Brigham And Women'S Hospital

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY This is a new application for a Ruth L. Kirschstein National Research Service Award Institutional Research Training Grant (T32) to train post-doctoral trainees in the field of oncopathology, defined broadly as research that seeks to improve our understanding of cancer pathogenesis and pathobiology, and in doing so provide the basis for the development of new approaches that improve cancer diagnosis, prognostication, and treatment. The overall goal of the Oncopathology Training Program (OTP) is to provide research training opportunities across the full spectrum of oncopathology research, from basic study of cancer pathogenesis using the tools of cell and molecular biology, genomics, proteomics, animal modeling, and bioinformatics, to translational research that involves the development and deployment of biomarker tests and the use of digital and computational pathology, approaches that are increasingly important in cancer research. The training program is designed to benefit the research career development of M.D. and M.D./Ph.D. trainees subsequent to their training in clinical and/or anatomic pathology, and Ph.D. trainees with educational backgrounds in computational fields interested in the emerging area of computational pathology. Support is requested for 4 postdoctoral trainees in years 1-2, and 5 trainees in years 3-5. The training program includes structured elements such as individual development plans, mentored research experiences, and customizable didactic training opportunities as well as workshops in grant writing, manuscript composition, and lab management. Each trainee will have Postdoctoral Advisory Committee comprised of the Program Director, primary research mentor, and one or more senior cancer researchers with relevant expertise that will review trainee progress, provide advice and constructive criticism, and serve as a review committee for external fellowship proposals. The OTP faculty includes 24 highly accomplished investigators, with extensive mentoring track records and substantial extramural grant support, who constitute a highly collaborative mentoring community whose expertise span a wide-range of multidisciplinary, oncopathology-relevant areas, including animal modeling of cancer, cancer immunology, cancer genomics, computational pathology and tissue imaging, cancer diagnostics, and translational cancer informatics. The OTP will also take advantage of the extensive complement of scientific and educational resources that are available at Brigham and Women’s Hospital (BWH), Harvard Medical School (HMS). and Dana Farber Cancer Institute (DFCI), including the NCI-funded Dana-Farber/Harvard Cancer Center.

NIH Research Projects · FY 2024 · 2020-08

Abstract Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disorder that results in premature and accelerated aging, while accumulation of progerin also occurs during physiological aging. Notably, one of the major targets of HGPS is the cardiovascular system, which are mechanically active tissues. Typical symptoms include hypertension, myocardial infarction, congestive heart failure, calcific aortic stenosis, and peripheral atherosclerosis. For example, autopsy findings have illustrated profound loss of vascular smooth muscle cells (SMCs) in the medial layer of large arteries, such as the aorta and carotid arteries, with replacement by collagen and extracellular matrix. Vascular wall echodensity is also increased in HGPS patients associated with exaggerated fibrotic adventitia formation. The overarching goal of this proposal is to optimize a biomimetic HGPS-on-a-chip system generated with patient-derived fibroblasts (FBs), SMCs, and endothelial cells (ECs) that allow application of relevant cyclic stretch for performing ‘clinical trials’ or informing clinical trial designs for HGPS patients. In particular, we will generate a whole-thermoplastic microfluidic system with a dual-layer blood vessel-mimicking structure. Based on our preliminary device previously already reported, the proposed system will be whole-thermoplastic consisting of two channels separated by a thin thermoplastic polyurethane (TPU) membrane. On top, layers will include a collagen hydrogel encapsulating FBs (adventitia) and an elastin hydrogel embedded with SMCs (media), post-seeded again with a monolayer of ECs. This methodology recreates the in vivo microenvironment of blood vessels to faithfully model pathological changes in HGPS.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY/ABSTRACT Su H. Chu, PhD, MS is a biostatistician and molecular epidemiologist with strong and substantial commitments to integrative omic research in respiratory and neurodevelopmental disease. Her career objective is to become an independent investigator with expertise in developing and applying novel statistical methods and multiomic network approaches that facilitate mechanistic understanding of the intersection of respiratory and neurodevelopmental disease. This proposal combines Dr. Chu’s extensive training in integrative omic methods development, along with her experience in genetic and metabolomic research, to examine the integrative metabolomic etiology of asthma and attention-deficit/hyperactivity disorder overlap (AAO). A wealth of epidemiological literature has revealed with remarkable consistency that asthmatic patients are at greater risk for ADHD, and vice versa, with a number of longitudinal studies of childhood asthma indicating excess risk of ADHD in adolescence and adulthood. However, no studies have directly interrogated the biological mechanisms by which these conditions may be related. The central hypothesis of this proposal is that childhood asthma and ADHD share both common and distinct dysregulated metabolic processes, some of which may have drivers that are genetic in origin. This will be explored using existing genetic and metabolomic data from the Childhood Asthma Management Program (CAMP), the Vitamin D Antenatal Asthma Reduction Trial (VDAART), and the Copenhagen Prospective Studies on Asthma in Childhood 2010 (COPSAC), three large prospective cohorts of children with extensive longitudinal phenotyping and multiple omic data types, by identifying metabolites and metabolic profiles associated with AAO (aim 1), constructing multiomic models and features that characterize dysregulated metabolic processes and their upstream drivers in AAO (aim 2), and validation and replication of all findings (aim 3). As Dr. Chu completes these aims, her career development program will facilitate the achievement of her primary training goals: 1) increase clinical understanding of ADHD and asthma diagnosis and treatment; 2) gain a nuanced understanding of best methods for, and practical experience in, the biological and statistical integration of genetic and metabolomic data; 3) develop skills in machine learning and integrative network methods for multiomic analysis; 4) strengthen current areas of experience to stay on the cutting edge of new analytic and study design techniques; and 5) enhance skills in mentorship, teaching, and the responsible conduct and communication of research. The support of a diverse mentoring team comprised of world experts in the fields of asthma and psychiatric genetics, metabolomics, integrative omics, and statistics, along with her strong quantitative training and the vibrant intellectual community of Harvard Medical School and the Brigham and Women’s Hospital, ensure the success of this proposal. Finally, this research will use state-of-the-art multiomic techniques to lay the initial groundwork for understanding the pathobiology of asthma and ADHD comorbidity, a major and well-established public health concern, from which new research programs will emerge.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY/ABSTRACT Obstructive sleep apnea (OSA) is a highly prevalent disorder with adverse neurocognitive and cardio-metabolic outcomes. Continuous positive airway pressure (CPAP) is the gold standard therapeutic option to treat airway obstructions during sleep and thus, prevent its adverse cardiovascular and neurocognitive outcomes. Previous clinical trials, however, have largely failed to show a consistent impact of CPAP on these health outcomes. One of the main limitations of these trials is, we believe, inadequate characterization of OSA and its acute physiological consequences. By characterizing OSA based on the “apnea-hypopnea index (AHI)”, there is a potential risk of negative results. We surmise that, by better characterization of OSA-related physiological consequences during sleep, we will be able to identify individuals at high risk for these adverse outcomes and those who would benefit most from therapy. We have developed physiologically driven metrics to capture the precise burden of OSA-related hypoxemia (“hypoxic burden”), autonomic response (“heart rate burden”), and sleep fragmentation (“arousal burden”). Our preliminary data from large observational studies suggest that these metrics outperform conventional sleep study parameters. In Aim 1, we seek to demonstrate that OSA patients with high hypoxic burden will exhibit greater improvements, after 12 weeks of CPAP therapy, in endothelial function (flow-mediated vasodilation) and oxidative stress markers than those with a low hypoxic burden. In Aim 2, we will investigate how heart rate burden determines the reduction in 24-hour mean blood pressure after 12 weeks of CPAP treatment. Finally, in Aim 3, we will seek to demonstrate that OSA patients with larger degrees of sleep fragmentation, quantified by arousal burden, will respond more favorably to CPAP, in terms of improvement in daytime sleepiness and attention, than those with low arousal burden. While the primary analysis will be the change in these outcomes after 12 weeks of CPAP, we will also assess these outcomes at 4 weeks to examine their time course. A total of 158 men and women with apnea-hypopnea index ≥15 events/hour will receive CPAP for 12 weeks. Adherence to therapy will be carefully monitored and encouraged by regular phone calls and in-person visits. Adverse events will also be closely monitored and recorded. Overall, our proposal is expected to demonstrate that prognostic markers of OSA that more strongly link with health outcomes will not only improve the diagnosis of OSA, but also provide a physiological basis for identifying those individuals most responsive to CPAP therapy. These results will have key mechanistic implications for “individualized medicine” in OSA by focusing on subgroups of patients who would most benefit from CPAP therapy. This personalized medicine approach will provide the scientific knowledge needed to progress towards larger studies in selected patients. Such results are of major importance because they have great potential to improve the quality of life and health outcomes of patients with OSA.

