Serum anti-pig antibodies as potential indicators of acute humoral xenograft rejection in pig-to-cynomolgus monkey kidney transplantation

BACKGROUND: Hyperacute rejection of solid organ pig xenografts in nonhuman primates has been overcome by using donors transgenic for human complement regulatory proteins, but grafts are still susceptible to humoral (antibody-mediated) rejection. We investigated whether circulating xenoreactive antibodies are a useful indicator of this xenograft rejection. METHODS: Five assays were employed in a retrospective analysis on 20 selected cynomolgus monkey recipients of renal xenografts transgenic for human decay-accelerating factor, with survival between 4 and 60 days. The assays included hemolytic and hemagglutination assays and the measurement of immunoglobulin (Ig)G and IgM binding to porcine endothelial cells and leukocytes, and to the Gal alpha 1-3Gal trisaccharide (Gal) antigen. To assess non-Gal-directed antibodies, sera were absorbed with a Gal-coated resin. A predictive value was defined as an increase in antibody levels before a decline in graft failure (>20% increase in creatinine levels) and humoral rejection in graft pathology. RESULTS: Data on hemolytic anti-pig antibody correlated with those on IgM antibody to endothelial cells, leukocytes, and Gal. In absorbed sera IgM and IgG antibody to endothelial cells and leukocytes correlated with each other, indicative for an elicited antibody response to non-Gal antigens. Sixteen animals showed humoral rejection, and in all but two animals one or more assays was considered of predictive value. On the other hand, increased antibody levels were noted in two animals without signs of rejection in graft pathology and in two cases with cellular xenograft rejection. CONCLUSIONS: It is recommended to use multiple assays (preferably hemolytic, anti-Gal, and anti-endothelial cell) to be able to fully monitor the peripheral antibody responses in pig-to-primate xenograft recipients.     

Detection of xenoantibodies using a simple flow cytometric assay

Abstract: Higher primates, including humans, have high levels of pre-existing naturally circulating antibodies that predominantly recognize the epitope Gal (1,3-Gal), which is highly expressed on the surface of xenogenic cells. Deposition of these antibodies on the endothelial cell surface of vascularized xenografts leads to an activation of the classical pathway of the complement system, resulting in tissue ischemia and necrosis with rapid demise of the xenograft. This hyperacute rejection (HAR) is always a major barrier in xenograft transplantation and should be minimized by accurately monitoring the naturally occurring antibodies. In the present study, we utilized a simple and rapid flow cytometric (FCM) assay to monitor the presence of these naturally occurring antibodies. We found that the FCM assay is very effective in measuring human antibodies bound to the xenogenic cells, which cause cytotoxicity. This assay could be useful in the pre- and post-xenotransplantation monitoring of xenoantibodies, thus, helping in the development of strategies to block the binding of preformed human antibodies to the xenograft in order to overcome the problem of HAR.     

The xenoantibody response and immunoglobulin gene expression profile of cynomolgus monkeys transplanted with hDAF-transgenic porcine hearts

