Cardiac xenotransplantation: recent preclinical progress with 3-month median survival

OBJECTIVES: Transplantation is limited by a lack of human organ donors. Organs derived from animals, most likely the pig, represent a potential solution to this problem. For the heart, 90-day median graft survival of life-supporting pig hearts transplanted to nonhuman primates has been considered a reasonable standard for entry into the clinical arena. Overcoming the immune barrier to successful cardiac xenotransplantation is most appropriately first explored with the non-life-supporting heterotopic model. METHODS: We performed a series of 7 heterotopic heart transplantations from CD46 transgenic pigs to baboons using a combination of therapeutic agents largely targeted at controlling the synthesis of anti-pig antibodies. Rituximab (anti-CD20) and Thymoglobulin (rabbit antithymocyte globulin [ATG]; SangStat Medical Corp, Fremont, Calif) were used as induction therapy. Baseline immunosuppression consisted of splenectomy, tacrolimus, sirolimus, steroids, and TPC (an anti-Gal antibody therapeutic). Rejection events were not treated. RESULTS: By using Kaplan-Meier analysis, median graft survival was 96 days (range, 15-137 days; 95% confidence interval, 38-99 days). Only 2 grafts were lost as a result of rejection, as defined by cessation of graft palpation. There was no evidence of a consumptive coagulopathy, infectious complications were treatable, and no posttransplantation lymphoproliferative disorders occurred. No cellular infiltration was observed. CONCLUSIONS: This study reports the longest median survival to date (96 days) of pig hearts transplanted heterotopically into baboons. Duplication of these results in the orthotopic life-supporting position could bring cardiac xenotransplantation to the threshold of clinical application.     

Thrombotic microangiopathy and graft arteriopathy in pig hearts following transplantation into baboons

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.     

Increased immunosuppression, not anticoagulation, extends cardiac xenograft survival

BACKGROUND: Cardiac xenograft function is lost due to delayed xenograft rejection (DXR) characterized by microvascular thrombosis and myocardial necrosis. The cause of DXR is unknown but may result from thrombosis induced by antibody-mediated activation of endothelial cells and/or by incompatibilities in thromboregulatory interactions. METHODS: To examine these issues, a series (Groups 1-6) of previous transgenic CD46 pig-to-baboon heterotopic cardiac transplants were reanalyzed for baseline immunosuppressive levels, graft survival and infectious complications with and without systemic anticoagulation. Groups 1-4 received low dose tacrolimus and sirolimus maintenance therapy, with splenectomy, anti-CD20 and daily alpha-Gal polymer. Group 1 recipients received no anticoagulation. Groups 2-4 were anticoagulated with aspirin and Plavix, Lovenox, or Coumadin, respectively. Group 5 was treated with Lovenox and high dose tacrolimus and sirolimus maintenance therapy. Group 6 recipients received no postoperative anticoagulation but the same immunosuppression as group 5. RESULTS: Median survival (15-22 days) within groups 1-4 was not significantly different. At rejection all tissues exhibited microvascular thrombosis, coagulative necrosis and similar levels of platelet and fibrin deposition. Groups 5 and 6 median survival (76 days) was significantly increased compared to groups 1-4. There was no significant difference in median survival between Lovenox treated recipients (68 days) and anticoagulant free recipients (96 days). Rejected tissues showed vascular antibody deposition, microvascular thrombosis, and myocyte necrosis. CONCLUSION: Significant prolongation in xenograft survival is achieved by improved immunosuppression. These results suggest that ongoing immune responses remain the major stimulus for DXR.     

Prolongation of second heart transplants in rats.