NIH Research Projects · FY 2024 · 2020-08

Project Summary/Abstract: This proposal details a five-year research and training plan with a scientific focus on defining the role of Notch signaling in driving pathogenic synovial fibroblast expansion in joints of patients with rheumatoid arthritis. The candidate spearheaded high-dimensional profiling studies of RA synovia that identified expansion of pathological sublining fibroblasts in RA (Mizoguchi, Slowikowski, Wei et al., Nature Communications, 2018 and Zhang*, Wei*, Slowikowski*, Fonseka*, Rao et al, Nature Immunology, 2019). The PI recently discovered that Notch signaling is a key regulator of pathogenic sublining fibroblasts differentiation. The long- term objective of the proposed study is to define how Notch signaling regulate pathological functions that contribute to RA pathology and determine if Notch receptors can be therapeutically targeted to treat arthritis. The specific aims proposed here utilize three complementary approaches to define how Notch regulates synovial fibroblast differentiation and function in RA. Aim 1 will identify Notch receptors critical for fibroblast differentiation towards a sublining fibroblast phenotype. Aim 2 interrogates the impact of global and fibroblast- specific Notch inactivation on synovitis in a mouse model of inflammatory arthritis. Aim 3 examines transcriptional control of Notch-driven fibroblast pathology. Using a combination of single cell technology, patient-derived tissues, and mouse model of arthritis, this study will provide the candidate with new training in bioinformatics, gene editing, and transgenic mouse model. The candidate’s immediate career development goals are to acquire expertise in bioinformatics and computational biology to guide analysis of single cell RNAseq data. A specific career development plan tailored to the candidate’s training needs is described by both the candidate and the mentors: Dr. Michael Brenner MD, an expert in fibroblast biology and rheumatoid arthritis, and Dr. Soumya Raychaudhuri MD PhD, an expert in single cell bioinformatics. The PI is an MD/PhD rheumatologist whose long-term goal is to become a tenure-track faculty focusing on developing novel treatments for rheumatic diseases. The proposed studies, training plan, and exceptional environment at Brigham and Women’s Hospital and Harvard Medical School will pave the way for the PI’s transition to an independent investigator and a leader in arthritis research.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY Chronic kidney disease is a powerful risk factor for subclinical left ventricular systolic dysfunction and incident heart failure, but the mechanisms of these relationships remain incompletely understood. The inflammasome drives renal and cardiac inflammation by activating the inflammatory cytokines interleukin-1β and interleukin-18 and the pyroptotic protein gasdermin-D. In patients with systolic heart failure or coronary artery disease, pharmacologic interleukin-1β blockade improves left ventricular systolic function and maximal exercise capacity and is associated with a trend towards fewer heart failure hospitalizations. Exogenous interleukin-18 administration to wild-type mice impairs left ventricular systolic function whereas blocking interleukin-18 after experimental myocardial infarction preserves left ventricular systolic function. Furthermore, interleukin-1β’s cardiodepressant effects are mediated in part by interleukin-18, suggesting that simultaneous blockade of both cytokines would confer additional benefit beyond targeting either cytokine alone. The broad goal of this application is to prepare the principal investigator, Dr. Leo Buckley PharmD, for a career as an independent, patient-oriented researcher who studies cardiovascular pharmacology with a specific interest in preventing and treating heart failure by identifying and targeting pathways that regulate myocardial structure and function. In addition to focused coursework and seminars, Dr. Buckley will complete a series of patient-oriented studies under the guidance of an expert mentoring committee to test the hypothesis that inflammasome activity contributes to incident heart failure risk in adults with chronic kidney disease by promoting left ventricular systolic dysfunction. He will address two specific aims: (1) that increased inflammasome activity associates with left ventricular systolic dysfunction and increased risk of incident heart failure in older adults; and (2) To test the hypothesis that colchicine improves left ventricular systolic function and reduces inflammasome activity in patients with uremic cardiomyopathy. These studies will improve our knowledge of and spur further investigations into the role of inflammatory cytokines in the pathogenesis of subclinical left ventricular dysfunction and heart failure. By the conclusion of the award, Dr. Buckley will have established an independent, patient-oriented cardiovascular pharmacology research program.

NIH Research Projects · FY 2024 · 2020-07

OVERALL – SUMMARY/ABSTRACT Organ transplantation remains a mainstay therapeutic strategy for patients with end organ diseases. One of the highest unmet needs to improve long-term transplant outcomes is devising more effective immune modulation. This requires innovative mechanistic studies of transplant alloimmunity. The lymph node (LN) is the quintessential organ of alloimmunity. While the recognition of alloantigens in the LN is fundamental to the generation of alloreactive T cells, our groups also have shown that the LN plays an important role in alloimmune-regulation and Treg-mediated tolerance. These multifaceted functions rest on the nature of LNs as extremely specialized organs with unique microvasculature, stromal fibers, and stromal cells (referred to as fibroblastic reticular cells [FRCs]). Our overarching hypothesis is that manipulating the microenvironment of LNs will provide a unique opportunity to direct the alloimmune reaction towards an anti-inflammatory tolerance response. Our major goals are to understand the cellular and molecular mechanisms that govern the microanatomical adaptation of the LN during immune activation or tolerance induction, and to develop highly innovative therapeutic strategies that promote a regulatory LN microenvironment and result in immune tolerance. This PPG sets forth a platform for connecting two teams (Drs. Abdi and Bromberg) with complementary skills and expertise in LN alloimmune-biology. Project 1 will test the hypothesis that sustained activation of FRCs of the LN during alloimmunity will result in FRC transformation to proinflammatory myofibroblasts creating an inflammatory milieu within the LN, which would further promote alloimmunity. Our corollary hypothesis is that restoration of the function of FRCs and microanatomy of the LNs will enhance their immunoregulatory function and promote tolerance. Aim 1 will examine the role of the HVEM/LIGHT pathway in the differentiation of FRCs into proinflammatory myofibroblasts, thereby creating an inflammatory milieu within the LN microenvironment and promoting transplant immunity. Aim 2 will investigate the mechanisms by which fibrotic FRCs promote a pro-inflammatory response in the LN. Aim 3 will reprogram the stroma of LNs via FRC delivery or LN-targeted delivery of senescence inhibitors to further promote alloimmune tolerance. Project 2 will test the hypothesis that FRCs regulate the LN laminin α4:α5 (LAMA4/LAMA5) ratio and control the fate of the immune response. Aim 1 will define the role of stromal cells in controlling the balance of LAMA4 and LAMA5. Aim 2 will define the role of LTβR as a key pathway in regulating the formation of LAMA5. Aim 3 will use targeted delivery of anti-CD40L and anti-LAMA5 mAbs to the LN to promote tolerance. An Administrative Core (Core A) and Nanoparticle and FRC Core (Core B) will provide the infrastructure and resources to support these two projects. The ultimate goal of these well-integrated and highly synergistic Projects and Cores is to generate transformative mechanistic data, which will lay the groundwork for developing highly targeted and innovative therapeutic strategies for transplantation.

