University Of Minnesota

NIH Research Projects · FY 2025 · 2026-01

PROJECT SUMMARY/ABSTRACT Type I interferons (IFN) are crucial to anti-viral immunity. Neutralizing autoantibodies (AAb) to IFN are found in the general population, increase in prevalence with age, and are linked to worse, often fatal, outcomes in some of the most lethal acute respiratory viral diseases known to date, including fulminant influenza and COVID-19 pneumonia. Despite this, the mechanisms behind the formation of IFN AAb remain unknown. Human data suggest that impairments in thymic tolerance—due to dysfunction of autoimmune regulator (AIRE) and medullary thymic epithelial cells (mTEC)—may be required for the development of IFN AAb. AIRE is a transcription factor expressed by mTEC that is essential for establishing T cell tolerance in the thymus. In mTEC, AIRE promotes the expression and presentation of antigens from extrathymic tissues to developing T cells (thymocytes). This allows for the elimination of auto-reactive thymocyte clones, thereby preventing autoimmunity. Interestingly, AIRE+ mTEC have been shown to express IFN at steady-state conditions in the thymus suggesting that, in this context, AIRE+ mTEC act as antigen-presenting cells to thymocytes to mediate T cell tolerance to IFN. Supporting this idea, individuals with Autoimmune Polyglandular Syndrome 1 (APS1), who lack AIRE and experience T cell tolerance loss, consistently develop IFN AAb. These AAb are isotype- switched and somatically hypermutated, supporting the notion that a failure of T cell tolerance, rather than solely B cell tolerance, is necessary for their generation. However, additional findings suggest that loss of thymic T cell tolerance alone is insufficient for IFN AAb to develop. First, APS1 patients do not typically present IFN AAb at birth or infancy; instead, they develop these AAb later in life after exposure to pathogens is likely to have occurred. Second, IFN AAb have not been observed in specific pathogen-free, Aire-deficient mice. Combined, these observations suggest that pathogen exposure, in addition to AIRE and mTEC dysfunction, may be required for IFN AAb to develop. This proposal aims to understand how infections, combined with AIRE deficiency, contribute to the loss of thymic tolerance to IFN. My central hypothesis is that in individuals with predisposing AIRE deficiency, infections that induce IFN expression act as a double hit, promoting the development of neutralizing IFN AAb. Until now, methods to detect neutralizing IFN AAb in mice have been lacking, which has hindered the field's ability to test this hypothesis. I have developed a novel, sensitive, reproducible, and high-throughput assay for detecting murine neutralizing IFN AAb. This new tool will serve as the basis for this proposal and will facilitate exploration of how microbial infections and thymic defects contribute to the development of IFN AAb in an animal model. The findings from this work will deepen our understanding of how tolerance to IFN is mediated and may inform strategies to prevent IFN AAb development in affected individuals.

NIH Research Projects · FY 2025 · 2025-09

Project Summary: The gradual loss of physical and cognitive ability has been taken as the near-universal consequence of growing older. Older adults slow down physically, with reduced gait speed and increased instability, and slow cognitively, with declining memory and executive function. Disease pathologies can accelerate these declines and are the focus of most aging research. Nonetheless, exceptions to these trajectories can be identified, most notably cognitive “SuperAgers,” who retain the working memory of people 30 years younger. Initial research on SuperAgers has focused only on cognition studied through static, MR- based neuroimaging and cytoarchitectural examination of post-mortem brains. However, as mounting evidence demonstrates fast movement is a distinguishing feature of health and longevity in older adults, our research emphasizes the dynamic interaction between brain neural and vascular function during physical movement. Our preliminary data illustrate that older adults age 75+ years with exceptionally fast walking speeds and who are free of cognitive decline – who we have termed fast-moving SuperAgers - display a robust capacity for neuroplasticity induction (measured with electroencephalography (EEG)) and remarkably high cerebral blood flow (measured with transcranial Doppler ultrasound (TCD)) during whole-body behavioral learning. Motivated by these captivating data, this DP2 project will test the novel hypothesis that fast-moving SuperAgers are enriched in unique neuroplasticity and cerebrovascular profiles that are neuroprotective and enable remarkably high physical and cognitive function as they near or enter their 9th and 10th decade. This project embraces a systems-neuroscience perspective to study neuroplasticity induction and interactions with real-time cerebrovascular function in the unique aging phenotype of fast-moving SuperAgers. We utilize a highly innovative, multimodal approach (EEG, biomechanics, TCD) to quantify the adaptation of cortical and biomechanical responses to repeated standing balance perturbations. We synergistically assess real-time cerebral blood flow velocity during this balance learning task and interactions with cortical plasticity induction. We will compare the state and longitudinal trajectory of neuroplasticity and cerebrovascular function in fast- moving SuperAgers to their normally- and slow-moving peers under the same learning conditions. These results could identify key functional neuroprotective mechanisms of brain aging to be leveraged and targeted with future interventions to prevent decline of cognition and physical function. The development of targeted, physiologically-informed interventions through a lens of brain resilience for neurodegenerative diseases such as dementia would be especially impactful because current pathology-focused treatments (e.g. anti-amyloid drugs) are extremely limited. Findings gleaned through our innovative conceptual and technical approach to study human aging neurobiology could pave the way towards a new resilience-focused framework, enabling us to make impactful scientific discoveries to improve the lives of aging Americans.

