After in vitro transfection in pig cells, the scFv antibody anti-pig alpha1,3-galactosyltransferase reduced the amount or function of enzyme by up to 70% as evidenced by immunofluorescence and measurement of cell-associated activity. Consequently, Gal alpha1,3Gal on cell membranes was reduced to the same extent. This led to a profound (more than 90%) reduction in the cytotoxicity involving anti-Gal antibodies and complement. CONCLUSION: Although not sufficient to knock out the overall human anti-pig natural xenoreactivity, intracellular expression of the scFv antibody anti-alpha1,3-galactosyltransferase in pig cells significantly decreases the amount of Gal alpha1,3Gal and could be important to protect cells from elicited antibodies as well as from innate effectors.     

The use of a stereological method to estimate the volume of Schistosoma mansoni granulomas: the effect of zinc deficiency

Kidney xenotransplantation is not yet a realistic clinical treatment modality. However, during the last decades more than 30 kidneys from other species have been transplanted into humans; some of the kidneys sustained some function up to 60 days. Recent progress in genetic engineering has raised the possibility to create large transgenic animals which express human complement regulatory proteins (CRP). Since early complement activation is believed to be the main triggering event for xenograft destruction, complement regulation by species-specific CRP should avoid hyperacute rejection in transspecies transplantation. The perfusion of hDAF-transgenic pig kidneys with human blood was not associated with the morphological signs of hyperacute rejection when compared to non-transgenic control organs. Specific immunohistology could demonstrate that the transgene was sufficient to regulate complement activation beyond C3 despite the endothelial deposition of xenoantibodies. In the future, these organs could be further optimized and ultimately tested in a clinical pilot protocol under appropriate immunosuppression.     

The differing impact of chloroquine and pyrimethamine/sulfadoxine upon the infectivity of malaria species to the mosquito vector

BACKGROUND: Mixed lymphohematopoietic chimerism can provide an effective means of inducing longterm immunological tolerance and has been documented in a monkey allograft model. A conditioning regimen including nonmyeloablative or myeloablative irradiation and splenectomy has been used to induce chimerism in a pig-to-primate transplantation model. Since the presence of anti-Gal(alpha)1-3Gal (alphaGal) natural antibodies leads to the hyperacute rejection of pig organs transplanted into primates, extracorporeal immunoaffinity adsorption (EIA) of anti-alphaGal antibodies is also included in the regimen. The effect of the tolerance induction protocol on the anti-alphaGal antibody response has been assessed. METHODS: Anti-alphaGal antibody was measured after the EIA of plasma through an alphaGal immunoaffinity column in baseline studies involving two unmodified baboons, one splenectomized baboon, and one baboon that received a challenge with porcine bone marrow (BM), and in three groups of baboons (n=2 in each group) that received different conditioning regimens for tolerance induction. Group 1 received a nonmyeloablative conditioning regimen without porcine BM transplantation. Group 2 received nonmyeloablative conditioning with pig BM transplantation and pig cytokine therapy. Group 3 received myeloablative conditioning, an autologous BM transplant (with BM depleted of CD2+ or CD2+/CD20+ cells), and pig BM transplantation. RESULTS: In the baseline studies, a single EIA of anti-alphaGal antibodies in an unmodified animal initially depleted anti-alphaGal antibody, followed by a mild rebound. Nonmyeloablative conditioning (group 1) in the absence of pig cell exposure reduced the rate of anti-alphaGal antibody return. Pig BM cells markedly stimulated anti-alphaGal antibody production in an unmodified baboon (alphaGal IgM and IgG levels increased 40- and 220-fold, respectively). This response was significantly reduced (to an only 2- to 5.5-fold increase of IgM and IgG) in baboons undergoing nonmyeloablative conditioning (group 2). A myeloablative conditioning regimen (group 3) prevented the antibody response to pig BM, with the reduction in response being greater in the baboon that received autologous BM depleted of both CD2+ and CD20+ cells. No new antibody directed against pig non-aGal antigens was detected in any baboon during the 1 month follow-up period. CONCLUSIONS: (i) EIA of anti-alphaGal antibody in unmodified baboons results in a transient depletion followed by a mild rebound of antibody; (ii) exposure to pig BM cells results in a substantial increase in anti-alphaGal antibody production; (iii) a nonmyeloablative conditioning regimen reduces the rate of antibody return and (iv) markedly reduces the response to pig BM cells; (v) the anti-alphaGal response is completely suppressed by a myeloablative regimen if CD2+ and CD20+ cells are eliminated from the autologous BM inoculum. Furthermore, (vi) challenge with pig BM cells appears to stimulate only an anti-alphaGal antibody response without the development of other (non-alphaGal) anti-pig antibodies. We conclude that regimens used for T-cell tolerance induction can be beneficial in reducing the anti-alphaGal antibody response to porcine BM.     