NIH Research Projects · FY 2024 · 2020-07

The objective of the Harvard Women's Reproductive Health Research (WRHR) Career Development Program is to provide an outstanding training environment for the development of Obstetrics and Gynecology (OB/GYN) Physician-Scientists leaders. The Program is based on a transdisciplinary scientific approach to understanding the molecular, cellular and structural bases of gynecologic health, fertility preservation, and healthy pregnancy, with attention to mechanistic factors contributing to racial and ethnic disparities in reproductive outcomes including exposures to environmental toxicants, stress hormones, metabolic and microbiome disturbances. The program capitalizes on the long tradition of world-class postgraduate training and women's reproductive health research with collaborative mentoring teams across Harvard and leading hospital centers in the Boston Area: Brigham and Women's Hospital, Massachusetts General Hospital, the Center for Glycoscience and the National Center for Functional Glycomics at Beth Israel- Deaconess Medical Center, the Boston Center for Endometriosis, Boston Children's Hospital, Harvard T.H. Chan School of Public Health, Harvard Medical School and Tufts Medical Center. The Harvard WRHR consortium offers cutting-edge facilities and equipment and unique resources including high risk patient populations and access to several large, well-curated and characterized biospecimen and data repositories. Each WRHR scholar will be assigned to a primary preceptor/mentor and a mentoring team including basic and clinical scientists, mentor in good laboratory practice, research integrity officer, epidemiologic, biostatistical, data management and health disparity advisor to guide education, thinking, and progress in the scholar's research topic of choice. Career Mentors will advise scholars to successfully navigate within the academic structures of career advancement. To develop Scholars who will successfully transition to an independent research career, remain and thrive in academic medicine, the Program will follow a carefully deigned plan and advisory committee recommendations to attract and nurture the most qualified candidates and the institutional commitment will guarantee 80% research time of sufficient duration in the Program for each WRHR Scholar. We are deeply committed to enhancing diversity developing the independent research careers of women and OB-GYN physicians from under-represented minority groups.

NIH Research Projects · FY 2024 · 2020-07

Project Summary/Abstract Growing evidence suggests an etiological link between asthma and chronic obstructive pulmonary disease (COPD) wherein mild-to-moderate persistent asthmatics are susceptible to persistent airflow obstruction, putting them at a higher risk for developing COPD. Furthermore, epigenetic modulation due to in utero smoke (IUS) exposure during fetal lung development may play a role in asthma and COPD. These and other data suggest a lifelong trajectory of lung impairment from prenatal lung development to childhood asthma to COPD in adulthood. Longitudinal and carefully phenotyped multi-omic data offer the opportunity to understand the molecular determinants of this disease trajectory. Systems and network biology methods hold the potential to effectively integrate, analyze and interpret multi-omics data. Multilayer networks, in particular, offer a feasible first step to model disease perturbations jointly across multiple molecular levels, from the genome to the proteome. In this application, we will simultaneously analyze the rich multi-omics data (SNP genotyping, DNA methylation, mRNA and miRNA expression) collected as part of long-standing asthma and COPD cohorts using multilayer network methods. We will first integrate IUS exposure, asthma and COPD multi-omics data into networks and develop their statistical framework to facilitate subsequent bioinformatics analyses. We will then track the developmental origins of asthma by the integrated temporal analysis of fetal lung and childhood asthma multi-omics data. Finally, we will identify key molecules and pathways of the phenotypic transition from asthma in early life to COPD in adulthood using multilayer networks. Dr. Halu’s training in statistical physics and complex networks has prepared him well for his proposed research. However, understanding the molecular basis connecting complex lung diseases such as asthma and COPD through the analysis of multi-omics data is a formidable task that will require further training in specific areas. Dr. Halu will leverage the excellent intellectual environment of Harvard Medical School (HMS) and its teaching hospitals, and will have access to extensive computational resources through the Channing Division of Network Medicine and HMS. Through formal coursework and workshops, and with the help of a mentoring and advisory team with complementary expertise, Dr. Halu will immerse himself in a training program focusing on statistical genetics, epigenetics, and omics integration, big data in medical informatics, and the biology of pulmonary diseases and clinical translation. Dr. Halu will also participate in regular meetings with his mentors and advisory board members, which will allow him to share his progress. Altogether, Dr. Halu’s training and research plan will enable him to expand his current skill set to include the ability to address the challenges of analyzing the complex genomic and epigenomic data of large epidemiological cohorts, identify open questions in the systems biology of asthma and COPD, and ultimately contribute to the precision medicine of lung disease.

NIH Research Projects · FY 2024 · 2020-07

Abstract Glioblastoma (GBM) remains a formidable cancer to treat with only anecdotal examples of long-term survivors. Recently, immunotherapy has seen multiple successes against various types of cancer, but several recent clinical trials of this modality in GBM have not been successful. It is evident that two inter-related factors in the complex immunobiology of GBM have thwarted therapeutic efficacy: the existence of multiple immunosuppressive mechanisms and the significant lymphodepletion in the GBM microenvironment. The overarching goal of the Program Project is to address the problem of insufficient T cell activation and marked T cell attenuation in the GBM microenvironment. We will test the overall hypothesis that promotion of CD8+ and CD4+ T cell functionality can overcome the highly immunosuppressive mechanisms of GBM. A corollary to this hypothesis is that preclinical and clinical trials of immunotherapy combinations will provide an effective approach to GBM treatment. We have assembled a highly interactive and interdisciplinary team of 11 investigators in 4 highly integrated Research Projects, supported by 4 Cores. This team (some of whom have been working together for more than two decades) brings deep expertise in immunobiology, neuro- oncology, clinical trials, genomics and computational analyses to mechanistically study these two critical factors. We plan to study how immune checkpoint blockade can be combined with other T cell activating immunotherapies both in a clinical trial (Project 1) and in preclinical mouse models (Project 2). We propose to study novel immunosuppressive pathways in human GBMs based on CD161/ Clec2D (Project 3), IL-27 and endogenous glucocorticoid signaling (Project 4) and understand how these can be overcome to improve the anti-tumor function of CD4 and CD8 effector T cells. Core services will provide sophisticated genomic (Core 1), biocomputational/ biostatistical (Core 2), and mouse modeling/ imaging (Core 3) approaches to these Projects. This Program Project will thus provide significant mechanistic insights into the immunosuppressive microenvironment of GBM, into preclinical avenues of how to activate T cells against these tumors and finally into a novel clinical trial to target personalized GBM neoantigens in humans.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY A small population of long-lived CD4 T cells harbors replication competent virus (the latent HIV reservoir) during effective antiretroviral therapy even when viremia is undetectable; this latent HIV reservoir is invariably associated with virus rebound when treatment is stopped. The latent HIV reservoir is a major barrier to curing HIV, but multiple technical challenges limit its investigation. We developed an ultra-high throughput droplet microfluidic workflow called PCR activated cell sorting (PACS) that detects, sorts and sequences single cells containing a single copy of intracellular HIV DNA. In preliminary studies we demonstrated the ability of PACS to 1) process millions of cells at ultra-high throughput, 2) detect HIV infected cells 3) single cell sort and sequence this rare population of latently infected CD4 T cells. Based on these findings, we believe that PACS provides a unique opportunity to overcome existing technical challenges and define, for the first time, the genomic mechanisms that control the HIV latent reservoir. We hypothesize that latently infected cells have unique properties that allow them to harbor replication competent HIV genomes without producing virus. Thus, we propose to combine PACS with methods for single cell RNA-seq, genome and integration site analysis, ATAC-seq and proteomics to define the genomic mechanisms that control the HIV latent reservoir in single infected cells from people with ART suppression of the virus. Our Specific Aims are as follows: Specific Aim 1: Define the transcriptome of single cells harboring latent virus. We propose to sequence the transcriptomes of single HIV positive CD4 T cells isolated from the blood of individuals on ART, while determining in parallel the HIV provirus sequence and insertion site. These studies will define the transcriptional program of latently infected cells harboring full-length replication competent virus. Specific Aim 2: Establish the chromatin landscape of single cells harboring latent virus. We propose to analyze the chromatin profile of single HIV positive CD4 T cells in order to define the relationship between host DNA chromatin status and HIV latency. These studies will determine the role of chromatin status in the control of HIV latency and identify regulators of the transcriptional program of latently infected CD4 T cells. Specific Aim 3: Identify surface markers of latently infected CD4 T cells. We propose to isolate HIV latently infected CD4 T cells using PACS and analyze their surface proteome using barcoded antibody sequencing to identify surface marker combinations that best define the HIV latent reservoir. Upon completion, these studies will define novel surface marker combinations to identify latently infected CD4 T cells. IN SUMMARY, these studies use a multi-omics approach based on our combined expertise in the genomic regulation of immune cells, HIV biology, and microfluidics to define the mechanisms that control HIV latency. Hence, these studies will guide the development of novel therapeutic interventions, while providing novel tools for the monitoring of the latent HIV reservoir in infected individuals undergoing ART.

