Prolonged Function of Macrophage, von Willebrand Factor-Deficient Porcine Pulmonary Xenografts

Porcine von Willebrand factor (vWF) activates human and primate platelets. Having determined the importance of pulmonary intravascular macrophages (PIMs) in pulmonary xenotransplantation, we evaluated whether, in the absence of PIMs, vWF might play a role in pulmonary xenograft dysfunction. Utilizing a left single-lung transplant model, baboons depleted of anti-alphaGal antibodies received lungs from either vWF-deficient (n = 2); MCP-expressing (n = 5); MCP PIM-depleted (n = 5); or vWF-deficient PIM-depleted swine (n = 3). Two out of three of the PIM-depleted, pvWF deficient grafts survived longer than any previously reported pulmonary xenografts, including PIM-depleted xenografts expressing human complement regulatory proteins. Depletion of PIM's from vWF-deficient lungs, like depletion of PIM's from hMCP lungs, resulted in abrogation of the coagulopathy associated with pulmonary xenotransplantation. Thus, in terms of pulmonary graft survival, control of adverse reactions involving pvWF appears to be equally or even more important than is complement regulation using hMCP expression. However, based on the rapid failure of PIM-sufficient, pvWF-deficient pulmonary xenografts, pVWF-deficient pulmonary xenografts appear to be particularly sensitive to macrophage-mediated damage. These data provide initial evidence that vWF plays a role in the 'delayed' (24 h) dysfunction observed in pulmonary xenotransplantation using PIM depleted hMCP organs.     

Disseminated intravascular coagulation in association with pig-to-primate pulmonary xenotransplantation

BACKGROUND: Profound coagulopathy has been proposed as a barrier to xenotransplantation. Disseminated intravascular coagulation (DIC) has been observed with the rejection of renal and bone marrow xenografts but has not yet been described in pulmonary xenografts. METHODS: This study examined the coagulation parameters in five baboons that received pulmonary xenografts and one baboon that was exposed to porcine lung during an extracorporeal perfusion. Platelet counts, prothrombin times (PT), and levels of fibrinogen, D-dimers, and thrombin-antithrombin III complex (TAT) were analyzed. In addition, serum levels of plasminogen activator inhibitor-1 (PAI-1), thrombomodulin (TM), tissue plasminogen activator (tPA), and tissue factor (TF) were determined. RESULTS: Hyperacute pulmonary xenograft dysfunction, which occurred within 0-9 hr of graft reperfusion, was associated with clinically evident DIC. This coagulopathy was characterized by thrombocytopenia, decreased fibrinogen levels, elevations in PT, and increases in D-dimers and TAT. Furthermore, transient increases in PAI-1, increases in TM, and increases in tPA were observed in the serum of some but not all recipients. None of the baboons demonstrated measurable increases in soluble TF. CONCLUSIONS: Although DIC in renal or bone marrow xenotransplantation develops over a period of days, DIC associated with hyperacute pulmonary xenograft dysfunction develops within hours of graft reperfusion. Thus, the DIC in pulmonary xenotransplantation may represent a unique and/or accelerated version of the coagulopathy observed with renal and bone marrow xenotransplantation.     

Disordered regulation of coagulation and platelet activation in xenotransplantation

Rejection of xenografts is associated with vascular-based inflammation, thrombocytopenia and the consumption of coagulation factors that may evolve into disseminated intravascular coagulation (DIC). Similarly, bone marrow-derived cellular xenotransplantation procedures are associated with endothelial cell activation and thrombotic microangiopathic injury. These complications generally develop despite the best available measures for depletion of xenoreactive natural antibody, inhibition of complement activation and suppression of T- and B-cell mediated immune responses. The mechanisms underlying the DIC and thrombotic microangiopathy associated with xenotransplantation are unclear. A proposed primary biological dysfunction of xenografts with respect to regulation of clotting could amplify vascular injury, promote immunological responses and independently contribute to graft failure. Disordered thromboregulation could have deleterious effects, comparable to unregulated complement activation, in the pathogenesis of xenograft rejection and may therefore represent a substantive barrier to xenotransplantation.     

