Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs.

A combination of genetic manipulations of donor organs and target-specific immunosuppression is instrumental in achieving long-term cardiac xenograft survival. Recently, results from our preclinical pig-to-baboon heterotopic cardiac xenotransplantation model suggest that a three-pronged approach is successful in extending xenograft survival: (a) α-1,3-galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal-specific antibody-mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co-stimulation blockade of CD40-CD40L pathways with anti-CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non-Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co-stimulation blockade-based immunomodulatory regimen.

CD4+CD25Hi FoxP3+ regulatory T cells in long-term cardiac xenotransplantation.

BACKGROUND: CD4+CD25Hi FoxP3+ T (Treg) cells are a small subset of CD4+ T cells that have been shown to exhibit immunoregulatory function. Although the absolute number of Treg cells in peripheral blood lymphocytes (PBL) is very small, they play an important role in suppressing immune reactivity. Several studies have demonstrated that the number of Treg cells, rather than their intrinsic suppressive capacity, may contribute to determining the long-term fate of transplanted grafts. In this study, we analyzed Treg cells in PBL of long-term baboon recipients who have received genetically modified cardiac xenografts from pig donors. METHODS: Heterotopic cardiac xenotransplantation was performed on baboons using hearts obtained from GTKO.hCD46 (n = 8) and GTKO.hCD46.TBM (n = 5) genetically modified pigs. Modified immunosuppression regimen included antithymocyte globulin (ATG), anti-CD20, mycophenolate mofetil (MMF), cobra venom factor (CVF), and costimulation blockade (anti-CD154/anti-CD40 monoclonal antibody). FACS analysis was performed on PBLs labeled with anti-human CD4, CD25, and FoxP3 monoclonal antibodies (mAb) to analyze the percentage of Treg cells in six baboons that survived longer than 2 months (range: 42-945 days) after receiving a pig cardiac xenograft. RESULTS: Total WBC count was low due to immunosuppression in baboons who received cardiac xenograft from GTKO.hCD46 and GTKO.hCD46.hTBM donor pigs. However, absolute numbers of CD4+CD25Hi FoxP3 Treg cells in PBLs of long-term xenograft cardiac xenograft surviving baboon recipients were found to be increased (15.13 ± 1.50 vs 7.38 ± 2.92; P < .018) as compared to naïve or pre-transplant baboons. Xenograft rejection in these animals was correlated with decreased numbers of regulatory T cells. CONCLUSION: Our results suggest that regulatory T (Treg) cells may contribute to preventing or delaying xenograft rejection by controlling the activation and expansion of donor-reactive T cells, thereby masking the antidonor immune response, leading to long-term survival of cardiac xenografts.

Consideration of appropriate clinical applications for cardiac xenotransplantation.

The field of cardiac xenotransplantation has entered an exciting era due to recent advances in the field. Although several hurdles remain, the use of rapidly evolving transgenic technology has the potential to address current allogeneic donor pool constraints and mechanical circulatory system device limitations. The success of xenotransplantation will undoubtedly be dependent on specific patient selection criteria. Defining these particular indications for xenotransplantation is important as we approach the possibility of clinical applications.

Postoperative acute kidney injury following intraoperative blood product transfusions during cardiac surgery.

INTRODUCTION: This study explored the nature of the association between intraoperative usage of red blood cell, fresh frozen plasma, cryoprecipitate or platelet transfusions and acute kidney injury. METHODS: A total of 1175 patients who underwent cardiac surgery between 2008 and 2013 were retrospectively analyzed. We assessed the association between: (1) preoperative patient characteristics and acute kidney injury, (2) intraoperative blood product usage and acute kidney injury, (3) acute kidney injury and 30-day mortality or re-hospitalization. RESULTS: In our cohort of 1175 patients, 288 patients (24.5%) developed acute kidney injury. This included 162 (13.8%), 69 (5.9%) and 57 (4.9%) developing stage 1, stage 2 or stage 3 acute kidney injury, respectively. Increased red blood cell, fresh frozen plasma or platelet transfusions increased the odds of developing acute kidney injury. Specifically, every unit of red blood cells, fresh frozen plasma or platelets transfused was associated with an increase in the covariate-adjusted odds ratio of developing ⩾ stage 2 kidney injury of 1.18, 1.19 and 1.04, respectively. CONCLUSIONS: Intraoperative blood product transfusions were independently associated with an increased odds of developing acute kidney injury following cardiac surgery. Further randomized studies are needed to better define intraoperative transfusion criteria.

