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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., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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 β4Gal 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., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

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., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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., (Published by Elsevier Inc.)

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.

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., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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 + T EM 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., Competing Interests: The authors declare no conflicts of interest.

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., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Vascularized composite allotransplantation is constrained by complications associated with standard immunosuppressive strategies. Vascularized thymus and bone marrow have been shown to promote prolonged graft survival in composite organ and soft-tissue vascularized composite allotransplantation models. We report development of a nonhuman primate vascularized thymosternal composite tissue transplant model as a platform to address donor-specific immune tolerance induction strategies., Methods: Vascularized thymosternal allograft (skin, muscle, thymus, sternal bone) was transplanted between MHC-mismatched rhesus monkeys (feasibility studies) and baboons (long-term survival studies), with end-to-side anastomoses of the donor aorta and SVC to the recipient common femoral vessels. A male allograft was transplanted to a female's lower abdominal wall, and clinically applicable immunosuppression was given. Skin biopsies and immunological assays were completed at regular intervals, and chimerism was quantified using polymerase chain reaction specific for baboon Y chromosome., Results: Four allo- and 2 xenotransplants were performed, demonstrating consistent technical feasibility. In 1 baboon thymosternal allograft recipient treated with anti-CD40-based immunosuppression, loss of peripheral blood microchimerism after day 5 was observed and anticipated graft rejection at 13 days. In the second allograft, when cutaneous erythema and ecchymosis with allograft swelling was treated with anti-thymocyte globulin starting on day 6, microchimerism persisted until immunosuppression was reduced after the first month, and the allograft survived to 87 days, 1 month after cessation of immunosuppression treatment., Conclusions: We established both allo- and xeno- composite vascularized thymosternal transplant preclinical models, which will be useful to investigate the role of primarily vascularized donor bone marrow and thymus.

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., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Specific blockade of T cell costimulation pathway is a promising immunomodulatory approach being developed to replace our current clinical immunosuppression therapies. The goal of this study is to compare results associated with 3 monoclonal antibodies directed against the CD40/CD154 T cell costimulation pathway., Methods: Cynomolgus monkey heterotopic cardiac allograft recipients were treated with either IDEC-131 (humanized αCD154, n = 9), 5C8H1 (mouse-human chimeric αCD154, n = 5), or 2C10R4 (mouse-rhesus chimeric αCD40, n = 6) monotherapy using a consistent, comparable dosing regimen for 3 months after transplant., Results: Relative to the previously reported IDEC-131-treated allografts, median survival time (35 ± 31 days) was significantly prolonged in both 5C8H1-treated (142 ± 26, P < 0.002) and 2C10R4-treated (124 ± 37, P < 0.020) allografts. IDEC-131-treated grafts had higher cardiac allograft vasculopathy severity scores during treatment relative to either 5C8H1 (P = 0.008) or 2C10R4 (P = 0.0002). Both 5C8H1 (5 of 5 animals, P = 0.02) and 2C10R4 (6/6, P = 0.007), but not IDEC-131 (2/9), completely attenuated IgM antidonor alloantibody (alloAb) production during treatment; 5C8H1 (5/5) more consistently attenuated IgG alloAb production compared to 2C10R4 (4/6) and IDEC-131 (0/9). All evaluable explanted grafts experienced antibody-mediated rejection. Only 2C10R4-treated animals exhibited a modest, transient drop in CD20 lymphocytes from baseline at day 14 after transplant (-457 ± 152 cells/μL) compared with 5C8H1-treated animals (16 ± 25, P = 0.037), and the resurgent B cells were primarily of a naive phenotype., Conclusions: In this model, CD154/CD40 axis blockade using IDEC-131 is an inferior immunomodulatory treatment than 5C8H1 or 2C10R4, which have similar efficacy to prolong graft survival and to delay cardiac allograft vasculopathy development and antidonor alloAb production during treatment.