NIH Research Projects · FY 2025 · 2025-09

ABSTRACT Project Title: Expanding Low-Barrier Buprenorphine Initiation and Post-Discharge Engagement in Emergency and Prehospital Settings Background: The opioid crisis continues to pose a significant public health challenge, with opioid- related overdose stubbornly high across the U.S. despite the availability of evidence-based treatment options. Emergency departments (EDs) and emergency medical services (EMS) serve as critical intervention points for patients experiencing opioid-related crises. However, despite strong evidence supporting ED-initiated medication for opioid use disorder (MOUD), implementation remains limited due to provider hesitancy, institutional barriers, and gaps in post-discharge care. This project seeks to address these challenges by integrating structured, high-dose buprenorphine induction across EMS and ED settings while ensuring sustained post-discharge engagement through a multidisciplinary mobile outreach team. Objectives: This study will evaluate the impact of a structured MOUD expansion model that spans prehospital, acute care, and community settings. The primary objectives are to: 1. Increase buprenorphine administration in ED and EMS settings to improve rapid access to MOUD. 2. Enhance provider adoption of high-dose buprenorphine induction protocols through structured training and technical assistance. 3. Improve patient retention at 7-day and 30-day post-discharge intervals via proactive mobile outreach and linkage to outpatient treatment. 4. Decrease overall ED utilization among patients receiving buprenorphine, reducing avoidable healthcare encounters and hospital burden. Methods: This study employs a mixed-methods evaluation framework, integrating both quantitative and qualitative assessments. Electronic health records (EHRs) will track buprenorphine administration rates, help monitor retention in treatment, and measure changes to ED utilization and hospital re-admissions. Provider surveys and structured interviews will assess confidence and knowledge in high-dose induction protocols. The ED-IMAT tool will systematically evaluate institutional readiness, training gaps, and care linkage challenges. Qualitative insights from Extension for Community Healthcare Outcomes (ECHO) sessions will further inform implementation barriers and real-time adaptation strategies. Significance: By embedding a sustainable, data-driven MOUD expansion model within acute and prehospital care settings, this initiative directly supports Healthy People 2030 objectives to increase the proportion of individuals receiving MOUD and reduce opioid-related mortality. Findings will contribute to national efforts to scale ED and EMS-based addiction treatment, inform state and municipal policy, and establish a replicable framework for integrating MOUD into emergency care workflows. Impact: This project aims to transform opioid overdose response by shifting from a reactive to a proactive, patient-centered model of care. Through structured capacity-building, real-time technical assistance, and post-discharge navigation, this initiative will optimize MOUD retention, improve health outcomes, and reduce opioid-related harms at the population level.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Major depressive disorder (MDD) is an epidemic, evident from its substantial global prevalence and burden, and is driving other major public health issues, including economic consequences. More than 30% of individuals living with MDD demonstrate no response to two or more antidepressant interventions and ultimately exhibit treatment resistance. Transcranial magnetic stimulation (TMS) has emerged as a safe, evidence-based, and non-invasive neuromodulation therapy for treatment-resistant depression. TMS utilizes electromagnetic induction to create an electric current in the brain, depolarizing cortical neurons and eliciting neurophysiological and behavioral effects. TMS works by inducing neuroplasticity, both within the stimulated cortex and its broader connected networks. However, the current efficacy rate of TMS for MDD remains up to 60%. This variability in treatment response may partially result from current clinical TMS approaches failing to incorporate the influence of brain dynamics at the time of stimulation on the neuroplastic effects and therapeutic outcomes. In line with this notion, it may be possible to reliably improve TMS response by enhancing the TMS-induced neuroplasticity through synchronizing the stimulation with ongoing neural activity. An oscillation-locked stimulation protocol delivers stimulation timed to a certain phase of an endogenous neural oscillation, which has been shown to modulate the ongoing brain activity in a more selective manner. Specifically, oscillation-locked stimulation can reliably produce opposing neuroplastic outcomes of potentiation or depression, depending on whether a pulse occurs at a negative-going or positive-going phase of a local oscillatory potential. Initial investigations and my Preliminary Data indicate that this effect is present in the human left dorsolateral prefrontal cortex, a critical hub in circuits governing cognitive and affective functions and the most common clinical TMS target. However, these findings have not been extensively examined, and the impact of oscillation-locked TMS on neuroplasticity has not been fully delineated. I will aim to bridge this knowledge gap by elucidating how precise, oscillation-locked TMS induces local and network neuroplasticity and by examining how these effects vary across the spectrum of depressive severity. My central hypothesis is that the phase of the ongoing oscillatory activity at the time of stimulation will differentially modulate both local (Aim 1) and network (Aim 2) neuroplasticity in adults without psychiatric diagnoses and those with MDD. This study will advance our theoretical understanding of clinically relevant neuroplasticity and investigate the potential of developing a brain state-dependent TMS protocol to effectively modulate relevant brain networks. In addition to completing the proposed research study under the Kirschstein-NRSA Fellowship, I will pursue rigorous clinical and career development activities to fulfill the requirements for my MD and PhD degrees and establish myself as an independent physician-scientist with expertise in neuromodulation.

NIH Research Projects · FY 2025 · 2025-09

Project Summary The lymphatic system plays an important role in maintaining tissue-fluid homeostasis, the absorption of dietary fats, and the transport of immune cells throughout the body to combat infection. Central conducting lymphatic anomaly (CCLA) is a disorder resulting from lymphatic network conduction abnormalities which can result in the accumulation of fluid in tissues. Despite recent advances in patient sample collection and more thorough genetic testing, only about 40% of patients with CCLA have a defined genetic cause. Identification of the underlying disease mechanism in these patients has resulted in the targeted trial of specific pathway inhibitors including MEK, ERK, and PI3K. Elucidating novel genetic causes of CCLA will inform the development of new custom therapies. Exome sequencing in a cohort of five families with children who presented with non-immune fetal hydrops revealed FZD6 variants. FZD6 has therefore emerged as a potential novel cause of CCLA. FZD6 is a WNT receptor and a core member of the planar cell polarity (PCP) signaling pathway. In humans, loss of FZD6 function causes nail dysplasia, which is also present in the identified cohort. The core PCP genes Celsr1 and Vangl2 are shown to be involved in lymphatic valve formation in mice and defects in PCP signaling due to pathogenic variants in CELSR1 have been implicated in lymphatic malformations in humans. However, the role of FZD6 in lymphatic network development and function is unstudied. We hypothesize that FZD6 is important for lymphatic system development, and the identified variants result in CCLA by disrupting PCP signaling. Further, in addition to the role PCP plays during valve formation, we propose PCP has an earlier role in shaping the lymphatic network by regulating lymphatic endothelial cell dynamics. I will test these hypotheses using PCP loss of function mouse models. I will assess the morphology of the developing lymphatic vessels and valves in PCP mutant embryos compared to littermate controls in the dermis and mesentery (Aim 1). I will then assess the molecular function of FZD6 variants identified in patients. Subsequently, I will determine how the loss of PCP function affects lymphatic network uptake and conductance capabilities by injecting a tracer molecule (Aim 2). Finally, I will determine which lymphatic endothelial cellular behaviors are influenced by PCP signaling through a live-imaging approach (Aim 3). Completing these aims will identify a novel genetic driver of CCLA and inform targeted treatments to rescue the effects of FZD6 loss. Further, the results of this study will fill the gaps in our understanding of the role of PCP signaling in lymphatic network formation during development.

