Mayo Clinic Rochester

NIH Research Projects · FY 2026 · 2024-08

Although cancer screening mammography saves lives, previous, relatively small studies suggest some individuals who are eligible for screening are not screened. For example, sexual minority (SM) groups of people generally remain inaccessible to research and are not screened for breast cancer. This K23 career development application seeks to learn how many of these screening-eligible individuals are not screened for breast cancer and why. In aim 1, we propose to acquire the necessary – but unknown -- benchmark data on the actual rates of cancer screening mammography in SM individuals who should be screened and to identify factors associated with screening or lack thereof. We propose to interrogate and analyze data from a 200 million-patient insurance claims database (the national Optum Labs Data Warehouse). To identify SM patients, we will explore medical visit coding information. We will report cancer screening mammography rates in these SM individuals and compare these rates to the general population. This first aim promises to provide robust data on cancer screening mammography in SM individuals and will be crucial in planning potential future studies to improve screening rates in this population. In aim 2, the principal investigator (PI) will undertake qualitative interviews of SM individuals who are 40+ years of age (40 years is the age when screening mammography typically starts) to understand these individuals’ thoughts on screening mammography (and on other cancer screening). These in-depth interviews will allow us to learn directly from these individuals how life-saving cancer screening procedures can be made more accessible. These 2 aims – in conjunction with the completion of relevant coursework and ongoing interactions with a multidisciplinary team of senior mentors (an endocrinologist; a medical oncologist; a statistician; a qualitative researcher; a primary care physician; and an epidemiologist who focuses on screening mammography) -- will enable the PI, a medical oncologist, to develop the necessary skills to emerge as a national leader in cancer care in underscreened individuals. The ultimate goal of this research is to help SM individuals receive the same lifesaving benefits of cancer screening as the general population.

NIH Research Projects · FY 2025 · 2024-08

PROJECT DESCRIPTION/ABSTRACT MDM2 amplification occurs in 3.5% of all malignancies and in approximately 10% of several highly aggressive malignancies, including glioblastoma. MDM2 is a critical negative regulator of the tumor suppressor p53, and high-level MDM2 amplification results in functional inactivation of p53 and is a genomic-driver event in tumorigenesis of these tumors. Even in tumors with ‘normal’ MDM2 expression, disruption of the MDM2/p53 pathway can trigger apoptosis. In this context, multiple pharmaceutical companies have developed potent MDM2 inhibitors that are in advanced clinical development for a variety of malignancies. In our studies with a highly potent MDM2 inhibitor from Boehringer Ingelheim (Brigimadlin; BI 907828), we found that MDM2- amplified glioblastoma (GBM) and sarcomas are exquisitely sensitive to this drug. Specific to MDM2-amplified tumors, drug treatment is associated with degradation of the pro-survival MCL1 protein and upregulation of other pro-apoptotic proteins. Moreover, brigimadlin treatment results in suppression of multiple DNA repair pathways that are critical for recovery from both endogenous and exogenous genotoxic stress. Based on these observations, we hypothesize that the combination of enhanced apoptosis and an inability to repair DNA damage creates a unique vulnerability of MDM2-amplified GBM to brigimadlin. We will test this in three Aims: Aim 1: Evaluate mechanism of exquisite MDM2 inhibitor sensitivity in MDM2 amplified GBM Aim 2: Define the effects of brigimadlin on cellular response to genotoxic stress Aim 3: Evaluate strategies to enhance the therapeutic window for MDM2 inhibitors in GBM

NIH Research Projects · FY 2025 · 2024-08

ABSTRACT Chronic pancreatitis (CP) and recurrent acute pancreatitis (RAP) continue to increase worldwide and in the U.S. and are associated with Diabetes Mellitus (CP-DM). Observational studies indicate higher morbidity in individuals with CP-DM compared to Type 2 Diabetes mellitus (T2DM). Observational mostly retrospective studies also describe worse glycemic control in CP-DM compared to T2DM; earlier and increased need for insulin, and an increased risk of severe hypoglycemia requiring assistance from third party in CP-DM when compared to T2DM significantly impacting the quality of life of these patients. Therefore, there is a need to test better and specific antihyperglycemic therapy for CP-DM with scientifically rigorous clinical trials. To our knowledge, very few or no randomized controlled trials (RCTs) have evaluated the efficacy of antihyperglycemic therapies when compared to control or to each other in CP-DM. Animal model data indicate that pioglitazone (PIO) may be a safe and effective treatment for hyperglycemia in pancreatitis. Extensive human investigations indicate that PIO improves insulin resistance and insulin secretion in T2DM. When tested in T2DM, RCTs of PIO have shown antihyperglycemic efficacy, durability, and favorable impact on cardiovascular (CV) disease and hyperlipidemia. PIO also decreases systemic inflammation. Safety of PIO has been extensively studied and confirmed though it should be used with caution in the long-term. SGLT2 inhibitors particularly Empagliflozin (EMPA) also improve hyperglycemia, decrease blood pressure, and body weight and decrease CV and renal adverse events in T2D. They could also be associated with unique adverse events. We are proposing a pilot, parallel group, randomized, dose escalation clinical trial of PIO versus EMPA lasting 24 weeks with dose escalation at 12 weeks or earlier based on pre-specified rules at 2 of the sites (Mayo Clinic, Rochester, MN and U of Pittsburgh, Pittsburgh, PA) in the NIH funded consortium for chronic pancreatitis, diabetes mellitus and pancreatic cancer (CPDPC). Specific Aim 1 will test the hypothesis that HbA1c lowering with PIO is non-inferior to EMPA in CP-DM with primary end point of the trial being improvement in Hemoglobin A1c. Secondary end points are multiple and include other measures of efficacy, mechanism, and safety. This is a pilot clinical trial for CP-DM without a precedent for this type of study; this is a challenge and precedent in itself for trial design but successful completion of this trial will enable the design and conduct of a multi-center RCT intervention to be conducted by the CPDPC in a larger CP-DM population to improve glycemic control.