The continual critical shortage of organs and cells from deceased human donors has stimulated research in the field of cross-species transplantation (xenotransplantation), with the pig selected as the most suitable potential source of organs. Since the US Food and Drug Administration concluded a comprehensive review of xenotransplantation in 2003, considerable progress has been made in the experimental laboratory to improve cell and organ xenograft survival in several pig-to-nonhuman primate systems that offer the best available models to predict clinical outcomes. Survival of heart, kidney, and islet grafts in nonhuman primates is now being measured in months or even years. The potential risks associated with xenotransplantation, for example, the transfer of an infectious microorganism, that were highlighted in the 2003 Food and Drug Administration guidance and subsequent World Health Organization consensus documents have been carefully studied and shown to be either less likely than previously thought or readily manageable by donor selection or recipient management strategies. In this context, we suggest that the national regulatory authorities worldwide should re-examine their guidelines and regulations regarding xenotransplantation, so as to better enable design and conduct of safe and informative clinical trials of cell and organ xenotransplantation when and as supported by the preclinical data. We identify specific topics that we suggest require reconsideration.

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 β2 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., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Anti-CD154 monotherapy is associated with antidonor allo-antibody (Ab) elaboration, cardiac allograft vasculopathy (CAV), and allograft failure in preclinical primate cell and organ transplant models. In the context of calcineurin inhibitors (CNI), these pathogenic phenomena are delayed by preemptive "induction" B cell depletion., Methods: αCD154 (IDEC-131)-treated cynomolgus monkey heart allograft recipients were given peritransplant rituximab (αCD20) alone or with rabbit antihuman thymocyte globulin., Results: Relative to previously reported reference groups, αCD20 significantly prolonged survival, delayed Ab detection, and attenuated CAV within 3 months in αCD154-treated recipients (αCD154 + αCD20 graft median survival time > 90 days, n = 7, vs 28 days for αCD154 alone (IDEC-131), n = 21; P = 0.05). Addition of rabbit antihuman thymocyte globulin to αCD154 (n = 6) or αCD154 + αCD20 (n = 10) improved graft protection from graft rejection and failure during treatment but was associated with significant morbidity in 8 of 16 recipients (6 infections, 2 drug-related complications). In αCD20-treated animals, detection of antidonor Ab and relatively severe CAV were anticipated by appearance of CD20 cells (>1% of lymphocytes) in peripheral blood and were associated with low αCD154 trough levels (below 100 μg/mL)., Conclusions: These observations support the hypothesis that efficient preemptive "induction" CD20 B cell depletion consistently modulates pathogenic alloimmunity and attenuates CAV in this translational model, extending our prior findings with calcineurin inhibitors to the context of CD154 blockade., Competing Interests: The authors declare no conflicts of interest.

A cynomolgus macaque received a heterotopic cardiac allograft as part of a transplant study, with monoclonal antibodies targeted to specific immune costimulation molecules (CD154, CD28) but no traditional immunosuppressive therapy after surgery. Clinical anemia was detected on postoperative day (POD) 35 and had worsened (Hgb, 2.3 g/dL; Hct = 7.3%) by POD 47, despite type-matched whole-blood transfusions. After a total of 4 blood transfusions, hematologic parameters were improved (Hgb, 5.9 g/dL; Hct, 18.7%). On POD 50, a peripheral blood smear revealed trypomastigotes, and qualitative RT-PCR of whole blood identified the organism as Trypanosoma cruzi. Although clinically stable initially, the macaque soon developed sufficient weight loss to necessitate euthanasia on POD 64. The final diagnosis was clinical anemia due to T. cruzi infection. This study represents the first reported case of Chagas disease after heart transplant in a NHP.