NIH Research Projects · FY 2025 · 2025-09

Functional decline of visceral adipose tissue, both immunologically and metabolically, contributes to age-related diseases. Aging induces phenotypic alterations in immune cells and adipocytes within adipose tissue, leading to chronic low-grade inflammation and metabolic dysfunction including impaired lipolysis and glucose resistance. Notably, aged adipose tissue macrophages (ATMs) exhibit increased mitochondrial burden, altered chromatin accessibility, and amplified secretion of inflammatory cytokines, collectively driving visceral adipose dysfunction through interactions with other immune cells and white adipocytes. In the pre-F99 phase, I demonstrated that GDF3, a cytokine secreted selectively by ATMs, activates the canonical SMAD2/3 pathway and shifts the chromatin landscape of ATMs toward an inflammatory profile with age. GDF3 also worsens glucose sensitivity in old mice, suggesting a GDF3-mediated interaction between ATMs and adipocyte dysfunction during aging. In the F99 phase, I will investigate how the GDF3- SMAD2/3 axis impairs lipolysis in aged adipocytes. Adipocytes are heterogeneous and can be classified into subsets based on functionality. Age-related imbalance in these subsets, such as a decline in lipolytic adipocyte subset, may impair lipolysis, leading to reduced exercise capacity, dampened cold stress resilience, and exacerbated inflammation during infections in the elderly. However, adipocyte complexity remains largely understudied, particularly in the context of aging, due to the lack of accessible tools for single cell resolution analysis. I will continue developing methods to explore adipocytes at the single cell level, aiming to identify, characterize and target the lipolytic adipocyte subset affected by GDF3-SMAD2/3 signaling. The pathophysiologic role of mitochondria during aging, specifically the link between mitochondrial dysfunction and chronic inflammation, has been increasingly recognized. In the K00 phase, I will explore how mitochondrial (mt) dysfunction originating from bone marrow hematopoietic stem cells (HSCs) impacts ATMs. With an increasing frequency of ATMs originating from HSCs during aging, HSC-derived mt dysfunction may drive the heightened inflammatory phenotype observed in their progeny ATMs. I propose targeting mt dysfunction and inflammatory ATMs by inhibiting the cGAS-STING pathway, which is activated by mt stress. Since HSCs give rise to macrophages in multiple tissues, this research may have broad applicability beyond adipose tissue and result in novel therapeutic approaches to reduce inflammation and metabolic decline in the elderly. This proposal will yield new insights into the molecular mechanisms regulating age-related alterations in macrophages and adipocytes. Furthermore, I aim to target these identified mechanisms using genetic and pharmaceutical tools to mitigate adipose functional decline, ultimately alleviating age-related diseases.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Advances in cancer prevention, diagnosis, and treatment have dramatically improved long-term survival of those diagnosed with breast cancer. However, this success has been tempered by a parallel increased incidence of adverse outcomes and chronic conditions in breast cancer survivors, including cardiotoxicity due at least in part to cardiotoxic treatment regimens. Current evidence-based guidelines for preventing and controlling cardiotoxicity in breast cancer survivors are broad, and we lack clear guidance for assessing individualized risks of cardiovascular events. Existing cardiovascular disease (CVD) risk prediction models focus on the general population and rely only on a limited number of variables. Thus, there is a critical need to develop novel AI- powered informatics frameworks to build, maintain, and enhance models predicting cardiovascular risk among breast cancer survivors across diverse health systems. Responding to the RFA-FD-25-015, the objective of this application is to develop and validate a scalable, generalizable, and explainable AI-powered informatics framework, CardioOnco-AI, which innovates on the curation and modeling of real world data (RWD) for individualized prediction of cardiotoxicity among breast cancer survivors. Towards this objective, we propose the following specific aims: 1) Data curation - extract and derive cancer phenotype and non-medical determinant of health (nMDoH) variables to create research datasets for cardiotoxicity risk prediction through novel AI- empowered informatics solutions; 2) EHR-based predictive modeling - develop and evaluate novel cardiotoxicity prediction models for breast cancer survivors across two sites; and 3) Evaluation - assess the generalizability of CardioOnco-AI in two large geographically diverse RWD consortia. This project will deliver a novel, generalizable framework that integrates structured and unstructured RWD to improve cardiotoxicity risk prediction in breast cancer survivors. This work directly aligns with FDA’s regulatory science priorities and supports safer post- treatment survivorship care. The tools and methods developed will be adaptable to other sites, data environments, and oncology drug safety surveillance efforts.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract A founder mutation in the ARTEMIS gene resulting in Severe Combined Immunodeficiency (SCID-A) is common in the Athabascan-speaking Navajo and Apache populations (a minority group) in the United States. One in 2,000 live births in this community have this devastating genetic disorder, resulting in a complete lack of mature T and B lymphocytes. The current standard of care for these patients is a bone marrow transplant, which often does not result in B cell reconstitution, carries risk of graft versus host disease, and necessitates harsh preconditioning regimens. Clinical trials are ongoing for ex vivo gene therapy using a lentiviral vector to insert additional copies of ARTEMIS in patient hematopoietic stem cells. We are pursuing a more targeted treatment using Adenine Base Editor (ABE) to convert two bases of ARTEMIS without causing double-stranded breaks in the genome. ABE can edit this mutation with a conservative amino acid substitution, replacing the premature stop codon. The effect of this edit on hematopoietic stem cells' immune reconstitution ability has not yet been examined. This proposed work aims to validate ABE conservative substitution as a viable therapy for SCID-A and establish in vivo delivery methods to hematopoietic stem/progenitor cells (HSPCs) for immune reconstitution using a novel, genetically humanized SCID-A mouse model that we recently developed. Aim 1 will characterize the efficacy and safety of the conservative substitution strategy with multiple in vitro Artemis assays in both a K562 SCID-A cell line and SCID-A murine hematopoietic stem cells. Aim 2 explores in vivo delivery of therapeutic guide RNA and base editor mRNA to HSPCs in our SCID-A mouse model. Novel cationic polymer micelles from the Reineke group will be tested in vivo along with recombinant adeno-associated virus (rAAV) serotype 6 to establish the feasibility of immune reconstitution by edited HSPCs in vivo. This work will lay the groundwork for potential in vivo base editor therapy clinical trials to treat SCID-A patients. This project describes the work that Ella will perform throughout the remainder of graduate studies. During the fellowship period, Ella will also undertake a career development plan to prepare for a future career as an independent researcher at an academic institution. This includes support from numerous collaborators to accomplish the research objectives, and training in new areas of biological research, such as polymer complex delivery methods, rAAV production, and rigorous in vivo studies to assess the efficacy of SCID-A treatment using ABE. These experiences, along with programming from the UMN’s Office of Graduate and Postdoctoral Studies will provide Ella with the skills and knowledge necessary to achieve her career goals and desire to perform impactful translational research.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Antimicrobials are critical for animal and human health, but antimicrobial resistance (AMR) is a threat to their continued effectiveness. Antimicrobial use (AU) is a modifiable risk factor for the emergence and spread of AMR, and tracking AU is needed to improve prescribing. Logistical challenges to AU measurement in the veterinary field include access to prescribing data held within diverse mutually exclusive electronic health record (EHR) systems and lack of standard diagnostic coding. The Companion Animal Veterinary Surveillance Network (CAVSNET), a successful public-private partnership that collects, analyzes, and reports companion- animal veterinary data, overcomes these obstacles through the compilation of non-standardized patient-level data into a standardized common data model (CDM). CAVSNET fulfills a critical need in the fight against AMR, providing data on AU at the point of use (i.e., veterinary clinics) to connect the clinical context, such as disease process, diagnostic tests, and patient demographics, with prescription data. In addition to discrete data fields, unstructured free-text data are available for classification with NLP-PIER, a natural language processing (NLP) platform that enables named-entity recognition and indexing in a secure computing environment; these notes can be mapped to structured categories for analysis. Point-prevalence survey (PPS) methodology, which relies on detailed manual review of medical records, is structured to collect standardized data from multiple sites over a specific period of time; PPS are used to provide granular data and augment automated CAVSNET data collection to better understand the complexities of relating AU data to clinical condition. The overarching goal of this project is to harness data from CAVSNET and complementary PPS to analyze AU for dogs, cats, and horses and provide actionable targets for antimicrobial stewardship. Longitudinal AU tracking provides information needed to target prescribing improvement interventions, can be used to motivate practitioners to change behaviors, and show the profession's progress over time. CAVSNET's public-private partnerships for ongoing and long-term data collection will 1) characterize and address AU data gaps, 2) collect, standardize, and provide annual summaries of AU data, including contextual information and data trends, while maintaining veterinary and client confidentiality, 3) share data with the veterinary community and the public, and 5) use a common data model that lends itself to interoperability. It will provide a comprehensive picture for U.S. companion animals by 1) providing yearly national estimates of systemic antibiotic use prevalence in companion animal practices, including contextual demographic information, using CAVSNET, 2) generating U.S. estimates of systemic antibiotic use for specific clinical conditions via CAVSNET, and 3) characterizing antibiotic use data gaps in companion animal practice and utilizing methods to address gaps, including NLP- PIER and national AU PPS for horses and select surgical procedures in dogs and cats.