Life-supporting pig-to-baboon heart xenotransplantation

BACKGROUND: The aim of this study was to investigate the effect of the expression of human decay-accelerating factor in transgenic pigs on hyperacute rejection in a pig-to-baboon heterotopic heart transplantation model and to assess the ability of such transgenic pig hearts in supporting the life of a primate when transplanted orthotopically. METHODS: Hearts from pigs transgenic for human decay-accelerating factor were transplanted heterotopically (n = 3) and orthotopically (n = 5) into the baboon. All animals received cyclosporine, steroids, and cyclophosphamide. Blood was sampled regularly for total antipig antibody titers, trough cyclosporine levels, full blood count, electrolytes, and creatinine. Rejection of the heterotopic hearts was defined as the absence of palpable cardiac pulsation. Explanted hearts were examined histologically with hematoxylin and eosin and with immunochemistry for complement components C3, C4, C9, and immunoglobulin M. RESULTS: None of the hearts were hyperacutely rejected. In the heterotopic group one heart underwent acute vascular rejection on day 13, and the remaining two recipients with beating xenografts were killed on days 2 and 21. In the orthotopic group, one recipient with a life-supporting xenograft was killed on day 9 because of poor general condition. Histologic examination demonstrated no evidence of rejection. Two xenografts stopped beating on day 5, and histologic study showed acute vascular rejection in both. There were also two graft failures for technical reasons in this group. CONCLUSIONS: Hyperacute rejection is abrogated in pig-to-baboon heart xenotransplantation with the expression of the human decay-accelerating factor transgene. The human decay-accelerating factor transgenic pig heart is able to support primate life for a prolonged period.     

Intact pancreatic islet function despite humoral xenorecognition in the pig-to-monkey combination

BACKGROUND: The aim of this study was to analyze humoral xenoreactivity of various Old World primate species sera against pig islets and the effects of these sera on pig islet viability and function after culture. METHODS: Freshly isolated or cultured adult pig islets were analyzed by immunohistology or by cytofluorimetry for Old World primate xenoreactive natural antibody (XNA) binding and complement deposition. Complement-mediated cytotoxicity was evaluated by 51Cr release assays. After 4 days of culture in 50% sera from Old World primates, the morphology and in vitro metabolic function of pig islets were also analyzed. RESULTS: Chimpanzee, Macaca mulatta (rhesus), or baboon XNA binding was detectable only on intra-islet endothelial cells (ECs). Incubation of pig islets with sera from all Old World primate species tested showed C3 and C4 deposition on ECs and on some surrounding endocrine cells. However, membrane attack complex (MAC) showed a pattern of positivity similar to XNA binding, i.e., restricted to ECs only. No deposition of factor B was detected. Although complement cascade was activated, no cytotoxicity was observed after incubation of islets with chimpanzee serum, whereas between 10% and 35% 51Cr specific release was obtained with rhesus, baboon, or Macaca fascicularis sera. Despite this cytotoxic effect, purified pig islets showed a normal morphology and a well-preserved insulin release in response to an acute glucose stimulus, after prolonged culture with 50% serum obtained from all primate species considered. CONCLUSIONS: Despite the fact that pig beta-cell function was not affected by the serum of any of the primate species tested, some of them yielded significant lysis of islet cells, presumably as a result of a cytotoxic effect on intra-islet ECs. These data show that Old World primate sera from different species do not have equivalent effect on pig islets; these differences should be taken into account in preclinical trials of pig islet xenotransplantation.     

