2019 ASTS Research Grants Recipients

2019 ASTS Faculty Development Grant

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Burcin Ekser, MD, PhD
Indiana University 

Scaffold-Free 3D-Bioprinting of an Advanced Human Liver Model

Synopsis: Drug-induced liver injury represents a serious problem worldwide. To date, no liver model (2D or 3D) has been established that can solidly maintain long-term physiological function. Therefore, there is a critical need to develop novel cellular approaches for drug toxicity/discovery and immunology testing. Our preliminary studies have successfully created scaffold-free 3D-bioprintedporcineliver models using genetically-engineered porcine cells and3D-bioprintedhumanliver models using human hepatocytes, liver sinusoidal endothelial cells (LSECs), and fibroblasts. The overall objective f this proposal is the optimization of scaffold-free 3D-bioprinted human liver model using above mentioned cells plus human hepatic stellate cells (HSC)and cholangiocytes in a novel perfusion bioreactor system in order to prolong survival of 3D human livers up to 1 month. In Aim 1, we will optimize the 3D-bioprinted liver model with the addition of HSC and cholangiocytes and continuously perfuse them with human blood. Using different ratios of human hepatocytes / LSECs/ fibroblasts/ HSCs / and cholangiocytes, we will create the most advanced 5-cell 3D-liver model. In Aim 2, we will measure liver-specific gene expression in advanced 3D-bioprinted human livers and perform cytotoxicity assessment using several different compounds. The creation of scaffold-free 5-cell 3D-bioprinted advanced human liver models and their continues perfusion with human blood will open new research avenues in transplant immunology, drug toxicity, and drug discovery in a physiological liver environment.  

2019 ASTS Collaborative Scientist Grant

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Helen Haskell Hobbs, MD
University of Texas Southwestern Medical Center at Dallas 

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Malcolm MacConmara, BCh, BAO, MB
University of Texas Southwestern Medical Center at Dallas 

Predicting Liver Transplant
Outcomes Using Donor Genetics: Utility of HSD17B13 as a Novel Biomarker

Synopsis: The role of donor genetics in predicting transplant outcomes has yet to be examined. Steatotic livers are increasingly common and now comprise one third of all deceased donor livers. As a consequence, there is an urgent need to establish biomarkers that can be used to improve donor selection and predict post-transplant outcomes within these livers. Our institution has characterized a number of key genes linked to non-alcoholic fatty liver disease (NAFLD) pathophysiology (1-3) and more recently reported the first protective genetic polymorphism within the 17-beta-hydroxysteroid dehydrogenase 13 (HSD17B13) gene (4). This study seeks to determine the potential role that donor genetic testing, specifically for HSD17B13 genotype has in liver transplantation. We hypothesize that donor HSD17B13 genotype will be an independent predictor of organ utilization as well as post-transplant outcomes.  

This study will focus on two primary objectives. Firstly, using a donor DNA biorepository that contains DNA from over 1500 donors over the past 20 years, we will determine the frequency of donor HSD17B13 genotypes in liver donors for both transplanted and discarded grafts. The donor HSD17B13 genotype will be compared in concert with a complete donor organ assessment through data available in the UNOS STAR files. In the second objective, we will determine the role of donor HSD17B13 genotype in predicting early and late recipient post-transplant outcomes.  

This study will establish the role of donor HSD17B13 as a biomarker for outcomes using livers with steatosis. The data will contribute clinically relevant information that will assist the transplant community to individualize and optimize post-transplant care.  

2019 ASTS-Veloxis Fellowship in Transplantation

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Hunter Moore, MD, PhD
University of Colorado 

Persistent Microvasculature Thrombosis Caused by Fibrinolysis Shutdown Driving Delayed Graft Function in Liver Transplant Recipients

Synopsis: Disseminated intravascular coagulation (DIC) causing multiple organ failure (MOF) in critically ill patients through microvascular thrombosis has been a hypothesis for over half a century. Data supports that DIC related organ failure is attributable to low fibrinolytic activity. Animal work has documented vessel thrombosis in poorly functioning organs following hemorrhagic shock, which improve with fibrinolytic therapy after resuscitation. Recently, thrombelastography (TEG) has been used to define pathologically low fibrinolysis [fibrinolysis shutdown (SD)] that is associated with increased mortality from organ failure in trauma and thrombotic complications in liver transplant patients. Preliminary data from our institution supports that TEG detected SD in liver transplant recipients is associated with early allograft dysfunction (EAD presumably through persistent microvascular thrombosis.  