NIH Research Projects · FY 2026 · 2024-08

There is now extensive evidence that cellular senescence drives age-related bone loss and multiple other aging co-morbidities. Characteristics of senescent cells include increased expression of p16Ink4a and p21Cip1 as well as resistance to apoptosis. In previous work, we have systematically identified senescent cells in the bone microenvironment and demonstrated a causal role, specifically for p16+ senescent cells, in mediating age- related bone loss in mice. It is becoming clear, however, that p16+ cells in the bone microenvironment are a heterogeneous population, and a better understanding of this heterogeneity is critical to developing new approaches to target these cells and prevent age-related bone loss, along with ameliorating other aging co- morbidities. To facilitate this, we have recently developed and validated multiparametric single-cell protein analysis by cytometry by time of flight (CyTOF) to perform a deep characterization of p16+ cells in mice. These studies identified a specific sub-population of p16+ cells that are growth-arrested (Ki67-) and express high levels of senescent-associated secretory phenotype (SASP) markers as well as the anti-apoptosis protein, BCL-2 (p16+/Ki67-/BCL-2+, “p16KB” cells), thus meeting all of the criteria for defining a senescent cell. Interestingly, we also identified non-senescent p16+/Ki67+ cells that express a SASP unique from that of the p16KB cells. These proliferating, inflammatory p16+ cells have also been identified by others in the lung using a highly sensitive p16Ink4a reporter, yet the role of this population in aging remains unclear. Taken together, these findings lead to the hypothesis that p16KB cells are truly senescent, while p16+/Ki67+ cells are inflammatory cycling cells, and perhaps “pre-senescent.” However, the role of each population in driving age-related disease remains unclear and Aim 1 proposes to characterize these populations in detail. In additional studies, we have developed and validated a novel mouse model, p16-LOX-ATTAC, capable of temporal- and cell-specific senescent cell clearance. Using these mice, we found that in contrast to global clearance of senescent cells using the (p16)INK- ATTAC model, clearance specifically of senescent osteocytes only partially replicated the beneficial skeletal effects of global senescent cell clearance, indicating an important role for other cells in the bone microenvironment in contributing to skeletal aging. Consistent with this, our CyTOF analyses have identified a novel p16+, highly inflammatory (SASP+) CD24high osteolineage population (negative for stem cell markers, e.g., Sca-1, CD200) in aged mice that, along with the late osteoblast/osteocytic cells, is robustly cleared by senolytic interventions. These studies indicate that although osteocyte senescence clearly contributes to age-related bone loss, p16+, CD24high osteolineage cells are also potential candidate cells that mediate skeletal aging and thus need to be further characterized, as proposed in Aim 2. Thus, the overall goal of our application is to leverage our novel tools and mouse models in order to better define the mechanisms driving cellular senescence in the bone microenvironment and to characterize the key senescent cell populations that contribute to skeletal aging.

NIH Research Projects · FY 2026 · 2024-08

ABSTRACT The studies in the proposal are focused on the neuromotor system in Alzheimer’s disease (AD) and natural aging. Increased age is associated with muscle atrophy and weakness (sarcopenia) and is a significant predictor of chronic disease and mortality in the elderly. Aging is a major risk factor for conditions such as AD and obstructive sleep apnea (OSA). In the elderly population, pneumonia incidence is 3-times higher than in younger age groups, with AD further increasing the incidence and severity of airway infections. The incidence of airway infection in aging and age-associated disorders is undoubtedly related to sarcopenia of the diaphragm muscles (DIAm) and discoordination of airway protective manoeuvres, which involve both DIAm and an assortment of other respiratory-associated muscles including individual tongue, chest wall and abdominal muscles. This proposal leverages the extensive experience of the PI in both respiratory neuromotor systems and neurodegeneration. Previously, we found that DIAm sarcopenia was related to a loss of larger phrenic motor neurons (MNs) and subsequent denervation, consistent with motor unit specific effects on maximum transdiaphragmatic pressure generation. Our preliminary observations in both intrinsic and extrinsic tongue and external abdominal oblique muscles also suggest sarcopenia in tongue may also be due to denervation. Despite the cause of age-related MN loss being unknown, clues from neurodegenerative conditions that affect MNs suggest that synaptic loss and mitochondrial disfunctions contribute to MN death, with disproportionate effects on larger MNs. The major conceptual advancement in this proposal is to comprehensively evaluate the entire motor unit: hypoglossal, thoracic and phrenic MNs – recruited to perform motor tasks; neuromuscular junctions; and tongue, chest, abdominal and DIAm muscles. We hypothesize that in old age and AD, motor impairments and loss of larger MNs (denervation) of respiratory muscles is underpinned by MN and NMJ synapse loss and mitochondrial dysfunction (reduced volume density, fragmentation, and activity). In addition, we will trial two approaches to ameliorate the contribution of synaptic loss (via SPG302) or mitochondrial dysfunction (via edaravone) to MN death in AD and aging. The proposed studies employ an array of innovative techniques, with assessments ranging from sub-cellular through to system level behavior in Fischer 344 rats and in an AD model (TgF344-AD) on the same genetic background. In Aim 1, we will assess excitatory and inhibitory synapse loss, dendritic and dendritic spine loss, and survival of hypoglossal, thoracic and phrenic MNs. Additionally, we will evaluate denervation, sarcopenia and functional impairments in tongue, chest, abdominal and DIAm muscles across aging and AD. In Aim 2, we will assess mitochondrial volume density (MVD), fragmentation and function (SDHmax) in respiratory MNs and in muscles in aging and AD. In Aim 3, we will assess whether mitigating synaptic dysfunction (by SPG302) and/or mitochondrial dysfunction (by edaravone) improves outcomes in respiratory MNs and muscles in aging and AD.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY Anomalies of the face invariably require some type of therapy, corrective surgery, and close follow-up while imposing a financial and emotional burden on patients and their families. Although the analysis of human data is critical, human studies pose particular problems, not the least of which is that critical times of prenatal development are not available for study. This proposal aims to identify the developmental and molecular processes underlying mandibulofacial anomalies using mouse mutants that we identified by systematically searching the current contents of the International Mouse Phenotyping Consortium (IMPC) in response to NIH PAR-20-137 for phenotyping IMPC embryonic and perinatal lethal KO mouse lines. Micro- or retrognathia are the most common terms used to describe mandibular phenotypes in mandibulofacial dysostosis, yet the current lack of precision in diagnoses of mandibular dysmorphology does not critically consider the potentially distinct etiology of these conditions and their influence on potential sequelae of anomalies. Micrognathia describes a mandible that is absolutely reduced in size, indicating that the mandible is primarily affected, while retrognathia refers to a normally sized mandible that is placed posteriorly relative to the upper jaw. Thus, micrognathia and retrognathia, while providing similar facial profiles, are produced by different primary developmental processes and each may integrate differently with tongue and palatal development. When mandibular dysmorphology occurs with glossoptosis, respiratory obstruction, and in some cases, a cleft palate, the condition is referred to as Pierre Robin (PR). A causative pathogenesis of a sequence of developmental events has been hypothesized for PR, but few clear causal relationships between discovered mutations, dysregulated gene expression, precise cellular processes, and PR-associated anomalies are documented. To test this hypothesis, we plan a carefully coordinated and fully collaborative set of analyses of IMPC mutant mouse lines identified based on genes known to be causative for PR in humans or on the presence of PR features recorded in these mouse lines. Our in-depth phenotyping will involve: Aim 1: quantitative 3D morphologic analyses of embryos using phosphotungstic acid-enhanced micro computed tomography; Aim2: differential gene expression analysis of relevant tissues and developmental time points between mutant and unaffected littermate controls using bulk RNA-seq and spatial transcriptomics; Aim 3: histologic studies using in situ hybridization or immunohistochemistry of relevant genes, signaling pathways, cellular processes, and differentiation states to determine the cellular and molecular events giving rise to dysmorphogenesis of the mandibulofacial complex. This multi-level, systems biology approach will provide precise definitions of the localized effects of genetic variants on mandibular and associated tongue, palatal, and upper airway phenotypes to identify the developmental and molecular functions involved in their production, using mouse lines that model the phenotypes associated with these conditions.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY Following HIV infection, a subset of T cells do not die and can revert to a latent state of infection. Much research over the past decade has consistently found that cells which persist are enriched for the antiapoptotic protein Bcl2. Consequently, many groups have been studying the selective Bcl2 antagonist Venetoclax and have now shown that Venetoclax augments death of HIV infected cells during acute HIV infection, IL-7 induced homeostatic proliferation, during reactivation from latency alone and in the presence of autologous HIV specific T cells, and in a humanized mouse model of acute HIV infection where Venetoclax monotherapy caused greater killing of HIV infected cells, normalized CD4:CD8 ratios, and reduced viral load and HIV reservoir size. Like many proteins, Bcl2 can undergo post translational modification by phosphorylation which alters the ability of Bcl2 to bind death inducing proteins such as Casp8p41, which is an HIV specific death stimulus. Bcl2 phosphorylation also alters the ability of Venetoclax to bind Bcl2 and promote HIV clearance. In this application, we present preliminary and novel data showing that during HIV infection, both in vitro and in lymphoid tissues from HIV infected subjects, Bcl2 is pervasively phosphorylated. To comprehensively study the effects of Bcl2 phosphorylation of HIV replication kinetics, cell killing and Venetoclax effects, we have generated for the first time a Bcl2 CRISPR knockout Jurkat cell line and have also generated Jurkats which stably express different Bcl2 variants with phospho mimic substitutions, or phospho resistant substitutions at residues known to be susceptible to phosphorylation (at positions 56, 69, 70, 74, 87). Initial results using those cells indicates that phospho Bcl2 cells are more resistant to HIV induced cell death and are resistant to the anti-HIV effects of Venetoclax. Given that Venetoclax has anti-HIV and anti-reservoir effects, it is highly relevant to understand the determinants of Venetoclax effectiveness and, more broadly, determinants of Bcl2 function. Studies proposed in this application will define the mechanism and sites of Bcl2 phosphorylation, the impact of those phosphorylation events of HIV replication kinetics, the number of HIV DNA positive cells, and the anti-HIV effects of Venetoclax. In addition, we will test clinically relevant strategies to overcome the functional consequences of phosphorylated Bcl2, enabling more HIV infected cells to die after reactivation from latency.