BACKGROUND: Recent work has indicated a role for anti-Gal alpha 1-3Gal (Gal) and anti-non-Gal xenoantibodies in the primate humoral rejection response against human-decay accelerating factor (hDAF) transgenic pig organs. Our laboratory has shown that anti-porcine xenograft antibodies in humans and non-human primates are encoded by a small number of germline IgV(H) progenitors. In this study, we extended our analysis to identify the IgV(H) genes encoding xenoantibodies in immunosuppressed cynomolgus monkeys (Macaca fascicularis) transplanted with hDAF-transgenic pig organs. METHODS: Three immunosuppressed monkeys underwent heterotopic heart transplantation with hDAF porcine heart xenografts. Two of three animals were given GAS914, a poly-L-lysine derivative shown to bind to anti-Gal xenoantibodies and neutralize them. One animal rejected its heart at post-operative day (POD) 39; a second animal rejected the transplanted heart at POD 78. The third monkey was euthanized on POD 36 but the heart was not rejected. Peripheral blood leukocytes (PBL) and serum were obtained from each animal before and at multiple time points after transplantation. We analyzed the immune response by enzyme-linked immunosorbent assay (ELISA) to confirm whether anti-Gal or anti-non-Gal xenoantibodies were induced after graft placement. Immunoglobulin heavy-chain gene (V(H)) cDNA libraries were then produced and screened. We generated soluble single-chain antibodies (scFv) to establish the binding specificity of the cloned immunoglobulin genes. RESULTS: Despite immunosuppression, which included the use of the polymer GAS914, the two animals that rejected their hearts showed elevated levels of cytotoxic anti-pig red blood cell (RBC) antibodies and anti-pig aortic endothelial cell (PAEC) antibodies. The monkey that did not reject its graft showed a decline in serum anti-RBC, anti-PAEC, and anti-Gal xenoantibodies when compared with pre-transplant levels. A V(H)3 family gene with a high level of sequence similarity to an allele of V(H)3-11, designated V(H)3-11(cyno), was expressed at elevated levels in the monkey that was not given GAS914 and whose graft was not rejected until POD 78. IgM but not IgG xenoantibodies directed at N-acetyl lactosamine (a precursor of the Gal epitope) were also induced in this animal. We produced soluble scFv from this new gene to determine whether this antibody could bind to the Gal carbohydrate, and demonstrated that this protein was capable of blocking the binding of human serum xenoantibody to Gal oligosaccharide, as had previously been shown with human V(H)3-11 scFv. CONCLUSIONS: DAF-transgenic organs transplanted into cynomolgus monkeys induce anti-Gal and anti-non-Gal xenoantibody responses mediated by both IgM and IgG xenoantibodies. Anti-non-Gal xenoantibodies are induced at high levels in animals treated with GAS914. Antibodies that bind to the Gal carbohydrate and to N-acetyl lactosamine are induced in the absence of GAS914 treatment. The animal whose heart remained beating for 78 days demonstrated increased usage of an antibody encoded by a germline progenitor that is structurally related, but distinct from IGHV311. This antibody binds to the Gal carbohydrate but does not induce the rapid rejection of the xenograft when expressed at high levels as early as day 8 post-transplantation.     

One-year monkey heart xenograft survival in cyclosporine-treated baboons. Suppression of the xenoantibody response with total-lymphoid irradiation.

The shortage of cadaveric human organs for transplantation could be alleviated by the use of xenografts. Long-term (> one-year) survival of xenografts in humans or experimental animals has not been achieved with previous immunosuppressive protocols. Poor results in xenotransplantation compared with allotransplantation have been attributed to a more potent antibody response rather than to cell-mediated responses. To investigate these issues a concordant heterotopic cardiac xenograft model was developed in conjunction with cyclosporine and/or total-lymphoid irradiation. Concordant models permit examination of xenoantigen induced antibody and cell-mediated responses since preformed humoral factors (in discordant models) do not cause hyperacute rejection. Four groups of baboon recipients received cervical heart transplants from cynomolgous monkeys. Group I (n = 2), untreated, mean survival (MS) = 6 days; group II (n = 5), CsA and methylprednisolone, MS = 25 days; group III (n = 3), preoperative TLI, MS = 29 days; group IV (n = 6), preoperative TLI and CsA+MP, MS = 255 days (> 77, > 108, > 142, 184, > 480, 540 days). Heart xenografts of CsA-MP-treated recipients appear to be destroyed predominantly by antibody (IgM)-mediated processes whereas cell-mediated rejection is likely the major reaction in TLI-treated recipients. CsA-MP-treated recipients had early immunohistochemical evidence of antibody and complement-mediated rejection (deposition of IgM and complement but not IgG on heart xenografts). In contrast IgM and complement deposits were not detected on heart xenografts in TLI- and TLI-CsA-treated recipients. IgG xenoantibodies were only detected on the two rejected heart grafts of TLI-CsA-treated recipients. CsA-MP-treated recipients rapidly developed high xenoantibody titers (1:256 to 1:512) that immediately preceded rejection. In contrast, TLI-treated animals developed lower levels of xenoantibody (< or = 1:8) and TLI-CsA-treated recipients had no detectable xenoantibody during the initial three months after transplantation (and titers no greater than 1:8 thereafter.) The lack of xenoantibody was likely not due to a generalized inhibitory effect of the immunosuppressants on B cell function since all classes of serum immunoglobulins were in the normal range. Intragraft cytolytic lymphocyte activity was detected in rejecting TLI- and TLI-CsA-treated recipients but could not be detected in xenografts of CsA-MP-treated recipients. One explanation for these data is that TLI (either directly or indirectly) induces a state of specific B cell unresponsiveness to monkey xenoantigens, thereby preventing IgM mediated rejection in the third week after transplantation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synergistic effect of anti-T cell receptor monoclonal antibody and 15-deoxyspergualin on cardiac xenograft survival in a mouse-to-rat model