Encouraging experience using multi-transgenic xenografts in a pig-to-baboon cardiac xenotransplantation model.

BACKGROUND: Innovations in transgenic technology have facilitated improved xenograft survival. Additional gene expression appears to be necessary to overcome the remaining immune and biologic incompatibilities. We report for the first time the novel use of six-gene modifications within a pig-to-baboon cardiac xenotransplantation model. METHODS: Baboons (8-15 kg) underwent heterotopic cardiac transplantation using xenografts obtained from genetically engineered pigs. Along with previously described modifications (GTKO, hCD46), additional expression of human transgenes for thromboregulation (endothelial protein C receptor, tissue factor pathway inhibitor, thrombomodulin), complement inhibition (decay accelerating factor), and cellular immune suppression (hCD39, hCD47) was used. Immunosuppression consisted of targeted T-cell and B-cell depletion and conventional anti-rejection agents. RESULTS: Heterotopic cardiac transplantations were performed without complication. Flow cytometry and immunohistochemistry on donor biopsies confirmed transgenic phenotype. In contrast to the prior three-gene generation, significant coagulopathy or consumptive thrombocytopenia has not been observed in the six-gene cohort. As a result, these recipients have experienced decreased bleeding-related complications. Pro-inflammatory responses also appear to be mitigated based on cytokine analysis. Baboons survived the critical 30-day post-operative period when mortality has historically been highest, with no evidence of graft rejection. CONCLUSIONS: The inclusion of additional human genes in genetically engineered pigs appears to confer superior xenograft outcomes. Introduction of these genes has not been associated with adverse outcomes. This multifactorial approach to genetic engineering furthers the prospect of long-term cardiac xenograft survival and subsequent clinical application.

Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein.

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 ß-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.

Role of anti-CD40 antibody-mediated costimulation blockade on non-Gal antibody production and heterotopic cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon model.

BACKGROUND: Recently, we have shown that an immunosuppression regimen including costimulation blockade via anti-CD154 antibody significantly prolongs the cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon heterotopic xenotransplantation model. Unfortunately, many coagulation disorders were observed with the use of anti-CD154 antibody, and recipient survival was markedly reduced by these complications. MATERIAL AND METHODS: In this experiment, we replaced anti-CD154 antibody with a more clinically acceptable anti-CD40 antibody while keeping the rest of the immunosuppressive regimen and the donor pig genetics the same. This was carried out to evaluate the antibody's role in xenograft survival and prevention of coagulopathies. Two available clones of anti-CD40 antibody were tested. One mouse anti-human CD40 antibody, (clone 3A8), activated B lymphocytes in vitro and only modestly suppressed antibody production in vivo. Whereas a recombinant mouse non-human primate chimeric raised against macaque CD40, (clone 2C10R4), blocked B-cell activation in vitro and completely blocked antibody production in vivo. RESULTS: The thrombotic complications seen with anti-CD154 antibody were effectively avoided but the graft survival, although extended, was not as prolonged as observed with anti-CD154 antibody treatment. The longest survival for the 3A8 antibody group was 27 days, and the longest graft survival in the 2C10R4 antibody group was 146 days. All of the grafts except two rejected and were explanted. Only two recipient baboons had to be euthanized due to unrelated complications, and the rest of the baboons remained healthy throughout the graft survival period or after graft explantation. In contrast to our anti-CD 154 antibody-treated baboons, the non-Gal antibody levels started to rise after B cells made their appearance around 8 weeks post-transplantation. CONCLUSIONS: Anti-CD40 antibody at the current dose does not induce any coagulopathies but while effective, had reduced efficacy to induce similar long-term graft survival as with anti-CD154 antibody perhaps due to ineffective control of B-cell function and antibody production at the present dose. More experiments are required to determine antibody affinity and effective dose for inducing long-term cardiac xenograft survival.

