University Of Minnesota

NIH Research Projects · FY 2026 · 2025-03

PROJECT ABSTRACT/SUMMARY Offspring of mothers with depression are at threefold risk of developing depression themselves, known as the intergenerational transmission of depression (IGTD). Self-regulation (SR) impairments are conceptualized as a vulnerability linking maternal and offspring depression. In infancy, SR develops in the context of infant- caregiver interactions requiring both infant and caregiver contributions. However, elevated maternal perinatal depressive symptoms—which affect one in five mothers—may hinder infant SR development. Specifically, prenatal symptoms may undermine infant physiological regulation while postnatal symptoms may undermine sensitive parenting, both of which are critical for infant-caregiver interactions that support SR. Thus, infants exposed to different timing patterns of perinatal depressive symptoms (prenatal, postnatal, or both) may experience different mechanisms of risk (i.e., prenatal symptoms may affect SR through impaired infant physiological regulation while postnatal symptoms may affect SR through reduced sensitive parenting). Importantly, these pathways likely interact to produce particularly heightened risk in the case of perinatal symptoms occurring across both the pre- and postnatal periods. The present study uses a leading-edge analytical approach to examine 1) the specific timing effects of prenatal only, postnatal only, or both pre- and postnatal depressive symptoms on infant SR and 2) the two potentially interacting pathways linking maternal perinatal symptoms to infant SR. In a longitudinal sample of 168 pregnant women, prenatal maternal depressive symptoms are assessed at 34-35 weeks gestation; postnatal maternal depressive symptoms, infant autonomic regulation, and maternal sensitivity at 2 months postpartum; and infant SR at 6 months postpartum to address my specific aims. With the support of the NRSA fellowship and my mentoring team, I will complete the proposed research and my training goals, which are to 1) integrate my existing knowledge of the perinatal and infancy periods and infant SR into models of the IGTD; 2) develop expertise in processing and interpretation of cardiorespiratory indices of autonomic functioning; 3) gain experience in behavioral coding of infant SR; 4) advance my expertise in quasi-experimental approaches for strengthening causal inference by integrating concerns about developmental timing and causal mediation; and 5) engage in professional development and ethics training. By identifying which dyads are most at risk for SR difficulties and how that risk is transmitted, the proposed project will inform targeted prevention and intervention efforts that reduce difficulties in SR during infancy and thus interrupt the IGTD, addressing a clear public health need. The training I will receive with the support of this NRSA fellowship will also prepare me to achieve my career goal of becoming a professor at an R1 research institution and continuing to pursue a research program aimed at understanding and interrupting the IGTD and reducing the substantial burden of depressive symptoms on individuals and our broader society.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY While cigarette smoking has declined nationally, its prevalence remains higher among those living in rural areas, who experience a disproportionate number of the deaths caused by smoking. Directly contributing to the rural-urban smoking disparity is a lower rate of smoking cessation among rural residing people who smoke (RPWS). Limited access to cessation treatments like nicotine replacement therapies (NRT) because of reduced heath care access is an important contributing factor. Concerns about the high costs of NRT, along with misperceptions about NRT safety and efficacy, are also common. A pragmatic strategy to increase access to cessation services is the provision of a free NRT starter kit (with NRT education) to RPWS—an approach that could be adopted by local public health departments, health organizations, and insurance companies and that has the potential to impact multiple domains of influence (i.e., behavioral, health care system). Previous research supports the potential efficacy of providing people who smoke with free NRT starter kits, but research is needed to evaluate the effectiveness of such an approach when implemented outside of primary care, since RPWS are less likely to have routine healthcare encounters, and when NRT education is offered via digital modalities, consistent with available resources and preferences voiced by RPWS. On the other hand, NRT and traditional cessation methods may not work for all RPWS. For these individuals, education about reduced harm and exposure (RHE) tobacco products (e.g. e-cigarettes, snus) may be a secondary approach to smoking cessation and to harm reduction. Overall, our goal is to inform the evidence base on what works for rural America to quit smoking and to advance health equity. We will use a randomized controlled trial (Aim 1) to determine the efficacy of providing an NRT starter kit to RPWS referred to digital smoking cessation resources. We will randomize RPWS (n=544) interested in a quit smoking program to weekly referrals to an existing digital resource (Smokefree.gov) alone or with a mailed NRT starter kit. We hypothesize that directly mailing an NRT starter kit (linked with digital NRT education) will increase smoking abstinence from combustible tobacco products at a 3-month follow-up. For those not successful in quitting using either method, we will explore the efficacy of providing education about RHE tobacco products as a potential secondary pathway towards cessation and harm reduction (Aim 2). Leveraging a SMART design, we will re-randomize these individuals to receive RHE product education messaging or continued referrals to Smokefree.gov, hypothesizing that RHE product education will increase accurate RHE product risk perceptions and intent to use them for smoking cessation at a 3-month follow-up. Finally, we will conduct interviews with a subset of participants in each trial to contextualize responses to NRT starter kits, digital cessation resources, RHE product education, and barriers/ facilitators to smoking abstinence among RPWS (Aim 3). Across aims, we will explore potential differences by factors including race, education, sex, and degree of rurality, and be guided by an advisory board of RPWS.