O-linked glycosylation and functional incompatibility of porcine von Willebrand factor for human platelet GPIb receptors

BACKGROUND: Xenograft rejection is associated with vascular inflammation, thrombocytopenia and the accelerated consumption of coagulation factors. Primary biological incompatibilities of the xenograft in the regulation of clotting appear to amplify pathological processes associated with rejection. The functional incompatibility of porcine von Willebrand factor (vWF) expressed within the xenograft vasculature may heighten interactions with the primate platelet receptor GPIb, hence augmenting formation of platelet microthrombi and vascular injury. Here, we address the functional impact of O-linked glycosylation of the vWF A1 domain on primate platelet activation. METHODS: Recombinant human or porcine vWF A1-domains were transiently over-expressed in COS-7 cells as FLAG-tagged fusion protein, linked to plasma membranes via GPI anchors. O-linked glycosylation was blocked by the addition of phenyl-alpha-GalNAc2 to cultures. Expressed vWF-A1 domains were characterized utilizing cytofluometric- and Western blot analyses. RESULTS: Cytofluometric analysis confirmed equivalent levels of human and porcine vWF A1-domain expression irrespective of the levels of O-linked glycosylation. Differential glycosylation patterns of vWF-A1 under these conditions were confirmed by Western blot analyses. Native porcine vWF A1-domains had enhanced human platelet activation potential when compared with human recombinant vWF A1. However, the loss of O-linked glycosylation abolished differences in aggregatory responses between human and porcine vWF A1 domains. CONCLUSIONS: Various degrees of O-linked glycosylation of vWF-A1-domains modulate levels of functional interaction with platelet receptor GPIb and consequent platelet aggregation responses in vitro. These data may have implications for outcomes of xenotransplantation. We speculate that alterations in glycosylation of vWF and other adhesion proteins associated with the targeting of the alpha1,3-Gal-epitope in mutant swine may have salutatory effects on the primate platelet activation observed in these xenografts.     

Systemic inflammation in xenograft recipients (SIXR)

Dysregulation of the coagulation system commonly develops in pig xenograft recipients, and remains an obstacle to successful pig organ xenotransplantation. Uncontrolled activation of coagulation leads to consumptive coagulopathy (CC) in the recipients, and thrombotic microangiopathy (TM) in the grafts. T cell‐directed immunosuppression successfully prevents the adaptive immune response to pig antigens after xenotransplantation and prolongs survival of organ xenografts. In some reports, T cell‐directed immunosuppression was able to delay (or prevent) the development of CC and/or TM. Recent reports have confirmed that inflammation can lead to activation of the coagulation system. Additionally, pro‐coagulant proteins, e.g. thrombin, are considered as pro‐inflammatory factors. In fact, an amplification loop is suggested to exist between inflammation and coagulation, leading to escalation of each other. Our in vitro data indicate that thrombin activation of pig endothelial cells is associated with upregulation of human T cell responses, suggesting that control of activation of coagulation and prevention of thrombin activation may facilitate the regulation of immune responses to xenografts in vivo. We hypothesize that a state of systemic inflammation develops after pig organ xenotransplantation, which is generated by both adaptive and innate immune responses. Even if T cell‐directed immunosuppression can control activation of coagulation induced by adaptive immune responses, pro‐inflammatory signals induced by the innate immune system can still promote activation of coagulation. We studied two models of xenotransplantation of different antigen loads; (i) pig aortic patch xenotransplantation, i.e. low antigen load, and (ii) pig organ (heart and kidney) xenotransplantation, i.e. high antigen load. We evaluated activation of coagulation, development of a T cell‐dependent immune response, and production of innate and adaptive pro‐inflammatory factors. In recipients of a low antigen load xenograft, effective prevention of the adaptive immune response by T cell‐directed immunosuppression (i.e. suppression of T cell proliferation in response to pig antigens and prevention of elicited antibody production) was associated with reduced thrombin activation. However, there was (i) upregulation of C‐reactive protein (CRP) and (ii) fibrinogen levels, (iii) increased IL‐6 production in the circulation, and (iv) an increase in the absolute number of innate immune cells (monocytes and neutrophils). Furthermore, (v) monocytes and dendritic cells showed significant upregulation of tissue factor expression, and aggregation with platelets after transplantation. In recipients with a high antigen load xenograft, short‐term organ survival was associated with high levels of CRP and IL‐6 early after transplantation. In long‐term organ survival, high levels of CRP and IL‐6 preceded the development of CC. There was intense CRP deposition in kidney xenografts (more than in heart xenografts) suggesting a stronger innate immune response. Additionally, CRP‐positive cells were detected in native lungs, suggesting an innate systemic inflammatory response. In conclusion, efficient blockade of the T cell‐dependent adaptive immune response in xenograft recipients is associated with systemic upregulation of inflammatory markers. Systemic inflammation in xenograft recipients (SIXR) is associated with upregulation of tissue factor expression on innate immune cells and their aggregation with platelets. As inflammation is known to break tolerance after transplantation, understanding the underlying mechanisms and regulation of SIXR may be necessary to achieve long‐term survival of organ xenografts (and T cell tolerance to pig antigens). Also, further genetic modifications of donor pigs to express anti‐inflammatory proteins may be essential.     

