Weill Medical Coll Of Cornell Univ

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract The aberrant incorporation or retention of ribonucleic acids (RNAs) in the genome is a common cause of genomic instability, rendering it susceptible to hydrolysis and downstream mutagenesis. The enzyme RNase H2 is one of the primary mechanisms protecting against this destabilization of the genome by removing these genome- embedded RNAs. Our lab recently uncovered a novel mechanism of regulation of RNase H2, by uncovering that replication termination factor 2 (RTF2) is involved in localizing and regulating the levels of RNase H2 at the replication fork. Further elucidation of this interaction is required to understand the basic biology behind the regulation and function of how RNase H2 prevents genome instability. Interestingly, copy number loss of RNase H2 is commonly found in Chronic Lymphocytic Leukemia (CLL), in over 30% of cases. In my preliminary work, I have developed various cellular models in which RNase H2 and RTF2 can be depleted, and I have expressed and purified recombinant RNase H2 and RTF2, allowing for both in vivo studies of which RNase H2 activities are regulated by RTF2 and in vitro studies of their interaction. Furthermore, I have developed a novel assay allowing a quantitative analysis of ribonucleotide incorporation in the genomes of human cells. This assay will be used to study the regulation of RNase H2 by RTF2, and will be assessed in its applicability to predict CLL responses to PARP-inhibitors. The direct mechanism behind tumor progression in the loss of RNase H2 has not been studied. In this proposal, building on my above preliminary work, I will test the hypothesis that RTF2 interacts directly with and regulates the activities of RNase H2 at the replication fork and examine the mechanism behind how loss of RNase H2 compromises genomic stability and leads to tumor progression. I am an MD/PhD student at the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional Program, where I am in the laboratory of Dr. Agata Smogorzewska at The Rockefeller University. My long-term goal is to become a physician scientist, practicing as a hematologist-oncologist as well as running an independent basic science lab as an academic university hospital. The plan outlined in this proposal, along with the support and mentorship of Dr. Agata Smogorzewska, my thesis research committee, and the Tri-Institutional MD-PhD program will help me achieve these career aspirations.

NIH Research Projects · FY 2025 · 2022-07

EBV-associated lymphomas are a heterogenous group of aggressive B-, T-, and NK- cell malignancies. In these lymphomas, EBV exists in a latent state where infectious virus is not produced but a limited number of viral proteins are expressed. One mechanism by which EBV+ lymphomas escape the immune response to EBV is through the latency I program where only the weakly immunogenic Epstein Barr nuclear antigen 1 (EBNA1) and non-coding RNAs are expressed. Latency I EBV+ lymphomas include Burkitt lymphoma and many cases of HIV-associated diffuse large B-cell lymphoma. Novel therapies are urgently needed in these subtypes where the outcome for patients with relapsed disease is dismal (OS<20% in Burkitt lymphoma, for example). In addition, many of the world’s cases arise in resource limited settings where upfront treatment with high dose chemotherapy is not feasible. Our approach to this unmet need in EBV+ lymphomas is to use epigenetic reprogramming to convert latency I tumors to the highly immunogenic latency II or III program, thereby rendering tumors sensitive to T-cell mediated killing. Using a high-throughput pharmacologic screen, we identified the hypomethylating agent decitabine as a potent inducer of latency II and latency III antigens in latency I EBV+ lymphomas. Furthermore, we observed that decitabine treatment in latency I tumors sensitizes resistant cells to killing by allogeneic EBV-specific T-cells, both in-vitro and in-vivo. Based on our preliminary findings, we hypothesize that epigenetic induction of latency switch in EBV+ lymphoma induces an anti-tumor immune response capable of eradicating disease through cytotoxic T-cell recognition of latency II/III viral epitopes. In the current proposal we will develop a rational approach to the use of epigenetic modulation to induce latency switching and immune mediated cell death in EBV+ lymphomas. We will determine which immune effector cells are required to eradicate latency switched EBV+ lymphomas including their activation status and function, and will characterize the predominant viral antigens to which they are responding. We will also explore mechanisms of potential resistance to decitabine mediated latency-switching and develop therapeutic combination strategies that maximize the percentage of cells that convert from latency I to latency II/III. Finally, we will establish therapeutic approaches that enhance the immune destruction of latency switched EBV+ lymphomas. Collectively, this proposal will accomplish the rational development of an entirely novel approach to the treatment of latency I EBV+ lymphomas, utilizing epigenetic reprogramming to induce immunogenic viral antigens in otherwise immunologically silent tumors. This work has implications beyond lymphomas to all EBV+ malignancies with restricted latency.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY The successful sequencing of the human genome and the evolution of human health services research in the post-genomic era combined with startling advances in imaging technology have offered unprecedented opportunities to transform patient care in cardiovascular diseases. Over the past decade, Weill Cornell Medicine (WCM) has made major investments in biomedical research, as well as research training and infrastructure. WCM requests support for the research training of 4 clinical and non-clinical postdoctoral fellows in a new multidisciplinary research training program (MRTP) in Cardiovascular Disease, with WCM providing funding for an additional training slot. The objective is to recruit outstanding candidates with solid foundations in basic science and/or clinical research and train them to apply their knowledge and skills towards addressing important clinically unmet needs in the prevention, diagnosis, and treatment of patients with cardiovascular diseases. MRTP trainees will receive program support for 3 years during which they will follow a structured and rigorous postdoctoral training program. The MRTP will be a joint effort of 20 eminent preceptors from WCM, Memorial Sloan Kettering Cancer Center (MSKCC) and the Rockefeller University (RU), which constitute a dense network of collaborative researchers, who are international leaders in fields directly relevant to cardiovascular physiology and disease. These preceptors have been organized into 2 themed, but interconnected units of research training: molecular and cell signaling and cardiovascular imaging. These training units will enable the development of content-specific educational programming, as well as increase the efficiency administration within the training program. Oversight will be provided by a Research Training Executive Committee consisting of the Program Director and two Associate Program Directors, who will also serve as leaders of the training units, as well as 3 additional members who will provide specific guidance on clinical/epidemiologic research, on achieving and maintaining diversity within the training program, and on mentoring. We have also assembled an outstanding roster of internal and external advisory board members with a mandate to evaluate the program and provide specific recommendations to improve both its quality and efficiency. The MRTP's highly personalized training program will include 1) individual development plans; 2) rigorous research training; 3) hands-on experience in cutting-edge methodologies; and 4) an integrated curriculum. Trainees will further benefit from the extensive institutional resources of WCM, MSKCC and RU. Based upon the levels of interest in our fellowship programs, we anticipate a substantial pool of highly qualified clinical and non-clinical candidates for the proposed MRTP, which will maintain a strong diversity focus. Through its rigorous, structured and highly personalized curriculum, this new training program seeks to train future leaders in cardiovascular disease research who are prepared to translate their findings towards improving patient care.