NIH Research Projects · FY 2025 · 2024-08

Abstract/Summary PAD is characterized by impaired blood flow to the lower extremities, causing claudication and exercise intolerance. Up to now, assessment of PAD is mainly performed by determination of stenosis or occlusion in the large arteries and does not focus on microcirculation. Impaired arteriolar endothelial function, microvascular responsiveness to specific stimuli in patients with PAD may play a central role in disease development and progression. The impaired microvascular reactivity can be assessed by hyperemic response to contraction and cuff occlusion as well as in response to submaximal exercise. Therefore, imaging microvascular flow reactivity at various scales using methods that are safe, noninvasive, reliably quantitative, and low-cost through application of already widely available technology is critically important for early diagnosis and management of PAD. Here, we propose a novel contrast-free ultrasound pressure-based method equipped with novel quantitative metrics that involves estimation of blood flow signals in the calf muscle through processing ultrasound data frames by means of coherent ensemble selection, clutter filtering using singular value decomposition, and background noise equalization, followed by quantitative analysis of the flow variations in response to external pressure and submaximal exercise. Our vision is to complement US with additional quantitative information of microvascular flow imaging that is relevant to disease progression. Our long-term goal is to develop a new noninvasive tool for early diagnosis of PAD. A secondary gain from such an imaging method will be monitoring the disease progression and the response to interventional treatment in PAD patients. The proposed contrast-free ultrasound-based technique is named Angio Flow Reactivity Analysis (AFRA). An advantage of this technique is that it does not require the use of contrast agents to produce high-resolution images of the microvasculature flow. The project includes 2 specific aims: Specific Aim 1- Determine the efficacy of the new contrast-free US-based method, AFRA, for assessment of PAD in patient volunteers with symptoms of claudication or atypical leg syndrome and correlate the results with ABI and CTA. Specific Aim #2- Determine the efficacy of the proposed contrast-free US-based method, AFRA, for assessment of disease progression and monitoring the response to interventional treatment in patients diagnosed with PAD and correlate and the results with ABI and CTA. This proposal is the result of collaboration among several experts in the field and benefits from the world-class clinical researchers and facilities at Mayo Clinic. The overall project aims to develop a new contrast-free ultrasound tool for evaluating microvascular flow changes in the lower leg of patients at risk for PAD, a disease that leads to common disabilities, amputation, and major cardiovascular events. Successful completion of this research will open the door for a new technology for this group of patients in the clinic.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT This application is a request for funding of a vaccine immunogenetics research program focused on identifying critical genetic and phenotypic determinants of COVID-19 infection and/or vaccination-induced immunity by examining associations between gene polymorphisms, HLA allelic variation, and clinical phenotypes and inter- individual variations in immune response to COVID-19. Our laboratory has done significant work delineating the effect of gene polymorphisms on mumps, measles, rubella, influenza, and smallpox vaccine immune responses. Our research demonstrates that variations in immune responses to viral vaccines are multigenic and not a single dominant allele model and that the genetic contribution to such variations in immune responses can be quantified. Informed by insights from these studies and given the public health importance of the ongoing SARS-CoV-2 pandemic, we now turn our attention to understanding genetic associations with COVID-19-induced immune responses (regardless of whether they are vaccine or infection-induced, or a result of hybrid immunity). The most thorough and efficient study for such purposes is a comprehensive discovery/replication genome-wide association study (GWAS), to which we will add a phenome association study (PheWAS) followed by a more complete characterization of distinct immune effector mechanisms believed to contribute to protective immunity. Our studies will identify gene polymorphisms and pathways having the largest or most critical impact on inter-individual variations in immunity among subjects, and how comorbidities (phenotypes) contribute to these variations. Our Specific Aims are to 1) perform a large, genome-wide association study to identify novel genetic associations between SNPs and HLA alleles and inter-individual variations in the humoral immune response to COVID-19 infection or vaccination; identify polygenic risk scores predictive of antibody response; replicate the findings in an independent cohort; evaluate significant findings in a cohort with documented infection and no vaccination; 2) conduct a phenome-wide association study to quantify the effect of additional subject characteristics (e.g., age, sex, race, ethnicity, BMI), comorbidities, and phecodes (clusters of ICD10 codes) on the antibody response to COVID-19 vaccines; and 3) Evaluate the effect of genetic variation and host factors on T cell and B cell markers of immune response following vaccination. These aims will allow us to comprehensively define how inter-individual variations in immune responses to COVID-19 vaccine are influenced by gene polymorphisms and host characteristics. Notably, despite the public health implications, there are no population-based studies identifying associations between COVID-19 vaccine immune response and genome-wide SNPs or clinical phenotypes. Our study is carefully designed to be rigorous and produce robust, unbiased, and reproducible results applicable to the US population.

