Cincinnati Childrens Hosp Med Ctr

NIH Research Projects · FY 2025 · 2022-08

Estrogen is prescribed for a variety of purposes in youth, and it may increase the risk of thrombosis. While he-matologists often evaluate patients before estrogen start who are at higher risk of thrombosis due to per-sonal/family risk factors, there are no guidelines or data to inform management in some situations. This lack of data leads to significant variation in clinical practice that may compromise care. Because this knowledge gap hinders the ability of clinicians caring for youth and hematologists to provide optimal care to patients, the objec-tives of this proposal are to prospectively examine biologic changes associated with thrombotic risk in a cohort of youth who are prescribed estrogen and to examine the attitudes and practices of clinicians who make deci-sions about thromboprophylaxis for youth on estrogen. The proposed work is relevant to the priorities of under-standing human biology (defining changes in thrombotic risk associated with estrogen) and reducing human disease (defining which youth may benefit from thromboprophylaxis). The proposed research is innovative in the focus on youth, comprehensive examination of changes in coagulation factors in youth taking estrogen, and determination of factors influencing the treatment recommendations of hematologists for thromboprophy-laxis among youth with personal and/or family risk factors for thrombosis. The specific aims are to: 1) prospec-tively determine changes in coagulation that would predispose to thrombosis over the first 24 months of estro-gen in a population of people who are up to age 22 years at estrogen start; and 2) characterize the attitudes, practices, and intentions of hematologists caring for youth toward recommending thromboprophylaxis to youth with personal and/or family risk factors for thrombosis. In Aim 1, 75 people up to 22 years of age who are start-ing estrogen will undergo thorough and systematic evaluation of hemostatic factors at baseline prior to estro-gen start, and then at 3, 6, 12, 18, and 24 months, with interim telephone study visits. Hormone usage and ad-herence, coagulation parameters, thrombophilia polymorphisms, and platelet activation will be assessed. In Aim 2, well-established qualitative and survey research methods will be used. Up to 20 adult and pediatric he-matologists will complete individual interviews. Data from these interviews will be used to generate items for a new survey that will undergo survey development methods (cognitive interviews, pilot testing) before being fielded to a sample of U.S. and Canadian hematologists to understand their attitudes, behaviors, and intentions to recommend thromboprophylaxis to youth taking estrogen who are at higher risk for thrombosis and factors associated with these intentions. The results of this work provide the foundation for 1) an intervention targeting physicians to improve knowledge of thrombosis risk in the setting of estrogen use and 2) development of clini-cal guidance to aid in referral, evaluation, and management of youth at higher risk for thrombosis.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Pathophysiologic changes during critical illness can affect drug pharmacokinetics (PK, how the body affects the drug) and pharmacodynamics (PD, how the drug affects the body), but the current paradigm of drug dosing is a standard “one-size-fits-all” approach, rather than a personalized approach. Emerging data in adults demonstrate increased risk of morbidity and mortality with standard doses of antibiotics in critically ill patients due to lack of PD target attainment. β-lactam antibiotics are a prime example of drugs demonstrating high PK and PD variability in critically ill patients. Thus, critically ill patients are at risk of having low antibiotic exposures leading to ineffective bactericidal activity or high antibiotic exposures resulting in toxicity. By using real-time drug concentrations, individual patient and disease factors, and population PK models, model-informed precision dosing (MIPD) can ensure adequate antibiotic exposure while avoiding toxicity. My proposed research program will address three critical knowledge gaps that are necessary to fill prior to implementation of antibiotic MIPD. First, the appropriate patient populations who will most benefit from precision dosing remain unknown. Implementation of MIPD for every patient admitted to the intensive care unit may be resource-intensive and costly. However, simply increasing antibiotic doses or frequency of administration without data-driven management can be dangerous as it carries risks for antibiotic-associated toxicity, including nephrotoxicity and neurotoxicity. Therefore, it is critical to optimize the benefit-to-risk ratio of therapeutic interventions for individual patients, a