Anti-pig antibodies in swine veterinarian serum: Implications for clinical xenotransplantation.

Recent clinical xenotransplantation and human decedent studies demonstrate that clinical hyperacute rejection of genetically engineered porcine organs can be reliably avoided but that antibody mediated rejection (AMR) continues to limit graft survival. We previously identified porcine glycans and proteins which are immunogenic after cardiac xenotransplantation in non-human primates, but the clinical immune response to antigens present in glycan depleted triple knockout (TKO) donor pigs is poorly understood. In this study we use fluorescence barcoded human embryonic kidney cells (HEK) and HEK cell lines expressing porcine glycans (Gal and SDa) or proteins (tetraspanin-29 [CD9], membrane cofactor protein [CD46], protectin, membrane attack complex inhibition factor [CD59], endothelial cell protein C receptor, and Annexin A2) to screen antibody reactivity in human serum from 160 swine veterinarians, a serum source with potential occupational immune challenge from porcine tissues and pathogens. High levels of anti-Gal IgM were present in all samples and lower levels of anti-SDa IgM were present in 41% of samples. IgM binding to porcine proteins, primarily CD9 and CD46, previously identified as immunogenic in pig to non-human primate cardiac xenograft recipients, was detected in 28 of the 160 swine veterinarian samples. These results suggest that barcoded HEK cell lines expressing porcine protein antigens can be useful for screening human patient serum. A comprehensive analysis of sera from clinical xenotransplant recipients to define a panel of commonly immunogenic porcine antigens will likely be necessary to establish an array of porcine non-Gal antigens for effective monitoring of patient immune responses and allow earlier therapies to reverse AMR.

Early and medium-term outcomes of Alfieri mitral valve repair in the management of systolic anterior motion during septal myectomy.

BACKGROUND: This report studies the early and medium-term clinical and echocardiographic outcomes of the Alfieri edge-to-edge mitral valve repair, as adjunctive therapy, to prevent and treat systolic anterior motion (SAM) at the time of septal myectomy (SM) for left ventricular outflow tract obstruction in hypertrophic cardiomyopathy. METHODS: From 2009-2015, 11 consecutive patients had a trans-atrial Alfieri repair, to prevent (n = 7) or treat (n = 4) SAM at the time of SM. RESULTS: No patients were lost to follow-up. There were no perioperative or late deaths. Pre-bypass, the mean left ventricular outflow tract gradient, measured directly by simultaneous needle insertion, was 40.7 ± 19.9 mmHg at rest and 115.8 ± 30.4 mmHg on provocation with Isoproterenol, which reduced after SM and Alfieri repair and discontinuation of bypass, to a mean gradient of 8.3 ± 9.8 mmHg at rest and 25.8 ± 9.2 mmHg on provocation. One patient who required mitral valve replacement on day 4, was hospitalized at 2.7 years with heart failure requiring diuresis and remains well at 6 years. One patient developed postoperative atrial fibrillation. There were no other early or late complications. At a median follow-up of 6.6 years (international quartile range 1.2-7.4), clinical and echocardiographic data demonstrated maintained improvement in mean New York Heart Association class from 2.6 ± 0.9 preoperatively to 1.7 ± 0.4 and reduction in mean grade of mitral regurgitation from 2.7 ± 0.8 preoperatively to 0.7 ± 0.6. CONCLUSIONS: The Alfieri repair, as adjunctive therapy, for the prevention or treatment of SAM at the time of SM demonstrates satisfactory early and medium-term clinical and echocardiographic outcomes supporting the ongoing utility of this approach.

A pig-to-mouse coronary artery transplantation model for investigating the pathogenicity of anti-pig antibody.

BACKGROUND: Rejection 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. METHODS: Wild-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. RESULTS: Dose-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. CONCLUSIONS: This 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.

Pre-clinical heterotopic intrathoracic heart xenotransplantation: a possibly useful clinical technique.