NIH Research Projects · FY 2026 · 2024-08

Project Summary The mammalian circadian system regulates metabolic processes over a 24-hour cycle and molecular circadian clocks control functional tissue specific and cell autonomous oscillations. Adipocytes, the primary cell type in adipose tissue, are central to regulating free fatty acid metabolism; dysregulation of free fatty acid release from adipocytes (i.e. lipolysis) is central to the pathophysiology of obesity and contributes to lipotoxicity in other tissues such as pancreas, liver, and muscle. Shift workers have disruptions in the circadian system and are at increased risk of obesity and metabolic disease. Adipocytes have a cell autonomous cycling molecular circadian clock and the literature suggests that adipocyte metabolism and lipolysis pathways are regulated by cell intrinsic circadian rhythms that are altered in obesity. However, adipocytes are highly responsive to both nutrient delivery and systemic hormonal signals driven by intermittent meal intake, making many secreted adipocyte products appear rhythmic over a 24 h fasting/feeding cycle. Therefore, the relative control of adipose tissue by feeding versus molecular clock driven mechanisms in humans is not understood. As such, the overall goal of this application is to define the molecular and systemic role of intrinsic adipocyte molecular circadian clock and how this is impacted by obesity in humans. In this context, Aim 1 will establish whether human subcutaneous adipose tissue exhibits autonomous systemic and molecular circadian rhythmicity independent of diurnal nutrient delivery in vivo in normal weight humans. Aim 2 will interrogate whether obesity alters adipocyte specific rhythms in circadian and lipolysis genes and is related to measures of systemic adipose tissue function. Finally, Aim 3 will interrogate the impact of feeding regime and obesity on the adipocyte specific cistrome of core circadian clock transcription factors allowing a more complete understanding of genomic regulation of adipocyte clock. This work will be the first work to address the relative contribution of intrinsic adipocyte circadian clock during continuous feeding versus adipocyte function driven by intermittent food intake in humans and interrogate circadian clock in the context of obesity. Answering these fundamental questions is essential to understanding whether disruptions in circadian molecular clocks contribute to metabolic abnormalities in obesity and these studies will provide a regulatory paradigm for circadian clock in adipocytes. Importantly, these proposed research activities with an outstanding mentorship team and the intellectually enriching environment at Mayo Clinic will build upon the applicant’s training and provide opportunities to expand her clinical translational knowledge and skillset culminating in the development of a research niche for the applicant.

NIH Research Projects · FY 2024 · 2024-07

Abstract Most gene-based vaccines are replication-defective mRNA, DNA, or adenovirus (Ad) vaccines. In each case, the vaccine delivers its one copy of an antigen gene and expresses "1X" of the antigens that are encoded by the vaccine. We developed single cycle Ad (SC-Ad) vaccines that replicate vaccine antigen genes up to 10,000-fold in every cell to amplify antigen production but do not produce infectious progeny viruses. When RD-Ad and SC- Ad6 expressing HIV-1 antigens or SARS-CoV-2 spike are compared, SC-Ad produces 100 times more antigen than RD-Ad and generates significantly higher antibodies than RD-Ad-Spike or mRNA vaccines. When spike- immunized animals were challenged 10.5 months after single immunization, SC-Ad reduced SARS-CoV-2 lung viral loads and damage and preserved body weights better than RD-Ad. During the COVID-19 pandemic, we tested SC-Ads expressing SIV gag and clade C HIV-1 Env in rhesus macaques by intramuscular (IM), intranasal (IN), and intravaginal (IVAG) routes of immunization in combination with IM co-immunization with adjuvanted clade C SOSIP protein. When the macaques were challenged vaginally with clade C SHIV.CH505.375H.dCT 1.5 years after last vaccine, PBS, IN, and IM vaccinated animals became infected with similar kinetics with only one IN animal resisting infection. In contrast, 50% of the IVAG macaques resisted vaginal challenge. These data suggest there are great merits to creating a mucosal barrier against incoming SHIV at the site of viral entry. Given this, this project will determine the reproducibility of this protection and examine how vaginal immunization drives local-regional immune responses in the female reproductive tract (FRT) and if these responses might be amplified by co-immunization in this site. Proof of concept here for being able to “push” immune responses into the FRT in macaques will allow these approaches to be translated for humans, to protect not just women, but all sexes and gender identities from vaginal, rectal, or penile exposures to HIV-1.

