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NIGMS - National Institute of General Medical Sciences

PROJECT ABSTRACT: The mechanism and regulation of protein synthesis determines the diversity and capacity of the cellular proteome. At the center of this regulation is the ribosome - a megadalton RNA-protein complex composed of two-subunits – which integrates a wide variety of cellular signals. The ribosome’s exquisite sensitivity to regulatory cues is underscored by the fact that the majority of clinically used antibiotics exert their effect by either dysregulating or blocking specific aspects of the protein synthesis mechanism. Understanding the kinetic and structural basis of protein synthesis promises to elucidate core paradigms of gene expression control and to inform strategies for addressing the global threat posed by drug-resistant and emerging pathogens. Moreover, given that loss of translational control is a hallmark of cancer, a mechanistic understanding of ribosome function holds significant promise for developing novel small-molecule therapies, which are currently lacking in the treatment of human disease. Historically, investigations into structure-function relationships governing the protein synthesis mechanism have focused on bacterial systems approaches. Comparable studies in human systems have been hindered by the demand for large amounts of homogeneous protein synthesis machinery. As a result, the molecular distinctions between bacterial and mammalian protein synthesis - which underpin antibiotic specificity and potential therapeutic windows – remain obscure. Current evidence suggests that the elongation phase of protein synthesis, during which messenger RNA (mRNA) is decoded into protein, is the most time consuming, physiologically regulated and small-molecule sensitive. We and others hypothesize that elongation is particularly susceptible to regulation because it involves transient, repetitive interactions of the ribosome with auxiliary factors, coordinated through finely tuned conformational transitions that are acutely sensitive to perturbation. Small effects on many individual steps compound to exert large impacts on protein production. The proposed research seeks to rigorously and robustly define and quantify the elemental reactions underpinning the elongation phase of protein synthesis in bacteria and human. Our objectives are to: 1) elucidate the conserved mechanistic principles and divergent features of protein synthesis across evolution to define paradigms for selective control; 2] identify novel strategies for more effective antibiotic interventions for the treatment of infectious disease; and 3] evaluate the therapeutic potential of targeting dysregulated translation in cancer. To achieve these goals, we will deploy a suite of advanced biophysical methodologies - including single- molecule fluorescence imaging and state-of-the-art cryo-electron microscopy - to establish comprehensive and integrated kinetic and structural frameworks defining the elemental steps of elongation in bacteria and humans. The insights gained will advance our understanding of translation control, reveal atomic-resolution descriptions of drug actions on bacterial and human ribosomes, and inform new strategies for improving the efficacy of clinical treatments for both microbial and human disease.

Imminent Mode

Vermillion is organizing a one-day art show event called: IMMINENT MODE: FAST FORWARD. The event will take place on August 8, 2019, it will be located at 1508 11th Ave. It will take place during the Capitol Hill Artwalk. The event will bring local artists together and is free and open to all.

NHLBI - National Heart Lung and Blood Institute

ABSTRACT People with HIV (PWH) are at a greater risk for chronic lung diseases compared to the general population, even with effective antiretroviral therapy (ART) and controlling for smoking and other risk factors. Diffusing capacity for carbon monoxide (DLCO) measures the ability of the lungs to transfer gas from inspired air to red blood cells. A reduced DLCO is the most common abnormality on pulmonary function testing (PFTs) in PWH and is associated with increased respiratory symptom burden and mortality. Our preliminary results show that 1) Using NanoString technology measuring 945 unique genes, a list of interferon pathway and inflammation genes are upregulated in PBMCs from PWH with impaired DLCO. 2) In spectral flow cytometry, cell surface expression of the Interferon γ -induced protein 10 (IP-10) receptor CXCR3 is lower in subsets of CD4 T cells and all CD8 T cell subtypes in PWH with impaired DLCO, including impaired DLCO with normal spirometry, a phenotype which we have termed iso↓DLCO, suggesting compromised type I T helper (Th1) CD4 function and CD8 T maturation defects. 3) In single cell RNAseq analysis, interferon α and γ pathways are upregulated in CD4 and CD8 cells, while TNF α signaling pathway is downregulated in CD4, CD8 and monocytes in PWH with impaired DLCO, suggesting immune exhaustion. 4) In bulk RNAseq analysis of cells from bronchoalveolar lavage (BAL), impaired DLCO is associated with an enriched oxidative phosphorylation pathway, potentially reflecting immune activation. 5) DLCO is positively correlated with telomere length in monocytes, suggesting that accelerated cellular aging/immune senescence in this specific cell type is associated with impaired DLCO. Taken together, these preliminary results support our central hypothesis that in PWH, immune exhaustion and senescence lead to defective immune functions and these defects contribute to impaired DLCO. These results lead us to propose that immune functional defects in circulating PBMCs (immune exhaustion, immune senescence, compromised Th1 function, CD8 differentiation defects, downregulation of TNFα pathway) lead to a systemic immune environment more permissive to fibrosis and endothelial dysfunction as mechanisms of impaired DLCO. We further propose that CD4, CD8 T cells and monocyte-derived macrophages (MDM) in BAL will have similar defects, and alveolar macrophages (AM) will have immune defects that decrease the ability to clear pathogen and toxin and promote pulmonary fibrosis and endothelial dysfunction. The specific aims are: Aim 1: To test the hypothesis that elevated levels of biomarkers of immune exhaustion in CD4, CD8 T cells and immune senescence in CD4, CD8 T cells and monocytes in PBMCs are associated with impaired DLCO, particularly iso↓DLCO. Aim 2: To test the hypothesis that CD4, CD8 T cells, and monocytes in PBMCs from PWH with impaired DLCO, particularly iso↓DLCO, have ex vivo functional defects. Aim 3: To test the hypothesis that biomarkers of exhaustion, senescence, and immune functional defects are associated with decline in lung function longitudinally (at 2 years). Both Aims 1&2 have a sub-aim to measure BAL samples to compare systemic vs. lung immune defects.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