Clinical trials and projected future of liver xenotransplantation

The trial and error of the pioneering xenotransplant trials over the past three decades has defined the limitation of the species used. Success was tantalizingly close with the chimpanzee, baboon, and other primates. The use of more disparate species has been frustrated by the xenoantibody barrier. Future attempts at clinical xenotransplantation will be hampered by the consideration of the species of animals and the nature of the organs to be transplanted. On one hand, primate donors have the advantage of genetic similarity (and therefore potential compatibility) and less risk of immunologic loss. On the other hand, pig donors are more easily raised, are not sentient animals, and may be less likely to harbor transmissible disease. It is recognized that the success of xenotransplantation may very with different organs. Because it is relatively resistant to antibody-mediated rejection, the liver is the organ for which there is the greatest chance of long-term success. Consideration of using xenotransplants on a temporary basis, or as a "bridge" to permanent human transplantation, may allow clinical trials utilizing hearts or kidney xenografts. Issues on metabolic compatibility and infection risks cannot be accurately determined until routine success in clinical xenotransplantation occurs. Based on a limited experience, the conventional approaches to allotransplantation are unlikely to be successful in xenotransplantation. The avoidance of immediate xenograft destruction by hyperacute rejection, achieved using transgenic animals bearing human complement regulatory proteins or modulating the antigenic target on the donor organ, is the first step to successful xenotransplantation. The ability to achieve tolerance by establishing a state of bone marrow chimerism is the key to overcoming the long-term immunologic insults and avoiding the necessarily high doses of nonspecific immunosuppression that would otherwise be required and associated with a high risk of infections complications. Xenotransplantation faces criticism that is strongly reminiscent of that leveled against human-to-human transplantation during the late 1960s and early 1970s. Yet with persistence, the field of human-to-human transplantation has proved highly successful. This success was the result of a stepwise increase in our understanding of the biology of rejection, improvements in drug management, and experience. It is possible that xenotransplantation may not be universally successful until further technologic advances occur; yet cautions exploration of xenotransplantation appears warranted to identify those areas that require further study.     

Hypoxic-ischaemic encephalopathy is associated with regional changes in cerebral blood flow velocity and alterations in cardiovascular function

Pig-to-primate cardiac xenografts surviving beyond the period of hyperacute rejection succumb after 3-4 days to a secondary immunologic response characterized by xenograft infiltration with NK cells and macrophages. Circulating baboon mononuclear cells contain NK cell precursors which mediate lysis of porcine endothelium by two distinct mechanisms: antibody-dependent cellular cytotoxicity and lymphokine activation. IL-2 activated NK lysis of porcine endothelium was 2.4-fold stronger than lysis occurring following engagement of FcRIII by xenoreactive IgG. IL-2 augmented NK lysis involved interactions between CD2 and CD49d on baboon NK cells and their respective ligands on porcine endothelium, since NK lysis was reduced either by using Mabs against CD2, CD49d, or porcine VCAM, or by treating endothelial cells with PIPLC to cleave GPI-linked molecules. These results imply that interactions between accessory molecule receptor-ligand pairs on primate NK cells, macrophages and porcine endothelium are of critical importance in delayed xenograft rejection.     

HLA-specific antibodies in highly sensitized patients can cause a positive crossmatch against pig lymphocytes

BACKGROUND: The presence of IgG HLA-specific antibodies in the serum of patients awaiting transplantation indicates T- and B-cell priming and would result in acute rejection of a poorly matched human allograft. Recent advances in xenotransplantation, with the amelioration of hyperacute rejection using transgenic pig kidneys, may benefit such patients. However, accelerated cellular rejection might result from the primed T-cell recognition of antigenic epitopes shared between pig and human MHC molecules. METHODS: We have compared the reactivity of IgG antibodies from 8 nonsensitized (NS) and 13 highly sensitized (HS) patients with human and pig lymphocytes by flow cytometry. Xenoreactive natural antibodies (XNA) were absorbed with pig red blood cells, and HLA class I-specific antibodies were further absorbed with pooled human platelets. RESULTS: Before XNA absorption, 20 of the 21 patients had a positive IgG crossmatch with pig lymphocytes, and there was no difference between NS and HS patients. In contrast, after XNA absorption, none of the 8 NS patients were positive, compared with 9 of the 13 HS patients (mean of the median channel fluorescence values of 7.7 and 86.5, respectively; P=<0.001). For XNA-absorbed HS patient sera, 20 of 30 (67%) pig lymphocyte crossmatch combinations were positive, with a mean median channel fluorescence value of 125 (range 31 to 294) compared with 9.5 (range 7 to 13) for the 10 crossmatch-negative combinations. Platelet absorption resulted in a concomitant reduction in antibody binding to pig lymphocytes in three of six HS patient sera, indicating that HLA class I-specific antibodies are responsible, at least in part, for the positive crossmatch. CONCLUSION: These results suggest that some IgG HLA-specific antibodies can bind to pig lymphocytes, analogous to a positive crossmatch with allogeneic donors.     

Organ xenotransplantation using human complement regulatory factor gene

For the success of xenotransplantation, hyperacute rejection must be controlled. Hyperacute rejection is induced by the activation of complement system via alternative pathway and/or classical pathway. Complement regulatory factors can inhibit activation of complement system via the both pathways. To overcome hyperacute rejection in organ xenotransplantation, gene engineering in the establishment of xeno-endothelial cells and transgenic animals (mouse and pig) using human complement regulatory factors are studied. In the near future, transgenic pig using human complement regulatory factor genes can be used as a potential donor in organ xenotransplantation.     