NIH Research Projects · FY 2025 · 2025-09

ABSTRACT: Over 1 million people with cancer live in African countries, 30-40% of whom are people with HIV (PWH). Chemotherapy-associated febrile illness is a frequent complication that occurs during cancer treatment and is associated with morbidity and mortality. Small-scale studies also suggest that the microbiology of chemotherapy-associated febrile illness differs significantly for those living in Africa. For example, our previous pilot study showed that 19% of Ugandans with solid tumors who developed chemotherapy-associated febrile illness tested positive for tuberculosis (TB). Furthermore, PWH have higher rates of chemotherapy-associated bone marrow suppression and are at increased risk for developing opportunistic infections when compared with their HIV-negative counterparts. Yet, international guidelines for diagnosis and treatment of chemotherapy- associated febrile illness do not reference any studies conducted in Africa and do not account for the patient’s HIV status. Point-of-care (POC) tests are a potentially cost-effective way to identify the microbiologic causes of febrile illness for patients living in low- and middle-income countries. Urinary lipoarabinomannan (LAM) is a first-line POC test for TB diagnosis among PWH. However, the sensitivity and specificity of urinary LAM has not been evaluated among patients with cancer. Given the high prevalence of HIV and TB throughout Africa, it is critical to develop guidelines that incorporate HIV and TB diagnostics among patients with cancer. In this study, we will use POC tests to develop a cost-effective diagnostic algorithm to guide antimicrobial management for patients with solid tumors receiving chemotherapy in Uganda. We proposed to use clinical microbiology diagnostics and next generation metagenomic sequencing to understand the association between HIV status and the microbiology of chemotherapy-associated febrile illness among Ugandan inpatients with solid tumors (Aim 1). We will assess the diagnostic accuracy of urinary LAM to detect TB among febrile patients with cancer (Aim 2). Finally, we will create a diagnostic algorithm that incorporates the patient’s HIV status and uses POC tests to guide antimicrobial management for patients with chemotherapy-associated febrile illness among Ugandan patients with solid tumors to assess an optimal, cost- effective diagnostic strategy (Aim 3). The proposed research aims will support Dr. Gulleen in meeting her career development training goals of 1) gaining proficiency in using metagenomic sequencing, 2) developing expertise in assessing the diagnostic accuracy of POC tests among novel populations, and 3) learning principles of cost-effectiveness analysis. The K23 award will facilitate her transition to an independent physician-scientist who evaluates and implements novel diagnostic algorithms for managing chemotherapy-related infections among PWH. Dr. Gulleen will conduct these activities with outstanding mentorship from experts in global health, metagenomic sequencing, clinical laboratory diagnostics, and cost-effectiveness analysis.

NIH Research Projects · FY 2025 · 2025-09

With continuous antiretroviral therapy (ART) and viral suppression, life expectancy for people with HIV (PWH) is approaching that of people without HIV. Some estimates suggest the mortality gap is 10 years or less, when comparing persons with and without HIV. However, with prolonged survival there have also been increases in non-AIDS defining age-related comorbid diseases. What has now emerged in contemporary HIV clinical care is a convergence of declining mortality with effective ART treatment, combined with the earlier onset of age- related comorbidities. This means a growing portion of the lifespan for PWH involves morbidity and disability from multiple diseases like obesity, diabetes, hepatic steatosis, cardiovascular diseases, and cancer. Several of these comorbidities are related to changes in metabolism, however the mechanism of these changes is not understood but may be unique to HIV. One common link between HIV and these metabolic diseases is that both are associated with chronic immune activation (IA). For example, people who are obese without HIV have elevated levels of circulating pro-inflammatory cytokines such as IL-6 and TNF. The same is true in treated HIV infection. There are several potential causes of IA including an altered microbiome, microbial translocation, and other infections like cytomegalovirus (CMV). However, we and others have shown that HIV RNA+ cells persist in lymphatic tissues despite fully suppressive ART, and we think this is a significant contributing cause of IA in PWH. Our primary hypothesis is that the size of this dynamic reservoir of HIV RNA+ cells is a primary driver of IA which, in turn, leads to development of these metabolic comorbidities. We will test this hypothesis in 2 ways. We will first explore the natural history of metabolic dysfunction in HIV using a large tissue repository of gut and lymph nodes (LN) we have collected over the last 20 years in multiple studies. We will measure the size of the HIV reservoir in gut and LN and frequency of hormone producing cells (e.g., GLP-1, PYY, GIP) in HIV negative people and PWH before and during ART and when viral recrudescence with treatment interruption. We will measure markers of IA and microbial translocation in mononuclear cells from those tissues as well as plasma that was collected at the time of the biopsy. Collectively, these data will provide insight into how metabolism is altered in HIV infection and the relationship to the size of the HIV viral reservoir. Our second approach is to focus on obesity as one of the most common comorbidities in PWH. We will enroll PWH with and without obesity (BMI30) and HIV-negative people with and without obesity into an 18-month prospective observational study. We will collect LNs, ileal, and colonic samples as well as PBMC and plasma at the baseline and 18-month timepoint. We will collect functional metabolic data with DXA and MRI scans to measure fat distribution throughout the body and we will do extensive metabolomics and functional genomics on these same tissues. We will apply machine learning algorithms to this extensive data set to determine the relationship of viral reservoirs to development of obesity as well as other predictors of obesity.

NIH Research Projects · FY 2025 · 2025-09

Life expectancy is a key metric of population health. Yet Black and low-income Americans exhibit much shorter life expectancy than their White, Hispanic, and higher-income counterparts. Housing and neighborhood context are key causes of these mortality differences via interlocking, accumulating social determinants of health at multiple levels across the life course. Yet literature examining how neighborhood context affects mortality remains weak methodologically. It relies on ecologic, cross sectional data; lacks a life course approach; ignores heterogeneity; rarely predicts population impact; and is divorced from policy solutions. Policy relevant levers are sorely needed that can disrupt the complex, upstream knot of family impoverishment. Our study of place-based factors leading to mid-life mortality addresses these limitations, leveraging data from a promising housing policy experiment: The Moving to Opportunity (MTO) Study. The MTO follows 4600 low-income families, living in public housing at baseline (1994-98), in five large US cities, who were randomized to receive one of three treatments: one of two types of housing vouchers to improve housing affordability and neighborhood context or an in-place control group that remained in public housing. The original MTO evaluation concluded in 2010, at which time adults randomized to the housing mobility voucher arm (vs. the other two arms) exhibited improved physical and mental health and improved neighborhood exposures. This study expands and enriches the MTO study by linking it to three administrative datasets through 2024, extending the study 15 additional years—(1) NCHS’s National Death Index, (2) Census Bureau/Social Security’s Numident, and (3) the Census Master Address File—allowing us to analyze the effects of housing policy on mid-life mortality and long-term residential mobility. An experienced and productive social epidemiologist and population health scholar leads this accomplished, interdisciplinary team. Our project has four specific aims: Aim 1: Test if the MTO low-poverty neighborhood housing mobility voucher treatment improves neighborhood opportunity in year 2024, vs. the in-place public housing or traditional voucher group; Aim 2: Test if the MTO low-poverty housing voucher treatment reduces mortality risk through mid-life versus the other 2 treatment groups; Aim 3: Test heterogeneity of MTO effects on mortality by baseline demographic factors (i.e., subgroup effects); Aim 4: Calculate the number of deaths that would be avoided and whether the mortality gap would narrow between groups, at the population level, if voucher-based housing policy were broadly implemented to promote opportunity moves. The proposed project aligns with the NIA Population and Social Processes Branch “Macro-Social Factors” priority area, by estimating population level impacts of a randomized social program. Our study presents a unique and important opportunity for population health: to understand whether housing mobility policy can promote sustained moves to high opportunity neighborhoods, and whether such effects may reduce mid-life mortality risk after 30 years.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Atherosclerosis is driven by the deposition of low-density lipoprotein (LDL) cholesterol within the aortic intima, forming a plaque. During plaque formation, monocytes infiltrate the sites of atherosclerotic lesions where they differentiate into highly heterogeneous macrophage populations, including lipid-loaded “foamy” macrophages which are a primary contributor to plaque progression. Foamy macrophages are defined by their expression of Triggering Receptor Expressed on Myeloid Cells 2 (Trem2), a known lipid sensor and key modulating factor of atherosclerosis disease outcomes. Recent findings from our lab implicated Trem2 as a key factor modulating atherosclerosis disease progression in vivo. We found that Trem2 mediated foam cell formation, and conditional deletion of Trem2 in myeloid cells resulted in increased foam cell death, reduced macrophage proliferation, and reduced plaque size. This study was followed by a Trem2 agonism approach, where we found that stimulating Trem2 signaling enhanced atherosclerotic lesion burden. Using a Trem2 blocking antibody, I will test the hypothesis that Trem2 blockade attenuates atherosclerotic plaque progression by limiting macrophage lipid uptake and maintaining cell function. Next, I will tease apart the molecular signaling downstream of Trem2 that mediates foamy macrophage formation. Ultimately, the proposed aims will determine mechanisms of Trem2 signaling that reprogram foamy macrophage function and illuminate the therapeutic potential of a Trem2 blocking antibody in atherosclerosis.

