Brigham And Women'S Hospital

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Hypertensive disorders of pregnancy (HDP) are the most common pregnancy complication and are associated with an increased long-term risk of cardiovascular disease (CVD) which is also the leading cause of death for women in the US. Emerging evidence suggests that environmental exposure during pregnancy may play an important role in the development of HDP. However, several limitations exist, including 1) the ignorance of residential mobility during pregnancy in exposure assessments, 2) the lack of studies considering different subtype, onset, and severity of HDP, and 3) the lack of considerations of the totality of environment (i.e. the exposome). While HDP have been linked to long-term CVD risk after pregnancy, limited data are available on early maternal CVD risk (within the first five years after delivery) complicated by HDP. More importantly, no study has focused on the impact of environmental exposures during and after pregnancy on maternal CVD risk after pregnancy. In addition, risk assessment models of HDP predominantly rely on biomarkers that are not ubiquitously tested among all pregnant women, and no predictive model has been developed to identify women at higher CVD risk after pregnancy. This study builds on our prior work in the external exposome and leverages our access to the unique statewide linked electronic health records-birth records data from the OneFlorida Clinical Research Consortium to address multiple challenges in the field by: 1) determining the association between pregnancy external exposome and risk of HDP, considering the subtype, onset, and severity of HDP and accounting for pregnancy residential mobility in exposure measurements; 2) assessing early risk of maternal CVD after pregnancy associated with HDP and the external exposome, and 3) developing predictive models of HDP and early risk of maternal CVD after pregnancy. This research project will be embedded in a comprehensive training program consisting of coursework, guided experiential and clinical learning, seminars, and mentorship by an established team of experts. The training program is designed to further my strengths and to develop new research skills while contributing to our scientific understanding of environmental impacts on cardiovascular health among pregnant and postpartum women. The training proposal details a five-year plan of formal and informal instruction in cardiovascular health, omics and exposome research, biomedical informatics and data science, and professional skills. My short-term career goals include completing coursework in all training areas, disseminating study findings through publications and presentations, engaging in career development activities, and applying for independent R01 funding in the third year of the award. My long-term career goal is to become an independent, collaborative, and productive epidemiologist and health data scientist leading research programs in cardiovascular health, with a focus on its intersection with perinatal and environmental epidemiology using big data.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract: Autoimmune diseases collectively affect over 23 million Americans and impose a huge burden of disease. Many autoimmune diseases are characterized by pathologic T cell-B cell interactions and production of autoantibodies. The T cell populations that help B cells in autoimmune diseases vary in phenotype and include T follicular helper (Tfh) cells, which reside in follicles of secondary lymphoid organs, as well as T peripheral helper (Tph) cells, which are B cell-helper T cells that migrate to inflamed peripheral tissues such as the rheumatoid joint. Tph cells and Tfh cells share the ability to recruit B cells via production of a B cell chemoattractant CXCL13 and then promote B cell differentiation through both surface interactions and secreted cytokines. The signals that regulate development and function of Tph cells and Tfh cells in autoimmunity remain incompletely described, and particularly little is known about T cell production of CXCL13. Our preliminary data reveal the aryl hydrocarbon receptor (AHR), a ligand-gated transcription factor, as a potent negative regulator of Tph and Tfh cell phenotype, with a dramatic effect on suppressing T cell CXCL13 production. In this project, we utilize human primary T cells and patient samples to evaluate the broad effects of AHR on Tph and Tfh cell development, function, and transcriptomic and epigenetic regulation. We will evaluate the direct targets of AHR in human T cells via ChIP-seq and seek to identify new transcriptional mediators downstream of AHR using CRISPR arrays. In addition, we will study synovial fluid and serum samples from patients with rheumatoid arthritis and comparator conditions to evaluate alterations in the extent of AHR agonist and antagonist activity in rheumatoid arthritis, a disease characterized by abundant accumulation of Tph cells in the target tissue. We expect that this project will reveal novel mechanisms mediated by AHR that regulate production of CXCL13 and key features of Tfh and Tph cell phenotypes. Understanding the molecular control of these key T cell functions may highlight new strategies to interfere with Tph and Tfh cells therapeutically.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract Dr Sharmila Dorbala is an Associate Professor of Radiology at Harvard Medical School and a physician/scientist at Brigham and Women's Hospital (BWH). She has devoted her career to patient-oriented research focusing on cardiac amyloidosis. She contributed independently to the field of cardiac amyloidosis through a succession of substantive studies utilizing molecular imaging to phenotype cardiac amyloidosis in order to diagnose and manage this condition. Her research program is impactful, both directly and through effective mentoring of the next generation of amyloidosis investigators. Dr. Dorbala has successfully mentored 26 pre- and post-doctoral trainees in patient- oriented research projects. She is currently PI of two active NIH R01 grants and also has funding from AHA and foundation/industrial sources. Her program provides a unique opportunity for POR due to the intersection of resources from programs in clinical investigation at the Harvard School of Public Health and the BWH cardiovascular imaging T32 program. Her mentees have presented their work nationally and internationally, published papers, and won research awards and pilot grants. The new research aims proposed in this award lay the foundation for adjunct interventions to enhance transthyretin cardiac amyloidosis (ATTR-CA) therapies and novel image-based personalized strategies to predict treatment failure. She will assess (1) sonoporation, a therapeutic ultrasound technique to increase myocardial perfusion and, consequently, improve delivery of antifibril drugs; (2) shotgun metagenomic sequencing of the gut microbiome to inform development of nutrition-based adjunct therapies; (3) radiomics of 99mTc- pyrophosphate SPECT/CT to assess treatment response, and (4) machine learning based CT-derived sarcopenia metric to predict survival. This research has important clinical implications. Currently approved TTR stabilizing/silencing drugs, though lifesaving for most, are not effective in about 30% of patients. Moreover, while these therapies improve clinical outcomes, they do not treat amyloid fibril. Fibril-targeted therapies have been unsuccessful to date, probably because of poor interstitial drug delivery due to the presence of fibril. The results of the proposed mentoring and research activities of this K24 project are likely to not only substantially improve outcomes and alleviate HF symptoms in these gravely ill patients, but also transform her patient- oriented research program into a formal individualized program of mentoring with a focus on preventing heart failure from amyloidosis.

