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.

Regulatory barriers to xenotransplantation.

PURPOSE OF REVIEW: There is a grave discordance between supply and demand for patients with failing organs largely due to an insufficient donor pool for transplantation. Xenotransplantation has been proposed as a solution to bridge this gap. RECENT FINDINGS: Recent success over the last decade in nonhuman primate models, due to emerging gene-editing technologies combined with novel immunosuppression regimens, has produced promising results in pancreatic islet cell, heart, lung, kidney and liver xenotransplantations. SUMMARY: As the prospect of xenotransplantation is realized, safety and ethical considerations have come to the forefront of discussion. The WHO and World Health Assembly have encouraged member states to form regulatory bodies to govern human xenotransplantation studies with the highest standards. Here, we summarize the current regulatory landscape governing preclinical advances toward the first human clinical trials.

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.

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.

Heart xenotransplantation.

PURPOSE OF REVIEW: Cardiac xenotransplantation has entered an exciting era, marked by numerous considerable advances in the field, and continues to be of great interest because of its potential ability in ameliorating the current constraints of limited allograft availability. This review aims to examine recent progress in this rapidly changing discipline. RECENT FINDINGS: Although several hurdles remain, the use of rapidly evolving transgenic technology, in combination with novel immunosuppression regimens, has the potential to address current allogenic donor pool constraints and mechanical circulatory system device limitations. Furthermore, innovative uses of biomarker tools, such as miRNA, serve as a method for improved monitoring of xenograft status. These tools may allow for more targeted immunosuppressive strategies as well as earlier interventions to mitigate xenograft rejection. Finally, coinciding with these remarkable advances, preliminary consideration of the clinical application for cardiac xenotransplantation has begun, particularly regarding specific patient criteria for these initial clinical trials. SUMMARY: The studies in this review highlight efforts in multiple disciplines to optimize perioperative and postxenotransplant outcomes. In examining these individual topics, this article reflects the exciting and ongoing progress in the field of cardiac xenotransplantation.

Early Tracheostomy May Be Performed Safely in Obese COVID-19 Patients Supported on VV-ECMO.

OBJECTIVE: Obese patients with coronavirus disease 2019 (COVID-19)-associated acute respiratory failure (ARDS) often require prolonged intubation. However, data are sparse regarding optimal tracheostomy timing in obese adults with COVID-19 requiring venovenous extracorporeal membrane oxygenation (VV-ECMO). This study retrospectively describes the course of obese patients with COVID-19 who underwent tracheostomy on VV-ECMO between March 2020 and December 2022. METHODS: There were 62 participants with a median age of 43 (interquartile range [IQR] 33 to 53) years and a median body mass index of 42 (IQR 34 to 50) kg/m2 who received VV-ECMO for COVID-19-associated ARDS. Of those, 42 underwent tracheostomy on VV-ECMO, and 50% (n = 21) of the 42 patients underwent early (within 14 days of initiated ventilatory support) tracheostomy. RESULTS: Among patients who received tracheostomies, the combined respiratory tract and lung parenchymal bleeding rate was 29% (n = 12), but only 7% required surgical intervention for bleeding from the tracheostomy site (n = 3). The hospital length of stay (LOS) was 42 (IQR 36 to 57) days, and mortality rate was 38% (n = 16). Tracheostomy timing was not associated with differences in respiratory tract bleeding, mechanical ventilatory support duration, VV-ECMO support duration, intensive care unit LOS, hospital LOS, mortality, or survival probability. CONCLUSIONS: Although an individualized and holistic approach to clinical decision making continues to be necessary, the findings of this study suggest that early tracheostomy may be performed safely in obese patients with COVID-19 on VV-ECMO.

Tetralogy of Fallot and Aortic Dissection: Implications in Management.

We present the case of a 61-year-old man with tetralogy of Fallot postrepair and mechanical aortic valve replacement with an aortic root/ascending/arch aneurysm with chronic type A aortic dissection. He underwent uncomplicated aortic root and total arch replacement. Continued surveillance for aortic aneurysm is necessary in the tetralogy of Fallot population. (Level of Difficulty: Intermediate.).

Intramyocardial Bone Marrow Stem Cells in Patients Undergoing Cardiac Surgical Revascularization.