NIH Research Projects · FY 2026 · 2025-03

Upregulation of the purine biosynthetic protein phosphoribosylformylglycinamidine synthase (PFAS) promotes liver cancer cell proliferation and is prognostic for lower liver cancer survival rates. There is a fundamental gap in knowledge about how PFAS contributes to the progression of liver cancer. Exacerbating this challenge is a lack of molecular tools for the study of the function of PFAS. This underlines the critical need for the development of specific inhibitors of PFAS activity, both as probes of function and to form the basis for drug development. The long-term goal of our research program is to develop new anti-cancer therapies that regulate metabolic enzymes to extend life and improve patient outcomes. The overall objective of the proposed studies is to identify and validate specific PFAS inhibitors. Our central hypothesis is that pharmacological knockdown of PFAS activity will slow tumor growth in a subset of cancers and lead to improved clinical outcomes. This hypothesis has been formulated on the basis of our own preliminary data that shows PFAS knockdown slows liver cancer cell proliferation and is also supported by substantial literature associating PFAS upregulation and/or overexpression with increased tumor growth and poorer overall prognoses. The rationale for this work is that a specific inhibitor of PFAS is an essential resource needed to address critical gaps in knowledge, rigorously validate this protein as a therapeutic target in cellular, tissue, and animal models, and to serve as the foundation for the development of a novel class of chemotherapeutic. Guided by strong preliminary studies, we will accomplish our overall objective by pursuing two specific aims: 1) To identify small molecule inhibitors of PFAS enzyme activity, and 2) validate the activity and assess the selectivity, mechanism of action (MoA), cytotoxicity, and initial structure- activity relationship (SAR) of the hit compounds. To facilitate this work, we have developed, miniaturized and piloted a new fluorescent assay for PFAS activity and solved a cryo-EM structure of the human PFAS protein. We will support our hit validation with several secondary enzyme- and cell-based assays which, along with extensive cheminformatics and structural studies, will be used to establish potency, selectivity, MoA and initial SAR. The proposed work is innovative, in our opinion, because it will generate the first-of-its-kind inhibitors of a central purine metabolic protein that is strongly correlated with liver cancer survival. Additional innovative aspects include a novel assay, developed for these studies, and EM structures that will be invaluable assets for structure- function studies and in support of drug development. The proposed studies are significant because they are expected to produce validated inhibitors for use in elucidating the role that PFAS, and nucleotide metabolism in general, plays in support and promotion of liver cancer progression. Ultimately, this has the potential to transform liver cancer treatment and improve patient survival by forming the basis for the development of a new class of therapeutic that reduces the activity of PFAS.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY/ABSTRACT Cancer remains a leading cause of mortality within the United States with overall cancer incidence predicted to rise. Immunotherapies have ushered in a new age of cancer treatment, harnessing the potency of the immune system to restrict cancer without many of the side effects of conventional therapies. These immunotherapies have largely targeted T cell responses that are specific for tumor antigens. While immunotherapies leveraging tumor-specific T cells have proven powerful tools in combatting cancer, their success if context-dependent, displaying limited efficacy in many solid tumors. Part of this phenomenon results from tumor-specific T cells receiving chronic T cell receptor (TCR) signaling (caused by recognition of cognate tumor antigens) within the tumor micro-environment (TME). Chronic TCR signaling in the TME permanently renders tumor-specific T cells functionally inert, suggesting a probable limit to which we can exploit these tumor-specific T cells before they are therapeutically unviable. However, T cells are diverse in human tumors, with many incapable of recognizing tumor or tumor-associated antigens. Though these “bystander” T cells have been discounted given their lack of TCR specificity for tumors, they are spared from TCR-mediated dysfunction within the TME. Thus, bystander T cells are a promising target for novel tumor therapies. Outside of tumors, bystander T cells can become activated into innate-like killers when exposed to combinations of pro-inflammatory cytokines (a phenomenon called bystander activation); but it remains unclear if this program can be harnessed intratumorally to elicit tumor killing. Using approaches incorporating TCR-defined bystander T cells, multiple solid tumor models, and in vitro and in vivo manipulations, I have found that intratumoral bystander activation is indeed possible but differs across tumor types. My objectives are 1) to identify the mechanisms of bystander activation (and subsequent cytotoxicity) that can be harnessed for tumor killing, 2) to pinpoint and overcome tumor-intrinsic regulatory mechanisms that blunt bystander activation, and 3) to translate these findings to other tumor types. To achieve these objectives, I will employ my expertise in high-parameter single-cell analyses and animal models to uncover the mechanisms that shape bystander T cell fate and function in the TME. Hypothesis: My central hypothesis is that bystander T cells can be therapeutically leveraged in solid tumors. AIM 1: In models that permit TME bystander activation, test the hypothesis that indirect- and direct-killing mechanisms of activated bystander T cells contribute to tumor restriction. AIM 2: In models that blunt TME bystander activation, test the hypothesis that immunosuppressive pathways curb bystander activation and can be blocked to promote anti-tumor responses. My proposed experiments will elucidate the role of bystander T cells in tumors and their therapeutic viability, with the goal of developing interventions to improve cancer outcomes, which is in alignment with the mission of the NCI and NIH. .

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY Breast cancer growth and progression require complex interactions between tumor cells and their surrounding environment. Increased numbers of infiltrating inflammatory cells, in particular macrophages, correlate with poor patient prognosis in breast cancer. Our studies have focused on identifying key signaling pathways within macrophages that drive their functions within the tumor microenvironment, including those that drive anti-tumor macrophage phenotypes. Specifically, we have found that treatment of macrophages with the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) drives an immunostimulatory expression profile in macrophages that is dependent upon signal transducer and activator of transcription 5 (STAT5). Furthermore, using conditional genetic knock-out approaches, we have found that loss of STAT5 signaling in macrophages leads to enhanced mammary tumor progression. This proposal focuses on further understanding the impact of STAT5 signaling in tumor associated macrophages and determining whether modulating STAT5 in macrophages impacts breast cancer growth and progression. Based on our preliminary results, we hypothesize that loss of STAT5 in macrophages promotes an immunosuppressive environment that enhances mammary tumor growth and metastasis and that restoration of STAT5 activity in macrophages will enhance anti-tumor immune responses, leading to decreased tumor growth and metastasis. Studies proposed in Specific Aim 1 will use genetic mouse models to define the effects of myeloid STAT5 deletion on mammary tumor growth and metastasis and determine the requirement for GM-CSF for STAT5 activation in tumor associated macrophages. Studies proposed in Specific Aim 2 will define the mechanisms through which modulation of STAT5 in myeloid cells impacts tumor progression by identifying direct STAT5 target genes in macrophages and assessing the roles of STAT5-regulated chemokines to modulation of the immune environment. Studies in Specific Aim 3 will develop new antibody-based approaches to determine the impact of restoring STAT5 function in macrophages on tumor growth and metastasis and use multiplex imaging to define the cellular neighborhoods associated with STAT5-activated macrophages in human breast cancer samples. The focus of these studies is to examine the impact of modulating the STAT5 pathway selectively in macrophages with a goal of enhancing anti-tumor immune responses. Obtaining a better understanding of the mechanisms through which macrophages impact tumor progression and respond to cancer therapies will ultimately lead to the development of approaches that exploit their potential anti-tumorigenic properties for therapeutic purposes.

NIH Research Projects · FY 2026 · 2025-01

ABSTRACT Neurodegenerative diseases like Alzheimer's and related dementias (ADRD) manifest with chronic inflammation and tau pathology. Passive immunotherapies have shown some success in clinical trials, but significant disease improvement has not been achieved to date. The rapid advancement and clinical application of immune cell based therapies in oncology has opened the door to developing innovative new cell therapies to treat neurodegeneration. Monocytes and derivative macrophages (MΦ) engineered with chimeric antigen receptors (CAR-MΦ) have emerged as a promising candidate for allogeneic cell-based cancer immunotherapy. MΦ are unique from other immune cells in their capacity for tumor and tissue infiltration, and ability to phagocytosis extracellular entities including proteins, pathogens, and whole cells. The use of primary MΦ derived from patient blood is challenging as these cells cannot be engineered and expanded ex vivo like T or NK cells, making complex genetic engineering maneuvers nearly impossible. We recently developed an optimized process for production of CAR MΦ from human induced pluripotent stem cells (iPSC). Human iPSC are amenable to complex genome engineering, and we have established a scalable process for production of genetically engineered MΦ (iMΦ) for oncology applications. Here we propose redirecting our platform toward treatment of Alzheimer's disease (AD). Specifically, we will evaluate a curated list of novel iMΦ-specific CAR designs for targeting the pathogenic Tau protein and evaluate their function using a battery of biochemical and in vitro assays. Additionally, as inflammation is a key contributor to AD pathology and also a potential risk associated with transfer of immune cells to the central nervous system, we will pursue a two-pronged approach to address these challenges. First, we will utilize multiplex base editing in iPSC to deactivate key inflammatory genes in derivative iMΦ, thus rendering them inert in their ability to instigate counterproductive inflammation. Second, we will incorporate a CAR activation-responsive genetic element in our vector that allows for selective production and secretion of our recently published high potency and high selectivity TNFR1 antagonist, thus achieving an additional layer of inflammatory control specifically at the sites of Tau mediated inflammation. The collective result will be a novel, scalable, allogeneic cell platform capable of addressing both Tau mediated pathology and associated inflammation.

