Statement on allogeneic and xenogeneic transplantation by Fifty-Seventh World Health Assembly, 22 May 2004.
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Biomedical subjects
D K C Cooper
Publications and source records attributed to D K C Cooper.
Spleen transplantation in miniature swine: surgical technique and results in major histocompatibility complex-matched donor and recipient pairs.
BACKGROUND: Spleen transplantation (Tx) between some strains of rodents can lead to donor-specific tolerance either spontaneously or after a short course of immunosuppression. This study developed a surgical technique for spleen Tx in miniature swine to investigate its immunologic impact in a large animal model. METHODS: The preferred surgical technique of spleen Tx (n=8) involved excision of the donor spleen with its vascular pedicle to the aorta and portal vein. Carrel patches of donor aorta and portal vein were anastomosed to the abdominal aorta and inferior vena cava, respectively, of the (splenectomized) recipient. The results in four major histocompatibility complex-matched pairs that were mismatched for the porcine allelic antigen are reported. Two recipients were untreated, one received a 12-day course of cyclosporine A (CsA) alone, and one received thymic irradiation (700 cGy) and CsA. Hematopoietic cell chimerism was followed by fluorescence-activated cell sorter, and graft survival was assessed by histology. RESULTS: Spleen Tx was technically successful. In two untreated pigs, chimerism was detected in the blood (maximum 5% for 17 and 25 days) and lymph nodes (maximum 6% for 28 and 56 days), but both grafts showed histologic rejection by day 28. In two treated pigs, chimerism was present in the blood for 47 and 57 days, and rejection was prevented, with follow-up for 57 and 217 days, respectively. CONCLUSION: Spleen Tx in major histocompatibility complex-matched pairs treated with CsA+/-thymic irradiation results in prolonged chimerism and is associated with the development of in vivo unresponsiveness to the transplanted spleen.
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An investigation of the effect of thalidomide on anti-gal antibody production in baboons.
Previous data suggest that natural anti-Galalpha1,3Gal (Gal) antibody (Ab) is produced by mature plasma cells. As thalidomide is effectively used in the treatment of patients with multiple myeloma, its effect on natural anti-Gal Ab production has been assessed in two baboons. During a 10-week course of thalidomide administration (until it reached toxic levels in one baboon), there was no major reduction in Ab levels or in their rate of return after a course of extracorporeal immunoadsorption. A significant reduction of CD3+ cells (75%), and particularly of CD4+ cells (65 to 95%), was measured in the blood by flow cytometry. This was associated with general hyporesponsiveness on mixed lymphocyte reactivity in one baboon, but not in the other. Thalidomide would appear to have little effect on the production of natural Ab, but these observations lend some support to previous work by others indicating that thalidomide may suppress the cellular response to an allograft or xenograft.
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An investigation of the specificity of induced anti-pig antibodies in baboons.
AIM: To provide information on the specificity of induced anti-pig antibodies (Abs) in baboons after exposure and sensitization to pig antigens. MATERIALS AND METHODS: Baboons (n=7) received either porcine mobilized peripheral blood progenitor cells (n=3), kidney (n=3) or heart (n=1) transplants. After rejection of these cells or organs, pre- and post-rejection sera were analyzed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry to detect and measure anti-Galactosealpha1,3Galactose (Gal) and anti-non-Gal Abs. To study the anti-non-Gal carbohydrate response, the sera were incubated with pig red blood cells pretreated with alpha-galactosidase (to remove Gal) and three other exoglycosidases to remove other potential oligosaccharide epitopes, and studied by flow cytometry. To study the anti-swine leukocyte antigen (SLA) response, non-Gal Abs from two baboons sensitized with kidneys from inbred miniature swine of dd or aa haplotype, respectively, were adsorbed on cells of aa, cc, or dd haplotypes, and binding to aa, cc or dd cells was measured by flow cytometry. Cytotoxicity of anti-non-Gal Abs was tested in vitro by a complement-mediated cytotoxicity assay, using pig cells as targets. RESULTS: In pre-transplant and pre-rejection sera, anti-Gal Abs were detected, but anti-non-Gal Abs were either absent or at minimal levels. After exposure to pig antigens, baboons developed induced anti-Gal and anti-non-Gal Abs. No anti-non-Gal Abs directed to the tested carbohydrate epitopes could be detected. Anti-non-Gal Abs showed minor evidence of specific SLA haplotype reactivity, suggesting that the major Ab response was to pan-pig determinants. Anti-non-Gal Abs showed a low level of complement-mediated lysis of pig cells in vitro. CONCLUSIONS: In this limited study, no Ab response to non-Gal carbohydrates was observed, and anti-SLA specificity was minor, indicating that most induced anti-non-Gal Ab was directed against non-specific pig proteins, including SLA-epitopes.
