HDAC activity is dispensable for repression of cell-cycle genes by DREAM and E2F:RB complexes.

Histone deacetylases (HDACs) play a crucial role in transcriptional regulation and are implicated in various diseases, including cancer. They are involved in histone tail deacetylation and canonically linked to transcriptional repression. Previous studies suggested that HDAC recruitment to cell-cycle gene promoters via the retinoblastoma (RB) protein or the DREAM complex through SIN3B is essential for G1/S and G2/M gene repression during cell-cycle arrest and exit. Here we investigate the interplay among DREAM, RB, SIN3 proteins, and HDACs in the context of cell-cycle gene repression. Knockout of SIN3B does not globally derepress cell-cycle genes in non-proliferating HCT116 and C2C12 cells. Loss of SIN3A/B moderately upregulates several cell-cycle genes in HCT116 cells but does so independently of DREAM/RB. HDAC inhibition does not induce general upregulation of RB/DREAM target genes in arrested transformed or non-transformed cells. Our findings suggest that E2F:RB and DREAM complexes can repress cell-cycle genes without relying on HDAC activity.

Impact of Gabapentin on Postoperative Hypotension in Enhanced Recovery after Surgery Protocols for Microvascular Breast Reconstruction.

Background: Enhanced recovery after surgery (ERAS) protocols have been associated with hypotensive episodes after autologous breast reconstruction. Gabapentin (Gaba), a nonopioid analgesic used in ERAS, has been shown to attenuate postoperative hemodynamic responses. This study assesses ERAS's impact, with and without Gaba, on postoperative hypotension after microvascular breast reconstruction. Methods: Three cohorts were studied: traditional pathway, ERAS + Gaba, and ERAS no-Gaba. We evaluated length of stay, inpatient narcotic use [morphine milligram equivalents (MME)], mean systolic blood pressure, hypotension incidence, and complications. The traditional cohort was retrospectively reviewed, whereas the ERAS groups were enrolled prospectively after the initiation of the protocol in April 2019 (inclusive of Gaba until October 2022). Results: In total, 441 patients were analyzed. The three cohorts, in the order mentioned above, were similar in age and bilateral reconstruction rates (57% versus 61% versus 60%). The ERAS cohorts, both with and without Gaba, had shorter stays (P < 0.01). Inpatient MME was significantly less in the ERAS + Gaba cohort than the traditional or ERAS no-Gaba cohorts (medians: 112 versus 178 versus 158 MME, P < 0.01). ERAS + Gaba significantly increased postoperative hypotensive events on postoperative day (POD) 1 and 2, with notable reduction after Gaba removal (P < 0.05). Across PODs 0-2, mean systolic blood pressure was highest in the traditional cohort, followed by ERAS no-Gaba, then the ERAS + Gaba cohort (P < 0.05). Complication rates were similar across all cohorts. Conclusions: Postmicrovascular breast reconstruction, ERAS + Gaba reduced overall inpatient narcotic usage, but increased hypotension incidence. Gaba removal from the ERAS protocol reduced postoperative hypotension incidence while maintaining similar stay lengths and complication rates.

The Young Investigator Committee of the International Xenotransplantation Association-Perspective of advancements in the field in 2023.

The 2023 IXA conference, hosted in San Diego, CA, brimmed with excitement against the backdrop of recent innovations in both the pre-clinical and clinical realms with several first-in-human applications of xenotransplantation. The theme, "Pigs are flying," alluded to the adage that xenotransplantation would only become a clinical reality "when pigs fly," suggesting a day that might never come. The event witnessed significant attendance, with 600 participants-the highest in the history of an IXA-IPITA joint congress. Among the attendees were members of the Food and Drug Administration (FDA), the National Institutes of Health (NIH), and corporate sponsors deeply engaged in the field. We summarize the latest topics from the congress, ranging from the pros/cons of decedent models of xenotransplantation and genetic engineering of porcine heart valves, solid organs, and cells for clinical translation and their regulatory and ethical landscape.

Enhanced Recovery after Surgery Protocol Decreases Length of Stay and Postoperative Narcotic Use in Microvascular Breast Reconstruction.