NIH Research Projects · FY 2025 · 2021-05

Sustained intra-articular delivery of disease modifying osteoarthritis drugs (DMOADs) holds promise for preventing the progression of osteoarthritis (OA). However, since (DMOADs) are intended for early OA, when patients are active, repeated mechanical loading of joints can be detrimental to the delivery system, causing rapid drug release. To our knowledge, none of the previously reported intra-articular platforms for DMOAD delivery have been evaluated in physically active animals or have considered the impact of activity induced mechanical stress on the delivery platform and the drug release. We have developed a hydrogel platform that can rapidly recover following mechanical stress relevant to running human knee joints, with no impact on sustained release of the encapsulated agents. Hydrogel loaded with cathepsin-K inhibitor (L-006235) – a small molecule DMOAD prevented OA progression in mice undergoing treadmill running. The overall objective of this application are to (i) develop variants of our hydrogel platform with different release kinetics of L-006235 to understand how local release kinetics/pharmacokinetics impacts therapeutic efficacy and (ii) further engineer the hydrogel platform for delivery of biologic DMOADs. Our long-term goal is to develop a versatile and mechanically stable drug delivery platform with tunable release kinetics for intra-articular delivery of DMOADs in active joints to prevent OA progression. Our central hypothesis is that a mechanically stable hydrogel platform can minimize the impact of joint-related mechanical stress on sustained release of DMOADs and therapeutic efficacy of this system can be maximized by tuning the local release kinetics of DMOADs. To achieve our objectives, we propose two specific aims: 1) Investigate the impact of release kinetics of L-006235 on therapeutic efficacy; and 2) Investigate the delivery of biologic DMOADs in active joints using hydrogel. Under the first aim, we will develop hydrogel variants with different release kinetics of L-006235 and will study the impact of mechanical stress relevant to human joints on hydrogel variants and L-006235 release. Next, we will validate the differences in release kinetics in treadmill running mice and evaluate the therapeutic efficacy and off-target effects in treadmill running mice with OA. For the second aim, we will identify formulation parameters, including TG-18 concentration and choice of solvent to maximize loading and stability of three different biologic DMOADs (IL-1Ra, FGF-18 and sTNFRII). Formulations will be evaluated for mechanical stability in vitro, release kinetics in treadmill running mice and efficacy and off-target effects in treadmill running mice with OA. The research proposed in this application is innovative, in our opinion, because it focuses on a novel hydrogel platform that is mechanically stable in joints, allows tunability of release kinetics and is versatile. We will be the first to (i) demonstrate therapeutic efficacy of a wide range of DMOADs in “physically active joints” and (ii) demonstrate that release kinetics of DMOADs defines their therapeutic efficacy. The proposed research is significant because it is expected to provide strong scientific justification for continued development and future clinical trials of this promising hydrogel that will enable us and others to compare the effect of different DMOADs on OA pathology in active joints, and identify the most promising DMOADs and their ideal release kinetics. Ultimately, such knowledge has the potential to offer paradigm shifting impact in OA therapy by enabling translation of promising DMOADs.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Excessive daytime sleepiness (EDS) affects 10-20% of the population and is associated with cardiovascular diseases (CVD) and mortality. Emerging data suggest that targeting EDS may provide a novel intervention for improving CVD. However, findings are limited by self-reported data and heterogeneity. There is a need to dissect and understand the underlying drivers of EDS subtypes, and to determine whether there are subtypes causally related to CVD and potentially modifiable. Our recent work identified two subtypes of EDS – sleep propensity (SP; characterized by objectively measured long sleep duration, high efficiency, and less fragmentation) and sleep fragmentation (SF; short sleep duration and low efficiency). Each of them is common in the population, associated with adverse cardiovascular outcomes and different genetic backgrounds. We hypothesize that SP is a novel sleep phenotype that reflects a property of the need of staying asleep; dissecting EDS into SP and SF subtypes will facilitate identification of genetic and physiological mechanisms for EDS, and improve understanding of pleiotropic or causal associations with CVD risk. In order to test these hypotheses, we will leverage macro- and micro- sleep architecture measurements, genomics, and other -omics data in population-based cohorts. We will address the following specific aims: 1) To identify demographic, behavioral, clinical and neurophysiological factors (assessed by actigraphy and electroencephalography) for SP and SF, and refine classification of EDS subtypes if needed; 2) To identify genetic variants and molecular pathways associated with EDS subtypes and generate robust polygenetic risk score for risk stratification; 3) To systematically evaluate the causal or non-causal associations between EDS subtypes and CVD traits; 4) To identify the modification effect of each EDS subtype on genetic susceptibility of CVD using gene-environment interaction analyses. This work will advance our understanding of the heterogeneity of EDS, reveal biological mechanisms and pathways linking to CVD, and provide information that will guide clinical and public health interventions as well as provide directions for future laboratory research.