fundamental concept of precision medicine. Second, there remains a knowledge gap on the association of precision dosing of antibiotics and clinical outcomes. Studies examining antibiotic exposure and outcomes in adults have had mixed results; some show improved outcomes with PD target attainment and some show no difference in outcomes with regards to target attainment. Third, many of the antibiotic population PK models needed for MIPD have not been prospectively validated in critically ill patients, so it is unknown which models should be used for precision dosing. To address these knowledge gaps, Project 1 will utilize innovative modeling and simulation to identify patient and disease factors associated with antibiotic under-exposure (risk of ineffective antibacterial activity) or over-exposure (risk of toxicity) and investigate mechanisms underlying toxicities. Project 2 will investigate the effect of precision dosing on clinical outcomes by evaluating the association between PD target attainment and clinical outcomes at the individual level. Project 3 will prospectively validate our models and previously published models to ensure accurate predictive ability in critically ill patients. With these prospectively validated models, we will lay the foundation to build MIPD decision support tools integrated with the electronic health record to generate individual patient PK and PD profiles for precision dosing in critically ill patients. These tools will be essential to implement antibiotic MIPD to guide clinicians in dosing regimen selection for critically ill patients.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY A widely conserved hallmark of aging is the decline in muscle mass and strength, promoting frailty, loss of independence and disability. Crucial for this is our gap in knowledge regarding mechanisms linking bioenergetic stimulation to muscle anabolism. Glucocorticoid steroids have pervasive effects on energy metabolism and muscle function. Dosing intermittence appears crucial to the benefits/risks ratio of these drugs in dystrophic animal models and patients, i.e. in genetic myopathies. Here we investigate whether intermittent glucocorticoids increase performance in aged muscle, where weakness and sarcopenia are not dependent on specific genetic insults. Our new results in 24 months-old WT mice show that a chronic regimen of intermittent once-weekly prednisone increased muscle performance in aging mice to levels comparable to young adult mice (4 months- old). Remarkably, treatment rescued both mitochondrial respiration and muscle mass in aging mice to young- like levels. Mechanistically, we found that bioenergetic, functional and anabolic effects of intermittent prednisone were blunted upon inducible ablation of PGC1a in adult muscle. While the role of PGC1a in boosting mitochondrial capacity in aged muscle is more established, its effects on age-related sarcopenia and weakness are still debated with conflicting results from constitutive knockout or overexpression models. Scattered reports have hinted at a role of PGC1a in activating growth pathways, including biosynthesis of amino acids like alanine, but this is still largely unknown in muscle aging. Here we will investigate the mechanisms through which a bioenergetic stimulation like intermittent prednisone rescues both oxidative metabolism and mass gain in muscle aging. In Aim 1, we will determine the extent to which intermittence discriminates deleterious versus beneficial effects of glucocorticoids in aging muscle. We hypothesize that dosing intermittence shifts exogenous glucocorticoid effects from a PGC1a-lowering pro-wasting program to a PGC1a-dependent pro-ergogenic program, i.e. balanced gain of performance and mass. We will test this through inducible muscle-specific PGC1a ablation after natural aging. In Aim 2 we will establish the role of muscle Lipin1 in PGC1a re-activation in muscle aging. We hypothesize that Lipin1 supports mitochondrial function in muscle aging and mediates the PGC1a re- activation in response to intermittent glucocorticoids. To test this, we will use our newly derived mice with muscle- restricted inducible Lipin1 ablation after natural aging. In Aim 3, we will elucidate the extent to which bioenergetics rescue sarcopenia in aging through amino acid biosynthesis. We hypothesize that mitochondrial reactivation fuels amino acid biogenesis, supporting protein synthesis during muscle aging. We will test this by tracing glucose-derived carbons into amino acids in oxidative versus glycolytic myofibers. We will also test the extent to which muscle PGC1a mediates these effects in young versus aging muscle. In summary, here we leverage the unprecedented effects of intermittent glucocorticoids to discern unrecognized mechanisms of aging physiology and rejuvenate muscle bioenergetics and anabolism.