NIH Research Projects · FY 2025 · 2022-07

Preterm birth indicates an end to the rich fetal supply of polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and Arachidonic Acid (ARA) which are a critical building blocks for brain and eye development and an important inflammatory modulator. Yet, for almost 40 years, enteral supplementation of DHA and ARA to replace lost fetal accretion has failed to translate into long-standing clinical benefit. The failure to understand the metabolism and induced molecular changes of fatty acid supplementation during the postnatal period has led to erroneous assumptions and replacement strategies that are, at best, not clinical beneficial and, at worst, harmful. The overall study objective of this proposal is to investigate the induced metabolism and downstream molecular mechanisms of enteral DHA and ARA supplementation from birth to 36 weeks' postnatal age in the extremely preterm infant. We have assembled a multidisciplinary team with expertise in neonatal-perinatal medicine and biostatistics/bioinformatics. The team of neonatologists are clinical and research leaders in nutrition, growth, and fatty acids and represents five level III/IV neonatal intensive care units with diverse demographic populations. We hypothesize that metabolism of DHA and ARA enteral supplementation is developmentally regulated and the impact of lipid derived metabolites on biology is dependent on the health state of the infant (context specificity). To interrogate our hypothesis, we propose a multi-center, randomized clinical trial to evaluate lipidomic actions of combined DHA and ARA supplementation from birth to 36 weeks postmenstrual age in 280 extremely low gestational age newborns born between 25 0/7 and 29 6/7 weeks of gestation. The following aims will be evaluated: Aim 1 – Identify the impact of combined enteral DHA and ARA supplementation on fatty acid metabolism including derivation of specialized pro-resolving mediators (SPMs) and oxylipin production and, Aim 2 – Determine the multisystemic change in circulating markers of inflammation and organ development and integrity as a function of enteral DHA/ARA supplementation. Successful completion of these aims will define developmentally regulated fatty acid metabolism in the immature host and impact of fatty acid supplementation on critical biological functions. Mechanistic data is required to bridge the scientific gap to appropriate clinical translation that is effective and safe. These data will inform a biologically rational approach to fatty acid delivery in preterm infants and identify molecular read-outs that may serve as biomarkers in future trials of clinical efficacy.

NIH Research Projects · FY 2024 · 2022-07

Project Summary/Abstract Cellular senescence is considered a “double-edged sword” in cancer and cancer therapy – while senescence- associated growth arrest and immune stimulation serve as potent anti-tumor mechanisms, chronic inflammation can be pro-tumorigenic and senescence bypass can contribute to therapy resistance and relapse. Many clinically used cancer therapies have been shown to trigger cellular senescence in tumor cells, so understanding the effects of senescent cells on the tumor microenvironment is critical. Gaining a clear understanding of the mechanism of senescence-inducing therapies will enable their improved clinical use and increase the likelihood for their success as cancer therapeutics. There is considerable evidence that senescent cells are proinflammatory and can be surveilled by T cells in vivo. This proposal will dissect the interplay between senescent cells and T cells in a mouse model of Hepatocellular Carcinoma in which senescence-induced T cell-mediated tumor regressions have been observed. Aim 1. Investigate senescence-induced T cell surveillance of senescent and proliferating tumor cells. I hypothesize that senescent tumor cells secrete chemokines that promote T cell infiltration and surveillance of both senescent and proliferating tumor cells. Aim 2. Identify strategies to potentiate T cell surveillance of senescent tumor cells. I hypothesize that senescent tumor cells employ resistance programs that diminish T cell recognition or killing. Validation of the hypotheses set forth in this proposal would have major implications in the fields of senescence biology and tumor immunology, as well as for the use of senescence-inducing therapies as clinical cancer therapeutics.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract The goal of the proposed K23 Career Development Award is to provide the PI with the mentorship, knowledge, and skillset to develop into an independent investigator studying neuroscience-inspired, targeted therapeutics for comorbid mood and cognitive dysfunction after stroke. The PI's mentoring team will provide him with training in the brain-based mechanisms of mood disorders, use of functional MRI to examine target engagement, and clinical trial design and data analytic approaches. The training will incorporate individualized tutorials, formal coursework, national and international workshops and conferences, research collaboration, manuscript preparation, and grant writing. The training is integrated in a research proposal that evaluates the efficacy and target engagement of a digital intervention for the depression-executive dysfunction syndrome (DED) after stroke. Stroke is a leading cause of disability worldwide. Post-stroke DED is associated with more persistent depressive symptoms and executive dysfunction, worse social functioning, and greater loss of independence than post-stroke depression or executive dysfunction alone. Existing interventions have limited evidence of efficacy, side effects, and can be difficult for stroke patients to access. Novel and scalable approaches that target the mechanisms underlying post-stroke DED may yield more efficacious treatment. To address these barriers, the PI proposes to study a remote intervention for post-stroke DED that combines an iPad-based digital therapy called AKL-T01 that intensively trains rapid multitasking skills, together with virtual metacognitive coaching. AKL-T01 is designed to target a common mechanism underlying post-stroke depressive symptoms and executive dysfunction, which is reduced intrinsic functional connectivity in the executive control network (ECN). Virtual metacognitive coaching is included to address patient unawareness of deficits to enhance transfer of training gains to daily functioning. In this pilot clinical trial, N=70 patients with a first-time stroke and DED will be randomized to receive the intervention, or to a control condition with general and non-targeted cognitive stimulation together with metacognitive coaching. Participants will complete measures of executive dysfunction, depressive symptoms, and disability at baseline, week 3 (mid treatment), and week 6 (end treatment). Participants will complete resting state fMRI scans at baseline and week 6. Linear mixed-effects models will test the hypotheses that the intervention will be associated with greater improvements in executive function, depressive symptoms, and disability relative to the control group. Also tested is the hypothesis that the intervention group will have greater change in intrinsic functional connectivity in the ECN from baseline to week 6, relative to the control group. This project has the potential to address, in a scalable manner, a highly debilitating consequence of stroke. It will generate data that can be used to optimize treatment efficacy, further refine and personalize the intervention, and further validate a target for treatment.

NIH Research Projects · FY 2024 · 2022-06

Project Summary Abstract Loss of heterozygosity (LOH) is the loss of the functional copy of a tumor suppressor gene in heterozygous individuals, leaving only the mutant copy, thereby contributing to cancer initiation and progression. Understanding how LOH arises in healthy cells is a vital component of cancer research, as it could lead to opportunities to prevent cancer in patients with increased early screening for individuals with mutations putting them at high risk for LOH, or with identification and targeting of cells that have undergone dangerous LOH. LOH can develop through deletions of whole chromosome arms or smaller regions of the genome, nondisjunction incidents, or homologous recombination events between homologous chromosomes (i.e., interhomolog homologous recombination, IH-HR). Despite decades of research, a critical need remains to identify the causes of LOH in normal or precancerous cells. Detecting these mechanisms in already established tumors is often difficult due to high levels of aneuploidy, ongoing chromosome instability, and DNA damage. We have recently developed a high throughput flow cytometry-based system which is sensitive for detecting LOH in normal diploid cells. Our objective is to exploit this system to identify and quantify mechanisms of LOH that arise from DNA double-strand breaks (DSBs), and the factors that modulate these events. We hypothesize that mechanisms of LOH which occur as a result of DSBs include copy number neutral LOH, as from IH-HR or non-disjunction associated with chromosome duplication and furthermore, that these events are modulated by the location of the DSB and DNA repair proteins, including proteins that act on recombination intermediates. We will address this hypothesis through the following specific aims: In Aim 1, we will investigate how proteins that act on recombination intermediates impact LOH arising from IH-HR, utilizing our flow-cytometry based system. We will assay the frequency of LOH that arises from IH-HR in the presence or absence of proteins that both prevent and contribute to crossover events, which can lead to long-range LOH. In Aim 2, we will take an unbiased approach to explore additional mechanisms of LOH arising from a DSB. We will determine how LOH mechanism is affected by the location of a DSB along a chromosome and by loss of different DSB repair pathways. This work will allow us to identify the factors that can contribute to or even prevent LOH, and give a better understanding of how these initiating events in cancer occur.