BACKGROUND: Successful xenograft transplantation faces several obstacles including the presence of xenoantibodies, natural killer cell- and macrophage-mediated rejection, and T lymphocyte activation. METHODS: A mouse-to-rat cardiac xenograft model was used to examine the synergistic effect of anti-T cell receptor (TCR) monoclonal antibodies (mAb) and 15-deoxyspergualin (DSG) on graft survival. RESULTS: Pretransplantation injections (days -5, -3, and -1) of anti-TCR mAb (500 microg/kg/day) combined with continuous i.p. infusion of DSG (5 mg/kg/day) from day -7 to 28 significantly prolonged graft survival compared to untreated controls (3.3+/-0.5 vs. 44.2+/-5.6 days, P<0.001). Postoperative splenectomy combined with discontinuation of all other treatment on day 28 enhanced graft survival in rats treated with anti-TCR mAb and DSG to 71.0+/-2.5 days. Histological examination of grafts showed characteristic signs of vascular rejection: interstitial edema and hemorrhage, and polymorphonuclear cell infiltration. Antimouse antibody titers in recipients were increased upon rejection in each group that received a xenograft. Flow cytometry analysis showed a markedly decreased T cell population and a relatively increased mature B cell population (IgM(bright)/IgD(dull)) in spleens of rats treated with anti-TCR mAb and DSG on day 28. CONCLUSIONS: The mechanism of prolonged xenograft survival in this model may include inhibition of antibody production by arrest of B-cell maturation during development from IgM(dull)/IgD(bright) mature B cells to antibody producing cells, and inhibition of T cell activation. The rejection seen in our model may be caused by xenoreactive antibodies and may be associated with T cells, natural killer cells, and macrophages.     

Efficacy of immunosuppressive drugs in islet xenotransplantation: A study in the pig-to-rat model

Abstract: Several immunosuppressive regime's were used in a pig-to-rat islet transplant model, with porcine islet-like cell clusters placed under the kidney capsule of Lewis rats. The animals were killed 12 days after the implantation. In untreated control rats, a massive infiltration of mononuclear cells occured in the xenograft and no morphologically intact endocrine cells could be seen. Most of the infiltrating cells were large, polygonal, macrophage-like cells. T-lymphocytes were found mainly in the peripheral parts of the xenografts. Some NK-cells were also observed. Treatment with cyclosporin A (CyA, 20 mg/kg BW, p.o.), cyclophosphamide (CYP, 20 mg/kg BW, p.o.), prednisolone (PRED, 20 mg/kg BW, p.o.) and mycophenolate mofetil (MMF, 40 mg/kg BW, p.o.) had little inhibitory effect on the xenograft rejection. In animals given cyclosporin and cyclophosphamide in combination with plasmapheresis there were only occasional infiltrating cells, still only endocrine cells were morphologically intact. Immunosuppression with CyA in combination with rapamycin (RPM, 5 mg/kg BW, i.p.) also had an inhibitory effect on xenograft rejection and there were only a few or a moderate number of cells infiltrating the grafts. However, in this treatment group many morphologically intact endocrine cells were present. The most effective treatment was CyA in combination with 15-deoxyspergualine (DSG, 10 mg/kg BW, i.m.). The animals so treated had hardly any infiltrating mononuclear cells and the islet xenograft revealed a morphological appearance similar to that observed after transplantation to nude mice-i.e., intact endocrine tissue, organized in duct-like structures or cell clusters. All rats had preformed, xenoreactive antibodies in serum directed against membrane antigens on pig ICC. In control animals and animals treated with CyA, a marked rise in antibody levels was found after transplantation. Treatment with CyA and DSG (10 mg/kg BW), CyA and CYP, and protocols including plasmapheresis, resulted in a significant decrease in antibody levels. With the other treatment protocols, the antibody levels remained unchanged.     

