Genetically engineered sheep: A new paradigm for future preclinical testing of biological heart valves.

BACKGROUND: Heart valve implantation in juvenile sheep to demonstrate biocompatibility and physiologic performance is the accepted model for regulatory approval of new biological heart valves (BHVs). However, this standard model does not detect the immunologic incompatibility between the major xenogeneic antigen, galactose-α-1,3-galactose (Gal), which is present in all current commercial BHVs, and patients who universally produce anti-Gal antibody. This clinical discordance leads to induced anti-Gal antibody in BHV recipients, promoting tissue calcification and premature structural valve degeneration, especially in young patients. The objective of the present study was to develop genetically engineered sheep that, like humans, produce anti-Gal antibody and mirror current clinical immune discordance. METHODS: Guide RNA for CRISPR Cas9 nuclease was transfected into sheep fetal fibroblasts, creating a biallelic frame shift mutation in exon 4 of the ovine α-galactosyltransferase gene (GGTA1). Somatic cell nuclear transfer was performed, and cloned embryos were transferred to synchronized recipients. Cloned offspring were analyzed for expression of Gal antigen and spontaneous production of anti-Gal antibody. RESULTS: Two of 4 surviving sheep survived long-term. One of the 2 was devoid of the Gal antigen (GalKO) and expressed cytotoxic anti-Gal antibody by age 2 to 3 months, which increased to clinically relevant levels by 6 months. CONCLUSIONS: GalKO sheep represent a new, clinically relevant advanced standard for preclinical testing of BHVs (surgical or transcatheter) by accounting for the first time for human immune responses to residual Gal antigen that persists after current BHV tissue processing. This will identify the consequences of immune disparity preclinically and avoid unexpected past clinical sequelae.

A Case Series of Long-Term Surgical Outcomes of Primary Pulmonary Artery Sarcomas With Opportunities for 3D-Printed Models in Surgical Planning.

There are limited data regarding the surgical management of primary pulmonary artery sarcomas (PPAS) because of their rarity and complicated diagnostic history. The objective of this study was to analyze our institution's long-term surgical management outcomes for PPAS in the absence of a care pathway. From May 1997 to June 2013, 8 patients (mean age 60.6 ± 11.8 years; range, 40-73 years; 5 women and 3 men) underwent surgical intervention for PPAS at our institution. The most common computed tomography finding was a luminal filling defect obstructing the pulmonary artery (PA), without evidence of extraluminal extension. Three patients underwent debulking/pulmonary endarterectomy alone and 5 patients underwent a more radical resection with PA patch angioplasty, PA resection and reconstruction, pulmonary valve replacement, and unilateral pneumonectomy. The mean postoperative survival in this series was 3.8 ± 3.6 years (range, 1-11.9 years), with 2 radical surgical resection patients alive at 4.9 and 11.9 years, respectively. For those patients with incomplete resection, 3-dimensional (3D) models were created to demonstrate the advantage of a preoperative guide for a more complete resection and what it would entail. Six patients had local recurrences with mean disease-free interval of 14 ± 10.9 months (range, 2 months-2.5 years), and 2 patients with re-resections had an overall postoperative survival of 2.8 and 11.9 years, respectively. In our small cohort of PPAS, patients treated with radical surgical resection had better survival. The small number of PPAS cases in this series makes proving this association unlikely but warrants consideration.

Proteomic Analysis of the Myocardium in Hypertrophic Obstructive Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is characterized by a complex phenotype that is only partly explained by the biological effects of individual genetic variants. The aim of this study was to use proteomic analysis of myocardial tissue to explore the postgenomic phenotype. METHODS: Label-free proteomic analysis was used initially to compare protein profiles in myocardial samples from 11 patients with HCM undergoing surgical myectomy with control samples from 6 healthy unused donor hearts. Differentially expressed proteins of interest were validated in myocardial samples from 65 unrelated individuals (HCM [n=51], controls [n=7], and aortic stenosis [n=7]) by the development and use of targeted multiple reaction monitoring-based triple quadrupole mass spectrometry. RESULTS: In this exploratory study, 1586 proteins were identified with 151 proteins differentially expressed in HCM samples compared with controls ( P<0.05). Protein expression profiling showed that many proteins identified in the initial discovery study were associated with metabolism, muscle contraction, calcium regulation, and oxidative stress. Proteins downregulated in HCM versus controls included creatine kinase M-type, fructose-bisphosphate aldolase A, and phosphoglycerate mutase ( P<0.001). Proteins upregulated in HCM included lumican, carbonic anhydrase 3, desmin, α-actin skeletal, and FHL1 (four and a half LIM domain protein 1; P<0.01). Myocardial lumican concentration correlated with the left atrial area (ρ=0.34, P=0.015), late gadolinium enhancement on cardiac magnetic resonance imaging ( P=0.03) and the presence of a pathogenic sarcomere mutation ( P=0.04). CONCLUSIONS: The myocardial proteome of HCM provides supporting evidence for dysregulation of metabolic and structural proteins. The finding that lumican is raised in HCM hearts provides insight into the myocardial fibrosis that characterizes this disease.