NIH Research Projects · FY 2025 · 2022-07

Abstract The cardiac myocyte has long been the primary focus of studies attempting to elucidate the regulatory aspects underlying cardiac development and disease. However, recently the involvement of nonmyocytes has emerged as potentially just as important as myocytes in contributing to and controlling cardiac remodeling during progressive pathogenesis associated with heart failure. More specifically, the cardiac fibroblast and its ability to convert to myofibroblasts in promoting extracellular matrix (ECM) production, ventricular remodeling and the fibrotic response is now viewed as an equally critical regulator of cardiac biology. Here we will address how fibroblasts in the heart function as key determinants of disease and pathologic remodeling. We have developed important genetic tools that specifically target the fibroblast in the heart so that we can manipulate the activity of these cells. Thus, here we can now test the novel hypothesis that activated fibroblasts (myofibroblasts) are critical regulators of cardiac disease processes, not only involving fibrosis but also the ability of cardiomyocytes in the heart to properly hypertrophy. We will test the more specific hypothesis that fibroblasts regulate the density and integrity of the cardiac ECM and collagen that cardiomyocytes must sense as increased structural support in order to effectively hypertrophy in vivo. Indeed, we further hypothesize that the tension sensing mechanism within the cardiomyocyte extends outward to the ECM and its integrity or stiffness. This application has 3 specific aims: 1) To increase the structural rigor of collagen in the heart to investigate the impact on cardiomyocyte hypertrophic growth potential in vivo, 2) To genetically impair type I collagen formation in the heart to reduce ECM structural rigor, and 3) To examine the actin filamentous network as a central signaling mechanism whereby ECM integrity or stiffness impacts myocyte growth in vivo. Collectively, these specific aims will uncover how fibroblasts communicate with cardiomyocytes in the heart through the ECM and its properties. Such an understanding will lay the foundation for future studies into specific therapeutic targets in treating longstanding fibrotic heart disease states or hypertrophy in general.

NIH Research Projects · FY 2025 · 2022-07

TITLE EFFICACY & SAFETY OF PHARMACOKINETICALLY-DRIVEN DOSING OF MYCOPHENOLATE MOFETIL FOR THE TREATMENT OF PEDIATRIC PROLIFERATIVE LUPUS NEPHRITIS - A DOUBLE-BLIND CONTROLLED CLINICAL TRIAL The Pediatric Lupus Nephritis Mycophenolate Mofetil (PLUMM) Study PROJECT SUMMARY: Meta-analyses in adults suggest equivalence of clinical efficacy of intravenous cyclophosphamide and mycophenolate mofetil when dosed based on patient weight or body-surface-area (MMFBSA) as is the current standard for the treatment of proliferative lupus nephritis (LN) treatments in the U.S. Pharmacokinetically- guided precision dosing of MMF (MMKPK) may offer a beneficial modification of the current standard treatment in that MMKPK promises over 30% higher LN response rates than MMFBSA. The objective of the proposed, adequately powered, randomized, double-blind controlled clinical trial is to compare the efficacy and safety of pharmacokinetically-guided precision dosing of MMF (MMFPK) with conventional dosing regimens of MMF (MMFBSA) among children with proliferative LN. The principal hypothesis to be tested in this 2-arm 104-week study is that, compared to MMFBSA, MMFPK results in significantly higher rates of renal remission in children with proliferative LN. The primary endpoint is the proportion of subjects achieving at least partial renal remission (PRR) at week 26 of the study in the intention to treat population. The key secondary endpoint is achievement of complete renal remission (CRR) at week 26 of the study. Our approach will be to enroll 105 pediatric subjects, ages 8 years or older, who have been newly diagnosed with proliferative LN plus have chosen MMF for induction therapy plus tolerate oral MMF. Randomization will occur at baseline (1:1) to the MMKPK arm or the MMFBSA arm, respectively. After week 26, non-responders will be discontinued from the active study intervention, and subjects randomized at baseline to the MMFBSA arm who achieved PRR but not CRR will cross over to the MMFPK arm. Volumetric Absorptive Microsampling (VAMS) devices will be used to facilitate estimation of the exposure to mycophenolic acid (MPA) in whole blood as is needed to personalize MMF dosing in the MMFPK arm. Use of corticosteroid will be standardized and closely regulated during the study, and adherence to MMF will be monitored. Patented biomarkers will be assayed in the urine in support of the superiority of MMFPK over MMFBSA in controlling LN activity. Upon completion of this trial, we expect to have unequivocal evidence of the superiority MMFPK therapy compared to MMFBSA use, and to show that MMFPK dosage is well tolerated and has an acceptable safety profile in children. RELEVANCE: The proposed trial is relevant to public health because therapies for LN are investigated, i.e. disease complications that concern the majority of children with cSLE. In this setting, optimizing drug use promises to improve long-term disease outcomes through rapid control of kidney inflammation, while minimzing unnecessary exposures to an immunosuppressive and teratogenic medication. This is relevant to the part of NIH’s mission that pertains to fostering research in treatment; and the dissemination of information on research progress in lupus. LN is central to NIAMS Strategic Plan and its Lupus Research Agenda in pursuance of improved public health and patient-centered personalized care.