Second clinical kidney grafts often survive longer than first grafts. Using a rat cardiac allograft model, we examined the conditions under which survival of a second graft can be longer than first graft survival. In the BUF-to-LEW combination, following rejection of the first transplant, second cardiac allografts from the same donor strain implanted immediately survived longer than the first grafts (P = 0.047). Although the mean survival time of first grafts was 9.0 days, second grafts implanted in the same animals survived 19.5 days. In contrast, when ACI donors were used for the same LEW recipients, the second grafts were rejected in 3 days compared with 6.6 days for first grafts. Donor-specific spleen cell transfusions in these combinations resulted in prolonged survival in the BUF to LEW combination, but had no effect when the donor strain was ACI. Second grafts from BUF had prolonged survival following rejection of the first graft. Thus the histocompatibility difference was a determining factor of whether or not prolongation would be obtained. Another factor was timing of the second transplant. If 7 days were allowed to elapse following rejection of the first graft before implantation of the second, the enhancement effect was lost. Moreover, in the LEW-to-ACI combination in which second grafts were rejected rapidly, removal of the first graft after 7 days (before rejection), resulted in prolonged survival of the second graft. There is, therefore, a window of time before rejection of first grafts and shortly thereafter, when the enhancement effect can be obtained. Passive transfer of serum in the BUF-to-LEW combination resulted in enhancement, but transfer of splenic cells was ineffective. We conclude that graft rejection can result in induction of enhancement during a specific period, after which this effect is lost; and that enhancement can be obtained only in certain strain combinations. This suggests that human patients with heart transplants that reject might benefit from a second graft, even from a donor with a mismatch similar to the first graft.     

Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs

A combination of genetic manipulations of donor organs and target‐specific immunosuppression is instrumental in achieving long‐term cardiac xenograft survival. Recently, results from our preclinical pig‐to‐baboon heterotopic cardiac xenotransplantation model suggest that a three‐pronged approach is successful in extending xenograft survival: (a) α‐1,3‐galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal‐specific antibody‐mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co‐stimulation blockade of CD40‐CD40L pathways with anti‐CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non‐Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co‐stimulation blockade‐based immunomodulatory regimen.     

Immunosuppressive effect of combination schedules of brequinar with leflunomide or tacrolimus on rat cardiac allotransplantation

Drug toxicity is one of the major problems in clinical immunosuppression. Combining two immunosuppressants in low or ineffective doses is an attractive strategy if it helps to reduce drug-related toxicity. We examined the immunosuppressive efficacy of brequinar (BQR) in combination with leflunomide (Lef) or tacrolimus (FK) in a heterotopic rat cardiac allotransplantation model. Abdominal heterotopic heart grafts (DA x LEW) were immunosuppressed from the time of transplantation and continued until the ninth posttransplant day (POD) in experiments examining prophylaxis of rejection treatment (PRT). In a separate series of experiments designed to test rescue treatment (RT), immunosuppression was begun on POD 4 and continued for 10 days; transplanted rats were sacrificed the following day intentionally. Cardiac rejection was monitored by palpation and documented by light microscopy. Immunosuppressive drugs (BQR 3 mg/kg and 12 mg/kg; BQR 3 mg/kg + Lef 5 mg/kg; BQR 3 mg/kg + FK 0.5 mg/kg) were given orally by gavage; thrice weekly according to the monotherapy or dual-therapy dosing protocol. Median survival time of the cardiac graft for controls (no treatment) was 5 days. BQR monotherapy 3 mg/kg (low dose) improved graft survival (P = 0.003); graft histology showed moderate acute rejection. BQR monotherapy 12 mg/kg (therapeutic dose) application in the PRT or RT treatment arms of the study design resulted in aortic-graft ruptures and clinical toxicity in each treatment arm due to overimmunosuppression; normal graft morphology was maintained. Successful rescue of rejecting grafts was histologically documented. Combining BQR with Lef or FK in the PRT protocol showed prolonged graft survival in both drug combination groups (median survival time, 14 days; P = 0.009 and 0.014, respectively). Using an identical combination protocol for RT, all grafts achieved a 14-day graft survival; cardiac histology showed reversible moderate acute rejection. BQR given in the presence of Lef or FK not only prevented acute rejection but intercepted it so long as it was administered; grafts were rejected within 4 days of stopping immunosuppression in the PRT study. These combinations using low or subtherapeutic doses may be important for controlling transplant rejection and rescuing ongoing graft rejection. The need for continuing treatment in this strongly allogeneic model is highlighted.     

