The Resurgence of Xenotransplantation.

There has been an upsurge of interest in xenotransplantation in recent years. This resurgence can attributed to a combination of factors. First, there has been a dramatic improvement in efficacy in several preclinical models, with maximum xenograft survival times increasing to 950 days for islets, 945 days for hearts, and 310 days for kidneys. Second, the rapid development of genome editing technology (particularly the advent of clustered regularly interspaced short palindromic repeats/Cas9) has revolutionized the capacity to generate new donor pigs with multiple protective genetic modifications; what once took many years to achieve can now be performed in months, with much greater precision and scope. Third, the specter of porcine endogenous retrovirus (PERV) has receded significantly. There has been no evidence of PERV transmission in clinical trials and preclinical models, and improved screening methods and new options for the treatment or even elimination of PERV are now available. Balancing these positive developments are several remaining challenges, notably the heavy and often clinically inapplicable immunosuppression required to prevent xenograft rejection. Nonetheless, the potential for xenotransplantation as a solution to the shortage of human organs and tissues for transplantation continues to grow.

CD39 overexpression does not attenuate renal fibrosis in the unilateral ureteric obstructive model of chronic kidney disease.

Chronic kidney disease has multiple etiologies, but its single, hallmark lesion is renal fibrosis. CD39 is a key purinergic enzyme in the hydrolysis of ATP and increased CD39 activity on regulatory T cells (Treg) is protective in adriamycin-induced renal fibrosis. We examined the effect of overexpression of human CD39 on the development of renal fibrosis in the unilateral ureteric obstructive (UUO) model, a model widely used to study the molecular and cellular factors involved in renal fibrosis. Mice overexpressing human CD39 (CD39Tg) and their wild-type (WT) littermates were subjected to UUO; renal histology and messenger RNA (mRNA) levels of adenosine receptors and markers of renal fibrosis were examined up to 14 days after UUO. There were no differences between CD39Tg mice and WT mice in the development of renal fibrosis at days 3, 7, and 14 of UUO. Relative mRNA expression of the adenosine A2A receptor and endothelin-1 were higher in CD39Tg than WT mice at day 7 post UUO, but there were no differences in markers of fibrosis. We conclude that human CD39 overexpression does not attenuate the development of renal fibrosis in the UUO model. The lack of protection by CD39 overexpression in the UUO model is multifactorial due to the different effects of adenosinergic receptors on the development of renal fibrosis.

Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

Altered glycosylation in donor mice causes rejection of strain-matched skin and heart grafts.

Differential protein glycosylation in the donor and recipient can have profound consequences for transplanted organs, as evident in ABO-incompatible transplantation and xenotransplantation. In this study, we investigated the impact of altered fucosylation on graft acceptance by using donor mice overexpressing human α1,2-fucosyltransferase (HTF). Skin and heart grafts from HTF transgenic mice were rapidly rejected by otherwise completely matched recipients (median survival times 16 and 14 days, respectively). HTF skin transplanted onto mice lacking T and B cells induced an natural killer cell-mediated innate rejection crisis that affected 50-95% of the graft at 10-20 days. However, in the absence of adaptive immunity, the residual graft recovered and survived long-term (>100 days). Experiments using "parked" grafts or MHC class II-deficient recipients suggested that indirect rather than direct antigen presentation plays a role in HTF skin graft rejection, although the putative antigen(s) was not identified. We conclude that altered glycosylation patterns on donor tissue can trigger a powerful rejection response comprising both innate and adaptive components. This has potential implications for allotransplantation, in light of increasing recognition of the variability of the human glycome, and for xenotransplantation, where carbohydrate remodeling has been a lynchpin of donor genetic modification.

Control of IBMIR in neonatal porcine islet xenotransplantation in baboons.

The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.

Protective effects of transgenic human endothelial protein C receptor expression in murine models of transplantation.

