Brigham And Women'S Hospital

NIH Research Projects · FY 2025 · 2024-08

Summary: ABO(H) blood group antigens and corresponding anti-ABO(H) alloantibodies were discovered over a century ago as the first polymorphisms in the human population and continue to be the most common immunological barrier to transfusion and transplantation. However, despite the fundamental nature of this discovery, very little is surprisingly known regarding the factors that govern anti-ABO(H) antibody formation or the fine details of the ABO(H) alloantigen targets responsible for hemolytic transfusion reactions (HTRs). As a result, current approaches designed to detect ABO(H) antigens and anti-ABO(H) antibodies largely rely on the same agglutination strategy leveraged by Landsteiner over 122 years ago. While ABO(H) incompatible RBC transfusion can result in a hemolytic transfusion reaction (HTR), only half of patients who receive ABO(H) incompatible RBCs experience this outcome. However, the factors that contribute to variable ABO(H) incompatible HTRs remain largely unknown. This limitation in our understanding is a direct consequence of the complexity of the post-translational modifications that comprise ABO(H) antigens and a historical limitation in the tools needed to study both ABO(H) antigens and the antibodies that develop against them. While a variety of highly novel tools have begun to revolutionize the field of glycosciences – the study of carbohydrate modifications – these tools have not been as thoroughly applied to perhaps the most common and arguably most clinically significant carbohydrate structures within the human population – ABO(H) blood group antigens. Fundamental questions surrounding anti-ABO(H) antibodies and their target antigens often require disparate areas of expertise, including glycosciences, immunology, hematology and microbiology, which has directly limited the study of this foundational discovery in transfusion medicine. To overcome this challenge, we have assembled a highly collaborative and integrated team of physicians and scientists with expertise in these fields. The combined collaborative history of the Project leaders (>50 papers and >15 years of collaboration) and the use of newly developed models and tools specifically designed to define factors that govern anti-ABO(H) antibody development and the specific ABO(H) targets on RBCs responsible for HTRs. In doing so, this program project grant (PPG) will provide a unique opportunity to define fundamental features of anti-ABO(H) antibody development and the ABO(H) targets that result in HTRs that have remained incompletely understood for over a century. This will be accomplished through 3 distinct Projects and the support of complementary Cores. Project 1: Examining the impact of microbial dynamics on B cells responsible for anti-blood group antibody formation (Leader: Stowell). Project 2: Convergence of innate immunity and microbial communities in the regulation of anti-blood group antibody development (Leader: Arthur). Project 3. Defining the distinct antigen targets and antibody specificities that govern ABO(H) RBC incompatibility (Leader: Cummings). By leveraging these Projects and Cores, this PPG will answer fundamental questions in transfusion medicine.

NIH Research Projects · FY 2026 · 2024-08

ABSTRACT Fifteen years after passage of the HITECH Act resulted in investment of billions of dollars into electronic health record (EHR) adoption, physicians and health system leaders face competing pressures related to the EHR. On one hand, widespread adoption of EHRs has had positive effects for patient safety and quality of care. On the other hand, use of EHRs has significantly worsened the physician work experience, particularly for primary care physicians (PCPs). In the context of ongoing threats to both the US primary care workforce and primary care quality and sustainability, health system leaders struggle to identify interventions that contribute to improved primary care quality while also enhancing PCPs’ EHR interactions and wellbeing. To address this gap, and in response to SEN-HS-22-011, we will evaluate the impact of three common or promising EHR-optimization interventions that are often considered and offered by health system leaders and adopted by primary care clinics and PCPs, either individually or in combination: 1) scribes, 2) advanced team- based inbox messaging support, and 3) artificial intelligence (AI)-assisted inbox messaging support. At present, health system leaders lack robust, actionable evidence on the benefits and drawbacks of these interventions, including their impact on patients’ quality of care and their relative cost effectiveness. Leveraging data and insights from three community-based and three academic primary care practice networks representing 800 PCPs caring for 1 million patients at 210 practice sites, we will: 1) test for associations between use of the 3 EHR-optimization interventions, individually or in combination, with PCPs’ EHR time and wellbeing, and patient-panel level quality of care and utilization of care; 2) describe PCP and team member experiences of adopting the 3 EHR-optimization interventions and the mechanisms by which the interventions are associated with EHR time, physician wellbeing, and quality and utilization of care; and 3) quantify the costs to health systems of implementing each of the 3 EHR-optimization interventions, either individually or in combination. This study blends quantitative, qualitative, and cost effectiveness methods to generate information about how to enhance PCPs’ EHR work while maintaining or enhancing quality of care. It brings together a strong, interdisciplinary team of experts in primary care operations, quality of care, informatics, EHR activity log research, and the clinician experience to generate rigorous evidence for healthcare leaders and policymakers regarding the impact of EHR-optimization interventions across PCP and patient outcomes. It will additionally generate evidence about the cost effectiveness of these interventions. The results of this study will guide health systems leaders towards interventions that both address EHR-based challenges negatively impacting the PCP work experience and maximize benefits of EHRs for physicians, patients, and health systems.

