Brigham And Women'S Hospital

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY The overall goal of this NCI K08 career development proposal is to facilitate Dr. Ko Un Park’s development into an independent investigator in cancer health services research focusing on implementation strategies that promote evidence-based care in surgical oncology and improve outcomes for cancer patients. Specifically, this proposal will apply insights from implementation science (IS) to the application of cancer surgery standards as set forth by the Commission on Cancer (CoC). The CoC is an accreditation organization that seeks to improve outcomes for individual patients with cancer through standardization of care delivery across 1500 hospitals providing care to some 70% of Americans with cancer. To date, the CoC has not paired its standards with implementation strategies; consequently, the standards’ implementation is often poor. The overarching goal of this project is to create stakeholder-driven strategies for adopting the use of the CoC’s new breast synoptic operative report (SOR) standard, by using tools from IS to identify and address barriers and facilitators to SOR implementation in diverse healthcare systems. To achieve this, in Aim 1 we will conduct key stakeholder interviews to identify barriers and facilitators to implementing breast SOR. In Aim 2, we will use evidence of implementation determinants to design strategies that facilitate breast SOR implementation. We will test the influence of the strategies on key implementation outcomes (e.g., feasibility, acceptability) in Aim 3. We hypothesize that we will identify both surgeon- and organization-level drivers of SOR implementation that can be leveraged to establish multi-level implementation strategies that will improve breast cancer outcomes. The planned training, proposed research project, multidisciplinary mentorship team, and exceptional research environment at The Ohio State University Comprehensive Cancer Center are ideally suited to address Dr. Park’s career goals. Her career development plan includes formal coursework in IS as well as directed readings and conferences. This education and mentored effort to execute the research proposal aid Dr. Park’s long-term career goal to become an independently funded implementation scientist-clinician to improve cancer patient outcomes by facilitating the implementation of professional self-regulation polices (i.e., CoC standards) in diverse health systems and cancer centers, and among physicians. With this training, Dr. Park will be able to successfully obtain funding for a subsequent prospective, multicenter trial testing the effectiveness of the strategies developed in the proposed project.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract: Humans emit an array of volatile organic compounds (VOCs) as part of normal metabolism. There are metabolic shifts in many disease states, and animals with highly sensitive olfactory systems can be trained to identify patients with certain diseases based on their characteristic scent. We seek to translate detection of these unique VOCs emanating through the skin to a more robust, standardized, and mechanized platform, readily adaptable for the screening, diagnosis, and monitoring of a variety of human disease states with distinct pathophysiologies. We hypothesize that systemic metabolic derangements in many high burden infectious, inflammatory, metabolic, malignant, psychiatric, and neurologic diseases can be identified via skin emissions. We will adapt a novel, portable gas chromatography- differential mobility spectrometry (GC-DMS) device that rapidly examines volatile samples directly at the point of care for the assessment of these skin volatile metabolite signatures. This device is highly sensitive, allowing a comprehensive and biologically representative assessment of the landscape of human volatile emissions, is more robust to confounding and environmental factors than many other gas sensing devices, has a long track record of successful use in various real-world sensing applications, and is already undergoing commercial development, which will greatly facilitate its rapid development for the skin VOC-based diagnosis of these 20 disease states. We propose further development and rigorous evaluation of this scent-based diagnostic approach to these disease states, (1) integrating additional process analytical technologies (PATs) to ensure instrumental accuracy and precision between samples and devices through repeated, high-volume patient testing over time and modifying the device inlet for skin volatile analysis, (2) identifying and validating GC- DMS signatures and the corresponding set of skin volatile metabolites that distinguish individuals with and without each of these 20 disease states using machine learning methods, integrating automated detection of these signatures on the GC-DMS device, and (3) developing a wearable sensor for at least one disease with a simple volatile signature, determining the test characteristics of this wearable diagnostic device. A lack of reproducibility is a major issue plaguing the field of volatile metabolite analysis, often due to confounding and study design flaws. We will make every effort to minimize and eliminate any sources of confounding, bias, and extraneous variability as we develop and evaluate this scent-based diagnostic approach, to yield generalizable signatures for each disease state. Ultimately, successful completion of these aims will yield rapid, noninvasive skin volatile metabolite assays for the screening, diagnosis, and monitoring of each of these diseases, with a clear, binary (yes/no) assessment of whether the individual has evidence of one or more of these diseases, facilitating early administration of appropriate therapeutics in these patients and mitigating the clinical consequences of each of these diseases.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Neurofibromatosis type 1 (NF1) is a prevalent familial cancer syndrome affecting 1 in 3500 individuals worldwide. The most commonly lethal feature associated with NF1 is malignant peripheral nerve sheath tumors (MPNST). These soft tissue sarcomas are highly aggressive and frequently metastasize. Despite radiation and chemotherapy, inoperable tumors rapidly progress and are universally lethal. As such, identifying effective treatments for MPNST is critical. The primary goal of this application is to establish a robust preclinical/clinical pipeline (bench-to-bedside and back) to rapidly develop and test new (combination) therapies for this deadly malignancy. This effort will harness the specialized expertise of clinical investigators at the NCI and Children’s National Medical Center, extramural experts in NF1 biology and therapeutic development, and will leverage the unique resources of the NIH Clinical Center. Specifically, new discoveries of mechanisms that drive NF1-related tumorigenesis together with recent insights into the immunoreactivity of MPNST will be used to develop rational combination therapies and will be tested in a robust preclinical MPNST mouse model (Karen Cichowski, BWH, extramural preclinical center). These insights will then be used to perform clinical trials in MPNST patients with an emphasis on evaluating more than one combination therapy within the same trial (Brigitte Widemann, NCI, Intramural NIH Clinical Center, AeRang Kim, Children’s National Medical Center). This will allow for more timely identification of active agents and will allow patients with this highly refractory disease to have more treatment options available to them. Furthermore, the preclinical to clinical translation will be complemented by comprehensive genomic and immunological analyses of tumor samples obtained prior to treatment and on treatment with novel agents in order to identify mechanisms of response and resistance and to identify additional potential targets for therapy (Jack Shern, NCI, Intramural NIH). As such, insight and samples from the clinic will serve as the foundation to develop new or improve existing therapies, thus highlighting the iterative and collaborative nature of this pipeline. Taken together, we have assembled a multi-disciplinary team of basic and clinical scientists from different fields to develop and translate promising therapies for individuals with MPNST. This effort includes experts in NF1 biology and therapeutic development, a diverse set of clinicians with expertise in MPNST and immunotherapy, and genomicists. Importantly, a subset of these investigators already have a track record of working together to develop new trials for MPNST patients. This grant will allow more effective and rapid translation of promising new therapies for MPNST and will expand the type of (combination) therapies that are developed, by bringing in additional expertise and leveraging the unique resources of the NIH Clinical Center. Ultimately, these studies have the potential to change the standard of care for the currently treatment refractory tumors associated with the common familial cancer syndrome NF1.