NIH Research Projects · FY 2025 · 2025-09

Project Summary The impairment in choline catabolism to its methyl-donor, betaine, is strongly related to the development of metabolic-associated liver disfunction (MASLD). In order for choline to be metabolized, it must be transported into the mitochondrial matrix. However, limitations in the understanding subcellular localization of choline have limited the understanding of choline metabolism in the development of MASLD. My work has identified SLC25A48 as a mitochondrial choline carrier (MChoC, SLC25A48) which regulates choline subcellular localization and metabolism. Elucidating the regulation choline metabolism under obese conditions may reveal novel mechanisms of treating MASLD. This proposal tests the hypothesis that diet induced obesity disrupts choline contribution to the methionine cycle which regulates metabolic health. A first set of studies focuses on the regulation of choline flux in the liver. I will map the fate of choline in the livers of control and MChoC liver- specific knockout (MChoC-LKO) mice through in vivo flux analysis with stable-labeled choline coupled with LC/MS. I will also determine whether overexpression of MChoC in livers of obese mice is sufficient to rescue fatty liver disease. A second set of studies will focus on choline dependent changes in epigenetics. This set of studies will expand my technical training with support from co-mentor Dr. Evan Rosen. To address the epigenetic modifications dependent on choline metabolism we will assess chromatin architecture regulated through histone methylation and translational control through polysome profiling in MChoC-LKO mice. A third set of studies will test the regulation of obesity on the choline metabolic pathway through proximal-labeling and in vivo complex assessment. Completion of these studies will expand the scope of my work, provide foundational datasets, and establish models that will be essential toward achieving my career goal of leading and developing scientists as an independent investigator.

NIH Research Projects · FY 2025 · 2025-09

ABSTRACT Digital base editors hold tremendous promise for precision gene therapies for monogenic diseases, such as severe combined immunodeficiency A (SCID-A), a primary immunodeficiency (PID). PIDs can be treated with allogeneic hematopoietic stem cell transplant (HSCT), however, >70% of patients lack suitable matched donors and of those transplanted, many encounter severe complications, such as graft-versus-host-disease and even death. An alternative and ideal approach would be genetic correction of hematopoietic stem/progenitor cells (HSPCs) in vivo, yet base editing therapies require tandem delivery of two component payloads (mRNA encoding base editor protein along with gRNA). Major clinical challenges include safe and effective development of delivery systems for two-component digital base editors, which involves packaging uniformity/stability, formulation optimization, safe/effective editing performance in vivo, and collective elucidation/prediction of structure-activity relationships. The objective of this application is to develop a build-test-learn-predict-refine closed loop machine learning (ML)-driven workflow of synthesis, formulation generation, and screening that will optimize micellar nanoparticles (MNPs) for binary base editor (gRNA and mRNA expressing adenine base editor, ABE) delivery in vitro with a novel engineered cell model and in vivo to HSPCs using a novel genetically humanized SCID-A mouse model (all developed by our team). To accomplish this, we will synthesize a novel uniform library of amphiphilic polymers for blended assembly into MNP vehicles. The proposed work is innovative and unique as a high throughput workflow will be combined with a novel multi-fidelity ML model to drive our synthesis/formulation strategy that will uniquely correlate chemistry/composition to size/stability (Aim 1), in vitro (Aim 2), and in vivo (Aim 3) performance relationships. The Specific Aims of this proposal are: Aim 1. Driven by ML, a novel library of precise amphiphilic block polymers will be synthesized, assembled into a library of compositions, formulated into MNPs with gRNA and mRNA expressing ABE, quantitatively characterized, and optimized for physicochemical properties. Aim 2. A novel engineered SCID-A cell model will enable quantitation and correlation of MNP cellular internalization, ABE editing, and toxicity to physicochemical properties via ML to identify and predict structural drivers of performance and safety in vitro. Aim 3. A novel genetically humanized SCID-A mouse model will enable quantitation and ML prediction of MNP physicochemical properties to functional in vivo ABE editing, immune system recovery, toxicity, and in vitro-in vivo performance trends. Our world-leading team is particularly well suited to undertake the proposed studies providing expertise in polymer synthesis and physicochemical characterization (Reineke), ML and computation (Sarupria), along with in vitro and in vivo model development, ABE therapies, and translational research (Moriarty and McIvor). Our pioneering and versatile MNP platform, ML, and biological discovery tools enable our long-term goals of achieving well-defined, safe, stable, and effective in vivo base editing therapy for PIDs currently treated by HSCT.

NIH Research Projects · FY 2025 · 2025-09

Abstract Cryptococcus is the most common cause of HIV-related meningitis with mortality >25%. Symptoms recur in 5–10% of survivors, typically due to either paradoxical immune reconstitution inflammatory syndrome (IRIS) with sterile cultures or culture-positive relapse of infection. Cerebrospinal fluid (CSF) cryptococcal antigen (CrAg) testing is highly effective to diagnose primary cryptococcal meningitis but cannot distinguish relapse from IRIS where culture is the gold standard. Culture’s slow turnaround (7–10 days) hinders timely clinical action. Thus, clinicians must choose empiric corticosteroids (IRIS) or amphotericin (relapse). Incorrect treatment means exposing patients to unnecessary toxicities without addressing the underlying illness, highlighting a clear clinical need for a rapid, accurate test to differentiate relapse from IRIS to improve outcomes and minimize drug-related harm. The BioFire Meningitis/Encephalitis PCR panel correctly identified 18 of 19 patients with relapse or IRIS. Yet, utility in cryptococcosis is limited by high cost, proprietary equipment, and poor sensitivity (29%) at low fungal burdens. Our Cryptococcus neoformans quantitative PCR assay showed excellent correlation with baseline CSF quantitative culture and fungal clearance rates in first-episode meningitis. Whether the assay can differentiate IRIS from relapse or detect early treatment failure has not been studied. Similarly, the roles of inadequate immune response (including due to poor antiretroviral therapy adherence), rising fluconazole minimum inhibitory concentrations (MICs), and insufficient fluconazole levels in relapse remain underexplored. Our overall objective is to reduce second episode cryptococcal meningitis mortality and morbidity by understanding the cause(s) and through cost-effective, rapid and accurate diagnostic tests. Our aims are: 1) To validate the diagnostic performance of a real-time CSF-based Cryptococcus qPCR assay for diagnosing culture-positive relapse in participants presenting with recurrent cryptococcal meningitis; 2) To determine the causes of culture-positive relapse by testing drug levels for antiretroviral and antifungal therapies, testing in vitro MICs to fluconazole, characterizing the host immune response and distinguishing new infection from recurrence by genomic sequencing of Cryptococcus yeast; and 3) To prospectively evaluate the diagnostic accuracy of Cryptococcus qPCR to detect real-time mycologic treatment failure with increasing quantitative CSF cultures. This proposal aims to transform the diagnosis of relapse from slow and often inaccurate to rapid, precise, and cost-effective. It also provides a tool to quickly identify mycological treatment failure for clinical care and trials. Additionally, the proposed work seeks to uncover relapse causes to inform future prevention strategies, such as higher fluconazole doses during secondary prophylaxis.

