RESUMEN
BACKGROUND: Xenografts from genetically modified pigs have become one of the most promising solutions to the dearth of human organs available for transplantation. The challenge in this model has been hyperacute rejection. To avoid this, pigs have been bred with a knockout of the alpha-1,3-galactosyltransferase gene and with subcapsular autologous thymic tissue. METHODS: We transplanted kidneys from these genetically modified pigs into two brain-dead human recipients whose circulatory and respiratory activity was maintained on ventilators for the duration of the study. We performed serial biopsies and monitored the urine output and kinetic estimated glomerular filtration rate (eGFR) to assess renal function and xenograft rejection. RESULTS: The xenograft in both recipients began to make urine within moments after reperfusion. Over the 54-hour study, the kinetic eGFR increased from 23 ml per minute per 1.73 m2 of body-surface area before transplantation to 62 ml per minute per 1.73 m2 after transplantation in Recipient 1 and from 55 to 109 ml per minute per 1.73 m2 in Recipient 2. In both recipients, the creatinine level, which had been at a steady state, decreased after implantation of the xenograft, from 1.97 to 0.82 mg per deciliter in Recipient 1 and from 1.10 to 0.57 mg per deciliter in Recipient 2. The transplanted kidneys remained pink and well-perfused, continuing to make urine throughout the study. Biopsies that were performed at 6, 24, 48, and 54 hours revealed no signs of hyperacute or antibody-mediated rejection. Hourly urine output with the xenograft was more than double the output with the native kidneys. CONCLUSIONS: Genetically modified kidney xenografts from pigs remained viable and functioning in brain-dead human recipients for 54 hours, without signs of hyperacute rejection. (Funded by Lung Biotechnology.).
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Rechazo de Injerto , Trasplante de Riñón , Trasplante Heterólogo , Animales , Animales Modificados Genéticamente/cirugía , Muerte Encefálica , Rechazo de Injerto/etiología , Rechazo de Injerto/patología , Rechazo de Injerto/prevención & control , Xenoinjertos/trasplante , Humanos , Riñón/patología , Riñón/fisiología , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/métodos , Porcinos/cirugía , Trasplante Heterólogo/efectos adversos , Trasplante Heterólogo/métodosRESUMEN
Thrombomodulin is important for the production of activated protein C (APC), a molecule with significant regulatory roles in coagulation and inflammation. To address known molecular incompatibilities between pig thrombomodulin and human thrombin that affect the conversion of protein C into APC, GalTKO.hCD46 pigs have been genetically modified to express human thrombomodulin (hTBM). The aim of this study was to evaluate the impact of transgenic hTBM expression on the coagulation dysregulation that is observed in association with lung xenograft injury in an established lung perfusion model, with and without additional blockade of nonphysiologic interactions between pig vWF and human GPIb axis. Expression of hTBM was variable between pigs at the transcriptional and protein level. hTBM increased the activation of human protein C and inhibited thrombosis in an in vitro flow perfusion assay, confirming that the expressed protein was functional. Decreased platelet activation was observed during ex vivo perfusion of GalTKO.hCD46 lungs expressing hTBM and, in conjunction with transgenic hTBM, blockade of the platelet GPIb receptor further inhibited platelets and increased survival time. Altogether, our data indicate that expression of transgenic hTBM partially addresses coagulation pathway dysregulation associated with pig lung xenograft injury and, in combination with vWF-GP1b-directed strategies, is a promising approach to improve the outcomes of lung xenotransplantation.