Pathogenesis and pathology of delayed xenograft rejection in pig-to-rhesus monkey cardiac transplantation

It has been recognized that delayed xenograft rejection (DXR) is the major barrier to the acceptance of xenotransplantation after overcoming hyperacute rejection. OBJECTIVES: This study sought to investigate the pathogenesis and pathology of delayed xenograft rejection following pig-to-rhesus monkey heart xenotransplantation. METHODS: Heterotopic xenogeneic heart transplants in the abdominal cavity were performed using piglet donors to four monkey recipients. Complete complement depletion was achieved in the recipients with repetitive doses of high-activity cobra venom factor (Y-CVF). The recipients were immunosuppressed with a combination of cyclosporine, cyclophosphamide, and steroids. Sera were analyzed for C3 and C4 levels and complement activity and anti-pig endothelial xenoantibody. The grafts were examined histopathologically and immunohistochemically for C3, C4;C5b-9, IgM, IgG, tumor necrosis factor-alpha (TNF-alpha), intercellular adhesion molecule-1(ICAM-1), CD57(NK cells), CD68 (macrophages), CD4, and CD8. RESULTS: Xenografts survived 8, 10, 13, and 13 days respectively, all developing DXR. Venous thrombosis was the outstanding feature within DXR xenografts, complicated by interstitial edema, local hemorrhage, myocardial necrosis, and mild to moderate cellular infiltration. The serum C3 levels and complement activity decreased to almost 0 from the day of transplantation due to treatment with Y-CVF. The C4 level began to decrease 2 to 4 days before the cardiac xenografts lost their function. Anti-pig endothelial xenoantibody also decreased after transplantation, slightly increasing during DXR. All rejected xenografts showed C3, C4, C5b-9, IgG, and IgM deposits to various degrees. Large numbers of macrophages (50% of total leukocytes) infiltrated the entire xenograft with a few natural killer cells (8% to 10%), as well as some CD4+ T cells (15%) and CD8+ T cells (25%). Upregulation of ICAM-1 on graft endothelial cells and TNF-alpha in the interstitium were also demonstrated in the rejected heart. CONCLUSION: Both humoral and cell-mediated immunologic reactions may play important roles in the pathogenesis of DXR. Besides C3, C4, C5b-9, IgM, and IgG destroying the xenograft, NK cells, macrophages, and CD4+ and CD8+ T cells may further aggravate the development of DXR.     

Mouse-to-rabbit xenotransplantation: a new small animal model of hyperacute rejection mediated by the classical complement pathway

BACKGROUND: Hyperacute rejection of porcine organs transplanted into primate recipients is initiated by the binding of preformed xenoreactive natural antibodies to the vascular endothelium of the graft and activation of the classical complement pathway. Several small animal models are currently employed to study various aspects of xenograft rejection; however, none has been shown to manifest hyperacute rejection mediated by the classical pathway of complement activation. METHODS: We performed heterotopic mouse heart transplants into weanling rabbits, adult rabbits, and C6-deficient rabbits. The recipients received no immunosuppression. Rejected grafts were subjected to histologic analysis and immunofluorescence staining for rabbit IgG, IgM, and C3. Levels of preexisting cytotoxic antibodies as well as classical and alternative complement pathway activities were determined in rabbit serum using mouse red cells as targets. RESULTS: Mean graft survival was 37+/-9.6 min for mouse-to-weanling rabbit transplants (n=10), and 40+/-11.1 min for mouse-to-adult rabbit transplants (n=5). Rejected grafts showed diffuse interstitial hemorrhage, endothelial cell damage, myocyte necrosis, moderate diffuse deposition of rabbit IgG, and dense deposition of rabbit IgM and C3 on the vascular endothelium of the graft, consistent with hyperacute rejection. One mouse-to-C6-deficient rabbit transplant was rejected at 21 hr with severe interstitial hemorrhage, cellular necrosis and a moderate cellular infiltrate consisting primarily of neutrophils and some mononuclear cells. A second transplant in a C6-deficient rabbit was functioning when the recipient died at 6.5 hr as a result of complications of surgery; the graft had normal myocytes and vasculature with minimal spotty interstitial hemorrhage. Both weanling and adult rabbit serum were found to have high titers of cytotoxic IgM anti-mouse antibodies and strong classical complement pathway activity with minimal alternative pathway activity towards mouse red cells. CONCLUSIONS: The mouse-to-rabbit species combination manifests hyperacute xenograft rejection. In vitro studies suggest that this process is mediated by IgM anti-mouse natural antibodies and activation of the classical pathway of complement.     