Background: Enhanced recovery after surgery (ERAS) protocols have demonstrated efficacy following microvascular breast reconstruction. This study assesses the impact of an ERAS protocol following microvascular breast reconstruction at a high-volume center. Methods: The ERAS protocol introduced preoperative counseling, multimodal analgesia, early diet resumption, and early mobilization to our microvascular breast reconstruction procedures. Data, including length of stay, body mass index, inpatient narcotic use, outpatient narcotic prescriptions, inpatient pain scores, and complications, were prospectively collected for all patients undergoing microvascular breast reconstruction between April 2019 and July 2021. Traditional pathway patients who underwent reconstruction immediately before ERAS implementation were retrospectively reviewed as controls. Results: The study included 200 patients, 99 in traditional versus 101 in ERAS. Groups were similar in body mass index, age (median age: traditional, 54.0 versus ERAS, 50.0) and bilateral reconstruction rates (59.6% versus 61.4%). ERAS patients had significantly shorter lengths of stay, with 96.0% being discharged by postoperative day (POD) 3, and 88.9% of the traditional cohort were discharged on POD 4 (P < 0.0001). Inpatient milligram morphine equivalents (MMEs) were smaller by 54.3% in the ERAS cohort (median MME: 154.2 versus 70.4, P < 0.0001). Additionally, ERAS patients were prescribed significantly fewer narcotics upon discharge (median MME: 337.5 versus 150.0, P < 0.0001). ERAS had a lower pain average on POD 0-3; however, this finding was not statistically significant. Conclusion: Implementing an ERAS protocol at a high-volume microvascular breast reconstruction center reduced length of stay and postoperative narcotic usage, without increasing pain or perioperative complications.

Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report.

BACKGROUND: A genetically engineered pig cardiac xenotransplantation was done on Jan 7, 2022, in a non-ambulatory male patient, aged 57 years, with end-stage heart failure, and on veno-arterial extracorporeal membrane oxygenation support, who was ineligible for an allograft. This report details our current understanding of factors important to the xenotransplantation outcome. METHODS: Physiological and biochemical parameters critical for the care of all heart transplant recipients were collected in extensive clinical monitoring in an intensive care unit. To ascertain the cause of xenograft dysfunction, we did extensive immunological and histopathological studies, including electron microscopy and quantification of porcine cytomegalovirus or porcine roseolovirus (PCMV/PRV) in the xenograft, recipient cells, and tissue by DNA PCR and RNA transcription. We performed intravenous immunoglobulin (IVIG) binding to donor cells and single-cell RNA sequencing of peripheral blood mononuclear cells. FINDINGS: After successful xenotransplantation, the graft functioned well on echocardiography and sustained cardiovascular and other organ systems functions until postoperative day 47 when diastolic heart failure occurred. At postoperative day 50, the endomyocardial biopsy revealed damaged capillaries with interstitial oedema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Increased anti-pig xenoantibodies, mainly IgG, were detected after IVIG administration for hypogammaglobulinaemia and during the first plasma exchange. Endomyocardial biopsy on postoperative day 56 showed fibrotic changes consistent with progressive myocardial stiffness. Microbial cell-free DNA testing indicated increasing titres of PCMV/PRV cell-free DNA. Post-mortem single-cell RNA sequencing showed overlapping causes. INTERPRETATION: Hyperacute rejection was avoided. We identified potential mediators of the observed endothelial injury. First, widespread endothelial injury indicates antibody-mediated rejection. Second, IVIG bound strongly to donor endothelium, possibly causing immune activation. Finally, reactivation and replication of latent PCMV/PRV in the xenograft possibly initiated a damaging inflammatory response. The findings point to specific measures to improve xenotransplant outcomes in the future. FUNDING: The University of Maryland School of Medicine, and the University of Maryland Medical Center.

Partial heart xenotransplantation: A research protocol in non-human primates.

Partial heart transplantation is a new type of transplant that delivers growing heart valve replacements for babies. Partial heart transplantation differs from orthotopic heart transplantation because only the part of the heart containing the heart valve is transplanted. It also differs from homograft valve replacement because viability of the graft is preserved by tissue matching, minimizing donor ischemia times, and recipient immunosuppression. This preserves partial heart transplant viability and allows the grafts to fulfill biological functions such as growth and self-repair. These advantages over conventional heart valve prostheses are balanced by similar disadvantages as other organ transplants, most importantly limitations in donor graft availability. Prodigious progress in xenotransplantation promises to solve this problem by providing an unlimited source of donor grafts. In order to study partial heart xenotransplantation, a suitable large animal model is important. Here we describe our research protocol for partial heart xenotransplantation in nonhuman primates.