NIH Research Projects · FY 2024 · 2020-07

Summary  Alzheimer’s  disease  (AD)  and  AD  related  dementias  (ADRD)  are  unpreventable,  incurable  and  remain  poorly  understood. Their hallmark pathology consists of misfolded proteins in characteristic “inclusions” within subsets  of neurons and glial cells of the brain. Misfolding of the membrane-­associated protein α-­synuclein (αS) is central  to ADRD. Inclusions rich in αS in cortical and dopaminergic (DA) neurons are the hallmark lesions of dementia  with Lewy bodies (DLB) and Parkinson disease with dementia (PDD). But αS inclusions are also found in >50%  of  AD  cases,  correlating  with  cognitive  decline  and  frequently  colocalizing  with  tau  pathology.  αS  pathology  is  strikingly  heterogeneous  and  poorly  understood.  Common  αS  pathology  comprises  vesicle-­rich  “pale  bodies”  (PBs),  amyloid-­rich  Lewy  bodies  (LBs),  or  combinations  of  these.    PBs  have  indeed  been  discussed  as  precursors  of  LBs,  but  what  gives  rise  to  PBs  and  how  they  may  convert  into  LBs  remains  enigmatic.  The  ultrastructural  features  of  PBs  and  LBs  parallel  enormous  interest  in  the  field  in  both  amyloid  and  vesicle-­ trafficking pathologies in PD. In experimental settings, the seeding of neurons with pre-­formed fibrils leads to LB-­ like  amyloid aggregates.  These  aggregates  can  under  certain  conditions  spread and  self-­template  in adjacent  cells. Different amyloid fiber conformers (“strains”) lead to different patterns of neurodegeneration, with differing  levels  of  phosphorylated  αS  and  tau.  Human  genetic  studies  have  repeatedly  implicated  perturbed  vesicle  trafficking and (membrane) lipid homeostasis as a fundamental and unifying feature in disparate forms of ADRD.  We  hypothesize  that  altered  cellular,  and  especially  lipid,  microenvironments  can trigger  αS  amyloid  formation  and the development of different strains and pathologies. An increasing body of evidence, including work from  our  groups,  indeed  suggests  that  αS  toxicity  and  aggregation  can  be  modulated  by  altering  cellular  fatty  acid  (FA) saturation or sphingolipid (SL) composition through manipulation of glucocerebrosidase (GBA) and stearoyl-­ coA  desaturase  (SCD),  respectively.  We  propose  to  dissect  the  influence  of  these  pathways  on  PB  and  LB  formation  and  transition  in  the  most  disease-­relevant  patient-­derived  induced  pluripotent  stem  cell  (iPSC)  models.  Importantly,  we  will  employ  patient  brain-­derived  “seed”  as  the  most  relevant  trigger  for  neuronal  αS  aggregation. The use of both patient-­specific cell types and misfolded protein conformers will allow us to capture  “in  the  dish”  both sides  of  the  toxic  equation  in  neurodegeneration.  We  recognize  the  importance,  but  also  the  limitations,  of  postmortem  end-­stage  pathology  in  delineating  disease  mechanisms,  and  propose  to  establish  cross-­correlation between in vitro assays, human induced pluripotent stem cell (iPSC) models and postmortem  brain tissue. We will focus on iPSCs derived from patients with familial and sporadic synucleinopathies that are  matched to postmortem brain, including cases with concomitant levels of AD (β-­amyloid and tau) pathology. Our  approaches not only promise to shed light on the formation and consequences of amyloid strains in ADRD, but  will also point at potential interventions centered around the transient interaction of αS with lipid membranes.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY/ABSTRACT This K08 Mentored Clinical Scientist Research Career Development Award describes a five-year research and training program to establish the principal investigator (PI) as an independent, R01-funded physician-scientist in the field of pulmonary vascular medicine. Co-mentorship by Bradley Maron, M.D., an expert in cysteinyl thiol biochemistry, vascular fibrosis, and PAH pathobiology and pathophysiology, and Joseph Loscalzo, M.D., Ph.D., an expert in redox biochemistry, network medicine, and proteomics, will oversee the PI’s research and professional development activities at Brigham and Women’s Hospital and Harvard Medical School. Scientific publication, guidance from an advisory committee, didactic education, and presentation at scientific conferences will support the scientific aims of the project and provide career development in three key training areas: the pathobiology of PAH inception, kinase ubiquitination, and experimental PAH models. The PI is guaranteed >80% protected academic time to dedicate to the proposed K08 program. PAH is characterized by endothelial dysfunction and oxidant stress that promotes fibrotic remodeling of the pulmonary arterioles, leading to right heart failure, and death. Mortality and hospitalization increase incrementally beginning at a mean pulmonary artery pressure (mPAP) below values that were previously thought to be pathogenic. Therefore, treatment of early-stage PAH may improve clinical outcome, but effective therapies do not exist because the pathobiology of endothelial dysfunction and vascular remodeling in early PAH is not known. We provide novel data that early-stage PAH is characterized by pulmonary arteriolar fibrosis, impaired right ventricle-pulmonary artery (RV-PA) coupling, and increased endothelial C-terminal src kinase (Csk) expression occurring prior to the development of elevated mPAP in vivo. Computational modeling predicts that the Csk binding-partner and E3 ubiquitin ligase Casitas b-lineage proto-oncogene (Cbl) is susceptible to oxidative modification at cysteinyl thiol 396 (Cys396). We hypothesize that oxidation of endothelial Cbl-Cys396 prevents Csk ubiquitination to increase Csk-dependent fibrosis and impair RV-PA coupling in early-stage PAH. To test this hypothesis, we propose the following specific aims: 1) Determine the impact of Cbl-Cys396 oxidation on Csk ubiquitination and expression in cultured human PAECs, 2) Assess the role of increased endothelial Csk on fibrillar collagen expression in cultured human PAECs, and 3) Test the effect of Csk inhibition on RV-PA coupling in early- and advanced-stage PAH in vivo. We anticipate that findings from this K08 will establish Csk- targeted therapy as a novel strategy by which to prevent or reverse endothelial fibrosis and vascular remodeling prior to the development of elevated mPAP and right heart failure in PAH. Furthermore, this research program will establish kinase-ubiquitin ligase dysregulation in early-stage PAH as a plane of separation from the PI’s current co-mentors.