Human organ transplantation has improved duration and quality of life for many people, but its full potential is critically limited by short supply of available organs. One solution is xenotransplantation, although this comes with its own set of challenges. Lungs in particular are highly sensitive to injury, during the transplantation process generally, and to multiple immune rejection mechanisms. Using pig lung donors, our lab has been working on lung transplants into baboons as a surrogate for a human recipient. Several ex vivo human blood perfusion models have also proven useful. The combination of these experiments allows us to test large animal models as well as whole organ or isolated endothelial reactions to perfusion with human blood. We have found that a multi-modality therapeutic approach to prevent various pathogenic cascades - such as antibody-driven complement activation, other immune pathway activation, thrombosis, and tissue ischemia-reperfusion injury - has met with progressively greater success to protect the xeno lung from injury. Pig gene knockout and human gene transfer has been perhaps the greatest contributor. This review will discuss mechanisms of xeno lung injury, relevant experimental models, as well as recent results and future targets for research., (Copyright © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.)

We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life-supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P < 0.01 vs. GalTKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri-transplant platelet activation (as β-thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Porcine xenografts are a promising source of scarce transplantable organs, but stimulate intense thrombosis of human blood despite targeted genetic and pharmacologic interventions. Current experimental models do not enable study of the blood/endothelial interface to investigate adhesive interactions and thrombosis at the cellular level under physiologic conditions. The purpose of this study was to develop and validate a live-cell, shear-flow based thrombosis assay relevant to general thrombosis research, and demonstrate its potential in xenotransplantation applications., Methodology/principal Findings: Confluent wild-type (WT, n = 48) and Gal transferase knock-out (GalTKO, which resist hyperacute rejection; n = 11) porcine endothelia were cultured in microfluidic channels. To mimic microcirculatory flow, channels were perfused at 5 dynes/cm2 and 37°C with human blood stained to fluorescently label platelets. Serial fluorescent imaging visualized percent surface area coverage (SA, for adhesion of labeled cells) and total fluorescence (a metric of clot volume). Aggregation was calculated by the fluorescence/SA ratio (FR). WT endothelia stimulated diffuse platelet adhesion (SA 65 ± 2%) and aggregation (FR 120 ± 1 a.u.), indicating high-grade thrombosis consistent with the rapid platelet activation and consumption seen in whole-organ lung xenotransplantation models. Experiments with antibody blockade of platelet aggregation, and perfusion of syngeneic and allo-incompatible endothelium was used to verify the biologic specificity and validity of the assay. Finally, with GalTKO endothelia thrombus volume decreased by 60%, due primarily to a 58% reduction in adhesion (P < 0.0001 each); importantly, aggregation was only marginally affected (11% reduction, P < 0.0001)., Conclusions/significance: This novel, high-throughput assay enabled dynamic modeling of whole-blood thrombosis on intact endothelium under physiologic conditions, and allowed mechanistic characterization of endothelial and platelet interactions. Applied to xenogeneic thrombosis, it enables future studies regarding the effect of modifying the porcine genotype on sheer-stress-dependent events that characterize xenograft injury. This in-vitro platform is likely to prove broadly useful to study thrombosis and endothelial interactions under dynamic physiologic conditions.

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., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Generation of an effective immune response against foreign antigens requires two distinct molecular signals: a primary signal provided by the binding of antigen-specific T-cell receptor to peptide-MHC on antigen-presenting cells and a secondary signal delivered via the engagement of costimulatory molecules. Among various costimulatory signaling pathways, the interactions between CD40 and its ligand CD154 have been extensively investigated given their essential roles in the modulation of adaptive immunity. Here, we review current understanding of the role CD40/CD154 costimulation pathway has in alloimmunity, and summarize recent mechanistic and preclinical advances in the evaluation of candidate therapeutic approaches to target this receptor-ligand pair in transplantation.