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.     

Effect of an anti-C5a monoclonal antibody indicates a prominent role for anaphylatoxin in pulmonary xenograft dysfunction

BACKGROUND: In contrast to renal or cardiac xenografts, the inhibition of complement using cobra venom factor (CVF) accelerates pulmonary xenograft failure. By activating C3/C5 convertase, CVF depletes complement while additionally generating C5a and other anaphylatoxins, to which pulmonary xenografts may be uniquely susceptible. The current study investigates the role of C5a in pulmonary xenograft failure in baboons. METHODS: Left orthotopic pulmonary xenografts using swine lungs expressing human CD46 were performed in baboons receiving: I) no other treatment (n=4), II) immunodepletion (n=5), and III) immunodepletion plus a single dose of mouse anti-human C5a monoclonal antibody (anti-C5a, 0.6 mg/kg administered intravenously) (n=3). The extent to which anti-C5a inhibits baboon C5a was assessed in vitro using a hemolytic reaction involving baboon serum and porcine red blood cells and by ELISA. RESULTS: Baboons in Group III exhibited significantly prolonged xenograft survival (mean=722+/-121 min, P=0.02) compared to baboons in Group I (mean=202+/-24 min) and Group II (mean=276+/-79 min). Furthermore, baboons in Groups I and II experienced pronounced hemodynamic compromise requiring inotropic support whereas those in Group III remained hemodynamically stable throughout experimentation without the need for additional pharmacologic intervention. CONCLUSIONS: These findings indicate that C5a exacerbates pulmonary xenograft injury and compromises recipient hemodynamic status. Moreover, blockade of anaphylatoxins, such as C5a, offers a promising approach for future investigations aimed at preventing pulmonary xenograft injury in baboons.     

Pre-treatment of porcine pulmonary xenograft with desmopressin: a novel strategy to attenuate platelet activation and systemic intravascular coagulation in an ex-vivo model of swine-to-human pulmonary xenotransplantation