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An exploratory investigation of the effect of arsenic trioxide on anti-Gal antibody production in baboons.
BACKGROUND: Arsenic trioxide (As2O3) is an anticancer drug that has been reported to induce apoptosis and inhibit differentiation in human plasmacytoma and normal plasma/B cells without significant myelosuppression. We assessed the ability of As2O3 as single therapy or in combination with an anti-CD20 monoclonal antibody (mAb) and whole body irradiation (WBI) to deplete B and plasma cells, both in vitro and in vivo, and to reduce the level of anti-alphaGal1-3Gal antibody (anti-Gal Ab) in baboons. METHODS: In vitro the effect of As2O3 on antibody secretion (anti-Gal IgM, total IgG and IgM) was measured by enzyme-linked immunospot assay (ELISPOT). Its inhibition of proliferation of baboon splenocytes and the NCI-H929 human plasmacytoma cell line was measured by tritiated thymidine uptake. In vivo: all baboons (n=7) had undergone splenectomy. The effects of As2O3 (0.18 to 0.36 mg/kg) on B/plasma cell depletion and anti-Gal Ab production were assessed in three baboons. For comparison, three baboons received either WBI (2 x 150 cGy) or anti-CD20 mAb (20 mg/kg x 4 doses), or both WBI and anti-CD20 mAb. A final baboon received As2O3 + WBI (150 cGy) + anti-CD20 mAb. Anti-Gal Ab levels were measured daily by ELISA. Depletion of B cells from blood and bone marrow (BM) was monitored by flow cytometry and by histology of lymph nodes (LN). Autopsy was performed in three baboons. RESULTS: In vitro: As2O3 (at 5 x 10-6 mol/l) reduced anti-Gal IgM and total IgM secretors by 76% (P=0.53) and 95% (P < 0.001), respectively, but did not reduce total IgG secretors. As2O3 inhibited in a dose-dependent manner the proliferation of activated splenocytes and of the NCI-H929 plasmacytoma cell line; complete inhibition was achieved at a dose of 1 x 10-5 mol/l. In vivo: As2O3 was found to be toxic at the doses given and was associated with the deaths of two of the four baboons that received it. Daily intravenous therapy with As2O3 alone reduced B cells (CD20+) in the blood (by 50 to 90%), BM (40%) and LN (20 to 30%), but anti-Gal Ab levels were not significantly decreased. Anti-CD20 mAb therapy alone or WBI alone depleted B cells by 100% in the blood and BM, and 80 to 100% in the LN. The combination of anti-CD20 mAb + WBI led to depletion of B cells in blood, BM and LN for 3 months, but reduction of anti-Gal Ab remained marginal. The combination of As2O3 + anti-CD20 mAb + WBI did not reduce anti-Gal Ab levels further. At autopsy in the latter baboon, B cells remained present in Peyer's patches and tonsils. CONCLUSIONS: In vitro: As2O3 reduced B/plasma cell numbers and suppressed IgM secretors, but not IgG secretors. In vivo: As2O3 was not as effective as either anti-CD20 mAb or WBI in depleting B/plasma cells, and was largely ineffective in reducing anti-Gal Ab levels. Its administration was associated with considerable toxicity. Autopsy in one baboon suggested that B cells in Peyer's patches and tonsils may be resistant to therapy and remain a source of continuing production of anti-Gal Ab.