NIH Research Projects · FY 2026 · 2022-07

ABSTRACT Cardiovascular disease (CVD) is the leading cause of death among U.S. women, accounting for approximately 1 of every 3 female deaths. Cumulative evidence has identified pregnancy complications as well as fertility measures, as CVD risk factors. However, there is very limited knowledge on the impact of environmental exposures during pregnancy and both short and long-term maternal cardiovascular and metabolic health. Environmental chemicals with potential cardiometabolic impact include phthalates and organophosphate (OP) flame retardants, which widespread use leads to ubiquitous general population exposure. Experimental studies demonstrated that both phthalates and OP flame retardants bind to human peroxisome proliferator-activated receptors (PPARs), a master nuclear receptor that is involved in lipid metabolism regulation. Among subfertile women enrolled in the Environment and Reproductive Health (EARTH) Study, we observed worse pregnancy outcomes, including decreased live birth rates, increased pregnancy loss and elevated glucose levels during pregnancy, with increasing urinary concentrations of phthalate and/or OP flame retardant metabolites. However, it is unstudied whether phthalate and OP flame retardant exposure during pregnancy is associated with long-term (midlife) maternal cardiovascular and metabolic health. We propose to evaluate associations of preconception, pregnancy and midlife urinary phthalate and OP flame retardant metabolite concentrations (individually and as a mixture) with long-term cardiometabolic health (anthropometry, glucose and lipid metabolism, blood pressure and inflammatory biomarkers). We will also identify the most important window(s) of exposure associated with cardiometabolic health, evaluate the joint and interactive effects of urinary metabolite concentrations and modifiable lifestyle risk factors for CVD (BMI, diet, physical activity, smoking) with cardiometabolic health, and investigate trajectories of cardiovascular health outcomes from pregnancy to midlife. We embed our proposal within the EARTH Study (2004-2021), a cohort of subfertile couples attending a single fertility center to identify environmental predictors of reproductive health. Strengths of EARTH Study include collection of urine samples both before and during pregnancy and its comprehensive and rich database of covariates. We will contact and re-enroll former female EARTH participants who will provide additional urine samples comprising the midlife window of exposure for phthalate and OP flame retardant metabolites and we will assess their current health status. Women with impaired fertility are at higher risk of CVD, thus this proposal provides an exceptional opportunity to explore cardiovascular and metabolic health among women within the well-established EARTH Study, which so far, has focused on fertility, pregnancy and offspring outcomes. Results from the proposed series of investigations will inform clinical care for women from the reproductive years through midlife, CVD guidelines regarding screening and long-term follow-up, and future research priorities.

