RESUMEN
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.
Asunto(s)
Antígenos CD/metabolismo , Diabetes Mellitus Experimental/cirugía , Diabetes Mellitus Tipo 1/cirugía , Trasplante de Islotes Pancreáticos , Receptores de Superficie Celular/metabolismo , Trasplantes/metabolismo , Animales , Apoptosis , Glucemia/análisis , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 1/sangre , Receptor de Proteína C Endotelial , Supervivencia de Injerto , Humanos , Insulina/análisis , Masculino , Ratones , Ratones Transgénicos , Sustancias Protectoras/metabolismo , Proteína C/metabolismo , PorcinosRESUMEN
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.
Asunto(s)
Ácidos Borónicos/uso terapéutico , Proteína Inhibidora del Complemento C1/uso terapéutico , Galactosiltransferasas/genética , Supervivencia de Injerto/fisiología , Xenoinjertos , Trasplante de Riñón , Intercambio Plasmático , Pirazinas/uso terapéutico , Animales , Animales Modificados Genéticamente , Enfermedades Autoinmunes , Bortezomib , Citomegalovirus/fisiología , Galactosiltransferasas/deficiencia , Técnicas de Inactivación de Genes , Inmunidad Innata/fisiología , Inmunosupresores/uso terapéutico , Riñón/cirugía , Riñón/virología , Modelos Animales , Papio anubis , Sus scrofa , Replicación Viral/fisiologíaRESUMEN
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.
Asunto(s)
Sangre , Rechazo de Injerto , Trasplante de Islotes Pancreáticos , Trasplante Heterólogo , Animales , Anticuerpos/sangre , Bovinos , PapioRESUMEN
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.
Asunto(s)
Fucosiltransferasas/metabolismo , Rechazo de Injerto/etiología , Trasplante de Corazón/efectos adversos , Complejo Mayor de Histocompatibilidad/fisiología , Trasplante de Piel/efectos adversos , Trasplante Heterólogo/efectos adversos , Animales , Presentación de Antígeno/inmunología , Femenino , Fucosiltransferasas/genética , Glicosilación , Rechazo de Injerto/mortalidad , Rechazo de Injerto/patología , Supervivencia de Injerto , Humanos , Técnicas para Inmunoenzimas , Células Asesinas Naturales/inmunología , Depleción Linfocítica , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Pronóstico , Factores de Riesgo , Tasa de Supervivencia , Linfocitos T/inmunología , Donantes de Tejidos , Trasplante Homólogo , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
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.
Asunto(s)
Antígenos CD/metabolismo , Endotelio Vascular/metabolismo , Glicoproteínas/metabolismo , Modelos Animales , Receptores de Superficie Celular/metabolismo , Animales , Receptor de Proteína C Endotelial , Citometría de Flujo , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Daño por Reperfusión/prevención & controlRESUMEN
Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.
Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Fucosiltransferasas/metabolismo , Trasplante de Riñón/inmunología , Trasplante Heterólogo/inmunología , Animales , Animales Modificados Genéticamente , Retrovirus Endógenos/metabolismo , Rechazo de Injerto , Supervivencia de Injerto , Humanos , Inmunoglobulina G/química , Inmunosupresores/uso terapéutico , Trasplante de Riñón/métodos , Papio , Porcinos , Factores de Tiempo , Trasplante Heterólogo/métodosRESUMEN
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.
Asunto(s)
Antioxidantes/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Estrés Oxidativo/fisiología , Procesamiento Proteico-Postraduccional/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Glutatión Peroxidasa/metabolismo , Peroxidación de Lípido/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
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.
Asunto(s)
Antígenos CD/biosíntesis , Apirasa/biosíntesis , Daño por Reperfusión/prevención & control , Adenosina/metabolismo , Animales , Isquemia Fría , Humanos , Necrosis de la Corteza Renal/prevención & control , Ratones , Ratones Transgénicos , Modelos AnimalesRESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Ciclosporina/farmacología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Ratas , Porcinos , Trombomodulina , Transgenes/efectos de los fármacosRESUMEN
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.
Asunto(s)
Proteína C/metabolismo , Trombina/metabolismo , Trombomodulina/metabolismo , Trasplante Heterólogo/efectos adversos , Animales , Carboxipeptidasa B2/metabolismo , Coenzimas/metabolismo , Activación Enzimática , Rechazo de Injerto/metabolismo , Humanos , Microcirculación , Unión Proteica , Porcinos , Trombosis/metabolismoRESUMEN
Delayed rejection of pig kidney xenografts by primates is associated with vascular injury that may be accompanied by a form of consumptive coagulopathy in recipients. Using a life-supporting pig-to-baboon renal xenotransplantation model, we have tested the hypothesis that treatment with recombinant human antithrombin III would prevent or at least delay the onset of rejection and coagulopathy. Non-immunosuppressed baboons were transplanted with transgenic pig kidneys expressing the human complement regulators CD55 and CD59. Recipients were treated with an intravenous infusion of antithrombin III eight hourly (250 units per kg body weight), with or without low molecular weight heparin. Antithrombin-treated recipients had preservation of normal renal function for 4-5 days, which was twice as long as untreated animals, and developed neither thrombocytopenia nor significant coagulopathy during this period. Thus, recombinant antithrombin III may be a useful therapeutic agent to ameliorate both early graft damage and the development of systemic coagulation disorders in pig-to-human xenotransplantation.