Heterotopic Porcine Cardiac Xenotransplantation in the Intra-Abdominal Position in a Non-Human Primate Model.

Heterotopic cardiac transplantation in the intra-abdominal position in a large animal model has been essential in the progression of the field of cardiac transplantation. Our group has over 10 years of experience in cardiac xenotransplantation with pig to baboon models, the longest xenograft of which survived over 900 days, with rejection only after reducing immunosuppression. This article aims to clarify our approach to this model in order to allow others to share success in long-term survival. Here, we demonstrate the approach to implantation of a cardiac graft into the intra-abdominal position in a baboon recipient for the study of transplantation and briefly highlight our model's ability to provide insight into not only xenotransplantation but across disciplines. We include details that have provided us with consistent success in this model; performance of the anastomoses, de-airing of the graft, implantation of a long-term telemetry device for invasive graft monitoring, and ideal geometric positioning of the heart and telemetry device in the limited space of the recipient abdomen. We additionally detail surveillance techniques to assess long-term graft function.

Intra-Abdominal Heterotopic Cardiac Xenotransplantation: Pearls and Pitfalls.

Heterotopic cardiac xenotransplantation in the intra-abdominal position has been studied extensively in a pig-to-baboon model to define the optimal donor genetics and immunosuppressive regimen to prevent xenograft rejection. Extensive investigation using this model is a necessary stepping stone toward the development of a life-supporting animal model, with the ultimate goal of demonstrating suitability for clinical cardiac xenotransplantation trials. Aspects of surgical technique, pre- and post-operative care, graft monitoring, and minimization of infectious risk have all required refinement and optimization of heterotopic cardiac xenotransplantation over time. This review details non-immunologic obstacles relevant to this model described by our group and in the literature, as well as strategies that have been developed to address these specific challenges.

Circulating cell-free DNA as a biomarker of tissue injury: Assessment in a cardiac xenotransplantation model.

BACKGROUND: Observational studies suggest that cell-free DNA (cfDNA) is a biomarker of tissue injury in a range of conditions including organ transplantation. However, the lack of model systems to study cfDNA and its relevance to tissue injury has limited the advancements in this field. We hypothesized that the predictable course of acute humoral xenograft rejection (AHXR) in organ transplants from genetically engineered donors provides an ideal system for assessing circulating cfDNA as a marker of tissue injury. METHODS: Genetically modified pig donor hearts were heterotopically transplanted into baboons (n = 7). Cell-free DNA was extracted from pre-transplant and post-transplant baboon plasma samples for shotgun sequencing. After alignment of sequence reads to pig and baboon reference sequences, we computed the percentage of xenograft-derived cfDNA (xdcfDNA) relative to recipient by counting uniquely aligned pig and baboon sequence reads. RESULTS: The xdcfDNA percentage was high early post-transplantation and decayed exponentially to low stable levels (baseline); the decay half-life was 3.0 days. Post-transplantation baseline xdcfDNA levels were higher for transplant recipients that subsequently developed graft loss than in the 1 animal that did not reject the graft (3.2% vs 0.5%). Elevations in xdcfDNA percentage coincided with increased troponin and clinical evidence of rejection. Importantly, elevations in xdcfDNA percentage preceded clinical signs of rejection or increases in troponin levels. CONCLUSION: Cross-species xdcfDNA kinetics in relation to acute rejection are similar to the patterns in human allografts. These observations in a xenotransplantation model support the body of evidence suggesting that circulating cfDNA is a marker of tissue injury.

Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.

Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

Regulatory T cells enhance mesenchymal stem cell survival and proliferation following autologous cotransplantation in ischemic myocardium.