NIH Research Projects · FY 2025 · 2022-07

Summary: Prior alloimmunization places patients at risk for anamnestic alloantibody formation following alloantigen re-exposure that can result in accelerated RBC clearance and lead to a potentially fatal delayed hemolytic transfusion reaction (DHTR). The inability to prevent DHTRs largely stems from a fundamental lack of understanding regarding key immune pathways that govern anamnestic RBC alloantibody responses capable of driving DHTRs. Our long-term goal is to identify and then target critical pathways that regulate the development of anamnestic alloantibody formation. Our central hypothesis is that anamnestic RBC alloantibody formation occurs through a distinct toll-like receptor (TLR), bridging channel dendritic cell (DC) and follicular (FO) B cell- dependent pathway that fundamentally differs from primary RBC alloimmunization. Our hypothesis is formulated on the basis of our discovery that unlike initial RBC alloimmunization, which requires marginal zone (MZ) B cells, MZ B cells are not required for anamnestic alloantibody formation, but are required for early priming events that occur following initial RBC alloantigen exposure. In addition to differences in MZ B cell requirements, while initial alloantibody formation is CD4 T cell independent (TI), increased alloantibody levels observed following RBC re- exposure is entirely CD4 T cell dependent (TD). Furthermore, while T follicular helper cell (TFH) and follicular (FO) B cells are not required for initial alloantibody formation, initial RBC transfusion does increase TFH and FO B cell numbers. Re-transfusion also increases 33D1+ DC activation and chemokine receptor expression. These results suggest that initial RBC transfusion primes recipients by generating distinct alloantigen-specific TFH and FO B cell populations that can, in turn, be activated by 33D1+ DCs following RBC alloantigen re-exposure independent of MZ B cells. In addition, while type I interferons (IFNab) are required for primary alloantibody formation, TLR signaling is dispensable for initial alloantibody development, yet is required for anamnestic alloantibody formation. Given the ability of MZ B cells to directly activate CD4 T cells and traffic antigen to the B cell follicle, our data suggest that initial priming events require MZ B cell-mediated CD4 T cell and FO B cell development through an IFNab-dependent process. However, as anamnestic alloantibody formation occurs through a MZ B cell-independent pathway and DCs can also traffic antigen to B cells and directly activate CD4 T cells, DCs likely orchestrate anamnestic alloantibody formation through a TLR-dependent pathway. To test this hypothesis, we will pursue the following specific aims: Specific Aim 1: Define the role of MZ B cells and IFNab in the development of CD4 T cells and FO B cells required for a subsequent anamnestic alloantibody response. Specific Aim 2: Define the role of TLRs and DCs in the development of an anamnestic alloantibody response following RBC alloantigen re-exposure. Successful completion of these aims will define key factors that regulate anamnestic alloantibody responses and in so doing provide an important framework to prevent alloimmunization that leads to DHTRs.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract Research: While effective measurement of the patient’s perspective on their illness and its treatment provides complimentary information to objective disease severity, patient-reported outcomes capturing the lived experience of chronic inflammatory skin disease are rarely used in routine dermatology practice. Item response theory techniques can enable more efficient administration of patient-reported outcomes in the clinic, which can address barriers such as time to administer these measures. The purpose of this study is to 1. prioritize the outcome domains that are most meaningful to individuals with chronic inflammatory skin diseases such as acne, psoriasis, and atopic dermatitis; 2. evaluate the content validity of a novel Patient-Reported Outcomes Measurement Information System (PROMIS) profile measuring these domains; 3. field test and evaluate the validity of a novel PROMIS profile to capture the lived experience of chronic inflammatory skin disease. The overall objectives of this K23 award proposal are to improve the care of patients with chronic inflammatory skin diseases by developing a practical patient-reported outcome to measure the lived experience of these skin diseases, which will enable increased use of patient-reported outcomes in routine clinical care. Candidate: Dr. Barbieri’s long-term career goal is to become an independent, patient-oriented physician- scientist, with a focus on chronic inflammatory skin diseases, patient-reported outcomes, and health services research. His prior training has provided him with a foundational background in these areas. Environment: During the award period Dr. Barbieri will devote 80% of his time to clinical research and 20% to the clinical care of patients with chronic inflammatory skin disease such as acne, atopic dermatitis, and psoriasis. Dr. Margolis and Dr. Pusic, the candidate’s primary mentors, have directly supervised the training of numerous successful junior faculty members and Dr. Barbieri will benefit from his experience and enthusiasm for cultivating the careers of junior scientists. The Brigham and Women’s Hospital offers a rich training environment, including resources and expertise available in the Clinical Unit for Research Innovation and Trials (CUReIT) and Patient-Reported Outcomes, Value & Experience (PROVE) Center. Combined with the knowledge and expertise available in the Department of Dermatology and in Dr. Margolis’s and Dr. Pusic’s labs, these opportunities will provide an unparalleled opportunity to begin a career as a physician scientist. Career Development: With the support of his advisory committee, Dr. Barbieri’s training will focus on advanced psychometric techniques and qualitative research to develop a strong knowledge base with respect to patient-reported outcome development and validation. Completion of the proposed research and career development plan will give Dr. Barbieri the knowledge and skills to achieve scientific independence, transition to a tenure track faculty position, and build a strong foundation for a career in patient-oriented clinical research.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY: Using brain lesions and deep brain stimulation to identify an epilepsy circuit Focal epilepsy is common in patients with brain lesions such as stroke, trauma, and tumors. Why some patients with brain lesions develop epilepsy while others do not is unknown. Deep brain stimulation (DBS) offers new therapeutic promise for patients with focal epilepsy, but seizure freedom is rare. Why some patients improve after DBS while others do not is also unknown. Current dogma focuses on the lesion location or DBS site alone. We hypothesize that connectivity of lesion locations and DBS sites to remote nodes distant from the lesion or DBS sites itself can explain variance in lesional epilepsy and DBS response. New techniques developed by the Fox lab (PI) can identify remote nodes connected to lesions causing and DBS sites treating brain diseases. These techniques combine the location of the lesion or stimulation site with a normative atlas of human brain connectivity termed the connectome. As such, they do not require connectivity data from the patients themselves and can be applied to almost any clinical lesion or DBS dataset. This approach has successfully mapped circuits involved in parkinsonism, amnesia, depression, and over 20 other brain diseases. Moreover, identifying circuit nodes connected to both lesions causing and DBS sites treating the disease can identify new or improved therapeutic targets. Our preliminary data in ischemic stroke suggests there is a common brain circuit involved in epilepsy. Lesion locations that cause epilepsy (n = 76) are more connected to the cerebellum and basal ganglia (‘Ce-BG’) compared to control lesions (n = 625). Connectivity of anterior thalamic DBS sites to these same circuit nodes is correlated with seizure reduction after DBS (n=30). While epilepsy is often considered a cortical disease, these subcortical Ce-BG nodes have previously been implicated in the modulation of seizures and cortical excitability. Although promising, further work is needed to determine if these results generalize across lesion types (Aim 1, n = 2,700), prospectively predict epilepsy risk (Aim 2, n = 6,000), and correlate with DBS response (Aim 3, n = 198). For each aim, we will focus on our Ce-BG nodes as an a priori hypothesis, but also perform data-driven (unbiased whole-brain) analyses to minimize risk of false positives or negatives. We will perform all analyses using a functional connectome (primary outcome) and structural connectome. Completion of these aims will determine whether connectivity of lesion locations and DBS sites to a common set of subcortical nodes can explain variance in lesional epilepsy and DBS response. Identifying this circuit could identify patients at high risk for epilepsy, guide DBS programming, and serve as a target for future brain stimulation trials. Collectively, these results could facilitate a shift in focus from the seizure-onset zone itself, which differs in every patient, to a brain circuit that may represent a new therapeutic target for focal epilepsy.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Genetic differences in cholesterol metabolism are major contributors to the risk of coronary artery disease (CAD), which is the leading cause of death in the USA. Unraveling the genetics of cholesterol has continued to yield promising therapeutics for heart disease. Nonetheless, the genetics of cholesterol levels are far from completely understood-- there are dozens to hundreds of genomic regions whose variation meaningfully alters cholesterol levels in the population, yet we can only explain the genetic basis of a small fraction of these loci. We have established a powerful approach combining CRISPR screening, gene network analysis, and human biobank coding variant burden analysis to dissect the genetics of cholesterol uptake and efflux. Through this pipeline, we have identified dozens of new genes that contribute to LDL cholesterol (LDL-C) uptake in cellular models and for which coding variants alter serum LDL-C levels in the population. In this proposal, we will refine and extend this pipeline to develop a coherent understanding of the variants, genes, and pathways underlying cholesterol uptake and efflux. In Aim 1, we pioneer a novel pipeline combining CRISPR screening, gene network analysis, and human biobank burden analysis to characterize ~500 genes we have found to alter cellular LDL-C uptake. We will develop a sensitive approach to extract the effects of rare coding variants on serum LDL-C levels using large exome sequencing biobank cohorts. We will group LDL-C uptake-altering genes into pathways through a combination of CRISPR screening and gene network analysis. We will perform mechanistic follow-up of novel candidate LDL- C-altering pathways in cellular and mouse in vivo models. In Aim 2, we will pioneer a new, more sensitive approach to CRISPR base editing screens to identify GWAS-associated variants that alter LDL-C uptake in liver cells. We will then use a suite of computational and experimental tools we have developed dissect the cis- regulatory mechanisms by which these variants act and connect them to trans-regulatory inputs controlling them. We expect to connect transcriptional drivers of hepatocyte LDL-C uptake with their cis-regulatory GWAS- associated variant targets and downstream LDL-C uptake-altering genes, shedding light on how human genetic variation influences serum LDL-C level. In Aim 3, we will use the pipeline of CRISPR-Cas9 screening and human biobank analysis to identify genes and pathways associated with monocyte/macrophage reverse cholesterol transport, a process thought to be important in CAD risk but which is incompletely understood at the genetic level. We will dissect disease-relevant genetic mechanisms involved in reverse cholesterol transport, helping to define the role of macrophage efflux in CAD risk. In sum, through high-throughput CRISPR screening followed by mechanistic follow-up, we will provide the most extensive experimental and computational analyses to date of the non-coding loci, genes, and pathways that underlie variation in human cellular cholesterol uptake and efflux.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY This proposal presents a five-year research and career development plan for Andrew Stern, MD, PhD, in the molecular basis of Alzheimer disease (AD), the leading cause of age-related cognitive failure worldwide. Dr. Stern is an Instructor in cognitive and behavioral neurology at Brigham and Women’s Hospital (BWH). The “amyloid cascade hypothesis” is the most studied theory of AD pathogenesis, positing that abnormalities in the metabolism of the Aβ peptide lead to the two defining pathologic lesions of AD (amyloid plaques of aggregated Aβ and neurofibrillary tangles of hyperphosphorylated tau), followed by loss of neurons. There are two central problems with this hypothesis: some patients have abundant amyloid plaques but little neuronal and cognitive loss; and clinical trials of antibodies that removed amyloid plaques have largely failed to slow decline. This proposal addresses these problems by beginning two answer two questions: 1) What biochemical and structural characteristics of the most toxic form of Aβ, soluble oligomers (oAβ), confer its toxicity? Novel monoclonal antibodies will be used to test if calcium-associated and low molecular weight oAβ subsets are correlated with tau abnormalities and dementia. CryoEM will allow structural characterization of immunoaffinity purified calcium-associated oAβ. 2) Do genetic differences cause some patients’ neurons to be more susceptible to toxic oAβ, and is oAβ from patients with high tau burden and dementia more toxic than oAβ from patients without? Induced pluripotent stem cell-derived neurons (iNs) and post mortem brain tissue, from patients with high amyloid pathology but varying tau and cognitive impairment will be compared using live cell imaging. An important innovation is to use naturally-occurring, human brain-derived oAβ, which may be more disease-relevant than synthetic oAβ. These experiments will help explain inconsistencies in our models of AD pathogenesis. In the long term, the results may assist clinical trial and drug design. The training goals of this proposal are for Dr. Stern to develop the necessary expertise to establish an independent laboratory researching the molecular pathogenesis of AD under R01 funding. Over the five years, Dr. Stern will acquire specific skills each with the one-on-one mentorship of an expert advisor: Aβ biochemistry (Dr. Selkoe), iN models (Dr. Young-Pearse), protein conformational chemistry and structural biology (Dr. Vos), neuropathology of neurodegeneration (Dr. Feany), and knowledge of AD clinical therapeutic development (Dr. Sperling). Dr. Stern has developed a training plan consisting of one-on-one meetings, in-person courses, and conferences. All mentors and advisors are renowned experts in their fields. Dr. Selkoe (primary mentor) is a leading AD researcher with decades of mentorship experience, including successful K08 awardees. Dr. Young- Pearse (co-mentor) is also a leading AD researcher, particularly in iN models of disease, and an accomplished mentor. The BWH Department of Neurology is committed to supporting Dr. Stern through protected research time (85%) and state-of-the-art facilities and equipment.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Rheumatoid arthritis (RA) is the most common autoimmune joint disease with over 15 treatment options, reflecting both advances in therapy as well as the heterogenous response to therapy. After the first line therapy methotrexate (MTX), patients and their rheumatologist proceed on a trial-and-error approach to identify the optimal treatment. A landmark randomized controlled clinical trial (RCT), RACAT, compared the effectiveness of triple therapy-MTX, sulfasalazine, and hydroxychloroquine vs MTX and a tumor necrosis factor inhibitor (TNFi). The RACAT subgroup analyses observed that some patients had a better response to one treatment strategy vs the other. However, like most RCTs, it was underpowered to better characterize these subgroups. Real-world data (RWD), such as electronic health record (EHR) and registry data, have a larger sample size but lack the randomization and precise clinical measurements performed as part of clinical trials. The objective of this proposal is to apply and rigorously test state-of-the-art methods that can combine the strengths of RCT and RWD to extend RCT findings. RACAT was a Veterans Affairs (VA) based clinical trial and thus many of their subjects also have EHR data in parallel, providing an ideal study design to test methods to understand how well we can replicated RCT using RWD. In Aim 1, we test methods using semi-supervised machine learning methods to impute RACAT clinical endpoints using EHR data; the linked RACAT data will be used as the gold standard comparison. Next, we apply causal inference modeling comparing triple therapy vs TNFi using EHR data with the imputed endpoints and validate results using the linked RACAT data. In Aim 2, we apply novel causal modeling methods that enable us to examine subgroup findings using RWD. We will identify subjects in the larger EHR and registries similar to RACAT subgroups, i.e. patients who benefitted more from triple therapy vs TNFi or vice versa, and subjects who remained on TNFi throughout the trial and did well. These larger populations will provide improved power to study potential predictors of treatment response. Moreover, the integration of EHR data allows us to study a broader set of potential predictors not collected in RCT or registry data. Our overarching hypothesis is that we will identify the clinical subgroups observed in RACAT with differing response to treatments within the larger populations of RA patients in EHR and registry. We will also identify novel predictors of response by using a broader set of clinical data available in EHR. This study is significant because it will provide a blueprint for studies for extending RCT findings in datasets with linked RCT and RWD, applicable to many treatments and conditions. This study is innovative because of its approach to maximize the data available from RCTs with existing RWD using linked datasets, powering studies to optimize RA therapy for different patients. This proposal also anticipates the growing ability of patients and institutions to access EHR data, enabling previously siloed datasets to become part of data-driven studies to advance clinical management of RA and other conditions.

