Expression of human thrombomodulin by GalTKO.hCD46 pigs modulates coagulation cascade activation by endothelial cells and during ex vivo lung perfusion with human blood.

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.

Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex-vivo perfusion of GTKO pig lungs with human blood.

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.

Knock-out of N-glycolylneuraminic acid attenuates antibody-mediated rejection in xenogenically perfused porcine lungs.

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 (∼40% 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.

Genetic modifications designed for xenotransplantation attenuate sialoadhesin-dependent binding of human erythrocytes to porcine macrophages.

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.

Human erythrocyte fragmentation during ex-vivo pig organ perfusion.

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.

hEPCR.hTBM.hCD47.hHO-1 with donor clodronate and DDAVP treatment improves perfusion and function of GalTKO.hCD46 porcine livers perfused with human blood.

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.

Pig-to-baboon lung xenotransplantation: Extended survival with targeted genetic modifications and pharmacologic treatments.

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.

Humanized von Willebrand factor reduces platelet sequestration in ex vivo and in vivo xenotransplant models.

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.

Progress Toward Cardiac Xenotransplantation.

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.

Xenogeneic Lung Transplantation Models.

Study of lung xenografts has proven useful to understand the remaining barriers to successful transplantation of other organ xenografts. In this chapter, the history and current status of lung xenotransplantation will be briefly reviewed, and two different experimental models, the ex vivo porcine-to-human lung perfusion and the in vivo xenogeneic lung transplantation, will be presented. We will focus on the technical details of these lung xenograft models in sufficient detail, list the needed materials, and mention analysis techniques to allow others to adopt them with minimal learning curve.

Multi-gene technical assessment of qPCR and NanoString n-Counter analysis platforms in cynomolgus monkey cardiac allograft recipients.

Quantitative gene expression profiling of cardiac allografts characterizes the phenotype of the alloimmune response, yields information regarding differential effects that may be associated with various anti-rejection drug regimens, and generates testable hypotheses regarding the pathogenesis of the chronic rejection lesions typically observed in non-human primate heart transplant models. The goal of this study was to assess interplatform performance and variability between the relatively novel NanoString nCounter Analysis System, ΔΔCT (relative) RT-qPCR, and standard curve (absolute) RT-qPCR utilizing cynomolgus monkey cardiac allografts. Methods for RNA isolation and preamplification were also systematically evaluated and effective methods are proposed. In this study, we demonstrate strong correlation between the two RT-qPCR methods, but variable and, at times, weak correlation between RT-qPCR and NanoString. NanoString fold change results demonstrate less sensitivity to small changes in gene expression than RT-qPCR. These findings appear to be driven by technical aspects of each platform that influence the conditions under which each technique is ideal. Collectively, our data contribute to the general effort to optimally utilize gene expression profiling techniques, not only for transplanted tissues, but for many other applications where accurate rank-order of gene expression versus precise quantification of absolute gene transcript number may be relatively valuable.

Thromboxane and histamine mediate PVR elevation during xenogeneic pig lung perfusion with human blood.

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.

Progress and challenges in lung xenotransplantation: an update.

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.

Pilot Study of Delayed ICOS/ICOS-L Blockade With αCD40 to Modulate Pathogenic Alloimmunity in a Primate Cardiac Allograft Model.

BACKGROUND: Inducible costimulator (ICOS) is rapidly upregulated with T-cell stimulation and may represent an escape pathway for T-cell costimulation in the setting of CD40/CD154 costimulation blockade. Induction treatment exhibited no efficacy in a primate renal allograft model, but rodent transplant models suggest that the addition of delayed ICOS/ICOS-L blockade may prolong allograft survival and prevent chronic rejection. Here, we ask whether ICOS-Ig treatment, timed to anticipate ICOS upregulation, prolongs NHP cardiac allograft survival or attenuates pathogenic alloimmunity. METHODS: Cynomolgus monkey heterotopic cardiac allograft recipients were treated with αCD40 (2C10R4, d0-90) either alone or with the addition of delayed ICOS-Ig (d63-110). RESULTS: Median allograft survival was similar between ICOS-Ig + αCD40 (120 days, 120-125 days) and αCD40 (124 days, 89-178 days) treated animals, and delayed ICOS-Ig treatment did not prevent allograft rejection in animals with complete CD40 receptor coverage. Although CD4+ TEM cells were decreased in peripheral blood (115 ± 24) and mLNs (49 ± 1.9%) during ICOS-Ig treatment compared with monotherapy (214 ± 27%, P = 0.01; 72 ± 9.9%, P = 0.01, respectively), acute and chronic rejection scores and kinetics of alloAb elaboration were similar between groups. CONCLUSIONS: Delayed ICOS-Ig treatment with the reagent tested is probably ineffective in modulating pathogenic primate alloimmunity in this model.