NIH Research Projects · FY 2026 · 2025-01

Infection with hepatitis B virus (HBV), is the most common risk factor for the development of hepatocellular carcinoma (HCC) worldwide. HBV infection can cause HCC before liver cirrhosis, thereby affecting younger persons. A recent study from our South American Liver Research Network (SALRN) found that HBV-related HCC occurs at earlier ages in Hispanics than HCC related to other liver diseases. This HBV complication is of particular concern as HCC occurs outside of the “standard” recommended age for starting HCC screening. Currently, individuals with HBV are advised to undergo ultrasonography of the liver every 6 months with the goal of “visually” identifying a tumor small enough that might be amenable to curative treatment. This visual screening approach has poor adherence due to a variety of issues, such as time commitment, and is dependent on the ultrasound operator expertise. We hypothesize that a non-visual screening approach with standardized immune-related blood biomarkers may be a sensible alternative approach. Our group recently identified a series of immune markers detected in serum of patients with hepatitis that were able to predict the future development of HCC, even when the cancer occurred two years later. In this project, we will investigate whether the hyper-immune environment, product of the continuous presence of the virus in the liver, could lead to further alterations in measurable immune analytes in serum so to predict early HCC development. Using our multinational on-going networks of SALRN and ESCALON in Latin America, we propose to cross-sectionally and prospectively evaluate peripheral immune variations in Hispanic HBV-infected individuals as markers to predict early HCC development. In Specific Aim 1, we will determine if novel immune signatures in the serum of persons with chronic HBV infection and hepatocellular carcinoma show differential expression compared to controls. We will evaluate a pre-defined panel of immune analytes via multiplex cytokine analysis comparing HBV- infected HCC cases with controls In Specific Aim 2, we will validate a novel immune signature in serum for identifying those who will progress to hepatocellular carcinoma in persons with chronic HBV infection. We will prospectively collect samples from HBV-infected Hispanics in two regions of high HBV prevalence in South America and validate our HCC-immune signature In Specific Aim 3, we will evaluate intrahepatic immune markers in HBV-infected livers at risk for HCC. We will use spatial genomics intersected with HBV staining in liver biopsies from Latin America to further understand the mechanisms behind immunomodulation of HBV-related HCC This study will provide innovative data that will impact Hispanic persons living with chronic HBV infection, seeking to create a new standard of care for liver cancer screening in this population.

NIH Research Projects · FY 2026 · 2025-01

Sudden cardiac death (SCD) resulting from malignant ventricular arrhythmia accounts for as much as one- third of all cardiac deaths in high-risk populations. Recently, we established a novel concept that organelles that take up and release Ca2+ could contribute to arrhythmic risk, especially during cardiomyopathy. Specifically, we showed that mitochondrial Ca2+ influx contributes to arrhythmic risk in nonischemic cardiomyopathy and that inhibiting mitochondrial Ca2+ influx prevents action potential prolongation, triggered activity, and arrhythmia. The ability of mitochondria to contribute to arrhythmic risk depended on: 1) organelle Ca2+ uptake and release, especially during cardiomyopathy, 2) approximation of mitochondria and the sarcoplasmic reticulum (SR), 3) amplification of mitochondrial diastolic Ca2+ release by activating SR Ca2+ release, and 4) increased sodium- calcium exchanger (NCX) current during diastole where the current effects are maximized by high membrane resistance. Cardiac lysosomes may contribute to arrhythmic risk during cardiomyopathy in a similar manner to mitochondria. Cardiac lysosomes are best known for their degradative role, but they possess all four criteria above for participation in arrhythmic risk. They maintain an intraluminal free Ca2+ concentration of ∼500 µmol/L. Lysosomal activity and interaction with SR increases in cardiomyopathy. Finally, lysosomal Ca2+ release can trigger diastolic SR Ca2+ release in noncardiac tissues. Lysosomal Ca2+ release is mediated by a two-pore channel (TPC2) and a transient receptor potential mucolipin channel (TRPML1) 13. We will present data that cardiac lysosomal TRPML1 but not TPC2 is increased in ischemic heart failure (HF). TRPML1 activation can initiate SR Ca2+ sparks and triggered activity. Conversely, TRPML1 inhibition can prevent triggered activity in HF. Finally, SR-lysosomal contact increases during HF. Hypotheses to be tested: The novel hypothesis is that lysosomes contribute to arrhythmic risk in cardiomyopathy by approximating the SR and by releasing Ca2+ during diastole causing SR diastolic Ca2+ release. We will test this hypothesis in three aims. Specific Objectives. Specific Aim 1: Determine whether a lysosomal-SR microdomain exists in ischemic HF. Specific Aim 2: Determine whether TRPML1 Ca2+ release can mediate SR diastolic Ca2+ release in ischemic HF. Specific Aim 3: Determine whether lysosomal Ca2+ release contributes to arrhythmic risk in ischemic HF. Significance. If correct, this application will establish the novel concept that lysosomes can contribute to arrhythmic risk, will reinforce the concept that Ca2+ handling organelles are important in arrhythmic risk, and will provide novel targets for antiarrhythmic therapy.