NIH Research Projects · FY 2026 · 2022-07

SUMMARY Strategies for achieving sustained HIV remission must target the long-lived reservoir of HIV-infected cells. These reservoirs remain a challenge to study because they make up a very small fraction of immune cells, can be located in difficult to sample anatomic sites (e.g., the central nervous system [CNS]), and are generally less well studied in individuals who undergo treatment interruption. Here we aim at answering three critical questions: (A) what are the drivers of clonal expansion, activation of HIV-1-infected cells, and viral rebound timing – is it viral factors (such as HIV-1 integration site) that provide survival benefit of the infected cells, or is it host factors (such as immune responses to antigen or HIV stimulation) that drive the proliferation of HIV-1- infected cells and viral rebound after treatment interruption (Project 1)? (B) How do HIV-1-infected cells persist and distribute between peripheral blood and the anatomical sanctuary of the CNS (Project 2)? (C) Do HIV-1 eradication strategies, such as broadly neutralizing antibodies (bnAbs), reprogram host immune effector responses, transcriptionally, epigenetically, and functionally (Project 3)? Overall, we aim at understanding the expansion dynamics, tissue distribution, and rebound predictors of HIV-1 persistence using several unique clinical cohorts and innovative methods to provide critical insight to mechanisms of HIV-1 persistence and strategies for HIV-1 eradication. We will use these samples to define the mechanisms that govern spontaneous HIV-1 reactivation during treatment interruption and the persistence of viremia despite effective antiretroviral therapy (ART), specifically exploring virus and immune mechanisms that may impact viral maintenance and rebound (Project 1). We focus on the establishment, persistence, clonal proliferation, and rebound competence in different stages of infection of HIV-1 brain reservoirs, a critically important virus sanctuary that has been a challenge to study in detail (Project 2). Lastly, we explore the immune mechanisms that impact virus reservoir dynamics and the role of host epigenetics and immune cell function in the control and pruning of the HIV-1 proviral landscape in the context of a first-in-human broadly neutralizing antibody (Project 3). These Projects will be supported by an Administrative Core and a Data Analytics & Modeling Core. 1

NIH Research Projects · FY 2023 · 2022-07

The critical neuropathology underlying the cognitive decline in Alzheimer's disease is the loss of synapses. A leading view of the pathogenesis of Alzheimer's disease is that synaptic abnormalities are produced that lead to enhanced synapse elimination. The synaptopathy in AD is thought to be due largely to the production of toxic soluble oligomers of the Aβ1-42 peptide (oAβ). Soluble Aβ oligomers, but not monomers, have been shown to cause synaptic dysfunction, manifest by inhibition of LTP, enhancement of LTD, loss of dendritic spines, biochemical abnormalities, and hyperactivity. The enhanced LTD and hyperactivity appear to be due to elevation of extracellular glutamate as a consequence of impaired glutamate reuptake. Although substantial evidence has accumulated to support this hypothesis, there are significant gaps in our understanding of how oAβ perturbs glutamate homeostasis. Specifically, the identity of the glutamate transporter or transporters targeted by oAβ to produce the defect in glutamate homeostasis is unknown, as are the molecular mechanisms by which glutamate transport function is compromised by oAβ. These gaps loom greater in light of recent evidence that monoclonal antibodies (aducanumab; BAN2401) targeting soluble Aβ oligomers in AD patients may slow cognitive decline. The major glutamate transporter in the forebrain is GLT- 1 (human homolog EAAT2), which represents 1% of brain protein. GLT-1 is expressed in both astrocytes and glutamatergic axon terminals. Recent work by the applicant using a conditional GLT-1 knockout (KO) has shown that GLT-1 expressed in axon terminals is the dominant transporter mediating glutamate uptake into crude synaptosome preparations, also known as plasma membrane vesicles (PMVs). GLT-1 expressed in presynaptic terminals has also been shown to play an important role in synaptic mitochondrial metabolism. Several studies suggest that in the human and in mouse models deficits in glutamate transporter expression and/or function occur in AD. In critical experiments, glutamate uptake into PMVs derived from brain slices was decreased when the slices were treated with oAβ, implicating neuronal GLT-1. The central hypothesis motivating this project is that GLT-1 is the primary mechanistic target of oAβ causing glutamate dyshomeostasis. Given these findings it is important to ascertain whether GLT-1 is the specific glutamate transporter targeted by oAβ, whether oAβ affects GLT-1 function in astrocytes or neurons, or both, and the molecular basis for the interaction of oAβ with GLT-1. The specific goals of this project are to: Aim 1: Identify the glutamate transporter whose function is impaired by oAβ. Aim 2: Determine the cellular localization of effects of oAβ on GLT-1 using a conditional GLT-1 KO. The pursuit of these goals will lead to a molecular understanding of how oAβ, the salient AD cytotoxins, perturb glutamate homeostasis in AD and ultimately lead to novel approaches to prevent and treat AD.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT This mentored career development K23 award is providing Dr. Katherine Scovner Ravi with the resources, additional training and protected time necessary to achieve her goal of becoming an independent clinical investigator. Dr. Ravi has completed her master’s degree in public health and continues to benefit from her outstanding institutional support and resources through the pursuit of didactic courses for continued formal education in biostatistics and study design through Harvard Catalyst and the Harvard Master of Medical Sciences in Clinical Investigation program, research conferences, leadership courses and training in the responsible conduct of research. These complement her strong mentoring plan and institutional support and are building upon her prior training in research investigation. Approximately 550,000 patients in the United States are treated with life-sustaining hemodialysis (HD) for end-stage kidney disease. These patients experience mortality rates of around 20% per year with nearly a quarter of their mortality attributed to sudden cardiac death (SCD). This K23 Mentored Career Development Award (PA-20-206), entitled “Association of Dialysate Bicarbonate with Hemodynamic Instability and Arrhythmia,” is investigating the role that dialysate bicarbonate plays in intradialytic hypotension and in causing cardiac rhythms which increase the risk for SCD. The goal of this research is to understand how dialysate bicarbonate interacts with serum pH and electrolytes to impact hemodynamics and cardiac rhythms and to identify how individualized dialysate prescriptions may reduce hemodynamic instability, arrhythmia and ultimately SCD in maintenance HD patients. In Aim 1, we are using cohort data to assess how dialysate bicarbonate is associated with intradialytic hypotension. In Aim 2, data from implanted loop recorders used in the Monitoring in Dialysis (MiD) study were analyzed to investigate the associations of serum and dialysate bicarbonate with clinically significant arrhythmias (results published; PMCID: PMC11556930). Aim 3 is a randomized, controlled double-blind trial in maintenance HD patients to measure how dialysate bicarbonate levels affect intradialytic QTc prolongation, ventricular ectopy, clinically significant arrhythmias and intradialytic hypotension to assess how HD prescriptions might be improved to prevent cardiac dysrhythmia and hemodynamic instability (NCT05814146; recruitment ongoing). During this award period, Dr. Ravi is employing her unsurpassed academic resources and mentorship at Brigham and Women’s Hospital to acquire the skills and expertise required to attain R01 and/or R03 funding (R03 submitted 10/16/24). This will allow her to continue her critically important research and to train future clinical investigators.