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Proteína C , Factor de von Willebrand , Animales , Porcinos , Humanos , Trasplante Heterólogo , Proteína C/metabolismo , Factor de von Willebrand/metabolismo , Células Endoteliales/metabolismo , Trombomodulina/genética , Animales Modificados Genéticamente/metabolismo , Pulmón/metabolismo , PerfusiónRESUMEN
INTRODUCTION: Expression of human complement pathway regulatory proteins (hCPRP's) such as CD46 or CD55 has been associated with improved survival of pig organ xenografts in multiple different models. Here we evaluate the hypothesis that an increased human CD46 gene dose, through homozygosity or additional expression of a second hCPRP, is associated with increased protein expression and with improved protection from injury when GTKO lung xenografts are perfused with human blood. METHODS: Twenty three GTKO lungs heterozygous for human CD46 (GTKO.heteroCD46), 10 lungs homozygous for hCD46 (GTKO.homoCD46), and six GTKO.homoCD46 lungs also heterozygous for hCD55 (GTKO.homoCD46.hCD55) were perfused with human blood for up to 4 h in an ex vivo circuit. RESULTS: Relative to GTKO.heteroCD46 (152 min, range 5-240; 6/23 surviving at 4 h), survival was significantly improved for GTKO.homoCD46 (>240 min, range 45-240, p = .034; 7/10 surviving at 4 h) or GTKO.homoCD46.hCD55 lungs (>240 min, p = .001; 6/6 surviving at 4 h). Homozygosity was associated with increased capillary expression of hCD46 (p < .0001). Increased hCD46 expression was associated with significantly prolonged lung survival (p = .048),) but surprisingly not with reduction in measured complement factor C3a. Hematocrit, monocyte count, and pulmonary vascular resistance were not significantly altered in association with increased hCD46 gene dose or protein expression. CONCLUSION: Genetic engineering approaches designed to augment hCPRP activity - increasing the expression of hCD46 through homozygosity or co-expressing hCD55 with hCD46 - were associated with prolonged GTKO lung xenograft survival. Increased expression of hCD46 was associated with reduced coagulation cascade activation, but did not further reduce complement activation relative to lungs with relatively low CD46 expression. We conclude that coagulation pathway dysregulation contributes to injury in GTKO pig lung xenografts perfused with human blood, and that the survival advantage for lungs with increased hCPRP expression is likely attributable to improved endothelial thromboregulation.
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Pulmón , Animales , Porcinos , Humanos , Animales Modificados Genéticamente , Trasplante Heterólogo , Xenoinjertos , PerfusiónRESUMEN
Galactosyl transferase knock-out pig lungs fail rapidly in baboons. Based on previously identified lung xenograft injury mechanisms, additional expression of human complement and coagulation pathway regulatory proteins, anti-inflammatory enzymes and self-recognition receptors, and knock-down of the ß4Gal xenoantigen were tested in various combinations. Transient life-supporting GalTKO.hCD46 lung function was consistently observed in association with either hEPCR (n = 15), hTBM (n = 4), or hEPCR.hTFPI (n = 11), but the loss of vascular barrier function in the xenograft and systemic inflammation in the recipient typically occurred within 24 h. Co-expression of hEPCR and hTBM (n = 11) and additionally blocking multiple pro-inflammatory innate and adaptive immune mechanisms was more consistently associated with survival >1 day, with one recipient surviving for 31 days. Combining targeted genetic modifications to the lung xenograft with selective innate and adaptive immune suppression enables prolonged initial life-supporting lung function and extends lung xenograft recipient survival, and illustrates residual barriers and candidate treatment strategies that may enable the clinical application of other organ xenografts.
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Supervivencia de Injerto , Pulmón , Animales , Animales Modificados Genéticamente , Rechazo de Injerto/tratamiento farmacológico , Humanos , Papio , Porcinos , Trasplante HeterólogoRESUMEN
INTRODUCTION: Platelet sequestration, inflammation, and inappropriate coagulation cascade activation are prominent in liver xenotransplant models and are associated with poor outcomes. Here, we evaluate a cassette of six additional genetic modifications to reduce anti-pig antibody binding (α-1,3-galactosyl transferase knockout [GalTKO]) and target coagulation dysregulation (human endothelial protein C receptor [hEPRC] and thrombomodulin [hTBM]), complement pathway regulation (human membrane cofactor protein, hCD46), inflammation heme oxygenase 1 [hHO-1]), and a self-recognition receptor (integrin-associated protein [hCD47]), as well as donor pharmacologic treatments designed to blunt these phenomena. METHODS: Livers from GaltKO.hCD46 pigs ("2-gene," n = 3) and GalTKO.hCD46 pigs also transgenic for hEPRC, hTBM, hCD47, and hHO-1 ("6-gene," n = 4) were perfused ex vivo with whole human blood. Six-gene pigs were additionally pretreated with desmopressin (DDAVP) and clodronate liposomes to deplete vWF and kupffer cells, respectively. RESULTS: The average perfusion times increased from 304 (±148) min in the 2-gene group to 856 (±61) min in the 6-gene group (p = .010). The average heparin administration was decreased from 8837 U/h in the 2-gene to 1354 U/h in the 6-gene group (p = .047). Platelet sequestration tended to be delayed in the 6-gene group (p = .070), while thromboxane B2 (TXB2, a platelet activation marker) levels were lower over the first hour (p = .044) (401 ± 124 vs. 2048 ± 712 at 60 min). Thrombin production as measured by F1+2 levels tended to be lower in the 6-gene group (p = .058). CONCLUSIONS: The combination of the hEPCR.hTBM.hCD47.hHO-1 cassette along with donor pig DDAVP and clodronate liposome pretreatment was associated with prolonged function of xenoperfused livers, reduced coagulation pathway perturbations, and decreased TXB2 elaboration, and reflects significant progress to modulate liver xenograft injury in a pig to human model.