Individualized surgical strategies for left ventricular outflow tract obstruction in hypertrophic cardiomyopathy.

OBJECTIVES: Surgical strategies to treat drug refractory left ventricular outflow tract obstruction (LVOTO) in hypertrophic cardiomyopathy include septal myectomy (SM) and, less frequently, mitral valve (MV) repair or replacement. The primary aim of this study was to report the surgical technique and management outcomes in a consecutive group of patients with variable phenotypes of hypertrophic cardiomyopathy in a broad national specialist practice. METHODS: A total of 203 consecutive patients, 132 men (mean age 48.6 ± 14.6 years) underwent surgery for the management of LVOTO. Surgical approaches included SM (n = 159), SM with MV repair (n = 25), SM with MV replacement (n = 9) and MV replacement alone (n = 10). Specific surgical approaches were performed based on the underlying mechanism of obstruction. Eleven (5.4%) patients had previous alcohol septal ablation for the management of LVOTO. Concomitant non-mitral cardiac procedures were carried out in 22 (10.8%) patients. RESULTS: Operative survival rate was 99.0% with 2 deaths within 30 days. The mean bypass time was 92.9 ± 47.8 min, with a mean length of hospital stay of 10.5 ± 7.8 days. Surgical complications included 3 ventricular septal defects requiring repair (1.5%), 1 Gerbode defect surgically repaired, 2 aortic valve repairs (1.0%), 2 transient ischaemic attacks (1.0%) and 4 strokes (2.0%). Thirty-nine (19.2%) patients had perioperative new-onset atrial fibrillation and 8 (3.9%) patients had unexpected atrioventricular block requiring a permanent pacemaker. Mean resting left ventricular outflow tract gradient improved from 70.6 ± 40.3 mmHg preoperatively to 11.0 ± 10.5 mmHg at 1 year postoperatively (P < 0.001). Mean New York Heart Association class improved from 2.6 ± 0.5 preoperatively to 1.6 ± 0.6 at 1 year after the procedure. CONCLUSIONS: In variable phenotypes of LVOTO in hypertrophic cardiomyopathy, an individualized surgical approach provided effective reductions in left ventricular outflow tract gradients and good symptomatic relief with acceptable mortality and morbidity.

Long-term outcomes for different surgical strategies to treat left ventricular outflow tract obstruction in hypertrophic cardiomyopathy.

AIMS: Surgical intervention is used to treat dynamic left ventricular outflow tract obstruction (LVOTO) in hypertrophic cardiomyopathy. This study assesses the effect of different surgical strategies on long-term mortality and morbidity. METHODS AND RESULTS: In total, 347 patients underwent surgical intervention for LVOTO (1988-2015). Group A (n = 272) underwent septal myectomy; Group B (n = 33), septal myectomy and mitral valve (MV) repair; Group C (n = 22), myectomy and MV replacement; and Group D (n = 20), MV replacement alone. Median follow-up was 5.2 years (interquartile range 1.9-7.9). The mean resting LVOT gradient improved post-operatively from 71.9 ± 39.6 mmHg to 13.4 ± 18.5 mmHg (P < 0.05). Overall, 72.4% of patients improved by >1 New York Heart Association (NYHA) class; 58.9% of patients undergoing MV replacement alone did not improve their NYHA class. There were 5 perioperative deaths and 20 late deaths (>30 days). Survival rates at 1, 5 and 10 years respectively were 98.4, 96.9, 91.9% in Group A; 97.0, 92.4, 61.6% in Group B; 100.0, 100.0, 55.6% in Group C; and 94.7, 85.3, 85.3% in Group D (log-rank, P < 0.05). Long-term (>30 days) complications included atrial fibrillation (29.6%), transient ischaemic attack/stroke (2.4%) and heart failure hospitalisation (3.2%). There were 16 repeat surgical interventions at 3.0 years. CONCLUSION: Septal myectomy is a safe procedure resulting in symptomatic improvement in the majority of patients. The annual incidence of non-fatal disease-related complications after surgical treatment of LVOTO is relatively high. Patients who underwent MV replacements had poorer outcomes with less symptomatic benefit in spite of a similar reduction in LVOT gradients.

Porcine to Human Heart Transplantation: Is Clinical Application Now Appropriate?

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.

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.

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.

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.

Recent investigations into pig antigen and anti-pig antibody expression.