Abstract The primary purpose of this Ruth L. Kirschstein NRSA F30 application is to provide the groundwork that will prepare the applicant for an academic medical career. More than 10% of the population in the United States meet the criteria for alcohol use disorder, and excessive alcohol use is one of the leading causes of preventable deaths. Specifically, alcohol-associated liver disease (ALD) is a leading cause of morbidity and mortality in people with alcohol misuse, and ALD-related deaths are projected to continue to rise 4.7% annually. Alcohol misuse causes significant alterations to the immune system that contribute to the development and progression of ALD. While the effects of alcohol on innate immune cells like Kupffer cells are well described, the role of adaptive immune cells on liver pathogenesis is not clear. Previous studies in the laboratory indicate that chronic alcohol treatment does not increase lipid content in hepatocyte spheroids. These results and published data suggest that cross talk with other cell types significantly contribute to lipid accumulation in hepatocytes, which is the first stage of ALD. Additionally, published work from the group demonstrate that alcohol increases differentiation of CD4 T cells to proinflammatory Th1 cells. Alcohol misuse increases inflammatory signaling from immune cells, including extracellular vesicles (EVs), microRNA-155 in EVs, and proinflammatory cytokines, which can contribute to hepatocyte injury. miR-155 targets lipid and inflammatory genes and has been implicated in ALD. Likewise, proinflammatory cytokine interferon gamma (IFN-g) is cytotoxic and contributes to bioenergetic dysfunction, reactive oxygen species production, and cell death of hepatocytes. Preliminary data generated for this proposal indicate plasma IFN-g significantly predicts AST/ALT ratios in people with recent alcohol misuse. Additional results suggest that coculturing HepaRG hepatocyte spheroids with naïve stimulated CD4 T cells or IFN-g significantly increases hepatocyte cell death, which is further increased in ethanol-treated spheroids. Taken together, preliminary data and published literature provide evidence for alcohol and T cell-mediated hepatocyte damage leading to end-organ injury. Thus, the overall hypothesis for this project is that alcohol misuse increases CD4 T cell secretion of both miR-155 in extracellular vesicles (EVs) and IFN-g that contribute to hepatocyte injury. The proposed study will employ a variety of state-of-the-art techniques to test the hypothesis that alcohol misuse (1) increases miR-155 in CD4 T cell-derived EVs, and (2) increases IFN-g secretion from CD4 T cells, which increases markers of cellular injury in hepatocyte spheroids. With a strong mentoring team dedicated to developing a well-rounded physician scientist, completion of the proposed training plan in alcohol misuse-related research will ensure that the applicant is equipped for a successful career in academic medicine.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT The UCSF Immunology Training Program (ITP) encompasses 49 laboratories engaged in molecular, cellular, and tissue immunology research and actively training ~215 graduate student and postdoctoral scientists. Of these trainees, 5-6 students per year (3 per cohort) and 2-3 postdocs per year from a range of educational backgrounds will be selected for the ITP’s T32-supported Immunology Leadership Path. Areas of ITP research encompass the full breadth of contemporary immunology including innate immunity; lymphocyte selection and differentiation; cytokine biology; immune cell signaling and migration; mechanisms of autoimmunity, allergy, and inflammatory diseases; tumor immunology; immune cell engineering; and immune defense against infectious agents. The ITP is designed to provide a strong background in genetics, cell biology, tissue biology, and quantitative approaches in biology, as well as deep training in modern immunology. The interdisciplinary nature of this training is enhanced by the tight affiliation of the ITP with the Biomedical Sciences (BMS) Program, a graduate program that also includes the study of other aspects of mammalian tissue/organ development, function, and disease. In addition to formal coursework and research training, the ITP includes a weekly immunology seminar series of outside speakers, a student-postdoc-faculty journal club, an annual immunology retreat (held jointly with UC Berkeley immunologists), and courses on advanced immunological topics. Postdoctoral training is additionally enhanced by a research-in-progress series. Students and postdocs receive extensive formal and informal training in methods for enhancing rigor and reproducibility and in the development of skills in evaluating, proposing and communicating research. The program fosters access to experiential training in a breadth of immunology-related career paths. Faculty receive regular training in mentoring practices to promote student and postdoc success. The ITP works in partnership with the UCSF ImmunoX Initiative that uses incentives for collaborative research, state-of-the-art technologies, and a unique data-sharing model to help basic immunologists and clinicians work together toward solutions to immunological diseases. The ITP’s Immunology Leadership Path will provide a select group of ITP students and postdocs with enhanced training in multiple areas, including support for attending meetings, hosting of invited speakers, in-depth feedback on oral and poster presentations, a postdoc secondary mentor program, a near-peer mentor program, a grant writing workshop, a semi-annual meeting that includes alumni presentations, increased teaching opportunities, and greater job search support. Selection for appointment to the Immunology Leadership Path is conducted through an application at the beginning of the 2nd year for students or early after arrival for postdocs, and deep engagement with the T32 program continues throughout their time at UCSF. The enhanced training environment enabled by the T32 will equip a select group of ITP graduate students and postdoctoral fellows with the knowledge and skills to become future leaders in the field of immunology.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT People living with Human Immunodeficiency virus (HIV) are up to 20 times more likely to develop active tuberculosis (TB) than people without HIV (PWoH). While anti-retroviral therapy (ART) has increased their health and lifespan, people with HIV (PWH) on ART are still at 4-5 fold greater risk for developing TB following infection with Mycobacterium tuberculosis (Mtb) compared to people without HIV (PWoH). However, why viral suppression and CD4 reconstitution following ART do not fully abrogate the risk of TB remains unclear. Residual inflammation present in PWH on ART has been linked to increased risk of co-infections and comorbidities. In addition, several lines of evidence show that PWH on ART have alterations in cellular metabolism, mainly within myeloid compartments, suggesting compromised abilities to mount effective immune responses to pathogens. A population of immunosuppressive cells termed myeloid-derived suppressor cells (MDSCs) has been implicated in dysfunctional immunity to HIV and Mtb infection. MDSCs were first described in cancer as negatively regulating tumor environments and suppressing NK and T cell functions. Higher frequencies of monocytic MDSCs (M- MDSC) were reported in PWH compared to PWoH, and M-MDSCs have been shown to expand after interruption of ART. Interestingly, M-MDSCs are also induced following Mtb infection but their role in anti-TB imunity in PWH on ART are not understood. We hypothesize that M-MDSCs suppress T cell functions and promote dysregulated immunometabolic responses to Mtb in PWH on ART. We propose to leverage bio-banked cryopreserved PBMC samples available through the Emory Center for AIDS research (CFAR) and our own archived samples, including from people with active and latent TB, with and without HIV. In Aim 1 we will employ multi-omic and immunologic approaches to derive an integrated MDSC biosignature associated with their suppressive functions and evaluate MDSC signature and frequencies in ART-naïve PWH, PWH on ART and PWoH. In Aim 2 we will test the hypothesis that expansion of M-MDSCs in PWH on ART contributes to ineffective immunity against Mtb-infection and promotes immunometabolic dysregulation. Our studies will bridge major knowledge gaps and provide a framework for targeting MDSCs to improve ART and advance HIV cure.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary The goal of this application is to request partial support for the 2026 Science Research Conference (SRC) on “Immunoreceptors and Immunotherapy” sponsored by Federation of American Societies for Experimental Biology (FASEB). This successful SRC has been running every two years since 1987 and has fostered a highly interactive community of international researchers with the goal of solving important problems in immunoreceptor and immunotherapy biology. The meeting in 2026 will be the 16th iteration of this highly successful series. Immunoreceptors play a central role in cancer, autoimmunity and infectious diseases, whilst immunotherapy is the rapidly expanding field focused on stimulating the immune system to fight cancer and other inflammatory diseases to change people’s lives in the clinic. Key highlights of this conference are: (1) A meeting program that is composed of the best science related to immune receptor function, immune cell signaling, and the physiology and pathology of immune diseases; (2) Addition of emerging topics in inhibitory receptors and antibody engineering, along with immunotherapy in cancer and autoimmunity, and organization of immunoreceptors on the membrane; (3) Speakers who are leaders in the field as well as early- and mid-career investigators, selected to feature cutting-edge aspects of immunoreceptor and immunotherapy biology, covering the range from basic science to translational applications; (4) A program that also includes state-of-the-art technologies in imaging, structural analysis, computational modeling, systems biology and therapeutic approaches; (5) Networking opportunities, including a “Meet the Expert” luncheon and Career Workshop to foster connections between junior and senior investigators; and (6) Financial support for trainees and early career investigators to attend and present at the conference. As such, this meeting brings together scientists from different fields, creating a dynamic and interdisciplinary environment for building networks and developing new collaborations. The FASEB SRC provides an ideal format for this meeting, because interactions between participants are guaranteed by the size of the meeting, an all-plenary format, multiple poster sessions and shared meals. The support requested through this application will enable the most promising trainees and early career faculty to share their work and interact with leaders in their field. With these features in mind, our specific aims are: Aim 1 – to disseminate the most recent advances and new paradigms in immunoreceptors and immunotherapy; and Aim 2 – to encourage intensive scientific discussion that promotes new collaborations among the international community engaged in immunobiology research. This meeting has received enthusiastic feedback from attendees of previous meetings, establishing a strong mandate for its continuation.