Interleukin-8 mediates neutrophil-endothelial interactions in pig-to-human xenogeneic models.

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.

Selective CD28 Inhibition Modulates Alloimmunity and Cardiac Allograft Vasculopathy in Anti-CD154-Treated Monkeys.

BACKGROUND: Selective CD28 inhibition is actively pursued as an alternative to B7 blockade using cytotoxic T lymphocyte antigen 4 Ig based on the hypothesis that the checkpoint immune regulators cytotoxic T lymphocyte antigen 4 and programmed death ligand 1 will induce tolerogenic immune signals. We previously showed that blocking CD28 using a monovalent nonactivating reagent (single-chain anti-CD28 Fv fragment linked to alpha-1 antitrypsin [sc28AT]) synergizes with calcineurin inhibitors in nonhuman primate (NHP) kidney and heart transplantation. Here, we explored the efficacy of combining a 3-week "induction" sc28AT treatment with prolonged CD154 blockade. METHODS: Cynomolgus monkey heterotopic cardiac allograft recipients received sc28AT (10 mg/kg, d0-20, n = 3), hu5C8 (10-30 mg/kg, d0-84, n = 4), or combination (n = 6). Graft survival was monitored by telemetry. Protocol biopsies and graft explants were analyzed for International Society of Heart and Lung Transplantation acute rejection grade and cardiac allograft vasculopathy score. Alloantibody, T-cell phenotype and regulatory T cells were analyzed by flow cytometry. Immunochemistry and gene expression (NanoString) characterized intra-graft cellular infiltration. RESULTS: Relative to modest prolongation of median graft survival time with sc28AT alone (34 days), hu5C8 (133 days), and sc28AT + hu5C8 (141 days) prolonged survival to a similar extent. CD28 blockade at induction, added to hu5C8, significantly attenuated the severity of acute rejection and cardiac allograft vasculopathy during the first 3 months after transplantation relative to hu5C8 alone. These findings were associated with decreased proportions of circulating CD8 and CD3CD28 T cells, and modulation of inflammatory gene expression within allografts. CONCLUSIONS: Induction with sc28AT promotes early cardiac allograft protection in hu5C8-treated NHPs. These results support further investigation of prolonged selective CD28 inhibition with CD40/CD154 blockade in NHP transplants.

P- and E-selectin receptor antagonism prevents human leukocyte adhesion to activated porcine endothelial monolayers and attenuates porcine endothelial damage.

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.

Synthetic liver function is detectable in transgenic porcine livers perfused with human blood.

In addition to immune barriers, molecular incompatibilities between species are predicted to limit pig liver survival in primate xenotransplantation models. Assessment and measurement of synthetic function of genetically modified porcine livers after ex vivo perfusion with human blood have not previously been described. Eight porcine livers from α1,3-galactosyl transferase knockout and human membrane cofactor (GalTKO.hCD46), six livers from GalTKO.hCD46 and N-glycolylneuraminic acid knockout (GalTKO.hCD46.Neu5GcKO), and six livers from GalTKO.hCD46 with humanized decay-accelerating factor (hCD55), endothelial protein C receptor (hEPCR), tissue factor pathway inhibitor (hTFPI), and integrin-associated protein (hCD47) (GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47) pigs were perfused with human blood under physiologic conditions. Timed blood samples were tested for liver enzymes and for pig-specific albumin production via Western blot. Porcine albumin levels increased with time in all experiments. By densitometry, GalTKO.hCD46.Neu5GcKO livers had the highest albumin levels, measured both as total produced, and when controlled for perfusion duration, compared to GalTKO.hCD46 (P = .068) and GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47 livers (P = .04). Porcine livers perfused with human blood demonstrated the synthetic ability to produce albumin in all cases. GalTKO.hCD46.Neu5GcKO pig livers demonstrated the most robust albumin production. This suggests that the Neu5GcKO phenotype provides a protective effect on the graft due to decreased human antibody recognition and graft injury.

The role of sialic acids in the immune recognition of xenografts.

Presentation of sialic acid (Sia) varies among different tissues and organs within each species, and between species. This diversity has biologically important consequences regarding the recognition of cells by "xeno" antibodies (Neu5Gc vs Neu5Ac). Sia also plays a central role in inflammation by influencing binding of the asialoglycoprotein receptor 1 (ASGR-1), Siglec-1 (Sialoadhesin), and cellular interactions mediated by the selectin, integrin, and galectin receptor families. This review will focus on what is known about basic Sia structure and function in association with xenotransplantation, how changes in sialylation may occur in this context (through desialylation or changes in sialyltransferases), and how this fundamental pathway modulates adhesive and cell activation pathways that appear to be particularly crucial to homeostasis and inflammation for xenografts.

N-glycolylneuraminic acid knockout reduces erythrocyte sequestration and thromboxane elaboration in an ex vivo pig-to-human xenoperfusion model.

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.