McGregor, Christopher G.A.

Experiments were designed to determine whether normal fluctuations in sex steroid hormones alter gene transcription for endothelial nitric oxide synthase (NOS) and preproendothelin-1 (prepro-ET-1). Aortic endothelial cells were removed from adult, gonadally intact male and female or ovariectomized Yorkshire pigs. Endothelial cells were prepared for Northern blot analysis, Western blot analysis or enzyme activity. Nitric oxide products (NOx) and endothelin-1 (ET-1) in plasma were measured by chemiluminescence and radioimmunoassay, respectively. Northern blot analysis identified single bands corresponding to endothelial NOS and prepro-ET-1. Quantification of the blots showed an increase in expression of mRNA for both endothelial NOS and prepro-ET-1 in ovariectomized pigs compared with gonadally intact male and female pigs. There were no differences in amount of endothelial NOS protein identified by Western blot analysis among groups. On the contrary, plasma concentrations of NOx were significantly decreased in ovariectomized pigs, and there were no differences either in the concentrations of ET-1 in the plasma or extracts from the coronary arteries. These results suggest that expression of endothelial NOS and prepro-ET-1 may be regulated at transcriptional level by ovarian hormones. In addition, the ovarian hormones may regulate production of these endothelium-derived factors at the posttranscriptional level.

Cardiac xenotransplantation (CXTx) is a promising solution to the chronic shortage of donor hearts. Recent advancements in immune suppression have greatly improved the survival of heterotopic CXTx, now extended beyond 2 years, and life-supporting kidney XTx. Advances in donor genetic modification (B4GALNT2 and CMAH mutations) with proven Gal-deficient donors expressing human complement regulatory protein(s) have also accelerated, reducing donor pig organ antigenicity. These advances can now be combined and tested in life-supporting orthotopic preclinical studies in nonhuman primates and immunologically appropriate models confirming their efficacy and safety for a clinical CXTx program. Preclinical studies should also allow for organ rejection to develop xenospecific assays and therapies to reverse rejection. The complexity of future clinical CXTx presents a substantial and unique set of regulatory challenges which must be addressed to avoid delay; however, dependent on these prospective life-supporting preclinical studies in NHPs, it appears that the scientific path forward is well defined and the era of clinical CXTx is approaching.

AbstractBackgroundAs a step towards clinical cardiac xenotransplantation, our experimental heterotopic intrathoracic xenotransplantation model offers a beating and ejecting donor heart while retaining the recipient′s native organ as a backup in case of graft failure. Clinically applicable immunosuppressive regimens (IS) were investigated first, then treatments known to be effective in hypersensitized patients or those with recalcitrant rejection reactions.MethodsConsecutive experiments were carried out between 2009 and 2013. Twenty‐one genetically modified pigs (GGTA1‐knockout/hCD46/± thrombomodulin, in one caseHLA‐E instead) were used as donors. In all experiments, two cycles of immunoabsorption reduced preformed antibodies. Recipient baboons were divided into two groups according toISregimen:In group one (n = 10), pre‐treatment started either one (anti‐CD20) or four weeks (anti‐CD20 plus the proteasome inhibitor bortezomib) prior to transplantation. The extended conventional (as for allotransplantation) immunosuppressive maintenance regimen included anti‐thymocyte globuline, tacrolimus, mycophenolate mofetil, methylprednisolone and weekly anti‐CD20.In group two (n = 11), myeloablative pre‐treatment as in multiple myeloma patients (long and short regimens) was added to extended conventionalIS; postoperative total thoracic and abdominal lymphoid irradiation (TLI; single dose of 600cGY) was used to further reduce antibody‐producing cells.ResultsIn the perioperative course, the surgical technique was safely applied: 19 baboons were weaned off extracorporeal circulation and 17 extubated. Nine animals were lost in the early postoperative course due to causes unrelated to surgical technique orISregimen.Excluding these early failures, median graft survival times of group 1 and 2 were 18.5 (12–50) days and 16 (7–35) days. Necropsy examination of group 1 donor organs revealed hypertrophy of the left ventricular wall in the six longer‐lasting grafts; myocardial histology confirmed pre‐clinical suspicion of humoral rejection, which was not inhibited by the extended conventional IS including intensified treatments, and signs of thrombotic microangiopathy.Grafts of group 2 presented with only mild‐to‐moderate features of humoral rejection and thrombotic microangiopathy, except in one case of delayed rejection on day 17. The other experiments in this group were terminated because of untreatable pulmonary oedema, recurring ventricular fibrillation, Aspergillus sepsis, as well as a combination of a large donor organ and late toxic side effects due toTLI.ConclusionsLonger‐term results were difficult to achieve in this model due to theISregimens used. However, we conclude that heterotopic intrathoracic heart transplantation may be an option for clinical xenotransplantation.