NIH Research Projects · FY 2025 · 2022-07

Vascular cells are present throughout the human body and contribute to risk of multiple diseases. Vascular dysfunction directly affects risk for arterial diseases (e.g. coronary artery disease and stroke) as well as manifestations of other diseases such as dementia, cancer, and diabetes. Single cell analysis of the human vasculature has already begun to identify the basic mechanisms of vascular dysfunction in the large number of associated diseases. Our group, and several other labs, have used single cell RNA-sequencing (scRNA-seq) to identify vascular cell heterogeneity. We performed scRNA-seq of the aorta to identify functionally distinct endothelial cell (EC) subpopulations, and multiple groups have identified activated myofibroblasts in diseased mouse and human vascular tissue. These studies prove heterogenous cell populations exist in the arterial wall, but it remains undetermined which populations play a causal role in early vascular dysfunction and disease risk. The Human BioMolecular Atlas Program (HuBMAP) provides a rich source of data to begin to establish a causal link for specific vascular cell subpopulations with disease. In HuBMAP data, ECs and vascular smooth muscle cells (VSMCs) comprise a large portion of the single cells identified from each organ. However, to establish the cell types and transcriptional pathways associated with disease it will be necessary to incorporate the new datasets and computational methods we propose in this application. We aim to use new computational methods to integrate data from diseased vascular tissue with normal HuBMAP data, to identify the disease- relevant features of vascular cells. New methods to integrate disease associated genes from GWAS will also help investigators prioritize causal cells for multiple common diseases. To achieve this, we will: 1) Use new software to identify organotypic features of vascular cells in HUBMAP reference data; 2) Identify disease-specific vascular cell signature by comparing HUBMAP reference data with samples from vascular disease; and 3) Build and share a computational program to identify disease-relevant cell populations and gene modules through integration with genetic association data. These analyses make use of existing vascular disease snRNA-seq data from a rich collection of diseased subjects we can share with the HuBMAP. All data from vascular disease subjects is available for open data sharing, and has been collected to include a collection of subjects with respect to sex and ancestry. Our methods and statistical software to perform this integration of multiple single cell datasets with genetic associations will establish a generalizable methodology to rapidly discover the disease-relevant cells and processes of the vasculature, and all other cell-types, for any diseases with genetic risk and available GWAS. Our team is immediately ready to undertake the proposed studies and share the software with the HuBMAP community. We have a track record of rapidly sharing single cell RNA-seq data, and have a team with expertise in vascular biology, statistical genetics, and computational biology.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Tumor associated macrophages (TAMs) are abundant in solid tumors and drive tumorigenesis and resistance to therapy. Strikingly, a commonly used cancer drug, poly (ADP-ribose) polymerase inhibitors (PARPi), drive development of suppressive TAMs through metabolic remodeling and induction of lipogenesis that restrict T- cell activation and function. Conversely, T-cells treated with PARPi exhibit bioenergetic fitness, superior viabil- ity, and heightened effector function. While there is enthusiasm for combining PARPi with immune checkpoint blockade (ICB), based on potential synergy of T-cell activation of each class of drug, early phase clinical trials have not yet demonstrated the combination to be superior to PARPi monotherapy, suggesting lipogenic TAMs may limit PARPi + ICB responses. Thus, there is a critical need to elucidate the dichotomy of PARPi-induced metabolic remodeling to generate deleterious TAMs while promoting superior antitumor T-cells. In the absence of such knowledge successful strategies to harness the power of PARPi to benefit cancer patients are unlikely. The long-term goal is to design clinically effective strategies through TAM modulation to promote T-cell activa- tion and weaken the immune-suppressive TME to improve therapy in breast cancer patients. The central hy- pothesis is that lipogenic TAMs promote tumorigenesis in part through T-cell inhibition and are catalyzed by PARPi therapy, offering a unique therapeutic opportunity to enhance PARPi + ICB. The overall objectives are to (i) characterize TAM and T-cell metabolic regulation during PARPi and (ii) determine clinically effective strat- egies to regulate TAM metabolism to enhance T-cell activation and PARPi responses. The central hypothesis will be tested by pursing the following specific aims: 1) Define the mechanism of lipogenic macrophage de- velopment. Human and murine monocytes and macrophages will be used to formally define TAM lipogenesis catalyzed by PARPi and determine the mechanism by which TAM lipogenesis is induced during TNBC therapy using unbiased lipidomic as well as functional studies. 2) Test that lipogenic TAMs promote tumorigenesis through suppression of PARPi-mediated metabolic remodeling of adaptive immunity. Aim 2 will employ in vitro and in vivo protein synthesis, proteomics and metabolomic analysis to detail how PARPi induces T-cell metabolic fitness and define optimal metabolic perturbation for anti-tumor therapy. 3) Determine the optimal treatment strategy of PARPi and metabolic remodeling for rapid translation to breast cancer patients. Multiple TNBC mouse models will be employed to test if depletion of TAMs clears a path for T-cells with im- proved bioenergetic fitness fashioned by PARPi, metabolic remodeling, or the therapies combined. Unique clinical trial samples from patients treated with PARPi and PARPi + ICB will be assessed using state-of-the-art, single cell imaging to identify immune phenotype and function. Successful completion will reveal novel thera- peutic strategies to circumvent lipogenic TAMs while simultaneously activating metabolically superior antitumor T-cells and has potential for rapid clinical translation to increase the effectiveness of PARPi + ICB therapy.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Mild-to-moderate iodine deficiency (ID) remains widespread worldwide among pregnant and lactating women who have higher dietary requirements than the general population. Iodine is required for the synthesis of thyroid hormone, which is essential for human brain development, particularly visual attention, fine motor development, language and memory. The benefits of iodine supplementation for severe ID during pregnancy and in childhood have been established. However, the effects of iodine supplementation in settings with mild- to-moderate ID and during lactation remain unclear. Furthermore, few trials to date have examined the effects of iodine supplementation in pregnancy or lactation on iodine-specific neural functions. In response to NICHD NOSI (NOT-HD-19-039), we present a unique opportunity to build upon an ongoing, randomized controlled trial (RCT) to address the consequences of iodine deficiency on fetal and infant neurodevelopment in a population of mild-to-moderate iodine deficiency in rural Ethiopia. In the funded parent pregnancy trial, women in early pregnancy are randomized to receive a package of strengthened nutrition support (including monthly supply of iodized salt) and/or a package of infection control. The funded parent pregnancy trial will determine the effect of the interventions on infant birth outcomes. With support from this award, we propose to add a lactation intervention utilizing an efficient 2x2 factorial design and randomize women after delivery to receive a) an intensive iodized salt intervention, including provision of a monthly supply of high-quality, adequately iodized salt and counseling regarding appropriate storage and use, or b) standard care, until 6 months post- partum. Our overall goal is to examine the effects of iodine in pregnancy and lactation on infant brain function, as well as maternal and infant iodine status and thyroid function. The specific aims of this project are to: (1) determine the effects of an intensive salt intervention in pregnancy and lactation on infant visual evoked potentials (primary outcome), as well as visual attention, motor function, and head circumference at 6 months of age; (2) determine the effect of the iodized salt intervention on maternal breast milk iodine concentration, as well as infant iodine status and thyroid function; and (3) examine interactions between iron and iodine on thyroid function in pregnancy and lactation. Globally, salt iodization is the primary intervention strategy for iodine delivery. To our knowledge, this trial will be the first rigorous, adequately powered RCT of high-fidelity iodized salt provision along the continuum of pregnancy and lactation in a setting of mild-moderate iodine deficiency. These discoveries will improve our understanding of the role of iodine in early human brain development in order to develop effective targeted intervention strategies to improve iodine status and neurodevelopmental outcomes in children worldwide.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Antibodies specific for pathogenic threats can provide immediate protection from infectious disease but longevity of an antibody responses after vaccination or infection can be highly variable. Responses induced by some live vaccines can persist for a lifetime, whereas protein-based vaccines are in general shorter lasting. However, antibody durability is not necessarily linked to the use of live virus as long-lived antibody responses have been shown to be induced by the human papilloma virus (HPV) vaccine, a non-live viral-like particle- based platform. This suggests that distinct immunological cues can be engineered to result in the generation of longer-lived antibody responses. While memory lymphocytes also provide a system of protective efficacy, strategies to maximize robust levels of protective secreted antibodies that are stable over time is an important goal in modern immunology. Understanding the capabilities of the immune system in this context, and how available vaccines can elicit durable secreted antibody responses will be important to decipher. This is relevant to the ongoing SARS-CoV-2 pandemic and for vaccine strategies more broadly. Preliminary data suggest that antibodies induced by natural infection harbor robust long-term stability at modest levels and greater polyclonal neutralizing breadth across viral variants compared to infection-naïve vaccinees. In addition, differential antibody durability trajectories tend to favor COVID-19 convalescent subjects with dual memory B cell features of greater antibody somatic mutation and cross-coronavirus reactivity. These findings support a hypothesis that high somatical mutation and cross-reactivity in antigen-binding memory B cell repertoires early after recovery predicts antibody durability and that recalled immunity may confer greater longevity of differentiated plasma cells. This hypothesis will be examined in two aims, (i) to illuminate factors influencing anti-SARS-CoV-2 antibody durability, and (ii) to chart the functional evolution of anti-CoV memory B cell over time. For aim 1, human and mouse studies will be used to illuminate potential mechanistic insights and features connected to durable antibody responses. For aim 2, the durability and evolution of memory B cell repertoire antigen recognition capacity will be charted over time to assess the evolving relationship between secreted polyclonal and memory B cell repertoires. This work is expected to shed light on factors that influence longevity and evolution of antibody responses, which will be important for ongoing improvement of vaccine strategies.