NIH Research Projects · FY 2025 · 2025-09

ABSTRACT: Globally, iron deficiency (ID) affects 40-50% of pregnant women, fetuses, and children. Fetal- neonatal ID acutely impairs cognitive, motor and social development in children. More troubling from a public health perspective is that neurobehavioral deficits persist despite neonatal iron treatment, causing increased risk of cognitive and neuropsychiatric disorders into adulthood. In mice, hippocampal neuron-specific ID causes long- term learning/memory neurocircuit dysfunction despite neonatal iron repletion, demonstrating the long-term effects are due to neuronal iron loss during development. The neurobiological mechanisms by which developing neurons metabolically adapt to early-life ID and that underly the long-term neurobehavioral deficits are unclear. Neuronal development depends on both iron and thyroid hormone (TH) to meet the high energy demands of rapid neuronal growth and maturation. Early-life TH deficiency (THD) causes similar neurodevelopmental impairments as ID. Excess iron and TH both cause mitochondrial stress and are toxic. ID is an independent risk factor for THD in pregnant women and children. However, the mechanistic basis for iron/TH interactions during brain development are understudied. We previously showed that fetal-neonatal dietary ID reduces serum and brain TH concentrations and impairs brain TH-regulated gene expression in neonatal rats. Reduced TH activity in the neonatal iron-deficient brain is concerning as concurrent deficits in both iron and TH could be maladaptive and cause a “double hit” to the developing brain and result in poorer outcome than either condition alone. However, an alternative hypothesis is that reduced TH action may be adaptive and protect the developing iron-deficient brain from metabolic stress by balancing the availability of iron, a critical metabolic substrate for mitochondrial ATP production with TH-mediated metabolic/growth rate. In support of this “metabolic matching” hypothesis, our published and preliminary data show that iron-deficient neurons have decreased mRNA levels for TH-regulated genes despite normal TH availability, and increased oxidative stress signaling when TH transcriptional activity is experimentally stimulated during ongoing ID. Understanding the neurobiological mechanisms underlying this iron/TH interaction and whether ID-induced THD is adaptive or maladaptive to the developing iron-deficient brain are critical gaps in knowledge that if addressed would lead to different clinical management strategies for early-life ID. In Aim 1, we will determine how iron mechanistically controls neuronal TH metabolism and activity during neuronal ID. Aim 2 will determine whether decreased TH activity is adaptive or maladaptive for the metabolic and structural development of iron-deficient neurons. Aim 3 will translate our competing “double-hit” and “metabolic matching” hypotheses to a clinically relevant rodent model of fetal- neonatal IDA to determine whether TH treatment is beneficial or detrimental to development and function of the iron-deficient brain. Our findings will either reveal a paradigm-shifting biological principle of iron/TH metabolic matching or provide a novel target, TH, for developing therapies beyond iron for early-life ID.

NIH Research Projects · FY 2025 · 2025-09

SUMMARY Type 1 diabetes (T1D) is an autoimmune disease caused by the immune mediated destruction of insulin-producing beta cells, significantly impacting human health. Tolerance to beta cells is normally maintained by regulatory T cells (Tregs). In T1D, Tregs stop protecting beta cells and leave them vulnerable to immune attack. Work in T1D models has shown that therapeutic restoration of Tregs can prevent disease progression, and clinical studies have shown the safety of this approach in humans. Despite these successes, infusion of polyclonal Tregs has limited clinical efficacy, representing key knowledge gaps. These challenges could be solved by creating “designer” Tregs that are engineered for islet antigen specificity. If successful, this technology would lead to a more personalized and effective T1D immunotherapy. We recently developed an efficient way to generate monoclonal antibodies that recognize peptides bound to MHC molecules. Using this approach we created a series of antibodies that are specific for islet-reactive peptide-MHCI and peptide-MHCII complexes, called ‘TCR-mimics’, relevant for autoimmunity in NOD mice and humans. We used these reagents as chimeric antigen receptors (CAR) to develop Tregs so they were antigen-specific. We demonstrated these CAR Tregs were specific and elicited suppressive functions. In preliminary studies we have shown in vivo feasibility by preventing autoimmune diabetes in mice. Our central hypothesis is that re-directing the specificity of Tregs can be harnessed to prevent and treat T1D. We hypothesize that engineered p:MHC CAR Tregs will provide superior suppression relative to TCR Tregs in autoimmune diabetes. We will test three specific aims; 1) Test efficacy of MHCI and MHCII CAR Treg-mediated suppression in vitro and in vivo, 2) Determine the mechanisms and differential gene expression mediating CAR Treg suppression, and 3). Evaluate synergy between CAR Tregs and anti-CD3 therapy in autoimmune diabetes. Our innovative approach, using p:MHC mAbs to generate CAR Tregs targeting islet antigens allows us to determine if engineered antigen-specific Treg will be optimal to prevent or reverse T1D pathogenesis. The results from this study will also determine the mechanism(s) for suppression for these engineered Tregs. Finally we will compare MHCI- or MHCII- restricted CAR Tregs and determine if both are required for optimal diabetes therapy either alone or in combination with anti-CD3 therapy. We are applying our knowledge to human CAR Tregs to test in preclinical models and are making additional CAR Tregs monthly. We strongly believe CAR Tregs are the next T1D therapy, and the time to focus on this therapeutic option is now.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Long COVID is a new chronic illness that represents an emerging public health crisis about which the medical community needs more information. The COVID-OUT trial was a phase 3, randomized, placebo-controlled clinical trial of early outpatient treatment of COVID-19 using a 2x3 factorial design to efficiently test 3 district treatments: metformin, ivermectin, and fluvoxamine. COVID-OUT continued follow-up assessments through 10 months after randomization. This unique long-term follow-up included monthly surveys to assess whether participants had been diagnosed with long COVID, had persistent or new symptoms, had new diagnoses, new medications, and repeat infections and vaccinations. The COVID-OUT trial also collected and quantified viral load from nasal swab samples at baseline, Days 5 and 10. This proposal seeks funding to conduct secondary analyses of already-collected data from the COVID-OUT trial to improve knowledge and understanding about long COVID. The COVID-OUT dataset is uniquely comprehensive with viral load samples and information on chronic disease development and progression, and it has less than 2% missingness for clinical outcomes during acute infection, and less than 5% missing long-term data through 9 months. Dr. Bramante started the COVID-OUT trial as a KL2 scholar, and the proposed R03 would support Dr. Bramante’s development into a fully independent translational researcher and conducting the proposed analyses will also give Dr. Bramante important insights into how to balance feasibility of clinical trial conduct and depth of data collected, which will inform future clinical trials. Aim 1 will replicate two newly emergent symptom-based definitions of long COVID, from ACTIV-6 and the RECOVER prospective cohort, in the COVID-Out monthly follow-up data. This will serve to understand whether the comparisons of medications versus placebo in the trial are supported across new definitions of long Covid. These are post-hoc, therefore hypothesis-generating analyses. Aim 1a will assess the specific symptom phenotypes identified in the RECOVER prospective cohort, and present descriptive analyses of symptoms at each month after randomization. Aim 2 will create a predictive model of Long COVID using this comprehensive dataset that includes: baseline demographic data, home medications and comorbidities, viral load data, outcomes and treatments received during initial acute COVID infection and subsequent infections, and vaccinations and boosters received before and after enrollment. This model can be repeated for the long COVID outcomes replicated in Aim 1, and will be important for adding information to the medical literature to better understand risks associated with developing long Covid. Aim 3 will assess whether changes in sleep, physical activity, and weight effect outcomes during acute COVID-19 infect or during long-term follow-up, as sleep, adiposity, and physical activity influence the immune system. The use of existing data for analyses proposed in this R03 would address communication and logistical roadblocks that exist when trying to define and efficiently research new diseases that arise in pandemic proportions.