NIA - National Institute on Aging

PROJECT SUMMARY Pelvic organ prolapse (POP) is an age-related disorder that affects up to 50% of women impacting quality of life and resulting in exorbitant healthcare costs. On a cellular level, the process of aging is termed cellular senescence and it has been associated with degenerative and age-related diseases. Despite current studies suggesting a link between POP and cellular senescence there is a lack of understanding of how senescence- associated changes contribute to the development of prolapse. In this application we will test the hypothesis that induction of cell senescence culminates in POP during aging. To accomplish this, we will use state-of-the art methodologies (single-cell and single-nuclei RNA sequencing; biomechanical phenotyping considering both contractile and non-contractile properties) to reveal new facets of cell senescence signaling pathways in the vagina with age and known development of prolapse. We will employ a translational approach that combines both human research and animal models, which will allow for longitudinal follow-up that is not feasible in humans. Our specific aims are to (1) identify the differential gene expression and extracellular matrix regulation of age- related POP using an animal model, (2) determine if vaginal contractile potential and biomechanical stability are rescued in a novel mouse model of prolapse and cellular senescence, and (3) link extracellular matrix composition with cell senescence in vaginal stromal tissues from women with and without prolapse. The research proposed here will help define the mechanism of cell senescence in initiating the path of loss of pelvic organ support during aging. Identification of cellular pathways and unique cell populations associated with the development of prolapse can result in the design of interventional strategies that can prevent or slow the progression of cell senescence and in turn prevent development of POP. Successful execution of the aims of the proposed study will transform pelvic floor medicine and inform other aspects of the biology of aging.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY Most HIV infections are initiated at mucosal surfaces, including the oral cavity, where transmission can occur through breastfeeding or oral–genital contact. Despite the effectiveness of antiretroviral therapy (ART), HIV persists in infected individuals as integrated, latent proviruses within long-lived reservoir cells, representing a major barrier to viral eradication. The tonsil is a secondary lymphoid organ that serves as a first line of immune defense against pathogens entering through oral and nasal routes. HIV-associated immune dysregulation is thought to compromise lymphoid tissue function, including that of the tonsils, leading to impaired antibody production, T cell dysfunction, cytokine imbalance, autoimmunity, and increased susceptibility to viral co- infections. Kaposi sarcoma–associated herpesvirus (KSHV), a human gamma-herpesvirus, is frequently co- infected with HIV and is a major contributor to AIDS-related malignancies. The oral cavity is a primary site of KSHV replication, where high levels of infectious virus are produced and shed in saliva, facilitating transmission through the salivary–mucosal route. B lymphocytes serve as the principal reservoir for lifelong KSHV persistence. Accumulating evidence suggests that HIV infection enhances oral acquisition of KSHV, promotes its persistent infection, and increases the risk of KSHV-associated tumorigenesis; however, the underlying immune mechanisms remain poorly defined. Recently, human tonsil organoids have emerged as highly physiologically relevant in vitro models for studying viral infection, immune responses, and vaccine evaluation. These three-dimensional culture systems preserve tissue architecture and immune cell diversity, offering significant advantages over traditional models while reducing reliance on animal studies. In this exploratory R21 project, we will leverage human tonsil organoid cultures to investigate how HIV-induced immune dysregulation impacts oral infection by the oncogenic gamma-herpesvirus KSHV. Specifically, we will establish chronic HIV infection in tonsil organoids and comprehensively characterize HIV-driven alterations in immune cell composition and function (Aim 1). In parallel, we will examine how HIV modulates KSHV co-infection within tonsils, with a focus on previously underappreciated immune mechanisms, including natural killer (NK) cell–mediated immunosurveillance (Aim 2).