Background: Scientists working in the field of xenotransplantation do not employ a uniform method to measure and report natural and induced antibody responses to non-Galα(1,3)Gal (non-Gal) epitopes. Such humoral responses are thought to be particularly pathogenic after transplantation of vascularized GalTKO pig organs and having a more uniform assay and reporting format would greatly facilitate comparisons between laboratories., Methods: Flow cytometry allows examination of antibody reactivity to intact antigens in their natural location and conformation on cell membranes. We have established a simple and reproducible flow cytometric assay to detect antibodies specific for non-Gal pig antigens using primary porcine aortic endothelial cells (pAECs) and cell culture-adapted pAEC cell lines generated from wild type and α1,3galactosyl transferase knockout (GalTKO) swine., Results: The consensus protocol we propose here is based on procedures routinely used in four xenotransplantation centers and was independently evaluated at three sites using shared cells and serum samples. Our observation support use of the cell culture-adapted GalTKO pAEC KO:15502 cells as a routine method to determine the reactivity of anti-non-Gal antibodies in human and baboon serum., Conclusions: We have developed an assay that allows the detection of natural and induced non-Gal xenoreactive antibodies present in human or baboon serum in a reliable and consistent manner. This consensus assay and format for reporting the data should be accessible to laboratories and will be useful for assessing experimental results between multiple research centers. Adopting this assay and format for reporting the data should facilitate the detection, monitoring, and detailed characterization of non-Gal antibody responses., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

Xenotransplantation has undergone important progress in controlling initial hyperacute rejection in many preclinical models, with some cell, tissue, and organ xenografts advancing toward clinical trials. However, acute injury, driven primarily by innate immune and inflammatory responses, continues to limit results in lung xenograft models. The purpose of this article is to review the current status of lung xenotransplantation--including the seemingly unique challenges posed by this organ-and summarize proven and emerging means of overcoming acute lung xenograft injury., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

Background: Although transplantation of genetically modified porcine livers into baboons has yielded recipient survival for up to 7 days, survival is limited by profound thrombocytopenia, which becomes manifest almost immediately after revascularization, and by subsequent coagulopathy. Porcine von Willebrand's factor (VWF), a glycoprotein that adheres to activated platelets to initiate thrombus formation, has been shown to constitutively activate human platelets via their glycoprotein Ib (GPIb) receptors. Here, we report our pig-to-primate liver xenoperfusion model and evaluate whether targeting the GPIb-VWF axis prevents platelet sequestration., Methods: Twelve baboons underwent cross-circulation with the following extracorporeal livers: one allogeneic control with a baboon liver, 4 xenogeneic controls with a GalTKO.hCD46 pig liver, 3 GalTKO.hCD46 pig livers in recipients treated with αGPIb antibody during perfusion, and 4 GalTKO.hCD46 pig livers pre-treated with D-arginine vasopressin (DDAVP) in recipients treated with αGPIb antibody during perfusion., Results: All perfused livers appeared grossly and macroscopically normal and produced bile. Xenograft liver perfusion experiments treated with αGPIb antibody may show less platelet sequestration during the initial 2 h of perfusion. Portal venous resistance remained constant in all perfusion experiments. Platelet activation studies demonstrated platelet activation in all xenoperfusions, but not in the allogeneic perfusion., Conclusion: These observations suggest that primate platelet sequestration by porcine liver and the associated thrombocytopenia are multifactorial and perhaps partially mediated by a constitutive interaction between porcine VWF and the primate GPIb receptor. Control of platelet sequestration and consumptive coagulopathy in liver xenotransplantation will likely require a multifaceted approach in our clinically relevant perfusion model., (© 2014 John Wiley & Sons A/S.)

The histopathology of cardiac xenograft rejection has evolved over the last 20 yr with the development of new modalities for limiting antibody-mediated injury, advancing regimens for immune suppression, and an ever-widening variety of new donor genetics. These new technologies have helped us progress from what was once an overwhelming anti-Gal-mediated hyperacute rejection to a more protracted anti-Gal-mediated vascular rejection to what is now a more complex manifestation of non-Gal humoral rejection and coagulation dysregulation. This review summarizes the changing histopathology of Gal- and non-Gal-mediated cardiac xenograft rejection and discusses the contributions of immune-mediated injury, species-specific immune-independent factors, transplant and therapeutic procedures, and donor genetics to the overall mechanism(s) of cardiac xenograft rejection., (© 2013 The Authors. Xenotransplantation Published by John Wiley and Sons Ltd.)