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Neurotoxicity of leflunomide in baboons.
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Depletion of anti-gal antibodies in baboons by intravenous therapy with bovine serum albumin conjugated to gal oligosaccharides.
BACKGROUND: Anti-Galalpha 1-3Gal (Gal) antibodies (Ab) play a key role in the rejection of pig cells or organs transplanted into primates. A course of extracorporeal immunoadsorption (EIA) of anti-Gal Ab using an immunoaffinity column of a Gal type 6 oligosaccharide depletes Ab successfully, but Ab returns during the next few days. Although therapy with an anti-CD154 monoclonal antibody (mAb) prevents an induced Ab response to Gal or non-Gal epitopes, T cell-independent natural anti-Gal IgM and IgG return to baseline (pretransplant) levels. We have investigated the capacity of continuous i.v. infusion of bovine serum albumin conjugated to Gal type 6 oligosaccharide (BSA-Gal) to deplete or maintain depletion of circulating anti-Gal Ab. METHODS: Porcine peripheral blood mobilized progenitor cells (PBPC) obtained by leukapheresis from MHC-inbred miniature swine (n=6) were transplanted into baboons. Group 1 baboons (n=4) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with antithymocyte globulin, complement depletion with cobra venom factor, short courses of anti-CD154 mAb therapy (20 mg/kg i.v. on alternate days), cyclosporine (CyA) (in two baboons only), mycophenolate mofetil, and porcine hematopoietic growth factors. Anti-Gal Ab depletion by EIA was carried out before transplantation of high doses (2-4x 1010 cells/kg) of PBPC. Group 2 baboons (n=3) received the group 1 regimen (including CyA) plus a continuous i.v. infusion of BSA-Gal. To prevent sensitization to BSA, anti-CD154 mAb therapy was continued until BSA-Gal administration was discontinued. RESULTS: In group 1, Gal-reactive Ab returned to pre-PBPC transplant levels within 15-21 days, but no induced Ab to Gal or non-Gal determinants developed while anti-CD154 mAb therapy was being administered. In group 2, anti-Gal Ab was either not measurable or minimally measurable while BSA-Gal was being administered. After discontinuation of BSA-Gal, Ab did not return to pre-PBPC transplant level for more than 40-60 days, and no sensitization developed even when all therapy was discontinued. In one baboon, however, Ab to Gal type 2, but not type 6, returned during BSA-Gal therapy. CONCLUSIONS: Prevention of the induced humoral response to Gal and non-Gal epitopes by anti-CD154 mAb therapy has been reported previously by our group, but our studies are the first to demonstrate a therapy that resulted in an absence of natural anti-Gal Ab for a prolonged period. The combination of BSA-Gal and T cell costimulatory blockade may facilitate survival of pig cells and organs transplanted into primates. The return in one baboon of Ab reactive with the Gal type 2 oligosaccharide, but not type 6, indicates some polymorphism of anti-Gal Ab and suggests that, to be effective in all cases, the infusion of a combination of type 6 and type 2 BSA-Gal may be required.
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Depletion of natural anti-pig antibodies by the continuous infusion of oligosaccharides in a pig-to-baboon model.
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Xenotransplantation--how far have we come?
The immunologic barriers to xenotransplantation are summarized and approaches to overcome them briefly reviewed. Intensive investigation is being directed to the problem of acute humoral xenograft rejection, which is the major current barrier. Although the induced antibody response appears to be prevented by combination therapy with an anti-CD154 monoclonal antibody and mycophenolate mofetil, deposition of natural anti-Gal antibody on the graft endothelial cells appears to be sufficient to lead to rejection or a state of consumptive coagulopathy. Approaches towards the induction of tolerance are described. The potential microbiologic risks and physiologic incompatibilities of pig-to-human organ transplantation are also briefly discussed.