NIH Research Projects · FY 2025 · 2022-06

Project Summary The landscape of aging and technology has changed dramatically. Uptake of technology is increasing among aging adults; more researchers are focusing on this topic (many of whom are CREATE progeny); technology is increasingly being considered as a solution for the support needs of aging adults; and more technology products are marketed to seniors. These trends underscore the continued significance of the Center for Research and Education on Aging and Technology Enhancement (CREATE). Deployment of technology in healthcare and day-to-day activities is increasing and advances in technology such as artificial intelligence (AI) are increasingly aimed at supporting older adults. Yet, aging adults are often ignored in design, and robust research evaluating the usability, safety, and efficacy of these systems with older adults is limited. Even among older technology adopters, rapid changes in technology pose challenges in terms of the constant need for adaptation and continual learning. Further, there remains a lag in uptake among many older adult sub-groups, including ethnic minorities, older cohorts, those of lower socio-economic status, those living in a rural location, or with a cognitive impairment such as Mild Cognitive Impairment (MCI) or Alzheimer's Disease/Alzheimer's Disease Related Dementias (AD/ADRD). This application is a request for continued support for CREATE, an established multidisciplinary, cohesive, highly productive, and innovative Center that focuses on aging adults and technology interactions. CREATE’s goal is to ensure that older adults are able to use and realize the benefits of technology. CREATE V has a new vision, expanded research program and teams, and target populations, such as those with MCI, expanded technical expertise, and access to an array of resources, community and clinical partners. Given that age is a significant risk factor for cognitive impairments such as MCI and AD/ADRD, and the criticality of cognition to everyday functioning, a thrust of our planned research is on using emerging technologies to help maintain cognitive health and provide support for those with cognitive impairments. Our Research Program includes three highly integrated cross-site projects focused on: 1) enhancing cognitive health and cognitive and social engagement and preventing cognitive impairment; 2) providing support for adults with MCI and using innovative techniques to assess further cognitive decline such as conversion to AD/ADRD; and 3) providing cognitive support for health-management activities for those with and without cognitive impairments. Our cross- site approach allows us to examine heterogeneity in response to our promising interventions. Our Program will also include an expanded Pilot Research Program to support new research and investigators. CREATE will involve three main sites: Weill Cornell Medicine, Florida State U. and the U. of Illinois Urbana-Champaign. J. Sharit will remain as a Co-Leader (U. of Miami). We will also draw on faculty support from other campuses and institutions. The Center will include Administrative, Data and Technical Development, and Dissemination Cores, External Scientific and Community Advisory Boards, and an Industry Advisory Council.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY Soil-transmitted helminth infections are estimated to infected two billion people worldwide, remaining one of the most neglected groups of infectious diseases and a significant public health and economic challenge. Infected individuals can suffer from malnutrition, growth retardation, impaired cognitive function, anemia and severe immunopathology as a result of chronic inflammation. Immunity to helminth parasites is dependent on type 2 inflammatory responses, characterized by activation of T helper type 2 (Th2) cells, group 2 innate lymphoid cells (ILC2), eosinophils, alternatively-activated macrophages, and B cell production of IgE and IgG. These protective type 2 responses are influenced by genetic and environmental factors, including diet and microbiota-derived metabolites. Therefore, further studies are urgently needed to understand the mechanistic basis of how these factors influence type 2 immune responses to improve strategies to treat and prevent allergic diseases and intestinal helminth infections. The microbiota is the source of various metabolites which can exert their effects at the site of absorption (intestine), as well at distant sites such as brain via bloodstream. Since various dietary components, including dietary fiber, influence the composition of the microbiota and the types of metabolites the microbiota produces, many of the effects of diet on immune cells can be mediated via the microbiota. However, the influence of dietary fiber on microbiota-derived metabolites and their roles in regulating type 2 immunity and inflammation in the context of allergic responses or helminth infection remain poorly defined. Our new preliminary studies in mice employing untargeted comparative metabolomic analyses identified that a high fiber diet drives a significant shift in the composition of the microbiota and remarkable changes in the levels of microbiota-derived metabolites. This metabolic reprogramming was associated with the development of a proinflammatory type 2 immune response, characterized by activation of group 2 innate lymphoid cells (ILC2s), accumulation of eosinophils, and accelerated parasite expulsion in a murine model of helminth infection. Based on these preliminary data, we hypothesize that high fiber diet-induced modulation of microbiota-derived metabolites promotes ILC2-induced type 2 inflammation and immunity to helminth parasite infection. Based on these preliminary data, studies outlined in Aim 1 will test which microbiota-derived metabolites activate ILC2s and trigger eosinophilia and type 2 inflammation to promote anti-parasite immunity. In Aim 2, we will employ chemical and genetic approaches to test how the microbiota-intrinsic bile acid metabolic pathway regulates dietary fiber-induced type 2 inflammation and immunity to infection. Upon successful completion of our proposed aims, we expect to contribute to a fundamentally new understanding of the biology of fiber diet, microbiota-derived metabolites, and ILC2s in regulating type 2 inflammation and anti-helminth immunity.