BACKGROUND: As 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. METHODS: Consecutive experiments were carried out between 2009 and 2013. Twenty-one genetically modified pigs (GGTA1-knockout/hCD46/± thrombomodulin, in one case HLA-E instead) were used as donors. In all experiments, two cycles of immunoabsorption reduced preformed antibodies. Recipient baboons were divided into two groups according to IS regimen: 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 conventional IS; postoperative total thoracic and abdominal lymphoid irradiation (TLI; single dose of 600 cGY) was used to further reduce antibody-producing cells. RESULTS: In 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 or IS regimen. 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 to TLI. CONCLUSIONS: Longer-term results were difficult to achieve in this model due to the IS regimens used. However, we conclude that heterotopic intrathoracic heart transplantation may be an option for clinical xenotransplantation.

Cloning and expression of porcine ß1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen.

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 cDNAs 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 ß1,4 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 B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes, respectively. The B4GALNT2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4GALNT2 in human HEK cells (HEK-B4T) results in increased binding of antibody to the B4GALNT2 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:CD55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non-Gal antibody, measured using GTKO PAECs, and the induction of antibodies which preferentially bind to HEK-B4T cells. CONCLUSION: The functional isolation of the porcine B4GALNT2 gene from a PAEC expression library, the pattern of B4GALNT2 gene expression and its sensitization of HEK-B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4GALNT2 produces a new immunogenic non-Gal glycan which contributes in part to the non-Gal immune response detected after pig-to-baboon cardiac xenotransplantation.

Development of a consensus protocol to quantify primate anti-non-Gal xenoreactive antibodies using pig aortic endothelial cells.

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.

Histopathologic insights into the mechanism of anti-non-Gal antibody-mediated pig cardiac xenograft rejection.

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.

Gal-knockout bioprostheses exhibit less immune stimulation compared to standard biological heart valves.

BACKGROUND AND AIM OF THE STUDY: Current biological heart valves (BHVs) contain the major xenogeneic antigen Gal. Recipient anti-Gal antibody binding to such an implanted BHV may contribute to valve degeneration. The study aim was to compare, by implantation in non-human primates, the immune consequences of BHVs from Gal-positive wild-type (WT) pigs and those from alpha-galactosyltransferase knockout (GTKO) pigs. METHODS: Recipients were immunized prior to implant with keyhole limpet hemocyanin (KLH) conjugated to alphaGal to match the anti-Gal levels and isotypes found in humans. Stented glutaraldehyde-fixed BHVs from WT (n = 4) and GTKO (n = 3) pigs were commercially manufactured and implanted in the mitral position in non-human primates. Recipients were treated with enoxaparin (1 mg/kg b.i.d.) for five weeks which was tapered, and then discontinued. Serum antibody levels to Gal and KLH were measured using ELISA. RESULTS: Overall anti-Gal and anti-KLH antibody levels were decreased in both WT and GTKO BHV recipients after implantation. Serum anti-Gal IgG levels in GTKO BHV recipients fell rapidly within one month, matching the loss of anti-KLH reactivity. There was no significant difference in retention of anti-KLH antibody between the groups. WT BHV recipients retained significantly elevated levels of anti-Gal IgG during the first year post implant. Area under the curve analysis showed that anti-Gal IgG was significantly increased in the WT BHV group compared to GTKO BHV recipients (p < 0.01). CONCLUSION: Persistent and significantly (p < 0.01) elevated levels of anti-Gal IgG were observed in WT but not GTKO BHV non-human primate recipients, and indicated a continuing BHV-specific immune stimulation to the alphaGal antigen. These data support the hypothesis that the clinical use of Gal-positive xenogeneic bioprosthetic materials can induce an anti-Gal antibody response. Bioprosthetic devices prepared from GTKO pig tissue would eliminate immune stimulation to this major xenoreactive antigen, which may reduce the potential of immune-mediated injury and degeneration.