Survival following orthotopic cardiac xenotransplantation between juvenile baboon recipients and concordant and discordant donor species: foundation for clinical trials

It has been more than a decade since the last clinical trial of cardiac xenotransplantation in a newborn infant. Since that event, laboratory research at Loma Linda University has focused on survival studies of orthotopically xenografted juvenile baboon recipients. Both concordant and discordant donor species have been used. Transgenic donors have not been explored at Loma Linda. Instead, simplified host immunoregulative protocols, consistent with those used in neonatal cardiac allografting, have been adapted to xenotransplant research. Xenograft bridge to alloengraftment was evaluated in a series of five juvenile baboon recipients. Heterotopically implanted cardiac xenografts stimulated host production of xenoreactive antibody. Orthotopic cardiac allografting was then carried out. Xenoantibody appeared to play little role in immediate or chronic survival of experimental hosts. A clinical protocol of xenobridging to allotransplantation would likely succeed. Two consecutive series of orthotopically xenotransplanted hosts using rhesus monkey cardiac donors demonstrated unprecedented long-term survival. Splenectomy combined with maintenance therapy consisting of FK-506 and methotrexate contributed to survival of up to 502 days in one series of xenografted baboon hosts selected for ABO blood grouping, mixed lymphocyte culture, and crossmatch compatibility. Survival beyond a year (maximum 515 days) among three consecutive juvenile baboon recipients of orthotopically implanted rhesus monkey hearts, in which splenectomy was omitted and cyclosporine was substituted for FK-506, represents a benchmark achievement. Commencing maintenance immunosuppression several weeks prior to transplantation appeared to improve chronic survival significantly. Investigation of discordant (pig-to-baboon) host survival has focused on adsorption of naturally occurring xenoreactive antibody at the time of transplantation. This strategy, combined with pretransplant total lymphoid irradiation and both pre- and posttransplant immunosuppression, succeeded in preventing hyperacute rejection and resulted in survival of up to 24 days, thereby permitting observation of the delayed xenograft rejection phase. Data support consideration of additional clinical trials of concordant neonatal cardiac xenotransplantation and offer promise for the development of discordant xenotransplantation as an ultimate therapeutic resource.     

Triple immunosuppression reduces mononuclear cell infiltration and prolongs graft life in pig-to-newborn baboon cardiac xenotransplantation

OBJECTIVE: Pig hearts transplanted into unmedicated newborn baboons do not undergo hyperacute rejection by preformed xenoantibody and complement. These grafts are rejected at days 3 to 4 in association with the infiltration of macrophages and natural killer cells. We investigated whether an immunosuppressive regimen used widely in cardiac allotransplantation could reduce this cellular response and prolong xenograft life. METHODS: Ten newborn baboons underwent heterotopic pig cardiac xenotransplantation. Five baboons were immunosuppressed with mycophenolate mofetil (100 mg/kg), methylprednisolone acetate (0.8 mg/kg), and cyclosporine A (INN: ciclosporin; 10 mg/kg). Xenograft rejection was studied by light microscopy and immunofluorescence. The induced humoral response to porcine xenoantigens was documented by enzyme-linked immunosorbent assay using synthetic alpha-1,3-galactosyl epitopes coupled to bovine serum albumin. RESULTS: Graft life was extended from a mean of 3.6 +/- 0.5 days (n = 5) to a mean of 6.2 +/- 1.1 days (n = 5, p = 0.01). In comparison with controls, explanted grafts from medicated baboons demonstrated reduced infiltration with natural killer cells and macrophages, but increased evidence of complement-mediated rejection substantiated by increased deposition of immunoglobulin M, complement, and fibrin. In all baboons receiving transplants, levels of both immunoglobulin M and immunoglobulin G anti-galactose were significantly increased after transplantation, with immunoglobulin G levels remaining persistently elevated. CONCLUSIONS: These results indicate that cyclosporine-based triple immunosuppression marginally prolonged xenograft survival and appears to have reduced the natural killer cell and macrophage infiltrates. The immunosuppressive protocol, however, was not adequate to prevent the induced immunoglobulin M humoral response and prevent complement-mediated graft injury.     