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Desamino Arginina Vasopresina , Trombocitopenia , Animales , Animales Modificados Genéticamente , Ácido Clodrónico/farmacología , Supervivencia de Injerto , Hemo-Oxigenasa 1/genética , Humanos , Inflamación , Hígado , Perfusión , Porcinos , Trasplante HeterólogoRESUMEN
BACKGROUND: Loss of barrier function when GalTKO.hCD46 porcine lungs are perfused with human blood is associated with coagulation pathway dysregulation, innate immune system activation, and rapid sequestration of human formed blood elements. Here, we evaluate whether genetic expression of human tissue factor pathway inhibitor (hTFPI) and human CD47 (hCD47), alone or with combined selectin and integrin adhesion pathway inhibitors, delays GalTKO.hCD46 porcine lung injury or modulates neutrophil and platelet sequestration. METHODS: In a well-established paired ex vivo lung perfusion model, GalTKO.hCD46.hTFPI.hCD47 transgenic porcine lungs (hTFPI.hCD47, n = 7) were compared to GalTKO.hCD46 lungs (reference, n = 5). All lung donor pigs were treated with a thromboxane synthase inhibitor, anti-histamine, and anti-GPIb integrin-blocking Fab, and were pre-treated with Desmopressin. In both genotypes, one lung of each pair was additionally treated with PSGL-1 and GMI-1271 (P- and E-selectin) and IB4 (CD11b/18 integrin) adhesion inhibitors (n = 6 hTFPI.hCD47, n = 3 reference). RESULTS: All except for two reference lungs did not fail within 480 min when experiments were electively terminated. Selectin and integrin adhesion inhibitors moderately attenuated initial pulmonary vascular resistance (PVR) elevation in hTFPI.hCD47 lungs. Neutrophil sequestration was significantly delayed during the early time points following reperfusion and terminal platelet activation was attenuated in association with lungs expressing hTFPI.hCD47, but additional adhesion pathway inhibitors did not show further effects with either lung genotype. CONCLUSION: Expression of hTFPI.hCD47 on porcine lung may be useful as part of an integrated strategy to prevent neutrophil adhesion and platelet activation that are associated with xenograft injury. Additionally, targeting canonical selectin and integrin adhesion pathways reduced PVR elevation associated with hTFPI.hCD47 expression, but did not significantly attenuate neutrophil or platelet sequestration. We conclude that other adhesive mechanisms mediate the residual sequestration of human formed blood elements to pig endothelium that occurs even in the context of the multiple genetic modifications and drug treatments tested here.
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Antígeno CD47 , Trombocitopenia , Animales , Antígeno CD47/genética , Antígeno CD47/metabolismo , Supervivencia de Injerto , Humanos , Integrinas/metabolismo , Lipoproteínas , Pulmón/metabolismo , Perfusión , Selectinas/metabolismo , Porcinos , Trasplante HeterólogoRESUMEN
Platelet sequestration is a common process during organ reperfusion after transplantation. However, instead of lower platelet counts, when using traditional hemocytometers and light microscopy, we observed physiologically implausible platelet counts in the course of ex-vivo lung and liver xenograft organ perfusion studies. We employed conventional flow cytometry (FC) and imaging FC (AMINS ImageStream X) to investigate the findings and found platelet-sized fragments in the circulation that are mainly derived from red blood cell membranes. We speculate that this erythrocyte fragmentation contributes to anemia during in-vivo organ xenotransplant.
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Trombocitopenia , Animales , Eritrocitos , Xenoinjertos , Humanos , Perfusión , Porcinos , Trasplante Heterólogo/métodosRESUMEN
BACKGROUND: Antibody-mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3-galactose (α1,3Gal) epitope, N-Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre-formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene knock-out (Neu5GcKO) in a xenogeneic ex vivo perfusion model METHODS: Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6 h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1-thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n = 18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre-defined time points throughout the perfusion RESULTS: All but one Neu5GcKO organs maintained adequate blood oxygenation and "survived" until elective termination at 6 h whereas two reference lungs failed before elective termination at 4 h. Human anti-Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (â¼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p = .007; IgG p < .001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p = .007, .005, and .037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p = .023), whereas complement activation (as C3a rise in plasma) was not altered. MCP-1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p = .007 and .032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p = .003) CONCLUSION: Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody-mediated inflammation and activation of the coagulation cascade. Knock-out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients.