NIH Research Projects · FY 2025 · 2021-04

SUMMARY Despite recent advances in therapeutic strategies, the prognosis for patients with highly malignant brain tumors, glioblastomas (GBM) remains poor, with a median survival of 12-19 months. Immunotherapy has emerged as a promising approach for different cancer types. However, its efficacy in GBM has been limited primarily by overall systemic immune suppression and the immune-suppressive tumor micro-environment. Recently, we have shown CRISPR/Cas9 engineered self-targeting re-purposed cancer cells specifically home to tumor cells and release targeted ligands that induce tumor cell killing which translates into survival benefits in mouse models of primary and metastatic tumors. Based on our exciting studies, we have gene edited and subsequently engineered syngeneic immunosuppressive GBM to express bi-functional immunomodulatory and cytotoxic protein, interferon (IFN)β and granulocyte macrophage stimulating factor (GMCSF), which is known to induce both innate and adaptive immunity. Our preliminary data reveal that repurposed immunosuppressive GBM cells do not proliferate in vivo and elicit an active immunity which prevents tumor recurrence. These results although promising, have raised fundamental questions for our tumor cell based gene edited therapy strategy to be characterized and tested extensively in immunocompetent mouse tumor models that mimic clinical settings of immunosuppressive, resected and recurrent immune-profiled GBM tumors. In this proposal, we will first develop and extensively characterize a platform of gene edited and engineered syngeneic immunosuppressive and active GBM therapeutic tumor cells (ThTC) and assess them for their mechanism based direct killing of parental GBM cells and their ability to elicit active anti-tumor immunity in primary and recurrent mouse GBMs. Based on our previous findings that GBM tumor resection promotes the recruitment of CD4/CD8 T cells and local delivery of synthetic extracellular matrix (sECM) encapsulated immunomodulators has therapeutic efficacy, we will test sECM-ThTC for their therapeutic efficacy in resected GBM mouse tumor models. We hypothesize that ThTC will lead to specific killing of residual GBM cells in the tumor resection cavity of primary and recurrent GBMs and elicit active immunity. To ease clinical translation, we will ultimately CRISPR/Cas9 gene edit and subsequently engineer patient derived resected primary tumor cells (hTC) to express human IFN and GMCSF (hThTC). These hThTC will be tested in recurrent GBM models generated from glioma stem cell (GSC) lines in humanized mice. The integration of the safety kill switch, HSV-TK in ThTC will ensure safety in our approach and the incorporation of genetically engineered imaging markers into both ThTC and GBMs will allow us to follow fate and efficacy in vivo and thus to fine tune the proposed approaches. We anticipate that our findings will have a major contribution towards developing novel ThTC based therapies for GBM and are likely to define a new treatment paradigm for patients with other cancers.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Pathological expansion of fibroblasts in the synovial tissue surrounding the joint drive disease in rheumatoid arthritis (RA) and osteoarthritis (OA). Recent studies have identified molecularly and functionally distinct phenotypes of synovial fibroblasts using single cell RNA sequencing (scRNAseq). One of the phenotypes, found exclusively in the lining compartment of the synovium and expanded in both RA and OA, has been implicated in tissue destruction in vivo. Previous studies have shown that synovial fibroblast phenotypes are plastic, making them potentially inducible with biological therapies but difficult to study in vitro, as they lose their phenotypes ex vivo. I propose two novel strategies to model the induction and maintenance of the synovial lining phenotype. Preliminary analyses prioritized TGF𝛽 , a cytokine known to drive fibroblast differentiation, in both strategies. The first strategy builds on the notion that fibroblast phenotypes are in dynamic equilibrium and exist at multiple stages of induction in human tissue. Aim 2 will model these states in over 100,000 fibroblasts from 108 synovial donor biopsies with the novel RNA velocity algorithm to infer lining fibroblast differentiations processes and nominate driver genes. Aim 2 will either perform 108 separate analyses combined through meta-analysis or do one joint analysis with Crescendo, to be developed in aim 1 as the first multi-donor RNA velocity analysis. The second strategy builds on preliminary data that show that genes activated in phenotype induction are inactivated during phenotype loss ex vivo. Aim 3 will directly experimentally assay the dynamics of phenotype loss ex vivo, profiling 150,000 fibroblast at multiple time points with scRNAseq. Aim 3a will test the efficacy of exogenous TGF𝛽 stimulation to maintain the lining phenotype ex vivo. Aim 3b will nominate and test more pathways from sophisticated analysis of the generated time-course data. Together, these aims will identify molecular drivers of the lining phenotype and fuel novel research on therapeutics to target lining fibroblasts. I have expertise in single cell computational biology and synovial fibroblast genomics. I developed the popular Harmony algorithm for single cell integration, published in Nature Methods and co-first authored a paper detailing the induction of a novel fibroblast subtype necessary for arthritic disease in vivo, in press at Nature. Completing the proposed research will help me build my analytical skills with time-course data analysis and develop invaluable skills in experimental fibroblast biology. I will train Dr. Soumya Raychaudhuri, in statistical analyses, co-mentor Dr. Michael Brenner, expert in synovial fibroblast biology, advisor Dr. Peter Kharchenko, developer of RNA velocity, advisor Dr. Fiona Powrie, director of the Kennedy Institute for Rheumatology, and advisor Dr. Christopher Buckley, expert in synovial fibroblast biology. With this multi- disciplinary training, I will be become a principal investigator applying computational and experimental methods to translational rheumatology research.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY / ABSTRACT This application for a Mentored Research Scientist Development Award (K01) is submitted by Katsiaryna Bykov, PharmD, ScD, in response to PA-20-190. Dr. Bykov is an Associate Epidemiologist and Instructor in Medicine in the Division of Pharmacoepidemiology and Pharmacoeconomics at Brigham and Women’s Hospital and Harvard Medical School. Her long-term goal is to generate clinically actionable evidence to guide the safe and effective use of medications in older adults in the complex milieu of polypharmacy and multimorbidity. Her recent research has focused on developing innovative methods for the detection and evaluation of drug-drug interactions and their clinical impact using electronic healthcare data. Dr. Bykov aims to acquire expertise in clinical geriatrics and diabetology, geriatric pharmacoepidemiology, and advanced statistical methods for data mining and signal detection to translate her methodological work into clinically relevant research. To achieve her aims, Dr. Bykov proposes a 5-year program of career development and mentored research centered on the development of a valid and efficient system for the detection and evaluation of drug-drug interactions in older adults with diabetes. Within the highly productive and supportive research environment of the Division of Pharmacoepidemiology, Dr. Bykov will work with an interdisciplinary team of mentors and collaborators drawn from across institutions at Harvard and University of Pennsylvania who have deep expertise and national/international reputations in the specific substantive areas of her proposed training: clinical geriatrics and diabetology, advanced statistical methods for data mining and signal detection, drug-drug interactions research, and pharmacoepidemiology. The overarching objective of the proposal is to develop a framework for the detection and evaluation of clinically relevant drug-drug interactions in older adults with diabetes. Dr. Bykov proposes a novel 2-stage screening that will identify potentially interacting drugs in electronic healthcare data and will be followed by a rigorous hypothesis-driven evaluation of identified interactions. The approach will use Medicare claims data, available in the Division and linked to electronic health records and outpatient laboratory test results for a subset of patients, and will be specifically tailored to address the heterogeneity and complexity of health status in older adults, including varying degrees of frailty and multimorbidity. The proposed work will provide high quality and clinically relevant evidence that will help older adults with diabetes and their clinicians to more fully assess the benefits and risks of adding a new medication in the presence of other medications and co-morbidities. The knowledge gained from this work will impact several million older adults with diabetes in the US and will provide the applicant with a solid background to become an independent investigator and, ultimately, a leader in the study of medication use and outcomes in aging populations.

NIH Research Projects · FY 2025 · 2021-04

Mental health conditions are common among pregnant and postpartum women as well as non-pregnant women of reproductive age. While many patients require pharmacologic treatment, the safety of psychotropic medications in pregnancy is an area with large evidence gaps. Historically, spontaneous reports, pregnancy exposure registries, and case-control designs were the main approaches used to evaluate the safety of psychotropic medications in pregnancy, all of which have well-known limitations. In recent years, the field has gained much expertise with the conduct of cohort studies nested in large healthcare utilization data. Across all these designs, studies tend to focus on a single or a few selected adverse pregnancy outcomes, and they are performed at a single time point many years after the drug has entered the market and has been used by many pregnant women. To avoid unnecessary exposure of mother and fetus to harmful medications or to avoid women being unnecessarily deprived of treatments for psychiatric disorders when no harmful effects exist, a new and systematic approach is needed to generate timely evidence on the safety of psychotropic medications in pregnancy with respect to all relevant maternal and fetal outcomes. We will develop and implement a TreeScan based approach to conduct active surveillance of antipsychotic medication safety in pregnancy. TreeScan is a novel method for drug safety surveillance, which scans hierarchical trees of specific outcomes as well as groups of clinically related outcomes for associations with the treatment of interest, while accounting for multiple testing of correlated hypotheses. We will first develop hierarchical trees for congenital malformations, maternal and other neonatal outcomes based on shared underlying disease processes, and modify the TreeScan approach to accommodate the unique challenges of drug safety evaluation in the context of pregnancy. Use of hierarchical trees increases power to detect clinically related outcomes, which would not be feasible by evaluating individual diagnoses only. We will then implement TreeScan to evaluate the risks of a broad range of adverse maternal and neonatal outcomes associated with antipsychotic medications. Antipsychotics are the mainstay of treatment for women with schizophrenia and bipolar disorder, but little is known regarding their safety profile in pregnancy, especially for the newer antipsychotics. We will use nationwide cohorts of over 3.5 million publicly and privately insured pregnancies in the US, nested in healthcare utilization databases that contain rich information on confounders. In the final aim, we will extend the approach to conduct near real-time prospective, sequential surveillance of newly approved antipsychotic and other psychotropic medications in order to detect potential safety signals as early as possible after approval. By providing the necessary information for healthcare providers to make evidence-based prescribing decisions and to counsel women about the use of specific antipsychotics during pregnancy, the proposed studies will have an immediate public health impact within the field of perinatal psychiatry.