We hypothesize that preexisting microvascular clot from the donor liver in combination with the SD in the recipient is a two hit event that drives EAD. Breaking down these theoretic clots in high risk donors with tissue plasminogen activator (t-PA) has demonstrated benefit in reducing ischemic cholangiopathy, and mechanistically supports why SD after transplant would be pathologic. Our most recent data supports that those recipients whom remain in SD on post-operative day-5 (POD) have the highest risk of EAD. This five-day window supports that a treatment for SD has clinical utility. Therefore, treating the donor and recipient to normalize the fibrinolytic system has the potential to reduce EAD and improve outcomes. Before effective therapeutic interventions can be implemented it is essential to define the mechanisms driving microvascular clot and persistent SD in liver transplantation. 

2019 Jonathan P. Fryer Resident Scientist Scholarship

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Yvonne Kelly, MD
University of California, San Francisco

Understanding the Immunogenicity of the Parathyroid Gland, a Novel Adjunct to Enhance the Survival and Engraftment of Pancreatic Islet Cell Transplants

Synopsis: Over the last decade, there has been increased use of pancreatic islet transplantation to restore normoglycemia in patients with Type 1 diabetes, however widespread application has been limited by several barriers, particularly the sensitivity of islets to ischemia. Conversely, another highly vascularized endocrine tissue, the parathyroid gland, engrafts at very high success rates (>90%) in thousands of patients every year in the intramuscular site, a site considered to be too harsh for islets given its lack of vascularity. The success in parathyroid autotransplantation has been attributed to an unusually high percentage (3-5%) of CD45-CD34+ vascular endothelial progenitor cells resident in these glands. Over the last year and a half, our lab has been exploring the novel idea of parathyroid and islet co-transplantation with both human islets and stem-cell derived insulin producing cells (SCIPCs) in the intramuscular and subcutaneous sites in preclinical mouse models of diabetes. We have seen statistically significant improvement in islet engraftment and function and unprecedented reversal of diabetes and restoration of normoglycemia. As the possibility of islet and parathyroid composite transplants moves from bench to bedside, it will be important to understand how the parathyroid is supporting and protecting the islet cells. There are sparse published data suggesting the parathyroid has little no MHC expression and can remain functional in an allotransplant setting for several months without immunosuppression. This study aims to further our knowledge of the interaction between the parathyroid and the immune system, a relationship which could prove key to islet protection and survival.

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Dimitrios P. Moris, MD, MSc, PhD
Duke University 

Establishing A Linear Program for the Development of Costimulation Resistant T-Cells

Synopsis: Belatacept is an alternative for calcineurin-inhibitors in transplantation. It binds B7 costimulation molecules, CD80 and CD86, blocking their interactions with CD28 and depriving naïve T-cells of signals required for activation. Although belatacept is effective in most patients, a sizable minority of patients experience T-cell-mediated rejection. A growing body of work suggests that these rejections stem from the actions of memory T-cells with diminished co-stimulatory requirements. Indeed, studies from our lab, and others, have identified phenotypic characteristics of maturing T-cells that associate with belatacept resistance, including complete loss of CD28 expression. We have shown that these cells proliferate and produce high levels of effector cytokines when stimulated with alloantigen. 

Our hypothesis is that upon stimulation, naïve T-cells begin down a path that progressively reduces their need for CD28-B7 costimulation, endowing them with a phenotype of belatacept resistance. We posit that this maturation is linear and anticipatable, and the movement from co-stimulation dependence to independence involves specific, time-dependent signals, which lead to tractable changes in surface molecule expression with prognostic and therapeutic value.  

We will test this hypothesis, focusing on the acquisition of a specific surface marker, CD57, which we have shown to associate with human costimulation resistant rejection. Aim 1 will define a linear maturation scheme leading to CD57 acquisition and relate this to belatacept resistance. Aim 2 will examine the impact of immunosuppressant exposure at various stages along the differentiation pathway mapped in Aim 1, with specific attention to the differential effects of calcineurin inhibition versus mTOR inhibition.  

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Brian Shaw, MD
Duke University 

 

T cell Receptor Diversity as a Determinant of Costimulation Blockade Resistance 

Synopsis: Kidney transplantation is the definitive treatment for end stage renal disease. However, the most common immunosuppression used, calcineurin inhibitor–based therapy, has significant toxicities. Ideally, an immunosuppressive regimen would permit allograft survival without adding morbidity. 