Abstract: Background: Von Willebrand factor (vWF) has been proposed as a major contributor to the development of coagulopathy in pulmonary xenotransplantation. Pretreatment of donor swine with 1‐deamino‐8‐d ‐arginine vasopressin (DDAVP), an analog of vasopressin, can reduce the content of vWF in pulmonary xenografts. Here, we investigate the effects of DDAVP pre‐treatment in an ex‐vivo perfusion model of pulmonary xenotransplantation. Methods: We set up and performed the ex‐vivo perfusion using porcine pulmonary accessory lobes and fresh human whole blood (n = 12). Half of the donor swine were given 3 μg/kg DDAVP intravenously for 3 days before ex‐vivo perfusion (DDAVP group) and half of them were left untreated (control group). The porcine lung was perfused with fresh blood for 1 h and changes in the following parameters were monitored: pulmonary arterial pressure, pulmonary vascular resistance, blood cell counts, fibrinogen, antithrombin, platelet factor 4, D‐dimer, C3a, C4d, and xenoreactive IgM. The release of Galα1‐3Gal xenoantigen (αGal) from porcine lung which had been perfused and retained for 30 min with human blood was assessed by enzyme‐linked immunosorbent assay using αGal‐binding lectin. Results: Both DDAVP and control groups showed typical findings of immediate pulmonary dysfunction: an increase of pulmonary vascular resistance and sequestration of leukocytes and platelets after ex‐vivo perfusion. However, in the DDAVP group, the increase of platelet factor 4, C3a, and C4d after perfusion was attenuated compared to that in the control group. The release of αGal after blood retention was significantly lower in the DDAVP group than that of the control group. Conclusion: Pre‐infusion of DDAVP to the donor swine was beneficial in attenuating platelet activation as well as complement/coagulation activation. These effects of DDAVP are likely to relate to the reduction of porcine vWF content in the xenograft. Therefore, the modulation of vWF secretion in donor lungs could be an additional therapeutic way to reduce systemic coagulopathy in pulmonary xenotransplantation.     

Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates

BACKGROUND: Efforts to achieve tolerance to transplanted pig organs in nonhuman primates by the induction of a state of mixed hematopoietic chimerism have been associated with disorders of coagulation and thrombosis. Activation of recipient vascular endothelium and platelets by porcine hematopoietic cells and/or activation of donor organ vascular endothelium and/or molecular differences between the species may play roles. Irradiation or drug therapy could possibly potentiate endothelial cell activation and/or injury. METHODS: We have investigated parameters of coagulation and platelet activation in nonhuman primates after (1) a regimen aimed at inducing mixed hematopoietic chimerism and tolerance (TIR that included total body irradiation, T cell depletion, and splenectomy; (2) pig bone marrow or pig peripheral blood mobilized progenitor cell transplantation (PCTx); and/or (3) pig organ transplantation (POTx). Five experimental groups were studied. Baboons were the recipient subjects in all groups except Group 1. Gp 1 Cynomolgus monkeys (n=6) underwent TIR + allotransplantation of hematopoietic cells and a kidney or heart or TIR + concordant xenotransplantation (using baboons as donors) of cells and a kidney; Gp 2 Baboons (n=4) underwent TIR with or without (+/-) autologous hematopoietic cell infusion; Gp 3 (n=12) PCTx+/-TIR; Gp 4 (n=5) POTx+/-TIR; Gp 5 (n=4) TIR + PCTx + POTx. Platelet counts, with plasma prothrombin time, partial thromboplastin time, fibrinogen levels, fibrin split products and/or D-dimer were measured. RESULTS: In the absence of a discordant (porcine) cellular or organ transplant (Groups 1 and 2), TIR resulted in transient thrombocytopenia only, in keeping with bone marrow depression from irradiation. PCTx alone (Group 3) was associated with the rapid development of a thrombotic thrombocytopenic (TTP)-like microangiopathic state, that persisted longer when PCTx was combined with TIR. POTx (+/-TIR) (Group 4) was associated with a gradual fall (over several days) in platelet counts and fibrinogen with disseminated intravascular coagulation (DIC); after graft excision, the DIC generally resolved. When TIR, PCTx and POTx were combined (Group 5), an initial TTP-like state was superseded by a consumptive picture of DIC within the first week, necessitating graft removal. CONCLUSIONS: Both PCTx and POTx lead to profound alterations in hemostasis and coagulation parameters that must be overcome if discordant xenotransplantation of hematopoietic cells and organs is to be fully successful. Disordered thromboregulation could exacerbate vascular damage and potentiate activation of coagulation pathways after exposure to xenogeneic cells or a vascularized xenograft.     