Late CD8+ T cell-dependent xenoantibody production in innate tolerant nude rats after hamster islet grafting but not after hamster heart grafting

BACKGROUND: Xenograft rejection can be provoked by both the innate and adaptive immune compartments and close reciprocal interactions exist between these two systems. We investigated the interdependent roles of T and B lymphocytes in vascularized (heart) and cellular (islet) xenograft rejection in a model with established xeno-nonreactivity of the innate immune system. METHODS: Specific innate xenotolerance was induced in nude rats bearing either a hamster heart or a hamster pancreatic islet graft by a tolerizing regimen consisting of donor antigen infusion, temporary natural killer cell depletion and a 4-week administration of leflunomide. One month after transplantation, syngeneic CD4 and CD8 T cells were adoptively transferred. RESULTS: Both vascular and cellular xenografts were rejected after CD4 T cell reconstitution, corresponding with production of high IgM and IgG xenoantibody titers. Deposition of xenoantibodies and complement was seen in the heart but not in the islet xenografts. After infusion of CD8 T cells, xenohearts underwent a delayed type of rejection without xenoantibody production and xenoislets were not rejected. In xenoislet recipients, CD8 dependent B cells were not tolerized, resulting in the production of IgG xenoantibodies belonging to Th2-dependent isotypes, known not to cause graft rejection, and deposited at the graft implantation site. CONCLUSIONS: We conclude that distinct mechanisms of immune activation underlie xenogeneic reactions against vascular and cellular grafts.     

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.     

ABO-incompatible kidney transplantation using both A2 and non-A2 living donors

BACKGROUND: Given the scarcity of cadaveric organs, efforts are intensifying to increase the availability of living donors. The current study assessed the feasibility of using ABO-incompatible living-donor kidneys to expand the donor pool. METHODS: The authors performed 18 ABO-incompatible living-donor kidney transplants between May 1999 and April 2001. Ten patients received living-donor kidneys from A2 and eight patients received kidneys from non-A2 blood group donors. Immunosuppression consisted of Thymoglobulin antibody induction, tacrolimus, mycophenolate mofetil, and prednisone. Eight non-A2 and two A2 kidney recipients also received a pretransplant conditioning regimen of four plasmapheresis treatments followed by intravenous immunoglobulin and splenectomy at the time of transplantation. Antidonor blood group antibody titer was measured at baseline, pretransplant, at 1- to 3-month and 1-year follow-up, and at the time of diagnosis of antibody-mediated rejection. RESULTS: No hyperacute rejection episodes occurred. One-year graft and patient survival rates in the 18 ABO-incompatible recipients were only slightly lower than those of 81 patients who received ABO-compatible kidney transplants during the same period (89% vs. 96% and 94% vs. 99%, respectively). Glomerular filtration rate and serum creatinine levels did not differ between the groups. Antibody-mediated rejection occurred in 28% of ABO-incompatible recipients, and was reversible with plasmapheresis, intravenous immunoglobulin, and increasing immunosuppression in all patients except one. CONCLUSIONS: ABO-incompatible living donor kidney transplants can achieve an acceptable 1-year graft survival rate using an immunosuppressive regimen consisting of Thymoglobulin induction, tacrolimus, mycophenolate mofetil, and prednisone combined with pretransplant plasmapheresis, intravenous immunoglobulin, and splenectomy.     