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Differential expression of Galalpha1,3Gal epitopes on fetal and adult porcine hematopoietic cells.
Galalpha1-3Gal (Gal) is the major epitope on pig tissues bound by human natural antibodies. Xenogeneic hematopoietic cell transplantation is being investigated to induce immunological tolerance to xenografts. We have investigated the level of Gal expression on pig hematopoietic cells. Cells were collected from pig fetal liver and bone marrow (BM), and also from adult BM and peripheral blood, before and after treatment with pig-specific hematopoietic growth factors. Fluorescent activated cell sorting (FACS) analysis was performed with the M86 monoclonal antibody (specific for Gal), lineage markers, and biotinylated stem cell factor (SCF) to detect c-kit expression. In fetal pig BM and liver, there was no significant difference in Gal expression between monocytes/macrophages (myeloid cells) and lymphocytes. In adult hematopoietic cells from all sources, Gal-positive subpopulations in T cells showed weak expression of Gal, whereas B cells demonstrated higher expression, and myeloid cells showed highest expression. Adult BM and mobilized peripheral blood progenitor cells contained small populations with very low or negligible expression of Gal. A very small population of c-kit-positive cells, indicating progenitor cells, were Gal-negative. The small Gal-negative population that exists in progenitor cells might explain why some pig colony forming units (CFU) can be resistant to human serum.
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Promotion of xenogeneic hematopoietic chimerism in rodents by mononuclear phagocyte depletion.
The successful establishment of tolerance toward pig tissues in primates through hematopoietic progenitor cell engraftment is restricted by the rapid disappearance of these cells in the recipient following infusion. We developed and tested the hypothesis that phagocytes of the reticuloendothelial system are responsible for the rapid clearance of infused pig hematopoietic cells using a mouse model. Mice received non-myeloablative conditioning and, on various days, were injected with medronate-encapsulated liposomes (M-L) or control blank liposomes, followed by the intravenous infusion of miniature swine hematopoietic cells. M-L were well-tolerated in mice (n=100) at levels that deplete mononuclear phagocytes. Depletion of mononuclear phagocytes in normal Balb/c mice as well as in severe combined immune deficient mice increased the accumulation of pig hematopoietic cells in the bone marrow (BM) by 10-fold when measured 24 h after the infusion of the cells. Colony-forming unit analysis showed an increased accumulation of pig hematopoietic progenitors in the BM of mice that were infused with medronate-liposomes. We conclude that depletion of mononuclear phagocytes by M-L has the potential to lower the barrier to the establishment of mixed chimerism and tolerance induction in xenotransplantation.
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Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade.