NIH Research Projects · FY 2026 · 2025-01

ABSTRACT Myelodysplastic syndromes (MDS), a group of blood disorders diagnosed in nearly 15,000 Americans annually, are characterized by patients with high-risk MDS having a median survival of less than two years. Roughly half of all MDS patients possess a mutation in an RNA splicing factor (SF) gene: most commonly U2AF1, SRSF2, or SF3B1. Mutations in U2AF1 and SRSF2 are associated with faster disease progression to secondary acute myeloid leukemia and with poorer patient outcomes. Complicating treatment, MDS-associated SF mutations are mutually exclusive and cause unique patterns of alternative splicing events with non-overlapping outcomes, based on the individual mutations. Overcoming this, previous work in the Nguyen lab identified R loops, three- stranded RNA:DNA hybrid transcription intermediates, as a unifying mechanism of disease pathogenesis induced by MDS-associated SF mutations. Recently, the lab further found that SF mutations sensitize cells to inhibitors of poly(ADP-ribose) polymerase (PARP) enzymes, and that PARP1 plays a vital role in regulating R loops and maintaining genomic stability in SF-mutant cells. The proposed project aims to expand on this prior work by elucidating the mechanism by which PARP2 regulates R loops in cells possessing MDS-associated SF mutations. Preliminary results show that PARP2 associates with R loops, and that loss of PARP2 leads to both a reduction in ADP-ribosylation signaling in SF-mutant cells, and R-loop accumulation. In contrast, loss of PARP2 had no effect on ADP-ribosylation levels following direct DNA damage alone. PARP2 was also found to interact with the R-loop-resolving helicase DDX41. The central hypothesis of this project is that PARP2 plays a unique role at R loops to prevent R-loop-associated genomic instability in SF-mutant cells. This hypothesis will be tested through two specific aims evaluating PARP2 function at R loops: 1) elucidate how PARP2 senses R loops in U2AF1S34F cells, and 2) determine the impact of DDX41 ADP-ribosylation at R loops in SF-mutant cells. Results obtained through completion of these aims will provide new insights into how cells maintain genomic stability in response to R-loop accumulation, explicate a mechanism by which PARP inhibitors sensitize SF-mutant cells, and potentially identify novel biomarkers to predict this sensitivity. This proposal will be completed at the University of Minnesota, under the co-mentorship of Dr. Hai Dang Nguyen, an expert in the DNA damage response, R-loop regulation, and hematologic disorders, and Dr. David Largaespada (co-sponsor), an expert in molecular-genetic mechanisms of pathogenesis. To complement this expertise, I will receive further guidance from my collaborators: Dr. Stanley Lee, who specializes in mouse models of splicing-factor mutant MDS, and Dr. Anthony Leung, an expert in ADP-ribosylation signaling. This mentorship team, paired with the collaborative research environment, provides a unique opportunity for the candidate to complete the research aims described in this proposal and achieve his goal of becoming a molecular translational scientist working to uncover mechanisms of pathogenesis in hematologic disorders.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY/ABSTRACT The overall long-term goal of this proposal is to provide the training, career development, and mentorship that will result in Dr. Wilhelm’s ability to develop an independent program of research that reduces tobacco-related health inequities among U.S. immigrants. The proposed study will modify an existing evidence-based parental tobacco cessation intervention (the Clinical Effort Against Secondhand Smoke Exposure, or CEASE) delivered in pediatric primary care clinics to address shared tobacco use determinants and barriers to smoking cessation treatment among Somali Americans, an immigrant population facing significant combustible tobacco use disparities. This new intervention, CEASE+, will increase access to and utilization of tobacco cessation resources for Somali parents. The goal of Aim 1 is 1a) to conduct focus groups with Somali parents who smoke and non-smokers who live with adults who smoke to identify how to modify clinic-based parental tobacco cessation treatment within pediatric primary care and 1b) to use these results to modify CEASE components and delivery to develop the new intervention, CEASE+. In Aim 2, we will conduct a pilot study of the new intervention, CEASE+, to evaluate its feasibility and acceptability for Somali parents. The goal of Aim 3 is to assess parent, clinician, and staff experiences with CEASE+ components and delivery strategies within pediatric primary care clinics to inform future modifications. Ultimately, the completion of this study will provide foundational data that will guide CEASE+ modifications for a future definitive test via a fully powered clinical trial in Somali immigrant populations. During the five years of this career development award, Dr. Wilhelm plans to develop expertise in 1) developing and refining tobacco prevention and control behavioral interventions using community-engaged research approaches, 2) using mixed methodologies within intervention development and testing, and 3) designing and conducting clinical trials. These areas of new training are essential for Dr. Wilhelm to be able to achieve her long-term career goal of becoming an independent investigator who develops and tests interventions aimed at reducing tobacco use within U.S. immigrant and refugee communities. The proposed research will be conducted within the M Health Fairview Health System, a large healthcare system affiliated with the University of Minnesota that serves a diverse population of immigrants and includes the site for Dr. Wilhelm’s clinical practice. Dr. Wilhelm has a strong mentorship team that includes world-renowned academic scientists in the fields of tobacco prevention and control and she has a rigorous career development plan that will support her continued development into a successful independent investigator. The result of this proposal and investment in Dr. Wilhelm’s continued training have the potential to dramatically reduce tobacco use disparities within U.S. immigrant populations and their downstream effects on immigrant families.

NIH Research Projects · FY 2026 · 2025-01

PROJECT ABSTRACT CD8+ T cells are pivotal players in the adaptive immune response, undergoing dynamic functional transitions crucial for mounting effective responses to antigens. Upon encountering pathogens, CD8+ T cells differentiate into effector cells responsible for pathogen clearance and memory T cells providing long-term protection against reinfections. Concurrently, migratory patterns of CD8+ T cells change with differentiation, where naïve T cells recirculate, effector and circulating memory T cells (TCIRCM) distribute into various tissues, and resident memory T cells (TRM) become localized long-term in tissues. Krüppel-like factor 2 (KLF2) emerges as a critical regulator of T cell trafficking, particularly through its control of Sphingosine-1-phosphate receptor 1 (S1PR1) expression. While expression of KLF2 or S1PR1 inhibits TRM formation, its role in regulating other aspects of T cell trafficking remains incompletely understood. Additionally, it is unclear whether KLF2 maintains memory T cell trafficking patterns beyond initial differentiation. This study aims to address these gaps by elucidating the mechanisms of S1PR1-independent KLF2-regulated CD8+ T cell trafficking and differentiation (Aim 1). Leveraging genetic manipulation and gene expression analysis, we will explore redundancy among S1PR factors and investigate non-S1PR molecules in effector and memory T cell trafficking. Furthermore, we will uncouple T cell trafficking from KLF2 expression to assess its influence on differentiation. In Aim 2, we will evaluate the role of KLF2 in reinforcing memory T cell recirculation versus tissue residency. Through inducible gene expression/deletion models, we will determine how KLF2 modulation affects TRM and TCIRCM migration characteristics post-establishment of memory. This investigation holds therapeutic implications for manipulating memory T cell localization, crucial in combating pathogens and tumors while resisting exhaustion. Overall, this comprehensive study will deepen our understanding of KLF2's multifaceted role in CD8+ T cell trafficking and function, offering insights into potential therapeutic strategies for immune modulation in various disease contexts.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract Periodontitis (PD) is one of the most common inflammatory diseases, affects hundreds of millions of people worldwide, and poses a significant global economic burden. Severe PD leads to oral mucosal inflammation, destruction of tooth-supporting bone and tooth loss. Aggregatibacter actinomycetemcomitans (Aa) is an oral microbe that is associated with severe disease, yet, how T cell recognition and the ensuing immune response to Aa contributes to immunopathology is not well understood. Thus, this proposal will explore the central hypothesis that Aa-specific T cell responses contribute to tissue destruction in Aa-associated periodontitis. In Aim 1, systemic T cell response to Aa in human PD will be assessed. Biobanked patient peripheral blood mononuclear cells (PBMC) will be co-cultured with heat-killed Aa. T cells will be isolated and assessed by flow cytometry and single cell RNA sequencing. In Aim 2, the role of Aa-specific T cells in periodontal immunopathology will be determined. T cells that respond to Aa antigen will be used to generate retrogenic T cell receptor (rgTCR) mice that produce T cells specific to Aa antigen. Aa-specific T cells will be transferred to mice followed by induction of Aa-associated experimental periodontitis. Immune response and pathology will be assessed using bone loss measurement, iterative immunostaining, flow cytometry, and single cell RNA sequencing. Successful completion of these aims will begin to uncover the role of adaptive immune response in oral disease and may pave the way for precise therapeutic interventions for PD. The proposed experiments will provide me with training in new methodologies (human and mouse T cell culture, iterative immunostaining, adoptive cell transfer) and concepts (host-microbe interactions, T cell biology), which will serve as a critical foundation to my independent research program that will study T cell response to indigenous and pathogenic microbes at the oral barrier in mice and humans. The mentored phase will occur in the laboratories of my primary mentor and co-mentor, Dr.’s Moutsopoulos and Belkaid at NIH, which have the combined expertise, equipment and facilities necessary to carry out the proposed work. Additional scientific, career, and technical support will be provided by a well-rounded team of mentor-advisors and consultants. I will meet regularly with my team to receive feedback on research and career progress. I will also complete additional training in grant/manuscript writing, lab management, mentorship, and academic job market preparation. The proposed training plan will provide necessary skills to establish and lead a competitive independent research program.