A Standardized Approach to Orthotopic (Life-supporting) Porcine Cardiac Xenotransplantation in a Nonhuman Primate Model.

Cardiac xenotransplantation from swine has been proposed to "bridge the gap" in supply for heart failure patients requiring transplantation. Recent preclinical success using genetically modified pig donors in baboon recipients has demonstrated survival greater than 6 mo, with a modern understanding of xenotransplantation immunobiology and continued experience with large animal models of cardiac xenotransplantation. As a direct result of this expertise, the Food and Drug Administration approved the first in-human transplantation of a genetically engineered cardiac xenograft through an expanded access application for a single patient. This clinical case demonstrated the feasibility of xenotransplantation. Although this human study demonstrated proof-of-principle application of cardiac xenotransplantation, further regulatory oversight by the Food and Drug Administration may be required with preclinical trials in large animal models of xenotransplantation with long-term survival before approval of a more formalized clinical trial. Here we detail our surgical approach to pig-to-primate large animal models of orthotopic cardiac xenotransplantation, and the postoperative care of the primate recipient, both in the immediate postoperative period and in the months thereafter. We also detail xenograft surveillance methods and common issues that arise in the postoperative period specific to this model and ways to overcome them. These studies require multidisciplinary teams and expertise in orthotopic transplantation (cardiac surgery, anesthesia, and cardiopulmonary bypass), immunology, genetic engineering, and experience in handling large animal donors and recipients, which are described here. This article serves to reduce the barriers to entry into a field with ever-growing enthusiasm, but demands expertise knowledge and experience to be successful.

Intraoperative Management of an Orthotopic Porcine-to-Human Cardiac Xenotransplant.

We report the intraoperative management of an orthotopic cardiac xenotransplant in a 57-year-old man with nonischemic cardiomyopathy requiring venoarterial extracorporeal membrane oxygenation. Transesophageal echocardiography was used for preharvest assessment. Continuous ex vivo perfusion of the heart was performed. Steps were taken to avoid potential xenozoonosis transmission to other patients and staff. Preclinical experience guided our intraoperative management in controlling hemodynamics and using prophylactic antiarrhythmic medications. Echocardiography aided in the diagnosis of aortic dissection in the patient after transplant. Intraoperative cardiac function was excellent. The patient was weaned from all mechanical support 4 days after transplant.

Preclinical rationale and current pathways to support the first human clinical trials in cardiac xenotransplantation.

Recent initiation of the first FDA-approved cardiac xenotransplantation suggests xenotransplantation could soon become a therapeutic option for patients unable to undergo allotransplantation. Until xenotransplantation is widely applied in clinical practice, consideration of benefit versus risk and approaches to management of clinical xenografts will based at least in part on observations made in experimental xenotransplantation in non-human primates. Indeed, the decision to proceed with clinical trials reflects significant progress in last few years in experimental solid organ and cellular xenotransplantation. Our laboratory at the NIH and now at University of Maryland contributed to this progress, with heterotopic cardiac xenografts surviving more than two years and life-supporting cardiac xenografts survival up to 9 months. Here we describe our contributions to the understanding of the mechanism of cardiac xenograft rejection and development of methods to overcome past hurdles, and finally we share our opinion on the remaining barriers to clinical translation. We also discuss how the first in human xenotransplants might be performed, recipients managed, and graft function monitored.

The growth of xenotransplanted hearts can be reduced with growth hormone receptor knockout pig donors.