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Antiretroviral therapy (ART) effectively suppresses HIV replication but fails to eliminate the viral reservoir—a heterogeneous population of infected cells that persist despite treatment. While most proviruses are defective, many retain the ability to express RNA, proteins, and even viral particles, contributing to persistent viremia, chronic immune activation, and complicating the interpretation of viral load measurements. Among these, proviruses with defects in the 5’ leader (5’L) region are common, and they decay more slowly than intact proviruses, are translation-competent, and are often implicated in persistent non-suppressible viremia (NSV), making them a critical component of HIV persistence and a significant source of residual antigenic stimulation. The long-term goal of this project is to define the mechanisms by which 5’L-defective proviruses evade negative selection, characterize their contribution to persistent viremia, and determine their impact on HIV-specific immune responses. Our central hypothesis is that 5’L-defective proviruses retain the ability to express viral genes while evading immune-mediated killing and cytopathic effects. To test this, we will (1) apply a novel digital PCR assay that distinguishes intact from 5’L-defective RNA without requiring sequencing to quantify their contribution of defective proviruses to plasma HIV RNA; (2) isolate and phenotype clonally expanded CD4+ T cells harboring 5’L-defective proviruses using two complementary approaches—stimulation with Tat-LNP followed by p24+ cell sorting, and limiting dilution culture ex vivo—to assess viral transcription and translation through multi-omic analyses (SMARTseq, Ribo-seq); (3) investigate susceptibility of 5’L defective proviruses to HIV-specific CTL, and leverage longitudinal samples from individuals with NSV to evaluate how antigen expression from defective proviruses shapes HIV-specific immune responses and clonotype dynamics over time. This research is innovative in developing a first-in-class assay to parse 5’L-defective RNA without sequencing and applying cutting-edge single-cell and immunologic profiling to study the interplay between defective proviral expression and host immunity. The proposed research is significant because it is expected to address critical gaps in our understanding of HIV persistence, improve the interpretation of HIV cure clinical trials, and inform new strategies aimed at targeting cells with intact and defective proviruses. Ultimately, this project has the potential to offer new tools to improve the clinical outcome of people living with HIV and opportunities for the development of innovative strategies to achieve HIV remission.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Significance: Providing antiretroviral therapy (ART) to pregnant women with HIV (WHIV) is a landmark global public health achievement, preventing millions of perinatal HIV infections. However, there are now >1 million children exposed to antiretrovirals (ARVs) born annually, a number expected to stabilize or increase over the next decade as WHIV and women exposed to HIV during conception and pregnancy are increasingly taking ARVs as treatment or prophylaxis against HIV (PrEP). To date, the effects and safety of ARVs taken in pregnancy are not fully established and prior research is limited by lack of objective measurement of ARV exposure, and data are lacking on the effects of ARVs on the placenta. Thus, there is a critical gap in knowledge about the impact of ARVs, including PrEP, and association with objective drug levels taken in pregnancy on the placenta and fetus, information needed for optimal ARV design and to advise women on the effects of ARVs taken as treatment or prevention during pregnancy to inform risk-benefit discussions. Innovation: We propose one of the first studies to simultaneously measure ARV levels in dried blood spots from pregnant women and their children’s hair to quantify drug exposure to PrEP and ART in utero and relate ARV levels to placental findings and birth weight. Distinct advantages of our proposed research over prior studies include 1) simultaneous collection and comparison of placentas from WHIV taking ART, HIV-uninfected women taking ARVs as PrEP, and HIV- uninfected women taking no ARVs, and 2) prospective enrollment and observation of pregnant women and children from these three groups to minimize bias, enhance rigor and reproducibility, and relate placental and birth outcomes to in utero exposures. Investigator team: PI Bebell has expertise in HIV epidemiology in pregnancy and placental effects. Co-I Ngonzi has expertise with HIV and pregnancy outcomes in Uganda. Biostatistician Correia has expertise in analyzing data from observational maternal-child outcomes studies in HIV-affected populations and mediation analysis. Approach: We will leverage stored dried blood spot and hair samples from the PI’s ongoing NIH-funded (R01HD112302) PACO cohort in Uganda, clinical and placental histopathology data from enrolled women and their children, established laboratory infrastructure at UCSF’s Hair Analytical Laboratory and AHRI’s pharmacology laboratory to elucidate the independent effects of HIV and ARV exposure on the placenta and birth weight through these Specific Aims: 1) Compare histologic placental abnormalities by ARV levels in neonatal hair and maternal DBS, and 2) Determine the effects of ARV exposure on birth weight and whether placental abnormalities mediate these effects. Determining the impact of ARVs in pregnancy on the placenta and birth weight and the possible mediating role of the placenta has great potential to improve child health through optimizing outcomes and inform choices for women accessing ARVs as treatment and prevention during pregnancy. By leveraging already-collected samples and data from an NIH-funded study, this proposal will inform practical strategies to improve ARV-related outcomes.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

Project Summary Highly effective antiretroviral therapy (ART) has significantly reduced the mother-to-child transmission of HIV but has also increased the number of children who are HIV exposed uninfected (HEU) and exposed to ART during gestation. Current national and international ART prescription guidelines now recommend maternal use of integrase strand transfer inhibitor (InSTI)–based regimens throughout pregnancy. Importantly, while InSTI use is associated with fat tissue expansion and impaired bone mass in adults and adolescence with HIV, the consequences of in utero exposure to InSTIs on offspring fat and bone development remain unknown. A key barrier to our understanding of these effects is the recent adoption of InSTI use during pregnancy, which limits the number and age of children exposed to InSTIs available for study. Therefore, to address this gap, our laboratory utilized a preclinical mouse model of gestational exposure to maternal InSTI-based ART and found long-lasting changes in offspring weight and bone length, suggesting that early InSTI exposure can disrupt normal developmental pathways. Building on these findings, the proposed study will investigate the mechanisms underlying impaired fat and bone development following exposure to maternal InSTI-based regimens. Due to the early exposure and the long-lasting tissue changes, we hypothesize that maternal and lactation exposure to InSTI-based ART causes epigenetic alterations associated with adverse fat and bone development in HEU children. To test our hypothesis, we will use our preclinical mouse model to investigate the longitudinal impact of various InSTI-containing regimens on key offspring development stages and evaluate the tissue level mechanisms, including epigenetic changes, associated with structural (mass) changes. To ensure clinical relevance, we will also leverage data and samples from the Pediatric HIV/AIDS Cohort Study (PHACS) and the International Maternal Pediatric Adolescent AIDS Clinical Trials Network (IMPAACT) to evaluate whether epigenetic changes in HEU children differ between InSTI-based and InSTI-sparing regimens and are dependent upon the antiretrovirals used in combination with InSTIs. If successful, results from this proposal will inform safer ART prescribing during pregnancy and guide strategies to optimize lifelong health among children exposed to HIV and ART in utero.