NIH Research Projects · FY 2026 · 2025-01

Lung cancer is the leading cause of cancer-related mortality in the United States and adenocarcinoma (ADC) is the most common histological type of lung cancer. Kras mutations occur in 20–40% of lung ADCs and, among Kras-mutant lung tumors (K tumors), concurrent mutations of Kras and the tumor suppressor gene LKB1 (KL tumors) ranged from 8% to 31%. Loss of function of LKB1 can also be induced through DNA methylation and phosphorylation of wild-type LKB1. Lung tumors induced by tobacco smoke and tobacco smoke carcinogens exhibit Kras mutation and methylation/phosphorylation of LKB1, thereby sharing several features with KL tumors. Tumors with alterations in both Kras and LKB1 are highly aggressive and not amenable to existing therapies, including immunotherapy. Therefore, the long-term goal of this application is to develop novel, safe and effective agents for the prevention and therapy of KL tumors. Since KL lung tumors are characterized by high levels of metabolic and oxidative stress, further enhancement oxidative stress through small molecules may preferentially induce the death of cancer cells due to their inherent vulnerability to oxidative stress. We hypothesize that mitomet, a mitochondria-targeted metformin analog, which selectively accumulates in the mitochondria of tumor cells and induces energetic and redox stress prevents malignant progression of lung tumors with co-occurring Kras and LKB1 alterations and sensitize these tumors to immune checkpoint blockade, in KL tumors. Our hypothesis is supported by extensive preliminary data. On the basis of these preliminary findings, we propose the following Specific Aims. Specific Aim 1: Assess the safety of mitomet and determine its efficacy to suppress malignant progression of NNK-induced lung tumors in mice. Specific Aim 2: Determine the efficacy of mitomet to suppress malignant progression of mouse lung tumors with co-occurring Kras and LKB1 mutations and to modulate the tumor immune microenvironment and thereby reverse the resistance of KL tumors to immunotherapy. Specific Aim 3: Compare the tumor propagating potential of CD44+ CD133+ and CD44—CD133— fractions of mouse KL tumor cells and assess if mitomet and/or immunotherapy suppress the tumor propagating capacity of CD44+ CD133+ mouse KL tumor cells. Tumor cells for these studies will be obtained from Aim 2. This proposal will elucidate whether Kras mutation and LKB1 loss-associated energetic and redox stress renders KL tumor cells differentially sensitive to mitomet. The results of these studies will lay the groundwork for future clinical trials of mitomet for the prevention and therapy of human KL lung cancer.

NIH Research Projects · FY 2025 · 2025-01

PROJECT SUMMARY In Alzheimer's disease, abnormalities in the morphology, transport, and functions of mitochondria are prevalent. Mitochondria are cellular organelles that generate energy to fuel cellular metabolism. Toxic Aβ42 is known to concentrate in mitochondria. However, how Aβ42 affects crucial mitochondrial processes that maintain mitochondrial health is still unclear. The long-term objective is to contribute to developing mechanism- based treatment strategies for Alzheimer's disease. The overarching goal of this application is to identify the mechanisms responsible for Aβ42's effects on the regulation of mitochondrial gene expression after transcription and mitochondrial dysfunction in a basic cell model. The central hypothesis is that Aβ42 interferes with mitochondrial liquid-liquid phase separation, causing morphological and functional mitochondrial defects in Alzheimer's disease. The rationale for the proposed research is that a mechanism-based determination of Aβ42's role in mitochondrial dysfunction will provide new opportunities for developing novel strategies to intervene in Alzheimer's disease. To attain the overall objective, the following two specific aims will be pursued: (Aim 1) Investigate the impact of Aβ42 on mitochondrial ribonucleoprotein granule dynamics in mitochondria.; (Aim 2) Optogenetic control of Aβ42 and mitochondrial ribonucleoprotein granules in mitochondria. At the completion of these aims, this project will provide insights into how to maintain mitochondrial health by modulating the mitochondrial liquid-liquid phase separation in Alzheimer's disease.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract Binge-spectrum eating disorders (BSED; bulimia nervosa, binge eating disorder) are associated with a high risk of mortality and debilitating health outcomes. Yet, over half of people who receive empirically- supported BSED treatments remain symptomatic and relapse is common. Existing treatments target symptoms (e.g., binge eating) vs. core BSED maintenance factors, such as poor interoception (the ability to accurately sense and connect with bodily sensations like hunger, satiety, heartbeat) and heartrate variability (HRV; a generalized neurobiological indicator of interoception and autonomic nervous system functioning). New treatments targeting these core biobehavioral mechanisms are urgently needed to reduce the burden of BSED. This research will be the first to use a multiphase optimization strategy (MOST) design to develop, refine, and assess whether a new mobile health (mHealth) interoceptive exposure intervention maps onto improvements in interoception and eating disorder (ED) symptoms among adults with BSED. Aim 1 of the K99 study is to test the feasibility, acceptability, and initial efficacy of targeting a generalized sensation (HRV) via a mHealth HRV biofeedback intervention (HRV-Bio) vs. sensations often feared by people with BSED (e.g., hunger, satiety, bloating) via a mHealth just-in-time adaptive intervention (ED-JITAI). Adults randomized to HRV-Bio (n=40) will learn to use their HRV data (assessed via sensors: Holter monitors, smartwatches) to improve their HRV via app-guided breathing tasks. Adults in ED-JITAI (n=40) will engage in body scans targeting feared sensations at times when they report worse interoception than usual (i.e., more distancing from feared sensations) via ecological momentary assessment (EMA). Data will be collected via EMA and sensors pre- and post- treatment. It is hypothesized that: (1) The feasibility and acceptability of both interventions will be evidenced by >75% treatment compliance and <20% attrition from pre- to post-treatment, with better rates expected for HRV-Bio vs. ED-JITAI; (2) Improvements in binge eating, purging, self-reported interoception (assessed via EMA), and HRV (via sensors) will be greater for ED-JITAI vs. HRV-Bio. K99 results will inform (e.g., by refining tasks) an optimized R00 study that will (Aim 2) examine the incremental benefits of targeting both generalized (HRV) and feared ED sensations among non-responders to initial interventions on short- and longer-term changes in interoception and ED symptoms. Under the guidance of expert mentors at a top-10 institution with robust resources, the applicant will work towards her career goals of launching a high-impact translational ED research program that aims to improve the understanding of associations among transdiagnostic self- regulatory processes (e.g., interoception) and ED symptoms, and translate this research into effective interventions. Training will focus on improving her skills in innovative methods (sensors, MOST design), advanced statistics, and developing mHealth interventions. This research may improve the health of adults with high morbidity and mortality risk, in line with NIMH Strategic Goal 3 (Strive for Prevention and Cures).