NIH Research Projects · FY 2025 · 2024-08

Project Summary: Chronic obstructive pulmonary disease (COPD) is a debilitating disease for which new, disease-modifying treatments are desperately needed. Since drug targets supported by human genetic evidence are more likely to lead to FDA-approved treatments, functional characterization of genome-wide association study (GWAS) loci is a translational research priority. COPD GWAS led by our group and others have identified more than 80 significant loci. We have identified the target genes for some loci, including FAM13A, ACRV1B, and TGFB2, using integration of GWAS and expression quantitative trait loci (eQTLs) to guide functional studies. However, most COPD GWAS loci remain uncharacterized due to 1) lack of QTL maps in appropriate cell types and 2) genetic effects other than eQTLs, such as alternative splicing. Splicing QTL (sQTLs) have been shown to be as important as eQTL for functional characterization of GWAS loci, and we have used sQTLs from blood and lung to identify FBXO38 and NPNT as COPD GWAS splicing genes. In this proposal, we will use a new resource of 185 primary human airway epithelial cells (HAEC-185) cultured and exposed to cigarette smoke (CS) or Air with associated genome-wide genotyping and RNA-seq measurements. HAEC-185 data will be used to identify alternative splicing related to COPD and/or CS exposure in HAECs, a highly relevant COPD cell type. In Aim 1, we will generate the first HAEC genome- wide sQTL maps for fully differentiated HAECs exposed to CS or Air. We will perform multiple colocalization analysis between COPD GWAS and these sQTL maps to identify genes whose splicing is altered by COPD GWAS variants. For two loci, genetic effects on splicing will be confirmed via long read RNA sequencing and isoform-specific protein mass spectrometry in genotype-selected HAECs. In Aim 2, we will perform state-of-the-art fine mapping of COPD GWAS sQTLs that integrates deep learning splicing model variant predictions with Bayesian fine mapping methods. We will train new, cell-type specific deep learning splicing models using data from HAEC-185 and 1,376 lung tissue samples from the Lung Tissue Research Consortium. In Aim 3, we will extend our previous work in gene regulatory network modeling to develop new methods to identify splicing regulatory networks. These methods will be applied to CS exposure data in HAEC-185 to provide a holistic view of CS-associated alternative splicing and its impact on gene expression pathways and cellular phenotypes. The network analysis will identify key splicing regulators, i.e. RNA binding proteins (RBPs), whose effects will be studied using shRNA knockdown of selected RBPs in HAECs. Our multi-disciplinary research team has the requisite expertise in COPD genomics, airway epithelial biology, computer science, gene network inference, long read sequencing, and proteomics to complete this important project to characterize COPD and CS-related alternative splicing in HAECs.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY/ABSTRACT Patient-reported outcome (PRO) measures, or PROMs, are powerful tools in cancer care to enhance clinician- patient communication, identify problematic symptoms and treatment priorities, facilitate shared decision-making, and prolong survival. Nonresponse significantly undermines the representativeness of PRO data that is routinely collected as part of clinical care, thereby compromising its utility for promoting high-value, equitable patient- centered care in cancer clinics. The lack of adequate representativeness also hampers the use of routinely collected PRO data for quality improvement and value-based healthcare. There is an urgent need to delineate the representativeness of PRO data collected in routine cancer care, identify the underlying causes of nonresponse, and develop novel methods to ensure data representativeness. Despite compelling evidence that expanding the reach of PROMs collection to underrepresented patients and improving their PROMs completions are critical to ensure the representativeness of routinely collected PRO data, most of the current research focuses on methods to increase collection instead of methods to enhance PRO data representativeness. The overarching goal of this proposal is to characterize and delineate the causes of nonresponse and devise mitigation strategies to improve the representativeness of PRO data collected in routine cancer care. By examining the data of the Patient-Reported Outcomes Measurement Information System Global-10 measure collected as part of routine care at seven Radiation Oncology clinics within the Mass General Brigham healthcare system, we will (Aim 1) characterize the nonresponse of the Global-10 across clinic, provider, and patient levels; (Aim 2) identify multilevel causes of nonresponse and potential strategies to improve representativeness in PROMs collection; and (Aim 3) develop effective modifications to missing-data methods to enhance the representativeness of pre-existing PRO data. Our expected outcomes are generalizable knowledge and strategies to address nonresponse in PROMs, enhancing PRO data representativeness in routine cancer care. The successful completion of this project will elucidate the characteristics of PROMs nonresponse and its intricate associations with multilevel factors of successful large-scale PROMs collection in diverse patient populations. Insights from this endeavor will also guide the evolution and development of PROMs collection programs to expand their reach to underrepresented cancer patients and improve data representativeness. This will, in turn, enable the utility of PRO data for quality improvement and high-value, equitable patient-centered cancer care.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT. Thymine DNA glycosylase (TDG) is a key enzyme in somatic cell reprogramming. TDG catalyzes DNA demethylation and reactivation of specific sets of genes to induce a pluripotent cell fate in somatic cells. However, there is a fundamental lack of understanding of how TDG is directed to its target genes to catalyze dynamic demethylation to facilitate their timely activation and thereby cell fate conversion. Thus, our understanding of epigenetic mechanisms of cell fate change remains incomplete. The long-term goal is to understand fundamental mechanisms by which factors such as TDG control processes requiring widespread epigenetic changes, including normal embryonic development and somatic cell reprogramming. Our recent studies have identified the nucleotide excision repair protein XPC as a potent activator of TDG-dependent DNA demethylation genome-wide and preferentially at gene enhancers bound by transcription factor HIF1a. The overall objective of this project is to determine how XPC and TDG together integrate signals to regulate active DNA demethylation and epigenetic reprogramming. The central hypothesis is that XPC is a novel regulator of dynamic DNA demethylation via TDG and thereby plays an essential role in pluripotent cell fate transitions. The rationale of our work is that by deciphering how XPC regulates TDG-dependent DNA demethylation in response to HIF1a, we will gain a new understanding of how the XPC-TDG-HIF1a axis integrates signals to coordinately regulate epigenetic changes that contribute to normal development and reprogramming. The central hypothesis will be tested by pursuing three Specific Aims: 1) Identify mechanisms by which XPC-TDG-HIF1a axis regulates DNA demethylation and metabolism in somatic cell reprogramming; 2) Identify mechanisms by which XPC promotes recruitment of TDG by HIF1a; and 3) Identify the role of post-translational modifications of TDG via SUMOylation in regulating DNA demethylation dynamics. Under the first aim, genome-wide and functional studies will be employed to identify how XPC and TDG regulate epigenetic changes at HIF1a-regulated gene loci to induce transcriptional and metabolic changes favorable for reprogramming. In the second aim, biochemical and loss-of-function studies will be performed to identify the role of XPC and novel associating factors in directing TDG to specific gene enhancers for demethylation. Under the third aim, the role of XPC in regulating post-translational modifications of TDG and the effect of TDG SUMOylation on demethylation dynamics and specificity will be directly measured in living cells using single particle tracking. The proposed research is innovative because new activities of XPC in DNA demethylation, through post-translational modification of TDG and novel TDG-HIF1a complex assembly mechanism, will be uncovered using new tools. This contribution is significant, because the regulatory components and epigenetic mechanisms we identify are likely to advance our fundamental understanding of epigenetic control of development, and how epigenetic dysregulation causes disease.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract. NUT-fusion oncoproteins, most commonly BRD4-NUT, drive NUT carcinoma (NC), an aggressive, poorly differentiated squamous cancer with a 6.5-month median survival and < 20% 3- year survival. NC predominantly affects adolescents and young adults and, with no routinely effective therapy, has a critical unmet need. BRD4-NUT drives growth by altering histone modifications and chromatin 3D structure to upregulate expression of pro-growth genes. We demonstrated that BET bromodomain inhibitors reverse these epigenetic modifications and block tumor growth. These findings led to the treatment of NC and numerous other malignancies with BET inhibitors. However, the clinical efficacy of BET inhibitor monotherapy is limited, indicating that BET inhibitors alone do not fully address NC biology. The goal of this proposal is to overcome the limitations of BET inhibition in NC and therefore improve patient outcomes by identifying targetable mechanisms of gene regulation that maintain NC growth. Using our combined expertise in translational NC cancer biology (CA French) and chromatin biology (KP Eagen) we will take a two-pronged approach to improve NC treatment through mechanism-based target discovery. Both prongs share the premise that identifying factors that cooperate with BRD4-NUT to promote NC growth will lead to new therapeutic targets. Our first Aim is based upon our recent discovery that repression of tumor suppressor genes by the histone methyltransferase EZH2 complements oncogene activation by BRD4-NUT. Simultaneous inhibition of EZH2 and BRD4-NUT synergistically blocks NC growth in vitro and in vivo. However, the precise mechanism of synergy remains unknown. Additionally, toxicity was observed in some xenograft models, and thus toxicity in humans is likely. Therefore, we propose to identify more precise therapeutic targets by determining key factors that mediate the oncogenic synergy between EZH2 and BRD4-NUT. Our second Aim is motivated by the discovery of a novel MLL1::NUTM1 fusion in a patient. Our proteomics data indicate that MLL1 and its key binding partner MENIN are both present in the BRD4-NUT oncogenic complex providing biochemical evidence that this fusion may reveal new factors that cooperate with BRD4-NUT. Accordingly, inhibiting the interaction between MLL1 and MENIN induces differentiation and blocks growth of NC cells. The combination of MENIN-MLL1 with EZH2 inhibition synergistically blocks NC growth in vitro. This data suggests that combination therapy with MENIN-MLL1 and EZH2 inhibitors could potentially replace BET inhibition, which we will test in pre-clinical models of NC. Aim 1. Determine the mechanism by which EZH2 cooperates with BRD4-NUT to drive NC growth. Aim 2. Elucidate the role of MENIN in BRD4-NUT oncogenic function.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY This K23 award will support the candidate’s training and development towards an independent clinical research career focused on behavioral sleep medicine and the use of remote technology-based delivery/assessment of mindfulness meditation (MM) to facilitate self-sleep management in patients with chronic insomnia disorder. BACKGROUND. Insomnia is highly prevalent and associated with a wide range of deleterious health consequences. According to well-established disease models, insomnia is characterized by bedtime stress, pre-sleep hyperarousal, or high sleep reactivity. MM is a promising intervention for patients with insomnia. The delivery of MM via mobile health has largely increased access to such interventions. Technology has allowed both remote delivery of intervention and home assessment with physiological recordings that increase access to real-world data. Existing studies have mostly focused on daytime MM practice and subjective sleep quality at night. It is not clear whether the temporal relationship of bedtime MM practice is important to sleep outcomes, what is the most effective way to deliver app-based MM for patients with insomnia disorder, or whether physiological changes correlated with mindfulness practice contribute to sleep improvement. SPECIFIC AIMS. This K23 proposes a proof-of-concept feasibility study with aims to: (1) refine content delivery of an app-based MM intervention (specifically for bedtime use); (2) evaluate the feasibility and acceptability of the app-based intervention in a pilot randomized controlled trial; and (3) explore patterns of change in sleep-related subjective/objective outcomes, including remote assessment. TRAINING. To develop expertise critical to the candidate’s overall career goals, Dr. Ma will receive training in three areas: (1) behavioral sleep medicine and clinical trials; (2) qualitative and mixed methods research; and (3) mobile health (mHealth), technology-based sleep interventions and assessment. These training aims will be supported by strong institutional resources, as well as coursework, trainings, research seminars/meetings, and scientific conferences. MENTORSHIP. The candidate will be supported by an expert mentoring team including primary mentors Janet Mullington, PhD and Gloria Yeh, MD, MPH, co-mentor/collaborators Peter Wayne, PhD, Suzanne Bertisch, MD, MPH, Ana-Maria Vranceanu, PhD, John Torous, MD, MBI, Pamela Rist, ScD, and Karen Kilgore, PhD. IMPACT. Findings from the pilot study will inform the design of a larger trial and form the necessary foundation to further investigate mobile-health MM approaches for insomnia disorder and understand links between pre-sleep MM and sleep quality. Building on the candidate’s existing skills in physiological signal analysis, this K23 award will allow the candidate’s development as a clinical/translational researcher with expertise in mobile technology-based MM intervention and assessment for management of sleep, and complex physiological signals to elucidate the effects of MM on sleep.