NIH Research Projects · FY 2025 · 2024-07

Our preliminary data suggest that inflamed adipose tissue may contribute to blunted exercise response in skeletal muscle of older adults. The objective of this project is to evaluate a hypothesis that inflamed adipose secretes factors that activate inflammatory cascades in skeletal muscle, which may interfere with exercise- responsive molecular pathways and contribute to dysfunctional muscle phenotypes with aging. Aim 1 will determine how adipose tissue influences skeletal muscle function and anabolic response to exercise in older adults. A combination of in vivo imaging and molecular phenotyping will be used to characterize abdominal adipose tissue (AAT) and intermuscular adipose tissue (IMAT) in young and older adults. Molecular response to acute exercise will be determined from protein synthesis rates, exercise- responsive mRNAs, and activation of signaling proteins in muscle. This aim will evaluate the relationship between adipose tissue inflammation and skeletal muscle phenotypes and function in older adults. Aim 2 will identify the molecular pathways by which AAT and IMAT influence muscle phenotype and exercise response in older adults. Primary muscle cultures and adipose conditioned media (AAT, IMAT) will be generated from tissue collected in aim 1. Myotubes will be exposed to adipose explant media or serum from inflamed or non-inflamed donors to evaluate their influence on molecular phenotype and response to exercise mimetics in vitro. Chemical inhibition of canonical inflammatory pathways in muscle will be used to determine their role in attenuating molecular response to exercise. Molecular profiling of conditioned media and serum will be used to identify candidate molecules for subsequent experiments to assess their individual influence on canonical exercise response pathways. The contribution of the proposed research is expected to be in the form of new insights into the paracrine/endocrine influence of adipose tissue on skeletal muscle responsiveness to exercise in older adults. The knowledge gained in the proposed study will have a positive impact because anabolic resistance is common in older adults and believed to contribute to sarcopenia, frailty, and loss of independence. This work will provide insight into the link between adipose tissue dysfunction and skeletal muscle biology in aging with a particular focus on distinct adipose tissue pools that are likely to have unique endocrine or paracrine influence on skeletal muscle. We will also gain new knowledge of how targeting inflammatory pathways in adipose tissue and skeletal muscle may enhance acute adaptations to exercise in older adults.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Binge-eating disorder (BED) and bulimia nervosa (BN) are potentially life-threatening eating disorders that share behavioral and brain similarities, genetic risk factors and higher-than-expected comorbidities with drug addiction – suggesting a common etiology. However, no mechanistic study has examined this possibility due in part to the lack of an animal model linking eating disorders and drug addiction. Like drug craving and use in drug addiction, food craving and eating in BED/BN persist despite adverse consequences (punishment). Our finding from rats indicates that extensive cocaine and alcohol histories, known to trigger addiction-like brain changes and punishment-resistant “compulsive” drug intake in rats, trigger punishment-resistant food intake or “compulsive appetite”. These results provide an animal model for studying the neurobiological mechanisms manifesting as compulsive behavior across eating disorders and drug addiction. Food motivation is thought to be regulated by both homeostatic (caloric) and non-homeostatic (hedonic/incentive) systems. The homeostatic system detects energy shortages and elicits food intake. However, like compulsive drug motivation, our finding suggests that compulsive appetite is driven by non-homeostatic ‘motivational/habitual’ dysregulation. Like cocaine and alcohol histories, obesogenic diet histories also led to compulsive appetite via non-homeostatic dysregulation. Thus, similarly common – rather than history-specific – changes in brain sites that control non- homeostatic regulation, such as reward circuits, likely cause compulsive appetite. Our collaborator Dr. Jhou’s group has found that punishments suppress appetitive behavior by recruiting neurons in the rostromedial tegmental nucleus (RMTg), which in turn inhibits reward circuits. Available evidence indicates that extensive drug histories [1] degrade excitatory afferents to RMTg, [2] decrease punishment-reactivity of RMTg neurons and [3] impair inhibitory control of RMTg efferents on reward circuits. Such brain changes would entail “less brakes” on non-homeostatic regulation, potentially manifesting as compulsive appetite. Accordingly, like extensive cocaine/alcohol/obesogenic diet histories, [4] RMTg inactivation results in punishment-resistant compulsive appetite. Based on the rigor of previous research and premise above, this project will test the central hypothesis that extensive cocaine/alcohol/obesogenic diet histories result in punishment-resistant compulsive appetite via decreased neural punishment-reactivity in the RMTg circuitry. RMTg contains neurons selectively reactive to punishments or rewards – likely exerting distinct behavioral functions. Each Aim is thus designed to selectively profile and interrogate punishment-reactive RMTg neurons/afferents/efferents (as Aims 1/2/3) using neural activity-specific methods based on the activation marker Fos. The results will reveal neural activity network reorganizations that are functionally linked to compulsive appetite – an overlapping ramification of extensive drug and obesogenic diet histories. Our hope is that such knowledge will help identify common therapeutic targets for compulsive behavior across drug addiction and eating disorders.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY Obesity is a chronic, relapsing, and multifactorial disease, with a prevalence of 42%. Obesity treatment is challenging in clinical practice because of the physiologic and behavioral adaptations that occur during the weight-reduced state to preserve energy. Our overall goal is to develop an evidence-based, phenotype-guided approach for obesity treatment that enhances weight loss and induces weight loss maintenance despite weight- reduced state adaptations. Our research has identified obesity phenotypes based on energy homeostasis and behavioral traits. Obesity phenotypes include abnormal satiation (i.e., requiring more calories at each meal to achieve fullness), abnormal postprandial satiety (i.e., accelerated gastric emptying and increased postprandial hunger), emotional eating (i.e., eating in response to positive or negative emotions), and abnormal resting energy expenditure (i.e., low resting energy expenditure). In pilot clinical studies, these obesity phenotypes have predicted weight loss response to anti-obesity medications and bariatric endoscopic devices. We recently published data from a 12-week proof-of-concept, non-randomized clinical trial of 165 patients in which 84 participants received lifestyle interventions designed for each phenotypic trait they had and 81 received standard lifestyle recommendations. The phenotype-tailored lifestyle intervention (PLI) resulted in greater weight loss compared to the standard lifestyle intervention (SLI) approach for obesity. Patients in the PLI showed improvement of their phenotype-defining trait(s). Improvement in these traits may explain the greater weight loss as they potentially counteract physiologic and behavioral adaptations of the weight-reduced state. To validate these data, we must study PLI in a longer-term and randomized clinical trial. Furthermore, the current methods used to identify phenotypes in our preliminary studies are time-consuming, invasive, expensive, limited to a few academic centers, and not accessible for most patients. In an academic-industry partnership, we have developed a novel biomarker test that predicts obesity phenotypes but that needs to be validated in a large prospective cohort. As such, we have formulated the following central hypothesis: “tailoring lifestyle recommendations to obesity phenotypes will enhance long-term weight loss outcomes in adult patients with obesity”. To test our central hypothesis, we propose a 12-month randomized, blinded, parallel clinical trial in adults with obesity to test three aims: 1) To compare the outcomes of PLI vs. SLI program; 2) After a 12-month weight loss program, to compare the long-term effect of PLI compared to SLI on physiological (i.e., satiation, postprandial satiety, energy expenditure) and behavioral (i.e., emotional eating) adaptations; and 3) To explore whether a phenotype biomarker predicts weight loss in response to PLI compared to SLI. Significance: Our study has the potential to introduce an individualized treatment that targets pathophysiological and behavioral phenotypes of energy balance to enhance and maintain weight loss outcomes.