NIH Research Projects · FY 2025 · 2022-06

Project Summary Chemical Biological Discovery of Lipid Virulence Factors in the Major Bacterial Pathogens For decades, the search for the causes of bacterial virulence has focused on genes rather than metabolites. Genetic approaches have been broadly successful, and modern infectious disease research relies fundamentally on genomic maps of the major pathogens. Owing to the lack of whole-organism chemical biology tools, bacterial lipids have not been systematically tested for their roles in virulence, even though lipids are the primary interface with the human host, where they control nutrient flow and trigger host immune response. We invented a mass spectrometry platform for lipid profiling to detect nearly all ionizable lipids in a bacterial cell within 2 hours. Experiments on Mycobacterium tuberculosis and Salmonella enterica serovar Typhi now provide clear evidence for the general insight that many, perhaps the majority, of lipids in the world's bacterial pathogens, are currently unknown as named compounds. Based on successes in identifying virulence factors in two major pathogens of worldwide significance, we will carry out a chemical biology approach known as forward lipidomics. Specifically, we will use whole organism mass spectrometry profiling to discover the lipids that are selectively expressed in virulent bacteria and are unknown in existing lipid catalogs. Then, we will chemically synthesize the virulence associated lipids and link them to their biosynthetic genes for deletion in bacteria using reverse genetic approaches. Using genetically modified bacteria that are deficient in defined lipids, we will determine the roles of virulence lipids during infection. Using nature identical synthetic lipids, we will determine the cellular mechanisms of generation of foamy macrophages and identify immune receptors that mediate host response. We will create lipid maps of the major Gram negative pathogen groups based on patient strains to build the overlooked field of chemical biology of bacterial virulence. These basic and translational studies will support the development new forward lipidomics approaches to the diagnosis and treatment of major infectious diseases.

NIH Research Projects · FY 2025 · 2022-06

Abstract The transplantation field has witnessed many major breakthroughs, including the development of immunoregulatory molecules (IRMs), which have been key to the success of organ transplantation. However, the use of IRMs is hindered by lack of efficiency and toxicity, and it is implicated in the pathogenesis of organ failure and accelerated cardiovascular disease, which is the leading cause of death in transplant recipients. Therefore, a substantial unmet medical need exists to develop novel strategies to increase the efficacy and reduce the toxicity of IRMs. The existing drugs are often adequately potent when directed specifically to their intended sites, so methods of targeted drug delivery could potentiate their safety and efficacy profiles significantly, while reducing the need for creating new drugs, a process that can be extremely expensive, labor- intensive, and time-consuming. Although targeted drug delivery using nanotechnology represents a highly promising and innovative strategy for site-specific drug delivery, its application to transplantation remains to be developed. The overall goal of this proposal is to develop a targeted drug delivery system for IRMs in transplantation, with the ultimate goal of increasing their efficacy and diminishing their toxicity. In transplantation, presentation of donor allo-antigens to recipient T cells in the draining lymph nodes (DLNs) is fundamental to the generation of alloreactive T cells that traffic to the allografts and cause allograft rejection. The overall hypothesis of this proposal is that targeted delivery of IRMs to the DLN would not only increase their efficacy, but also decrease their toxicity by significantly reducing systemic dosage. In Aim 1, we plan to devise a clinically applicable active targeted method of delivering IRMs to the DLNs to promote heart allograft acceptance. We will focus primarily on murine heart allograft survival by devising a combinatorial therapeutic strategy with our targeted delivery platform to address the immediate unmet need for safer and more efficacious therapies in transplantation. In Aim 2, we plan to evaluate the mechanism of prolongation of heart allograft survival by our active targeted delivery platform to DLNs. Mechanistic studies will also permit improvement of the design of our targeted delivery method. These experiments will employ murine heart transplant models, established functional assays, and sophisticated imaging studies to understand better the biodistribution of IRMs and their nanocarriers. In Aim 3, we plan to pursue our preliminary data to generate proof-of-concept data in devising a method of targeting IRMs to DLNs in non-human primates. This multidisciplinary, collaborative approach sets forth a novel targeted delivery platform that could potentially shift the paradigm of the approach to immunosuppressive therapy in transplantation.