Life supporting function for over one month of a transgenic porcine heart in a baboon.

BACKGROUND: Inhibition of hyperacute rejection (HAR) and sustained graft survival have been demonstrated in a pig-to-primate model of heterotopic cardiac xenotransplantation using pigs transgenic for human Decay Accelerating Factor (hDAF). Building on this work, an orthotopic model has been developed. This case records 39-day cardiac xenograft function in a life-supporting capacity with clinically applicable immunosuppression. METHODS: Using a heart from an hDAF transgenic pig, an orthotopic cardiac transplant was performed on an adult baboon. The immunosuppressive regimen consisted of induction with a short course of cyclophosphamide, followed by maintenance therapy with cyclosporine A, mycophenolate mofetil and a tapering course of corticosteroids. Post-operative monitoring included daily anti-pig hemolytic antibody titer surveillance and endomyocardial biopsy. RESULTS: The animal survived 39 days and was active and energetic throughout its postoperative course, remaining free of signs of cardiopulmonary failure. Endomyocardial biopsy performed on post-operative Day 36 revealed only patches of sub-endocardial fibrosis with no signs of active rejection. The baboon succumbed to an acute cardiopulmonary decompensation immediately following administration of medication via oral gavage. Post-mortem histopathology demonstrated well-preserved myocardial architecture with small foci of mild humoral rejection. CONCLUSIONS: This case documents the longest survival recorded to date of a discordant orthotopic cardiac xenograft and illustrates that the hDAF transgene combined with a clinically acceptable maintenance immunosuppressive regimen enables sustained, life-supporting function of porcine cardiac xenografts in non-human primates. The inhibition of hyperacute rejection and the subsequent control of humoral and cellular rejection for over 1 month demonstrated in this experiment represent significant progress in the development of a viable strategy for clinical xenotransplantation.     

CTLA4-Ig-based conditioning regimen to induce tolerance to cardiac allografts

BACKGROUND: Transplant rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem. Mixed hematopoietic chimerism has been shown to produce tolerance to solid organ transplants. However, currently available protocols to induce mixed hematopoietic chimerism invariably require toxic pre-conditioning. In this study, we investigated a non-toxic CTLA4-Ig-based protocol to induce donor-specific tolerance to cardiac allografts in rats. METHODS: Fully mismatched, 4 to 6 week old ACI (RT1.A(a)) and Wistar Furth (RT1.A(u)) rats were used as cell/organ donors and recipients, respectively. Recipients were treated with CTLA4-Ig 2 mg/kg/day (on days 0, 2, 4, 6, 8), tacrolimus 1 mg/kg/day (daily, from days 0 to 9), and a single dose of anti-lymphocyte serum (10 mg) on day 10, soon after total body irradiation (300 cGy) and donor bone marrow (100 x 10(6) T-cell depleted cells) transplantation (BMT). Six weeks after BMT, chimeric animals received heterotopic heart transplants. RESULTS: Hematopoietic chimerism was 18.8 +/- 10.6% at day 30, and was stable (24 +/- 10%) at 1 year post-BMT; there was no graft versus host disease. Chimeric recipients (RT1.A(u)) permanently accepted (>360 days) donor-specific (RT1.A(a); n = 6) hearts, yet rapidly rejected (<9 days) third-party hearts (RT1.A(l); n = 5). Graft (heart) tolerant (>100 days) recipients accepted donor-specific secondary skin grafts (>200 days) while rejected the third-party skin grafts (<9 days). Lymphocytes of graft tolerant animals demonstrated hyporesponsiveness in mixed lymphocyte cultures in a donor-specific manner. Tolerant graft histology showed no obliterative arteriopathy or chronic rejection. CONCLUSIONS: The CTLA4-Ig based conditioning regimen with donor BMT produced mixed chimerism and induced donor- specific tolerance to cardiac allografts.     