Background: Cardiac allograft vasculopathy (CAV) remains the main cause of long-term transplant rejection. CAV is characterized by hyperproliferation of vascular smooth muscle cells (VSMCs). Canonical β-catenin signaling is a critical regulator of VSMC proliferation in development; however, the role of this pathway and its regulation in CAV progression are obscure. We investigated the activity of β-catenin signaling and the role for a putative activating ligand, transglutaminase 2 (TG2), in chronic cardiac rejection., Methods: Hearts from Bm12 mice were transplanted into C57BL/6 mice (class II mismatch), and allografts were harvested 8 weeks after transplantation. Accumulation and sub-cellular distribution of β-catenin protein and expression of several components of β-catenin signaling were analyzed as hallmarks of pathway activation. In vitro, platelet-derived growth factor treatment was used to mimic the inflammatory milieu in VSMC and organotypic heart slice cultures., Results: Activation of β-catenin in allografts compared with isografts or naïve hearts was evidenced by the augmented expression of β-catenin target genes, as well as the accumulation and nuclear localization of the β-catenin protein in VSMCs of the occluded allograft vessels. Expression of TG2, an activator of β-catenin signaling in VSMCs, was dramatically increased in allografts. Further, our ex vivo data demonstrate that TG2 is required for VSMC proliferation and for β-catenin activation by platelet-derived growth factor in cardiac tissue., Conclusions: β-Catenin signaling is activated in occluded vessels in murine cardiac allografts. TG2 is implicated as an endogenous activator of this signaling pathway and may therefore have a role in the pathogenesis of CAV during chronic allograft rejection., (Copyright © 2012 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Barriers to successful lung xenotransplantation appear to be even greater than for other organs. This difficulty may be related to several macro anatomic factors, such as the uniquely fragile lung parenchyma and associated blood supply that results in heightened vulnerability of graft function to segmental or lobar airway flooding caused by loss of vascular integrity (also applicable to allotransplants). There are also micro-anatomic considerations, such as the presence of large numbers of resident inflammatory cells, such as pulmonary intravascular macrophages and natural killer (NK) T cells, and the high levels of von Willebrand factor (vWF) associated with the microvasculature. We have considered what developments would be necessary to allow successful clinical lung xenotransplantation. We suggest this will only be achieved by multiple genetic modifications of the organ-source pig, in particular to render the vasculature resistant to thrombosis. The major problems that require to be overcome are multiple and include (i) the innate immune response (antibody, complement, donor pulmonary and recipient macrophages, monocytes, neutrophils, and NK cells), (ii) the adaptive immune response (T and B cells), (iii) coagulation dysregulation, and (iv) an inflammatory response (e.g., TNF-α, IL-6, HMGB1, C-reactive protein). We propose that the genetic manipulation required to provide normal thromboregulation alone may include the introduction of genes for human thrombomodulin/endothelial protein C-receptor, and/or tissue factor pathway inhibitor, and/or CD39/CD73; the problem of pig vWF may also need to be addressed. It would appear that exploration of every available therapeutic path will be required if lung xenotransplantation is to be successful. To initiate a clinical trial of lung xenotransplantation, even as a bridge to allotransplantation (with a realistic possibility of survival long enough for a human lung allograft to be obtained), significant advances and much experimental work will be required. Nevertheless, with the steadily increasing developments in techniques of genetic engineering of pigs, we are optimistic that the goal of successful clinical lung xenotransplantation can be achieved within the foreseeable future. The optimistic view would be that if experimental pig lung xenotransplantation could be successfully managed, it is likely that clinical application of this and all other forms of xenotransplantation would become more feasible., (© 2012 John Wiley & Sons A/S.)

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 are briefly reviewed and two different experimental models, the ex vivo porcine-to-human lung perfusion and the in vivo xenogeneic lung transplantation, are presented. We 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.