Evidence that deoxyspergualin prevents sensitization and first-set cardiac xenograft rejection in rats by suppression of antibody formation

This study provides evidence of antibodies playing an important role in hamster-to-rat cardiac xenograft rejection and discusses the use of 15-deoxyspergualin (DSG) to suppress this first-set rejection, as well as hyperacute rejection induced by sensitization. The effect of recipient splenectomy (Spx) as an adjuvant to DSG to control first set xenograft rejection was also examined. When hyperimmune serum was taken from control recipients at rejection time and injected i.v. into new recipients of cardiac xenografts, hyperacute graft rejection resulted. Survival depended on the amount of serum injected and ranged from 14.7 +/- 2.5 min with 3 ml of serum to 233.3 +/- 61.1 min with 0.5 ml. Experiments on first-set xenograft rejection revealed that a dose of 2.5 mg/kg/day DSG could prolong xenograft survival from 3.4 +/- 0.5 days in untreated controls to 9.5 +/- 2.6 days (P less than 0.01). A dose of 5 mg/kg/day DSG, though it increased graft survival to 16.4 +/- 5.9 days, proved to be toxic to the recipients. Spx alone prolonged xenograft survival to 5.2 +/- 0.4 days, and, when combined with 2.5 mg/kg/day DSG administration, prolonged graft survival to 22.1 +/- 5.5 days (P less than 0.01 vs. DSG alone). The appearance of cytotoxic antibodies was delayed, and titers decreased from 1:256 in untreated controls to 1:16-1:64 both in the group that underwent Spx only and in the group that received 2.5 mg/kg/day DSG. Combined treatment suppressed antibody response for more than two weeks. Experiments on hyperacute rejection induced by sensitization revealed that 1 ml of hamster whole blood transfused into prospective heart recipients 1 week before grafting resulted in graft loss in 18.2 +/- 6.1 min. Pretransplant transfusion and concomitant daily administration of 5 mg/kg/day DSG until one day after grafting not only prevented hyperacute rejection but prolonged graft survival to 7.0 +/- 0.7 days. This survival was significantly longer than with DSG alone (4.2 +/- 0.8 days, P less than 0.01). We concluded that the marked suppression of antibody formation by DSG plays a major role in preventing first-set xenograft rejection and hyperacute rejection induced by sensitization.     

Safe transplantation of blood type A2 livers to blood type O recipients

BACKGROUND: Transplantation of blood type A subgroup 2 (A2) livers into non-A recipients has not been reported previously. A2 to O renal transplantation has been reported, with early results including some accelerated rejections and graft losses. This has led some to selectively offer A2 renal transplantation only for patients with low anti-A titers. Given the different clinical behavior of liver allografts to preformed antibody, we felt that such restriction was unnecessary. METHODS: We performed six cases of A2 to O liver transplantation with no augmented immunomodulation or restriction with regard to antibody titers. Clinical courses, anti-A titers, rejection rates, and graft and patient survival were evaluated. RESULTS: All six patients had high pretransplant anti-A titers (>1:8), and all six grafts functioned normally. There were nine rejections in the six patients, of which three were severe (steroid-resistant) and five were late (>90 days). No rejection was vascular, and no grafts were lost, with mean follow-up of 665 days. In one patient who had anti-A antibody measured at the time of rejection IGM titers increased from baseline. Currently all patients are home with normal function. CONCLUSIONS: We found that transplantation of blood group A2 livers into blood group O recipients is safe and can be performed without graft loss and without regard to anti-A titer level. The rate of acute cellular rejection is high in this small series, and a significant proportion of these events were late or required OKT-3. We did not rely on plasmapheresis or anti-A titer determinations. However, the potential for late rejection prompts us to consider the addition of a third immunosuppressive agent. The transplantation of A2 livers into O recipients can partially compensate for the more frequent use of O livers in recipients from other blood groups.     

The prospects for renal xenotransplantation

Summary: Xenotransplantation of non-human organs into human recipients has long been proposed as a possible strategy to overcome the acute shortage of donor organs. However, vascular organ transplants to humans from phylogenetically disparate species such as the pig are not currently possible due to a rapid rejection process termed hyperacute rejection. This process is initiated by the binding of host pre-formed 'natural antibodies' to the donor vascular endothelium, activation of the host complement system and activation or injury of the donor endothelial cells, leading to intravascular coagulation and loss of the graft due to ischaemic necrosis within minutes to hours of engraftment. Prevention of natural antibody binding and complement activation is viewed as paramount to preventing hyperacute rejection. Even if hyperacute rejection can be prevented, further barriers to successful discordant xenografts such as delayed xenograft rejection and a donor-directed cell-mediated rejection process will still represent major obstacles. This review examines recent advances being made in the various areas of xenograft research and the potential clinical application of pig-to-human xenografts that these strategies may bring.     