BACKGROUND: Bone marrow stromal or stem cells (BMSCs) remain a promising potential therapy for ischemic cardiomyopathy. The primary objective of this study was to evaluate the safety and feasibility of direct intramyocardial injection of autologous BMSCs in patients undergoing transmyocardial revascularization (TMR) or coronary artery bypass graft surgery (CABG). METHODS: A phase I trial was conducted on adult patients who had ischemic heart disease with depressed left ventricular ejection fraction and who were scheduled to undergo TMR or CABG. Autologous BMSCs were expanded for 3 weeks before the scheduled surgery. After completion of surgical revascularization, BMSCs were directly injected into ischemic myocardium. Safety and feasibility of therapy were assessed. Cardiac functional status and changes in quality of life were evaluated at 1 year. RESULTS: A total of 14 patients underwent simultaneous BMSC and surgical revascularization therapy (TMR+BMSCs = 10; CABG+BMSCs = 4). BMSCs were successfully expanded, and no significant complications occurred as a result of the procedure. Regional contractility in the cell-treated areas demonstrated improvement at 12 months compared with baseline (TMR+BMSCs Δ strain: -4.6% ± 2.1%; P = .02; CABG+MSCs Δ strain: -4.2% ± 6.0%; P = .30). Quality of life was enhanced, with substantial reduction in angina scores at 1 year after treatment (TMR+BMSCs: 1.3 ± 1.2; CABG+MSCs: 1.0 ± 1.4). CONCLUSIONS: In this phase I trial, direct intramyocardial injection of autologous BMSCs in conjunction with TMR or CABG was technically feasible and could be performed safely. Preliminary results demonstrate improved cardiac function and quality of life in patients at 1 year after treatment.

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.

Novel Direct Annuloplasty Fastener System for Minimally Invasive Mitral Valve Repair.

The development of less invasive approaches for mitral valve repair remains an important objective, particularly in patients with multiple comorbidities. We describe a novel method to affix a mitral valve annuloplasty ring in a minimally invasive manner. A delivery apparatus for an annuloplasty fastener system was designed. Two channels were created, one for advancing the annuloplasty ring, and another to accommodate the fastener applicator. Custom designed fasteners, either with a helical-shaped screw or a strap-shaped tack structure, were tested. Fasteners were primed within an application device and automatic alignment of fasteners was achieved to allow accurate firing of the fixators securing the ring. The delivery apparatus was constructed to be deployed within a 10 mm trocar through a left atrial approach. Using a cadaveric swine heart model, access to the mitral valve from the left atrium was obtained with insertion of a trocar. The delivery apparatus was accurately directed to the mitral annulus under echocardiographic guidance. Fasteners were placed along the annular plane to secure the annuloplasty ring. Both fastener designs achieved considerable fixation force; the helical-shaped screw was found to have significantly greater fixation force compared to the strap-shaped tack design. The annuloplasty ring remained intact and did not experience any structural deformity during the fixation process. The use of a novel fastener system was successful in deploying and securing a mitral valve annuloplasty ring. These promising results may have further application for minimally invasive mitral valve repairs. Additional evaluation of this procedure with pre-clinical in vivo animal studies is necessary.

A Multidisciplinary Protocol-Driven Approach to Improve Extubation Times After Cardiac Surgery.

BACKGROUND: Prolonged intubation after cardiac surgery is associated with significant morbidity. A fast-track extubation protocol primarily driven by bedside providers was instituted for all postoperative cardiac surgery patients to facilitate safe and expeditious extubation. METHODS: A retrospective review of 1,581 cardiac surgery patients over an 8-year period was performed. Before 2011, nonprotocolized standard perioperative management was utilized (n = 807). From 2011 onward, a fast-track extubation (FTE) protocol directed by bedside providers was instituted (n = 774). Postoperatively, patients were placed on pressure-regulated volume control and titrated down to minimal support to maintain peripheral capillary oxygen saturation greater than 94%. For patients deemed ready for weaning (no evidence of hypoxia, hemodynamic instability, and so forth), a 30-minute continuous positive airway pressure trial was performed. Patients meeting all neurologic, respiratory, and cardiovascular criteria were extubated. The impact of the FTE algorithm on timely extubation, clinical outcomes, and safety was assessed. RESULTS: Baseline preoperative and intraoperative characteristics were similar between pre-FTE and FTE groups. Before instituting the FTE protocol, the rate of early extubation (less than 6 hours) was 43.7%, and increased to 64.1% during the FTE era (p < 0.001). Median time to extubation was also found to be significantly decreased: 295 minutes (interquartile range: 288) versus 385 minutes (interquartile range: 362, p = 0.041). There was no statistically significant difference in reintubation rates or 30-day mortality. CONCLUSIONS: The institution of a bedside provider-directed FTE pathway reduced overall intubation times and increased the rate of early extubation, without an increase in reintubation or mortality. This program-wide multidisciplinary approach appears to promote safe and expeditious extubation of cardiac surgery patients.