NIH Research Projects · FY 2025 · 2025-09

PROJECT ABSTRACT Compared with dialysis, kidney transplant recipients have significantly better survival and quality of life. In the last 2 decades, short-term posttransplant patient and kidney transplant survival have improved. However, there has not been a similar improvement in long-term survival. Within 10 years after a kidney transplant, about 47% of deceased donor and about 30% of living donor transplants have failed. The 2 most common cause of late kidney transplant failure are: a) premature death despite adequate kidney transplant function; and b) slow progressive decline in function of the transplant, eventually resulting in kidney failure. Our goal is to improve long-term outcomes after kidney transplantation. In the general population, for patients with cardiovascular disease, heart failure and/or chronic kidney disease, a new class of drugs, sodium-glucose cotransporter-2 inhibitors (SGLT2i), significantly reduce death, cardiovascular disease and progression of native kidney disease. Thus, these drugs may have a major impact on the two most common reasons for late posttransplant kidney failure. This class of drugs are now recommended for persons in the general population with heart disease and/or chronic kidney disease. However, for immunosuppressed kidney transplant recipients there has been concern about whether these drugs will have similar benefits, and whether there will be an unacceptable risk of side effects. As a result, kidney transplant recipients have been excluded from large randomized trials of SGLT2is. Because of the concerns about the efficacy and side effects of these drugs in transplant recipients, and the lack of information from large trials, reviews and national and international medical guidelines state that before recommending SGLT2is for transplant recipients, large long-term randomized trials need to be done. We propose a prospective randomized trial (n=900) of a SGLT2i (dapagliflozin), compared with placebo, to study efficacy and safety of this drug in kidney transplant recipients. Enrolled participants will be followed for a minimum of 3 years. Our hypothesis is that dapagliflozin will be safe in kidney transplant recipients and will reduce death and cardiovascular disease while prolonging kidney transplant function. To maximize enrollment and diversity, we plan a pragmatic trial with no extra visits to the transplant center and minimal study-related laboratory tests. This will facilitate participation for those at long distances from a transplant center and/or those with difficulty in finding transportation. This trial represents a unique opportunity to profoundly impact the care of kidney transplant recipients. Showing that dapagliflozin improves survival after a transplant and reduces cardiovascular disease and progression of kidney disease will transform clinical care in this high-risk population.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract Resistance to chemotherapy is a major contributor to cancer relapse and treatment failure. Of many molecular mechanisms of cancer therapeutic resistance, increased drug efflux, mediated by ATP-binding cassette (ABC) transporters, results in multidrug resistance (MDR) and poses a huge barrier to effective cancer therapy. Small molecule inhibitors of efflux could be used to sensitize cancer cells toward chemotherapeutic treatment and improve treatment quality. Significantly, we have identified a highly potent lead compound, a deazaflavin analog which at single digit nanomolar concentrations drastically increased the intracellular concentration of etoposide (ETP), and to a lesser extent, that of teniposide and pemetrexed, all are important cancer chemotherapeutics. Our prior studies revealed that it is a potent inhibitor of MRP1. Notably, it displayed excellent selectivity over other major ABC transporters, including P-gp, BCRP, MRP2 and MRP3, and fully reversed the MRP1-mediated drug resistance to ETP and doxorubicin in H69AR small cell lung cancer cells. In addition, it exhibited desirable PK in preliminary mice testing without altering distribution of co-administered ETP to normal tissues in healthy mice, and hence may have limited toxicological consequences. These preliminary findings strongly suggest this lead and its analogs could be a highly valuable lead series for developing combination treatments with clinical TOP2 poisons or other anticancer drugs. The objectives of this grant application are to examine a library of deazaflavin analogs for MRP1 inhibiting activity and to further evaluate their potential for cancer chemotherapy sensitization in in vitro and in vivo models, by pursuing these specific aims: Aim 1. to screen and characterize deazaflavin analogs for MRP1 inhibition using membrane vesicle-based and cell-based drug transport assays. In this aim, we will screen an in-house deazaflavin analog library (~30 synthetic analogs) using vesicular transport assay and cell-based calcein retention assay for MRP1 inhibitory activity and characterize their potency, selectivity as well as examine the structure-activity relationship (SAR). We will also perform site-directed mutagenesis on MRP1 to identify residues that may be critical for the inhibitory effect of the analogs. Aim 2. to evaluate the deazaflavin analogs for cancer chemo- sensitization in in vitro and in vivo models. In this aim, we will first evaluate advanced analogs for chemo- sensitization in a panel of lung cancer and neuroblastoma cells toward selected chemotherapeutics and establish correlations to the MRP1 protein expression and inhibition. We will then profile the in vivo toxicity of our top analogs in mice, and further evaluate the anti-tumor effect and toxicological consequence of the analogs in combination with TOP2 poison ETP in a xenograft mouse model of multidrug resistant lung cancer. At the conclusion of the studies outlined in this proposal, a better understanding of the mechanism of action and the chemotherapeutic sensitizing potential of our lead compounds will be gained, which will lay a solid foundation for future preclinical studies.