单核细胞、NK细胞和T细胞在猪-猕猴延迟性异种移植排斥反应中的作用

目的观察单核细胞、NK细胞和T细胞在猪-猕猴延迟性异种移植排斥反应(DXR)中的作用。方法建立湖北白猪-云南猕猴的腹腔异位心脏移植模型，实验分为2组：对照组(n=5)，不使用中华眼睛蛇毒因(Y-CVF)；实验组(n=4)应用Y-CVF完全清除受者体内补体。2组受体猴均采用环孢素A(CsA)，环磷酰胺(CTX)和甲基强的松龙(MP)三联免疫抑制治疗。免疫组织化学方法检测移植心组织中细胞间黏附分子(ICAM)-1、肿瘤坏死因子(TNF)-α、单核细胞、NK细胞和T细胞的表达。结果对照组3个移植心在15～60min内发生超急性排斥反应(HAR)，另2个分别存活22h及6d，移植心均未见明显的炎性细胞浸润及ICAM-1和TNF-α的表达。实验组移植心存活时间分别为8、10、13和13d，移植物浸润细胞中可见大量的单核细胞(50％)，少量的NK细胞(8％～10％)，CD4^ T细胞(15％)和C08^ T细胞(25％)。移植物血管内皮细胞表面出现ICAM-1的表达上调，移植物间质中出现TNF-α的表达增加。结论单核细胞、NK细胞和T细胞介导的移植物损伤，在应用Y-CVF处理的猪-猕猴DXR发生中发挥重要作用。     

The Role of Antibodies and Von Willebrand Factor in Discordant Pulmonary Xenotransplantation

Pulmonary xenotransplantation is one potential solution to the paucity of donors but is currently limited by rapid failure of the graft. Unlike cardiac and renal xenotransplants, pulmonary xenografts release large quantities of swine von Willebrand factor (vWF). Swine vWF binds xenoreactive antibodies and is capable of activating primate platelets. The contribution of swine vWF to lung xenograft dysfunction is not entirely clear. To probe the role vWF plays in xenograft dysfunction, we traced the fate of xenoantibodies in vWF+ and von Willebrand factor-deficient (vWFD) swine lungs. These studies showed that the vast majority of xenoantibodies bind the vWF released from the vWF+ swine lung, and thus do not remain bound on lung endothelium. The vWF complexed to xenoantibody remained capable of aggregating primate platelets. With this information, we performed swine-to-baboon lung transplants using vWF+ and vWFD donors. Without vWF present to complex xenoantibodies, a picture of hyperacute rejection more typical of heart and kidney xenografts, with antibody deposition along the graft endothelium, interstitial hemorrhage, and edema occurred. These findings suggest that porcine vWF plays a major role in the pathogenesis of pulmonary xenograft dysfunction, and suggests promising strategies to treat lung xenograft dysfunction.     

Renal transplantation despite a positive antiglobulin crossmatch with and without prophylactic OKT3

The antiglobulin crossmatch (AGXM) is a sensitive technique employed by many transplant centers to enhance detection of preformed antibody to donor antigens that may cause hyperacute rejection. However, positive AGXM may detect irrelevant or very low titers of anti-HLA antibody precluding transplantation in suitable recipients. To investigate the significance of a positive AGXM, cadaveric renal transplantation was carried out despite a weakly positive AGXM (defined as cell killing above background but not greater than 20%) in 48 recipients. In an initial group (n = 10), maintained on triple therapy (cyclosporine, azathioprine, and prednisone), accelerated acute rejection occurred in 4 recipients and 3 grafts were lost. A subsequent group (n = 38) was treated with a prophylactic course of OKT3 then triple therapy. There were no episodes of accelerated acute rejection (P less than 0.01) although clinical hyperacute rejection claimed one graft and the incidence of delayed graft function was high (75%). The prophylactic OKT3 group had a reduced incidence of acute rejection (0.5 versus 1.0) per recipient and the onset of first episodes was delayed (mean onset: 13 versus 35 days after transplantation). One year actuarial primary graft survival was 88% in the prophylactic OKT3 group as compared with only 50% in the initial group. The outcome in the positive AGXM group was similar to a concurrent group (n = 32) with a negative AGXM and immediate graft function. On the other hand, the subset of positive AGXM regraft recipients treated with prophylactic OKT3 fared poorly, with a 36% (4/11) incidence of primary nonfunction. In summary, a positive AGXM, as defined in this report, is not a contraindication to primary renal transplantation--in fact, the use of the AGXM will identify recipients that would benefit from prophylactic OKT3.     