NIH Research Projects · FY 2025 · 2021-04

Abstract After years of infection, a small subset of people with HIV can develop broadly neutralizing antibodies (bnAbs), defined as antibodies that neutralize a diverse range of HIV isolates. While eliciting bnAbs is a central focus of HIV-1 vaccine research, bnAbs may have additional roles as long-acting biologic antiretroviral therapy or as an immune effector arm in virus eradication studies. Broadly neutralizing antibodies target the HIV-1 envelope glycoproteins, heterotrimers of surface gp120 and transmembrane gp41 molecules, that are 50% glycan by mass. The cloning of antibody genes from individuals with HIV identified new bnAbs with increasing potency and breadth of neutralization that have been studied clinically as HIV-1 treatment. Whereas single infusions of bnAbs can reduce plasma virus loads in people with HIV, virus variants resistant to the individual bnAb emerge quickly and limit the activity and therapeutic potential of bnAb monotherapy. Classically, an antibody molecule contains an Fc region linked to two Fab regions with identical antigen binding sites. Recently, an antibody was engineered that combined three distinct Fab regions into a single molecule. SAR441236 is a tri-specific bnAb that combines the CD4bs specificity of VRC01-LS, the V1/V2 glycan-directed binding of PGDM1400, and the gp41 MPER binding of 10E8v4 into one antibody molecule. ACTG A5377 is a phase I first-in-human study of SAR441236 that investigates the safety, pharmacokinetics (PK), and anti-HIV-1 activity of this novel trispecific bnAb. A maximum of thirty viremic participants will be studied in a single infusion dose de-escalation trial design with 24 weeks of follow-up. The goal of this proposal is to leverage samples from A5377 to determine if HIV-1 decay in response to “triple” biologic ART differs from conventional combination ART and to define the mechanisms of virus escape from a trispecific bnAb. The purpose of this proposal is to combine innovative experimental and mathematical approaches with classic molecular virology to characterize the decay of viremia and define the mechanisms of HIV-1 escape from this first-in-class trispecific bnAb. We hypothesize that the trispecific bnAb, SAR441236, clears cell-free and cell-associated virus from blood, induces large and dynamic population shifts in the HIV-1 env quasispecies, and selects for virus escape variants at the protein and glycan level to maintain infectivity in the presence of bnAb. Specific Aims of this proposal are to determine the kinetics of SAR441236-induced HIV- 1 decay, understand the effects of SAR441236 on HIV-1 env quasispecies, and define the HIV-1 env and glycan shield determinants of SAR441236 resistance. Our approaches test hypotheses that are central to understand the dynamics and evolution of HIV-1 under a trispecific bnAb selection pressure.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract Sepsis is a significant public health concern with substantial financial burden in the USA. Mortality is alarmingly high in sepsis recurrence. Whether by trauma or nosocomial infection, microbes can give rise to uncontrolled infectious inflammation that impacts millions. Therefore, a deeper knowledge is needed of the endogenous resolution mechanisms as well as their potential failure(s) to resolve sepsis. The acute inflammatory response is protective; yet, when uncontrolled, inflammation is associated with many diseases, trauma, and surgical interventions that can lead to sepsis and loss of life. Resolution of inflammation was widely held to be a passive response and today is considered an exciting and essentially untapped terrain for new interventions. In self-limited inflammation, the PI first mapped and elucidated the structures, biosynthesis and functions of novel families of resolution phase mediators collectively termed specialized pro-resolving mediators (SPM). The SPM superfamily include lipoxins, resolvins, protectins and maresins where each family is proven to actively stimulate the resolution of inflammation, infections and are organ protective (i.e. lung, heart, neuroprotective) in pre-clinical animal models. In human tissues, cellular and molecular understanding of resolution programs for infectious inflammation is critically needed to harness the endogenous chemical signals that resolve innate responses to bacterial challenge. SPM target both human neutrophils and macrophages that are central in initiating the inflammatory response for defense as well as its timely resolution. In this R35 MIRA application, the PI shall focus on addressing critical gaps and challenges in the field of resolution of inflammation relevant to human infectious inflammation, sepsis and recurrence. The main overarching question and challenge to be addressed focuses on the general mission of determining whether failed resolution mechanisms in inflammation contribute to poor outcomes in sepsis or its recurrence and to identify these new components. This information is critically needed and must be obtained from accessible human tissues such as blood so that they can be swiftly implemented. Results from these will help stratify and shape the basis of new strategies for monitoring resolution mechanisms and pathways as well as their potential failure in human sepsis. Addressing these fundamental questions on the resolution of inflammation is the thrust of this MIRA application and are designed using new innovative approaches and technologies in place in the PI’s laboratory from NIGMS support. The PI has a record of innovation, and the flexibility of a MIRA will enable obtaining critical new information on mechanisms of SPM in resolution of infectious inflammation needed in the long-term, to carry out well-informed new treatment approaches for sepsis and other maladies that involve and will require taking into account resilience and the resolution response in inflammation.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Contraceptive patterns are changing and the implications for ovarian cancer risk are unknown. Oral contraceptives (OCs), which are an established protective factor for ovarian cancer, are being used less frequently and intrauterine device use (IUD) is on the rise. However, the impact of changing contraceptive patterns on ovarian cancer incidence is unclear. While most studies of IUDs and ovarian cancer risk suggest an inverse association, some studies report no association or even a slight increase in risk. Differences between these studies could be attributable to IUD type, timing, or molecular features of tumors including histotypes. Preliminary data from the New England Case Control (NEC) Study suggest the association between IUDs and ovarian cancer risk vary by histotype, with a non-significant decrease in risk of low-grade serous and clear cell tumors but not for other histotypes. Furthermore, we observed an increased risk of ovarian cancer with low tumor stromal CD163 expression, reflecting heme scavenger receptor expression decreased M2-type macrophage infiltration. These observations suggest the impact of IUD use on ovarian cancer risk may differ by subtype and subgroup, but larger samples sizes are needed. Inflammation is known to play a role in ovarian cancer pathogenesis, and IUDs exert their physiologic effect through local inflammation. However, IUD-associated inflammation may be accompanied by an immune response that could lead to clearance of premalignant cells or local infection. An appreciation of how ovarian cancer risk varies by expression of immune markers within tumors may inform the biologic mechanisms possibly involved (e.g. CD3, CD8, CD4, CD69, FOXP3, CD163). Here we propose to evaluate the association between IUD use and ovarian cancer risk in 17 case control studies and 7 cohort studies with a total of more than 20,000 cases that collected, with varying degrees of detail, data on IUD type and timing of use (e.g. age at use and before/after first birth), as well as detailed histologic data critical for distinguishing ovarian cancer subtypes. Importantly, our proposed research includes a case-control study of African American women enrolled between 2010-2015, reflecting recent contraceptive trends and increasing diversity. The inclusion of cohort studies with updated contraceptive use data and a case-control study which recently completed enrollment will provide information on the contemporary contraceptive use. Furthermore, we will utilize unique resources and innovative platforms to examine the potential mechanisms through which contraceptive choice influences ovarian cancer risk. In more than 3,000 cases with detailed contraceptive data, we will simultaneously measure a panel of immune markers by multiplex immunofluorescence which shows co-localization of marker expression. Both the high prevalence and modifiable nature of contraceptive use make this an important public health question with a significant impact on the population burden of ovarian cancer.