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Galactosiltransferasas , Histamina , Animales , Porcinos , Humanos , Trasplante Heterólogo , Animales Modificados Genéticamente , Galactosiltransferasas/genética , Ácido N-Acetilneuramínico , Supervivencia de Injerto , Inmunoglobulina G , Rechazo de InjertoRESUMEN
The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec-1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild-type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and "rosettes" (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near-complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation.
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Ácido N-Acetilneuramínico , Lectina 1 Similar a Ig de Unión al Ácido Siálico , Humanos , Porcinos , Animales , Lectina 1 Similar a Ig de Unión al Ácido Siálico/genética , Trasplante Heterólogo/métodos , Ácido N-Acetilneuramínico/metabolismo , Macrófagos , Eritrocitos/metabolismoRESUMEN
Consistent survival of life-supporting pig heart xenograft recipients beyond 90 days was recently reported using genetically modified pigs and a clinically applicable drug treatment regimen. If this remarkable achievement proves reproducible, published benchmarks for clinical translation of cardiac xenografts appear to be within reach. Key mechanistic insights are summarized here that informed recent pig design and therapeutic choices, which together appear likely to enable early clinical translation.
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Supervivencia de Injerto , Trasplante de Corazón , Corazón , Animales , Xenoinjertos , Humanos , PorcinosRESUMEN
The transplantation of organs across species offers the potential to solve the shortage of human organs. While activation of human platelets by human von Willebrand factor (vWF) requires vWF activation by shear stress, contact between human platelets and porcine vWF (pvWF) leads to spontaneous platelet adhesion and activation. This non-physiologic interaction may contribute to the thrombocytopenia and coagulation pathway dysregulation often associated with xenotransplantation of pig organs in nonhuman primates. Pigs genetically modified to decrease antibody and complement-dependent rejection (GTKO.hCD46) were engineered to express humanized pvWF (h*pvWF) by replacing a pvWF gene region that encodes the glycoprotein Ib-binding site with human cDNA orthologs. This modification corrected for non-physiologic human platelet aggregation on exposure to pig plasma, while preserving in vitro platelet activation by collagen. Organs from pigs with h*pvWF demonstrated reduced platelet sequestration during lung (p ≤ .01) and liver (p ≤ .038 within 4 h) perfusion ex vivo with human blood and after pig-to-baboon lung transplantation (p ≤ .007). Residual platelet sequestration and activation were not prevented by the blockade of canonical platelet adhesion pathways. The h*pvWF modification prevents physiologically inappropriate activation of human or baboon platelets by porcine vWF, addressing one cause of the thrombocytopenia and platelet activation observed with xenotransplantation.
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Trombocitopenia , Factor de von Willebrand , Animales , Plaquetas , Agregación Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria , Porcinos , Trasplante HeterólogoRESUMEN
Cardiac xenotransplantation has recently taken an important step towards clinical reality. In anticipation of the "first-in-human" heart xenotransplantation trial, we propose a set of patient characteristics that define potential candidates. Our premise is that, to be ethically justified, the risks posed by current state-of-the-art options must outweigh the anticipated risks of a pioneering xenotransplant procedure. Suitable candidates include patients who are at high immunologic risk because of sensitization to alloantigens, including those who have exhibited early onset or accelerated cardiac allograft vasculopathy. In addition, patients should be considered (1) for whom mechanical circulatory support would be prohibitively risky due to a hypercoagulable state, a contraindication to anticoagulation, or restrictive physiology; (2) with severe biventricular dysfunction predicting unsuccessful univentricular left heart support; and (3) adults with complex congenital heart disease. In conclusion, because the published preclinical benchmark for clinical translation of heart xenotransplantation appears within reach, carefully and deliberately defining appropriate trial participants is timely as the basis for ethical clinical trial design.