Use of durable mechanical circulatory support on outcomes of heart-kidney transplantation.

OBJECTIVES: Previous studies have demonstrated that preheart transplant mechanical circulatory support (MCS) can lead to a small but significant increase in mortality. However, data on outcomes of patients with MCS who require simultaneous heart-kidney transplant are limited. METHODS: A retrospective review of simultaneous heart-kidney transplantations (HKTxs) performed at a single institution over a 5-year period was performed. Patients were divided based on the preoperative use of durable MCS. Renal graft-related end points were evaluated, including glomerular filtration rate following transplantation, prevalence of delayed renal graft function and freedom from antibody and cellular-mediated graft rejection. Patient-specific outcomes, including survival and frequency of non-fatal major adverse cardiac events at 1 year, were additionally assessed. RESULTS: During the study period, 50 HKTxs were performed, 14 of which had preoperative MCS. HKTx patients with and without MCS implantations had a similar prevalence of delayed graft function (57.1% vs 50.0%; P = 0.757). A numerical trend was observed towards a reduced glomerular filtration rate 1-month post-transplant in patients without an MCS device (81.2 ± 32.8 vs 64.4 ± 27.5; P = 0.072), but no significant difference was observed at 6 and 12 months. No significant difference was observed on the need for post-transplant renal replacement therapy, non-fatal major adverse cardiac events, freedom from graft rejection and overall survival at 1 year. CONCLUSIONS: The use of preoperative MCS in patients undergoing combined HKTx was not found to affect renal graft function post-transplantation and does not seem to be associated with increase in morbidity or mortality.

Characterizing Predictors and Severity of Vasoplegia Syndrome After Heart Transplantation.

BACKGROUND: Vasoplegia is characterized as a severe vasodilatory shock after cardiac surgery, and can be associated with substantial morbidity. Increased systemic inflammation and endothelial dysfunction, often related to prolonged cardiopulmonary bypass times, anesthesia, or mechanical circulatory support have been shown to be associated with the development of vasoplegia. We sought to identify risk factors and the impact of various degrees of vasoplegia after heart transplantation. METHODS: A retrospective review was conducted of 244 consecutive patients who underwent heart transplantation over a 3-year period. Patients were divided into three groups: no vasoplegia, mild vasoplegia (requiring one vasopressor), and moderate/severe vasoplegia (more than two vasopressors). One-year survival, freedom from rejection, and postoperative complication rates were assessed. Risk factors for vasoplegia subgroups were retrospectively identified. RESULTS: Vasoplegia syndrome was observed in 34.3% of patients after heart transplantation (mild, 74.1%; moderate/severe, 25.9%). Cardiopulmonary bypass time was significantly longer and pretransplant creatinine was significantly higher in the moderate/severe vasoplegia group. There was a strong trend toward greater use of mechanical circulatory support among moderate/severe vasoplegia patients compared with mild and no vasoplegia patients. After heart transplantation, 1-year survival, freedom from rejection, and need for hemodialysis were not significantly different between groups. CONCLUSIONS: Vasoplegia syndrome is common after heart transplantation. Risk factors for increased severity include longer cardiopulmonary bypass times and elevated preoperative creatinine. Although higher rates of mortality or graft rejection were not detected, vasoplegia was associated with prolonged intubation, greater blood product usage, and lengthened hospital stay. Further studies involving larger cohorts are warranted.

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.

Routine Use of Topical Bacitracin to Prevent Sternal Wound Infections After Cardiac Surgery.