AbstractBackgroundRejection of Gal‐free (GTKO) donor pig cardiac xenografts is strongly associated with vascular non‐Gal antibody binding, endothelial cell (EC) injury, and activation and microvascular thrombosis. We adopted a pig‐to‐SCID/beige small animal transplant model to compare the pathogenicity of baboon and human anti‐pig antibody.MethodsWild‐type (GT+) or GTKO porcine coronary arteries (PCAs) were transplanted into the infrarenal aorta of SCID/beige mice. Three days after transplant, recipients were infused with anti‐pig antibody (anti‐SLA class I, an isotype control, naive or sensitized baboon serum, or naive human serum). PCAs were recovered 24 h after antibody infusion and examined using histology, immunohistochemistry, and in situ hybridization.ResultsDose‐dependent intragraft thrombosis occurred after infusion of anti‐SLA I antibody (but not isotype control) in GT+ and GTKO PCA recipients. Naive baboon serum induced thrombosis in GT+ grafts. Thrombosis was significantly reduced by pre‐treating naive baboon serum with Gal polymer and not observed when this serum was infused to GTKO PCA recipients. Naive human serum caused dose‐dependent intragraft thrombosis of GTKO PCAs. In all cases, thrombosis involved graft‐specific vascular antibody and complement deposition, macrophage adherence, EC delamination, and subendothelial thrombus formation.ConclusionsThis study provides the first direct in vivo comparison of the pathogenicity of naive human and baboon serum. The results suggest that human preformed non‐Gal antibody may have increased pathogenicity compared to baboon. This model, which showed a rejected graft histopathology similar to antibody‐mediated rejection in cardiac xenotransplantation, may be useful to assess the pathogenicity of individual protein or carbohydrate specific non‐Gal reactive antibodies.

Abstract 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‐ G alα(1,3) G al (non‐ G al) epitopes. Such humoral responses are thought to be particularly pathogenic after transplantation of vascularized Gal TKO 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‐ G al pig antigens using primary porcine aortic endothelial cells ( pAEC s) and cell culture‐adapted pAEC cell lines generated from wild type and α1,3galactosyl transferase knockout (Gal TKO ) 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 Gal TKO pAEC KO :15502 cells as a routine method to determine the reactivity of anti‐non‐ G al 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‐ G al antibody responses.

Abstract Background Xenograft rejection of pigs organs with an engineered mutation in the GGTA‐1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non‐Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNA s which encode pig antigens that bind to IgG induced after pig‐to‐primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N‐acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. Methods The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK‐B4T) was produced. Expression of porcine B4GALNT2 in HEK‐B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK‐B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK‐B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non‐Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. Results The porcine B4 GALNT 2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4 GALNT 2 genes, respectively. The B4 GALNT 2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4 GALNT 2 in human HEK cells ( HEK ‐B4T) results in increased binding of antibody to the B4 GALNT 2 enzyme, and increased reactivity with anti‐Sd a and DBA . HEK ‐B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO : CD 55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non‐Gal antibody, measured using GTKO PAEC s, and the induction of antibodies which preferentially bind to HEK ‐B4T cells. Conclusion The functional isolation of the porcine B4 GALNT 2 gene from a PAEC expression library, the pattern of B4 GALNT 2 gene expression and its sensitization of HEK ‐B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4 GALNT 2 produces a new immunogenic non‐Gal glycan which contributes in part to the non‐Gal immune response detected after pig‐to‐baboon cardiac xenotransplantation.

AbstractThe 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.

The heterotopic thoracic pig‐to‐baboon heart transplantation has been established by our group as a safe preclinical model. Since the recipient's own heart remains in place, it is possible to evaluate immunological reactions of various types and the symptoms of the thrombotic microangiopathy under working heart conditions. However, these experiments are complex and costly. In order to limit the number of transplants, we tested our multi‐transgenic porcine donor hearts in an ex‐vivo perfusion model first.Ex‐vivo model: Both beating ventricles were perfused with heparinized freshly drawn human whole blood using a centrifugal pump and a membrane oxygenator. During 3 h of observation, cardiac function parameters were obtained continuously; specimens from the perfusate, coronary sinus blood, and myocardial biopsies were assessed. Various genetically modified porcine donor hearts were tested; we are currently evaluating the impact of human thrombomodulin on the porcine microcirculation; the efficacy of complement regulation is another work package.Pig‐to‐baboon heterotopic cardiac xenotransplantation: In our latest group, seven baboons received five double (Gal‐KO/hCD46), and two triple (Gal‐KO/hCD46/hTM resp./HLA‐E) transgenic pig hearts. Our immunosuppressive regimen included preoperative anti‐CD20‐antibody, bortezomib, dexamethasone and cyclophosphamide; postoperatively, ATG, tacrolimus, MMF, anti‐CD20‐antibody, bortezomib and dexamethasone were administered. Total lymph node irradiation (6 Gy) was applied on postoperative day five.The triple transgenic hearts survived 35 and 37 days respectively. Both hearts maintained function throughout the experiment. Both recipients succumbed to fungal infections. Humoral rejection was seen only once. The next barrier appears to be the complex event of thrombotic microangiopathy that needs to be addressed with the additional expression of human thrombomodulin strategies.

This study is a retrospective analysis of the influence of previous cardiac surgery on the outcome of heart transplantation in 118 consecutive recipients from January 1988 to December 1996. Group 1 consisted of 67 patients who had no previous sternotomy, group 2 was 33 patients who had one previous sternotomy, and group 3 was 18 patients who had two or more previous sternotomies. The three groups were demographically similar apart from the indication for transplantation and the sex distribution. Preoperative hemodynamic data, except systolic pulmonary artery pressure, were similar among the three groups. There was no significant difference in hospital mortality, 1-year and 5-year actuarial survival rates, incidence of renal dysfunction requiring dialysis, prolonged respiratory support, elevated total bilirubin, re-exploration for bleeding, incidence of mediastinitis, or postoperative hospital stay between the groups. The rejection-free and infection-free survival rates were similar in the three groups. However, previous sternotomy resulted in significantly longer cardiopulmonary bypass times and increased requirement for blood and blood products. The use of a cell saver limited the need for stored red blood cells. This study demonstrated no survival disadvantage of previous sternotomy in patients undergoing heart transplantation.