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Cardiopatías , Trasplante de Corazón , Adulto , Animales , Contraindicaciones , Humanos , Complicaciones Posoperatorias , Porcinos , Trasplante HeterólogoRESUMEN
BACKGROUND: Elevated pulmonary vascular resistance (PVR), platelet adhesion, coagulation activation, and inflammation are prominent features of xenolung rejection. Here, we evaluate the role of thromboxane and histamine on PVR, and their contribution to other lung xenograft injury mechanisms. METHODS: GalTKO.hCD46 single pig lungs were perfused ex vivo with fresh heparinized human blood: lungs were either treated with 1-Benzylimidazole (1-BIA) combined with histamine receptor blocker famotidine (n = 4) or diphenhydramine (n = 6), 1-BIA alone (n = 6) or were left untreated (n = 9). RESULTS: Six of the nine control experiments (GalTKO.hCD46 untreated), "survived" until elective termination at 4 hours. Without treatment, initial PVR elevation within the first 30 minutes resolved partially over the following hour, and increased progressively during the final 2 hours of perfusion. In contrast, 1-BIA, alone or in addition to either antihistamine treatment, was associated with low stable PVR. Combined treatments significantly lowered the airway pressure when compared to untreated reference. Although platelet and neutrophil sequestration and coagulation cascade activation were not consistently altered by any intervention, increased terminal wet/dry weight ratio in untreated lungs was significantly blunted by combined treatments. CONCLUSION: Combined thromboxane and histamine pathway blockade prevents PVR elevation and significantly inhibits loss of vascular barrier function when GalTKO.hCD46 lungs are perfused with human blood. Platelet activation and platelet and neutrophil sequestration persist in all groups despite efficient complement regulation, and appear to occur independent of thromboxane and histamine antagonism. Our work identifies thromboxane and histamine as key mediators of xenolung injury and defines those pathways as therapeutic targets to achieve successful xenolung transplantation.
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Supervivencia de Injerto/fisiología , Xenoinjertos/inmunología , Histamina/farmacología , Resistencia Vascular , Animales , Animales Modificados Genéticamente , Plaquetas/inmunología , Humanos , Pulmón/metabolismo , Trasplante de Pulmón/métodos , Porcinos , Trasplante Heterólogo/métodos , Resistencia Vascular/fisiologíaRESUMEN
BACKGROUND: Alongside the need to develop more effective and less toxic immunosuppression, the shortage of human organs available for organ transplantation is one of the major hurdles facing the field. Research into xenotransplantation, as an alternative source of organs, has unveiled formidable challenges. Porcine lungs perfused with human blood rapidly sequester the majority of circulating neutrophils and platelets, which leads to inflammation and organ failure within hours, and is not significantly attenuated by genetic modifications to the pig targeted to diminish antibody binding and complement and coagulation cascade activation. METHODS: Here, we model the interaction of freshly isolated human leukocytes with xenotransplanted vasculature under physiologic flow conditions using microfluidic channels coated with porcine endothelial cells. Both isolated human neutrophils and whole human blood were perfused over transgenic pig aortic endothelial cells that had been activated with rhTNF-α or rhIL-4 using the BioFlux system. Novel compounds GMI-1271 and rPSGL1.Fc were tested as E- and P- selectin antagonists, respectively. Cellular adhesion and rolling events were tracked using FIJI (imageJ). RESULTS: Porcine endothelium activated with either rhTNF-α or rhIL-4 expressed high amounts of selectins, to which isolated human neutrophils readily rolled and tethered. Both E-and P-selectin antagonism significantly reduced the number of neutrophils rolling and rolling distance in a dose-dependent manner, with near total inhibition at higher doses (P < .001). Similarly, with whole human blood, selectin blocking compounds exhibited dose-dependent inhibition of prevalent leukocyte adhesion and severe endothelial injury (Untreated: 394 ± 97 PMNs/hpf, 57 ± 6% loss EC; GMI1271+rPSGL1.Fc: 23 ± 9 PMNs/hpf, 8 ± 6% loss EC P < .01). CONCLUSIONS: Selectin blockade may be useful as part of an integrated strategy to prevent neutrophil-mediated organ xenograft injury, especially during the early time points following reperfusion.