NIH Research Projects · FY 2025 · 2020-07

Project Summary/Abstract: Advanced cutaneous T cell lymphoma (CTCL) and peripheral T cell lymphoma (PTCL) have very poor survival due to limited effective treatments. Three fundamental barriers have stalled drug development: 1) the low incidence of each T--cell lymphoma (TCL) subtype, which limits sample availability and clinical trial enrollment; 2) heterogeneity across subtypes, which further complicates biologic interrogation; and 3) a lack of faithful model systems for in vitro and in vivo studies, which also cannot adequately assess immunotherapies. Thus, there is a major scientific gap to direct rational clinical trial efforts for novel therapeutic regimens. Here, we propose the use of an innovative implantable microdevice (MD) for in situ drug screening in patients to identify the most effective cancer drugs and novel agents to direct future personalized treatment of TCL. Our MD delivers up to 20 drugs of any class into spatially distinct regions of a tumor. It is delivered percutaneously and retrieved 72 hours later by excising the device along with surrounding tissue with a standard skin biopsy tool. This device allows testing of a range of relevant drugs directly inside the living tumor, thereby preserving the native tumor physiology and, importantly, avoiding systemic toxicities. Drug sensitivity is determined by histologic assessment of the tumor surrounding the drug chambers with immunohistochemistry (IHC). The MD and the method for processing and analyzing tissue has been validated across multiple cancer types in murine models and is being employed in an ongoing pilot study in breast cancer. The specific aims of these studies will be to: i) determine the safety and feasibility implanting and retrieving the MD in skin lesions of TCL; ii) assess local responsiveness of TCL to cancer treatments delivered via the MD through IHC and use highly multiplexed cyclic immunofluorescence, a novel platform, to characterize changes in the native immune microenvironment following drug treatment, and iii) correlate genomic features of TCL with drug response to nominate potential therapeutic biomarkers and to correlate these responses with a patient’s clinical and molecular response to systemically administered treatment. We hypothesize that in situ pharmacological profiling of drugs in TCL has the ability to predict systemic responses to standard of care therapies and therefore can serve as a rapid screen for multiple investigational single or combination treatments. Additionally, genomic and immunophenotypic analysis of tumors may assist in identification of predictive biomarkers of treatment. The results of our studies will direct clinical trial efforts evaluating the most promising treatment strategies and usher in the era of precision medicine in TCL.

NIH Research Projects · FY 2024 · 2020-07

Summary: Red blood cell (RBC) alloimmunization can make it difficult to procure compatible RBCs for future transfusion, which can directly increase morbidity and mortality in transfusion-dependent individuals. While patients who develop multiple alloantibodies against distinct alloantigens are particularly challenging to manage, the immune events during initial alloimmunization that may increase the likelihood of generating additional alloantibodies following subsequent transfusion remain unknown. Our long-term goal is to identify immune factors that enhance subsequent alloimmunization events in previously alloimmunized individuals in order to prevent the accumulation of multiple alloantibodies in transfusion dependent individuals. Our central hypothesis is that initial alloimmunization events directly enhance subsequent RBC alloimmunization by inducing CD4 T cells that possess the ability to directly activate B cells against a completely unrelated RBC alloantigen following subsequent transfusion. Our hypothesis is formulated on the basis of our recent discovery that B cells specific for one antigen (the HOD (HEL, OVA and Duffy) antigen) not only internalize HOD following RBC engagement, but likewise remove and internalize additional RBC components, suggesting that B cells may possess the ability to remove multiple antigens following engagement of the target antigen. Consistent with this, adoptive transfer of CD4 T cells primed by KEL RBC transfusion in the presence of poly I:C, which induces viral-like inflammation, directly enhances alloantibody formation against the completely distinct HOD antigen following subsequent transfusion of RBCs expressing HOD and KEL. Depletion of marginal zone (MZ) B cells, a unique B cell population previously shown to be critical in the initiation of alloantibodies, inhibits KEL RBC priming and the HOD RBC boost following HOD x KEL RBC transfusion, suggesting that MZ B cells work in concert with previously recognized bridging channel 33D1+ dendritic cells (33D1+ DCs) shown to be critical in the initial activation of CD4 T cells following HOD RBC transfusion. In contrast, while KEL RBC-induced alloimmunization requires type I interferons (IFNab) and HOD RBC-induced alloimmunization requires toll-like receptor (TLR) signaling, KEL-induced alloimmunization in the presence of PIC requires both IFNab and TLRs, suggesting that while innate immune pathways may differ for KEL and HOD RBC-induced alloimmunization, PIC allows KEL RBCs to engage TLRs and prime a subsequent HOD boost. We will use a series of pre-clinical models to define the key priming and subsequent boosting pathways by testing the following specific aims: Aim 1: Define the role of MZ B cells, 33D1+ DCs, IFNab and TLRs in PIC/KEL RBC-induced priming. Aim 2: Define the role of MZ B cells, 33D1+ DCs, and TLRs in subsequent KEL-mediated HOD RBC boost. We think that successful completion of these aims will define key immunological priming and boosting events that facilitate alloimmunization and therefore will provide an important framework to develop rational approaches to prevent the development of RBC alloantibodies against multiple alloantigens in chronically transfused individuals.

NIH Research Projects · FY 2024 · 2020-07

Project Summary/Abstract: Immunomodulatory drugs (IMiDs) are a mainstay of myeloma therapy. They work by binding to the substrate recognition surface of the cereblon (CRBN) E3 ubiquitin ligase and, in so doing, alter its substrate specificity such that it now targets the critical myeloma transcription factors IKZF1 and IKZF3 for proteasomal degradation. A majority of patients develop resistance to IMiDs, and in some patients, the myeloma cells fail to make CRBN and become pan-IMiD resistant. Such patients have a very poor prognosis. To address this issue, I have developed a novel positive-selection screen for the identification of small molecule degraders of any protein of interest. As a proof-of-concept, I have applied this screen to IKZF1. I have identified Spautin-1, a novel degrader of IKZF1 that acts via an unknown mechanism that is distinct from that of IMiDs. In Aim 1, I propose to identify the minimal Spautin-1 responsive degron. In Aim 2, I propose complementary genetic and biochemical experiments to identify the proteins necessary for the action of Spautin-1. Aims 1 and 2 should together help elucidate the mechanism of action of Spautin-1. In Aim 3, I propose SAR studies to find Spautin- 1 derivatives with increased potency against IKZF1 and minimal off-target effects, and that destabilize IKZF1 in vitro and in vivo models. These studies will help identify a novel pathway for destabilizing IKZF1, and may yield novel treatments for IMiD-refractory myeloma. Finally, if successful, these studies will point the way towards the eventual application of this strategy to undruggable transcription factors in other neoplasms. I'm a medical oncologist with a research background in cancer biology, applying for a K08 award with the long-term goal of becoming a tenure-track, independent laboratory investigator. I envision developing an independent research program aimed at understanding the pathways that regulate the activity and stability of critical oncogenic transcription factors in hematologic malignancies, and harnessing that knowledge to develop new therapies for patients. During my proposed K08 research training, I will perform mentored research in the lab of Dr. William Kaelin at the Dana-Farber Cancer Institute (DFCI) on the mechanism by which Spautin-1 degrades IKZF1. I will spend 90% of my time on research and 10% on patient care and teaching duties while seeing patients with hematologic malignancies. I have assembled an expert scientific advisory committee to help guide my development including: Dr. Kenneth Anderson (Harvard Medical School, DFCI), Dr. Eric Fischer (DFCI), Dr. Sara Buhrlage (DFCI), and Dr. Wade Harper (HMS). My clinical mentor is Dr. Daniel Deangelo, a national expert on leukemia. I believe that training in a world-class clinical and research environment, along with additional coursework and conferences will help me achieve my long-term career goals.