NIH Research Projects · FY 2026 · 2024-12

ABSTRACT Medical nutrition therapy can be an accessible, cost effective and powerful tool for treatment of type 2 diabetes mellitus (T2DM), but this potential has not been realized for all patients. A common shortcoming of dietary strategies is failure to provide personalization, despite increasing evidence showing interpersonal variability in blood glucose response to meals. This disconnect diminishes the ability of dietary interventions to optimize glycemic control and may lessen patient satisfaction, diabetes self-efficacy, and long-term diet adherence. Continuous glucose monitoring (CGM) systems give real-time glucose data that could provide a solution; data from the literature support the efficacy of CGM use during lifestyle interventions for diabetes and suggest use of CGM can improve hemoglobin A1c (HbA1c), reduce glycemic variability, and increase diabetes self-efficacy. The overall objective of this proposal is to compare medical nutrition therapy personalized by CGM feedback to three control interventions in participants with T2DM. A 12-week randomized controlled trial will enroll 72 adult participants with T2DM and baseline hemoglobin A1c of 6.8-8.5% in a factorial design (18 per intervention group): 1) Unblinded CGM/Nutrition Therapy, 2) Blinded CGM/Nutrition Therapy, 3) Unblinded CGM/No Nutrition Therapy, or 4) Blinded CGM/No Nutrition Therapy. The central hypothesis is that CGM-informed personalized nutrition therapy is superior to improve glycemic control in T2DM compared to medical nutrition therapy alone or unblinded CGM use alone. A secondary hypothesis is that CGM-informed personalized nutrition will improve patient satisfaction and diabetes self-efficacy. Specific aims are 1) to determine if medical nutrition therapy with active adjustment based on CGM data is superior to control interventions for improvement of glycemic control of T2DM, and 2) to test if this new approach is acceptable to participants with T2DM, and if it improves treatment satisfaction, diet satisfaction, and diabetes self-efficacy scores measured by previously validated questionnaires. It is anticipated that this project will demonstrate feasibility and produce preliminary data for a future fully powered effectiveness trial testing our CGM-informed personalized nutrition approach. Potential benefits of this approach include improved glycemic control and increased diabetes self-efficacy for patients with T2DM. An additional advantage is that this strategy could be easily adapted to individual and cultural food preferences, making it broadly acceptable and readily adaptable to a wide range of patients with T2DM. This project is a critical next step to plan for a longer, larger multicenter trial to definitively test the effectiveness of this new and exciting approach.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY There is an urgent and unmet need for pathophysiologically relevant, clinically validated biomarkers for SCD. We know from numerous studies and decades of experience that the lack of validated biomarkers for SCD severity results in suboptimal care and a lack of objective outcome metrics for clinical research. Even as therapeutic development is rapidly advancing, the lack of robust quantitative biomarkers also severely impedes optimization of proposed treatments, prioritization of distinct treatment modalities, as well as assessment, approval, implementation, and comparison of candidate treatments. We have known for more than a century that polymerization of sickle hemoglobin (HbS) and subsequent RBC biomechanical change is the fundamental pathologic event in SCD. Despite the dominant role of HbS polymerization in disease pathology and treatment mechanisms of action, there are no clinically validated assays to measure single RBC HbS polymerization and stiffness increases that drive the disease. Our team developed the first assay to quantify the distribution of HbS polymer among a population of RBC over the physiologic range of oxygen concentration. Moreover, we recently demonstrated the clinical utility of these measurements to distinguish patients who received gene therapy in a clinical trial from those receiving standard of care hydroxyurea even when the two groups have similar levels of fetal hemoglobin (HbF). To further the potential sensitivity of these measurements, we recently demonstrated that we could combine high throughput measurement of single cell HbS polymer content with high throughput measurements of single cell mechanical properties. Thus, we have developed a platform capable of characterizing blood samples based on the key mechanisms that drive the downstream pathologies in the disease. We hypothesize that this combination of RBC biochemical and biophysical properties could serve as a more robust clinical predictor of adverse outcomes than existing measurements. In the proposed studies, we will (1) Define the relationship between common SCD adverse clinical outcomes and single cell biochemical and mechanical properties to identify the most promising biomarkers; (2) Define the relationship between favorable and unfavorable response to SCD treatments and single cell biochemical and mechanical properties to identify the most promising biomarkers; (3) Develop a platform to measure kinetic changes in single RBC properties during deoxygenation and identify kinetic metrics that correlate with clinical outcomes in SCD. With a set of validated biomarkers we can proactively predict odds of an adverse event from steady state values obtained in clinic and monitor response to a new therapy in a rapid, objective and quantitative manner.

NIH Research Projects · FY 2026 · 2024-12

Abstract Ovarian clear cell carcinoma (OCCC) is a rare and highly lethal gynecological cancer. The majority of OCCC patients carry inactivating mutations in ARID1A, a component of the SWI/SNF chromatin- remodeling complex, leading to a lack of response to standard chemotherapy and poor prognosis. To address this unmet medical need, recent research, including from our own laboratory, has suggested that targeting mitochondrial respiration may be a promising approach for treating ARID1A-mutated tumors. However, there is still a significant knowledge gap in understanding the specific mitochondrial Electron Transport Chain (mETC) components that contribute to the dependency of ARID1A-deficient cancer cells on mitochondrial respiration. Current mitochondrial inhibitors lack specificity and often cause severe side effects. This grant proposal aims to identify unique proteomic and functional mitochondrial signatures associated with ARID1A deficiency in OCCC tumors. Additionally, it seeks to perform a genetic screen of mETC drop-out in ARID1A-proficient and deficient cells. The results from this research could reveal potential therapeutic vulnerabilities within the mETC for ARID1A- deficient tumors, not only in OCCC but also in other cancers with ARID1A mutations. The innovative aspect of this grant lies in conducting a comprehensive analysis of mETC in ARID1A wild-type and mutated tumors and performing the first-ever mETC genetic screen in ARID1A-proficient and deficient cells. The findings from this research have the potential to uncover novel therapeutic targets for ARID1A-deficient OCCC tumors and may have broader implications for unresponsive tumors in different contexts.