Thrombosis and inflammation are major obstacles to successful pig-to-human solid organ xenotransplantation. A potential solution is genetic modification of the donor pig to overexpress molecules such as the endothelial protein C receptor (EPCR), which has anticoagulant, anti-inflammatory and cytoprotective signaling properties. Transgenic mice expressing human EPCR (hEPCR) were generated and characterized to test this approach. hEPCR was expressed widely and its compatibility with the mouse protein C pathway was evident from the anticoagulant phenotype of the transgenic mice, which exhibited a prolonged tail bleeding time and resistance to collagen-induced thrombosis. hEPCR mice were protected in a model of warm renal ischemia reperfusion injury compared to wild type (WT) littermates (mean serum creatinine 39.0 ± 2.3 µmol/L vs. 78.5 ± 10.0 µmol/L, p < 0.05; mean injury score 31 ± 7% vs. 56 ± 5%, p < 0.05). Heterotopic cardiac xenografts from hEPCR mice showed a small but significant prolongation of survival in C6-deficient PVG rat recipients compared to WT grafts (median graft survival 6 vs. 5 days, p < 0.05), with less hemorrhage and edema in rejected transgenic grafts. These data indicate that it is possible to overexpress EPCR at a sufficient level to provide protection against transplant-related thrombotic and inflammatory injury, without detrimental effects in the donor animal.

Antioxidant network expression abrogates oxidative posttranslational modifications in mice.

Antioxidant enzymatic pathways form a critical network that detoxifies ROS in response to myocardial stress or injury. Genetic alteration of the expression levels of individual enzymes has yielded mixed results with regard to attenuating in vivo myocardial ischemia-reperfusion injury, an extreme oxidative stress. We hypothesized that overexpression of an antioxidant network (AON) composed of SOD1, SOD3, and glutathione peroxidase (GSHPx)-1 would reduce myocardial ischemia-reperfusion injury by limiting ROS-mediated lipid peroxidation and oxidative posttranslational modification (OPTM) of proteins. Both ex vivo and in vivo myocardial ischemia models were used to evaluate the effect of AON expression. After ischemia-reperfusion injury, infarct size was significantly reduced both ex vivo and in vivo, ROS formation, measured by dihydroethidium staining, was markedly decreased, ROS-mediated lipid peroxidation, measured by malondialdehyde production, was significantly limited, and OPTM of total myocardial proteins, including fatty acid-binding protein and sarco(endo)plasmic reticulum Ca(²+)-ATPase (SERCA)2a, was markedly reduced in AON mice, which overexpress SOD1, SOD3, and GSHPx-1, compared with wild-type mice. These data demonstrate that concomitant SOD1, SOD3, and GSHPX-1 expression confers marked protection against myocardial ischemia-reperfusion injury, reducing ROS, ROS-mediated lipid peroxidation, and OPTM of critical cardiac proteins, including cardiac fatty acid-binding protein and SERCA2a.

Anti-inflammatory and anticoagulant effects of transgenic expression of human thrombomodulin in mice.

Thrombomodulin (TBM) is an important vascular anticoagulant that has species specific effects. When expressed as a transgene in pigs, human (h)TBM might abrogate thrombotic manifestations of acute vascular rejection (AVR) that occur when GalT-KO and/or complement regulator transgenic pig organs are transplanted to primates. hTBM transgenic mice were generated and characterized to determine whether this approach might show benefit without the development of deleterious hemorrhagic phenotypes. hTBM mice are viable and are not subject to spontaneous hemorrhage, although they have a prolonged bleeding time. They are resistant to intravenous collagen-induced pulmonary thromboembolism, stasis-induced venous thrombosis and pulmonary embolism. Cardiac grafts from hTBM mice to rats treated with cyclosporine in a model of AVR have prolonged survival compared to controls. hTBM reduced the inflammatory reaction in the vein wall in the stasis-induced thrombosis and mouse-to-rat xenograft models and reduced HMGB1 levels in LPS-treated mice. These results indicate that transgenic expression of hTBM has anticoagulant and antiinflammatory effects that are graft-protective in murine models.

Transgenic overexpression of CD39 protects against renal ischemia-reperfusion and transplant vascular injury.