Specific depletion of preformed IgM natural antibodies by administration of anti-mu monoclonal antibody suppresses hyperacute rejection of pig to baboon renal xenografts

BACKGROUND: The elimination of circulating anti-porcine preformed antibodies is crucial for avoiding hyperacute vascular rejection (HAVR) of primarily vascularized xenograft in discordant pig to baboon model. Previously described methods used for eliminating natural antibodies, however, constantly removed both anti-porcine IgM and IgG antibodies, as well as often complement proteins. To study specifically the role of preformed anti-porcine IgM antibodies, a specific anti-IgM monoclonal antibody (mAb) has been designed and evaluated in vivo. METHODS: Iterative injections of anti-IgM mAb (LO-BM2) at high dose (20 mg/kg) depleted to undetectable level the circulating IgM and therefore anti-porcine IgM antibodies but did not change the concentration of anti-pig IgG antibodies. The serum concentration of IgM and IgG antibodies was assessed by ELISA and the level of anti-pig natural IgM and IgG antibodies by flow cytometry (FC). Anti-rat sensitization was assessed by specific ELISA as well as the serum concentration of LO-BM2. RESULTS: Iterative injections of LO-BM2 allowed to specifically eliminate the anti-porcine IgM antibodies to undetectable levels at ELISA. Despite a normal serum level of anti-porcine IgG and complement proteins, HAVR was avoided. Without immunosuppression, the specific elimination of preformed anti-porcine IgM prolonged the survival of a renal xenograft in baboon up to 6 days, whereas without IgM antibody elimination, the renal xenografts were hyperacutely rejected within hours. The lost of activity of LO-BM2 after 10 days was concomitant to an IgM and IgG antibody rebound, which caused an acute vascular rejection of the xenograft. CONCLUSION: Specific elimination of natural anti-porcine IgM antibodies allows to avoid HAVR of a pig to baboon renal xenograft, whereas anti-porcine IgG antibodies and complement proteins were present in the serum. This result confirms previous in vitro reports and demonstrates for the first time in vivo that preformed IgM antibodies alone are responsible for HAVR, while preformed anti-porcine IgG antibodies are unable alone to cause HAVR. Anti-IgM therapy appears as an important tool to transiently but completely eliminates xeno-IgM antibodies in vivo.     

Recipient intramuscular cotransfection of transforming growth factor beta1 and interleukin 10 ameliorates acute lung graft rejection

OBJECTIVE: Multiple gene transfer might permit modulation of concurrent biochemical pathways involved in acute lung graft rejection. We investigated whether gene cotransfection into the recipient reduces acute lung graft rejection. METHODS: Brown Norway rats were used as donors, and F344 rats were used as recipients. Recipient animals were injected with saline (groups I/VI) or 1 x 10(10) pfu of adenovirus encoding beta-galactosidase (groups II/VII), transforming growth factor beta1 (groups III/VIII), interleukin 10 (groups IV/IX), or both transforming growth factor beta1 and interleukin 10 (groups V/X) into both leg muscles 2 days before transplantation (groups I-V) or at the time of harvest (groups VI-X). The Kruskal-Wallis test for rejection score and 1-way analysis of variance were used to compare groups. RESULTS: Oxygenation was significantly improved in the cotransfected groups treated 2 days before transplantation and at the time of harvest. Rejection scores were also reduced in the cotransfected groups. In group V cotransfection suppressed endogenous interleukin 2 but not interferon gamma and tumor necrosis factor alpha. CONCLUSION: Recipient intramuscular cotransfection of transforming growth factor beta1 and interleukin 10 suppressed interleukin 2 expression and provided a synergistic effect that reduced acute lung graft rejection. This approach might be applied to the clinical setting because transplant recipients could be treated at the time of implantation.