Genetic engineering of donor pigs to eliminate expression of the dominant xenogeneic antigen galactose α1,3 galactose (Gal) has created a sea change in the immunobiology of xenograft rejection. Antibody mediated xenograft rejection of GGTA-1 α-galactosyltransferase (GTKO) deficient organs is now directed to a combination of non-Gal pig protein and carbohydrate antigens. Glycan analysis of GTKO tissues identified no new neo-antigens but detected high levels of N-acetylneuraminic acid (Neu5Gc) modified glycoproteins and glycolipids. Humans produce anti-Neu5Gc antibody and in very limited clinical studies sometimes show an induced anti-Neu5Gc antibody response after challenge with pig tissue. The pathogenicity of anti-Neu5Gc antibody in xenotransplantation is not clear however as non-human transplant models, critical for modelling anti-Gal immunity, do not produce anti-Neu5Gc antibody. Antibody induced after xenotransplantation in non-human primates is directed to an array of pig endothelial cells proteins and to a glycan produced by the pig B4GALNT2 gene. We anticipate that immune suppression will significantly affect the T-cell dependent and independent specificity of an induced antibody response and that donor pigs deficient in synthesis of multiple xenogeneic glycans will be important to future studies.

Current status of pig heart xenotransplantation.

Significant progress in understanding and overcoming cardiac xenograft rejection using a clinically relevant large animal pig-to-baboon model has accelerated in recent years. This advancement is based on improved immune suppression, which attained more effective regulation of B lymphocytes and possibly newer donor genetics. These improvements have enhanced heterotopic cardiac xenograft survival from a few weeks to over 2 years, achieved intrathoracic heterotopic cardiac xenograft survival of 50 days and orthotopic survival of 57 days. This encouraging progress has rekindled interest in xenotransplantation research and refocused efforts on preclinical orthotopic cardiac 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.

A retrospective analysis of myocardial preservation techniques during coronary artery bypass graft surgery: are we protecting the heart?

BACKGROUND: Retrograde perfusion into coronary sinus during coronary artery bypass graft (CABG) surgery reduces the need for cardioplegic interruptions and ensures the distribution of cardioplegia to stenosed vessel territories, therefore enhancing the delivery of cardioplegia to the subendocardium. Peri-operative myocardial injury (PMI), as measured by the rise of serum level of cardiac biomarkers, has been associated with short and long-term clinical outcomes. We conducted a retrospective analysis to investigate whether the combination of antegrade and retrograde techniques of cardioplegia delivery is associated with a reduced PMI than that observed with the traditional methods of myocardial preservation. METHODS: Fifty-four consecutive patients underwent CABG surgery using either antegrade cold blood cardioplegia (group 1, n = 28) or cross-clamp fibrillation (group 2, n = 16) or antegrade retrograde warm blood cardioplegia (group 3, n = 10). The study primary end-point was PMI, evaluated with total area under the curve (AUC) of high-sensitivity Troponin-T (hsTnT), measured pre-operatively and at 6, 12, 24, 48 and 72 hours post-surgery. Secondary endpoints were acute kidney injury (AKI) and inotrope scores, length of intensive care unit (ICU) and hospital stay, new onset atrial fibrillation (AF) and clinical outcomes at 6 weeks (death, non-fatal myocardial infarction, coronary artery revascularization, stroke). RESULTS: There was evidence that mean total AUC of hsTnT was different among the three groups (P = 0.050). In particular mean total AUC of hsTnT was significantly lower in group 3 compared to both group 1 (-16.55; 95% CI: -30.08, -3.01; P = 0.018) with slightly weaker evidence of a lower mean hsTnT in group 3 when compared to group 2 (-15.13; 95% CI -29.87, -0.39; P = 0.044). There was no evidence of a difference when comparing group 2 to group 1 (-1.42,; 95% CI: -12.95, 10.12, P = 0.806). CONCLUSIONS: Our retrospective analysis suggests that, compared to traditional methods of myocardial preservation, antegrade retrograde cardioplegia may reduce PMI in patients undergoing first time CABG surgery.

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.

Somatic MYH7, MYBPC3, TPM1, TNNT2 and TNNI3 mutations in sporadic hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a clinically heterogeneous genetic heart disease characterized by left ventricular hypertrophy in the absence of another disease that could explain the wall thickening. Elucidation of the genetic basis of HCM lead to the identification of several genes encoding sarcomeric proteins, such as MYH7, MYBPC3, TPM1, TNNT2, and TNNI3. Sarcomeric genes are mutated in approximately 40% of HCM patients and a possible explanation for the incomplete yield of mutation-positive HCM may be somatic mutations. METHODS AND RESULTS: We studied 104 unrelated patients with non-familial HCM. Patients underwent clinical evaluation and mutation screening of 5 genes implicated in HCM (MYH7, MYBPC3, TPM1, TNNT2, and TNNI3) in genomic DNA isolated from resected cardiac tissue; 41 of 104 were found to carry a mutation, but as several patients carried the same mutations, the total amount of different mutations was 37; 20 of these mutations have been previously described, and pathogenicity has been assessed. To determine the effect of the 17 new mutations an in silico assay was performed and it predicted that 4 variants were damaging mutations. All identified variants were also seen in the DNA isolated from the corresponding blood, which demonstrated the absence of somatic mutations. CONCLUSIONS: Somatic mutations in MYH7, MYBPC3, TPM1, TNNT2, and TNNI3 do not represent an important etiologic pathway in HCM.

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.