NIEHS - National Institute of Environmental Health Sciences

PROJECT SUMMARY Micro- and nanoplastics (MNPs) are emerging as a ubiquitous and persistent environmental contaminant. Human exposure to MNPs is widespread, with ingestion being the main exposure route. Research addressing the potential impact of MNPs on human health is urgently needed. MNPs can reach and accumulate in the heart. However, the impact of MNPs on the heart is very poorly understood. Notably, a recent epidemiologic study has shown that higher exposure to MNPs is associated with increased cardiovascular events in human patients. This new evidence highlights the potential cardiovascular toxicity of MNPs in humans and the critical need to understand the effects of MNPs exposure on the heart. In preliminary studies in human inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and/or rats, we found that exposure to MNPs caused cardiac toxicity including reduced cardiac myocyte viability, increased reactive oxygen species (ROS), and decreased left ventricular mass. Remarkably, in rat exposure studies, we found that MPNs exposure significantly increased myocardial infarction size and cardiac tissue damage following cardiac ischemia injury. Supported by compelling preliminary results, we propose to address the central hypothesis that exposure to environmental MNPs causes mitochondrial dysfunction and oxidative stress in the heart, leading to increased susceptibility of the heart to damage; such cardiac toxicity is manifested as worsened infarction and heart dysfunction following ischemia injury (ie, heart attack). The proposed study will be carried out by an interdisciplinary team that comprises researchers in cardiac toxicology, chemistry, clinical cardiology, and biostatistics. The study uses both an in vivo rat model and human iPSC-derived cardiomyocytes and human cardiac organoid models, and are of strong relevance to human heart health. Importantly, taking advantage of our breakthroughs in producing “true-to-life” MNPs that mimic real-life environmental MNPs, we will use such “true-to-life” MNPs in the entire study, making the study highly relevant to real-life environmental MNPs exposure. In whole animal exposure studies, internal MNPs exposure levels and tissue distribution will be analyzed using state-of-the-art analytical chemistry approaches. Three aims are proposed. Aim 1 examines the impact of MNPs on cardiac physiology and function; Aim 2 addresses the impact of MNPs on cardiac damage and adverse outcomes following ischemia injury; Aim 3 examines the mechanism underlying MNPs-induced cardiac toxicity, focusing on the autophagy-lysosome pathway and mitochondria dysfunction. The proposed studies are significant because they are expected to provide critical knowledge on MNPs-induced cardiac toxicity in human-relevant experimental models, thus having strong environmental health significance. Further, the studies will contribute to our recognition of the role of MNPs in affecting the outcomes of heart attack - a top cause of death and morbidity in the US, thus having strong clinical and translational impact.

NINDS - National Institute of Neurological Disorders and Stroke

Project summary Despite the success of antiretroviral therapy (ART) in extending the life expectancy of people with HIV (PWH), nearly half of this population continues to experience chronic HIV-related conditions, including HIV-associated neurocognitive disorders (HAND). HAND persists even with effective viral suppression and is recognized as a chronic neurological condition. Emerging evidence suggests that the gut microbiome and its metabolites play key roles in shaping host immunity and neurological health in PWH. However, our understanding of the microbial metabolites involved in HAND pathogenesis remains limited due to limited functional studies and incomplete annotation of the metabolome. During my postdoctoral research, I identified thousands of previously uncharacterized N-acyl lipids and bile acids–classes of microbially derived molecules that are significantly altered in PWH and individuals with HAND. However, due to the widespread use of ART in human cohorts, it is still unclear whether these molecular changes are driven by HIV, ART, or their interaction. Moreover, their microbial producers and biological functions in the context of HAND remain largely unknown. Therefore, the objective of this proposal is to investigate the functional roles of microbial metabolites in HAND by integrating metabolomics, metagenomics, in vitro and in vivo assays, and computational approaches. In Aim 1, I will use the HIV-1 transgenic (HIV-1Tg) rat model to determine how ART affects the metabolome, microbiome, behavior, and tissue distribution of microbial metabolites, particularly N-acyl lipids and bile acids. In Aim 2, I will evaluate the neuroinflammatory potential of N-acyl lipids and bile acids using stem cell–derived microglia (iPSC-MG), identify their microbial sources by culturing a synthetic gut community (111 strains) and individual strains, and integrate multi-omic data to establish microbe-metabolite correlations in different cohorts. Finally, in Aim 3, I will expand this approach to discover additional classes of microbial metabolites relevant to HIV and HAND and build a publicly accessible, web-based functional atlas to systematically catalog microbial metabolites associated with HIV and HAND. Together, these aims will address a critical knowledge gap in neuroHIV by defining specific microbial metabolites that contribute to HAND and providing tools for biomarker discovery and mechanistic insight. During the K99 phase, this work will be conducted at the University of California San Diego, where I will receive training from Dr. Pieter Dorrestein (expert in metabolomics and data mining), Dr. Rob Knight (expert in microbiome research), Dr. Karsten Zengler (expert in microbial communities), and Dr. Ronald Ellis (expert in neuroHIV), along with a team of multidisciplinary collaborators with large expertise in the in vivo and in vitro experiments proposed. Their combined expertise and strong record of transitioning postdoctoral researchers to faculty positions make them an ideal mentorship team for my goal of becoming an independent group leader working on the microbiome-brain axis.