Cardiac xenotransplantation in primates

Successful cardiac xenotransplantation would alleviate the severe shortage of donor organs that presently limits the availability of cardiac transplantation. Early attempts at human xenotransplantation achieved minimal success. However, the effectiveness of cyclosporine in nonhuman xenotransplant models has received little experimental investigation. We have therefore studied the effect of cyclosporine-based immunosuppression in primate cardiac xenograft models using cynomolgus monkey donors and baboon recipients. Donor hearts were transplanted heterotopically into the necks of recipients or in the orthotopic position. Recipients were treated with no immunosuppression (controls), cyclosporine and steroids, or cyclosporine, steroids, azathioprine, and antithymocyte globulin. Statistically significant prolongation of graft survival compared to the control group was observed in the heterotopic groups. Mean survival time of the cyclosporine-treated and steroid-treated heterotopic grafts was 61 days compared to 6 days for grafts in the control group (p = 0.01); the addition of azathioprine and antithymocyte globulin yielded a mean survival of 84 days (p less than 0.01). No significant increase in graft survival was noted in the orthotopic groups treated with either immunosuppressive regimen. Although long-term use of human xenografts as an alternative for heart replacement is not supported by these data, further investigation of the orthotopic model is clearly justified.     

Hyperhomocyst(e)inemia Induces Accelerated Transplant Vascular Sclerosis in Syngeneic and Allogeneic Rat Cardiac Transplants

Chronic rejection (CR) and transplant vascular sclerosis (TVS) cause the majority of graft failures in cardiac transplantation. Hyperhomocyst(e)inemia [hH(e)] is associated with human TVS without a proven causal relationship. This study investigated the effect of hH(e) on graft survival and TVS in allogeneic and syngeneic rat cardiac transplants. Lewis recipients of heterotopic F344 heart allografts, received normal or hH(e)-inducing (folate, methionine) diets [controls: syngeneic transplanted [+/- hH(e), + CsA] and nontransplanted rats [+/- hH(e), +/- CsA]]. Serial plasma homocyst(e)ine [H(e)] levels were measured. TVS was assessed in clinically rejected grafts and a subset of pre-rejection normal diet allografts (day 64) (neointimal index, NI). The hH(e) diet elevated plasma H(e) levels. When compared with normal diet controls (n = 9), hH(e) diet allografts (n = 9) had decreased time to onset of CR (40 +/- 9 vs. 72 +/- 10d, p = 0.02), and graft failure (64 +/- 10 vs. 107 +/- 12d, p = 0.009). hH(e) diet allografts at rejection (n = 9, 64d) had more severe TVS (NI = 68 +/- 2) than both time-matched normal diet allografts (NI = 49 +/- 6, n = 8, 64d, p <0.001) and normal diet allografts at rejection (NI = 58 +/- 5, n = 9, 107d, p = 0.007). hH(e) induced TVS in syngeneic grafts (NI=50 +/- 3, n = 10 vs. NI = 5 +/- 3, n = 10, 130d, p <0.001). hH(e) accelerated rejection and increased the severity of TVS in allogeneic cardiac transplants, and induced TVS in syngeneic cardiac transplants.     

The role of tumor necrosis factor in allograft rejection. III. Evidence that anti-TNF antibody therapy prolongs allograft survival in rats with acute rejection