NIH Research Projects · FY 2025 · 2021-04

Immunity to viral infections of the lung require cooperation between humoral and cellular arms of the immune system. A newly defined subset of CD8 T cell, called CXCR5+ CD8 T cells, have been identified. CXCR5+ CD8 T cells can gain access to the B cell follicle of lymph nodes and interact with B cells. In settings of chronic infection these CXCR5+ CD8 T cells may have stem-like potential and can differentiate further into effector CD8 T cells. Therefore, CXCR5+ CD8 T cells may have unique polyfunctionality to control both humoral and cellular arms of the immune system. However, a fundamental understanding of the functions of CXCR5+ CD8 T cells is lacking due to a paucity of tools to study these cells in vivo. The object of the proposed studies is to elucidate the precise roles of CXCR5+ CD8 T cells in regulating cellular and antibody mediated anti-viral immunity during acute respiratory infections, and to assess intrinsic and extrinsic mechanisms controlling these functions. We hypothesize that polyfunctional CXCR5+ CD8 T cells restrain humoral immunity yet are essential for cellular immunity and T cell memory. We also hypothesize that the immune system can fine-tune immunity by altering CXCR5+ CD8 T cell fates through extrinsic signals from effector Tfh cells, and intrinsic factors such as the expression of the transcription factor Tbet. To test these hypotheses, we will: 1) assess the roles of CXCR5+ CD8 T cells to control humoral and cellular immunity at distinct times in vivo during viral infection and vaccination with Influenza and SARS-CoV-2, and 2) assess the roles of extrinsic signals from Tfh cells and intrinsic signals from Tbet to control CXCR5+ CD8 T cell memory and polyfunctionality. We will pursue these aims using innovative strategies to identify and perturb CXCR5+ CD8 T cell subsets in vivo during viral infection and vaccination in intact, polyclonal, mice. These strategies include newly developed CXCR5+ CD8 T cell deleter and Tfh cell deleter models which facilitate the assessment of functionality during the distinct stages of viral infection and vaccination. The expected outcome of these studies is an elucidation of the precise functions of, and mechanisms controlling, CXCR5+ CD8 T cell regulation of humoral and cellular immunity. These studies are significant because they will lead to a deeper understanding of how the immune system mediates anti-viral immunity and will provide framework for the development of new therapeutics to enhance anti-viral immunity and promote vaccine efficacy to influenza and SARS-CoV-2.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY More than 40% of children under 5 years in low- and middle-income countries (LMICs) are at risk of not reaching their developmental potential. One of the most effective and proven strategies for supporting early child development in these settings is to empower caregivers and communities to support their children’s development. However, evidence on the effectiveness and the barriers and facilitators to implementation of interventions to promote this nurturing care are need. In this study, we will use an implementation science framework to evaluate an individualize intervention to promote nurturing care—the international Guide for Monitoring Child Development (GMCD), in rural India and Guatemala. We will conduct a hybrid effectiveness/implementation cluster randomized trial, where children aged 0-2 years will receive individualized visits from frontline health workers administering the GMCD. The study will have three parts: In Part 1, we will randomize clusters to receive either the GMCD or usual care for 12 months. After 12 months, control clusters will cross-in and all children will receive an additional 12 months of intervention. We will assess the impact of the intervention at 12 and 24 months on developmental outcomes and on the home care environment. In Part 2, we will use the RE-AIM implementation science framework to assess the Reach, Effectiveness, Adoption, Implementation, Maintenance of the intervention. We will also use the CFIR (Consolidated Framework for Implementation Research) framework to conduct in-depth complementary qualitative evaluation of implementation barriers and facilitators in high- and low- performing clusters from Part 1. In Part 3, we will assess cost effectiveness of the intervention. In conclusion, the study will answer three important questions: (1) Is the GMCD effective at improving developmental outcomes and the home care environment for children at risk in rural India and Guatemala? (2) What real-world institutional and contextual factors influence the impact of the intervention and might affect its potential ability to sustainably reach children and families? (3) Is the intervention cost-effective? The project will generate globally relevant evidence on community-based early child development interventions in LMICs.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Aabida Saferali, PhD, is an academic researcher with a focus on the genetics of respiratory diseases, whose overarching career goal is to become an independent investigator with the skill set to utilize integrative ‘omic techniques for the improved biological understanding and clinical management of chronic obstructive pulmonary disease (COPD), one of the leading causes of death worldwide. The proposed research combines her prior experience in genetics and transcriptomics with training in long-read sequencing, advanced integrative ‘omics, and biostatistics, in order to address the fundamental issue of identifying disease causing genes in GWAS loci. The hypothesis of this proposal is that transcriptional regulation through RNA splicing plays an important role in COPD development and pathogenesis. This will be explored by leveraging existing data from two well-phenotyped studies of lung disease: COPDGene and the Lung Tissue Research Consortium. Specific Aims: (1) characterizing gene expression and splicing in smokers and testing for associations with COPD case-control status and lung function; (2) discovering expression quantitative trait loci (QTLs), splice QTLs and transcript ratio QTLs and using these data to explain GWAS findings; and (3) using long-read RNA sequencing to characterize full-length isoforms associated with disease and definitively identifying splicing patterns. The innovative research plan, which utilizes novel methodologies to characterize splicing, will identify COPD GWAS loci involved in alternative splicing. It is accompanied by a training plan, which will provide Dr. Saferali with the skills to complete the research aims, as well as the experience to transition to independence and submit an R01 expanding upon the characterization of isoform diversity in COPD using long-read sequencing. In particular Dr. Saferali has four training goals which build upon her existing background in respiratory genetics. (1) To strengthen her knowledge of biostatistics and statistical genomics; (2) to expand her integrative ‘omics skill set; (3) to gain experience and expertise in the generation, analysis and interpretation of long-read sequencing data; and (4) to deepen her understanding of study design and mentoring. She is supported by a mentoring team with complementary skillsets and successful mentoring careers, which, together with her experience and training, will guarantee the success of this proposal. The findings may pave the way for the development of targeted therapeutics and personalized approaches, while exploring a novel methodology to address one of the most important analytic challenges in the field today: characterization of isoform diversity using RNA sequencing.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract This K23 award will establish Ishani Ganguli MD, MPH as a clinician-investigator focused on how care delivery and payment innovation can advance high-value outpatient care and better outcomes for older adults. Dr. Ganguli is an Assistant Professor of Medicine and primary care physician at Harvard Medical School and Brigham and Women’s Hospital. Building on her clinical training, methods coursework, and experiences studying care innovation and leading it at an Accountable Care Organization (ACO), this award will allow her to develop expertise in (1) how aging needs, especially for those with multimorbidity and Alzheimer’s Disease (AD) and AD-related dementias (AD/ADRD), can be met through outpatient care innovation, (2) advanced quantitative methods, and (3) claims-linked electronic health record (EHR) data analysis. To achieve these goals, Dr. Ganguli has assembled a team with expertise in health services research on care innovation (Drs. Ateev Mehrotra, Thomas Sequist), geriatrics (Dr. Christine Ritchie), primary care (Drs. Meredith Rosenthal, Joshua Metlay), quantitative methods (Drs. E. John Orav, J. Michael McWilliams, Craig Pollack), and EHR data analysis (Dr. Christine Vogeli). Primary care access and continuity of care improve outcomes for multimorbid older adults, so a recent decline in primary care office visit rates – likely due in part to barriers attending these visits – raises concerns. Virtual care may enable access and continuity. But we do not know which groups faced office visit barriers pre-dating the Covid-19 pandemic nor how post- pandemic expansion of virtual care will affect primary care access or continuity for these groups. To address these critical questions, Dr. Ganguli will first use the claims-linked Medicare Current Beneficiary Survey to describe visit use and visit continuity of care (COC) associated with reported trouble getting to the doctor’s office and whether adults with AD/ADRD or social risk factors were at greater risk of this barrier (Aim 1). In Aim 2, using difference-in-differences analysis of claims-linked EHR data in a large Medicare ACO, she will assess how post- pandemic expansion of virtual care affects primary care use in these populations. In Aim 3, she will use these claims-linked EHR data to develop a novel application of the COC measure (“touch continuity,” which includes virtual care) and determine if it is associated with risk of preventable hospitalizations. This work and training will provide the basis for a future R01 proposal of a quasi-experimental, multi-health system study of virtual primary care for multimorbid older adults. This K23 award and future R01 will inform how to pay for and deliver virtual care to promote high-value outpatient care and better outcomes for older adults.