NIH Research Projects · FY 2024 · 2024-07

ABSTRACT Acute and chronic effects of viral diseases often involve immune-mediated responses, including inflammation, immune activation, and autoimmunity. Long-COVID immunopathogenesis is complex but its understanding is advancing rapidly. The consequences of immune-mediated damage in long COVID share similarities (and differences) with other post-viral diseases like Ebola and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). In the case of HIV, ongoing activation of the inflammatory response is a major concern, and immune reconstitution remains limited in some patients even long after treatment initiation. In addition to SARS-CoV-2 and HIV, other viral infections such as CMV are known to result in inflammation- related pathology. Viruses can also trigger autoimmunity. Epstein-Barr Virus is tightly linked with the onset of Multiple Sclerosis through several autoimmune mechanisms. Viral encephalitis also often has an autoimmune background. Common underlying mechanisms imply the possibility of employing similar therapeutic interventions, such as neutralizing antibodies, antiviral drugs, and therapeutic vaccines to eliminate the persistent virus or anti-inflammatory drugs to ameliorate chronic inflammation. Therapeutic interventions must often target both ends of the virus-immune system spectrum. The prevalence of post-acute infectious syndromes is concerning. The severity of the syndrome disables daily activities, posing a significant burden on health, economy, and society. Despite intensive research on cellular and molecular mechanisms, scientific platforms to share data and discuss new concepts are limited to sessions at major international conferences or scientific meetings focusing on a specific condition. This annual workshop links leading international scientific investigators in a discussion of cutting-edge research developments on underlying inflammatory mechanisms related to viral infection, and its clinical implications. Specific aims of the meeting include: 1) providing a global cross-disciplinary platform to exchange knowledge on underlying mechanisms of virus-mediated inflammation; 2) gathering basic, translational, and clinical researchers, and clinicians to stimulate discussion on remaining unknowns; 3) fostering future collaborations among participants; 4) translating the data into clinical guidance; and 5) increasing the visibility of early career investigators and investigators from underrepresented minority (URM) groups and their research, thereby facilitating a generation of future leaders in the field.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract: Mast cells (MCs) expand within the airway epithelium and sub-epithelium during prevalent and burdensome human respiratory disease, including asthma and nasal polyposis, where they are thought to play a central role in disease pathobiology. MCs take on discrete protease expression profiles in each location, with sup-epithelial MCs co-expressing tryptase and chymase (MCTC) while epithelial MCs express tryptase alone (MCT). Neither the mechanisms underlying this expansion, the signals directing the epithelial and sub-epithelial MC phenotypes, nor the differential contribution of MC phenotypes to tissue inflammation are well understood. This application focuses on understanding the mechanisms through which airway tissue structural cells differentially direct the expansion and differentiation of MC progenitors (MCps), based on preliminary studies identifying a central role for fibroblasts and epithelial cells in both processes. This proposal tests the central hypothesis that MC hyperplasia in inflamed human airway mucosa is driven by the recruitment of MCps exhibiting a robust proliferative capacity, and that the proliferation and differentiation of these recruited MCps towards the histochemically recognized MCT and MCTC subsets are driven by key signals secreted by airway structural cells. A related hypothesis is that the resulting intraepithelial and subepithelial MC phenotypes are maintained through a network of transcription factors regulated by these tissue stromal-derived signals, and that signal nucleotide polymorphisms in these transcription factors increase risk of developing airway disease. Aim 1 of this proposal evaluates MCp concentration in human sinonasal tissue across disease endotypes, determining the relationship between recruited MCp and the proliferative population of MCs we previously identified in nasal polyposis. Aim 2 of this proposal tests the impact of a series of candidate stromal-derived ligands on directing MCp proliferation and differentiation using CRISPR/Cas9 gene editing. Aim 3 probes the transcription factor network underlying MC polarization via shRNA knockdown and evaluates the impact of key asthma- associated single nucleotide polymorphisms in one such transcription factor on intraepithelial MC differentiation. Completion of these aims will greatly expand our understanding of the pathways through which MCs are capable of influencing tissue inflammation and the mechanism(s) through which these pathways are regulated by their tissue microenvironment.