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Mesenchymal stromal cells (MSCs) have regenerative, immunosuppressive properties, but using MSCs to treat autoimmunity has resulted in mixed clinical results at best. Lackluster therapeutic efficacy has been attributed to insufficient trafficking to the tissue of interest and suboptimal immunosuppression. Our preliminary data indicate that using lentiviruses with specific enhancers, we can successfully engineer MSCs to express chimeric antigen receptors (CARs) and become CAR-MSC. We also have demonstrated that incorporating specifically designed CARs targeting gut integrins into MSCs results in their enhanced immunosuppression and trafficking to the colon in preclinical models. Engineering MSCs maintain their stemness and CAR-MSCs are safe in canine models. This led to our central hypothesis that engineering MSCs with a CAR enhances their efficacy in graft versus host disease (GVHD) and autoimmune inflammatory bowel disease (IBD) and colitis. In this project, we will leverage our available tools of viral vectors, CRISPR constructs, mouse models, and established biobanks to develop CAR-MSCs that demonstrate enhanced therapeutic efficacy over unmodified MSCs in the treatment of GVHD. In Aim 1 of this proposal, we will determine the mechanisms by which CAR-MSCs exert their enhanced immunosuppression. In Aim 2 of this proposal, we will study cellular trafficking in preclinical models. In Aim 3 of this proposal, we will optimize signaling of CAR-MSCs. At the conclusion of this project, we will have developed a new, translatable cellular therapeutic strategy to treat GVHD and IBD by engineering CAR-MSCs to have improved targeting and immunosuppression over unmodified MSCs. This platform can be applicable in different autoimmune diseases.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Prevalence of pulmonary disease due to non-tuberculous mycobacteria in the United States has doubled in the last 10 years, and Mycobacterium avium pulmonary disease (MAC-PD) is the most common. Treatment requires ≥3 antibiotics for >12 months and efficacy remain low. Given this intense treatment regimen and limited efficacy, antibiotic therapy is not always advisable. In fact, ~50% of patients will attain culture conversion with observation alone or with bronchial hygiene treatments. There is currently no way to predict which patients will improve with or without antibiotic treatment. The result of this uncertainty is prolonged ineffective treatment which risks side effects, drive drug resistance and extend treatment duration. We propose an interdisciplinary approach that combines clinical, immunological and pharmacological data. We will integrate these datasets from retrospective and prospective cohorts using machine learning and mechanistic computational simulations with the goal of developing a disease progression risk score (DP-RS) for patients managed without antibiotics (Aim 1), and a treatment failure risk score (TF-RS) for patients treated with guidelines-based antibiotic regimens (Aim 2). Such risk scores can help to predict patients at risk of disease progression without antibiotics or treatment failure with antibiotics, so they can be prioritized for closer observation or intensified treatment, respectively. For patients who do not receive antibiotics (Aim 1) we will a) use retrospective clinical data from Indiana, Florida and Minnesota to train and test random forest algorithms to predict disease progression and validate our predic- tions against prospective clinical cohorts from Florida and Minnesota; b) identify antigen specific T cells re- sponses associated with disease progression; c) predict tissue-level disease progression based on patient-spe- cific T-cell responses using mechanistic computational models; and d) train and test simulation assisted random forest (SARF) algorithms that integrate clinical, immunological and computational data to predict disease pro- gression and derive the DP-RS. For patients who receive antibiotics (Aim 2) we will a) use retrospective clinical data (Indiana, Florida, Minnesota) to train and test random forest algorithms to predict treatment failure and validate our predictions against pro- spective clinical cohorts (Florida, Minnesota); b) identify antigen specific T cells responses associated with treat- ment failure; c) identify serum and bronchoalveolar lavage pharmacokinetics associated with treatment failure; d) predict tissue-level treatment response based on patient-specific T-cell responses and pharmacokinetics us- ing mechanistic computational models; and d) train and test SARF algorithms that integrate clinical, immunolog- ical, pharmacological and computational data to predict treatment failure and derive the TF-RS. Together, this work will provide fundamental insights into clinical, immunological and pharmacological contribu- tors to MAC-PD disease progression and treatment response, as well as provide predictive risk scores (DP-RS and TF-RS) that will translate into precision medicine management tools, helpful for clinicians and patients.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Prostate cancer (PC) is a leading cause of male cancer-related deaths. Once disseminated PC is incurable as it gradually progresses to all therapeutic options. The mechanisms underlying aggressive features and lethality of metastatic PC remain an unmet clinical need. We and others have reported that metastatic PC displays the highest levels of chromosomal instability (CIN), and we have shown that transcriptional reprogramming allows PC CIN-adaptation and survival in advanced disease. Yet, the molecular underpinnings of CIN impact on PC remain poorly understood. Using genetic, proteomic, epigenomics and high-resolution microscopy, we found that CIN is linked to transcriptional rewiring and bookmarking suggesting a role of CIN in cell reprogramming in lethal PC. High CIN PC remains highly dependent on mitotic fidelity programs like the ones controlled by MASTL kinase to restrain lethal catastrophic CIN levels. We find that MASTL regulates new late mitotic processes involved in centrosome biology and cytokinesis in PC cells. However, the substrates and effectors by which MASTL regulate these functions remain unknown. Finally, our in vivo data strongly suggest a relevant role of CIN impacting anti- tumor immunity in PC. In this application we will use unique genetic models to identify the substrates and cofactors by which MASTL kinase regulates new functions in high CIN PC (Aim 1). We will investigate the molecules and mechanisms underlying CIN-induced transcriptional reprogramming and gene bookmarking in PC (Aim 2) and will explore the tumor immune cell intrinsic and extrinsic mechanisms triggered by CIN in PC that may enhance immunotherapy efficacy (Aim 3). Through these studies we will mechanistically uncover novel aspects of CIN consequences for PC and may identify new potential treatment strategies for this lethal disease.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Aspirin-exacerbated respiratory disease (AERD) is a clinical syndrome characterized by asthma, nasal polypo- sis, and acute respiratory reactions to known cyclooxygenase-1 inhibitors, including the commonly used non- steroidal anti-inflammatory drugs (NSAID), such as aspirin. Patients with AERD suffer a disproportional burden of the nasal polyposis and asthma: most AERD patients need repeated sinus surgeries, they tend to have a more severe asthma, and are more likely to develop upper and lower airway remodeling. There is no labora- tory diagnostic test available for AERD, often delaying its diagnosis. Polyp growth is associated with a strong type 2 pro-inflammatory state characterized by high systemic levels of prostaglandin D2 (PGD2) and leukotriene E4 (LTE4), eicosanoids derived from arachidonic acid. Cyclooxygenase-1 (COX-1) inhibition in AERD causes a multifold increase in PGD2 and LTE4, while it should instead decrease PGD2 and only minimally change LTE4 levels. This discrepancy suggests dysregulation of arachidonic acid metabolism, likely due to as yet unidenti- fied factors in AERD patients. To uncover the role of nasal polyps in AERD pathophysiology, we have studied Staphylococcus (S.) aureus as a frequent colonizer in the nasal polyp tissue of AERD patients. S. aureus exotoxins are known to activate leu- kotriene biosynthesis in neutrophils, and the bacteria have also been shown to invade mast cells in nasal polyps. In the proposed research, we will extend these observations by exploring the effects of S. aureus on arachidonic acid metabolism, including changes in AERD and non-AERD polyp tissue in response to COX-1 inhibition. Because S. aureus is a pathogen that often evades immune defenses, it is plausible that S. aureus can impact arachidonic acid metabolism to avoid the host’s antibacterial immune response. The proposed research will test whether colonization or infection of nasal polyps by S. aureus contributes to abnormal metabolism of arachidonic acid in AERD patients. Aim 1: To determine if S. aureus exotoxins increase pro-inflammatory eicosanoid PGD2 and LTE4, levels in AERD polyp tissue. Aim 2: To determine the mast cell transcriptional program upregulated in AERD and identify if it is induced by S. aureus. The proposed research will potentially yield a new paradigm of AERD pathogenesis in which S. aureus acts as a major environmental factor that dysregulates eicosanoid metabolism and contributes to AERD. In addition, understanding this mechanism may lead to the development of novel diagnostic markers and of therapeutic strategies to target S. aureus or mast cells to limit the extent of type-2 inflammation and improve health