Reduction of consumptive coagulopathy using porcine cytomegalovirus-free cardiac porcine grafts in pig-to-primate xenotransplantation

BACKGROUND: Xenotransplantation using pigs as the source species for organs carries a potential risk for transmission and activation of porcine herpesviruses. Activation of porcine cytomegalovirus (PCMV) in pig-to-baboon xenotransplantation is associated with xenograft injury and possibly an increased incidence of consumptive coagulopathy (CC). METHODS: To further investigate the role of PCMV activation in the occurrence of CC, a strategy to exclude PCMV from the donor was developed. To exclude PCMV, piglets were early-weaned and raised separated from other swine. These piglets were used as donors in an experimental protocol of pig-to-baboon heart xenotransplantation. RESULTS: Early weaning of piglets was successful in excluding PCMV. Use of PCMV-free cardiac porcine xenografts in baboons resulted in prolonged graft survival and prevented consumptive coagulopathy in all recipients. CONCLUSIONS: The use of PCMV-free cardiac grafts is beneficial in reducing the direct effects of PCMV activation in the graft (tissue damage) and the indirect effects of PCMV activation in the recipient (consumptive coagulopathy).     

Human platelets activate porcine endothelial cells through a CD154-dependent pathway.

BACKGROUND: Delayed xenograft rejection is associated with endothelial cell activation, platelet sequestration, and subsequent thrombosis. We evaluated whether human platelets could directly activate porcine endothelium (PEC), and if so, whether this was mediated by an interaction between platelet-bound CD154 and PEC CD40. METHODS: Platelet activation was achieved by thrombin exposure and confirmed by evaluation of up-regulated CD62P and CD154. Co-incubation of platelets or D1.1 cells with PEC was performed, and PEC activation was evaluated by up-regulation of CD62E. RESULTS: Co-incubation of resting platelets that lacked significant expression of CD62P and were void of CD154 did not activate PEC. In contrast, thrombin-activated human platelets expressing considerable amounts of both CD62P and CD154 induced PEC activation. This activation could be completely inhibited by coincubation with a humanized monoclonal antibody directed at human CD154 (hu5c8). Similarly, human D1.1 cells expressing CD154 were shown to activate PEC in a CD154-dependent manner. CONCLUSION: Human CD154 expressed on activated human platelets or on T cells interacts with CD40 expressed on PEC leading to PEC activation. This interaction can be inhibited by a monoclonal antibody directed against CD154, suggesting that an interaction between human CD154 and PEC CD40 is at least in part responsible for PEC activation seen in delayed xenograft rejection. These data strengthen the rationale for the use of CD154-directed therapy in discordant xenotransplantation.     

Coagulopathy in α-galactosyl transferase knockout pulmonary xenotransplants

Bush EL, Barbas AS, Holzknecht ZE, Byrne GW, McGregor CG, Parker W, Duane Davis R, Lin SS. Coagulopathy in α‐galactosyl transferase knockout pulmonary xenotransplants. Xenotransplantation 2011; 18: 6–13. © 2011 John Wiley & Sons A/S. Abstract: Background: After substantial progress on many fronts, one of the remaining barriers still opposing the clinical application of xenotransplantation is a disseminated intravascular coagulopathy (DIC) that is observed in the pre‐clinical model of porcine‐to‐primate transplantation. The onset of DIC is particularly rapid in recipients of pulmonary xenografts, usually occurring within the first days or even hours of reperfusion. Methods: In this study, we describe the results of two porcine‐to‐baboon transplants utilizing porcine lungs depleted of macrophages, deficient in the α‐1,3‐galactosyltransferase gene, and with the expression of human decay‐accelerating factor, a complement regulatory protein. Results: In both cases, evidence of DIC was observed within 48 h of reperfusion, with thrombocytopenia and increases in levels of thrombin–antithrombin complex evident in both cases. Depletion of fibrinogen was observed in one graft, whereas elevation of D‐dimer levels was observed in the other. One graft, which showed focal lymphocytic infiltrates pre‐operatively, failed within 3 h. Conclusions: The results indicate that further efforts to address the coagulopathy associated with pulmonary xenotransplantation are needed. Further, evidence suggests that resident porcine immune cells can play an important role in the coagulopathy associated with xenotransplantation.     