NIH Research Projects · FY 2026 · 2022-06

Project Summary/Abstract The mitochondrial protein Prohibitin (PHB) is essential for life. Its importance to cellular activities is attested by the fact that deletion of PHB is embryonic lethal in mice and that to date no mutation has been found in the coding region of PHB in any neurological disease conditions, indicating that PHB integrity is essential and that somatic mutation is detrimental. Our recent work has demonstrated that PHB has remarkable neuroprotective potential against ischemic brain injury with an underlying mitochondrial associated mechanism. Its expression is critical for mitochondrial function in stress situations. However, how this important protein that are stable at both mRNA and protein levels, is functionally regulated in neuroprotection, as well as how it is dysregulated in other neurological conditions, remain surprisingly unknown. In exploring the mechanisms of PHB regulation, we discovered that nitric oxide (NO) is required for PHB expression mediated neuroprotection. Therefore, we investigated the interaction between NO and PHB and found that NO modifies PHB post-translationally, through protein s-nitrosylation, a novel regulatory mechanism similar to protein phosphorylation. In this application, we propose to study the effects of PHB S-nitrosylation and the mechanisms underlying functional regulation of PHB by NO. Our central hypothesis is that nitrosylation is critical for PHB’s neuroprotective function and, consequently, disturbances of PHB nitrosylation are detrimental and contribute to pathology. We will use a novel mutant knock-in mouse, in which the sole cysteine residue of PHB protein is mutated so that PHB cannot be nitrosylated, to analyze the mechanisms of NO regulation and the effects of loss of PHB nitrosylation on PHB function, in the settings of brain ischemic injury in association with mitochondria structural alterations. Three specific aims will systematically test the hypothesis. The results of the proposed studies will reveal a previously unrecognized regulatory mechanism of PHB which we believe is crucial to facilitate the design of potential therapies that could ultimately benefit patients at risk of stroke and other neurological diseases.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY A major gap in cancer diagnostics is that state-of-the-art imaging and other existing methods fail to reliably detect low levels of cancer known as minimal residual disease (MRD), which remain following surgical resection of early-stage tumors or treatment of advanced disease. Left untreated, MRD can proliferate and result in lethal cancer recurrence. Hence, there is a critical need to sensitively detect MRD in order to optimize adjuvant therapies or precision immunotherapy. Liquid biopsy offers the ability to noninvasively monitor MRD by detecting circulating tumor DNA (ctDNA) originating from cancer cells. Nonetheless, detection of ctDNA is challenging due to extremely low levels of ctDNA in low-burden disease. The prevailing paradigm argues for deep targeted sequencing of informative loci. However, we have shown that this approach faces fundamental barriers to sensitivity due to the low amount of available DNA in typical plasma samples, which imposes a physical ceiling on depth of sequencing. To overcome this challenge, our interdisciplinary team of geneticists, computer scientists, and oncologists developed MRDetect, an orthogonal approach for ctDNA detection based on genome-wide mutation aggregation of single-nucleotide variants (SNVs) and copy number variants (CNVs) using whole-genome sequencing (WGS) of plasma. MRDetect enables ultra-sensitive MRD detection down to one part in a hundred thousand, and we have demonstrated its ability to detect MRD shortly after surgery or treatment in colorectal cancer, melanoma and non small-cell lung cancer (NSCLC). Our objective in this project is to develop crucial advances that will foster broad-based adoption of this technology across cancer settings. First, we propose to incorporate advanced machine learning (ML) framework known as ‘deep learning’ (DL) into the MRDetect platform to enable SNV identification in plasma WGS in low tumor burden settings (Aim 1). This will yield MRDetect-DL, which we anticipate will significantly improve cancer detection at low tumor levels through a >100-fold improvement in signal to noise enrichment compared to MRDetect. MRDetect-DL performance will be tested in high-risk post-operative melanoma to define the need for adjuvant therapy, as well as in advanced melanoma treated with immunotherapy for precision immunotherapy applications. Critically, MRDetect-DL will obviate MRDetect’s need for a matched tumor sample, ensuring broad adoption across different clinical settings. Second, we posit that in addition to SNV-based advances, MRDetect’s sensitivity can be increased by enhanced detection of CNVs, as these are broadly observed in solid tumors. We propose to develop MRDetect-CNV, an ML-denoising technique to ultra-sensitively detect small CNVs using plasma WGS (Aim 2). We will test MRDetect-CNV on NSCLC plasma samples from patients undergoing neoadjuvant immunotherapy to define its ability to predict treatment response. Impact: Pairing MRDetect-DL with MRDetect-CNV will significantly improve low burden cancer detection in adjuvant, neoadjuvant, and systemic immunotherapy, enabling broad clinical application in oncology.

NIH Research Projects · FY 2025 · 2022-06

ABSTRACT Follicular lymphomas (FL) are germinal center (GC) B-cell derived, slow-growing tumors. Although initially indolent, FLs are essentially incurable with many cases undergoing progression and a relapsing course during which they become increasingly resistant to therapy. Additionally, as many as 45% of cases undergo histologic transformation to an aggressive form of B-cell lymphoma, that is generally refractory to chemo-immunotherapy. Hence there remains a critical unmet need to understand how low-grade FLs survive and are maintained, and to develop rational therapeutic regimens able to prevent disease progression and transformation and eradicate these tumors. The genetic hallmark of FLs include BCL2 translocations and somatic mutations of epigenetic modifier genes such as EZH2. Histologically, FLs typically feature a rich microenvironment, most notably featuring extensive follicular dendritic cell (FDC) networks with dendrites making extensive contact with lymphoma cells. In recent work we showed that the main effect of EZH2 gain-of-function mutations in GC B-cells is to enable them to become less dependent of T-cell help and strengthen their immune synapse formation with FDCs, which induces aberrant proliferation and survival of GC centrocytes and hence formation of FLs and their unique lymphoma-permissive immune niche. It is notable that even though GC B-cells are highly T-cell dependent, FLs are generally resistant to T-cell augmentation therapies such as checkpoint inhibitors. EZH2 mutant GC B-cells do not require T-cell help and are unable to form stable interactions with T-cells that might otherwise suppress these tumors (which might explain checkpoint inhibitors failure). However, we find that EZH2 inhibitors can recruit CD4 and CD8 cells back into these lymphomas, which we propose may represent the major anti-tumor mechanism of this now FDA-approved treatment in FLs. Moreover we have shown that EZH2 inhibitors reduce apoptotic thresholds in primary human EZH2 mutant lymphoma cells and are highly synergistic with BH3 mimetics in vivo and are implementing a clinical trial combining Tazemetostat and Venetoclax for FL and DLBCL patients. Based on these considerations and other preliminary data we hypothesize that EZH2 mutant FLs are dependent on signals received from FDCs, most notably BAFF receptor. We propose that therapeutic targeting of the FDC-FL B-cell immune synapse will yield a lethal blow to FLs, especially when combined with EZH2 inhibitors to restore T-cell anti-lymphoma immunity and BH3 mimetics such as Venetoclax. We expect these treatments to prevent FL progression and transformation. Our goals for this proposal are to determine whether EZH2 mutant FL B-cells depend on FDCs for their survival, whether EZH2 inhibitors act through restoring interactions of FL B-cells with T-cells, and to leverage this information to test novel combination of therapeutic approaches to prevent progression of EZH2 mutant FLs and transformation to aggressive lymphoma.

NIH Research Projects · FY 2026 · 2022-06

Abstract The goals of this project are to determine the molecular mechanisms that control activity of the tandem pore (K2P) family of potassium channels, with a focus on how K2Ps integrate a diverse set of incoming signals to regulate channel function. K2Ps are ion channels primarily responsible for producing background “leak” currents that set cellular resting membrane potential. Modulation of K2P activity directly affects cellular excitability and K2P channels have been implicated to play important roles in cardiac, neuronal, endocrine, and vascular biology. The mechanosensitive subfamily of K2Ps that are the focus of this proposal have been identified as potential drug development targets for treatment of cardiac arrhythmia, depression, and chronic pain, though efforts to develop small molecule modulators that target K2Ps have largely failed to produce high affinity and subtype selective agents. Meanwhile, endogenous lipids are known to modulate many K2P channels and show strong subtype specificity. The overall aim of this proposal is to examine the basic biology underlying K2P modulation by lipids and the related effects of membrane tension, with the long-term goal of using this knowledge to develop a framework for development of improved pharmacology against K2P channels. To achieve these goals, we will pursue a multifaceted approach that includes cryoEM structural studies, native mass spectrometry to define K2P/lipid interactions, and electrophysiological functional studies of K2P behavior. Our first aim will examine the mechanisms by which positive and negative allosteric membrane phosopholipids or free fatty acids are sensed by K2P channels, with a focus on the molecular details of lipid binding sites in the K2P structure. We will also address the basis for the interrelated impacts of allosteric lipids and membrane tension on K2P gating behavior. In aim2, we will explore the mechanism by which lipids, mechanosensitivity, and other K2P input signals control channel output at the ion conducting potassium selectivity filter, defining the molecular connectivity within the structural architecture of the K2P channel. Taken together, our studies will provide a detailed structural model of K2P gating and modulation that is broadly relevant to the basic biology of this important family of channels.