The role of apoptosis in the thymic involution during growth of the syngeneic transplanted tumor in mice]

BACKGROUND: Natural antibodies (NAbs) against a terminal alpha1-3 galactosyl (alphaGal) epitope have been identified as the major human anti-pig NAbs. METHODS AND RESULTS: We used two synthetic alphaGal trisaccharides--type 6 (alphaGal6) and type 2(alphaGal2)--linked to an inert matrix to remove NAbs from human plasma in vitro. Flow cytometry indicated that an average of 85% of the NAb binding activity was depleted by adsorption with alphaGal6. By measuring the binding of NAbs to pig peripheral blood mononuclear cells and bone marrow cells, we demonstrated that alphaGal6 was more effective than alphaGal2 in removing NAbs, and the combination of alphaGal6 + alphaGal2 did not further increase removal of NAbs. The specificity of the removal of NAbs (IgM and IgG) reactive with the alphaGal epitope by alphaGal6 matrix was shown by enzyme-linked immunosorbent assay. In vivo studies in nonhuman primates compared plasma perfusion through a alphaGal6 immunoaffinity column with hemoperfusion through a pig liver for changes in blood pressure, hematocrit, platelets, and NAb adsorption. CONCLUSIONS: Both methods reduced the level of anti-pig IgM and IgG xenoreactive antibodies to nearly background, but column perfusion caused less hypotension and reduction in platelets than liver perfusion. Four pig kidneys transplanted into monkeys after column perfusion did not undergo hyperacute rejection, remaining functional for 2-10 days, with a mean functional period of 7 days, demonstrating that a pig kidney can support renal function in a primate.     

Pretransplantation immunoadsorption therapy in patients immunized with human lymphocyte antigen: effect of treatment and three years' clinical follow-up of grafts

Sensitization against human lymphocyte antigen (HLA) occurs frequently in previously transplanted patients that lose a first cadaveric graft. To shorten their time on the waiting list and reduce the incidence of early rejection in such patients, we performed immunoadsorption therapy by a tryptophan column in 10 patients as an attempt to remove circulating antibodies prior to regrafting. Resynthesis of antibodies was suppressed with cyclophosphamide and prednisolone. Following the course of immunoadsorption therapy, the panel reactive antibodies (PRA) decreased by more than 50% from the pretreatment values. In the present study, 8 patients were transplanted with cadaveric renal grafts. At the time of follow-up, graft survival was 63% in these patients (2-36 months post-transplantation, mean 23 months). There was one incidence of acute rejection, one graft was lost within 48 h owing to renal artery thrombosis, and one was lost within 2 weeks as a result of stenosis. The serum creatinine levels were down to near normal during the first 3 weeks in hospital (p < 0.0001) and remained at this level during the period of follow-up. We conclude that immunoadsorption might be a beneficial pretransplantation therapy and an alternative to plasmapheresis in HLA-immunized patients awaiting kidney transplantation.     

Novel markers for constitutive secretion used to show that tissue plasminogen activator is sorted to the regulated pathway in transfected PC12 cells

Once hyperacute rejection has been prevented, the pig-to-human xenograft might be exposed to vascular cell-mediated rejection directed against vascular structures. In order to evaluate the relative importance of direct and antibody-dependent anti-endothelial cell-mediated cytotoxicity in different individuals, freshly isolated human blood leukocytes were incubated with confluent porcine aortic endothelial cells (PAEC) in a 4 h Cr-release cytotoxicity assay. Peripheral blood mononuclear cells (PBMC) and lymphocytes (PBL) of all subjects tested (but not monocytes or neutrophils) directly killed PAEC, with wide interindividual variations (from 2.8% to 32%). The addition of heat-inactivated autologous serum to PBMC and PBL (but not to myeloid cells) always enhanced cytotoxicity. This antibody-dependent cell-mediated cytotoxicity (ADCC) was also observed in the presence of adult pooled serum and cord blood pooled serum and was eliminated after adsorption of adult pooled serum to insoluble protein A, demonstrating that IgG is the only class of immunoglobulin involved in this phenomenon. Moreover, blocking Fc gamma RIII with an anti-CD16 mAb eliminated ADCC without affecting direct cytotoxicity. When the ADCC exerted by the PBL of all subjects was assessed with the same preparation of purified IgG, wide interindividual variations were again observed. Surprisingly, there was no correlation between direct cytotoxicity and ADCC although, as depletion experiments demonstrated, both were due to CD16+ natural killer (NK) cells. These results argue that CD16+ NK cells could play an important role in early vascular rejection of porcine discordant xenografts, by both a direct and an IgG xenoreactive natural antibody-dependent cell-mediated cytotoxicity.