Depletion of pulmonary intravascular macrophages prevents hyperacute pulmonary xenograft dysfunction

BACKGROUND: Recent years have brought dramatic progress in the field of xenotransplantation, with the development of transgenic swine and various other means of overcoming the rejection mediated by xenoreactive antibodies. Although progress has been rapid with kidney and heart xenografts, progress with pulmonary xenografts has lagged behind. Recent findings have suggested that donor pulmonary intravascular macrophages may play a critical role in the hyperacute dysfunction of pulmonary xenografts. METHODS: The function of pulmonary xenografts from pigs depleted of pulmonary intravascular macrophages was compared with the function of xenografts from normal pigs. RESULTS: Pulmonary xenografts from pigs from which pulmonary intravascular macrophages were depleted survived (23.5+/-0.9 hours) about five times longer than normal (macrophage sufficient) xenografts (4.4+/-1.41 hours) (P< 0.0001). At 21 hours post-reperfusion, the left pulmonary arterial flow was 225.0+/-34 ml/min in lungs depleted of pulmonary intravascular macrophages, whereas all normal xenografts had failed. CONCLUSIONS: These findings indicate that donor macrophages play a critical role in pulmonary xenograft dysfunction. This finding has broad implications for xenotransplantation, suggesting that porcine macrophages might pose a barrier to the engraftment and function of a variety of porcine organ xenografts.     

The role of antibodies in dysfunction of pig-to-baboon pulmonary transplants

OBJECTIVE: Pulmonary transplantation has become the preferred treatment for end-stage lung disease, but application of the procedure is limited because of a paucity of donors. One way to solve donor limitations is to use animal organs as a donor source or xenotransplantation. The current barrier to pulmonary xenotransplantation is the rapid failure of the pulmonary xenograft. Although antibodies are known to play a role in heart and kidney xenograft rejection, their involvement in lung dysfunction is less defined. This project was designed to define the role of antibodies in pulmonary graft rejection in a pig-to-baboon model. METHODS: Orthotopic transgenic swine left lung transplants were performed in baboons depleted of antibodies by one of three techniques before transplantation: (1) ex vivo swine kidney perfusion, (2) total immunoglobulin-depleting column perfusion, and (3) ex vivo swine lung perfusion. Results were compared with those of transgenic swine lung transplants in unmodified baboons. RESULTS: All three techniques of antibody removal resulted in depletion of xenoreactive antibodies. Only pretransplantation lung perfusion improved pulmonary xenograft function compared with lung transplantation in unmodified baboons. CONCLUSIONS: The pathogenesis of pulmonary injury in a swine-to-primate transplant model is different from that in renal and cardiac xenografts. Depletion of antibodies alone does not have a beneficial effect and may actually be detrimental.     

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.     

Potential of an injectable polymer to prevent hyperacute rejection of ex vivo perfused porcine lungs

BACKGROUND: Removal of xenoreactive antibodies in pig-to-human lung transplantation by columns or organ perfusions proofed to be unsatisfactory and associated with adverse effects. In an ex-vivo lung perfusion model, we evaluated the potential of a soluble trisaccharide polymer (GAS914) to bind alpha-Gal antibodies and to protect a pulmonary xenograft from hyperacute rejection (HAR) and pulmonary xenograft dysfunction. METHODS: Porcine lungs were perfused with fresh human blood for 240 min. In the GAS914 treated group (n=6) the polymer was applied in three different concentrations. The control group (n=6) received no GAS914. Survival and function of perfused xenografts were monitored, and alpha-Gal antibodies as well as cytolytic anti-porcine antibodies analyzed. RESULTS: In the GAS-treated group survival of lungs was significantly prolonged, pulmonary vascular resistance reduced, pulmonary edema prevented, and oxygenation improved. On histopathological evaluation application of GAS resulted in minimal graft injury and significantly less deposition of the terminal complement complex C5b-9. Following application of GAS914, up to 89.8% of IgG alpha-Gal, 79.5% of IgM and 73.6% of anti-porcine antibodies in the human blood were bound by the polymer. Subsequent perfusion of porcine lungs resulted in absorption of only 3% of the baseline IgG alpha-Gal antibodies in the GAS914 group, compared to 87% in the controls. CONCLUSIONS: In this ex-vivo lung perfusion model, a trisaccharide polymer prevented immediate HAR, due to effective removal of alpha-Gal antibodies. In combination with additional strategies GAS914 may be a valuable tool in overcoming HAR and dysfunction of pulmonary xenografts.     