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY Abnormality of the brachial plexus (BP), i.e. brachial plexopathy, can result in profound functional, psychological and economic consequences. Dedicated peripheral nerve MRI, or MR neurography (MRN), is an important adjunct to the physical exam and electrodiagnostic testing to evaluate brachial plexopathies, and influences clinically decision making, including surgical planning, and outcomes. MRN affords direct visualization of individual nerves and their relationship to osseus and soft tissue structures but suffers from insufficient spatial resolution (~1.0mm-isotropic) resulting from poor signal-to-noise ratio (SNR). This is largely due to the inherently concave anatomy of the neck-shoulder junction that precludes close proximity of conventional MRI coils to the skin. The inherent, complex branching and intertwining anatomy of the BP requires higher spatial resolution (~0.5 mm-isotropic) than possible with current radiofrequency (RF) coils. Current RF coils are either rigid or not adequately flexible, and do not conform to the curvatures of the neck, shoulder and axillary regions. We will develop novel, non-toxic, robust liquid metal RF coil technology to enable the design of a conformal and flexible neck-BP array. This design will ensure that coil elements conform to the body contour (to maximize SNR) in their entirety and with the arm in different positions. The characteristics of bendability and form-fitting stretchability are feasible with liquid metal technology, but this technology has not been previously implemented commercially. This project proposes the design and construction of a dedicated RF coil array for brachial plexus MRN, to enable higher spatial-resolution and 3D imaging, with unprecedented detail, in patients with clinically suspected thoracic outlet syndrome (TOS). We will systematically evaluate liquid metal coils against standard coils for BP MRN. We hypothesize that the achievable spatial resolution will be ~0.5 mm isotropic, greater than the ~1 mm isotropic currently achieved with commercial coils, and will therefore better depict regional anatomy and pathology. Impact: The proposed research will not only address TOS but will also facilitate evaluation of (1) other brachial plexopathies and more peripheral neuropathies (of traumatic, inflammatory, iatrogenic etiologies, e.g.), and (2) other complex/curved anatomies including the breast/chest wall region, perineal/groin region, and digits. This technology would also facilitate dynamic imaging of the extremities to elucidate pathology such as femoroacetabular impingement (hip), ligamentous laxity (multiple joints), and meniscal incompetence (knee), not borne out with conventional, static MRI.

NIH Research Projects · FY 2025 · 2022-04

Project Summary The remarkable ability of pluripotent stem cells (PSCs) to give rise to a plethora of fully functional somatic cell types becomes progressively impaired upon ex vivo culture by aberrant gain or loss of DNA methylation marks at essential developmental gene loci. This pervasive epigenetic instability represents a significant hurdle for biomedical applications using PSCs, but the underlying molecular reasons remain almost entirely unknown. An important example for epigenetic instability in PSCs is loss of genomic imprinting, an essential gene regulatory mechanism in mammals. By combining transgenic reporter alleles for imprint stability with mouse genetics, we found that imprint dysregulation in PSCs is caused by identifiable genetic variants that affect trans acting chromatin regulators. The goal of this proposal is to develop a comprehensive molecular understanding on how specific genetic variants affect the stability of vital epigenetic marks in PSCs. To work towards this goal, we will first functionally dissect the causal variants within a genomic susceptibility region that we discovered and which drives locus-specific DNA hypermethylation and loss of developmental potential. This will allow us to pinpoint gene regulatory features that predispose imprinted and other genes to aberrant gain of DNA methylation (Aim 1). We will then use genetic mapping in hybrid PSCs to discover quantitative trait loci that can protect against detrimental DNA hypomethylation and validate the function of associated gene products in both mouse and human pluripotent cells (Aim 2). Finally, we will model the genetics of imprint stability in a panel of PSCs with highly diverse yet well-characterized genomes. This will enable us to dissect the molecular regulation of epigenetic stability in complex genetic backgrounds resembling the human population and help us to establish new biomarkers for the identification of culture conditions that preserve developmental potential in PSCs of unknown genetic background (Aim 3). Our work will establish novel conceptual and mechanistic frameworks for the detrimental epigenetic vulnerability of pluripotent cells, which will aid the safe and reliable use of these cells to study developmental and disease processes. In addition, our findings will have relevance for a wide range of human diseases and developmental syndromes characterized by epigenetic instability.