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Selectina E/metabolismo , Células Endoteliales/inmunología , Leucocitos/inmunología , Selectina-P/metabolismo , Animales , Animales Modificados Genéticamente , Adhesión Celular/fisiología , Humanos , Neutrófilos/inmunología , Porcinos , Trasplante Heterólogo/métodosRESUMEN
BACKGROUND: Human neutrophils are sequestered by pig lung xenografts within minutes during ex vivo perfusion. This phenomenon is not prevented by pig genetic modifications that remove xeno-antigens or added human regulatory molecules intended to down-regulate activation of complement and coagulation pathways. This study investigated whether recipient and donor interleukin-8 (IL-8), a chemokine known to attract and activate neutrophils during inflammation, is elaborated in the context of xenogeneic injury, and whether human or pig IL-8 promote the adhesion of human neutrophils in in vitro xenograft models. METHODS: Plasma levels of pig, human or non-human primate (NHP) IL-8 from ex vivo pig lung perfusion experiments (n = 10) and in vivo pig-to-baboon lung transplantation in baboons (n = 22) were analysed by ELISA or Luminex. Human neutrophils stimulated with human or pig IL-8 were analysed for CD11b expression, CD18 activation, oxidative burst and adhesion to resting or TNF-activated endothelial cells (EC) evaluated under static and flow (Bioflux) conditions. For some experiments, human neutrophils were incubated with Reparixin (IL-8/CXCL8 receptor blocker) and then analysed as in the in vitro experiments mentioned above. RESULTS: Plasma levels of pig IL-8 (~6113 pg/mL) increased more than human (~1235 pg/mL) between one and four hours after initiation of ex vivo lung perfusion. However, pig IL-8 levels remained consistently low (<60 pg/mL) and NHP IL-8 plasma levels increased by ~2000 pg/mL after four hours in a pig-to-baboon lung xenotransplantation. In vitro, human neutrophils' CD11b expression, CD18 activation and oxidative burst all increased in a dose-dependent manner following exposure to either pig or human IL-8, which also were associated with increased adhesion to EC in both static and flow conditions. Reparixin inhibited human neutrophil activation by both pig and human IL-8 in a dose-dependent fashion. At 0.1 mg/mL, Reparixin inhibited the adhesion of IL-8-activated human neutrophils to pAECs by 84 ± 2.5%. CONCLUSIONS: Pig IL-8 increased in an ex vivo model of pig-to-human lung xenotransplantation but is not detected in vivo, whereas human or NHP IL-8 is elevated to a similar degree in both models. Both pig and human IL-8 activate human neutrophils and increase their adhesion to pig aortic ECs, a process significantly inhibited by the addition of Reparixin to human neutrophils. This work implicates IL-8, whether of pig or human origin, as a possible factor mediating in lung xenograft inflammation and injury and supports the evaluation of therapeutic targeting of this pathway in the context of xenotransplantation.
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Células Endoteliales/inmunología , Xenoinjertos/metabolismo , Interleucina-8/metabolismo , Neutrófilos/inmunología , Trasplante Heterólogo , Animales , Quimiocinas/metabolismo , Humanos , Inflamación/inmunología , Papio , PorcinosRESUMEN
PURPOSE OF REVIEW: Recent progress in genetic engineering has facilitated development of transgenic donor animals designed to overcome the known barriers to discordant xenotransplantation, and greatly accelerated progress in the field of xenotransplantation. Here we review and summarize recent progress in lung xenotransplantation, and discuss possible additional genetic modifications and other interventions that may further advance the use of pulmonary xenografts towards clinical applications based on known mechanisms of xeno lung injury. RECENT FINDINGS: Ex-vivo lung perfusion experiments have shown that the addition of human complement (hCD46, hCD55), coagulation (hEPCR, hVWF, hTBM, hTFPI, hCD39), or anti-inflammatory pathway regulatory genes (HO-1, HLA-E), and the knockout (KO) of major porcine carbohydrates (GalT, Neu5Gc, B4Gal) have each protective effects on lung survival and function. The use of these transgenes in multitransgenic donor organs, targeting several known xenogeneic rejection mechanisms, combined with drug treatments addressing remaining known rejection pathways, have led to prolonged recipient survival of up to 31 days with in some cases preserved live-supporting organ function of the transplanted graft for several days. Pulmonary vascular resistance elevation, which has been found to be associated with high thromboxane levels and has been the major failure reason of xenogeneic lung grafts in the past years, has been successfully attenuated by the addition of a thromboxane synthase inhibitor (1-Benzylimidazole). Currently, the predominant failure mechanism of xenogeneic lung grafts is an inflammatory process, leading to vascular barrier function injury with interstitial and trachea edema. Work with other pig organs in primate models show that regimens based on costimulatory pathway blocking antibodies prolong xenograft function for months to years, suggesting that once initial lung inflammation mechanisms are fully controlled, clinically useful application of pig lung xenografts may be feasible. SUMMARY: The use of multitransgenic donor pigs coupled with drugs targeting complement activation, coagulation, and inflammation have significantly improved the survival of xenogeneic pig lungs both during ex vivo human blood perfusion and in life-supporting in vivo models, and for the first time allowed consistent life-supporting function of lung xenografts. Overcoming delayed loss of vascular barrier function injury appears to be within reach, and will be essential to make lung xenografts a clinically relevant treatment option.