Thrombotic microangiopathic glomerulopathy in human decay accelerating factor-transgenic swine-to-baboon kidney xenografts

Models of pig-to-baboon xenografting were examined to identify the mechanisms and pathologic characteristics of acute humoral xenograft rejection (AHXR). Thymus and kidney (composite thymokidney) from human decay accelerating factor-transgenic swine were transplanted into baboons (n = 16) that were treated with an immunosuppressive regimen that included extracorporeal immunoadsorption of anti-alphaGal antibody and inhibition of complement activation. Morphologic and immunohistochemical studies were performed on protocol biopsies and graftectomy samples. All renal xenografts avoided hyperacute rejection. However, graft rejection coincided with the increase of anti-alphaGal antibody in the recipient's circulation. The 16 xenografts studied were divided into two groups dependent on the rapid return (group 1) or gradual return (group 2) of anti-alphaGal antibody after immunoadsorption. In group 1 (n = 6), grafts were rejected to day 27 with development of typical AHXR, characterized by marked interstitial hemorrhage and thrombotic microangiopathy in the renal vasculature. In group 2 (n = 10), grafts also developed thrombotic microangiopathy affecting mainly the glomeruli by day 30 but also showed minimal evidence of interstitial injury and hemorrhage. In the injured glomeruli, IgM and C4d deposition, subsequent endothelial cell death and activation with upregulation of von Willebrand factor and tissue factor, and a decrease of CD39 expression developed with the formation of fibrin-platelet multiple microthrombi. In this model, the kidney xenografts, from human decay accelerating factor-transgenic swine, in baboons undergo AHXR. In slowly evolving AHXR, graft loss is associated with the development of thrombotic microangiopathic glomerulopathy. Also, anti-alphaGal IgM deposition and subsequent complement activation play an important role in the mechanism of glomerular endothelial injury and activation and the formation of multiple microthrombi.     

Platelet-mediated activation of endothelial cells: implications for the pathogenesis of transplant rejection

BACKGROUND: Platelets exert their normal functions at sites of endothelial disruption by plugging discontinuities in blood vessels and secreting products that promote thrombosis, inflammation, and the healing of wounds. Whether platelets might induce these changes in xenograft blood vessels, leading to development of acute vascular rejection, has been uncertain. METHODS: To examine the role of human platelets in modulation of xenograft endothelium, pig endothelial cells were treated with human platelets. RESULTS: Treatment of quiescent porcine endothelial cells with human platelets modulated the endothelial cells. Whereas resting human platelets caused little change in normal porcine endothelial cells, platelets activated with small amounts of thrombin induced striking changes in the endothelial cells, including the induction of tissue factor activity, the expression of E-selectin, and the secretion of endothelin-1. These changes were induced, at least in part, by interleukin-1 (IL-1) associated with the platelet surface and were modified by the secretion of transforming growth factor-beta (TGF-beta). CONCLUSION: These findings may explain how the activation of platelets at an early point in the rejection of vascularized organ xenografts or in chronic diseases might contribute to thrombotic, ischemic, and inflammatory changes characteristic of an organ xenograft undergoing rejection.     

C1 inhibitor for prophylaxis of xenograft rejection after pig to cynomolgus monkey kidney transplantation