NIH Research Projects · FY 2025 · 2024-07

Background: RSA has one of the highest rates of human mobility on the continent, and mobile men are at high risk of HIV acquisition. Pre-exposure prophylaxis (PrEP) is effective at preventing HIV. Mobile men face barriers to PrEP use, including lack of PrEP knowledge, stigma at clinics, and unfamiliarity about health services in new settings. To decrease the incidence of HIV, new interventions are needed to promote PrEP use. The goal of this proposal is to develop and pilot test a “Healthy Welcome” intervention to support mobile men in starting and adhering to PrEP by linking them to community pharmacies. Candidate: I am an Internal Medicine physician and an Associate Scientist at Brigham and Women’s Hospital (Harvard Medical School). I am applying for a five-year K23 Career Development Award to obtain the training and research experience that will allow me to become an independent investigator at the R01 level and an expert in developing interventions to promote engagement in HIV services. Mentoring: Drs. Ingrid Katz and Jessica Haberer will serve as my co-primary mentors. Dr. Katz brings expertise in behavioral components of intervention development and strategies to track hard-to-reach populations. Dr. Haberer brings expertise in PrEP clinical trials, including implementation science, the concept of “Prevention-Effective” (P-E) adherence to PrEP, and mobile health strategies. Co-mentor Prof. Terris-Prestholt will provide mentorship on discrete choice experiment (DCE) methodology. Profs. Pascoe and Setswe, RSA-based co-mentors, will provide mentorship on local application of DCE methods and behavioral economics (BE) strategies for intervention development (Pascoe) and community engagement and local implementation (Setswe). Profs. Miot and Venter, RSA-based members of my Scientific Advisory Board (SAB), will offer guidance on integrating my proposed study within an existing Gates-funded project to offer PrEP through community pharmacies and on policy implications of study findings. SAB member Prof. Lurie will offer guidance on engaging mobile men. SAB member Prof. Thirumurthy will guide me on BE theoretical frameworks. SAB member Smeaton will provide statistical expertise to guide the pilot randomized controlled trial (RCT). Training: Training in DCE methodology, BE strategies, and pilot RCTs for PrEP will be achieved through intensive direct mentorship and coursework. Research: The specific aims are to 1) elicit preferences for PrEP services among mobile men through a DCE; 2) design a “Healthy Welcome” intervention for mobile men to promote linkage to pharmacies for PrEP uptake and adherence; and 3) evaluate the feasibility, acceptability, appropriateness, and fidelity of the intervention through a pilot RCT. I will use this formative research to apply for an R01-level grant to evaluate the efficacy and effectiveness of the intervention.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract: Mast cells (MCs) expand within the epithelium and sub-epithelium during prevalent and burdensome human mucosal disease, including asthma, nasal polyposis, food allergy and eosinophilic esophagitis, where they are thought to play a central role in disease pathobiology. MCs take on discrete protease expression profiles in each location, with sup-epithelial MCs co-expressing tryptase and chymase (MCTC) while epithelial MCs express tryptase alone (MCT). Although MCT preferentially expand during human type 2 mucosal inflammatory disease, very little is known about how these cells influence tissue inflammation in part because all existing systems for in vitro study of MCs give rise to MCTC. We have recently developed a novel cell culture system for directing the selective in vitro differentiation of MCT or MCTC from human peripheral blood CD34+ cells, allowing in-depth study of MCT effector function of phenotypes for the first time. This proposal seeks to use our culture system to test the central hypothesis that MCTs within the epithelium have a discrete effector phenotype and that a more comprehensive understanding of the effector function linked to this phenotype will enhance our understanding of disease endotypes in nasal polyposis. A related hypothesis is that in vitro activation profiles of MCTC poorly predict. In support of these hypotheses, preliminary findings indicate that in MCT and MCTC have differential capacity to generate the pro-inflammatory eicosanoids and type 2 inflammation-linked cytokines IL-5 and IL-13 following activation, mimicking transcriptional differences between the two phenotypes in vivo, and that IL-4 differentially regulates upregulation of the high affinity IgE receptor FceR1a in each population. Aim 1 of this proposal seeks to comprehensively define production of cytokines, chemokines and growth factors in vitro-derived MCT vs MCTC in response to a range of activating stimuli, as well as determine the transcriptional changes associated with each. Aim 2 characterizes the differential effects of several inflammation-associated mediators on MCT vs MCTC activation, based on differential expression of cell surface receptors and signal transduction components between the two subsets. Aim 3 datasets generated through aim 1 and 2 to probe existing scRNA-seq data and datasets generated by a collaborator assessing eicosanoids in the nasal lavage of nasal polyposis patients at baseline and following a series of therapeutic interventions, in an attempt to use our in vitro findings to better understand MC effector function in vivo. Completion of these aims will greatly expand our understanding MC effector capacity differential effector capacity of human MC subsets in human disease and is a critical first step towards a more comprehensive understanding of the role of MCs in human disease pathobiology.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Overview: The goal of this project is to assess the efficacy of a tailored phone-delivered positive psychological intervention (PATH) for improving anxiety and depression symptoms, and quality of life (QOL) in hematopoietic stem cell transplantation (HSCT) survivors. Background: Allogeneic HSCT survivors experience high rates of treatment-related toxicities, a prolonged recovery, substantial risk of life-threatening complications, and long-term morbidity and mortality. Hence, HSCT survivors experience tremendous psychological symptoms (e.g., anxiety, depression) and low levels of positive psychological well-being (e.g., flourishing, gratitude), which undermines various health-related outcomes such as quality of life (QOL). However, most existing psychosocial interventions are not accessible to the majority of HSCT survivors because they are 1) delivered in-person, 2) entail long 60-90-minute sessions, 3) require delivery by palliative care clinicians or psychologists with extensive and costly training, and 4) not focused on positive psychological well-being. To address these gaps, we developed PATH, an accessible, phone-delivered positive psychological intervention with deliberate and systematic activities to cultivate gratitude, strengths, and meaning. Interventions like PATH can buffer against distress to improve anxiety and depression symptoms, and QOL in HSCT survivors and entails 15–20-minute sessions delivered by interventionists from diverse backgrounds, including nursing, social work, or psychology. With a single site randomized controlled trial (RCT) of PATH, we showed it was feasible and preliminarily led to clinically significant improvement in patients’ anxiety symptoms. As the first RCT to show the feasibility and preliminary efficacy of a tailored HSCT positive psychology intervention, the next step is to assess the efficacy of PATH on anxiety and depression symptoms, and QOL. Research Plan: We will conduct an RCT in 400 HSCT survivors to test the efficacy of PATH for improving anxiety and depression symptoms, and QOL (Aim #1). We will also assess the impact of PATH on positive well- being (e.g., gratitude, flourishing) and self-management targets (e.g., coping, physical activity) (Aim #2). Finally, we will assess mediators and moderators of the effect of PATH on anxiety symptoms and QOL (Aim #3). Environment: This project will be conducted at the Mass General Brigham/Dana-Farber Cancer Institute (MGB/DFCI), Duke Cancer Institute, and Moffitt Cancer Center, which individually conduct 200-250 allogeneic HSCTs per year. Dr. Amonoo and the research team at MGB/DFCI, Duke, and Moffitt have extensive proficiency in developing and conducting multi-site psychosocial interventions for patients with hematologic malignancies who are HSCT survivors to ensure the successful execution of the proposed RCT across study sites. Relevance of Research: If efficacious at improving anxiety and depression symptoms and QOL, PATH can potentially change the paradigm of psychosocial care for HSCT survivors and vulnerable cancer populations.