NIH Research Projects · FY 2026 · 2022-04

αSynuclein (αSyn) plays an important role at the synapse, to maintain neurotransmitter release via clustering synaptic vesicles (SV) and chaperoning SNARE-complex assembly. Aggregation of αSyn is a key pathological feature in multiple age-driven neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease related dementias (ADRD) such as Lewy body dementia. Despite the involvement of βSyn and γSyn in synucleinopathies including Lewy body dementia, Gaucher’s disease, and PD, virtually nothing is known about their physiological functions in the brain. Understanding their function is the first step to finding out how their dysfunction causes PD and Alzheimer’s disease related dementias including Lewy body dementia, and how these diseases can be prevented or delayed. The objective here is to determine the effects of βSyn and γSyn on αSyn’s synaptic function. The central hypothesis is that interaction of βSyn and γSyn with αSyn causes a reduction in αSyn’s activity, leading to reduced SV clusters and SNARE-complex assembly, and altered neu- ronal activity. This hypothesis will be tested in 3 specific aims: 1) Assess the effect of βSyn and γSyn on SNARE-complex assembly; 2) Assess the effect of βSyn and γSyn on SV clustering; and 3) Determine the im- plications of synuclein interactions on SV cycling. Under aim 1, SNARE-complex assembly will be quantified in select brain areas and neurons from mice lacking βSyn and/or γSyn, in heterologous cells and using recombi- nant proteins. Under aim 2, synaptobrevin-2 binding and multimerization of αSyn, and SV pools will be quanti- fied in mice lacking βSyn and/or γSyn and using recombinant proteins. Under aim 3, SV exocytosis and cycling will be quantified in hippocampal brain slices and in neurons from mice lacking βSyn and/or γSyn with or with- out lentivirally increasing βSyn or γSyn levels. This research is innovative because it (1) tests the novel hypoth- esis that βSyn and γSyn affect the synaptic function of αSyn, (2) uses a multidisciplinary approach combining biophysical, biochemical, electrophysiological and whole animal approaches, and (3) analyzes new mouse models lacking βSyn and/or γSyn that were generated from αβγSyn triple knockout mice, that not only enable a direct comparison of the synucleins but may serve as models for synucleinopathies and Alzheimer’s disease related dementias including Lewy body dementia. Our work is significant because it (1) will clarify the im- portance of βSyn and γSyn for neuronal function, (2) will provide new insights into the molecular mechanism underlying SV binding of αSyn, (3) may uncover the contributions of βSyn and γSyn to synucleinopathies and Alzheimer’s disease related dementias including Lewy body dementia, and (4) has translational importance for the targeted development of new treatment strategies for the above-mentioned age-driven dementias aimed at targeting all synucleins instead of focusing solely on αSyn. With synuclein pathology and co-pathologies com- mon to PD and Alzheimer’s disease related dementias including Lewy body dementia, our study has the poten- tial to contribute a mechanistic understanding of the role of the three synucleins in these synucleinopathies.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY Cellular metabolic pathways exhibit remarkable plasticity across different cell types in both development and disease. In addition to accompanying changes in cell state, metabolic rewiring has been shown to drive cell fate decisions programs by altering the chromatin landscape. The deposition of chemical modifications that decorate chromatin requires the intermediates of metabolic pathways, and several enzymes that remove these marks use metabolites as part of their enzymatic reaction. Therefore, fluctuations in metabolite levels have the capacity to shape chromatin to effect cell fate-specific gene expression, but the metabolic changes that drive chromatin reorganization and the enzymes that mediate metabolic control of cell fate during early development remain largely unknown. We have previously identified specific metabolites that control self-renewal of mouse embryonic stem cells (ESCs). Whether metabolism is altered as ESCs exit the self-renewing pluripotent state, and whether these metabolic changes are required for multi-lineage differentiation remains an open question. The goal of this research proposal is to characterize the metabolic rewiring that occurs during exit from naïve pluripotency and to determine the mechanisms by which this rewiring controls mouse ESC differentiation. Our preliminary data indicate that exit from naïve pluripotency is accompanied by an increase in the mitochondrial export of citrate. In Aim 1, we will use genetic and pharmacologic approaches to target the mitochondrial citrate transporter SLC25A1 or the downstream citrate-catabolizing enzyme ATP-citrate lyase to test the hypothesis that mitochondrially-derived citrate is required for early differentiation. We will investigate whether this metabolic change regulates cell fate through the deposition of citrate-derived histone acetylation marks. Preliminary data also shows changes in cellular redox state marked by an increase in the cytosolic NAD+/NADH ratio during early differentiation. In Aim 2, we will determine if this metabolic change is required for exit from naïve pluripotency by modulating the NAD+/NADH ratio using pharmacological or genetic tools. Further experiments will identify the mechanism by which cellular redox state signals to the chromatin landscape to dictate cell fate. These studies will reveal the mechanisms of metabolic control during exit from naïve pluripotency and will provide critical insight into how metabolic regulation contributes to changes in cell identity during embryonic development. The work and training plan outlined in this proposal will be completed in the laboratory of Dr. Lydia Finley with the co-advisement of Dr. Kristian Helin at Memorial Sloan Kettering Cancer Center and will ideally prepare the applicant for further clinical training and a career as an independent physician-scientist.

NIH Research Projects · FY 2026 · 2022-04

Our main objective is to use quantitative susceptibility mapping (QSM) in establishing reliable noninvasive MRI for identification and risk stratification of unstable carotid atherosclerotic plaques. Currently, decisions about carotid revascularization to prevent stroke, such as carotid endarterectomy or carotid artery stenting, are based on whether there is ?50% carotid artery stenosis. However, this strategy uses only one feature of vulnerable plaque and frequently misclassifies patients. Using imaging to identify other features of rupture-prone carotid plaques with high risk for thromboembolic stroke, in combination with stenosis assessment, proves to be a more effective approach for risk evaluation. Of these features, intraplaque hemorrhage (IPH) is associated with a 4 to 6-fold higher risk of stroke, while calcification is associated with a 50% lower stroke risk. In the conventional approach, IPH and calcification are defined as hyperintensity and hypointensity, respectively, in a plaque region on the T1-weighted (T1w) image acquired as part of the multi-contrast MRI (mcMRI) protocol. However, T1w hyperintensity only captures the transient methemoglobin phase of hemorrhage. In the ensuing hemosiderin phase, IPH appears hypointense due to the strong susceptibility-induced dephasing effects of the superparamagnetic hemosiderin (susceptibility>150 ppm), which can be misinterpreted as calcification, although calcification is strongly diamagnetic (-2.3 ppm). The key scientific premise of this proposal is that QSM can reliably resolve T1w hypointensity into IPH hemosiderin versus calcification based on their different magnetic property, and therefore will significantly improve imaging characterization and risk stratification of patients with atherosclerotic carotid plaques. We have pioneered QSM development and demonstrated the exquisite sensitivity of QSM for hemorrhage and calcification in carotid plaque. In this project, we will further improve the utility of carotid plaque QSM for routine clinical imaging by developing a multi-contrast QSM (mcQSM) approach which can provide not only QSM but also traditional mcMRI contrasts in 5 min scan time. We will develop a nonlinear QSM reconstruction algorithm which is robust against noise and motion and can separate co-existing IPH and calcification to improve IPH detection in calcified vessels. We will then establish the improvement in diagnostic accuracy of mcQSM over mcMRI for detecting IPH and calcification in patients who are scheduled for carotid endarterectomy. Finally, we will test the hypothesis that mcQSM will provide significantly higher discrimination for stroke than mcMRI. A successful outcome of this proposal will make carotid plaque QSM ready for widespread and routine clinical use in the emerging era of personalized medicine to reduce the individual and societal burden of stroke.

NIH Research Projects · FY 2025 · 2022-04

ABSTRACT Intensive Care Units (ICUs) are stressful places fraught with grief and potentially traumatic exposures for those witnessing a critically ill family member in pain, struggling to breathe, maintain consciousness, and stay alive. Compounding their distress, family caregivers are often thrust into the position of patient “surrogate,” needing to make life-and-death decisions on the patient’s behalf. We have shown that end-of-life (EoL) decision-making is compromised by elevated symptoms of distressing and disabling grief, resulting in family surrogates often opting for costly EoL care that has consumed a quarter of annual Medicare expenditures, prolonged suffering and exacerbated surrogates’ grief, trauma, and regrets. Prior efforts to address the plight of these family surrogates have proved disappointing, with some significantly worsening surrogates’ psychological trauma. Yet these were not psychological interventions, much less ones using psychological techniques with proven efficacy. To address these shortcomings, we developed a brief, flexibly administered intervention applying empirically supported cognitive-behavioral and acceptance-based techniques. In an R21 pilot, this intervention, EMPOWER (Enhancing & Mobilizing the POtential for Wellness & Emotional Resilience), dramatically reduced experiential avoidance, grief, and traumatic stress, and was associated with higher rates of advance care planning. The proposed multisite, mixed-methods trial will randomize 172 family surrogates to receive EMPOWER (N=86) or a standardized supportive conversation (SC; N=86) delivered via videoconferencing. Surrogate symptoms will be assessed pre-intervention, immediately post-intervention, and 3- and 12-months post-intervention. The primary aim of this study is to compare the efficacy of EMPOWER to SC. We hypothesize that, compared to SC, EMPOWER will yield significantly greater declines in H1a. surrogate grief and posttraumatic stress (primary outcomes) and H1b. experiential avoidance, depression, regrets, and increase patients’ receipt of value concordant care (secondary outcomes). The secondary aim of this study is to contextualize quantitative RCT results with qualitative findings. H2. Qualitative interviews will provide complementary data on perceived barriers to and facilitators of symptom improvement, dissemination, and implementation of EMPOWER in critical care settings. The third aim will explore experiential avoidance as a mediator of intervention effects. H3. Reductions in experiential avoidance will mediate reductions in grief and posttraumatic stress. This study is expected to confirm EMPOWER’s efficacy and enhance understanding of ways to improve its delivery to psychologically vulnerable surrogates. If successful, EMPOWER will address the urgent need for effective interventions for distressed surrogates, preventing adverse mental and physical health outcomes for these vulnerable caregivers as well as poor EoL outcomes among patients, decreasing related healthcare spending.