NIH Research Projects · FY 2025 · 2024-12

Project Abstract Defining mechanisms of CD8+ T cell immune dysfunction is critical to treating immune-mediated diseases. Bystander memory CD8+ T cells, in particular, are known to be protective to the host by supporting pathogen clearance and infection control early in an infection by producing and responding to pro-inflammatory cytokines and direct killing of infected cells. However, in some human disease contexts, dysregulation of bystander memory CD8+ T cells has led to immunopathology and poor disease outcomes. The factors that drive this phenomenon are unclear. We developed a mouse model to explore the pathological contexts and consequences of bystander memory CD8+ T cells. In our model, immunity to lymphocytic choriomeningitis virus (LCMV) leads to impaired control of an infection with the unrelated bacteria, Listeria monocytogenes (Lm). Our preliminary data indicate that mice carrying increased frequencies of LCMV-specific memory CD8+ T cells (P14 TCR transgenic cells) die following wild type (WT) Lm. Additionally, our preliminary data suggest that exaggerated inflammation is not the cause of this pathology, as LCMV-immune mice did not have improved bacterial clearance compared to naïve controls and they had markedly elevated levels of serum IL- 10, an anti-inflammatory cytokine. We hypothesize that elevated IL-10 and LCMV-specific bystander memory CD8+ T cell sensitivity to IL-10 is central to dysregulated Lm control in our model. Following this, abundant bystander memory CD8+ T cells cause fatal tissue damage by inappropriate cytolysis of uninfected cells, via innate-like mechanisms. I propose the following aims to test these hypotheses: Aim 1 will identify the function of IL-10 during a bystander Lm infection of LCMV-immune mice, by IL-10RA blockade and by ablating IL-10R on bystander memory CD8+ T cells. Aim 2 will determine the basis for bystander memory CD8+ T cell induced lethal immunopathology by modulating P14 TCR CD8+ T cells to ablate genes encoding key proteins involved in bystander immune responses. Together this proposal addresses an important knowledge gap into mechanisms of immune dysfunction induced by bystander memory CD8+ T cells.

NIH Research Projects · FY 2026 · 2024-11

PROJECT SUMMARY / ABSTRACT Metabolic pathways that Mycobacterium tuberculosis (Mtb) requires for growth represent promising targets for development of novel antibiotics to shorten tuberculosis (TB) treatment and combat multidrug resistant TB. Mtb requires the de novo riboflavin synthesis metabolic pathway for growth in vitro, but whether Mtb requires riboflavin synthesis during infection or can scavenge sufficient riboflavin from host tissues has not been directly tested. Knowledge of the Mtb riboflavin synthesis and transport mechanisms and their importance during infection will be critical to exploit these pathways for development of new TB drugs. The overall objective of this proposal is to define the importance of riboflavin synthesis and transport to Mtb survival in the host. The central hypothesis is that Mtb requires de novo riboflavin synthesis for replication and/or survival during all stages of mammalian infection, despite the existence of dedicated riboflavin transporters, because it cannot acquire sufficient riboflavin from host tissues. Preliminary data using a Mtb strain that conditionally expresses RibA2, which catalyzes the first step of riboflavin synthesis, provide compelling support for this hypothesis, which will be tested in three specific aims. Aim 1 will use the RibA2 conditional expression strain to determine the impact of riboflavin starvation on Mtb physiology and drug susceptibility and develop a panel of Mtb strains with hypomorphic expression of riboflavin synthesis enzymes to enable future drug discovery efforts. Aim 2 will determine the extent to which Mtb requires riboflavin synthesis in macrophage and mouse infection models using the RibA2 conditional expression strain. Aim 3 will use a synthetic lethal genetic screen to identify Mtb riboflavin transporters. The approach uses innovative genetic strategies to identify mechanisms of riboflavin transport and to identify synergy between riboflavin synthesis inhibition and existing anti-TB drugs. The proposed research is significant because it is expected to establish riboflavin synthesis as a viable target for development of new anti-TB drugs by demonstrating that riboflavin synthesis inhibition kills Mtb in mammalian infection models and sensitizes Mtb to existing antibiotics. The proposal will also lay the foundation for high throughput screens to identify small molecule riboflavin synthesis inhibitors with antibiotic activity.

NIH Research Projects · FY 2026 · 2024-11

PROJECT SUMMARY Endstage heart failure is a common cause of death in the U.S. and worldwide. The only curative therapy for end-stage heart disease is heart transplantation. Millions of patients (worldwide) could benefit from such therapy but are not eligible for transplantation due to limited donor organ availability. Therefore, there is an urgent need to develop alternative organ sources for cardiac transplantation. The long range goal and the clinical significance of this proposal are to use our newly engineered NKX2-5/TBX5/HAND2 knockout pigs as hosts ultimately for the production of personalized human hearts for clinical applications. The goal of the current application is to establish a nonhuman primate platform in a pig that would provide the feasibility for engineering a humanized heart in a gene edited pig. Our previous publications demonstrate the rationale and feasibility for our approach. Using CRISPR/Cas9 gene editing technology, we have established that NKX2- 5/TBX5/HAND2 mutant porcine embryos lack a heart and are lethal early during development. Using blastocyst complementation and SCNT we have further demonstrated that we can rescue the null phenotype and produce a viable chimeric pig with normal cardiac function. In these proposed studies, we will utilize a number of emerging technologies to engineer a paradigm shifting nonhuman primate heart in a genetically modified porcine surrogate. To examine our hypotheses, we will address the following specific aims: Specific Aim #1: To examine the capacity of GFP-labeled MHC mismatched porcine stem cells to rescue the porcine NKX2-5/TBX5/HAND2 null embryo; Specific Aim #2: To examine the capacity of interspecies (macaque iPSCs) stem cells to rescue the NKX2-5/TBX5/HAND2 null porcine host in vitro and Specific Aim #3: To generate a macaque heart in the NKX2-5/TBX5/HAND2 null porcine host. In these studies, we will use state-of-the-art gene technologies and macaque GFP-labeled stem cell populations to engineer a nonhuman primate heart in a gene edited pig. This nonhuman primate large animal model will be an important resource for regenerative medicine and will serve as a platform for generating personalized humanized porcine models. This strategy has the capacity to have a profound impact on the development of emerging therapies for endstage heart failure and transplantation. Given the tremendous morbidity and mortality of cardiovascular disease in our society, this proposal could have a transformative impact on the field of cardiac transplantation and the democratization of organ availability for our patients.

NIH Research Projects · FY 2026 · 2024-11

Project Summary/Abstract It is paradoxical that vertebrates do not develop immunity to their food because many of the proteins in it are completely foreign to their immune systems. This conundrum is particularly relevant for CD4+ T cells, which use T cell antigen receptors (TCRs) to recognize peptides bound to major histocompatibility complex class II (MHCII) molecules and can cause gliadin-induced celiac disease in some people. A failure to reach consensus about how the CD4+ T cell system tolerates food in most individuals but not those with celiac disease is limiting current approaches to treatment. We recently addressed this problem by using peptide:MHCII tetramer-based cell enrichment and flow cytometry to show that CD4+ T cells respond to MHCII-bound food peptides by proliferating weakly in the gut associated lymphoid tissues (GALT). The population that manages to proliferate contains regulatory T (Treg) cells and a hyporesponsive (anergic) conventional T cell population consisting of cells that lack markers of canonical CD4+ T cell subsets (Thlin–) but resemble central memory T (Tcm) cells or follicular helper T (Tfh) cells. In this proposal, we seek to understand how exposure to MHCII-bound food peptides causes cognate T cells to form the Treg and Thlin– subsets and how this process is subverted in celiac disease. We will use gene knockout approaches to identify how the Tfh-like cells form and cell transfer approaches to resolve the precursor-product relationships between the Tfh-like, Tcm-like, and Treg cells. We will use DQ8-Dd-villin-IL-15tg mice that overexpress IL-15 in the gut and develop CD4+ T cell-dependent celiac disease after gliadin ingestion to determine how gliadin peptide:HLA-DQ8-specific T cells become pathogenic. We will use peptide immunotherapy and a Treg cell-promoting IL-2 mutein to see if celiac disease can be prevented and treated. Completion of this research will fill basic knowledge gaps about CD4+ T cell tolerance of food antigens and point to possible therapies for failures of this process that lead to celiac disease.