The aim of the present study was to assess the survival of adult porcine islets transplanted into baboons receiving either (I) conventional triple drug immunosuppressive therapy or (2) a non-myeloablative regimen and an anti-CD154 monoclonal antibody (mAb) aimed at tolerance-induction. Group 1 baboons (n = 3) were pancreatectomized prior to intraportal injection of 10,000 porcine islet equivalents (IE)/kg and immunosuppressed with anti-thymocyte globulin (ATG), cyclosporine and azathioprine. In Group 2 (n = 2), non-pancreatectomized baboons underwent induction therapy with whole body and thymic irradiation, and ATG. Extracorporeal immunoadsorption (EIA) of anti-Galalpha1,3Gal (Gal) antibody was carried out. Maintenance therapy was with cobra venom factor, cyclosporine. mycophenolate mofetil, methylprednisolone and anti-CD154 mAb. Porcine islets were injected intraportally (14,000 and 32,000 IE/kg, respectively) and high-dose pig mobilized peripheral blood progenitor cells (3 x 10(10) cells/kg) were infused into a systemic vein. Porcine islets were also implanted in the sternomastoid muscle to facilitate subsequent biopsies. In both groups. porcine C-peptide was measured, and histological examination of liver or sternomastoid muscle biopsies was performed at regular intervals. In Group 1, total pancreatectomy reduccd human C-peptide to < 0.1 ng/ml and induced insulin-requiring diabetes. The transplantation of porcine islets was followed by normalization of glycemia for 15-24 h. Porcine C-peptide was detected only transiently immediately after porcine islet injection (maximum 0.12 ng/ml). Histological examination of liver biopsies taken between days 2 and 19 did not reveal viable islets, but necrotic cell structures with mononuclear cell infiltrates were identified in portal venules. In Group 2, injection of porcine islets into non-pancreatectomized recipients induced a transient hypoglycemia (2-4 h) requiring concentrated intravenous dextrose administration. Porcine C-peptide was detectable for 5 and 3 days (maximum 2.8 and 1.0 ng/ml), respectively. Baboon #4 died on day 12 from small bowel intussusception. Liver and sternomastoid muscle biopsies showed well-preserved porcine islets, staining positive for insulin and glucacon, without signs of rejection. In baboon #5, viable islets were detected in the sternomastoid muscle biopsy on day 14, but not on day 28 or thereafter. A progressive mononuclear cell and macrophage infiltration was seen in the biopsies. In conclusion, conventional immunosuppression allowed survival of porcine islets in baboons for < 24 h. The non-myeloablative regimen prolonged survival of porcine islets for > 14 days. However, despite depletion of T cells, anti-Gal antibody and complement, and CD154-hlockade, porcine islets were rejected by day 28. These results suggest that powerful innate immune responses are involved in rejection of discordant xenogencic islets.
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Therapeutic interventions in xenotransplantation.
Xenotransplantation, involving the transplantation of pig organs into humans, would resolve the current shortage of organs. It involves, however, a new therapeutic approach to organ transplantation. The presence of natural antibody in primates directed against Galalpha1,3Gal epitopes on pig vascular endothelium leads to early antibody-mediated rejection. An elicited antibody response against the same target epitopes as well as against nonGal antigens intensifies the immune destruction of the organ. Even the minimal deposition of antibody appears to lead to the development of a consumptive coagulopathy that can be fatal. Approaches being investigated to overcome these barriers include depletion and inhibition of natural antibody and complement, and suppression of the elicited T cell-dependent antibody and cellular responses. In addition, however, physiologic incompatibilities between human and pig, particularly those relating to coagulation, may enhance or complicate the immune process, and may require additional therapeutic measures. Current approaches aimed at achieving successful xenotransplantation also include investigation of agents that prevent potential xenozoonotic infection of the recipient. At present, therefore, the therapeutic interventions required to attempt to overcome the barriers to xenotransplantation are multiple. Work indicating progress in the breeding of pigs that do not express the critical Galalpha1,3Gal epitopes, however, is encouraging. The introduction of these pigs may greatly reduce the therapy required, and may ultimately allow the development of methods to induce tolerance to the transplanted pig organ.
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Potential of aspirin to inhibit thrombotic microangiopathy in alpha1,3-galactosyltransferase gene-knockout pig hearts after transplantation in baboons.
Hearts from alpha1,3-Galactosyltransferase gene-knockout (GaIT-KO) pigs were transplanted heterotopically into 8 baboons that received an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen and heparin. Three baboons died or were euthanized with beating grafts on 16, 23, and 56 days, respectively, and the remaining 5 grafts functioned for 59-179 days. Hyperacute rejection did not occur, and classical features of acute humoral xenograft or acute cellular rejection were rare. However, thrombotic microangiopathy (TM) developed in all cases; its onset was delayed in 2 baboons that received aspirin. Function of a pig organ in a baboon for a period approaching 6 months has not been reported previously and lends encouragement that the barriers to xenotransplantation will be overcome, but TM requires investigation.
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