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of hematopoietic derived cell precursors that can suppress immune responses in a variety of inflammatory settings. Here we review recent studies detailing expansion of phenotypically and functionally disparate MDSC. Findings related to MDSC accumulation, activation, and mechanisms utilized in immune suppression are presented. Further, we discuss recent reports that suggest MDSC are expanded during transplantation and that modulation of MDSC can participate in preventing graft rejection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Galactosyl transferase gene knock-out (GalTKO) swine offer a unique tool to evaluate the role of the Gal antigen in xenogenic lung hyperacute rejection., Methods: We perfused GalTKO miniature swine lungs with human blood. Results were compared with those from previous studies using wild-type and human decay-accelerating factor-transgenic (hDAF(+/+) ) pig lungs., Results: GalTKO lungs survived 132 ± 52 min compared to 10 ± 9 min for wild-type lungs (P = 0.001) and 45 ± 60 min for hDAF(+/+) lungs (P = 0.18). GalTKO lungs displayed stable physiologic flow and pulmonary vascular resistance (PVR) until shortly before graft demise, similar to autologous perfusion, and unlike wild-type or hDAF(+/+) lungs. Early (15 and 60 min) complement (C3a) and platelet activation and intrapulmonary platelet deposition were significantly diminished in GalTKO lungs relative to wild-type or hDAF(+/+) lungs. However, GalTKO lungs adsorbed cytotoxic anti-non-Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points., Conclusions: In summary, GalTKO lungs are substantially protected from injury but, in addition to anti-non-Gal antibody and complement, platelet adhesion and non-physiologic intravascular coagulation contribute to Gal-independent lung injury mechanisms., (© 2011 John Wiley & Sons A/S.)

Chronic rejection currently limits the long-term efficacy of clinical transplantation. Although B cells have recently been shown to play a pivotal role in the induction of alloimmunity and are being targeted in other transplant contexts, the efficacy of preemptive B cell depletion to modulate alloimmunity or attenuate cardiac allograft vasculopathy (CAV) (classic chronic rejection lesions found in transplanted hearts) in a translational model has not previously been described. We report here that the CD20-specific antibody (alphaCD20) rituximab depleted CD20+ B cells in peripheral blood, secondary lymphoid organs, and the graft in cynomolgus monkey recipients of heterotopic cardiac allografts. Furthermore, CD20+ B cell depletion therapy combined with the calcineurin inhibitor cyclosporine A (CsA) prolonged median primary graft survival relative to treatment with alphaCD20 or CsA alone. In animals treated with both alphaCD20 and CsA that achieved efficient B cell depletion, alloantibody production was substantially inhibited and the CAV severity score was markedly reduced. We conclude therefore that efficient preemptive depletion of CD20+ B cells is effective in a preclinical model to modulate pathogenic alloimmunity and to attenuate chronic rejection when used in conjunction with a conventional clinical immunosuppressant. This study suggests that use of this treatment combination may improve the efficacy of transplantation in the clinic.

Transplantation is the treatment of choice for patients with end-stage organ failure. Its success is limited by side effects of immunosuppressive drugs, such as inhibitors of the calcineurin pathway that prevent rejection by reducing synthesis of interleukin-2 by T cells. Moreover, none of the existing drugs efficiently prevent the eventual rejection of the organ. Blocking the CD28-mediated T cell costimulation pathway is a nontoxic alternative immunosuppression strategy that is now achieved by blockade of CD80/86, the receptor for CD28 on antigen-presenting cells. However, interaction of CD80/86 with cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is required for immune regulation. Therefore, CD28 blockade, instead of CD80/86 blockade, might preserve regulatory signals mediated by CTLA-4 and preserve immune regulation. By using monovalent antibodies, we identified true CD28 antagonists that induced CTLA-4-dependent decreased T cell function compatible with regulatory T (Treg) cell suppression. In transplantation experiments in primates, blocking CD28 augmented intragraft and peripheral blood Treg cells, induced molecular signatures of immune regulation, and prevented graft rejection and vasculopathy in synergy with calcineurin inhibition. These findings suggest that targeting costimulation blockade at CD28 preserves CTLA-4-dependent immune regulation and promotes allograft survival.