NIH Research Projects · FY 2025 · 2022-04

Protein glycosylation is essential in all eukaryotes, from disease-causing protists such as malaria, to yeast and mammals. Secretory proteins are N-glycosylated, O- and C-mannosylated, and/or glycosylphosphatidylinositol (GPI)-anchored as they enter the lumen of the endoplasmic reticulum (ER). Yeast that cannot synthesize N- glycoproteins or GPI-proteins are inviable, and mice with the same defects die as embryos. Glycosylation is important in dengue and SARS-CoV-2 viral infections, and defects in glycosylation cause human disease. Thus, deficient O-mannosylation of dystroglycan is a cause of muscular dystrophy and GPI deficiency in hematopoietic human stem cells underlies the hemolytic disease paroxysmal nocturnal hemoglobinuria. Congenital Disorders of Glycosylation (CDGs) are severe inherited diseases with neurological symptoms. Protein glycosylation reactions require the glycolipids mannosyl- and glucosyl-phosphoryl dolichol (MPD, GPD) to act as sugar donors in the lumen of the ER. As these lipids are synthesized on the cytoplasmic side, they must be flipped across the ER membrane to function in the lumen, a process requiring specific transporters, termed scramblases, that have yet to be identified. Assays of the two scramblases in microsomes and reconstituted vesicles, using natural lipids and short-chain analogs as reporters, reveal that transport is bidirectional, ATP-independent, and highly structure specific, discriminating between structural isomers. We will identify the MPD and GPD scramblases using chemo-proteomic and bioinformatic approaches. Deploying novel photo-clickable probes synthesized by the Häner group (University of Bern) we will determine the MPD and GPD interactomes, that we hypothesize will include the scramblases. Our preliminary results validate this approach: the MPD probe functions in ER mannosylation and photo-identifies specific yeast microsomal proteins. Photo-adducted proteins will be identified by quantitative proteomics and tested for scramblase activity in our reconstitution-based assays. Promising candidates will be validated in vivo by evaluating phenotypes of yeast mutants. For GPD scramblase we will also identify candidates via phylogenetic profiling, a bioinformatics method for assignment of protein function. This approach complements the photo- identification strategy and has already yielded a list of GPD scramblase candidates for testing. This is a consequential proposal to discover critical players in ER protein glycosylation. Our extensive experience in studying scramblases puts us in a strong position to tackle this objective. We discovered the scramblase activity of Class A GPCRs and were the first to show lipid scrambling by a TMEM16 ion channel. We now deploy in silico, biochemical and biophysical methods to elucidate their mechanism. We will use this expertise in future work to reveal the molecular mechanism of structure-specific lipid scrambling mediated by the MPD and GPD scramblases that we predict to be distinct from that of the currently known phospholipid scramblases. At a biological level, our discoveries will reveal new genetic loci associated with CDGs.

NIH Research Projects · FY 2026 · 2022-04

ApoE is a lipid transport protein enriched in brain and present in three allelic variants (e2, e3, e4). Homozygosity for the e4 allele (e4/e4) is the main genetic risk factor for Alzheimer’s disease, but ApoE4 carriers also have increased risk for white matter lesions in both vascular cognitive impairment and Alzheimer’s disease and related dementias. Subcortical and periventricular white matter damage is a major cause of age-related cognitive impairment, but the mechanisms remain elusive. Located at the borderzone between separate arterial territories, the deep white matter is highly vulnerable to hypoxia-ischemia. ApoE4 carriers have reduced cerebral blood flow, whereas mice expressing human ApoE4 exhibit a profound disruption of key mechanisms regulating the delivery of blood flow to the brain. These findings raise the possibility that such cerebrovascular dysregulation renders the deep white matter more susceptible to hypoxia-ischemia. Perivascular macrophages (PVM), brain resident myeloid cells closely apposed to the outer wall of cerebral pial and penetrating vessels, can produce ApoE, are enriched in ApoE receptors, and are a powerful source of vascular oxidative stress and inflammation. Therefore, we hypothesize that PVM-derived ApoE4 acts in an autocrine manner on PVM to produce vascular oxidative stress and inflammation, leading to neurovascular dysfunction and increased susceptibility to white matter injury. Since NOX2 is the main source of reactive oxygen species in macrophages and TRPM2 channels are critical for macrophage activation and neurovascular dysfunction, we will also examine their role. We will test the following hypotheses: (a) PVM are both a source and target of the ApoE4 mediating neurovascular dysfunction; (b) PVM TRPM2 channels and NOX2 mediate ApoE4-induced vascular oxidative stress and inflammation leading to neurovascular dysfunction; (c) PVM-derived ApoE4 is responsible for the increased susceptibility to oligemic WM damage through NOX2 and TRPM2 channels. Studies are conducted in young and old mice of both sexes with targeted replacement of mouse ApoE with human ApoE3 or 4. Deep white matter injury is produced in the corpus callosum by bilateral carotid artery stenosis using microcoils. Cutting-edge approaches are used, including 3-photon excited fluorescence to image the deep white matter and a novel mouse model enabling conditional gene targeting in PVM. These approaches allow us to assess microvascular perfusion and damage in the deep white matter in mice with ApoE4, NOX2, or TRPM2 deletion in PVM. These studies will provide insight into the mechanisms underlying the impact of ApoE4 on white matter damage and may unveil new therapeutic targets for one of the leading causes of cognitive impairment and dementia.