Costimulation blockade-based regimens hold promise to decrease immunosuppressive toxicity but have been plagued by higher rates of acute rejection, termed Costimulation Blockade Resistant Rejection (CoBRR). Previous work has suggested that CoBRR is mediated by mature T cells that have become costimulation independent. We have shown that therapeutic T cell depletion capitalizes on differential proliferation characteristics of terminally differentiated cells leading to a more naïve and costimulation sensitive repertoire. IL-7 is critical in this differentiation. Consequently, we have demonstrated that patients may be weaned safely to belatacept monotherapy without rejection when induced with alemtuzumab and a course of sirolimus(ABR regimen).Though the immediate lack of CoBRR after induction may be explained by the change in memory phenotype, we hypothesize that repertoire diversity (specificity and maturation diversity) among costimulation resistant cells is a tractable metric for predicting CoBRR.  

In the present study, we propose to examine TCR diversity in our cohort of patients under the ABR regimen. Our specific aims seek to:  

  1. Determine overall changes in TCR diversity and memory status following T cell depletion and subsequent repopulation under the ABR regimen
  2. Determine qualitative changes in TCR diversity among donor specific alloreactive T cells and “3rdParty” specific alloreactive T cells under the ABR regimen with or without mesenchymal stem cell infusion 

    2019 ASTS Presidential Student Mentor Grant

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    Daniel P. Allen, Jr., BS
    Medical University of South Carolina 

    The Impact of aCT1 nebulization on Ischemia Reperfusion Injury in Lung Transplantation

    Synopsis: The Transplant Immunobiology Laboratory (TIBL) at the Medical University of South Carolina (MUSC)has focused research towards two main goals; evidence-based paradigm improvements in transplantation and widening the donor pool to treat more patients. One method the TIBL has studied to do this is the use of pretreatment therapies that are applied to the donor organs before transplantation rather than treating only with immunosuppressive drugs after transplantation, which is the current standard of care. Donor organs are already primed for an inflammatory response due to the insults received through brain death, organ procurement, hypoxia, and cold storage to name a few. In lung transplantation, we have a unique opportunity for nebulized treatment due to the organ’s exposure to the external environment. Additionally, nebulization is a highly economical and almost universally available delivery system that can be used to treat the lungs while they are still being perfused within the donor’s body. Our previous findings have shown positive results in mitigating damage propagated through ischemia reperfusion injury (IRI) by nebulizing a gap junction stabilizing agent, aCT1, in a living donor murine lung transplantation model. Previous data shows that by stabilizing the alveolar capillary barrier, we reduce immune cell infiltration, pulmonary edema, pulmonary hemorrhage, and a number of inflammatory cytokines. Our focus for this proposal is to determine the efficacy of this treatment in the more clinically relevant scenario of brain-dead donors and any consequences of nebulized aCT1 on other transplantable donor organs. 

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    Clare Grady, BS
    University of Florida College of Medicine 


    Use of Nanodiamonds for Localized Immune Modulation

    Synopsis: Ischemia/reperfusion injury (IRI) is a fundamental component of the pathophysiology of organ transplantation. Reperfusion of ischemic organs leads to tissue injury through a multifactorial process triggered by activation of the innate immune system; this process includes the generation of reactive oxygen species. The oxidative stress of IRI and resulting tissue injury have been inhibited by inducing heme oxygenase 1 (HO-1) and its protective effects through treatment with doxorubicin. However, no current therapies to prevent or treat IRI with doxorubicin are used in clinical practice due to its systemic toxicity. One potential approach to overcome the problems of systemic toxicity is targeted drug delivery to the affected organs using nanodiamond-modified drugs. This study seeks to characterize the uptake, localization, and toxicity of ND-adsorbed doxorubicin (NDX). This will be followed by evaluating the efficacy of NDX in stimulating HO-1 expression and downregulating the production of inflammatory cytokines in vitro. We hypothesize that ND-modified inhibitors of IRI can be targeted to specific organs and tissues and that they will have greatly improved efficacy and toxicity profiles compared to unmodified versions of the same molecules. 

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    Sumner E. Kilmarx, AB
    Massachusetts General Hospital 

    Validation of the Prognostic Ability of a Novel Imaging Methodology in Advanced Heart Failure and Transplant Patients: The proECMO Study

    Synopsis: The research project I will pursue with Drs. Joren Madsen and David D’Alessandro in the Massachusetts General Hospital Cardiac Surgery Department entails the investigation and development of a novel patient-evaluation technology. Before matriculating to medical school, I worked with Dr. D’Alessandro as a research coordinator using this technology to predict outcomes for ECMO patients. The Prognostic ECMO technology (“proECMO”) is a novel method to evaluate the peripheral microvasculature of patients with advanced heart failure, with the aim of validating the technology’s ability to predict patient outcomes and allow family members and healthcare providers to make better-informed decisions. The method involves spectroscopy and light microscopy non-invasively (over the skin) to measure peripheral oxygenation levels and capillary flow. The light microscopy is able to capture in real-time the quantity and quality of capillary circuits. Dr. D’Alessandro and I have previously worked extensively with this technology and we are excited to apply it to an expanded patient population.  