NIH Research Projects · FY 2025 · 2024-07

Abstract Epstein-Barr virus (EBV) causes lymphomas at greatly increased frequency in people living with HIV/AIDS, including diffuse large B-cell lymphoma, primary CNS lymphoma, Hodgkin lymphoma, Burkitt lymphoma, and post-transplant lymphomas. Consequently, most AIDS-related lymphomas are EBV-infected. Yet, EBV transformation sensitizes infected B-cells to attack by natural killer (NK) cells, which exert key roles in control of EBV-driven B-cell malignancies in immunocompetent hosts. Much remains unknown about how NK recognize latently EBV-infected B lymphoblastoid cells and how EBV+ B cells evolve resistance to NK control. To gain insights, we performed the first human genome-wide CRISPR screen of EBV-transformed B-cell escape from NK surveillance using a lymphoblastoid cell line (LCL), a key model for EBV-driven immunoblastic lymphomas including the AIDS defining cancer primary CNS lymphoma. Our screen identified factors known to be important for EBV+ B-cell recognition by NK, including CD48, which is highly EBV-induced. Unexpectedly, multiple CRISPR screen hits centered on the Human Silencing Hub (HUSH) complex, an epigenetic repressor that induces heterochromatin formation to silence target gene expression. HUSH is implicated in silencing of HIV genes, but has not been studied in the contexts of EBV infection, B-cell immunobiology or NK surveillance. Protocadherin gamma (PCDHG) family members, which are adhesion molecules typically expressed only by neurons, were highly enriched amongst LCL genes de-repressed by HUSH KO. Furthermore, PCDHG are necessary for NK resistance driven by HUSH KO and sufficient for inhibition of LCL lysis by NK. We therefore hypothesize that PCDHG expression represents a ‘don’t kill me’ signal to protect neurons from NK attack, but which is subverted to support EBV+ lymphomagenesis. Our central hypothesis is that NK surveillance of EBV transformed B-cells requires B-cell intrinsic HUSH complex activity, in the absence of which gamma- protocadherin are de-repressed and interact with a novel NK inhibitory receptor to prevent NK lysis. Our specific aims are: (1) Characterize key proto-cadherin gamma properties in EBV transformed B-cell evasion of NK attack, including in tumor samples from HIV+ individuals. (2) Identify the Proto-cadherin Gamma NK Counter-Receptor and Characterize its NK Inhibitory Roles. Collectively, these studies promise to identify a novel NK cell inhibitory pathway subverted by EBV+ tumors. Our studies may lay the foundation for novel therapeutic approaches that could re-sensitize tumors that subvert this pathway to NK attack.

NIH Research Projects · FY 2026 · 2024-07

ABSTRACT Cell type-specific transcriptional programming enables a single fertilized egg to make the remarkable transition to become a multicellular organism. The requisite sequence of gene expression transitions is the result of a collaboration between cell type-specific transcription factors and broadly expressed chromatin regulators. Historically, my group has made significant contributions to understanding the targeting and spreading of chromatin domains, and currently we are probing the ability of modular chromatin factors to respond to local concentrations of transcription factors and their co-activators to specify cell type. Our studies focus on the dynamic competition between Polycomb (PcG) and Trithorax group (TrxG) proteins, and due to the high conservation of these key regulatory factors we move between fly embryos and mammalian cells with ease. We have recently contributed to the recognition of the ancient conservation of alternative forms of PcG complexes, and over the next funding period we will investigate the distinct molecular roles that must underly this remarkable conservation. To understand when, where, and how variant Polycomb complexes function, the Drosophila melanogaster embryonic and germline systems harbor special promise for key aspects of this work. In particular, emerging approaches in the germline will allow us to dissect the earliest events in the establishment of PcG/TrxG regulation. A key challenge will be to adapt current methods to integrate alternative configurations of protein complexes with their local binding patterns and genetic knockdown phenotypes. For example, we have discovered that a Pho/Sfmbt protein module can interact with co- activators as well as variant PRC1.6 complexes in Drosophila embryos, and we will test whether these are modular interactions that vary on a gene-by-gene basis during normal development. We will also analyze aberrant epigenetic programs in mammalian cells, in light of the important role that a dynamic PcG/TrxG competition plays in human malignancies. Focusing on chromatin-driven cancers caused by aberrant TrxG fusion oncoproteins such as MOZ-TIF2 will complement our analyses of normal cell type transitions. This topic is of key biomedical significance, as plasticity of transcriptional programming is thought to underlie the devastating facility of cancer sub-populations to acquire drug resistance and to metastasize. Important benchmarks for our work will be i) identifying the initial steps that poise developmental genes for activation or repression, ii) providing a mechanistic understanding for how resolution occurs, and iii) contributing to an understanding of cell state switching in response to cancer drivers. More broadly, our work will highlight regulation at the level of dynamic protein interactions, which is likely universal to all biological systems.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY The past two decades have witnessed several air-borne viruses (SARS-CoV1, MERS, SARS- CoV2) that led to significant mortality. There is always a lag time before a vaccine is introduced. This lag creates an urgent need for a universal antiviral therapy that can be deployed rapidly as a prophylactic measure. Here, we propose to address this gap by engineering a novel intranasal- delivered nanoparticle that exerts an universal antiviral effect by activating a unique evolution- encoded innate immune state in nasal epithelial cells to provide early protection. There is a strong rationale for targeting nasal epithelium for an antiviral prophylaxis for air-borne viruses: (1) Nasal epithelium is the most common portal of entry of these pathogens; (2) The nasal cavity and nasopharynx contain some of the highest viral loads; and (3) Neutralizing the virus in the nasal epithelium has been shown to not only decrease viral load in both the nasal cavity and in distant sites, but potentially reduce transmission from asymptomatic individuals. While these studies validate nasal delivery as an ideal route of administration for viral prophylaxis, an effective universal antiviral therapy remains an unmet need. We hypothesize that activating an evolutionary conserved antiviral mechanism, i.e. the induction of IFN-Induced proteins with tetratricopeptide repeats 1 (IFIT1), can emerge as an effective universal prophylaxis against airborne viruses. This can be achieved by inhibiting CMTR2, a novel target, in the nasal epithelium. We will: (Aim 1) Engineer antiViral Response-Activating Nanoparticles (V-RANs) for intranasal delivery and test for inhibition of CMTR2 and induction of IFIT1; (Aim 2) Evaluate the antiviral efficacy and safety of V- RANs in mouse models of severe SARS coronavirus or pandemic flu infections; and in (Aim 3) Elucidate the molecular mechanism of V-RAN-induced antiviral response. Published reports and our preliminary results demonstrate the feasibility of this project. This study can lead to new insights into intranasal nanodelivery, and a novel antiviral prophylaxis that can shift the paradigm for early interventions in future pandemics.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY: The sudden loss of smell and taste are among the defining features of COVID-19 that set it apart from other viral respiratory syndromes with an incidence of up to 70% with some SARSCoV2 variants. Importantly, in some patients smell and taste disturbances persist for longer than 12 months after recovery from COVID-19. Furthermore, classical anti-inflammatory treatment therapies are ineffective for COVID19 chemosensory dysfunction. To inform targeted treatments, it is critical to elucidate the specific pathways that lead to persistent disruption of chemosensory function. Our preliminary data show that persistent COVID19 chemosensory dysfunction involves persistently disordered neuroepithelial composition and infiltration with immune cells with features of immunosuppressive macrophages. The dysregulated inflammatory milieu prominently features linoleic acid metabolites and glioma-associated proteins with direct effect on olfactory stem cell proliferation and differentiation and olfactory sensory neuron survival. This study will determine how the inflammatory but steroid-resistant immune cells drive chemosensory dysfunction that plagues patients with persistent COVID19 disruption of smell and taste. In Aim 1 we will define the phenotype of the infiltrating immune cells using both transcriptional and mediator studies. In Aim 2, we will determine how immune mediators defined in Aim 1 disrupt olfactory stem cell and sensory neuron development and apoptosis trajectories using a mouse in vitro system and relate these findings to the disordered epithelial composition of subjects with persistent COVID19 chemosensory dysfunction. Thus, we use complementary approaches with molecular tools and cell systems and samples from carefully phenotyped human subjects. The studies should reveal new potential strategies for therapeutic development that are based on the novel underlying mechanism that we identified here.