NIH Research Projects · FY 2025 · 2025-09

Abstract Histoplasma capsulatum causes histoplasmosis, which is estimated to occur in ~500,000 persons per year in the U.S. alone and is most common cause of HIV-related mortality in Central and South America. While many cases are asymptomatic or mildly symptomatic, disseminated histoplasmosis (particularly in those with immune compromise) leads to significant morbidity and mortality. Current therapy for moderate-severe histoplasmosis consists of induction with daily liposomal amphotericin B (3mg/kg) for 2 weeks followed by at least 12 months of itraconazole consolidation therapy. Despite these treatments, 6-month mortality remains ~25%. Further, these toxic therapies commonly cause drug-drug interactions, kidney injury, electrolyte disturbances which can lead to fatal cardiac arrythmias, liver toxicity and GI symptoms, which commonly cause early discontinuation. Thus, there is a clear clinical need for improved therapies in moderate to severe histoplasmosis. Our team led a phase II clinical trial comparing single high-dose (10mg/kg) liposomal amphotericin B to daily standard dose liposomal amphotericin B for induction therapy in histoplasmosis and found similar efficacy but improved safety in the experimental arm. Posaconazole has excellent in vitro activity against Histoplasma capsulatum, case series’ showing good outcomes in a small number of human cases, and favorable tolerability compared to itraconazole. Lastly, 12 months of consolidation therapy for people with HIV is a remnant from a time when anti-retroviral therapy access and potency were poor – these have improved and six months may be adequate with lower costs and toxicity for patients. We need a large phase III trial to answer these questions. Our overall objective is to reduce morbidity and mortality due to moderate to severe histoplasmosis while improving safety and tolerability of therapy through a phase III, factorial design clinical trial. Our aims are: 1. To assess the safety and efficacy of a single high-dose liposomal amphotericin B (10mg/kg) compared to the standard of care (SOC) daily dosing (3mg/kg) for induction therapy in moderate to severe histoplasmosis. 2. To assess the safety and efficacy posaconazole for consolidation therapy compared to SOC itraconazole in moderate to severe histoplasmosis. 3. To assess the safety and efficacy of 6 months of consolidation therapy compared to the SOC 12 months of consolidation therapy in persons with HIV on appropriate antiretroviral therapy. This proposal has the potential to completely change every aspect of therapy for moderate to severe histoplasmosis. If successful, this trial would lead to histoplasmosis therapies that are less toxic, more tolerable and more affordable, translating to better outcomes in histoplasmosis and shorter hospitalizations. These results would change WHO, PAHO and U.S. guidelines for histoplasmosis.

NIH Research Projects · FY 2025 · 2025-09

ABSTRACT The Src-family kinase Lyn is critical for maintaining immune homeostasis and protecting against autoimmunity, and deficits in Lyn function are linked to systemic lupus erythematosus (SLE). However, cellular studies report conflicting (paradoxical) inflammatory and immunosuppressive functions, varying by cell and perturbation. We have discovered that the two Lyn splice forms (A, B) are differentially regulated, suggesting non-overlapping interactions and roles The apparently paradoxical positive and negative effects might be explained by isoform- specific functions that vary with expression, immune compartment, and environment. We generated single-iso- form LynAKO and LynBKO mice and discovered (1) a dominant role for LynB in protecting against autoimmunity and (2) a female-specific role for LynA in immune regulation. Our preliminary data point to overlapping roles of LynA and LynB in steady-state ITIM signaling but a specific role for LynB in inducing antimicrobial hemi-ITAM signaling. To this toolkit we add a reagent that biases splicing toward increased LYNB production in human cells. We hypothesize LynB uniquely functions in ITAM and TLR pathways, while LynA suppresses ER (estrogen- receptor) signaling, accounting for the sexual dimorphism. Alterations in LynA and LynB expression may explain the apparently paradoxical observations of net positive and negative functions and suggest a path forward for therapeutic development. We aim to 1: Assess roles of LynA and LynB in ITAM, TLR, and ER signaling in human and murine myeloid cells. As myeloid cells drive autoimmunity and have been targets of immunomod- ulatory therapies, they will be the focus of signaling studies. Candidate and unbiased kinase-substrate mapping and interactomics in macrophages and DCs will provide a comprehensive profile of LynA and LynB substrates and functions in receptor activation. Predictions: LynB will interact stably with ITAM and TLR signaling complexes and downstream mediators, whereas LynA will uniquely suppress ER signaling. LynA and LynB will be found to localize differently at the cell membrane, explaining why upregulation of the other isoform fails to rescue signal- ing. 2: Probe isoform- sex-, and cell-specific mechanisms of Lyn dysregulation in lupus progression. We will assess progression of lupus in spontaneous and inducible models in male and female mice from our Lyn knockout series. WT and LynKO experiments will be repeated mice with ovariectomy and with pharmacological antagonism/agonism of estrogen and progesterone signaling to pinpoint contributors to sexual dimorphism. We will assess cell-specific contributions to disease in bone-marrow chimeras. Finally, we will test the ability of a splice-altering reagent to suppress disease. Predictions: Upregulation of a single isoform will partially suppress lupus, cell-specificity will follow LynA/B expression patterns, sex hormones will drive lupus in females, and alter- ing LynA/B balance will modulate disease. With our Lyn knockout series, we are poised to resolve longstanding paradoxes in Lyn signaling. Our Lyn splice reagent adds translational value, testing a new therapeutic avenue.

NIH Research Projects · FY 2025 · 2025-09

Program Director/Principal Investigator (Last, First, Middle): Naselaris, Thomas PROJECT SUMMARY We propose to amass a large sampling brain activity as humans internally generate mental images. The proposed Mental Imagery Database (MID) will use high-resolution 7T fMRI to achieve large-scale sampling of brain activity during mental imagery, a timely project directly motivated by the rise of generative AI. Of the many use cases we envision, we see two as especially urgent: (1) MID will be used by neuroscientists and AI researchers to gain insights into how the brain transforms text into novel visual representations. This operation is central to current AI research and distinguishes humans from other biological intelligences. Although AI image generators have become better at making pictures than most humans, they still struggle with visual interpretations of written information that humans find easy. Very little is currently known about how human brains do this. MID will provide the needed neuroimaging data at a scale that is matched to the complexity of the problem. (2) MID will advance recent work on visual decoding that has made great strides reconstructing seen images. Such work is hampered by the lack of data for cross-generalization to mental imagery. Using MID to fine-tune vision decoders for mental imagery would bring us closer to viable technology for externalizing visual thoughts. This technology could be deployed by clinicians to help patients interrogate and control intrusive mental images of traumatic events, and aid diagnosis and communication for unresponsive patients whose consciousness is not readily apparent through standard behavioral assessments. Decoding such patients' mental images could, in principle, affirm consciousness and aid in accurate diagnoses. RELEVANCE We expect this work to deliver a method for producing high-quality reconstructions of visual mental images. This work will impact the diagnosis and treatment of mental health disorders that are driven by dysregulated visual mental imagery. Little is known about the brain systems that mediate mental imagery's role in mental health, in part because of the paucity of high-quality mental imagery data, which the proposed Mental Imagery Database will provide.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract My laboratory works at the interface of chemistry and biology on technology development for post-translational modification enzyme activity measurement. We use a broad variety of techniques including peptide chemical biology, spectroscopy, proteomics, cell biology, and analytical chemistry to create tools that help answer biological questions with molecular precision. We have spent the most time on studying and building a toolkit for measuring kinase activity, both in vitro and in live cells. We are currently working on building out our platform technology called KINATEST-ID to characterize kinase preferences, design novel substrates, and apply them in kinase assays with innovative read-outs. Our goals for the next five years are (1) to build up our understanding of kinase-substrate interactions using multiple avenues: in vitro phosphoproteomics experiments to characterize the substrate profiles of many different kinases, comparing substrate preference motifs to try to dissect differences, designing novel artificial substrate tools that could distinguish between the activities of different kinases, and using cutting-edge artificial intelligence-based structure-based computational methods like Rosetta and AlphaFold to characterize and predict determinants of substrate selectivity; and (2) to develop and implement assay read-out detection methods that can meet different levels of technical needs for drug screening, drug target validation, pharmacodynamic monitoring for companion diagnostics, as well as basic study of fundamental questions about enzyme function and interaction with substrates in cells.