Aims: Clinical studies suggest that intake of omega-3 polyunsaturated fatty acids (omega-3 PUFA) may lower the incidence of heart failure. Dietary supplementation with omega-3 PUFA exerts metabolic and anti-inflammatory effects that could prevent left ventricle (LV) pathology; however, it is unclear whether these effects occur at clinically relevant doses and whether there are differences between omega-3 PUFA from fish [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] and vegetable sources [alpha-linolenic acid (ALA)]., Methods and Results: We assessed the development of LV remodelling and pathology in rats subjected to aortic banding treated with omega-3 PUFA over a dose range that spanned the intake of humans taking omega-3 PUFA supplements. Rats were fed a standard food or diets supplemented with EPA+DHA or ALA at 0.7, 2.3, or 7% of energy intake. Without supplementation, aortic banding increased LV mass and end-systolic and -diastolic volumes. ALA supplementation had little effect on LV remodelling and dysfunction. In contrast, EPA+DHA dose-dependently increased EPA and DHA, decreased arachidonic acid in cardiac membrane phospholipids, and prevented the increase in LV end-diastolic and -systolic volumes. EPA+DHA resulted in a dose-dependent increase in the anti-inflammatory adipokine adiponectin, and there was a strong correlation between the prevention of LV chamber enlargement and plasma levels of adiponectin (r = -0.78). Supplementation with EPA+DHA had anti-aggregatory and anti-inflammatory effects as evidenced by decreases in urinary thromboxane B(2) and serum tumour necrosis factor-alpha., Conclusion: Dietary supplementation with omega-3 PUFA derived from fish, but not from vegetable sources, increased plasma adiponectin, suppressed inflammation, and prevented cardiac remodelling and dysfunction under pressure overload conditions.

We have previously shown that high-sugar diets increase mortality and left ventricular (LV) dysfunction during pressure overload. The mechanisms behind these diet-induced alterations are unclear but may involve increased oxidative stress in the myocardium. The present study examined whether high-fructose feeding increased myocardial oxidative damage and exacerbated systolic dysfunction after transverse aortic constriction (TAC) and if this effect could be attenuated by treatment with the antioxidant tempol. Immediately after surgery, TAC and sham mice were assigned to a high-starch diet (58% of total energy intake as cornstarch and 10% fat) or high-fructose diet (61% fructose and 10% fat) with or without the addition of tempol [0.1% (wt/wt) in the chow] and maintained on the treatment for 8 wk. In response to TAC, fructose-fed mice had greater cardiac hypertrophy (55.1% increase in the heart weight-to-tibia length ratio) than starch-fed mice (22.3% increase in the heart weight-to-tibia length ratio). Treatment with tempol significantly attenuated cardiac hypertrophy in fructose-fed TAC mice (18.3% increase in the heart weight-to-tibia ratio). Similarly, fructose-fed TAC mice had a decreased LV area of fractional shortening (from 38+/-2% in sham to 22+/-4% in TAC), which was prevented by tempol treatment (33+/-3%). Markers of lipid peroxidation in fructose-fed TAC hearts were also blunted by tempol. In conclusion, tempol significantly blunted markers of cardiac hypertrophy, LV remodeling, contractile dysfunction, and oxidative stress in fructose-fed TAC mice.

Objective: Sugar consumption affects insulin release and, in hypertension, may stimulate cardiac signaling mechanisms that accelerate left ventricular hypertrophy and the development of heart failure. We investigated the effects of high-fructose or sucrose diets on ventricular function and mortality in hypertensive Dahl salt-sensitive rats., Methods: Rats were fed chows that were either high starch (70% starch, 10% fat by energy), high fat (20% carbohydrates, 60% fat), high fructose (61% fructose, 9% starch, 10% fat), or high sucrose (61% sucrose, 9% starch, 10% fat). Hypertension was induced by adding 6% salt to the chow (n = 8-11/group)., Results: After 8 weeks of treatment, systolic blood pressure and left ventricular mass were similarly increased in all rats that were fed high-salt diets. Hypertension caused a switch in mRNA myosin heavy chain isoform from alpha to beta, and this effect was greater in the high-salt sucrose and fructose groups than in starch and fat groups. The cardiac mRNA for atrial natriuretic factor was also increased in all high-salt groups compared to respective controls, with the increase being significantly greater in the hypertensive sucrose fed group. Mortality was greater in the sucrose group (44%) compared to all the other hypertensive groups (12-18%), as was cardiomyocyte apoptosis. Left ventricular ejection fraction was lower in the high-salt sucrose group, which was due to an increase in end-systolic volume, and not increased end-diastolic volume., Conclusion: Diets high in sugar accelerated cardiac systolic dysfunction and mortality in hypertension compared to either a low-carbohydrate/high-fat or high-starch diet.