The vascular ectonucleotidases CD39[ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1), EC 3.6.1.5] and CD73[EC 3.1.3.5] generate adenosine from extracellular nucleotides. CD39 activity is critical in determining the response to ischemia-reperfusion injury (IRI), and CD39 null mice exhibit heightened sensitivity to renal IRI. Adenosine has multiple mechanisms of action in the vasculature including direct endothelial protection, antiinflammatory and antithrombotic effects and is protective in several models of IRI. Mice transgenic for human CD39 (hCD39) have increased capacity to generate adenosine. We therefore hypothesized that hCD39 transgenic mice would be protected from renal IRI. The overexpression of hCD39 conferred protection in a model of warm renal IRI, with reduced histological injury, less apoptosis and preserved serum creatinine and urea levels. Benefit was abrogated by pretreatment with an adenosine A2A receptor antagonist. Adoptive transfer experiments showed that expression of hCD39 on either the vasculature or circulating cells mitigated IRI. Furthermore, hCD39 transgenic kidneys transplanted into syngeneic recipients after prolonged cold storage performed significantly better and exhibited less histological injury than wild-type control grafts. Thus, systemic or local strategies to promote adenosine generation and signaling may have beneficial effects on warm and cold renal IRI, with implications for therapeutic application in clinical renal transplantation.

Pig thrombomodulin binds human thrombin but is a poor cofactor for activation of human protein C and TAFI.

Incompatibility between pig thrombomodulin (TM) and primate thrombin is thought to be an important factor in the development of microvascular thrombosis in rejecting pig-to-primate xenografts. To examine this interaction at the molecular level, we cloned pig TM and measured its ability to bind human thrombin and act as a cofactor for the activation of human protein C and TAFI. The 579-residue pig TM protein showed approximately 69% sequence identity to human TM. Within the EGF domains necessary for binding of thrombin (EGF56), protein C (EGF4) and TAFI (EGF3), all of the amino acids previously identified as critical for the function of human TM, with the exception of Glu-408 in EGF5, were conserved in pig TM. Comparison of transfected cells expressing pig or human TM demonstrated that both proteins bound human thrombin and inhibited its procoagulant activity. However, pig TM was a poor cofactor for the activation of human protein C and TAFI, with domain swapping showing that EGF5 was the most important determinant of compatibility. Thus, while pig TM may be capable of binding thrombin generated in the vicinity of xenograft endothelium, its failure to promote the activation of human protein C remains a significant problem.

Human Endothelial Protein C Receptor Overexpression Protects Intraportal Islet Grafts in Mice.

Islet transplantation can potentially cure type 1 diabetes mellitus, but it is limited by a shortage of human donors as well as by islet graft destruction by inflammatory and thrombotic mechanisms. A possible solution to these problems is to use genetically modified pig islets. Endothelial protein C receptor (EPCR) enhances protein C activation and regulates coagulation, inflammation, and apoptosis. We hypothesized that human EPCR (hEPCR) expression on donor islets would improve graft survival and function. Islets from an hEPCR transgenic mouse line strongly expressed the transgene, and hEPCR expression was maintained after islet isolation. Islets were transplanted from hEPCR mice and wild-type (WT) littermates into diabetic mice in a marginal-dose syngeneic intraportal islet transplantation model. The blood glucose level normalized within 5 days in 5 of 7 recipients of hEPCR islets, compared with only 2 of 7 recipients of WT islets (P < .05). Transplanted hEPCR islets had better preserved morphology and more intense insulin staining than WT grafts, and they retained transgene expression. The improved engraftment compared with WT islets suggests that inflammation and coagulation associated with the transplant process can be reduced by hEPCR expression on donor tissue.

Anti-xenograft immune responses in alpha 1,3-galactosyltransferase knock-out mice.

Although originally generated to test the effect of eliminating the alpha-Gal epitope on HAR, it is becoming increasingly clear that GalT KO mice offer a convenient and inexpensive model to investigate many aspects of the anti-xenorgraft immune response. Clearly, not all aspects of anti-xenograft rejection responses are identical in mice and primates, which should be kept in mind when interpreting results of GalT KO mouse studies. However, with this and other mouse models it is possible to test a large number of variables, which is impractical for both logistical and financial reasons with primates. Furthermore the short gestation time and large litter size of mice means that genetic strategies targeting different aspects of the anti-xenograft immune response can be combined and subsequently tested to identify the optimal combination of genetic and therapeutic approaches to achieve long term xenograft survival. In this regard the GalT KO mouse has been and will continue to be a valuable small animal model for the study of all facets of xenograft rejection involving anti-Gal antibodies.