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Trasplante de Pulmón/métodos , Trasplante Heterólogo/métodos , Animales , Humanos , PorcinosRESUMEN
BACKGROUND: Lung xenografts remain susceptible to loss of vascular barrier function within hours in spite of significant incremental advances based on genetic engineering to remove the Gal 1,3-αGal antigen (GalTKO) and express human membrane cofactor protein (hCD46). Natural killer cells rapidly disappear from the blood during perfusion of GalTKO.hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature. Here we asked whether porcine expression of the human NK cell inhibitory ligand HLA-E and ß2 microglobulin inhibits GalTKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models. METHODS: Lungs from pigs modified to express GalTKO.hCD46 (n=37) and GalTKO.hCD46.HLA-E (n=5) were harvested and perfused with human blood until failure or elective termination at 4 hours. Airway pressures and pulmonary artery hemodynamics were recorded in real time. Blood samples were also collected throughout the experiment for analysis. Porcine aortic endothelial cells (PAECs) from each genotype were cultured in monolayers in microfluidic channels and used in fluorescent cytotoxicity assays using human NK cells. RESULTS: HLA-E expression on GalTKO.hCD46 PAECs was associated with significantly decreased antibody-dependent and antibody-independent NK-mediated cytotoxicity under in vitro conditions simulating physiologic shear stress. Relative to GalTKO.hCD46 pig lungs perfused with human blood on an ex vivo platform, additional expression of HLA-E increased median lung survival (>4 hours, vs 162 minutes, P=.012), and was associated with attenuated rise in pulmonary vascular resistance, and decreased platelet activation and histamine elaboration. As expected, HLA-E expression was not associated with a significant difference in NK cell adhesion to endothelial cells in vitro, or NK cell and neutrophil sequestration during organ perfusion. CONCLUSIONS: We conclude human NK cell activation contributes significantly to GalTKO.hCD46 pig endothelial injury and lung inflammation and show that expression of HLA-E is associated with physiologically meaningful protection of GalTKO.hCD46 cells and organs exposed to human blood.
Asunto(s)
Células Endoteliales/inmunología , Supervivencia de Injerto/inmunología , Antígenos HLA/inmunología , Xenoinjertos/inmunología , Leucocitos/inmunología , Lesión Pulmonar/terapia , Proteína Cofactora de Membrana/inmunología , Animales , Animales Modificados Genéticamente , Citotoxicidad Inmunológica/inmunología , Galactosiltransferasas/genética , Supervivencia de Injerto/genética , Antígenos HLA/genética , Humanos , Células Asesinas Naturales/inmunología , Lesión Pulmonar/inmunología , Proteína Cofactora de Membrana/genética , Porcinos , Trasplante Heterólogo/métodosRESUMEN
BACKGROUND: Wild-type pigs express several carbohydrate moieties on their cell surfaces that differ from those expressed by humans. This difference in profile leads to pig tissue cell recognition of human blood cells causing sequestration, in addition to antibody-mediated xenograft injury. One such carbohydrate is N-glycolylneuraminic acid (Neu5Gc), a sialic acid molecule synthesized in pigs but not in humans. Here, we evaluate livers with and without Neu5Gc in an ex vivo liver xeno perfusion model. METHODS: Livers from pigs with an α1,3-galactosyl transferase gene knockout (GalTKO) and transgenic for human membrane cofactor (hCD46) with (n = 5) or without (n = 7) an additional Neu5Gc gene knock out (Neu5GcKO) were perfused ex vivo with heparinized whole human blood. A drug regimen consisting of a histamine inhibitor, thromboxane synthase inhibitor, and a murine anti-human GPIb-blocking antibody fragment was given to half of the experiments in each group. RESULTS: Liver function tests (AST and ALT) were not significantly different between livers with and without the Neu5GcKO. GalTKO.hCD46.Neu5GcKO livers had less erythrocyte sequestration as evidenced by a higher mean hematocrit over time compared to GalTKO.hCD46 livers (P = .0003). The addition of Neu5GcKO did not ameliorate profound thrombocytopenia seen within the first 15 minutes of perfusion. TXB2 was significantly less with the added drug regimen (P = .006) or the presence of Neu5GcKO (P = .017). CONCLUSIONS: The lack of Neu5Gc expression attenuated erythrocyte loss but did not prevent profound early onset thrombocytopenia or platelet activation, although TXB2 levels were decreased in the presence of Neu5GcKO.