    This summer, Dr. D’Alessandro and I look forward to investigating the use of proECMO in both LVAD and heart transplant patients. My research proposal is for a single-center observational study of advanced heart failure patients who undergo LVAD placement or orthotopic heart transplant at the Massachusetts General Hospital. Patients who provide informed consent for the trial will be evaluated pre-and post-operatively with the proECMO technology. The results will be compared with established clinical metrics such as length of stay and morbidity to test proECMO’s prognostic ability.  

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    Katharine L. Krupp, BA
    University of Pennsylvania Perelman School of Medicine

    Characterizing Maternal T-cell Responses to the Fetus to Advance the Care of Parous Transplant Patients

    Synopsis: Exposure to alloantigen during pregnancy represents a common immune-sensitizing event that can carry significant consequences for parous women who require transplants. The purpose of our research is to employ emerging T-cell profiling techniques to characterize the changes that occur in the maternal T-cell repertoire over the course of pregnancy to evaluate the potential implications of these cells in the context of transplantation. Our current research has two aims. First, we will use TCR sequencing to document changes in the maternal regulatory T-cell repertoire over the course of pregnancy, then we will assess the utility of Nur77GFP mice as a superior model system for studying the full spectrum of T cell responses to fetal alloantigen. By providing novel insight into this exceedingly common, but still poorly understood immunological event, this work will help to improve the care of parous transplant patients, and aid in the identification of novel opportunities for therapeutic intervention.  

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    Danae Olaso, BS 
    Duke University School of Medicine 

    Measuring the impact of alloantibody on antibody-mediated rejection

    Synopsis: We propose to use previous existing data on over 50 sensitized monkey kidney transplants to test the hypothesis that MHC class I antibody levels correlate with the severity of antibody-mediated rejection (AMR)of renal allografts. Immunization to major histocompatibility (MHC) antigens, such as through blood transfusions, pregnancy, or previous organ transplant, is a major inhibiting factor for many patients in need of a kidney transplant. In order to overcome this problem, prevent acute AMR, and increase the length and quality of life through kidney transplantation, we must advance ways to desensitize patients. My mentor, Dr. Stuart Knechtle, has developed a sensitized nonhuman primate kidney transplant model, where maximally MHC mismatched pairs of monkeys are sensitized and produce high levels of alloantibody, receive desensitization treatments, and undergo a kidney transplant to clinically assess the outcomes of desensitization. In this project, the first aim of this study is to analyze the correlation between AMR with Banff scores used to clinically measure acute AMR. We will perform a meta-analysis of AMR and pathological outcomes, such as serum creatinine, serum antibody, and graft survival time. The second aim of this study is to evaluate the contribution of Mamu class I antibody to AMR pathology. Since beads coated with HLA antigens are not currently available for nonhuman primates, we propose to generate new target cell lines expressing single Mamu class I in collaboration with Dr. Garnett Kelsoe in order to evaluate how Mamu-specific alloantibody contributes to AMR and graft loss.  

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    Elisabeth Seyferth, BA
    Duke University School of Medicine 

    Investigating Damage Associated Molecular Patterns in Solid Organ Transplantation: Next Steps in Reducing Transplant Liver Dysfunction and Expanding the Donor Pool 

    Synopsis: The main obstacle facing the field of liver transplantation is a critical shortage of donor organs. The transplant community is attempting to expand the donor pool by using “marginal” organs, and ex vivo normothermic machine perfusion (NMP) is a promising emerging technology to allow use of these high-risk organs by reducing the ischemic injury they sustain during storage. However, damage associated molecular patterns (DAMPs) released during NMP are known to activate inflammatory signaling and contribute to poor outcomes. Our project aims to measure levels of DAMPs and their inflammatory effects during NMP and relate them to donor traits. NMP perfusate samples from a phase III clinical trial of NMP will be analyzed for DAMPs, cytokines, and TLR activation, and graft biopsies taken after the preservation period ends will be analyzed for inflammatory gene expression. These data will be correlated with donor characteristics and outcomes. In addition, NMP may be used as a platform to assess ECD organ viability before implantation, but biomarkers of viability have yet to be fully characterized. We will therefore measure levels of coagulation proteins and other proteins in perfusate as well as levels of ischemia-reperfusion-injury-associated microRNAs in perfusate that may potentially serve as markers of graft function. This project will allow us to optimize NMP protocols, develop interventions to reduce the effects of DAMPs, and improve selection of functional organs. Ultimately, our results will help both to improve post-transplant outcomes and to expand the donor pool through salvage of marginal organs.