OBJECTIVE: Genetically engineered pigs are thought to be an alternative organ source for patients in end-stage heart failure unable to receive a timely allograft. However, cardiac xenografts exhibit growth and diastolic heart failure within 1 month after transplantation. Grafts function for up to 6 months, but only after administration of temsirolimus and afterload-reducing agents to reduce this growth. In this study we investigated the growth and hemodynamics of growth hormone receptor (GHR) knockout xenografts, without the use of adjuncts to prevent intrinsic graft growth after transplantation. METHODS: Genetically engineered pig hearts were transplanted orthotopically into weight-matched baboons between 15 and 30 kg, using continuous perfusion preservation before implantation (n = 5). Xenografts included knockout of carbohydrate antigens and knockin of human transgenes for thromboregulation, complement regulation, and inflammation reduction (grafts with intact growth hormone, n = 2). Three grafts contained the additional knockout of GHR (GHR knockout grafts; n = 3). Transthoracic echocardiograms were obtained twice monthly and comprehensively analyzed by a blinded cardiologist. Hemodynamics were measured longitudinally after transplantation. RESULTS: All xenografts demonstrated life-supporting function after transplantation. There was no difference in intrinsic growth, measured using septal and posterior wall thickness and left ventricular mass, on transthoracic echocardiogram out to 1 month in either GHR knockout or GHR intact grafts. However, hypertrophy of the septal and posterior wall was markedly elevated by 2 months post transplantation. There was minimal hypertrophy out to 6 months in GHR knockout grafts. Physiologic mismatch was present in all grafts after transplantation, which is largely independent of growth. CONCLUSIONS: Xenografts with GHR knockout show reduced post-transplantation xenograft growth using echocardiography >6 months after transplantation, without the need for other adjuncts.

CD40-CD40L Blockade: Update on Novel Investigational Therapeutics for Transplantation.

Effective immune responses require antigen presentation by major histocompatibility complexes with cognate T-cell receptor and antigen-independent costimulatory signaling for T-cell activation, proliferation, and differentiation. Among several costimulatory signals, CD40-CD40L is of special interest to the transplantation community because it plays a vital role in controlling or regulating humoral and cellular immunity. Blockade of this pathway has demonstrated inhibition of donor-reactive T-cell responses and prolonged the survival of transplanted organs. Several anti-CD154 and anti-CD40 antibodies have been used in the transplantation model and demonstrated the potential of extending allograft and xenograft rejection-free survival. The wide use of anti-CD154 antibodies was hampered because of thromboembolic complications in transplant recipients. These antibodies have been modified to overcome the thromboembolic complications by altering the antibody binding fragment (Fab) and Fc (fragment, crystallizable) receptor region for therapeutic purposes. Here, we review recent preclinical advances to target the CD40-CD40L pair in transplantation.

Stable binding to phosphatidylserine-containing membranes requires conserved arginine residues in tandem C domains of blood coagulation factor VIII.

At sites of vascular damage, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated platelet surfaces with activated factor IX (fIXa) to form the intrinsic "tenase" complex. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through ß-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex. In this study, we devised a novel platform for generating recombinant C1, C2, and C1C2 domain constructs and performed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Binding measurements between phosphatidylserine (PS)-containing lipid membrane surfaces and fVIII C domains demonstrated an ionic strength dependence on membrane binding affinity. Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. Lastly, we determined the X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of PS, which binds to a basic cleft and makes charge-charge contact with R2320. We conclude that basic clefts in the fVIII C domains bind to PS-containing membranes through conserved arginine residues via a C domain modularity, where each C domain possesses modest electrostatic-dependent affinity and tandem C domains are required for high affinity binding.

The road to the first FDA-approved genetically engineered pig heart transplantation into human.

We have been testing genetically engineered (GE) pig hearts and optimizing immunosuppression (IS) in non-human primates (NHPs) since 2005. We demonstrate how we translated this preclinical investigation into a US Food and Drug Administration (FDA)-approved clinical cardiac xenotransplantation. First, genetically engineered (GE) pig hearts were transplanted into the abdomen of NHP along with IS, which included anti-CD20 and anti-CD40-based co-stimulation blockade antibodies. We reported 945 days of survival of three gene GE pig hearts in NHPs. Building on this proof-of-concept, we tested 3-10 gene-modified GE pig hearts (in order to improve the immunocompatibility of the xenograft further) in a life-supporting orthotopic model, but had limited success due to perioperative cardiac xenograft dysfunction (PCXD). With novel non-ischemic continuous perfusion preservation (NICP), using the XVIVO Heart solution (XHS), life-supporting survival was extended to 9 months. We approached the FDA under an application for "Expanded Access" (EA), to transplant a GE pig heart in a patient with end-stage non-ischemic cardiomyopathy. He was without other therapeutic options and dependent on VA-ECMO. A team of FDA reviewers reviewed our preclinical research experience and data and allowed us to proceed. This clinical cardiac xenotransplantation was performed, and the patient survived for 60 days, demonstrating the translational preclinical investigation of cardiac xenotransplantation from bench to bedside. The ultimate etiology of graft failure is currently a topic of investigation and lessons learned will progress the field forward.