NIH Research Projects · FY 2026 · 2021-03

Allergen-specific antibodies promote and exacerbate allergic inflammation. Antibody responses are controlled by T follicular helper (Tfh) cells that promote B cells to produce pathogenic antibodies both in lymphoid organs and in inflamed tissues. Alterations in Tfh cells can lead to pathogenic antibody responses and disease. Tfh differentiation is a multi-step process that not only produces generic effector cells but also disease-specific effector cells such as Tfh13 cells. This process gives the immune system multiple opportunities to regulate disease through controlling Tfh fate decisions. However, how this occurs in tissues is poorly understood We hypothesize that stepwise Tfh differentiation occurs in lungs during allergic airway inflammation. Moreover, we hypothesize that the inflamed lung reprograms Tfh cells to unique transcriptional and functional states. By elucidating mechanisms controlling Tfh fate decisions in inflamed tissues, new strategies can be developed that specifically target pathogenic responses during allergies without affecting broad immune responses. To test these hypotheses, we have developed a number of novel mouse models for the in-depth study of the roles of specialized subsets of Tfh cells in allergies. Our aims are to 1) identify the Tfh subsets required for in situ pathogenic B cell responses in allergic lungs; 2) Determine the key transcriptional programs enabling Tfh13 differentiation in allergic lungs; and 3) Assess essential pathways inducing pathogenic IgE B cells during allergic lungs. Our goals are to elucidate mechanisms by which Tfh promote allergies in tissues, and to identify targeted strategies to limit allergic inflammation.

NIH Research Projects · FY 2025 · 2021-03

Project Summary/Abstract Organ transplantation is a critical therapy for patients with irreversible organ damage. Short-term outcomes are excellent, but most patients lose their organs eventually due to chronic immune-mediated injury over time. Ischemia reperfusion injury (IRI) is unavoidable in transplantation and the primary activator of the innate immune response in the early post-transplant period, which enhances the rates of acute and chronic allograft rejection subsequently. Furthermore, a critical worldwide shortage of organs available for transplant exists, which has prompted clinicians to use organs from donors who are older or have greater comorbidity. These organs have much greater susceptibility to ischemic injuries. Therefore, the association of IRI with increased allograft immunogenicity has very broad clinical implications. Costimulatory blockade (CB) has emerged recently as a highly promising therapeutic approach in transplantation with far superior microvascular and metabolic safety profile than calcineurin inhibitors. However, CB is associated with increased rates of acute allograft rejection during the early post-transplantation phase. Our data indicate that IRI abrogates the tolerogenic effect of CB. Therefore, a key unmet need in transplantation is to understand better the mechanisms by which IRI and its activation of the innate immune response potentiates transplant rejection, as novel therapeutic regimens to prevent or ameliorate IRI-induced alloimmunity could assist in reducing chronic rejection. Our main goal is to reveal the underlying mechanisms of augmentation of transplant rejection by IRI. Pursuant to our data, our main hypothesis is that IRI activates alloimmunity by A) increasing the early intra- graft inflammatory response and B) priming the draining lymph node (DLN) of the graft recipient through distinctive microanatomical changes. We have shown that early intra-graft inflammatory responses play a critical role in augmenting alloimmunity. We also propose here for the first-time the use of nanoparticles for targeted delivery of immune therapeutics to the DLN for the reduction of IRI-induced alloimmunity. In AIM 1, we will examine how induction of autophagy in donor dendritic cells by IRI creates a pro-inflammatory milieu within the organs that augments alloimmunity. In AIM 2, we will examine the mechanism by which IRI of the grafts primes the DLN microanatomically to amplify the alloimmune response. In AIM 3, we will develop nanoparticles for the targeted delivery of CB to the DLN for augmentation of their tolerogenic effects in reducing the deleterious effects of IRI.

NIH Research Projects · FY 2025 · 2021-03

Project Summary Volumetric muscle loss (VML) usually occurs following traumatic injury and results in a composite loss of muscle mass. These injuries manifest in decreased strength and functional impairments. Clinically, these injuries often heal with fibrosis, as opposed to skeletal muscle regeneration. Current existing therapeutic options are also insufficient for VML treatment, and complications are often associated with surgical repair including nerve injury, excessive immune response, infection, scarring, and limitations of tissue graft supply. Indeed, natural healing and surgical procedures are inefficient in restoring the functionality of injured muscles, resulting in a poor quality of life. Therefore, developing clinically relevant three-dimensional (3D) tissue using patient-specific genetically identical cells has emerged as a potential solution to address the above issues. To achieve this aim, there are two existing main challenges. The first challenge is obtaining large amounts of patient-specific genetically identical cells. The use of human pluripotent stem cells (hiPSCs) differentiated to the muscle lineage represents a promising candidate to build upon personalized therapy. However, directing the differentiation of hiPSCs to the muscle fate along with reproducible differentiation schemes has proven to be challenging. The second challenge is developing a highly organized and vascularized 3D skeletal muscle tissue to maintain the viability of cells inside thick tissue constructs via engineered vessel networks. Furthermore, the fabricated tissues have to strongly integrate into injured site via surgical methods. To address these challenges, we plan to develop a suturable 3D vascularized muscle tissue from hiPSC-derived myogenic precursor cells (hiPSC-MPCs) embedded in biomaterials using bioprinting techniques. We will optimize the recently developed protocols allowing efficient production of functional myofibers from hiPSCs in hydrogels with tunable mechanical properties and degradable profiles, which mimic the extracellular matrix (ECM) of native skeletal muscle tissue. To create biomimetic vascularized muscle constructs, a multi-material embedded bioprinting technique will be used to precisely control the positions of the vascular network and aligned muscle fibers with biologically relevant architectures. With the conventional bioprinting system, it is difficult to precisely control the materials’ position in Z directions to create freestanding hydrogel architectures. Also, to achieve prolonged retention of implants into the injured site and to improve muscle regeneration, a muscle growth factor (IGF-1) laden suturable graft will be developed. hiPSC-MPCs-laden constructs will be printed on the suturable graft consisting of IGF-1-laden PGS/GelMA substrates using electrospinning.