NIDA - National Institute on Drug Abuse

Use of antiretroviral therapy (ART) has considerably increased life expectancy for people with HIV (PWH) resulting in increased risk of age-related non-AIDS-defining health burden, including inflammaging, a low-grade chronic proinflammatory immune response, among the older population of PWH compared to the general population. A large proportion of PWH are often found to be addicted to synthetic or non-synthetic opioids, resulting in opioid use disorder (OUD), which can also add to proinflammatory immune response. However, little is known about the consequences of long-term ART and OUD on inflammaging in this aging PWH. Interestingly, a common denominator for aging, HIV and OUD is that they all cause mitochondrial dysfunction in immune cells as indicated by various previous reports. Mitochondria are highly dynamic organelles, essential for cellular metabolism, stress responses and homeostasis maintenance and are considered as the hub for biochemical processes including oxidative phosphorylation (oxphos), adenosine triphosphate (ATP) production, intracellular reactive oxygen species (ROS) generation, fatty acid synthesis and calcium (Ca2+) homeostasis. Our previously published data have shown that mitochondrial ROS increased with mitochondrial depolarization in people with HIV. Moreover, our exciting preliminary data indicated that mitochondrial ROS in immune cells is further increased in older PWH with age ≥50years compared to younger PWH with age <50 years. In addition, PWH with OUD had more mitochondrial ROS+ and reduced mitochondrial membrane potential in immune cells compared to PWH without OUD. Also, we found increased inflammatory immune markers in plasma of PWH with OUD. In view of this evidence, we hypothesize that OUD aggravates inflammatory immune response by increased mitochondrial dysfunctions in aging PWH. To test our hypothesis, we propose to evaluate the extent of OUD induced mitochondrial dysfunction in aging PWH, by studying mitochondrial mass, mitochondrial superoxide levels, mitochondrial membrane potential, mitochondrial fusion/fission, mitophagy, expression of mitochondrial stress response genes, oxidative phosphorylation and mitochondrial NADH levels in peripheral blood mononuclear cells (PBMC) of ART-treated PWH of age ≥50years, with or without OUD. In addition, we will identify cell specific molecular targets contributing to mitochondrial dysfunction, cellular senescence and inflammaging in PWH with OUD by transcriptomic analysis using single cell RNAseq. Finally, we will investigate if treatment with ROS a scavenger or a NAD+ booster can improve mitochondrial function in immune cells and reduce proinflammatory immune response in aging PWH with OUD. This proposal aims to advance the foundational knowledge about the role of mitochondria in the age-related altered immune cell function in older ART receiving PWH with OUD, which could be leveraged to develop targeted therapies and improve outcomes in people aging with HIV, opioid use and addiction.

NIA - National Institute on Aging

PROJECT SUMMARY/ABSTRACT Epidemiological evidence links peripheral inflammation to an increased risk of dementia, yet the mechanisms underlying this association remain unclear. Although peripheral immune challenges can amplify neuroinflammation and accelerate cognitive decline, we still do not understand the thresholds required to elicit neuroinflammatory responses that alter neuronal activity, their underlying cellular mechanisms, or how these thresholds and mechanisms change with age. This gap stems from viewing neurons as a passive recipient of neuroinflammatory signals, but also from the use of models with limited relevance. Our long-term goal is to understand how peripheral inflammation interacts with aging to increase the risk of dementia. Prior studies showed that aging sensitizes microglia, making them highly responsive to immune signals and driving exaggerated responses that disrupt synaptic circuits and lead to cognitive deficits. Furthermore, increasing evidence-including our preliminary data-indicates that peripheral inflammation can also directly alter neuronal activity and connectivity. Our proposal builds on this foundation and advances a novel hypothesis: while chronic inflammation directly activates microglia, in acute inflammation, the flow of events begins with neurons. Our preliminary data show that acute inflammation initially alters the activity of inhibitory neurons in key cortical regions. This change may then be detected by microglia, which respond according to their current, age-dependent state by further altering inhibitory synapses and the excitation-inhibition balance. To test this hypothesis, we will use mouse models that mimic common human inflammatory conditions: house dust mite (HDM}-induced respiratory allergy and dextran sulfate sodium (DSS}-induced colitis. Aim 1 will define microglia responses to acute and chronic inflammation as a function of age and will assess whether T cells contribute to increased responses of microglia to acute inflammation in aged mice. Aim 2 will test whether changes in inhibitory activity following acute inflammation trigger microglia activation in aged mice and will define the associated microglial molecular responses. Aim 3 will examine if microglia respond to changes in inhibitory activity by further altering cortical circuits in an age-specific manner. With expertise in molecular, cellular, and circuit neuroscience, inflammation, and imaging, our team is uniquely positioned to carry out this interdisciplinary project. The Pl's past discovery of specific microglia-inhibitory neuron interactions is an additional strength. We will use an innovative approach that combines relevant mouse models, advanced immunological techniques (e.g., adoptive transfer, immune cell depletion), and state-of-the-art neuroscience methods (e.g., calcium imaging, chemogenetics) to test our hypothesis. This research addresses a significant biomedical challenge-understanding how peripheral inflammation affects the aging brain-and has the potential to transform our understanding of neuroimmune interactions in dementia. We will identify key features of microglia-PV neuron interactions following peripheral inflammation and define how they shift with aging. Ultimately, our findings could reveal age-specific therapeutic strategies to reduce the risk of dementia.

NIDA - National Institute on Drug Abuse

HIV infection and nicotine addiction are significant global health issues, particularly among young adults, with increasing co-occurrence. Despite successful antiretroviral therapy (ART), 30–60% of people living with HIV (PLWH) develop HIV-associated neurocognitive disorders (HAND). Nicotine negatively impacts HIV⁺ individuals, potentially contributing to disease progression by facilitating neuroinvasion through CD4⁺ T cells that cross the blood-brain barrier (BBB). These cells, once in the central nervous system (CNS), release neurotoxic particles and inflammatory cytokines, leading to synaptic damage. Chronic nicotine activates nicotinic acetylcholine receptors (nAChRs) on T cells, enhancing their proliferation and cytokine release. Our hypothesis is that chronic nicotine enhances HIV⁺ CD4⁺ T cell neuroinvasion through convergent chemotactic signaling involving HIV gp120 and nicotine receptor activation. We will test this hypothesis through three aims. Aim 1 is to investigate how HIV infection promotes chemotaxic signaling in CD4⁺ T cell neuroinvasion, utilizing an in vitro BBB transmigration assay and measuring the phosphorylation of cytoskeletal regulators. Aim 2 is to assess chronic nicotine's effects on HIV⁺ CD4⁺ T cell proliferation and pro-inflammatory responses, focusing on nAChR expression and activation of chemotactic signaling pathways. Aim 3 is to evaluate the effects of chronic nicotine and HIV infection on CD4⁺ T cell behavior in an HIV humanized mouse model, monitoring inflammatory cytokines, viral reservoirs, and neuroinvasion in brain regions susceptible to T cell infiltration. This study is proposed by a strong team of co-PIs with complementary expertise in HIV signaling, nicotine research, T cell immunology, and drug abuse animal models, ensuring a comprehensive approach to understanding the interplay between HIV and nicotine addiction in neuroinvasion.