NIH Research Projects · FY 2025 · 2024-07

Brain imaging has long been criticized for revealing correlations rather than causes. In recent years, there has been a rapid growth of focal brain stimulation technology, which can bring us closer to studying causes. If stimulating a specific brain circuit causes a specific behavioral change, then that circuit can be inferred to have a causal role in that behavior. However, the number of potential stimulation sites is practically infinite. This makes it difficult to test the causal relevance of every possible stimulation target and every possible behavior. There is a need for a systematic wiring diagram that narrows the field of potential options, enabling researchers and clinicians to make informed decisions about stimulation targets. In this proposal, we will develop this wiring diagram. Using highly-sampled resting-state functional MRI scans as well as cutting-edge brain stimulation protocols, we will randomly apply stimulation to different targets in each participant. To address the problem of a near-infinite target space, we will narrow it down by mapping whole-brain connectivity of each target, not just its location. As a result, we will have information about every brain region for every participant, enabling us to draw whole-brain-level contrasts. Using this information, we will map the circuitry connected to TMS sites that modulate a wide range of different behaviors relevant to mental illness, including transdiagnostic behaviors and diagnosis-specific symptoms. We will include patients with major depression, obsessive- compulsive disorder, schizophrenia, and generalized anxiety disorder. If successful, the study will yield a multidimensional atlas of causal brain circuitry, which can be used by future researchers and clinicians to select a stimulation target that is appropriate for the a specific patient based on their symptoms and objective emotional testing.

NIH Research Projects · FY 2024 · 2024-07

Abstract: We propose to acquire an Orion HT multiplex fluorescent imaging system (Rarecyte inc.) to support research at Brigham and Women’s Hospital (BWH) and neighboring institutions. Immunofluorescence (IF) is a standard tool for microscopy research and is used routinely to identify cell types and study their organization and function in tissues. However, the overlapping emission spectra of common fluorophores means that standard IF is limited to a few colors, which is a major limitation for studying cell interactions in complex tissues and disease states. Many cell types can only be identified using combinations of several markers, and to understand their interactions it is often necessary to visualize multiple markers simultaneously. Thus, multiplex immunolabeling methods are essential. One approach is to perform multiple labeling cycles, and various methods have been developed to achieve this. However, these cyclical methods are slow and labor-intensive, and specimens can become degraded after multiple cycles. The Orion instrument was developed to solve these problems, and to achieve highly multiplex imaging in a single scan. Antibodies are conjugated to a palette of up to 20 fluorophores, excitation is produced by seven high-power lasers, and fluorescence is measured through a series of tunable narrow bandpass filters. Following staining, multiple fluorescence images are captured automatically, and individual labels are computationally separated using the company’s SpectralEdge algorithm. The instrument can scan an entire slide at subcellular resolution, which is especially important when studying large complex structures such as brain or tumors, whose spatial heterogeneity is not adequately captured by smaller fields of view. The Orion can also capture a H&E image on the same section, meaning that multiplex molecular data can be correlated with existing H&E datasets from clinical pathology. The reliability of the color separation has been extensively validated, and this novel technology holds great promise for a wide range of applications. The PI, Sandro Santagata, has previously collaborated with Rarecyte to develop the Orion technology, with support from a NIH SIBR grant. He also collaborates closely with Peter Sorger at Harvard Medical school, a current advisor to Rarecyte. Our users will benefit greatly from their expertise and connections to the company, and from a growing list of validated antibodies and computational tools that have been developed to support the Orion platform. The requested instrument will be installed in a shared core facility at BWH, where it will support a large community of NIH-funded researchers studying cancer, autoimmunity, inflammation, neurodegeneration and many other disorders.

NIH Research Projects · FY 2025 · 2024-07

Atherosclerosis, a chronic arterial disease, is markedly accelerated in individuals with diabetes, accounting for a 2- to 4-fold increased risk of ischemic cardiovascular events. One of the hallmarks in this process involves the recruitment and accumulation of intimal macrophages, a process that is more pronounced in diabetes than without diabetes. The presence of pro-inflammatory macrophages facilitates the progression of atherosclerosis and impairs the ability of lesions to regress despite optimal medical therapy. Therefore, modulating monocyte/macrophage chemotaxis and accumulation in the vessel wall may provide a novel therapeutic approach to limit atherosclerotic progression and facilitate regression. Long non-coding RNAs (lncRNAs) are an emerging class of regulators of epigenetic modifiers, RNA, or protein-coding genes that has garnered attention for impacting diverse biological processes relevant to atherosclerosis. However, the identity and roles of specific lncRNAs within atherosclerotic lesions, especially in diabetes, are not well defined. Using RNA-Seq profiling to identify lncRNAs derived specifically from the atherosclerotic intima, we identify the lncRNA MERRICAL (Macrophage-Enriched lncRNA Regulates Inflammation, Chemotaxis, and AtheroscLerosis (MERRICAL). MERRICAL was the highest expressed lncRNA with a 249% increase concomitant with atherosclerotic progression under a high fat sucrose-containing (HFSC) diet and decreased with regression after a high cholesterol diet, but not after a HFSC diet. MERRICAL is a macrophage-enriched and nuclear-specific lncRNA. Our preliminary data demonstrate that delivery of inhibitors to MERRICAL in LDLR-/- HFSC-fed mice strongly reduced lesion size by 86%, independent of effects on circulating lipid profile, but rather by decreased macrophage accumulation in lesions. Preliminary studies show that MERRICAL in-cis regulates its neighboring genes Ccl3 and Ccl4 and macrophage chemotaxis mediated via interaction with a complex of the histone modifying enzyme H3K4me3. In addition, MERRICAL knockdown functions in-trans to regulate IL-1b-associated inflammasome signaling. These observations provide the central hypothesis that MERRICAL deficiency, via regulatory effects on H3K4me3 and macrophage chemokines and IL-b inflammasome signaling, reduces macrophage chemotaxis, lesional macrophage inflammatory signaling, and suppresses diabetes-associated atherosclerosis. To address this further, in Aim1 we examine the role of MERRICAL and the in-cis regulation of CCL3/CCL4 in macrophage chemotaxis and in-trans regulation of IL- 1b-associated inflammatory signaling; in Aim2, we assess how alterations of MERRICAL expression affects recruitment of lesional macrophages on lesion progression and regression in vivo; and in Aim3, we examine the role of the MERRICAL-H3K4me3 signaling axis in human cells and atherosclerotic lesions. Our studies will address a major gap in our understanding of lncRNAs in atherosclerosis and diabetes and inform how MERRICAL-mediated control of lesional macrophages may provide new targets for therapy.