Complex organisation of the 5'-end of the human glycine tRNA synthetase gene.

Glycine tRNA synthetase (glyRS) catalyses the addition of the amino acid glycine to its cognate tRNA molecules. In the silk moth worm Bombyx mori, this gene is subject to complex transcriptional regulation because of the predominance of glycine in silk. In vertebrates, glycine is a major constituent of collagen but there have been no studies of glyRS regulation. In this study we have isolated and mapped a genomic clone containing the 5'-end of glyRS. Primer extension studies identified only one transcriptional start point (TSP) in three different cell lines. Expression of the transcript identified may be regulated translationally because it contains five potential initiation codons, three of which are in good context for initiation. The most 3' of the potential initiation codons has previously been predicted to be the initiating codon for cytoplasmic glyRS. Two of the upstream codons are in-frame with this codon, and both are predicted to extend the N-terminus of glyRS to include a mitochondrial targeting sequence. Sequencing of genomic DNA surrounding the TSP showed features common to the promoters of housekeeping genes, as well as a canonical TATA box at the unusual position of +9. Surprisingly, promoter activity in vitro was not specified by a 1.9 kb genomic fragment containing the TSP and TATA box, but by a contiguous 0.4 kb fragment immediately downstream. These studies suggest that the transcription of glyRS from a single start point requires downstream promoter elements.

Targeting gene expression to endothelial cells in transgenic mice using the human intercellular adhesion molecule 2 promoter.

Genetic engineering of donor animals in xenotransplantation research has been directed largely toward obtaining expression of various immunoregulatory molecules on vascular endothelium, the initial target of recipient antibody and complement. However, specific high-level expression of transgenes throughout the vascular tree in adult animals has proved difficult to achieve, perhaps because of the inherent heterogeneity of endothelium. Using the promoter of the gene for intercellular adhesion molecule 2 (ICAM-2), which is constitutively expressed on all vascular endothelium, we have developed a system for endothelial cell gene targeting in vivo. A 334-basepair fragment from the 5' flanking region of the human ICAM-2 gene was used to drive the expression of human CD59 in transgenic mice. Strong and uniform expression of CD59 was observed on the endothelial cells of all blood vessels in the heart, kidney, lung, liver, and pancreas in the three lines of mice examined. Little or no expression was seen in other cell types, with the exception of neutrophils and monocytes. These results suggest that this small promoter region contains most of the signals necessary to endow it with endothelial cell specificity, making it a potentially valuable tool in areas ranging from xenotransplantation to gene therapy.

High-level endothelial expression of human CD59 prolongs heart function in an ex vivo model of xenograft rejection.

BACKGROUND: Hyperacute rejection of discordant xenografts is dependent on activation of the complement system of the recipient. Transgenic expression of recipient complement regulatory factors in donor tissue has proved to be a promising approach to dealing with hyperacute rejection, although the relationship between the level of complement regulatory factor expression and the degree of protection is not well established. Here, we examine this relationship using CD59 transgenic mouse hearts in an ex vivo model of xenograft rejection. METHODS: The level of expression of CD59 in two lines of transgenic mice, in which CD59 is expressed under the control of either the murine H2Kb (MHC class I) promoter (line CA-17) or the endothelium-specific human intercellular adhesion molecule-2 promoter (line 237-7), was compared by immunohistochemistry and flow cytometry. Hearts from both groups and wild-type controls were perfused ex vivo with human plasma, and mean heart work for each group was compared over a 60-min period. RESULTS: CD59 expression on cardiac endothelial cells isolated from homozygous CA-17 mice was 25- to 30-fold lower than that on cardiac endothelial cells from heterozygous 237-7 mice. CA-17 hearts perfused with 6% human plasma exhibited a reduction in deposition of the membrane attack complex, but not a prolongation of function, compared with nontransgenic mouse hearts. In contrast, 237-7 hearts showed significantly prolonged function during perfusion with 20% plasma. CONCLUSIONS: High-level endothelial-specific expression of CD59 was effective in prolonging the function of mouse hearts perfused with 20% human plasma, whereas low-level, broader expression did not provide protection from 6% plasma.