NIH Research Projects · FY 2025 · 2022-06

PROJECT ABSTRACT Inflammation contributes substantially to atherosclerotic cardiovascular disease (CVD) in the general population. Epidemiology, basic science and randomized clinical trial data support the importance of this relationship. Patients with systemic inflammatory conditions, such as rheumatoid arthritis (RA), can provide important insights into this relationship because of their more extreme systemic inflammatory phenotype. Investigators have appreciated the elevated risk of CVD experienced by RA patients: the risk of MI and stroke are both elevated in RA compared with the general population, contributing to a shortened lifespan. CVD risk stratification in RA is imprecise and general population tools are not accurate. Most attempts at improving CVD risk stratification have added clinical RA factors to existing population risk tools. Easily assessed protein biomarkers would likely enhance CVD risk prediction. The literature strongly suggests relationships between > 20 biomarkers shared by RA and CVD. These relationships have never been studied systematically across diseases. The overarching goal of this proposal is to identify protein biomarkers for CVD in RA patients, leveraging the structure of a controlled trial and rigorous methods for deriving and validating a risk score. We complement the robust biomarker analyses with high-dimensional cellular immuneprofiling, which has the potential to link specific cell types mechanistically to protein biomarkers and to identify new cellular biomarkers. We conducted a randomized controlled trial, the TARGET trial, to examine whether specific treatments for RA produce reductions in CV risk as measured by FDG PET/CT. This trial, funded by NIH (U01 AR068043) allowed us to prospectively characterize RA patients, collect biospecimens before and after treatment, and conduct baseline and 24-week FDG PET/CT scans to assess vascular inflammation. Analyses are still ongoing to determine whether different RA treatments translate into differential changes in CV risk. We propose to leverage the TARGET study cohort, dataset and biorepository for the following aims. Aim 1: To use a comprehensive biomarker panel to derive and validate a CV risk score for patients with RA. The TARGET trial provides biospecimens, patient phenotypes, and a broad biomarker discovery panel that will have been run as an in-kind donation. We hypothesize that adding biomarkers to the Pooled Cohort Equation and variables related to RA disease activity will significantly improve prediction of CV outcomes in RA patients. Aim 2: To elucidate cellular immune mechanisms linking RA and CVD through scRNA-seq profiling. We will use single cell transcriptomic and surface proteomics (CITE-seq) to study PBMCs from a subset of TARGET patients, including both responders and non-responders based on FDG PET/CT, to identify circulating immune cell populations associated with CV risk and CV biomarkers. We hypothesize that specific immune cell populations will associate with CV risk at baseline and will decrease in abundance or activation state after treatment in parallel with CV risk. Further, these treatments will differ in their effects on relevant cell populations.

NIH Research Projects · FY 2025 · 2022-06

Gut symbiotic microbiota–derived CD1d ligands and their immunomodulatory mechanisms PROJECT SUMMARY The symbiotic microbiota has co-evolved with the mammalian host for millennia, and the host has developed a sophisticated system for distinguishing pathogens from commensal microbes. Unlike pathogens, many of whose molecules trigger robust inflammatory and immune reactions, symbiont-derived molecular factors have been believed to be “silent,” even though they reside within the host at a very high density. However, recent studies strongly suggest that molecular factors of symbiotic origin actively contribute to host immune regulation and protection from excessive inflammation. We have previously identified and characterized a unique class of lipids (alpha-galactosylceramides) from the human gut symbiont Bacteroides fragilis that can modulate host immune development early in life. Our preliminary results show that these molecules (BfaGCs) are presented by the nonclassical MHC class I–like molecule CD1d in a structurally conserved manner similar to that documented for prototypic CD1d ligands. Of considerable interest, unlike CD1d agonists such as KRN7000, BfaGCs function as a regulator of natural killer T (NKT) cells, a specific T cell subtype restricted by the CD1d–lipid antigen complex. Synthetic BfaGC molecules can induce distinct immunomodulatory signals from NKT cells and can function as a regulator of NKT cell proliferation in the colon. Furthermore, targeted lipidomic profiling of gut symbionts has identified lipid species structurally related to BfaGCs in multiple gut symbionts, implying that gut symbionts can collectively synthesize potential NKT cell regulators. We propose an investigation of molecular immunomodulatory mechanisms that underlie the activity of gut symbiont–derived lipid ligands. We aim to determine (1) the distinct NKT population recognized by the CD1d-BfaGC complex and their immunomodulatory responses to BfaGCs, (2) the specific molecular-level interactions between CD1d and BfaGCs, (3) the structure and immunomodulatory activity of previously uncharacterized lipid species of symbiont origin and (4) the modulation of inflammatory responses by symbiont-derived CD1d ligands in vivo. The proposed studies will provide valuable knowledge of the molecular mechanisms by which symbiotic microbiota-derived molecules modulate the host immune system and help the development of potential immunotherapeutics.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY Dynamic cell-cell interactions are crucial for healthy cell behavior and proper intercellular communication. Impaired intercellular communication has been implicated in the pathologies of various diseases including cancer, neurodegenerative diseases, bacterial and viral infections, autoimmune diseases, and cardiovascular diseases. As a result, probing cell-cell interactions is essential to many areas of biomedical research. It can lead to a more detailed understanding of various diseases and the development of novel therapeutic strategies, such as personalized immuno-oncology. However, current single-cell analysis techniques are slow and require potentially harmful physical contact with cells of interest, hindering the progress in elucidating these phenomena. Recently, we invented Harmonic Acoustics for Neighboring cell Dynamic studies (HANDs), an acoustic- based, automated, contact-free, cell-cell-pairing technology, which overcomes the key obstacles associated with the existing technologies. In this R01 project, we will develop and validate the HANDs platform with the following features: (1) Contactless nature and high biocompatibility: Instead of requiring direct contact with solid substrates or beads, the proposed HANDs technology is a contactless method. In addition, rather than exposure to large shear forces, strong pressures, or powerful optics, which can cause physiological damage, the cells in our setup are manipulated gently with low-power acoustic waves. The proposed HANDs platform allows long- term (>24 hours) cell-cell interaction studies. This feat cannot easily be achieved using existing state-of-the-art technologies such as atomic force spectroscopy. (2) High-throughput reversible cell-cell interactions and precise quantitative analysis at the single-cell level: The multi-trapping nature of the HANDs technology enables the simultaneous and parallel study of numerous (>20,000) cell pairs with single-cell precision. Existing single-cell techniques are either limited to studying a single pair of cells at any given time or lack the precision needed to control cell pairing and separation, and precise quantitative analysis. (3) Automated operation: Unlike existing cell pairing technologies which require complicated procedures and tools to achieve operation, the proposed technology automatically aligns cell-cell pairs using acoustic traps. Additionally, once the control signal is specified, cells can be brought into contact and separated in whatever automated and prescribed contact pattern is desired for testing. (4) High resolution (~100 nm): Using single-phase unidirectional transducers and harmonic frequency modulation, we will improve the spatial resolution of our HANDs technology from ~1 μm to ~100 nm. We will validate the performance of our HANDs platform across two well-established models: interactions between T cells and cancer cells, and interactions between stem cells and macrophages. In this regard, we aim to demonstrate the far-reaching potential of HANDs to enable improved research in areas ranging from fundamental biology to personalized immuno-oncology and drug discovery.