NIAID - National Institute of Allergy and Infectious Diseases

Project Abstract: Significant advances in antiretroviral therapy (ART) has enabled functional suppression of HIV, allowing individual individuals on ART to live relatively normal lives. However, viral rebound from a persistent reservoir of provirus-harboring cells following ART cessation demands a lifelong reliance on ART. ART-free control of this persistent reservoir therefore represents the current focus of HIV cure strategies. Recent work from our group and others has identified HIV-infected CD4 T cells that exhibit cell-intrinsic resistance to CD8 cytotoxicity, despite antigen expression and recognition. While identification of resistance factors previously annotated in cancer (such as overexpression of the granzyme inhibitor SERPINB9 and the anti-apoptotic protein BCL-2) has provided some insight concerning underlying mechanisms, the factors leading to this resistance phenotype remain largely unknown. To address this, we have developed a novel genome-wide screening platform that allows identification of genes involved in primary human CD4 T cell resistance and susceptibility to CD8 T cell cytotoxicity. In this proposal, we aim to maximize the potential of this newly developed CD4 resistance screening model (CRSM). Additional screens in different contexts will be conducted to further enrich the dataset. We will also employ a methodical pipeline for hit validation and therapeutic characterization. This pipeline will begin with promising hits identified in the initial screen, including the lipid metabolism-associated PPAR family proteins. This proposal will produce a robust functional genomic dataset thoroughly characterizing the mechanisms underlying CD4 resistance to CD8 killing. With these datasets, we envision the nomination of a range of promising targets for small-molecule sensitization of HIV-infected CD4s to CD8 killing.

NIMH - National Institute of Mental Health

PROJECT SUMMARY This research proposal aims to enhance the effectiveness of machine learning (ML) in improving adherence to antiretroviral therapy (ART) among people living with HIV (PWH). Adherence to ART is critical for viral suppression, a key goal in the Ending the HIV Epidemic Plan for the United States. While modern ML methods have been shown to identify individuals at risk of missing their visits with HIV providers and potentially discontinuing ART, these ML models are only as effective as the data they are trained on. As a result, ML models can be ineffective when applied to different patient populations or different healthcare settings, potentially worsening health outcomes for PWH. To address this challenge, this project will study synthetic data augmentation (SDA), a method that uses generative artificial intelligence (AI) to create additional data to train ML models. This approach has shown promise in various applications of ML, but has yet to be evaluated in, or adapted for, the domain of HIV care. My hypothesis is that SDA can improve ML models' ability to predict retention in HIV care, ensuring the benefits of these technologies extend to all PWH. However, there are limitations to SDA that must first be addressed. First, SDA is a new method and can be performed with many different computational approaches. Thus far, these varied methodologies for SDA have produced mixed results, making it unclear what the precise mechanism is behind how SDA works and in what circumstances SDA will be most effective. Second, the generative AI models that SDA utilizes do not have any external knowledge, learning only what they observe in the data. This makes generative AI models prone to creating unrealistic or biomedically implausible synthetic data. While a well-trained generative model does this infrequently, this can still occur and harm the performance of ML models trained via SDA. The proposal is structured around three specific aims. Aim 1: develop a foundational framework for understanding the impact of SDA on ML performance, particularly in the presence of issues common to HIV datasets. Aim 2: enhance the quality of synthetic HIV data by embedding biomedical knowledge into generative AI models. Aim 3: apply SDA to a large cohort dataset of PWH in the United States and evaluate the effectiveness of SDA in improving ML performance across different healthcare sites and populations. Successful completion of this project will lead to more reliable and generalized ML systems, ensuring that advancements in ML technology benefit all individuals living with HIV.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Despite major advances in antiretroviral therapy (ART) treatment and distribution, latently infected “reservoir” cells remain a major barrier to developing a cure for human immunodeficiency virus (HIV) infections. Replication- competent HIV reservoirs are rare and remain stable for decades during ART, providing opportunities for viral rebound in the absence of ART. Multiple strategies have been explored in the field to eliminate or permanently silence HIV reservoirs, including the “kick and kill” cure approach. This two-pronged approach consists of inducing HIV reactivation from latency (kick) using a latency reversing agent (LRA), enabling a subsequent “kill” through immune mechanisms or viral cytopathic effects. However, no LRAs that completely reactivate all latent HIV have been identified, and the most successful introduce potentially harmful side effects such as proinflammatory cytokine induction. Thus, there remains a need to not only identify improved LRAs that balance HIV reactivation and immune activation, but also develop a greater understanding of the pathways involved in maintaining HIV latency. In this project, we propose to improve “kick and kill” LRAs through two approaches. Previous studies demonstrate protein kinase C modulators (PKC) are a particularly potent LRA class that strongly reverse HIV from latency but simultaneously induce high levels of proinflammatory cytokines upon activating T cells. Synthesized analogs of the natural PKC modulator bryostatin-1 have also been shown to possess improved tolerability and in vivo HIV reservoir reduction, highlighting the potential of optimizing compounds for the “kick and kill” approach. Building upon this, in Aim 1, we will evaluate promising next- generation PKC modulators for in vitro and in vivo latency reversal. To explore new molecular mechanisms of HIV latency, we propose to evaluate a novel polyadenylation-driven pathway using JTE-607, a small molecule with anti-inflammatory effects. In Aim 2, we will evaluate JTE-607 as a HIV LRA in vitro and in vivo and to complement strong but inflammatory PKC modulators by downregulating proinflammatory cytokine expression. By designing and working on this project, I will contribute enhanced LRAs that advance the “kick and kill” approach towards wider, safer implementation while enhancing fundamental knowledge of HIV gene regulation. Simultaneously, when completed, this project will yield improved HIV reservoir depletion strategies, complementing other cure approaches and bringing the field closer to a definitive HIV cure.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract. South Florida is an epicenter of the United States HIV epidemic, yet evidence-based prevention and treatment does not reach South Floridians most in need. Mental health and substance use problems fuel the HIV epidemic, yet mental health and substance use treatment reach is suboptimal in South Florida. Our team found that HIV (e.g., PrEP, PEP, ART), mental health, and substance use services in South Florida are complicated to access and not coordinated across organizations, which drives their suboptimal reach. Our partners (Care Resource, Florida Department of Health) asked our team to centralize information about local services to facilitate community reach. With these partners, we developed the “Joining Under-connected Networks To Optimize Salud” (JUNTOS) Referral Network. The JUNTOS (“together”) app and website provides information about >60 organizations to enable community members to find HIV-relevant clinical services. Our partners house HIV self-testing programs and are eager to improve client linkage to PrEP, PEP, ART, and mental health/substance use treatment, as well as new interventions such as doxyPEP. HIV self-testing provides a powerful context in which JUNTOS could function as an adjunctive intervention – one that promotes engagement in other HIV-relevant clinical interventions – to address the gap between the availability of these interventions and their community reach. We propose a three-year R01 with the following Aims: In Aim 1, we will develop a scalable implementation blueprint for integrating JUNTOS into HIV self-testing programs. The blueprint will include client-level reach strategies to promote engagement with JUNTOS and program-level adoption and implementation strategies to support our partners in integrating JUNTOS into their self-testing programs. We will use co-design workshops and service mapping to develop the blueprint. In Aim 2, we will conduct a randomized controlled trial to evaluate JUNTOS and its implementation blueprint in our partners’ HIV self-testing programs. Self-testing clients (N=210) will be randomized 1:1:1 to three different reach strategy packages from Aim 1: (a) standard (palm cards in test kits); (b) enhanced (e.g., standard strategy + consistent text/email reminders); and (c) tailored (enhanced strategy + JUNTOS usage feedback, e-coach). We will compare JUNTOS reach across the three strategies, as well as HIV-relevant clinical intervention reach (e.g., PrEP/PEP or ART, mental health or substance use treatment) and evaluate appropriateness and acceptability using mixed methods. In Aim 3, we will evaluate self-testing program staff implementation outcomes, including acceptability, appropriateness, and feasibility of JUNTOS and the blueprint, as well as adoption and implementation using mixed methods. The project responds directly to our partners’ priorities of increasing client linkage to HIV-relevant clinical interventions using an adjunctive intervention that they co-developed in service of immediate public health and organizational goals. Findings will inform refinements to JUNTOS and the blueprint in preparation for a fully powered implementation trial of JUNTOS and its implementation blueprint to achieve national EHE goals.