Knock out of alpha1,3-galactosyltransferase or expression of alpha1,2-fucosyltransferase further protects CD55- and CD59-expressing mouse hearts in an ex vivo model of xenograft rejection.

BACKGROUND: Organs from transgenic animals with high-level endothelial expression of the human complement regulatory factors CD55 and CD59 are significantly protected from human complement-mediated injury. Elimination or reduction of the major xenoepitope alphaGal, achieved by knocking out the alpha1,3-galactosyltransferase gene (Gal KO) or expressing human alpha1,2-fucosyltransferase (H transferase or HTF), also affords protection, although to a lesser degree. In this study, we examined whether the protection provided by strong CD55 and CD59 expression can be augmented by the Gal KO or HTF modifications. METHODS: Hearts from four groups of mice (wild type, CD55/CD59, CD55/CD59/HTF, and CD55/CD59/Gal KO) were perfused ex vivo with 40% human plasma. Mean heart work for each group was compared over a 60-min period. RESULTS: Wild-type hearts ceased to function effectively within 15 min of plasma addition. CD55/CD59 hearts displayed prolonged survival and maintained approximately 10% maximum work at the end of perfusion. Introduction of Gal KO or HTF onto the CD55/CD59 background resulted in a further prolongation, with work maintained at 20-30% of the maximum level. CONCLUSIONS: We used an ex vivo model to demonstrate that eliminating alphaGal expression further prolongs the function of mouse hearts expressing high levels of CD55 and CD59. In addition, we showed that reducing alphaGal by expressing HTF is equally as effective in prolonging CD55/CD59 heart function as knocking out Gal transferase, thus providing a feasible strategy for translating these advances to the pig.

The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement.

BACKGROUND: It has been proposed that hyperacute rejection (HAR) of pig-to-primate vascularized xenografts is due in large part to ineffective regulation of recipient complement by pig complement regulatory proteins (CRPs), and indeed transgenic expression of human CRPs in pigs can prevent hyperacute rejection. However, at least one pig CRP (CD59) efficiently regulates human complement in vitro, suggesting that it is the level of expression of a particular CRP(s) rather than cross-species incompatibility that explains the HAR of porcine xenografts. We investigated the relative effectiveness of transgenically expressed pig and human CD59 in providing protection of mouse hearts from human complement in an ex vivo setting. METHODS: Transgenic mice expressing pig CD59 or human CD59 under the control of the human ICAM-2 promoter, which restricts expression in tissues to vascular endothelium, were used. Hearts from mice expressing similar levels of pig CD59 or human CD59 were perfused ex vivo with 10% human plasma and heart function was monitored for 60 min. Sections of perfused hearts were examined for deposition of the membrane attack complex (MAC). RESULTS: Control nontransgenic hearts (n=5) were rapidly affected by the addition of human plasma, with mean function falling to less than 10% of the initial level within 15 min. In contrast, hearts expressing either pig CD59 (n=6) or human CD59 (n=8) were protected from plasma-induced injury, maintaining 31 and 35% function, respectively, after 60 min of perfusion. MAC deposition was markedly reduced in both pig CD59 and human CD59 transgenic hearts compared to nontransgenic control hearts. CONCLUSIONS: When highly expressed on endothelium in transgenic mice, pig CD59 provided equivalent protection to human CD59 in a model of human complement-mediated xenograft rejection. Thus supranormal expression of endogenous porcine CRPs may be a feasible alternative to the expression of human CRPs in preventing HAR of pig-to-primate xenografts.

Expression and functional analysis of glycosyl-phosphatidyl inositol-linked CD46 in transgenic mice.