NIH Research Projects · FY 2025 · 2021-03

ABSTRACT The tumor ecosystem plays a critical role in tumor development, progression and therapeutic response. Previous studies have utilized dissociative and single-cell omics technologies to profile the tumor ecosystem, specifically to understand therapeutic resistance and identify predictive biomarkers for precision cancer medicine. Yet, very few of these biomarkers have adequate performance characteristics for adoption in clinical practice. We hypothesize that a fundamental facet of the tumor ecosystem, i.e., the spatial organization of cells, which encodes key information involving paracrine and juxtracrine interactions that drive “neighborhood- level” biology, can further inform predictive models. Recent technological breakthroughs in highly multiplexed imaging and spatial transcriptomics offer an unprecedented opportunity to delineate the therapeutic consequences of spatial relationships within clinical tumor samples. Quantitative spatial features can provide independent valuable information, which is unlikely to be captured by clinical, genetic and bulk-transcriptional predictors. Hence, we propose to integrate highly multiplexed imaging data with omic approaches to delineate mechanisms of resistance and build predictive models of response for patients with T-cell lymphoma, who have a desperate unmet clinical need. In Aim 1 (K99 phase), I will build automated computational tools to robustly quantify spatial features from highly multiplexed imaging data and integrate it with exome and RNA- Seq. I will utilize >100 primary specimens collected pre-, on- and after-treatment with the PI3K-δγ inhibitor duvelisib to nominate mechanisms of de novo and acquired resistance. In Aim 2 (K99 phase), I will build an integrated machine-learning model to predict which patients are most likely to benefit from duvelisib and evaluate the impact of spatial features towards model performance. In Aim 3 (R00 phase), I will validate the model in an independent cohort and extend to samples from patients treated with additional agents, to identify consistent and parsimonious signatures of spatial features that could be developed for broader use. My extensive background in computational biology and experimental biology puts me in a unique position to accomplish this proposal. During the K99 phase, I will be supported by an outstanding and interdisciplinary team of advisors and collaborators (Drs. David Weinstock, Peter Sorger, Jon Aster, Allon Klein, Peter Park, and Steven Horwitz) with expertise in all aspects of the proposed research. I will acquire new skills in (1) computational analysis of highly multiplexed imaging to model molecular and spatial information, (2) data integration methods to delineate regulatory programs for designing effective drug combinations and (3) analysis of predictive biomarkers in clinical trial samples from clinical trials. Together with institutional support from Dana Farber Cancer Center and formal coursework and training, I will bridge my knowledge gap in cancer biology and gain the communication and leadership skills vital to transition into an independent position and establish an independent, data science-driven, translational research program.

NIH Research Projects · FY 2026 · 2021-02

Endometrial cancer (EC) is the most common gynecologic cancer in the US. Incidence is increasing, especially for aggressive, understudied tumors that confer poor prognosis and are more often seen in African Americans (AAs). EC has one of the largest survival differences of all cancers: AAs have >2-fold higher mortality vs. other populations. This difference remains after accounting for stage, histology, comorbidities, and treatment. The etiology of aggressive tumors and 2-fold higher mortality are large knowledge gaps in EC. The Cancer Genome Atlas (TCGA) achieved milestones in clarifying endometrial tumor biology. Using exome sequence data, TCGA defined 4 new tumor subtypes with prognostic significance and showed these data can refine subtype classification beyond classic histology. But TCGA used mostly good prognosis endometrioid tumors (>90%) and tumors in white women—with only 46 AAs—to define these subtypes. Our pilot analysis of AA vs. non-AA tumors in these sparse data suggested AAs more often had mutational features suggestive of poor outcomes. We hypothesize somatic differences in AA vs. non-AA tumors may help explain the large survival differences. Here, we will use the largest population to date—including 1,011 AA and 2,043 non-AA cases in the Epidemiology of Endometrial Cancer Consortium (E2C2)—to study genomic variation across the full spectrum of endometrial tumors, distinct risk factor profiles across tumor types, and the role of underlying tumor biology in driving the 2-fold higher mortality. We will: define the mutational landscape and novel tumor subtypes using whole-exome sequence data in 3,054 endometrial tumors and compare these in AA vs. non-AA cases. This will use exhaustive genomic profiling of point mutations, indels, and copy number alterations. Next, we will identify differences in risk factor associations by tumor molecular subtypes in 3,054 cases and 3,054 matched controls. Despite many known EC risk factors, TCGA was not designed to study these in concert with somatic changes. We will combine tumor profiling data in cases with information on known germline genetic and epidemiologic risk factors in cases and controls to study distinct risk factor profiles by tumor subtypes. Finally, we will 3) determine the extent to which tumor molecular subtypes explain the 2-fold survival difference in AA and non-AA cases: Having characterized tumor genomes, we will use mediation analysis to determine the extent to which tumor molecular profiles in AAs and non-AAs explain the difference in outcomes. Leveraging E2C2 resources and collaborations, we will characterize the biology and risk profiles of the component subtypes of EC, including aggressive tumors, and somatic differences that drive outcomes across populations. Long-term this can lead to refined risk prediction tools, improved targeting of disease prevention and treatment, and strategies to reduce EC burden and mortality. Our study will also build a unique platform on which to perform future population-based -omics studies of EC

NIH Research Projects · FY 2025 · 2021-02

Abstract: Clostridioides difficile, the etiology of pseudomembranous colitis, causes substantive morbidity, mortality and close to $5 billion/year in US healthcare costs. Commensals provide primary protection against C. difficile infections though the underlying mechanisms of action remain ill-defined. We have identified individual bacterial species that provide long-term survival against virulent C. difficile strains, and other species that can make the infection worse. Our proposed aims will define specific commensal activities and commensal genes mediating these effects on the pathogen, and test their functions in vivo, in mice carrying mouse vs human complex microbiota, for the purposes of developing defined bacteriotherapeutics and biomarkers to predict successful therapy.