We have previously demonstrated that TNF-alpha levels are elevated in liver transplant patients experiencing acute rejection. In addition, prophylactic administration of anti-TNF-alpha or anti-TNF-beta antibodies prolonged graft survival in a rat heterotopic cardiac transplant model. This experiment was designed to evaluate anti-TNF therapy in the treatment of acute allograft rejection. Heterotopic cardiac transplants were performed using Buffalo donors and Lewis recipients. Histologic sections of transplanted grafts from untreated animals revealed significant rejection at day 4 with terminal rejection occurring on day 10.8 +/- 0.4. Animals in the experimental groups received antirejection therapy from postoperative days 4-13. Treatment with cyclosporine at 2 mg/kg/day prolonged graft survival to 16.5 +/- 2.0 days (P = 0.01 versus controls). Administration of polyclonal anti-TNF-alpha in combination with polyclonal anti-TNF-beta increased graft survival to 14.6 +/- 0.4 days (P less than 0.001 versus controls). Use of a monoclonal anti-TNF-alpha antibody was even more effective, with graft survival of 17.4 +/- 0.7 days (P less than 0.001 versus controls). Combination immunotherapy with monoclonal anti-TNF-alpha in conjunction with CsA extended survival to greater than 30 days. In contrast, recombinant TNF-alpha (5 micrograms/day, i.p.) markedly accelerated the time to graft failure (7.4 +/- 0.2 days, P less than 0.001 versus controls). Examination of explanted graft tissue on postoperative day 9 from animals treated with anti-TNF showed decreased mononuclear cell infiltrate when compared to untreated animals. Treatment with TNF-alpha markedly increased the inflammatory process. These results suggest that TNF may play a role in the pathogenesis of acute rejection.     

Improvement in rejection of human decay accelerating factor transgenic pig-to-primate renal xenografts with administration of rabbit antithymocyte serum

BACKGROUND: Survival in pig-to-baboon kidney xenotransplantation is currently limited by acute humoral xenograft rejection (AHXR). We hypothesized that the administration of rabbit antithymocyte serum (RATS) would delay or prevent AHXR as compared with a cyclophosphamide (CyP)-based immunosuppressive regimen. METHODS: Nine baboons received life-supporting heterotopic single-kidney transplants from human decay accelerating factor transgenic pigs. Immunosuppression consisted of GAS (a galactosyl alpha-1,3-galactose analog), cyclosporine, and steroids. Group 1 (n=2) was also treated with CyP and a rapamycin derivative (RAD), group 2 (n=4) received RATS and RAD, and group 3 (n=3) received only RATS. Animals were maintained until death or sacrifice because of uncontrollable rejection or other complications. Graft histopathology was assessed at the study endpoint. RESULTS: Mean survival was 28+/-11.3 days, 23+/-2.5 days, and 20+/-2.5 days for groups 1, 2, and 3 (not significant). Graft rejection was the cause of death in both CyP-treated animals. One RATS-treated animal died of rejection; the others died of infections or bleeding. Two RATS-treated animals developed posttransplant lymphoproliferative disorder, and one died of cytomegalovirus pneumonitis. Histopathology revealed severe AHXR in group 1 kidneys, involving 100+/-0% of the tissue examined. In contrast, AHXR was reduced in groups 2 and 3, involving 21+/-14% and 18+/-28%, respectively, of the tissue examined (P<0.01). CONCLUSIONS: Substitution of RATS for CyP was well tolerated and resulted in reduced severity of AHXR in this model. Complications seen in RATS-treated animals may be preventable through the use of standard prophylaxis for infections. Our data suggest that further studies are warranted to explore the use of antilymphocyte agents in xenotransplantation.     

Co-stimulation blockade targeting CD154 and CD28/B7 modulates the induced antibody response after a pig-to-baboon cardiac xenograft