NIH Research Projects · FY 2025 · 2020-06

Abstract Non-invasive, multi-parametric characterization of prostate cancer (PC), with magnetic resonance imaging (MRI) methods, is an active area of research with great potential for providing improved diagnosis and treatment monitoring. The PI-RADSv2 assessment system that was established by an international team of experts recognizes the value of quantitative images in PC diagnosis, but relies largely on qualitative evaluation of weighted images. Although this grading approach achieves reasonably good separation be- tween normal and abnormal prostate tissue, it does not achieve adequate separation between indolent and aggressive disease, with the risk that more unnecessary and costly surgery is performed with poten- tially dire consequences on patient quality of life. High-value protocols, without need for an invasive and costly endo-rectal radio-frequency coil are being investigated. This comes at the cost of extended scan time and reduced image quality in terms of spatial resolution, signal-to-noise ratio and signal bias, which negatively impacts sensitivity and specificity of multi-parametric MRI. With the pronounced increase of multi-parametric MRI exams, there is also the desire to integrate the support by the most recent revolution in diagnostic imaging, i.e., machine learning. It becomes increasingly clear, that in order to avoid having to train neural networks for each specific system and protocol, reproducible and thus preferably quantitative imaging protocols are essential. To overcome these limitations, we propose both pulse sequence develop- ment, investigation of ADC validity and reproducibility and novel post-processing strategies. The overall objective is to demonstrate the added value of lesion characterization with quantitative values and at the same time understand and minimize the influence of protocol choices and scan hardware, hence improve overall reproducibility. Specific Aim 1 will focus on the development of a low distortion MR imaging sequence for rapid concurrent quantification of T2 and diffusion signal decay. Specific Aim 2 will examine ADC variations that result from changes in diffusion time over a range that is typical with present day clinical MR systems. Specific Aim 3 introduces advanced handling of low noise diffusion data, which will be indispensable for achieving high accuracy and precision with non-invasive and economic external coils. Specific Aim 4 introduces a novel ADC computation approach that fully captures the complex diffusion signal decays in tissues and at the same time is largely protocol and system independent. Moreover, re- sulting images and quantitative maps processed according this approach, exhibit considerably lower noise, which can be traded for higher spatial resolution or shorter scan duration. In combination, the consis- tently quantitative nature of the data and its ubiquitous validity and comparability will greatly facilitate the establishment of recommendations for disease-related thresholds. Ultimately this may permit much more reliable differentiation of aggressive from indolent disease.

NIH Research Projects · FY 2025 · 2020-06

The Brigham and Women’s Hospital (BWH)/Harvard Medical School (HMS) Reproductive Outcomes of Stress and Aging (ROSA) SCORE focuses on stress exposures and how neural regulation transmits stress to worsen women’s health during and after menopause. The overarching scientific goal of the ROSA SCORE renewal is to investigate the impact of both individual- and neighborhood-level stress exposures on vasomotor symptoms (VMS) and problems with sleep, mood, memory, and cognition during menopause. Disturbances in these neurocognitive domains greatly disrupt the health and activities of women during this pivotal reproductive health transition and may accelerate cognitive aging, including dementia, which is more common in aging women than men. Stress has been linked with many health conditions in women, but important gaps remain related to menopause and neurocognitive aging. One vital exposure the ROSA SCORE will investigate is artificial light at night (ALAN) at both neighborhood and individual levels, which appears elevated in menopausal women due to VMS-related sleep disruption, with consequences for stress responsivity, mood, and cognition (including memory). ROSA SCORE research will include innovative, inter-related animal and human projects; human projects that share a study cohort and neurocognitive outcomes; and an innovative Sleep and Light Research Core that supports all projects. Project A (epidemiology) leverages a 25+ year cohort study with rich neighborhood- and individual-level data. Project B (clinical) uses a randomized controlled trial to test the impact of ALAN exposure on sleep, mood, and cognitive outcomes in women with VMS. Project C (pre-clinical) uses a mouse model of menopause to characterize the roles of hypothalamic KNDy neurons in the arcuate nucleus and neurokinin B receptor-expressing neurons in the median preoptic area in sensing and transmitting stress signals to modulate core body and skin temperature changes of VMS, sleep, and cognition. A robust Career Enhancement Core will continue our objective of catalyzing research on aging women’s neurocognitive health by funding, mentoring, and educating early-career investigators through pilot grants and educational programming. The ROSA SCORE has engaged an outstanding interdisciplinary team of renowned scientific leaders who continue to be supported by the BWH Connors Center for Women’s Health Research, the leading women’s health research program at BWH/HMS, which will contribute significant institutional resources, including supplemental funding for ROSA Center pilot grants, and which interfaces with synergistic research programs focused on women’s brain health, including on dementia, to augment the impact of the ROSA Center locally and across the SCORE Consortium. The ROSA Center, thus, is uniquely well positioned to contribute critical knowledge about the impact of stress on neurocognitive performance in aging women.

NIH Research Projects · FY 2024 · 2020-04

Mast cells (MCs) play important roles in allergic responses. Recent studies suggest that MCs are also essential to other inflammatory diseases by releasing inflammatory cytokines, chemokines, and the MC- specific proteases chymase and tryptase after degranulation. Pharmacological inactivation of MCs prevents or slows disease progression. Alzheimer’s disease (AD) is the most common cause of dementia and disability in the elderly. It is the sixth leading cause of death in the U.S., affecting more than 5 million Americans alone, according to the Alzheimer’s Association. One definitive diagnosis of AD is based on the presence of extracellular deposition of neurotoxic β-amyloid (Aβ) into senile plaques. Human AD brains have elevated protease expression, neuronal death and synapse loss, blood-brain barrier (BBB) leakage, and activation of inflammatory cells such as microglia, astrocytes, and T cells. MCs also present in human AD brains, mainly in the hippocampus, cerebral cortex, and thalamus, but studies have yet to test whether these cells participate directly in the pathogenesis or serve merely as another inflammatory hallmark. Our preliminary data demonstrated that the plasma levels of MC activator IgE and MC granular contents tryptase and histamine were elevated in patients with early stage AD, indicating enhanced systemic MC activation. Anti-tryptase and CD117 antibodies detected MC accumulation in the cortex and hippocampus from human and murine AD brains. Using MC-deficient KitW-sh/W-sh mice and over-the-counter (OTC) MC inhibitor ketotifen, we demonstrated that the absence or pharmacological inhibition of MCs reduced Aβ deposition and senile plaque formation in the hippocampus and cerebral cortex, and reduced the numbers of total Iba-1-positive microglia and CD68-positive phagocytic microglia in these regions in APPSWE-PS1∆e9+/– (APP-PS1) mice that develop cerebral amyloidosis. Brain tissue extract ELISA showed that the absence of MCs reduced the production of pathological Aβ species (Aβ1-40 and Aβ1-42). Adoptive transfer of in vitro-prepared MCs into KitW-sh/W-shAPP-PS1- recipient mice restored cortical and hippocampal Aβ deposition, microglia infiltration and activation, and AD brain cortex Aβ1-40 and Aβ1-42 contents. A preliminary water T-maze behavior test suggested that MC depletion improved cognitive decline in APP-PS1 mice. We hypothesize that MCs play a pathogenic role in AD by releasing pro-inflammatory cytokines and proteases, and MC inhibition with the anti-allergy drugs may become a novel therapy of human AD. We propose three aims to examine whether MC depletion or inhibition protects mice from Alzheimer’s disease; to examine whether genetic deficiency of FcεR1 or anti-IgE antibody therapy protects mice from Alzheimer’s disease and, to establish the mechanistic link between mast cell activation and mouse Alzheimer’s disease.