Anatomical Differences Between Human and Pig Hearts and Their Relevance for Cardiac Xenotransplantation Surgical Technique.

Cardiac xenotransplantation has been proposed to bridge the gap between supply and demand for patients with end-stage heart failure requiring transplantation. However, differences in pig anatomy compared with human anatomy require modification of the surgical approach. In addition, careful consideration should be given to size matching before transplantation. (Level of Difficulty: Advanced.).

Genetically Modified Porcine-to-Human Cardiac Xenotransplantation.

A 57-year-old man with nonischemic cardiomyopathy who was dependent on venoarterial extracorporeal membrane oxygenation (ECMO) and was not a candidate for standard therapeutics, including a traditional allograft, received a heart from a genetically modified pig source animal that had 10 individual gene edits. Immunosuppression was based on CD40 blockade. The patient was weaned from ECMO, and the xenograft functioned normally without apparent rejection. Sudden diastolic thickening and failure of the xenograft occurred on day 49 after transplantation, and life support was withdrawn on day 60. On autopsy, the xenograft was found to be edematous, having nearly doubled in weight. Histologic examination revealed scattered myocyte necrosis, interstitial edema, and red-cell extravasation, without evidence of microvascular thrombosis - findings that were not consistent with typical rejection. Studies are under way to identify the mechanisms responsible for these changes. (Funded by the University of Maryland Medical Center and School of Medicine.).

Adiponectin receptors sustain haematopoietic stem cells throughout adulthood by protecting them from inflammation.

How are haematopoietic stem cells (HSCs) protected from inflammation, which increases with age and can deplete HSCs? Adiponectin, an anti-inflammatory factor that is not required for HSC function or haematopoiesis, promotes stem/progenitor cell proliferation after bacterial infection and myeloablation. Adiponectin binds two receptors, AdipoR1 and AdipoR2, which have ceramidase activity that increases upon adiponectin binding. Here we found that adiponectin receptors are non-cell-autonomously required in haematopoietic cells to promote HSC quiescence and self-renewal. Adiponectin receptor signalling suppresses inflammatory cytokine expression by myeloid cells and T cells, including interferon-γ and tumour necrosis factor. Without adiponectin receptors, the levels of these factors increase, chronically activating HSCs, reducing their self-renewal potential and depleting them during ageing. Pathogen infection accelerates this loss of HSC self-renewal potential. Blocking interferon-γ or tumour necrosis factor signalling partially rescues these effects. Adiponectin receptors are thus required in immune cells to sustain HSC quiescence and to prevent premature HSC depletion by reducing inflammation.

Cardiac Xenotransplantation: Progress in Preclinical Models and Prospects for Clinical Translation.

Survival of pig cardiac xenografts in a non-human primate (NHP) model has improved significantly over the last 4 years with the introduction of costimulation blockade based immunosuppression (IS) and genetically engineered (GE) pig donors. The longest survival of a cardiac xenograft in the heterotopic (HHTx) position was almost 3 years and only rejected when IS was stopped. Recent reports of cardiac xenograft survival in a life-sustaining orthotopic (OHTx) position for 6 months is a significant step forward. Despite these achievements, there are still several barriers to the clinical success of xenotransplantation (XTx). This includes the possible transmission of porcine pathogens with pig donors and continued xenograft growth after XTx. Both these concerns, and issues with additional incompatibilities, have been addressed recently with the genetic modification of pigs. This review discusses the spectrum of issues related to cardiac xenotransplantation, recent progress in preclinical models, and its feasibility for clinical translation.

Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months.

We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with six gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (three gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted in the baboons. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the requirement of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective "multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.