BACKGROUND: The induced antibodies against Galalpha1,3Gal (Gal) and non-Gal epitopes may contribute to delayed xenograft rejection (DXR). We asked whether blockade of the CD40/CD154 and CD28/B7 co-stimulatory pathways modulates the baboon elicited antibody response to pig Gal and non-Gal antigens. METHODS: Eighteen baboons received heterotopic heart transplants from pigs transgenic for human decay-accelerating factor (n = 13) or membrane cofactor protein (n = 5). Ten reference 'conventional therapy' animals received cyclosporin A, cyclophosphamide and mycophenolate mofetil, with (n = 4) or without (n = 6) anti-CD20. Eight 'co-stimulation blockade' animals received anti-CD154 mAb (IDEC-131) and anti-thymocyte globulin, with (n = 4) or without (n = 4) anti-CD20; two of these animals also received CTLA4-Fc. Anti-alphaGal IgG and IgM, anti-non-Gal antibodies and graft histology were assessed serially. RESULTS: Excluding two early graft failures, median graft survival with conventional therapy was 15 days (range 6 to 36 days, n = 8). Anti-Gal IgG antibody remained low through day 6 to 10, only one graft failure was accompanied by significant rise in anti-Gal IgG, and the anti-non-Gal response was weak (n = 2) or absent (n = 7). However many recipients succumbed with infection (n = 4) or coagulopathy (n = 2); DXR and ICOS+ T cells were prevalent in long-surviving grafts. With co-stimulation blockade, excluding three early graft failures, median graft survival was 7 days (range 6 to 11 days, n = 5). This regimen was very well tolerated, but increased anti-Gal antibody titer within 14 days was associated with graft failure in four of six animals. Although an anti-non-Gal response was present in three of six animals during IDEC-131 monotherapy (one strong, two weak), it was absent in both cases with additional CTLA4-Fc treatment. CONCLUSIONS: As used here, CD154 blockade alone does not completely prevent induction of Gal and non-Gal anti-pig antibodies. Our preliminary data suggest that other co-stimulation pathways, including CD28/B7 and ICOS, are sufficient to mediate high-titer anti-non-Gal antibody to porcine antigens in baboons, and contribute significantly to the pathogenesis of DXR.     

Intragraft delivery of 16, 16-dimethyl PGE2 induces donor-specific tolerance in rat cardiac allograft recipients

The stable prostaglandin E2 analogue, 16,16-dimethyl PGE2 (di-M-PGE2) was continuously infused by osmotic pump directly into rat heterotopic cardiac allografts. Intragraft delivery of 20 micrograms/kg/day di-M-PGE2 for 2 weeks completely prevented graft rejection for more than 150 days (n = 10), while untreated Buffalo recipients rejected Lewis cardiac allografts within 8 days after transplantation (mean survival time = 7.4 +/- 0.5 days, n = 5). When given for only 1 week, 20 micrograms/kg/day had a partial effect, since 60% of recipients accepted grafts long-term and 40% experienced rejection by day 14 (n = 5). In contrast, systemic intravenous administration of 20 micrograms/kg/day di-M-PGE2 for 2 weeks could not prolong graft survival (MST = 7.0 +/- 0.0 days, n = 3), and the higher dose of 200 micrograms/kg/day resulted in death by day 2 (n = 5). Long-term BUF recipients of LEW cardiac allografts accepted LEW donor strain skin grafts for more than 35 days while rejecting third-party Wistar Furth skin grafts in a normal fashion (MST = 7.3 +/- 0.5 days, n = 3), indicating the induction of donor-specific tolerance. Long-surviving LEW cardiac allografts retransplanted into naive BUF recipients were rejected within 7 days (MST = 6.7 +/- 0.5 days, n = 3), indicating no change in graft immunogenicity. Therefore, a 14-day infusion of di-M-PGE2 directly into a strongly MHC-mismatched cardiac allograft uniformly has resulted in long-term engraftment and the development of recipient donor-specific tolerance.     