NIH Research Projects · FY 2024 · 2020-04

PROJECT SUMMARY Depression and anxiety are highly comorbid and costly diseases. Evidence-based psychotherapy is the first-line treatment but is underutilized and not scalable. Digital mental health interventions (DMHIs), delivered via the internet and/or mobile apps, have evolved as efficacious and potentially scalable treatments. To date, however, effectiveness in routine care is limited by insufficient patient engagement. In order to achieve the transformative potential of DMHIs, we must identify strategies to keep patients engaged without adding human support in a form that would limit scalability. Automated motivational push messaging (AMM) and light-touch human coach support (CS) offer two such strategies. The proposed research tests these strategies, while drawing preliminary conclusions about a hypothesized model of DMHI engagement based on the technology adoption and treatment adherence literature. The model posits that two systems-level constructs (social influence and facilitating conditions) and three patient-level constructs (attitude, self-efficacy, habit strength) drive DMHI engagement. In Study 1 (N=20), I will employ user-centered design to develop and refine a set AMMs targeting the three hypothesized patient-level engagement-driving constructs (Aim 1). In Study 2, I will recruit N=76 primary care patients with depression and/or anxiety via provider referral to an 8-week 2x2 factorial clinical trial whereby participants will all receive access to a DMHI with known efficacy and be randomized to an engagement strategy condition (i.e., a previously-validated CS protocol, newly-developed AMM, both or neither). To further understand how AMMs function, message delivery in the AMM arms will be micro-randomized: each day participants will be randomized to receive a message or not, such that they receive an average of 4.2 messages/week. Micro- randomization allows causal inference about the near-term impact of message delivery (i.e., are AMMs a cue to action) and the relationship between message impact and context (e.g., time of day the message is delivered). Measured outcome data will comprise level of engagement (operationalized as minutes of DMHI use), weekly self-reports on the five engagement-driving constructs, and weekly self-reports of clinical outcomes. I will test im pacts of each strategy on m easured outcom e data (Aim 2) and explore the hypothesized relationships between engagement-driving constructs and DMHI engagement (Aim 3). Clinical outcomes will be assessed, however, consistent with the experimental therapeutics model, this research leverages a DMHI with known efficacy, allowing the focus to be an upstream target (patient engagement) rather than the clinical outcomes themselves. The overarching goal is to influence the target so as to ultimately enhance clinical effectiveness. This project will build my expertise in clinical trial design and build my proficiency in user-centered design (i.e., rapid, prototype testing via field studies) and data science (i.e., analysis of intensive, correlated longitudinal data) methods commonly applied in DMHI optimization research. Findings will lay a foundation for R01s aimed at optimizing DMHIs for engagement, and ultimately effectiveness, when integrated into routine care.

NIH Research Projects · FY 2025 · 2020-04

The prevalence of opioid use disorder (OUD) in pregnancy has risen markedly in the United States over the past two decades, reflecting trends observed in the general population. Guidelines recommend that pregnant patients with OUD should receive medications for opioid use disorder (MOUD) during pregnancy to reduce the risk of illicit opioid use and to improve fetal and maternal outcomes. In the context of R01 DA049822, we found that buprenorphine use during pregnancy compared to methadone is associated with a lower risk of neonatal abstinence syndrome, preterm delivery, small-for-gestational age, as and most congenital defects. However, we also found that retention in MOUD treatment during pregnancy and the postpartum period is suboptimal, particularly for buprenorphine, and lack of treatment in the postpartum period is associated with a markedly higher risk of maternal overdose death. Co-exposure to psychotropics is highly prevalent in pregnant patients treated with MOUD, with 54% being co-exposed. Likewise, non-opioid substance use disorders are common in those treated with MOUD: 8% are diagnosed with illicit stimulant use, 8% with cocaine use disorder, 14% with cannabis use disorder, and 7% with alcohol use disorder. Given plausible pharmacodynamic and pharmacokinetic mechanisms by which these co-exposures might affect the comparative safety and effectiveness of MOUD treatments, there is a need to better understand risks associated with co-exposure to psychotropic medications and non-opioid substance use disorders and how they should impact the choice of MOUD. The recent surge of highly potent fentanyl in the drug supply also introduces complexity into treatment decisions regarding type of MOUD and optimal treatment protocols that needs to be evaluated. The suboptimal retention in MOUD treatment points to the need to identify modifiable factors that can improve treatment retention (including provider, medication dosing and dispensing schedule, and care delivery model), and thus maternal and neonatal outcomes. Finally, the use of naltrexone to treat OUD has increased rapidly in recent years in women of reproductive age, leading to a rapid rise in pregnancy exposure. Yet very few data are available regarding the safety of use during pregnancy, creating a critical gap in the evidence needed to guide the growing number of pregnant patients (or those planning pregnancy) who have been successfully treated with naltrexone. In this competing renewal application, we propose to build on the team’s significant experience studying OUD treatments to address these newly emerging critical gaps in evidence. We will use state-of-the-art and innovative epidemiological methods applied to large nationwide cohorts of publicly and commercially insured pregnancies that are continuously updated. The resulting evidence will help steer providers toward the optimal treatment choice for individual patients and will identify factors that can be intervened upon directly to improve retention in MOUD, a critical aspect of successful substance use disorder treatment.

NIH Research Projects · FY 2025 · 2020-04

ABSTRACT Depression is debilitating, costly, and highly prevalent, representing the leading cause of ill health and disability worldwide. A majority of US households includes pets. Pets offer a source of emotional support and previous research has shown that they may help ease depression, anxiety, and stress. However, longitudinal studies to establish causal relationships or explore mechanistic pathways are still lacking. The objective of this proposal addressing PAR-18-213, ‘Human-Animal Interaction (HAI) Research’, is to, for the first time longitudinally, examine the effects of pets in the home on depression and psychological distress in humans, and to explore potential pathways by studying stress biomarkers (salivary cortisol-DHEA ratio) and the direct and mediating effects of the gut microbiome and metabolome in these associations. We will leverage three ongoing large population-based studies – the Nurses’ Health Study 2 (NHS2, n=116,430), NHS3 (n=47,435), and the Growing Up Today Study (GUTS), 12,413 children of the NHS2 women, who were 9-14 years old at enrollment in 1996 (88% with pets). With biennial follow-up, they offer a wealth of repeatedly assessed lifestyle, environmental, and psychosocial assessments allowing to finely control for their potential confounding in our analyses. Our primary study aims include an ongoing collection of stool samples from 35,000 participants in the NHS2, who we will follow after stool collection for depression to include 400 women with/without pets and incident depression in the current application. In addition, a subcohort of 226 NHS2 participants (62% with pet) collected detailed, repeated psychosocial assessments including information on pet attachment, pet owner’s personality dimensions, and stress biomarkers as well as participants’ gut microbiome; thus, we leverage these microbiome data at no additional cost. Further, embedded in NHS3, we will initiate a longitudinal ‘Nurses’ Pets Study’ (NPS), enrolling pets of nurses into active follow-up for future HAI studies. Our chief hypotheses are that pet ownership/attachment reduces risk of incident depression and psychological distress, and that primary pathways include an improved cortisol-DHEA ratio and alterations in the gut microbiome, offering the potential for new therapeutic approaches and evidence- based advice regarding the introduction of a pet into one’s home.