BACKGROUND: The development of sternal wound infections remains a serious complication after cardiac surgery. A number of studies have assessed the use of topical antibiotics placed on the sternal edges. We evaluated the routine use of bacitracin ointment applied over the sternotomy skin incision as a prophylactic measure against sternal wound infections. METHODS: A retrospective review of all coronary artery bypass graft and valve surgery cases performed at a single institution between 2006 and 2015 was performed (n = 1,495). Appropriate preoperative intravenous antibiotics were administered for all patients. Bacitracin topical antibiotic ointment was routinely applied to the sternal surgical incision after skin closure for all patients during this period. The incidence of sternal wound infection was assessed. RESULTS: During this 9-year experience, no episodes of deep sternal wound infections were observed, compared with a predicted rate of 0.29% (interquartile range: 0.19% to 0.46%) based on The Society of Thoracic Surgeons National Database risk calculator. Four episodes of superficial sternal wound infections were noted; gram-positive organisms were cultured in the majority of these cases. Bacitracin ointment was well tolerated by patients, with no serious adverse effects reported. CONCLUSIONS: This study suggests that the routine application of topical bacitracin over the surgical skin incision is safe after cardiac surgery. Deep sternal wound infections were notably absent during this period. As a readily available and inexpensive therapy, this simple intervention may be a useful adjunct strategy in preventing sternal wound infections.

Selection of Patients for Initial Clinical Trials of Solid Organ Xenotransplantation.

Several groups have reported extended survival of genetically engineered pig organs in nonhuman primates, varying from almost 10 months for life-supporting kidney grafts and more than 2 years for non-life-supporting heart grafts to less than 1 month for life-supporting liver and lung grafts. We have attempted to define groups of patients who may not have an option to wait for an allograft. These include kidney, heart, and lung candidates who are highly-allosensitized. In addition, some kidney candidates (who have previously lost at least 2 allografts from rapid recurrence of native kidney disease) have a high risk of further recurrence and will not be offered a repeat allotransplant. Patients with complex congenital heart disease, who may have undergone previous palliative surgical procedures, may be unsuitable for ventricular assist device implantation. Patients dying of fulminant hepatic failure, for whom no alternative therapy is available, may be candidates for a pig liver, even if only as a bridge until an allograft becomes available. When the results of pig organ xenotransplantation in nonhuman primates suggest a realistic potential for success of a pilot clinical trial, highly selected patients should be offered participation.

Vasoplegia after heart transplantation: outcomes at 1 year.

OBJECTIVES: Vasoplegia syndrome is a potentially life-threatening condition that can occur following cardiopulmonary bypass. Heart transplantation is a recognized risk factor for developing this vasodilatory state. The objective of this study was to determine the effects of vasoplegia syndrome on 1-year heart transplant outcomes. METHODS: A retrospective review of orthotopic heart transplants at a single institution between November 2010 and December 2014 was performed. Of the 347 consecutive adult patients, 107 patients (30.8%) met criteria for vasoplegia syndrome. Preoperative factors and intraoperative variables were collected and compared between vasoplegia and non-vasoplegia cohorts. The incidence of postoperative complications, transplant rejection and patient survival within 1 year were evaluated. RESULTS: Demographics and preoperative medication profiles were similar in both groups, while mechanical circulatory support device use was associated with vasoplegia syndrome (30.8% vs 20.0%; P = 0.039). Perioperative characteristics such as longer cardiopulmonary bypass [165.0 (interquartile range [IQR] 74) min vs 140.0 (IQR 42.7) min; P < 0.001] and increased blood product usage (24.7 ± 17.2 units vs 17.7 ± 14.3 units; P < 0.001) were associated with vasoplegia. Non-vasoplegia patients were more likely to be extubated [42.9 (IQR 37.3) h vs 66.8 (IQR 50.2) h; P < 0.001] and discharged earlier [10.0 (IQR 6) days vs 14.0 (IQR 11.5) days; P < 0.001]. One-year patient survival (92.0% vs 88.6%; P = 0.338) and any-treated rejection rates (82.7% vs 84.3%; P = 0.569) were not significantly different between groups. CONCLUSIONS: Although vasoplegia syndrome was associated with an increase in perioperative morbidity, including greater mechanical ventilation time and hospital length of stay, no significant differences in survival or allograft rejection at 1 year was demonstrated.