NIH Research Projects · FY 2025 · 2021-02

X-linked hypophosphatemia (XLH) is the most common form of inheritable rickets characterized by mutations in PHEX. These mutations result in elevated serum levels of FGF23, which leads to hypophosphatemia, rickets, and osteomalacia. FGF23 also inhibits vitamin D 1-a-hydroxylase (Cyp27b1), thus blocking 1,25 dihydroxyvitamin D (1,25D) production. Pathologic mineralization of the enthesis (tendon-bone attachment site), referred to as enthesopathy, is a debilitating complication of XLH that causes significant pain and impaired mobility in affected individuals. Common sites affected include the patellar and Achilles entheses. The pathogenesis of XLH enthesopathy is poorly understood. We previously demonstrated that Achilles entheses from mice with XLH (Hyp) have an expansion of hypertrophic appearing cells (HECs) that exhibit an aberrant chondrogenic phenotype with enhanced BMP/IHH signaling by P14. Treatment of Hyp mice with 1,25D or a FGF23 blocking antibody (FGF23Ab) early in development (P2) similarly prevented enthesopathy despite the dramatic increase in FGF23 expression in bone, suggesting impaired 1,25D action underlies the enhanced BMP/IHH signaling observed in Hyp enthesopathy. In both mice and humans with XLH, 1,25D therapy cannot reverse enthesopathy, supporting the hypothesis that early restoration of 1,25D is needed to prevent enthesopathy. The increase in serum 1,25D levels wane post-initiation of FGF23Ab in both mice and humans with XLH, suggesting FGF23Ab may not be effective in preventing enthesopathy in XLH patients. There is no data on the effects of optimized 1,25D monotherapy or FGF23Ab on enthesopathy in XLH patients. Therefore, given the similar responses of mice and humans to 1,25D and FGF23Ab, studies on the hormonal regulation of XLH enthesopathy are essential to guiding future clinical studies on enthesopathy prevention. Preliminary data show that XLH enthesopathy results from impaired 1,25D action, not actions specific to FGF23 or consequences of the Hyp mutation. Studies in Aim I will examine mice with global deletions of Cyp27b1, FGF23, or both with or without the Hyp mutation to address the hypothesis that impaired 1,25D action leads to enhanced BMP/IHH signaling and enthesopathy. Studies will also elucidate if decreased local 1,25D action leads to enthesopathy. Since our data demonstrates increased BMP signaling is accompanied by enhanced GDF5 expression in Hyp entheses, studies in Aim II will identify a pathogenic role for GDF5/BMP signaling in Hyp enthesopathy development. Studies will determine the time course of GDF5 expression in WT and Hyp entheses. Ablation of GDF5 in Hyp entheses will define the role of GDF5 in the activation of BMP/IHH signaling in XLH enthesopathy. Inhibition of BMP signaling in Hyp mice will show that IHH signaling is activated by BMP signaling in entheses and enhanced BMP/IHH signaling directly leads to enthesopathy. Taken together, these studies will identify novel hormonal and molecular regulators of XLH enthesopathy and normal enthesis development. They will also identify targets for the design of new therapies to prevent enthesopathy.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY ALS is a neurodegenerative disease affecting motor neurons with limited treatment options and a median survival of 3-5 years. We submit a significantly revised proposal that addresses the comments of the reviewers and provides new preliminary data. Our hypothesis is that the intestinal microbiome and its metabolites play an important role in in ALS by modulating peripheral and CNS immunity and by affecting ALS disease pathways. Our hypothesis is strengthened by a recent paper in Nature by Blacher showing an important role of the gut microbiome and metabolites in ALS. In new preliminary data we show: 1) Antibiotics that worsen survival also downregulate microglia homeostatic genes while upregulating inflammatory genes. 2) Changes in the microbiome in 68 ALS patients vs. 61 healthy controls (largest microbiome study to date), including a decrease in butyrate producing bacteria E. rectale and R. intestinalis, are robust when controlled for ALS clinical confounders. 3) Administering these bacteria or Akkermansia reverses SOD1-disease associated transcriptional changes in the spinal cord; including Fus, Oxr1, and Smn1, and protein degradation (Ubqln1). 4) Transferring SOD1 microbiota to WT mice modulates microglia pathways involved in ALS related to RNA processing (Fus), protein degradation (HSPa1b and USP2) and lysosomal transport (CD68 and Lyz2). We believe there is compelling evidence to support the investigation of the microbiome in ALS. We will address these aims: AIM 1. Which microbial components are associated with protection in SOD1 and TDP-43 models? We will deplete the microbiota with specific low-dose antibiotics, orally administer Akkermansia components and a unique micro-RNA and identify brain, serum, and stool metabolites associated with disease protection. AIM 2. Which human microbiota components contribute to disease pathogenesis? It is unknown whether the ALS microbiota can also drive disease pathogenesis. We will transfer microbiota from patients with ALS to the SOD1 and TDP-43 models and measure motor function and survival time. We will identify microbial populations, functions, and metabolites that are associated with protection or worsening of disease. We will also confirm and expand our findings in the ALS microbiome in a newly recruited cohort. AIM 3. Investigate the immune mechanisms by which the gut microbiota modulates disease progression in ALS animal models. We will sort microglia, monocytes, and T cells from mice treated with individual antibiotics, colonized with ALS microbiota, or specific bacterial strains and characterize transcriptional signatures by RNAseq. Utilizing WT mice to investigate how the microbiota affect microglia, we will transfer specific microbes identified associated with ALS and screen for changes in genes involved in ALS pathogenesis.

NIH Research Projects · FY 2025 · 2021-01

Project Summary/Abstract The molecular mechanisms that explain the potent anti-diabetic effects of bariatric surgery remain elusive. The rapid nature of type 2 diabetes mellitus (T2D) remission after surgery have led to the suggestion that unidentified small molecules are responsible. For sleeve gastrectomy (SG), the most common bariatric operation performed today, knockout mouse studies have shown that bile acid receptors are critical for surgery’s metabolic benefits. The key ligand(s) that are changed post-SG to engage these bile acid receptors is unknown. Work from our laboratory has identified a bile acid metabolite, cholic acid 7-sulfate (CA7S), that is induced in the intestine by SG. We have found that CA7S is a potent TGR5 agonist that improves glucose handling in diabetic mice, and the production of CA7S occurs in the liver by sulfation of cholic acid in response to the gut microbial product, lithocholic acid (LCA), that signals via the hepatic vitamin D receptor (VDR). Our long-term goal is to understand and replicate less invasively the anti-diabetic mechanisms of bariatric surgery. The overall objective of this application is to define the anti-diabetic properties of CA7S, the microbiome-dependent mechanisms of CA7S production, and CA7S contribution to T2D remission following SG. Our central hypothesis is that CA7S is produced in response to gut microbial metabolites and improves T2D following SG via intestinal TGR5 activation. We will test this hypothesis in the following specific aims. In Aim 1, we will determine the effects of long-term CA7S administration on insulin sensitivity, glucose tolerance, and weight in diet induced obese (DIO) mice and TGR5 deficient mice to understand the global metabolic effects of CA7S and sustained intestinal TGR5 activation. In Aim 2, we will determine how the microbiome induces CA7S production by (1) quantifying LCA- producing Clostridia bacterial species and expression of LCA-producing enzymes post-SG in mice and humans, and (2) generating DIO mice with and without intestinal LCA and assessing their metabolic phenotype and response to SG. In Aim 3, we will determine the role of CA7S in T2D improvement post-SG. We will perform SG in VDR deficient mice, which lack endogenous CA7S, or in mice with knockdown of SULT2A1, the key enzyme responsible for CA7S production. We will reconstitute CA7S by exogenous replacement in CA7S deficient animals to determine the contribution of CA7S to surgical improvements in glucose metabolism. This work will characterize the effects of a natural, gut-restricted TGR5 agonist, CA7S, on T2D and lay the foundation for its translation as a therapeutic. By characterizing specific metabolite-receptor interactions within the intestine, portal vein, and liver, we will define a novel, microbiome-dependent, gut-liver signaling pathway that explains improvement in glucose metabolism after SG. This work will significantly advance our molecular understanding of the causal mechanisms of bariatric surgery and identify multiple novel targets for the treatment of T2D.