Asunto(s)
Galactosiltransferasas/genética , Xenoinjertos/efectos de los fármacos , Ácidos Neuramínicos/farmacología , Trasplante Heterólogo , Animales , Animales Modificados Genéticamente , Técnicas de Inactivación de Genes/métodos , Supervivencia de Injerto/inmunología , Humanos , Proteína Cofactora de Membrana/genética , Porcinos , Trombocitopenia/terapiaRESUMEN
PURPOSE OF REVIEW: This article reviews recent progress in the field of lung xenotransplantation, including mechanisms of xenograft injury, and the influence of mechanism-directed genetic modifications and other interventions that may soon enable therapeutic use of pig lungs in humans. RECENT FINDINGS: An extensive series of lung xenotransplantation experiments demonstrates that multiple genetic modifications targeting known xenogeneic lung injury mechanisms are associated with incremental improvements in lung survival or function. Addition of human complement (hCD46, hCD55), coagulation (hEPCR, hTBM, hTFPI, hCD39), or anti-inflammatory pathway regulatory genes (HO-1, HLA-E), and GalT and Neu5Gc gene knockout has each demonstrated protective effects on lung survival or function. In addition, drug treatments targeting key inflammatory and clotting pathways have been shown to attenuate residual mechanisms of lung injury. Work with other pig organs in primate models show that regimens based on costimulatory pathway blocking antibodies prolong xenograft function for months to years, suggesting that once initial lung inflammation mechanisms are fully controlled, clinically useful application of pig lung xenografts may soon be feasible. SUMMARY: Genetic modification of pigs coupled with drugs targeting complement activation, coagulation, and inflammation have significantly increased duration of pig lung function in ex-vivo human blood perfusion models, and life-supporting lung xenograft survival in vivo.
Asunto(s)
Trasplante de Pulmón/métodos , Trasplante Heterólogo/métodos , Animales , Animales Modificados Genéticamente , Humanos , PorcinosRESUMEN
BACKGROUND: Genetically modified pigs are a promising potential source of lung xenografts. Ex vivo xenoperfusion is an effective platform for testing the effect of new modifications, but typical experiments are limited by testing of a single genetic intervention and small sample sizes. The purpose of this study was to analyze the individual and aggregate effects of donor genetic modifications on porcine lung xenograft survival and injury in an extensive pig lung xenoperfusion series. METHODS: Data from 157 porcine lung xenoperfusion experiments using otherwise unmodified heparinized human blood were aggregated as either continuous or dichotomous variables. Lungs were wild type in 17 perfusions (11% of the study group), while 31 lungs (20% of the study group) had one genetic modification, 40 lungs (39%) had 2, and 47 lungs (30%) had 3 or more modifications. The primary endpoint was functional lung survival to 4 h of perfusion. Secondary analyses evaluated previously identified markers associated with known lung xenograft injury mechanisms. In addition to comparison among all xenografts grouped by survival status, a subgroup analysis was performed of lungs incorporating the GalTKO.hCD46 genotype. RESULTS: Each increase in the number of genetic modifications was associated with additional prolongation of lung xenograft survival. Lungs that exhibited survival to 4 h generally had reduced platelet activation and thrombin generation. GalTKO and the expression of hCD46, HO-1, hCD55, or hEPCR were associated with improved survival. hTBM, HLA-E, and hCD39 were associated with no significant effect on the primary outcome. CONCLUSION: This meta-analysis of an extensive lung xenotransplantation series demonstrates that increasing the number of genetic modifications targeting known xenogeneic lung injury mechanisms is associated with incremental improvements in lung survival. While more detailed mechanistic studies are needed to explore the relationship between gene expression and pathway-specific injury and explore why some genes apparently exhibit neutral (hTBM, HLA-E) or inconclusive (CD39) effects, GalTKO, hCD46, HO-1, hCD55, and hEPCR modifications were associated with significant lung xenograft protection. This analysis supports the hypothesis that multiple genetic modifications targeting different known mechanisms of xenograft injury will be required to optimize lung xenograft survival.