Background: It is not known how carbohydrate and fat intake affect the development of left ventricular (LV) hypertrophy and contractile dysfunction in response to pressure overload. We hypothesized that a low-carbohydrate/high-fat diet prevents LV hypertrophy and dysfunction compared with high-carbohydrate diets., Methods and Results: Rats were fed high-carbohydrate diets composed of either starch or sucrose, or a low-carbohydrate/high-fat diet, and underwent abdominal aortic banding (AAB) for 2 months. AAB increased LV mass with all diets. LV end-diastolic and systolic volumes and the ratio of the mRNA for myosin heavy chain beta/alpha were increased with both high-carbohydrate diets but not with the low-carbohydrate/high-fat diet. Circulating levels of insulin and leptin, both stimulants for cardiac growth, were lower, and free fatty acids were higher with the low-carbohydrate/high-fat diet compared with high-carbohydrate diets. Among animals that underwent AAB, LV volumes were positively correlated with insulin and LV mass correlated with leptin., Conclusion: A low-carbohydrate/high-fat diet attenuated pressure overload-induced LV remodeling compared with high-carbohydrate diets. This effect corresponded to lower insulin and leptin concentrations, suggesting they may contribute to the development of LV hypertrophy and dysfunction under conditions of pressure overload.

The purpose of this study was to develop a nonhuman primate model for heterotopic composite tissue facial transplantation in which to study the natural history of facial transplantation and evaluate immunosuppressive regimens.A composite oromandibular facial segment transplant based on the common carotid artery was evaluated. Flaps from 7 cynomolgus monkeys were transplanted to the groins of 7 recipients at the superficial femoral artery and vein. The immunosuppressive regimen consisted of thymoglobulin, rapamycin, and tacrolimus. Allograft survival ranged from 6 to 129 days. Histology performed in the long-term survivor at the time of necropsy revealed extensive inflammation and necrosis of the allograft skin; however, muscle and bone elements were viable, with minimal inflammation. This heterotopic facial transplantation model avoids the potential morbidity of mandibular resection and orthotopic facial transplantation. Our work also concurs with the work of other groups who found that the skin component is the most antigenic.

Background: Dietary lipid content effects activation of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) and may accelerate cardiac hypertrophy and dysfunction in response to pressure overload. This study investigated the effects of a high-fat diet on the development of cardiac hypertrophy., Methods and Results: C57BL/6J mice (n = 14-16/group) underwent transverse aortic constriction (TAC) or sham surgery and were fed either standard low-fat diet (STD; 10% fat) or a high-fat diet (HFD; 60% fat) for 16 weeks. Sham mice showed no differences between STD and HFD for heart mass or echocardiographic parameters despite greater plasma free fatty acid and leptin concentrations with HFD. TAC increased heart mass and decreased ejection fraction similarly in both groups. Left ventricular end systolic and diastolic diameters with TAC were increased compared with shams on the HFD (P < .05), but were not different from STD TAC mice. High-fat feeding increased expression of PPAR-alpha-regulated genes. The activity of medium chain acyl-coenzyme A dehydrogenase (MCAD), a marker of fatty acid oxidation capacity, was increased in HFD TAC mice compared with STD, consistent with PPAR-alpha activation., Conclusions: Increased fat intake prevented the fall in MCAD activity and did not exacerbate the hypertrophic response to TAC compared with a low-fat diet.