NIH Research Projects · FY 2026 · 2022-03

Project Summary/Abstract Female genital schistosomiasis (FGS), caused by the parasitic worm Schistosoma haematobium, affects 40 million girls and women in Africa. Parasite eggs migrate through mucosal tissue, inducing a host immune reaction that leads to erosions and mucosal breaches of the female genital tract with symptoms including genital discharge, bleeding, pain, and infertility. Chronic genital tract damage and symptoms persist after praziquantel therapy in ~70% of women, even though praziquantel effectively kills parasite worms, reduces excretion of eggs in urine, and resolves most tissue pathology in the bladder. In contrast, parasite eggs remain trapped in genital tissue post-treatment where, from autopsy studies, they are known to induce a mucosal immune response characterized by granuloma formation and fibrosis. FGS is a neglected tropical disease and there are important knowledge gaps in our understanding of its cellular and molecular pathophysiology. We do not know the profiles or functions of immune cells that respond to S. haematobium eggs in genital tissue, the effects of FGS on the epithelial cell barrier, and if FGS-related cellular and molecular changes increase susceptibility to viral genital tract infections. The rationale for this proposal is that addressing these knowledge gaps could lead to targeted immunomodulatory, tissue reparative, or viral suppressive interventions to restore damaged genital mucosa. Based on our preliminary data, our central hypothesis is that S. haematobium eggs in the genital mucosa modulate cervical immunity and decrease anti-viral immune cells, cause breakdowns in the epithelial barrier, and increase recurrences of HSV-2, resulting in the morbidity and persistent symptoms of FGS even after praziquantel therapy. To test this hypothesis, we will study 90 women with S. haematobium infection and 90 controls without. Women with S. haematobium will receive praziquantel treatment at baseline and during 12 months of follow up if persistent or recurrent S. haematobium is detected. We will pursue three specific aims: 1) Define the genital mucosal immune cell composition in S. haematobium infection, before and after praziquantel; 2) Determine the molecular mechanisms linked to breakdown of genital epithelial integrity in women with S. haematobium infection; and 3) Quantify the effect of S. haematobium infection on the frequency, intensity, and duration of genital HSV-2 reactivation. In the first aim, we will collect cervical cells by brush and characterize cells by flow and mass cytometry. In the second aim, we will isolate epithelial cells collected by cervical brush and perform RNA-Seq to elucidate genes and pathways specific to epithelial integrity. In the third aim, we will quantify HSV-2 viral shedding over one month in women from the cohort who are HSV-2 seropositive (n=90). In an exploratory analysis, we will also examine the vaginal virome by metagenomic sequencing. The proposed research is significant because it may identify new therapeutic targets for millions of girls and women with FGS. Further, it advances novel studies of parasites and viruses to expand our understanding of interactions between helminths, mucosal immunity, and viral infections.

NIH Research Projects · FY 2025 · 2022-03

PROJECT SUMMARY Inflammatory bowel diseases (IBD), which include both ulcerative colitis and Crohn's disease, are estimated to affect three million individuals in the United States, and the number of people living with IBD continues to rise. Currently available medications are costly, ineffective for some patients, and associated with serious risks including opportunistic infections, hepatic inflammation, pancreatitis, and cancer. Thus, there is an urgent need to improve our understanding of the modulators of intestinal inflammation and repair in order to identify novel therapeutic targets to treat and prevent IBD. The microbiota is the source of various metabolites which can exert their effects at the site of absorption (intestine), and at distant sites such as brain via bloodstream. In this regard, the microbiota mimics an endocrine organ, and its output has only begun to be understood. Since various dietary components, such as dietary fiber, influence the levels of microbiota-derived metabolites, many of the effects of diets on immune cells could be mediated via the microbiota. Although dietary fiber has some anti-inflammatory effects, IBD patients are often instructed to limit their fiber consumption to reduce the frequency and severity of disease flares. However, the mechanism behind dietary fiber-induced exacerbation of IBD-associated symptoms is poorly understood. In new preliminary studies, we identified that a fiber-rich diet activates ILC2s in the colon and significantly increases the levels of eosinophils, a type 2 inflammatory immune cell regulated by ILC2s. The effects of dietary fiber on the eosinophil responses are dependent on the microbiota and are associated with remarkable changes in microbiota-derived metabolites. Furthermore, the high fiber diet increased disease severity in a murine model of intestinal damage and inflammation. Despite these observations, the mechanisms through which dietary fiber regulates the ILC2-eosinophil axis and colonic inflammation remain unknown. Based on our new preliminary data, we hypothesize that microbial metabolites regulate colonic ILC2s and that alterations of these metabolites by a high fiber diet induce pathologic activation of ILC2s and severe intestinal inflammation. We propose to generate a detailed understanding of how microbial metabolites influence inflammatory pathologies in murine models of intestinal inflammation and examine these pathways in human IBD patient samples. In Aim 1, we will determine what microbial metabolites and host metabolite receptors are involved in the high fiber diet-induced type 2 inflammation and their role in intestinal damage and inflammation. In Aim 2, we will employ a novel CRISPR-based microbial gene-editing technique to directly test the microbial metabolic pathways through which a high fiber diet mediates intestinal inflammation. In Aim 3, we will translate these findings to human disease and determine how alterations in microbial metabolites and the ILC2-eosinphil axis correlate with clinical and endoscopic measures of IBD disease activity. In addition to uncovering novel immunoregulatory mechanisms of diet and microbiota and their unique roles in IBD, these studies will provide preclinical justification for development of novel therapeutics to target this pathway.

NIH Research Projects · FY 2025 · 2022-03

PROJECT SUMMARY/ABSTRACT: When we navigate a new environment, we have an internal understanding of where we are located in that environment. To build this internal sense of space, our brains integrate over time our estimate of how far we are walking and in which direction. I propose to study how brains integrate sensory information over time to form a working spatial memory. This question is pertinent to human health because impairments of spatial working memory are hallmarks of neurological and psychiatric disorders, like Alzheimer's disease and schizophrenia. A more detailed understanding of the mechanisms underlying spatial cognition should ultimately lead to more rational treatments for these conditions. I will study neural integration in the Drosophila central complex, an emerging model for spatial navigation that has notable similarities with the mammalian navigational system. Building on recent work that has characterized neural signals that track the direction and speed in which a fly travels, I will analyze central complex cells for processes that might integrate such signals into a working memory of a fly's position in space. The long-term objective of this project is to build a cellular- and circuit-level understanding of how neural signals are integrated over time to form working memories of spatial variables.

NIH Research Projects · FY 2026 · 2022-03

Abstract Immune cells at the maternal/fetal interface play dual roles of orchestrating immune tolerance required for pregnancy maintenance, while also protecting against placental pathogens, such as cytomegalovirus (CMV). Specialized inhibitory immune cells, known as natural killer (NK) cells, are predominant and dynamic immune cells populating the decidua throughout gestation. Yet, there is a major gap in our understanding of the role of NK cells in protection of the fetus against immune rejection and pathogen invasion. The role of NK cells, including recently identified cytomegalovirus (CMV)-specific memory NK cells, in the interplay between fetal tolerance and protection against congenital CMV transmission is poorly defined. This study aims to define the role of maternal NK cell populations in regulation of fetal tolerance and protection against placental CMV transmission in the setting of chronic maternal CMV infection. Our group is uniquely equipped to dissect the immunologic functions at the maternal-fetal interface with significant expertise in both a nonhuman primate (NHP) model of pregnancy and placental CMV transmission as well as investigations of immune cells at the maternal-fetal interface in human placenta and cord blood. The NHP model affords the opportunity to study the immunology and physiology of pregnancy across the gestational stages through elective fetal harvest and access to the immune cells at the maternal-fetal interface, as well as validated strategies for peripheral and tissue depletion of effector NK cells in vivo. We hypothesize that maternal NK cells are critical to both pregnancy maintenance and prevention of CMV reactivation in early pregnancy. Understanding this intersection between maintaining immune tolerance while protecting against placental pathogens is critical to developing immune-based strategies to reduce the morbidity and mortality resulting from adverse pregnancy outcomes.