Effective antiplatelet therapy does not prolong transgenic pig to baboon cardiac xenograft survival

BACKGROUND: Microvascular thrombosis is a prominent characteristic of delayed xenograft rejection, therefore the effects of antiplatelet therapy with aspirin and clopidogrel on long-term cardiac xenograft function was investigated in a heterotopic pig-to-baboon cardiac transplant model. METHODS: Donor hearts from human CD46 transgenic pigs were transplanted heterotopically to baboons. The recipients received immunosuppression that included tacrolimus, sirolimus, corticosteroids, anti-CD20 monoclonal antibody and TPC, an alpha-galactosyl-polyethylene glycol conjugate. In group 1 (n = 9) in addition to immunosuppression, the recipients received combination therapy consisting of aspirin (80 mg/day) and clopidogrel (75 mg/day) beginning 2 days after transplant and continuing until cessation of graft function. Antiaggregatory efficacy was evaluated by platelet aggregation assay. In group 2 (n = 9) antiplatelet drugs were not given. RESULTS: Functional assays confirmed inhibition of platelet aggregation in group 1 suggesting sufficient systemic effects of the treatment. However, anticoagulant therapy did not result in significant prolongation of xenograft function (group 1: median survival 22 days, range 15 to 30 days; group 2: median survival 15 days, range 4 to 53 days). Histologic analysis at rejection revealed no difference in the level of platelet containing thrombi between the groups. CONCLUSIONS: Inhibition of platelet aggregation by a combination of aspirin and clopidogrel did not have a significant impact on the length of xenograft survival or on the development of microvascular thrombosis in this pig-to-primate model.     

Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies

BACKGROUND: We have previously demonstrated that blockade of either CD80/86-CD28 or CD40-CD154 costimulatory pathways by using adenovirus vector coding CTLA4Ig (AdCTLA4Ig) or CD40Ig (AdCD40Ig) genes induced donor-specific tolerance in rat liver transplantation. In this study, we asked whether these gene-therapy-based costimulation blockade would induce tolerance in cardiac transplantation. METHODS: Heterotopic heart transplantation was performed in a full major histocompatibility complex (MHC) barrier combination of ACI (RT1avl) to Lewis (LEW, RT1l) rats. Vector (1 x 10(9) plaque forming unit [PFU]), AdLacZ, AdCTLA4Ig, or AdCD40Ig, was administered intravenously to recipient animals immediately after grafting, and graft survival, serum CTLA4Ig/CD40Ig levels, and graft histology were assessed. Tolerance was determined by secondary skin-graft challenging. RESULTS: Allografts of both untreated and AdLacZ controls were promptly rejected within 7 days, whereas a single treatment with AdCTLA4Ig or AdCD40Ig significantly prolonged median graft survival to 55.5 and 28.5 days, respectively. In contrast, the combined AdCTLA4Ig and AdCD40Ig gene therapy maintained high CTLA4Ig and CD40Ig levels through the posttransplant period and allowed long-term cardiac allograft survival for more than 270 days. However, both donor and third-party skin grafts were rejected in the animals who harbored cardiac grafts over 150 days. Also, typical features of chronic rejection were evident in the long-term surviving grafts. CONCLUSION: Simultaneous blockade of CD28 and CD154 pathways by AdCTLA4Ig plus AdCD40Ig induces a strong immunosuppression that allows long-term acceptance of full MHC mismatched cardiac graft in rats. This strategy, however, was not enough to induce tolerance to skin grafts and to avoid chronic rejection, as shown in the liver-transplantation model.     

Vulnerability of allografts to rejection by MHC class II-restricted T-cell receptor transgenic mice

BACKGROUND: Examination of the in vivo activation and function of CD4+ T cells in response to allografts may advance our understanding of the rejection process. We analyzed the capacity of transgenic class II-restricted CD4 T cells to reject skin, cardiac, and islet transplants. METHODS: TS1 mice possess a high frequency of CD4+ T cells specific for the immunodominant epitope of the viral hemagglutinin (HA) protein. We analyzed the kinetics of rejection of skin, heart, and islet grafts by na?ve and sensitized TS1 mice and by adoptively transferred TS1 lymphocytes. RESULTS: Rejection of heart transplants was more rapid than skin grafts (mean survival time, 12.9 vs. 26.6 days), and islet grafts survived indefinitely in TS1 mice. These findings may be partly attributable to the supranormal frequency of HA-reactive cells in TS1 mice. In support of this, we found that adoptive transfer of 5 x 10(5) TS1 lymphocytes to Balb/c hosts effected consistent rejection of HA-bearing skin transplants, whereas a significantly greater number (3 x 10(6)) was required for heart transplant rejection. The in vivo proliferative response of HA-specific T cells to heart and skin was found to be robust and predominantly localized to the draining lymph nodes. CONCLUSION: We developed a model of allograft rejection in which the responding T cells and relevant graft antigen are specifically defined. Adoptive transfer of carboxy-fluorescein succinimidyl ester-labeled transgenic T cells allowed us to visualize a robust proliferative response in vivo to heart and skin allografts, which in both cases was localized to regional lymph nodes.     