NIH Research Projects · FY 2025 · 2020-03

Glioblastoma is the most common primary brain cancer in adults and remains a deadly disease. Despite intense research and clinical efforts, its survival has not significantly improved for the past several decades. A major reason for this recalcitrant nature is the tumor’s ability to escape toxicity from currently available therapies, including Temozolomide (TMZ), radiation, and immune-modulation. This resistance depends on the activation of defense mechanisms in response to damage, and is mediated epigenetically by a complex, multi- component machinery that governs the transcriptional status of the tumor cells. Our studies have defined oncogenic proteins EZH2, BMI1 and LSD1 as crucial components of this epigenetic response. We have also demonstrated that they are regulated by specific microRNAs (miR-124, miR-128 and miR-137), co-expressed in normal brain, and simultaneously lost in glioblastoma. The re-expression of these three microRNAs in glioblastoma provides a strong inhibition against the three proteins and results in a very relevant sensitization of tumor cells to stress, both in vitro and in vivo. The overall goal of this proposal is two-fold: first, to characterize the mechanism whereby the co-activation of EZH2, BMI1 and LSD1 mediates tumor survival, establishing its necessary role in this response; Second, to develop a microRNA-mediated strategy to abate this epigenetic shield that is vital to the tumor, to support a gene therapy approach synergistic with current standard therapies. AIM1 proposes experiments to dissect the role of the EZH2/BMI1/LSD1 epigenetic complex in tumor responses against Temozolomide, radiation, and immunotherapy. AIM2 provides novel solutions to design and construct artificial genes that multiply the payload of microRNAs, and which thus function as an ideal gene therapy platform for multi-targeting this epigenetic response. AIM3 investigates the applicability of multi-microRNA-based therapy in animal models of glioblastoma, to address the vital problem of tumor resistance in vivo. INNOVATION: The proposed study to use artificial microRNA clusters for the control of the complex epigenetic tumor survival response is an innovative and heretofore unexplored approach. Also, this proposal introduces strategies to exploit the unique features of microRNAs biology, processing and intercellular trafficking in the perspective of an integrated gene therapy approach. LONG-TERM OBJECTIVE: We seek to meaningfully impact the care of glioblastoma patients through the application of principles of microRNA clustering, epigenetic interference, and synergism with other therapies.

NIH Research Projects · FY 2026 · 2020-02

PROJECT SUMMARY: First cloned in our laboratory, TIM-3 is a hallmark of T cell exhaustion and dysfunction in murine and human chronic viral infections and cancer. Since its discovery, TIM-3 has been recognized as an important negative regulator of T cell functions, which has formed the basis for checkpoint blockade immunotherapies being developed for cancers. Our recent studies suggest that TIM-3 also antagonizes the expression of TCF1, a crucial transcription factor expressed by a recently discovered subset of T cells with stem-like properties that are the source of effector T cells. However, TIM-3 is also highly expressed on dendritic cells (DCs). Despite the high levels expressed on DCs, the role of TIM-3 in these cells remains unclear. The focus of this renewal is on TIM-3 expression in DCs and how this co-inhibitory molecule regulates DC functions and orchestrates the priming and fate of T cells. To interrogate TIM-3-mediated effects on DC functions, we have generated mice in which TIM-3 deficiency is restricted to DCs. Initial studies with these conditional knockout mice revealed that TIM-3 expressed on DCs is pivotal in immune regulation, including in autoimmune diseases and cancer. While we have known that mice deficient in TIM-3 develop more severe experimental autoimmune encephalomyelitis (EAE) and are better at suppressing tumor growth, our published and preliminary findings indicate that TIM-3 expressed on DCs, has profound effects on these disease outcomes. In these experimental models, we found that mice with DC-specific loss of TIM-3 generated an expanded pool of stem-like CD4+ T cells. Further investigation revealed that multiple pathways, including inflammasome and cGAS-STING, were perturbed in TIM-3- deficient DCs upon activation. These observations led us to hypothesize that TIM-3 curbs the activation of DCs with functional implications for the generation and maintenance of autoreactive stem-like CD4+ T cells. Uncovering the molecular mechanisms by which TIM-3 exerts its effects on DC functions and influences T-cell-mediated immunity has fundamental and therapeutic importance. Thus, to test this hypothesis, we propose two specific aims to: 1) elucidate the functions of TIM-3 in DCs that impact the generation of stem-like autoreactive CD4+ T cells driving CNS autoimmunity, and 2) uncover the molecular mechanisms underlying TIM- 3:Inflammasome crosstalk and its impact on CNS autoimmunity. Using an extensive suite of mutant mouse lines, we outline experiments that will tease apart the TIM-3- mediated molecular interactions between DCs and T cells during CNS autoimmunity and build on the advances made during the previous grant cycle. By combining the power of sequencing and CRISPR/Cas9 genetic screening with in vitro functional assays and in vivo disease models, we will explore how TIM-3 signaling regulates DC function and generation of autoreactive stem-like T cells. The proposed studies will provide a greater understanding of the contribution of cell-intrinsic and -extrinsic effects of TIM-3 in DCs on the development and persistence of autoimmunity.

NIH Research Projects · FY 2025 · 2020-01

PROJECT SUMMARY Among the top priorities of the HIV field is the search for therapeutic interventions that can lead to sustained antiretroviral therapy (ART)-free HIV remission. Although the majority of HIV-infected persons will experience rapid viral rebound after ART interruption, there are rare individuals, termed post-treatment controllers (PTCs), who demonstrate sustained virologic suppression for months or years after treatment cessation. These individuals are considered an ideal example of durable HIV control, with direct implications for HIV cure research. However, our understanding of the virologic determinants of HIV remission remains incomplete. This is in part due to the scarcity of PTCs identified through any one research center or clinical trial, and in part because of the limited scope of viral reservoir studies that have been performed to date. The main goal of this proposal is to perform an in-depth virologic analysis of PTCs and our overarching hypotheses are that HIV reservoir characteristics can provide mechanistic insights behind their ability to achieve HIV remission. Through an international effort, we have established the Control of HIV after Antiretroviral Medication Pause (CHAMP) study, the largest study of PTCs world-wide. Our initial studies of PTCs from the CHAMP cohort has already yielded intriguing findings, including the identification of the first HIV reservoir biomarker, total proviral genome numbers, that predicts which individuals will become PTCs after treatment interruption. While these results are promising, the HIV reservoir is far more complex than just the total number of proviral genomes in the peripheral blood mononuclear cells (PBMCs). The activity and impact of the HIV reservoir must take into account a constellation of factors in a conceptual framework that we call the HIV reservoir ecology. This paradigm incorporates the combined proviral, cellular and molecular circuits that contribute to HIV persistence, including 1) the chromosomal environment of the HIV integration sites in the host genome, 2) susceptibility to host immune responses, and 3) molecular pathways underlying viral persistence and reactivation. Until now, technical challenges have hindered our ability to comprehensively characterize the HIV reservoir, but we have overcome these technical hurdles with novel assays in our laboratories and propose to take a holistic approach in characterizing the HIV reservoir ecology in both PTCs and NCs. The results of such studies will advance our understanding of the mechanisms of HIV control in PTCs, with implications for predicting post-treatment control and determining which of these circuits may be the best targets in the design of strategies for all individuals living with HIV.