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NIMH - National Institute of Mental Health

PROJECT SUMMARY Stable HIV reservoirs remain a significant challenge for the eradication of HIV-1 (HIV). While several anti- HIV latency strategies have been proposed to control HIV reservoirs, few have proven effective in reducing the size of the HIV reservoir in clinical settings. This suggests that our current understanding of how HIV achieves its persistent infection is limited and underscores the need for the development of alternative innovative tools for HIV cure. Recently, we and others characterized a unique integrated stress response (ISR)/ATF4 signaling pathway that is essential for HIV transcription and early seeding of HIV. Conversely, when ATF4 is knocked down, HIV transcription is inhibited. We further defined ATF4 as a new transcription factor of HIV, exploited by HIV for its own transcription after the recruitment of ATF4 to the ATF/CREB consensus site at the 5’ HIV long-terminal repeat. ATF4 induction leads to HIV activation from latency, indicating that ISR/ATF4 signaling activation promotes HIV transcription while the suppression of ISR signaling is associated with the establishment of HIV latency. Of note, prolonged activation of ISR/ATF4 signaling also induces cell death, mediated by ATF4 binding to the promoter of the CHOP gene, an essential protein driving apoptosis during persistent ISR/ATF4 activation. In a primary CD4+ T cell model of latency, prolonged ISR/ATF4 signaling activation disrupts latent HIV and selectively induces apoptosis in HIV+ CD4+ T cells, without affecting bystander HIV-negative CD4+ T cells. Notably, using viral outgrowth assays, we discovered that prolonged ISR/ATF4 signaling activation reduces replication-competent HIV by up to 2,300-fold in resting CD4+ T cells isolated from people with HIV receiving suppressive antiretroviral therapy (ART). Therefore, we hypothesize that the suppression of ISR/ATF4 signaling is essential for HIV persistence. This will be tested through three specific aims: Aim 1: Investigate how activation of the ISR/ATF4 signaling pathway eliminates HIV-latently infected T cells and brain microglia. Aim 2: Elucidate the unique mechanisms by which ISR/ATF4 signaling controls the stable HIV reservoir for the establishment of HIV latency. Aim 3: Determine the effectiveness of activating ISR/ATF4 signaling in eliminating HIV reservoirs in the hu-BLT mouse model of HIV latency in vivo.

NCI - National Cancer Institute

PROJECT SUMMARY Obesity is a risk and progression factor for ≥13 cancers including, including endometrial cancer (EC, the most common obesity-related cancer in women), and colorectal cancer (CC, the most common obesity-related cancer in men). Incretin mimetics, which are agonists at one or more satiety-related receptors, can induce sustained weight loss. Limited epidemiological and preclinical data on these medications suggest potential for preventing obesity-related cancers, albeit via unclear mechanisms. We propose to test the novel central hypothesis that incretin mimetics offer protection from common obesity-related cancers via interacting mechanisms involving direct effects on CD8+ T cells as well as systemic effects on diet-regulated hormones, particularly leptin. We will leverage rigorous, complementary lines of research (preclinical models of obesityassociated cancers and a medically supervised human weight loss cohort) with state-of-the-art approaches, robust informatics data integration, and an accomplished transdisciplinary team to (a) identify immunologic, molecular, metabolic and anticancer effects of semaglutide v tirzepatide, 2 FDA-approved incretin mimetics for weight loss that differ in their anti-obesity effectiveness, across mouse models of EC and CC; (b) extend in multiple preclinical models our preliminary finding that CD8+ T cells play a central mechanistic role in TZP’s anticancer effects; and (c) establish concordance of incretin mimetic-associated anticancer effects between mouse models and women at high risk for EC undergoing medically supervised weight loss. Three specific aims are proposed to test our hypothesis: 1. Determine the effects of SEM v TZP v vehicle on phenotypic, immunologic, and molecular features and tumor outcomes in mouse models of obesity-driven EC and CC; 2. Establish the interacting roles of CD8+ T cells and leptin in SEM v TZP’s anticancer effects in models of EC and CC; and 3. Determine the effects of SEM and TZP on phenotypic and molecular features systemically and in the uterus of women at risk of EC undergoing medically supervised weight loss. The proposed studies in mice and humans will clarify whether, and how, 2 incretin mimetics, differing in their receptor targets and antiobesity effectiveness, inhibit EC development and progression in individuals. The proposed studies propose the use of vertebrate animal models in parallel with human studies because the mechanistic effects of incretin mimetics on obesity-associated tumor development, systemic metabolism, leptin signaling, and CD8⁺ T-cellmediated immunity cannot be adequately interrogated in humans alone, or replicated using non-animal systems such as cell culture or organoids. Mouse models are essential to enable controlled manipulation of diet, obesity status, pharmacologic exposure, and longitudinal tissue analyses across the full course of tumorigenesis, while also providing translationally relevant physiologic and immune responses that closely parallel those observed in humans.