Histopathology of cardiac xenograft rejection in the pig-to-baboon model.

BACKGROUND: The use of pig organs transgenic for human decay accelerating factor (hDAF) has largely overcome the problems of hyperacute rejection. With improved immunosuppressive protocols, life supporting grafts are showing greater survival times bringing the possibility of clinical xenotransplantation closer. Examination of the histopathology of the rejection process provides insight into the underlying mechanism and may suggest ways in which new immunosuppressive strategies should be directed. METHODS: 44 baboons (Papio anubis) underwent heart transplants of which 39 were from transgenic donors. The transplanted organs were examined histologically and stained for evidence of immunoglobulin and complement deposition as well as cellular infiltrates. RESULTS: In the transgenic animals survival times were 2 to 99 days (mean 23.5) and the heterotopic group and 1 to 39 days (mean 11.7) in the orthotopic group. There were 3 cases of hyperacute rejection between the 2 groups. Rejected organs showed areas of old and recent myocardial infarction associated with vascular thrombosis. There was widespread deposition within vessels of immunoglobulins IgM and IgG together with complement fractions C3 and C5b to 9 in those organs that were rejected. The amount of complement positive in the longer surviving organs was less than those rejecting early. Cellular infiltate was predominantly macrophage with some later appearing T or natural killer cells. CONCLUSIONS: The histopathological changes support the importance of immunoglobulin and complement in delayed xenograft or acute vascular rejection. With time there is an increase in cellular infiltrate predominantly macrophages and these findings suggest an increasingly important role for the cells and the rejection process. The presence of areas of infarction and underlying vascular thrombosis is in keeping with endothelial activation and the establishment of procoagulant phenotype which may be due to immunoglobulin, complement, secreted cytokines and direct cellular effects.     

The effects of size and site of origin of intestinal grafts on small-bowel transplantation in the rat

This study was initiated to evaluate the effects of varying the length and site of origin of small-intestine transplants on rejection and on graft-versus-host disease (GVHD). Eighty rats had heterotopic transplants performed with systemic venous drainage of the grafts. The host native bowel was left in situ and no immunosuppressive agents were used. Twenty male Lewis inbred (LEW) rats who received isogenic grafts survived without any evidence of rejection or GVHD. When intestine from Lewis X Brown Norway hybrid rats (LBN) was transplanted into LEW rats, rejection occurred between day 6 and 9 and the time of onset of rejection was not influenced either by the length of transplanted bowel (10 to 80 cm, n = 6 each) or by whether the graft was from the jejunum or the ileum. However, rates of survival for 100 days from rejection were significantly better if 10 cm (100%) or 20 cm (84%) was transplanted than if the grafts were 40 cm or more in length (56%). The LBN recipients of LEW allografts developed GVHD on days 7 through 9, and this response was similarly unrelated to the length or segment of bowel transplanted. However, host survival was quite dependent on graft segment length and site of origin. All animals who received 20 cm or less of proximal bowel survived (with GVHD but no evidence of rejection). While 50% of the animals that received proximal intestinal grafts 40 cm in length survived GVHD, none who received identical-sized grafts from the distal ileum survived (all were dead by day 20). Our data document that the results of small-intestine transplantation is dependent on the length and site of origin of the grafts.