NIH Research Projects · FY 2026 · 2024-07

Project Summary/Abstract Early detection and sensitive tracking of cognitive changes related to Alzheimer’s disease (AD) are critical for identifying individuals at-risk for decline and assessing treatment response more rapidly. Current gold standards (e.g., paper and pencil measures administered semi-annually) are insufficient, particularly as the field has shifted towards prevention at the preclinical stage of AD where cognitive changes are subtle and where widespread screening at much larger scales is needed. To meet this need, we will leverage web-based cognitive testing which both offers both scalability and which allows for relatively understudied, but promising cognitive paradigms to be explored. More specifically, our preliminary data suggests that diminished learning associated with preclinical AD is observable over several days using a Multi-Day Learning Curve (MDLC) i.e., the trajectory of daily learning on the same memory and processing speed tests administered 10 min/day for 7 days on a personal device. Diminished learning curves, collectible with frequent, repeated assessments may reflect early aberrations in memory consolidation- that is, difficulty transforming temporary, labile memories into more stable, lasting forms. We will use the Boston Remote Assessment for Neurocognitive Health (BRANCH), an investigator-developed, non-proprietary, web-based platform, to capture high-resolution MDLC data. We will capture an initial baseline MDLC (10 min/day over 7 days) and longitudinal MDLCs (10min/day over 7 days every 6 months) for up to 3 years. In Aim 1, we will develop a summary MDLC score (e.g., accuracy and reaction time across several MDLC tests, Day 1 performance, “area under” the learning curve) honed to evidence of AD (i.e., Aβ (PiB) and tau (FTP) deposition on PET imaging) leveraging well-characterized participants (Imaging Cohort; n=250) who span the continuum from cognitively unimpaired (CU; 65+), to subjective cognitive decline (SCD; 60+), to Mild Cognitive Impairment (MCI; 55+). Second, to test the generalizability of this early detection approach, we will replicate findings in a diverse Community Cohort (n=400; >24% from under-represented groups; 65+) with novel AD plasma biomarkers (e.g., ptau217, Aβ42/40). In Aim 2, we will determine whether an individual’s initial learning curve (baseline MDLC) is predictive of their clinical trajectory over years. In Aim 3, we will determine whether change in an individual’s learning curve (longitudinal MDLCs evaluating new learning every 6 months), can sensitively track AD-related decline. Ultimately, we seek to provide a rapidly obtainable, repeatable, high-resolution snapshot of clinically relevant memory declines across the very early AD continuum to facilitate early detection of worrisome memory changes in the general population and to provide a more sensitive method to monitor cognitive change.

NIH Research Projects · FY 2025 · 2024-07

Summary: While anti-ABO(H) antibodies represent the most common immunological barrier to transfusion and transplantation, variation in anti-ABO(H) antibody levels and overall specificity may play a critical role in dictating the clinical consequence of these naturally occurring antibodies on adverse events following ABO(H) incompatible transfusion and transplantation. However, the factors that regulate this variation, and development of anti-ABO(H) antibodies in general, remain relatively unknown. Given this, our long-term goal is to define key factors that regulate the production and clinical outcome of naturally occurring anti-ABO(H) antibodies. Our central hypothesis is that innate immune factors target blood group expressing microbes, which shapes the specificity and clinical significance of naturally occurring anti-ABO(H) blood group antibodies. Our results also demonstrate that a series of innate immune lectins called galectins possess the ability to specifically bind and kill blood group positive (BG+) microbes through engagement of their carbohydrate BG antigens. Using a new preclinical model that leverages the murine equivalent of ABO(H) blood group antigens, we have also shown that these same microbes can drive the formation of anti-BG antibodies capable of causing ABO(H) incompatible hemolytic transfusion reactions (HTRs). Microbial populations within galectin knockout strains demonstrate significantly increased numbers of BG+ microbes and exposure of recipients to the non-metabolizable pan galectin inhibitor, thiodigalactoside (TDG), likewise results in increased colonization by BG+ microbes. However, galectin absence or inhibition blunts the ability of BG+ microbes to stimulate anti-blood group antibody formation, suggesting that galectin-mediated microbial killing enhances anti-blood group antibody formation. Importantly, we have also shown that galectins do not target all BG+ microbes equally. Thus, a dynamic interplay between the composition of distinct blood group positive microbes in an individual’s microbiota and the ability of galectins to target these microbes exists that may ultimately shape the specificity and overall levels of anti-BG antibodies. As studies to address this intriguing possibility require a unique combination of expertise, I will leverage my current expertise in glycobiology and microbiology with key training in immunology and transfusion medicine, obtained during the K99 phase of this proposal. I will also leverage our new preclinical model and series of recently developed glycan microarrays and build on these tools with establishment of key galectin KO lines and continued microarray expansion to address the following specific aims. Aim 1: Define the role of galectins in shaping anti-blood group antibody levels. Aim 2: Define the consequence of anti-blood group antibody repertoire on HTRs. Completion of these aims will provide unprecedented insight into the fundamental and previously unrecognized role of the interplay between innate immune factors and BG+ microbes, and how it shapes the most common immunological barrier to transfusion and transplantation. Furthermore, these studies will provide important foundation for the launch of my independent career during the R00 phase of this proposal.

NIH Research Projects · FY 2025 · 2024-07

The objective of these studies is to better understand submucosal biology as it relates to implantation of cell therapy devices. Endoscopic submucosal dissection (ESD) is employed to access the gastrointestinal (GI) submucosa for excision of lesions, therapeutic myotomy, and even peritoneal access. Expansion of this technique to include device implantation could enable novel approaches to long-term delivery of therapeutics, including cell therapies, to the GI tract. However, the biology of the submucosal space as a host for implantable devices is not well understood. The long-term goal is to leverage the GI submucosa as a host for regenerative therapies in the treatment of degenerative or autoimmune GI pathologies. The central hypothesis of the proposed work is that the submucosa can serve as an effective host for cell therapies. A model involving pancreatic islet transplantation into the gastric submucosa for the treatment of type 1 diabetes (T1D) will be employed, inspired by the finding of subepithelial heterotopic pancreatic tissue (or “pancreatic rests”) incidentally found on biopsies of the stomach. The pancreatic islet is a compelling cell model as there are existing animal models for T1D and validated assays to quantify islet survival and function. The rationale is that evaluation of the gastric submucosa as a potential host for cell therapies may not only lead to breakthroughs in the treatment of T1D, but also localized treatment of degenerative GI disorders in which normal tissue has been compromised. The central hypothesis will be tested by pursuing 3 specific aims: 1) Evaluate endoscopic procedure technique and safety for submucosal implantation in a swine model; 2) Characterize in vivo host response to submucosally implanted materials and devices; and 3) Determine encapsulated islet survival and function as well as phenotypic rescue. For Aim 1, a swine model will be used to evaluate outcomes and complications of ESD with and without device implantation, using devices of various geometries. To achieve Aim 2, tissue histology, immunofluorescent staining, flow cytometry, RT-PCR, and scRNA-seq will be used to evaluate the host response to ESD/device implantation as compared to the more common methods of subcutaneous and intraperitoneal implantation. For Aim 3, devices containing a test dose of allogeneic islets will be implanted into the gastric submucosa to evaluate islet survival and function (using immunofluorescent staining, RT-PCR, and scRNA-seq), and if successful, therapeutic doses will be implanted in a T1D swine model to evaluate for phenotypic rescue. The proposed research is significant as it will demonstrate a novel approach to GI device delivery and improve understanding of submucosal biology as it pertains to device implantation. Ultimately, this will contribute not only to the field of T1D and cell therapies, but also offer valuable insights for the future development of submucosally implanted localized treatment of other disorders, including degenerative GI disorders. This project will provide unparalleled training experience to the applicant in the translational aspects of implantable device design, large animal models, and submucosal biology.