NIH Research Projects · FY 2026 · 2024-11

ABSTRACT: STROMAL-IMMUNE CELL CROSSTALK PROMOTES AUTOIMMUNE VALVULAR CARDITIS Understanding how immune cells and stromal cells interact and communicate with one another is critical in both normal physiology and pathophysiologic states. Such interactions dictate whether, in the face of a perturbation, the tissues will eventually return to normal physiologic function or whether the tissue will experience chronic changes such as fibrosis that may impact tissue and organ function. This proposal focuses on immune-stromal cell interactions in the cardiac valves. In human diseases including rheumatic heart disease (RHD), systemic lupus erythematosus, and related immune-driven conditions, the cardiac valves become inflamed. Over time, chronic inflammation leads to fibrosis, resulting in dysfunction of the valves and culminating eventually in heart failure. Therapies are currently limited, involving primarily surgical valve repair or replacement. Our laboratory has pioneered a mouse model of autoantibody-driven valvular carditis resembling these human conditions. We have shown key roles for particular immune cells (macrophages and B cells) as well as stromal cells (fibroblasts and endothelial cells). This proposal focuses on defining the pathways by which these immune and stromal cells communicate to perpetuate disease. In the first Aim, we will investigate whether fibroblasts – termed valve interstitial cells (VICs) – promote macrophage-mediated inflammation by producing the growth factor CSF-1. We will also determine if PDGF, produced primarily by immune cells, promotes VIC proliferation and activation to drive valve fibrosis. The second Aim builds on our recent description of new lymphatic endothelial cells and vessels emerging in the inflamed cardiac valves in this mouse model. The function of these lymphatic vessels remains incompletely defined. Building on our preliminary data, we will explore the hypothesis that these lymphatic endothelial cells produce the chemokine CCL21 that interacts with CCR7-expressing B cells to perpetuate local autoantibody-driven pathology in the cardiac valves. The proposed experiments involve conditional gene knockout approaches designed to provide cell-type specific mechanistic insight regarding these stromal-immune cell crosstalk pathways. Our studies will be the among the first to investigate these cellular communication pathways in the context of immune- mediated valvular carditis and to explore the function of the valve lymphatics we recently characterized. Furthermore, we have focused on pathways for which novel therapies are currently being developed, with the hope that identification of key molecular pathways could be translated rapidly to improve care for patients with RHD and related conditions.

NIH Research Projects · FY 2026 · 2024-11

Project Summary The blacklegged tick lxodes scapularis is the major vector of tick-borne disease agents in North America, transmitting seven known human pathogens. However, despite its capability as a vector and its geographical overlap with other ticks that host pathogenic spotted fever group rickettsiae (SFGR), /. scapularis transmits no disease-causing Rickettsia species, and instead is widely infected by the endosymbiont Rickettsia tamurae subspecies buchneri (Hardt et al. 2020; hereafter R. buchnen). Presence of this symbiont has been proposed as the primary reason that/. scapularis is rarely infected by nor transmits pathogenic members of the SFGR. Intriguingly, the R. buchneri genome encodes putative antibiotic synthesis operons and a toxin/antidote module, which may contribute to the symbiont's role in preventing rickettsial superinfection of its tick host. However, beyond bioinformatic analyses, no investigation of the antibiotic activity of the products of these genes has been attempted. In tick cell culture the presence of R. buchneri has been shown to prevent infection with the human pathogen Rickettsia parkeri, and reduce infection by Anaplasma phagocytophilum and Rickettsia monacensis. Besides the effect of the encoded genes for antibacterial antagonism, we also hypothesize that the endosymbiont plays a role in modulating the immune response of its tick host to further reduce infection by tick-borne pathogens. Both these mechanisms driven by R. buchneri may play important roles in determining the tick's vector competence for human-pathogenic bacteria and shaping tick borne disease epidemiology. Furthermore, identification of antimicrobial products from R. buchneri may result in important leads for new drug development for the treatment of human infections. To improve understanding of tick-symbiont interactions, research will focus on the following Aims: Aim 1: Investigate regulation of the activity of antibiotic synthesis gene clusters and toxin/antidote genes in R. buchneri under conditions of challenge with tick-borne pathogens in vitro, and assessment of tick cell responses. Using qRT-PCR, the expression of R. buchneri's putative antibacterial antagonism genes will be analyzed in tick cells when the symbiont is present only by itself or when tick cells are additionally challenged with pathogenic R. parkeri. The expression of genes from multiple immune pathways of the tick host will also be examined through RNAseq and single-cell genomics. Aim 2. Discover the antibiotic natural products encoded in Rb genome. Homologous and heterologous expression strategies will be employed to identify and produce the target natural products for characterization. This research will clarify to what extent R. buchneri's antibacterial machinery is employed during tick infection, whether the symbiont is involved in shaping /. scapularis immunity, and will seek to identify and isolate the natural products synthesized by the symbiont.

NIH Research Projects · FY 2026 · 2024-11

Project Summary/Abstract The mechanism of DNA packaging for double-stranded DNA viruses will be studied in the Bacillus subtilis bacteriophage φ29, the most efficient in vitro viral packaging system known. Using an integrated genetic, biochemical, single-molecule and structural approach, we will characterize protein conformational change and movement in the transiently assembled packaging motor during DNA encapsidation. The mechanism of packaging in φ29 will serve as a model for animal virus packaging in the analogous herpesvirus and adenovirus systems, and aid in the search for new antiviral therapies. Due to similarities between the φ29 ATPase and other ring translocases, insights gained from the study of φ29 packaging will also provide insight into the basic principles of macromolecular motor function in higher organisms. As part of our long-term and multicomponent efforts to interrogate the mechanism of DNA packaging, here we will: 1 – Characterize the structure, dynamics, and energy landscape of DNA packaging motors. In Aim 1.1, we will use standard cryoEM SPA and nested highly focused reconstructions to test structural predictions of the helical to planar model. In Aim 1.2: we will utilize advanced image processing approaches to characterize the dynamics and energy landscape of packaging motors, and 2 – Complete single- molecule studies of ring motor mechanism. In Aim 2.1 we will use single molecule optical laser tweezers to dissect how key residues participate in critical functions during DNA translocation. Further, we will utilize combined single molecule imaging and force measurements to characterize DNA rotation during DNA packaging (Aim 2.2) and to correlate changes in motor conformation with DNA translocation (Aim 2.3).