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY Monogenic autoimmune diseases, while rare, offer a unique opportunity to develop models that enable a more mechanistic understanding of the disease process, in particular, if the mutation is in the coding region and the penetrance is high. We have identified a family, in which father and two daughters have Immunoglobulin G4- related disease (IgG4-RD) while the mother is healthy. Exome sequencing identified shared mutations in the E3 ubiquitin ligase UBR4 (Cys4179Ter) and the transcription factor IKZF1 (IKAROS, Arg183His) genes in affected family members. IgG4-RD is a systemic autoimmune disease manifesting as a fibrosing inflammation of major salivary glands, orbits, pancreas, and retroperitoneal soft tissue, frequently associated with elevated serum IgG4 and IgE, increased peripheral eosinophilia and high rates of atopy/allergies. The pathogenesis of the disease is essentially unknown. IKZF1 is a gene that has been associated with a variety of autoimmune manifestations, without that pathogenetic pathways have been identified. Specifically, loss of function mutations cause immune deficiencies combined with tissue inflammation, while IKZF1 haplotypes have been associated with SLE and other autoimmune diseases in GWAS studies. The IKZF1 mutation identified here is unique in that it is a gain- of-function (GOF) due to increasing DNA binding affinity. We propose that an understanding of the effect of the GOF effects and their interaction with the proteome changes due UBR4 haploinsufficiency provides an opportunity to define mechanisms that lead to defective tolerance and excessive TH2 polarization. We propose a two-pronged approach. In Aims 1 and 2, we will build on our preliminary data that UBR4 haploinsufficiency increases CD45 expression while GOF IKAROS increases FYN transcription. Aim 1 will determine whether UBR4 and IKFZ1 GOF gene variants increase TCR signaling and break tolerance by upregulating CD45 and FYN. Aim 2 will examine the model that FYN drives TH2 polarization by phosphorylating ITCH2 and preventing the degradation of JunB. In these aims, we use a combination of human in vitro and mouse in vivo studies. Therapeutic implications will be examined in models of allergic airway inflammation by pharmacological depletion of IKAROS, as recently done for human SLE. In addition to these hypothesis-driven studies based on a large body of preliminary data, we propose to continue an unbiased screening of the proteomic and transcriptomic changes due to the gene variants to identify molecules that can be built into the current model. Aim 3 will determine whether the biomarkers developed from studying this digenic disease have broader implications for non-familial IgG4-RD disease as well as for other diseases that are characterized by an unopposed TH2 immunity.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY Binge-eating disorder (BED) and bulimia nervosa (BN) are potentially life-threatening eating disorders that share behavioral and brain similarities, genetic risk factors and higher-than-expected comorbidities with drug addiction – suggesting a common etiology. However, no mechanistic study has examined this possibility due in part to the lack of an animal model linking eating disorders and drug addiction. Like drug craving and use in drug addiction, food craving and eating in BED/BN persist despite adverse consequences (punishment). Our pilot data from rats indicate that extensive cocaine and alcohol histories, known to trigger addiction-like brain changes and punishment-resistant “compulsive” drug intake in rats, trigger punishment-resistant food intake or “compulsive appetite”. These results provide an animal model for studying the neurobiological mechanisms manifesting as compulsive behavior across eating disorders and drug addiction. Food motivation is thought to be regulated by both homeostatic (caloric) and non-homeostatic (hedonic/incentive) systems. The homeostatic system detects energy shortages and elicits food intake. However, like compulsive drug motivation, our data suggest that compulsive appetite is driven by non-homeostatic “motivational/habitual” dysregulation. Like cocaine and alcohol histories, obesogenic diet histories also led to compulsive appetite via non-homeostatic dysregulation. Thus, drug/diet-induced changes in brain sites that control non-homeostatic regulation, such as reward circuits, likely cause compulsive appetite. We previously found that appetitive behavior is, in part, controlled by ‘food-reactive’ neurons (as indicated by the activation marker Fos) in the infralimbic cortex (IL) – a part of reward circuits thought to regulate drug and food motivation independently of energy homeostasis; these neurons thus appear to function as “accelerators” for non-homeostatic appetite regulation. We have also found that extensive drug histories increase neural food-reactivities in IL and other brain sites within reward circuits while inducing gene expression changes linked to aberrant neural plasticity and addiction preferentially in food-reactive – rather than non-reactive – neurons. Such brain changes would entail more “acceleration” on food motivation via non-homeostatic dysregulation, thereby likely manifesting as compulsive appetite. Based on the rigor of previous research and premise above, this project will test the central hypothesis that extensive cocaine/alcohol/obesogenic diet histories induce compulsive appetite via gene expression changes unique to food-reactive neurons in the reward circuits. The reward circuits contain neurons selectively reactive to each specific behaviorally relevant stimuli – likely exerting different behavioral functions. We will thus utilize neural activity-specific gene expression profiling (Aim 1) and rescuing (Aim 2) to target food-reactive neurons. The expected results will determine genes expression changes functionally linked to compulsive appetite. Such knowledge may help identify novel therapeutic targets to counter compulsive behavior across eating disorders and drug addiction.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY The mechanisms involved in lung repair and regeneration are still poorly understood and currently no clinical techniques are available to specifically induce lung repair. In young tissues, the lung is appreciated to repair and regenerate in response to damage. However, lung diseases such as idiopathic pulmonary fibrosis, are largely considered diseases of aging and subsequent disordered repair. Current treatment options available are limited, and mainly provide an option to slow disease progression, but none offer the ability to repair existing damage. Improved understanding of repair and regenerative mechanisms could drastically improve the treatment options available for these terminal fibrotic conditions. Of the cells studied in repair and regeneration, mesothelial cells are largely excluded, presenting a gap in the knowledge base. While pleural mesothelial cells (PMCs) are largely quiescent in healthy conditions, our preliminary data shows recruitment and migration of PMCs in a mouse model of transient fibrotic injury. Compelling data also exists for PMC recruitment and migration in a mouse model of lung regeneration. Contradicting this, other work has suggested that PMCs serve a role to stimulate fibrotic progression, leading to confusion surrounding the function of these cells. The lack of study and contradictory evidence regarding PMC functions in lung injury, fibrosis and repair sets the stage for detailed study of this cell population and the signaling and functional roles it may play in these contexts. Using a combination of careful lineage-tracing experiments, we will investigate PMCs in the bleomycin injury model, as well as in the pneumonectomy compensatory growth and regeneration model. We will subsequently collect these cells for further genomic analysis to assess expression of genes consistent with injury progression or controlled repair. Our initial investigations into the signaling of PMCs has capitalized on the various lung single-cell RNA sequencing dataset that are publically available. In multiple human and mouse datasets, the cell clusters identified as mesothelium represent the exclusive lung cell type that expresses the Wnt mediator R-spondin 1 (Rspo1). Our preliminary data confirms PMC to be highly expressive of Rspo1. As Wnt signaling is a pathway that has been well explored in contexts of injury and repair, this proposal will explore the ability of PMCs to modulate Wnt signaling in the local environment but using organoid co-culture. The goal of these studies is to provide context for the effect of these cells on specific cell types involved in lung injury and repair. Overall, the studies may reveal a unique and unexplored potential to target pleural mesothelial cells and explore their expanded role in lung function/dysfunction.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract: The hallmark features of heart failure with preserved ejection fraction (HFpEF) are exercise intolerance and exertional symptomology. It is now clear that the pathophysiology of exercise intolerance involves multiple physiological systems in HFpEF with peripheral (‘non-cardiac’) abnormalities having a critical role. Locomotor muscle afferent feedback reflexes are necessary for the normal locomotor muscle blood flow and ventilatory responses to exercise in healthy adults. However, these locomotor muscle afferent reflexes appear to be ‘overactive’ with HFpEF impairing locomotor blood flow and exaggerating the ventilatory response. Additionally, pulmonary system alterations and inspiratory muscle metabolic inefficiency in HFpEF necessitate an exaggerated inspiratory muscle blood flow demand during exercise. Importantly, HFpEF patients are unable to meet this exaggerated inspiratory muscle blood flow demand during exercise. This exaggerated deficit between inspiratory muscle blood flow demand and blood flow response to exercise in HFpEF may predispose them to exercise intolerance and exertional dyspnea. Our scientific premise is that locomotor and inspiratory muscle pathophysiologic mechanisms contribute to the well described exercise intolerance and exertional symptoms in HFpEF patients. The Specific Aims that will be explored in this proposal include: 1) To test if locomotor skeletal muscle afferent feedback reflexes contribute to the abnormal cardiovascular and ventilatory function during exercise in HFpEF, and 2) To test if inspiratory muscle training reduces the exaggerated inspiratory muscle blood flow demand in HFpEF and improves exercise tolerance and exertional symptomology in these patients. Both Aims are framed with testable hypotheses and clearly associated with the experimental protocol and statistical analysis plan. Our integrative, highly collaborative research team has the intellectual and technical expertise, established infrastructure, and clearly demonstrate high feasibility in performing all facets of these studies to address and interpret the Aims we have proposed. Our proposal addresses an important problem by focusing on ideas that are a significant departure from current paradigms on exercise intolerance and exertional symptomology in HFpEF patients. Our preliminary data and review of the rigor of prior research supporting our Aims provide strong justification for our innovative experimental design, gold standard techniques, and inspiratory muscle training (an intervention with high clinical utility) to reveal how locomotor and inspiratory muscle dysfunction impair exercise tolerance and exertional dyspnea in patients with HFpEF. Finally, we have aligned our scientific premise, aims, and associated hypotheses with the NHLBI Research Priorities and the current NIH review criteria that emphasizes significance, impact, and innovation for R01 applications.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY - OVERALL COEQUaL, the COllaboration for EQuity in Uterine Leiomyomas, builds on longstanding collaborations among women with uterine fibroids (UF) and multidisciplinary researchers at Mayo Clinic, Fibroid Foundation, University of Mississippi Medical Center and University of Florida, Jacksonville to place the woman with UF at the center of this project with the overarching goals to improve outcomes for all women with UF and eliminate health disparities for Black/African American (BAA) women. Our prior research demonstrates that most women and significantly more BAA women prioritize uterine sparing treatments, yet hysterectomy continues to dominate UF care. Thus, this proposal aims to identify affected women earlier, understand and meet their needs, delineate internal and external barriers to care, optimize communication between women and a variety of health care providers, and use innovative tools to eliminate barriers to individualized care. Our longstanding collaboration has pioneered the study of health disparities and UF for over two decades. Our collaboration with the Fibroid Foundation, the leading UF patient advocacy organization, began at its founding in 2012 and has been present in the conception and design of this proposal. Three projects are proposed: 1) Targeted Awareness and Education on Options in Uterine Fibroids 2) Articulating provider and insurer factors that limit outcomes for all women and disparities for BAA women. 3) Developing Innovative Tools to Overcome Factors that Limit Outcomes for All Women and Disparities for BAA women. The Community Partnership, Education and Outreach Core, led by the Founder of the Fibroid Foundation, will represent woman with UF at every stage and be the authoritative educational source of information about ethical, psychological, scientific, legislative, and advocacy issues related to uterine fibroids via a weekly briefing summary, alerts, podcasts and multiple modalities of dissemination and will mentor and train the next generation of policy leaders through a student internship program. The Administrative Core will provide logistic, communication and scientific support to all work and train the next generation of academic and lay leaders through selection of pilot projects to bring diverse early-stage investigators into the field and via succession planning within the collaboration. The findings of COEQUaL will impact every facet of UF care and will improve experience for all women but especially focus on the health disparities experienced by BAA women. This work will also change the clinical paradigm for clinical care by creating tools for women, healthcare providers, and healthcare systems to empower personalized and evidence-based decision making. 1