NIH Research Projects · FY 2025 · 2022-06

Surgical resection is the initial treatment for nearly all brain tumors and the extent of resection is strongly correlated with prognosis. However, because brain tumors, especially gliomas, are intimately involved in surrounding functioning brain tissue, aggressive resection must be balanced against the risk of causing new neurological deficits. Modern advances in anatomical and functional imaging and the widespread adoption of neuro-navigation now help neurosurgeons to plan and execute an optimal surgical approach. Unfortunately, changes in the shape of the brain during surgery, known as brain shift, invalidate the assumption of all commercial neuro-navigation systems that preoperative data can be mapped to patient coordinates using rigid registration. Because brain shift progresses during surgery, the rigid registration of neuro- navigation systems is least accurate at the critical final stages of resection when the marginal tissue is being removed. There has been more than 20 years of research invested in measuring, modeling and compensating for brain shift with the goal of providing neuro-navigation systems with an accurate nonrigid registration from preoperative image data to the patient’s brain in the presence of brain shift. While results are promising, they are not yet accurate enough to be incorporated into commercial systems. Nonrigid registration is subject to both measurement and modeling uncertainty that varies throughout 3D space. Most nonrigid registration methods do not attempt to quantify this uncertainty and, to our knowledge, there have been no attempts to present this uncertainty to the surgeon. We believe that it is important to make surgeons aware of this uncertainty so that they can make informed decisions, particularly in locations where uncertainty is high. In this project, we plan to investigate nonrigid registration algorithms that model registration uncertainty explicitly, semi-automatic and fully-automatic nonrigid registration methods that utilize registration uncertainty to iteratively guide registration improvements, and visualization paradigms for effective presentation of registration uncertainty to surgeons in the surgical environment. We hypothesize that effective representation and visualization of registration uncertainty for brain shift correction in neuro-navigation will 1) lead to iterative semi-automatic and fully-automatic nonrigid registration methods that improve registration accuracy and 2) allow neurosurgeons to make more informed decisions during tumor resections that will lead to increased clinical impact of image-guided neurosurgery. We will carry out the following Aims: 1. Develop novel feature- based image registration algorithms that represent uncertainty explicitly; 2. Use registration uncertainty maps to guide semi- and fully-automatic nonrigid registration; 3. Evaluate the utility of nonrigid registration with uncertainty visualization in a clinical setting.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY Immune checkpoint blockade has elicited unprecedented clinical responses in patients with metastatic melanoma and other cancers. A promising new checkpoint under investigation in cancer therapeutic trials is T- cell immunoglobulin and mucin domain 3 (Tim-3). Tim-3 blockade reverses T-cell impairment, thereby reinvigorating antitumor T-cell immunity. However, we found that Tim-3 inhibitors, including those in clinical trials, not only target T-cell-Tim-3, but also have varying affinity for Tim-3 on dendritic cells (DCs), macrophages (MΦs), NK and melanoma cells. Clinical benefit might thus not exclusively rely on antagonism of T-cell-Tim-3, but also on inhibition of these additional Tim-3-expressing cell types. In support, blockade of T- cell-Tim-3 suppressed, while melanoma-directed Tim-3 inhibition enhanced tumor growth in murine melanoma models, thereby counteracting desired efficacy of Tim-3 therapy. Consistently, enforced expression of Tim-3 on melanoma cells suppressed tumorigenesis, metastasis formation, and proliferative pathway activity. Our preliminary studies thus identify melanoma cell-intrinsic, DC-, MΦ-, and NK-cell-Tim-3 as unexpected variables and/or potential confounders of treatment outcome. They further highlight the need to define therapeutic consequences of Tim-3 antibody (Ab) responses at the level of specific cell types. The Tim-3 protein bears multiple N- and O-glycostructures that differ dramatically in composition, size, and charge between cell lineages and which might explain the marked variations in Tim-3 Ab clone reactivity we found between cell types. For example, the clinical Tim-3 trial candidate, TSR-022, avidly bound T-cell- and melanoma-, but not NK-, DC-, or MΦ-Tim-3, while other Tim-3 Abs showed high affinity for Tim-3 on T-cells, MΦs, DCs, and/or NK, but not melanoma cells. Notably, glycan-modifying regimens shifted inhibitor binding towards desired T-cell- Tim-3 recognition and reduced melanoma-Tim-3 reactivity. Our preliminary data highlights the critical need for dissecting immune- vs. melanoma cell-Tim-3 glyco-epitopes, Ab affinity, signaling, and immunobiology. Results will help optimize Tim-3 therapeutic efficacy by validating regimens that preferentially target immune cell-Tim-3 glycans, while avoiding unwanted blockade of melanoma cell-Tim-3. Our aims are to 1) define cell type- associated Tim-3 glycan moieties, ligands, Ab affinities, and signaling networks, 2) examine immune cell- vs. melanoma-intrinsic effects of existing Tim-3 antagonists and their relevance to interpreting therapeutic benefit, and 3) identify new Tim-3 targeting strategies that accentuate immune cell-Tim-3 inhibition. We will use state- of-the-art gain and loss of Tim-3 function and glycan-modifying strategies, Tim-3 inhibitors with variable tissue- associated affinities, and immune and melanoma model systems to define cell type-specific Tim-3 functions and glycomolecular targets. Our initiative also implements clinical tumor biospecimens from patients receiving immune checkpoint inhibitors. Together, these studies will pave the way for next generation biomarkers and treatment modalities that discriminate immune- from cancer cell-Tim-3 for optimized immunotherapy outcomes.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY This proposal directly addresses a high-priority research topic, “Enabling precision medicine for Alzheimer’s disease and related dementias through deep molecular phenotyping”, for PAR-19-070, Research on Current Topics in Alzheimer's Disease and Its Related Dementias, by beginning to develop precision dietary approaches for AD prevention through integrating multiple molecular data types. The potential for healthy dietary patterns to maintain cognitive health is supported by cohort studies and a randomized trial. However, these healthy dietary patterns were developed based on population averages and may not be best suited for a given individual. Preliminary data from our group and others support the rationale to study personalized approaches tailored to individual gut microbiomes to improve dietary prevention of Alzheimer’s dementia (AD). However, no study has examined modifying and mediating roles of the gut microbiome in the diet-cognition association. The few human studies of the gut microbiome in AD were limited by small size, cross-sectional design, and lack of high-resolution microbial functional profiling. This background supports our central hypotheses that 1) healthy dietary patterns contribute to cognitive health partly through modulating the gut microbiome; and 2) associations of the dietary patterns with cognitive function vary by individuals’ gut microbial profiles. This proposal represents a highly cost-efficient, prospective study leveraging existing fecal samples/microbiome data and cognitive function assessments in three studies with complementary strengths in study design, and diet and outcome assessments: the Nurses’ Health Study II (NHSII, n =1,500) with decades-long repeated dietary assessments and extended follow-up, the Mediterranean-DASH Intervention for Neurodegenerative Delay (MIND) Trial with randomized dietary interventions (n =604), and the ASPREE-XT Study (n =13,000) with adjudicated incident AD endpoints. To gain more advanced mechanistic insights, we will take a multi’omic approach to combine shotgun metagenomics and metatranscriptomics to profile the microbial composition and enzymatic function, and fecal metabolomics to measure the microbiome metabolic activity. We will investigate roles of gut microbial composition and enzymatic function in the associations of the Mediterranean diet (MedDiet) and the MIND Diet with cognitive function (Aim 1) and examine the interrelationships among the two healthy dietary patterns, the metabolic activity of the gut microbiome and cognitive function (Aim 2) in the NHSII. We will replicate findings from Aims 1 and 2 in ASPREE-XT and the MIND Trial (Aim 3). This project will generate reproducible, translational evidence on gut microbial and fecal metabolomic features that explain inter-individual heterogeneity in response to healthy dietary patterns and provide foundational knowledge for maximizing the benefits of dietary approaches, discovering novel predictive biomarkers, and ultimately contributing to precision prevention of AD.