BACKGROUND: Early rejection of discordant porcine xenografts in primate recipients is initiated by the intragraft binding of either preformed (hyperacute xenograft rejection) or induced (acute vascular rejection) antiporcine recipient antibodies with subsequent complement activation via the classical pathway. We have investigated the efficacy of the supplemental administration of C1-inhibitor (C1-INH), a specific inhibitor of the classical complement activation pathway, for prophylaxis of xenograft rejection in a pig to primate kidney xenotransplantation setting. METHODS: Based on the results of pharmacokinetic studies performed in two nontransplanted monkeys, supplemental C1-INH therapy was administered daily to three Cynomolgus monkeys receiving a life-supporting porcine kidney transplant together with cyclophosphamide-induction/cyclosporine A/mycophenolat-mofetil/steroid immunosuppressive therapy. RESULTS: In the three monkeys receiving porcine kidney xenografts and continuous C1-INH treatment none of the grafts underwent hyperacute rejection; all xenografts showed initial function. Recipient survival was 13, 15, and 5 days. No graft was lost due to acute vascular rejection. All animals died with a functioning graft (latest creatinine 96, 112, and 96 micromol/liter) due to bacterial septicemia. CONCLUSION: We conclude that, in our model, supplemental C1-INH therapy together with a standard immunosuppressive regimen can be helpful for prevention of xenograft rejection in a pig to primate kidney xenotransplantation setting. The optimal dose and duration of C1-INH treatment, however, has yet to be determined.     

The immunobiology of pig‐to‐nonhuman primate islet xenotransplantation: insights,innovation, and impact

Previous studies of pig‐to‐non‐human primate (NHP) islet xenotransplantation have provided important insights into the immune recognition and effector pathways operative in this relevant preclinical model. The specifics of the xenograft product, microenvironment at the implantation site, and the immunosuppressive regimen significantly influence the mechanisms underlying the rejection of xenogeneic islets. Our current understanding of the immunological barriers to survival of pig islets in NHPs is largely based on studies on intraportal islet xenografts and on comparisons with islet allografts. The demonstration of cell‐mediated rejection of intraportal porcine islet xenografts at about 1 month posttransplant in monkeys immunosuppressed with the same protocols that prevent monkey islet allograft rejection indicates that islet xenograft rejection involves cellular mechanisms that are not present in acute islet allograft rejection. While these mechanisms remain poorly defined the demonstration of long‐term diabetes reversal after intraportal islet xenotransplantation in non‐human primates immunosuppressed with anti‐CD40L but not with anti‐CD40 antibody‐based protocols suggests that the therapeutic efficacy of anti‐CD40L in this transplantation setting likely involves the depletion of donor‐reactive, activated T cells besides CD40:CD40L costimulation blockade. Rejection of intraportal islet xenografts in NHPs immunosuppressed with CTLA4‐Ig and rapamycin was mediated largely by IL‐15‐primed, CXCR3+CD8+ memory T cells recruited by IP‐10 (CXCL10) positive pig islets and macrophages that showed staining for IL‐12 and iNOS. Adding basiliximab induction and tacrolimus maintenance therapy to this protocol prevented rejection in 24 of 26 recipients followed for up to 275 days. Comparison of both groups suggests, though by no means conclusive, that prolongation of graft survival in this large cohort was associated with reduced direct T cell responses to xenoantigens, reduced proportion of intrahepatic (intragraft) B cells and IFN‐γ+ and IL‐17+ CD4 and CD8 T cells, and increased local production of immunoregulatory molecules linked with Tregs, including TGF‐β, Foxp3, HO‐1, and IL‐10. Anti‐pig non‐Gal IgG antibody elicitation was suppressed in both groups. We are currently exploring the concept of negative vaccination to markedly minimize the need for immunosuppression in islet xenotransplantation. Peritransplant administration of donor apoptotic cells extended pig‐to‐mouse islet xenograft survival to >250 days when combined with peritransplant B cell‐depletion and rapamycin. This costimulation blockade‐sparing, antigen‐specific immunotherapy is expected to cause rapid pretransplant clonal deletion of indirect and anergy of direct xenospecific T cells while inducing regulatory T cells. As anti‐CD40L antibodies, B cell depleting antibodies are expected to interfere with indirect antigen presentation, costimulation, and cytokine production required for optimal T cell proliferation, memory formation, and intragraft CD8+ effector function. It is conceivable that additional strategies must be employed in NHPs and eventually in diabetic patients to achieve – as previously with anti‐CD40L antibodies – more complete, yet selective depletion of donor‐reactive, activated T‐cells for the purpose of stable xenograft acceptance.