BACKGROUND: Complement activation plays a pivotal role in hyperacute xenograft rejection. In humans, activation of complement is regulated by a number of cell surface regulatory proteins. Membrane cofactor protein (CD46) is one such regulator that protects cells by acting as a cofactor for the factor I-mediated cleavage of C3b and C4b. Transgenic animals expressing human CD46 may provide organs that are resistant to complement attack. However, attempts to generate mice expressing human CD46 using cDNA-based constructs have been largely unsuccessful. METHODS: Transgenic mice expressing a glycosylphosphatidyl inositol (GPI)-linked form of CD46 were generated by microinjection of a hybrid CD46/CD55 cDNA under the control of the human intercellular adhesion molecule-2 promoter. Expression of CD46-GPI on the vascular endothelium was determined by immunohistochemistry. The ability of CD46-GPI to protect mouse tissues from human complement attack was determined using an ex vivo isolated perfused heart model. RESULTS: Three founder animals expressing CD46-GPI were identified. Histological analysis showed strong and uniform expression of CD46-GPI on the vascular endothelium of all organs examined. Ex vivo perfusion of transgenic mouse hearts with human plasma showed a reduction in C3c deposition and a slightly prolonged function compared with controls. CONCLUSIONS: High-level expression of CD46-GPI was achieved in transgenic mice by using a modified cDNA-based construct. The CD46-GPI was functional, providing some protection from complement-mediated damage in the ex vivo model, and may be useful in xenotransplantation if expressed in combination with CD55 and CD59.

Renal xenografts from triple-transgenic pigs are not hyperacutely rejected but cause coagulopathy in non-immunosuppressed baboons.

BACKGROUND: The genetic modification of pigs is a powerful strategy that may ultimately enable successful xenotransplantation of porcine organs into humans. METHODS: Transgenic pigs were produced by microinjection of gene constructs for human complement regulatory proteins CD55 and CD59 and the enzyme alpha1,2-fucosyltransferase (H-transferase, HT), which reduces expression of the major xenoepitope galactose-alpha1,3-galactose (alphaGal). Kidneys from CD55/HT and CD55/CD59/HT transgenic pigs were transplanted into nephrectomised, nonimmunosuppressed adult baboons. RESULTS: In several lines of transgenic pigs, CD55 and CD59 were expressed strongly in all tissues examined, whereas HT expression was relatively weak and did not significantly reduce alphaGal. Control nontransgenic kidneys (n=4) grafted into baboons were hyperacutely rejected within 1 hr. In contrast, kidneys from CD55/HT pigs (n=2) were rejected after 30 hr, although kidneys from CD55/CD59/HT pigs (n=6) maintained function for up to 5 days. In the latter grafts, infiltration by macrophages, T cells, and B cells was observed at days 3 and 5 posttransplantation. The recipients developed thrombocytopenia and abnormalities in coagulation, manifested in increased clotting times and an elevation in the plasma level of the fibrin degradation product D-dimer, within 2 days of transplantation. Treatment with low molecular weight heparin prevented profound thrombocytopenia but not the other aspects of coagulopathy. CONCLUSIONS: Strong expression of CD55 and CD59 completely protected porcine kidneys from hyperacute rejection and allowed a detailed analysis of xenograft rejection in the absence of immunosuppression. Coagulopathy appears to be a common feature of pig-to-baboon renal transplantation and represents yet another major barrier to its clinical application.

Blockade of the CD28 and CD40 pathways result in the acceptance of pig and rat islet xenografts but not rat cardiac grafts in mice.

Previously, we demonstrated that combination CTLA4-Fc and anti-CD40L mAb treatment results in tolerance to concordant, cellular islet xenografts. The aim of this study was to determine its effectiveness in a model of fetal pig pancreas (FPP) xenotransplantation. Survival of FPP fragment grafts were compared to the survival of rat islet or cardiac xenografts following short term CTLA4-Fc and anti-CD40L mAb treatment. Rat islet and FPP fragment grafts survived long-term. However, rat cardiac grafts were rejected by 52-91 days. Both rat islet and FPP grafts showed similar histology with intact islet structures and adjacent 'nests' of lymphocytes. Concordant vascularised rat hearts showed extensive polymorphonuclear infiltrate, concentric vasculitis and a perivascular infiltrate predominantly of CD8+ T cells. This suggests that this therapy is effective for prolonging islet xenografts and demonstrates that the cellular mechanism of rejection for vascularised and non-vascularised xenografts are different.