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Severe immune-related adverse events (irAEs) occur in up to ~60% of melanoma patients treated with combination (anti-PD1 / anti-CTLA4) immune checkpoint inhibitors (ICIs), and cause treatment- related morbidity and mortality. However, the pathophysiology underlying severe irAE development remains unclear and there is no way in clinical practice to predict who will develop severe toxicities and who will not. Based on our preliminary data, we hypothesize that clonally diverse activated CD4 memory T cells, and more specifically CXCR5–PD1hi peripheral helper T (Tph) cells, specifically underpin ICI-mediated toxicity in melanoma patients. To address this hypothesis, we will perform flow cytometry, CyTOF, scRNA-seq, scV(D)J-seq and immunoSEQ® to broadly assess T and B cell states in peripheral blood to 1) determine whether Tph levels in pretreatment blood are predictive of severe irAE development in melanoma patients treated with combination immunotherapy (Aim 1), and 2) determine whether Tph clonotypes preferentially expand in on-treatment blood and are enriched in irAE skin lesions during combination immunotherapy in patients who develop severe toxicity (Aim 2). While the prediction of severe irAEs from peripheral blood will be important clinically, patients who experience some degree of toxicity have also been shown to have better durable immunotherapy response rates. Therefore, it will be challenging to make clinical decisions regarding immunotherapy without also considering the probability of durable response. We will thus utilize cell-free DNA methylation sequencing to predict 1) immunotherapy toxicity and 2) durable immunotherapy response concurrently from pre-treatment plasma using both cell-state signatures and an agnostic machine learning approach, which we will validate in held-out cohorts (Aim 3). By doing so, we will lay the foundation for future clinical trials where immunotherapy decision-making is guided by the risk versus benefit of combination immunotherapy using the liquid biopsy biomarkers defined here. In summary, this study will reveal determinants of irAE development which will form the basis for liquid biopsy technology to predict both immunotherapy response and toxicity to make treatment safer and more personalized in the future.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ ABSTRACT This is an application for a K08 award for Dr. Nikolaos Skartsis, an Assistant Professor of Medicine at Mayo Clinic College of Medicine and Science, Rochester, MN. Dr. Skartsis is establishing himself as a young investigator in clinical science research with a translational focus on the impact of inflammation on regulatory T cells (Tregs) with an overarching goal to design the next generation Treg-enhancing therapies for transplantation. This K08 award will provide Dr. Skartsis with the support necessary to accomplish the following goals: (1) to become an expert translational researcher in Treg biology as it relates to inflammation; (2) to develop all the tools necessary to have an independent translational research career. To achieve these goals, Dr. Skartsis has assembled a multidisciplinary mentoring team of experts comprised of his primary mentor, Dr. William Faubion, expert in basic and translational research in Treg biology and epigenetics; co-mentors are: Dr. Cornelia Weyand, expert in immunometabolism; Dr. Virginia Shapiro, expert in T cell activation and maturation; Dr. Mark Stegall, expert in clinical trials in kidney transplantation, and Dr. Vesna Garovic, expert in immunological mechanisms of preeclampsia. He has established collaborations with Dr. Sun, expert in bioinformatics, and Dr. Wangensteen, expert in CRISPR screens. The impact of inflammatory signals on Tregs is not well understood despite the pivotal contribution of Tregs to terminate immune responses and maintain immune system homeostasis. The central hypothesis for this proposal is that human Tregs can positively response to proinflammatory cytokines by stabilizing their cell lineage, while clonally expand to scale to inflammation. Molecular mechanisms that control human Treg lineage stability and proliferation in inflammation are not fully understood, which will be the focus of his research in the next 5 years. In Aim 1, Dr. Skartsis will study the transcriptional and epigenetic changes induced by TNFR2 that promote FOXP3 expression in Tregs. In Aim 2, he will analyze the transcriptional and metabolomic control of Treg proliferative fitness under inflammatory conditions. This proposal represents an innovative approach to studying Treg biology because it incorporates CRISPR gene editing, functional genomics, and in-vivo disease modeling in humanized NSG mice to dissect the impact of inflammatory signals on Treg homeostasis that have not previously applied together in Treg studies in transplantation or autoimmune diseases. This project addresses a major gap in our knowledge of the impact of inflammation on Tregs, and the training plan it requires will form the basis for a compelling R01 grant application to harness the molecular pathways that promote Treg survival and function under inflammatory conditions to design the next generation of Treg enhancing therapies in transplantation and autoimmune diseases.