NIH Research Projects · FY 2025 · 2024-07

The neuronal protein alpha-synuclein (αS) plays a key role in the group of neurodegenerative diseases known as synucleinopathies: Parkinson disease (PD), Parkinson disease dementia (PDD), and Dementia with Lewy bodies (DLB). The prevalence and associated societal and personal burdens of these disorders is increasing rapidly, yet no disease modifying treatment exists. Thus, new therapeutic strategies are urgently needed. Prior work of the PI has shown that targeting palmitoylation may be such a strategy. Palmitoylation is the post- translational modification of proteins by fatty acids, usually palmitate. Palmitoylation plays a key role in protein and vesicle trafficking. While αS itself is not palmitoylated, under pathologic conditions it disrupts trafficking. Thus, palmitoylation facilitates normal trafficking, while disease-associated αS disrupts it. In accord, the PI has found that enhancing palmitoylation by inhibiting the depalmitoylase acyl protein thioesterase-1 (APT1), which increases palmitoylation of its unknown brain substrate(s), ameliorates multiple measures of αS cytopathology including toxicity and inclusions. Further, treating genetically modified PD/DLB model mice with a pharmacologic APT1 inhibitor, ML348, improves their motor and dementia-like symptoms on validated measures such as the rotarod, pole climbing, Y-maze, and Morris water maze tests. While compelling, in the context of clinical translation these results are incomplete: the brain substrates of APT1 are unknown, and this is a major obstacle to developing palmitoylation-based treatments for PD/DLB. In particular, information on the APT1 substrate(s) responsible for these benefits can 1) ensure specificity of any potential drug by targeting individual pathways and 2) uncover new palmitoylation-based therapeutic targets. Furthermore, it is not known how αS itself may alter palmitoylation profiles (the “palmitoylome”) in the brain. This information would provide insight into whether APT1 inhibition corrects an underlying αS-dependent change in palmitoylation, or compensates via a separate pathway. Our Aims address each of these questions and together represent a crucial step in the pre-clinical assessment of palmitoylation as a therapeutic strategy for PD/DLB, using both physiologic and cellular approaches. In Aim 1, we identify physiologic APT1 substrates by isolating the palmitoylome in rat and human iPSC derived neurons with and without APT1 inhibition. We then validate the effect of potential substrates on αS pathology by assessing for somatic and synaptic trafficking defects. In Aim 2, we compare the brain palmitoylomes of wild type versus mutant αS transgenic PD/DLB model mice to evaluate for potential αS-dependent changes in palmitoylation. As in Aim 1, we then validate the role of this second set of proteins in synucleinopathy trafficking defects. Together, our Aims address important questions crucial to the development of palmitoylation-based therapeutics for PD/DLB while providing new leads for mechanistic insights into the intersection of palmitoylation and αS pathophysiology. To that end, they pursue research priorities pertaining to the Alzheimer’s disease related dementias (ADRD), specifically PDD and DLB.

NIH Research Projects · FY 2025 · 2024-07

Project Summary: Alzheimer's disease (AD), prevalent among the elderly, is a fatal neurodegenerative disorder marked by memory deficits and cognitive decline. It currently afflicts approximately 6.5 million Americans with an estimated 500,000 new cases diagnosed annually. One challenge in improving AD treatment is delayed intervention, as AD develops years before symptoms are evident. Objective, accurate diagnosis of AD is critical for effective intervention but remains an unmet clinical need. Existing diagnoses involve either lengthy and subjective clinical tests with neuroimaging or expensive and invasive procedures like lumbar punctures. This work aims to engineer an ultrasensitive blood-based diagnostic tool that can accurately determine the presence and severity of AD, to inform effective treatment. The accuracy of blood tests is partly hampered by scarce presence of neurological proteins, as the blood-brain barrier (BBB) restricts their presence. Blood extracellular vesicles (EVs) can reflect protein profiles in inaccessible brains, as they can traverse the BBB. Utilizing markers in EVs improves accuracy and reliability of current blood tests. Yet, developing cost-effective, streamlined, and scalable EV isolation tools remains a challenge. Integrating analysis of novel and robust AD biomarkers in EVs may further enhance AD diagnosis. One such marker could be the collective phosphorylation changes in tau, as an increase in tau phosphorylation correlates with tau aggregation. However, a highly selective and quantitative assessment of protein phosphorylation is lacking. The diagnostic challenge lies in the low concentrations of AD-specific biomarkers present in EVs, particularly the minute fraction of phosphorylated proteins. Therefore, the proposed diagnostic tool will utilize single molecule detection to measure low abundance AD protein biomarkers in EVs. Single Molecule Array (Simoa) technology, developed by the Walt lab, is the current state of the art for ultrasensitive protein detection, using digital enzyme-linked immunosorbent assay (ELISA) to isolate and count single molecules in femtoliter-sized microwells, achieving 1000-fold higher sensitivity than conventional ELISA. An integrated pipeline designed to efficiently isolate EVs from plasma and quantitatively uncover the phosphorylation state of AD diagnostic biomarkers is required to advance AD diagnosis. This pipeline can be achieved through multiplexed Simoa assays developed for a panel of robust AD protein biomarkers, enabling a simple, accurate, minimally invasive, and cost-effective diagnostic tool for AD, using plasma EVs. This project will provide Dr. Stephanie Zhang rigorous scientific training for an independent academic research career. She will work closely with Dr. David Walt in a highly interdisciplinary, collaborative environment at Brigham and Women’s Hospital and the Wyss Institute, and develop an extensive skillset in analytical chemistry, diagnostics, and technology development and translation. The training plan also provides opportunities to expand her scientific expertise and professional skills through coursework, seminars, conferences, and grant writing workshops.

NIH Research Projects · FY 2025 · 2024-07

Project Summary I am committed to a career as a physician-scientist and have a keen interest in receptor biology, with the goal of translating fundamental findings into improved care of patients. G protein–coupled receptors (GPCRs) are some of the most established drug targets. However, the clinical successes within the GPCR super-family are not evenly distributed. Certain receptor families have many successful drugs, such as adrenergic GPCRs in cardiac diseases and dopamine GPCRs in psychiatric diseases. Other GPCR families, such chemokine receptors, have very few approved drugs. I recently discovered a new GPCR signaling pathway that has relevance in chemokine signaling. This pathway is differentially regulated by the three chemokines that bind to CXCR3, which is expressed on activated T cells and drives Th1-mediated inflammation in the skin and other tissues. The goal of my project is to biochemically define this new GPCR signaling pathway and discover how this pathway signals in T cells. My mentor, Dr. Andrew Kruse, Professor of Biological Chemistry and Molecular Pharmacology, was one of the first in the world to obtain high-resolution structures of GPCRs. He has an established track record of excellent trainees and leading discoveries. Through this project, I will acquire critical skills in protein purification and structural biology that I currently lack, but which will be instrumental in establishing my own independent research program. Through Dr. Kruse and my broader advisory committee, I will also obtain training in skills necessary for my independence, including written and oral communication, grant writing, leadership and management, and mentoring/teaching. My clinical expertise is in autoimmune skin and connective tissue diseases and in the effects of immunomodulatory medications. Many of the diseases I treat appear to be driven in part by dysfunctional T cell signaling as highlighted by abnormal cytokine and chemokine profiles. The chemokines CXCL9, CXCL10, and CXCL11 bind to the chemokine receptor CXCR3. These, and other chemokines, are abnormally altered in diseases such as cutaneous lupus and psoriasis. My recently published work and preliminary data show that CXCL9, CXCL10, and CXCL11 cause different post-translational modifications on CXCR3 and lead to divergent transcriptional responses. Furthermore, CXCL11, but neither CXCL9 nor CXCL10, appears to signal through this new GPCR signaling pathway. These findings challenge the current paradigm of GPCR signaling. I believe that therapeutically targeting chemokine receptors has been challenging in-part because we are overlooking key intracellular signaling pathways that have therapeutic relevance. I believe this project is novel, challenges established paradigms of receptor signaling, and is clinically significant. Understanding the different chemokine signaling pathways at the molecular level will help us design new therapeutics with the desired effects.