OBJECTIVES: We sought to investigate if autologous freshly isolated regulatory T cells (Tregs) provide a protective and supportive role when cotransplanted with mesenchymal stem cells (MSCs). METHODS: In a porcine model of chronic ischemia, autologous MSCs were isolated and expanded ex vivo for 4 weeks. Autologous Treg cells were freshly isolated from 100 mL peripheral blood and purified by fluorescence-activated cell sorting. MSCs and Treg cells were then cotransplanted into the chronic ischemic myocardium of Yorkshire pigs by direct intramyocardial injection (1.2 × 10(8) MSCs plus an average of 1.5 million Treg cells in 25 injection sites). Animals were killed 6 weeks postinjection to study the fate of the cells and compare the effect of combined MSCs + Treg cells transplantation versus MSCs alone. RESULTS: The coinjection of MSCs along with Tregs was safe and no deleterious side effects were observed. Six weeks after injection of the cell combination, spherical MSCs clusters with thin layer capsules were found in the injected areas. In animals treated with MSCs only, the MSC clusters were less organized and not encapsulated. Immunofluorescent staining showed CD25+ cells among the CD90+ (MSC marker) cells, suggesting that the injected Treg cells remained present locally, and survived. Factor VIII+ cells were also prevalent suggesting new angiogenesis. We found no evidence that coinjections were associated with the generation of cardiac myocytes. CONCLUSIONS: The cotransplantation of Treg cells with MSCs dramatically increased the MSC survival rate, proliferation, and augmented their role in angiogenesis, which suggests a new way for future clinical application of cell-based therapy.

Genetically engineered pigs and target-specific immunomodulation provide significant graft survival and hope for clinical cardiac xenotransplantation.

OBJECTIVES: Cardiac transplantation and available mechanical alternatives are the only possible solutions for end-stage cardiac disease. Unfortunately, because of the limited supply of human organs, xenotransplantation may be the ideal method to overcome this shortage. We have recently seen significant prolongation of heterotopic cardiac xenograft survival from 3 to 12 months and beyond. METHODS: Hearts from genetically engineered piglets that were alpha 1-3 galactosidase transferase knockout and expressed the human complement regulatory gene, CD46 (groups A-C), and the human thrombomodulin gene (group D) were heterotropically transplanted in baboons treated with antithymocyte globulin, cobra venom factor, anti-CD20 antibody, and costimulation blockade (anti-CD154 antibody [clone 5C8]) in group A, anti-CD40 antibody (clone 3A8; 20 mg/kg) in group B, clone 2C10R4 (25 mg/kg) in group C, or clone 2C10R4 (50 mg/kg) in group D, along with conventional nonspecific immunosuppressive agents. RESULTS: Group A grafts (n = 8) survived for an average of 70 days, with the longest survival of 236 days. Some animals in this group (n = 3) developed microvascular thrombosis due to platelet activation and consumption, which resulted in spontaneous hemorrhage. The median survival time was 21 days in group B (n = 3), 80 days in group C (n = 6), and more than 200 days in group D (n = 5). Three grafts in group D are still contracting well, with the longest ongoing graft survival surpassing the 1-year mark. CONCLUSIONS: Genetically engineered pig hearts (GTKOhTg.hCD46.hTBM) with modified targeted immunosuppression (anti-CD40 monoclonal antibody) achieved long-term cardiac xenograft survival. This potentially paves the way for clinical xenotransplantation if similar survival can be reproduced in an orthotopic transplantation model.

Repair of anomalous origin of right coronary artery from the left sinus of Valsalva.

Anomalous origin of the right coronary artery from the left sinus of Valsalva is a rare congenital anomaly but a relatively frequent cause of sudden death in the young. The medical records of 4 consecutive patients with this anomaly were retrospectively reviewed. The first 2 patients underwent coronary artery bypass and had early graft failure. The next 2 patients underwent coronary reimplantation and unroofing and have done well. This limited series suggests that bypass procedures used to treat anomalous origin of the right coronary artery from the left sinus of Valsalva are prone to early graft failure.