NIH Research Projects · FY 2024 · 2022-07

Shared decision making (SDM) occurs when patients and clinicians collaboratively make medical decisions that align with a patient’s values and preferences. It is a core component of the patient-centered medical home. It is especially important for adolescents with chronic conditions and their parents or guardians (hereafter parents) given the frequent need for medical decisions among these patients and their families. Yet, there is a critical gap: no SDM measure has been developed to measure adolescent SDM (aSDM). Without a reliable and valid measure of aSDM, it is impossible to evaluate the extent to which: aSDM has occurred, aSDM impacts outcomes, or interventions designed to facilitate aSDM are successful. In alignment with PA-16-424 (Developing Measures of Shared Decision Making), our objective is to create the first SDM measure to explicitly incorporate the pediatric patient’s role in aSDM and measure their perspective. An SDM measure developed specifically for use with adolescent patients is needed because SDM with adolescents is fundamentally different than SDM with adult patients or with parents of younger children. Unlike most SDM, which occurs between two individuals (the adult patient or young patient’s parent and the clinician), aSDM with adolescents occurs among at least three people (the adolescent patient, their parent, and the clinician). Thus, our measure will explicitly evaluate, from the patient’s and parent’s perspectives separately, the extent to which each of these three individuals engages in aSDM. To develop our measure of aSDM, we will employ PROMIS measure development methods. These methods have never been used to develop and validate any SDM measure. Our specific aims are to: Aim 1: Refine the dimensions of aSDM from adolescent, parent, and pediatric clinician perspectives. Aim 2: Develop a measure of aSDM that incorporates aspects of the decision triad (adolescent, parent, clinician) and separately measures aSDM from the adolescent and parent perspectives. Aim 3: Demonstrate the aSDM measure’s reliability, validity, acceptability, and ability to feasibly measure SDM in clinical settings. We will conduct qualitative interviews with adolescents with chronic conditions, parents of adolescents with chronic conditions, and clinicians who care for adolescents to refine the dimensions of aSDM. We will demonstrate the measure’s reliability, validity, feasibility, and acceptability in a sample of 500 adolescents with chronic conditions and 500 parents. The expected outcome of our proposal is a reliable and valid measure of aSDM that is feasible for use in clinical settings for research, quality improvement, and performance measurement. Our proposal is innovative because 1) it will be the first SDM measure developed specifically for adolescents and 2) the first study to apply PROMIS standards to the development of any SDM measure. Our proposal directly addresses multiple AHRQ priority populations: children, adolescents, and individuals with special health care needs.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY PRDMs regulate chromatin accessibility to determine alveolar maturation. Pulmonary surfactant is a complex mixture of lipids and proteins produced by AT2 (Alveolar Type 2 Cells) which is secreted into alveolar spaces to reduce surface tension and prevent alveolar collapse during the ventilatory cycle. Lack of pulmonary surfactant underlies the pathogenesis of neonatal respiratory distress syndrome (RDS) in preterm infants and contributes to the pathogenesis of acute lung injury (ARDS) in adults. The fetal lung undergoes dramatic changes in architecture, cell differentiation and gene expression in preparation for birth, increasing surfactant lipid and protein production required for postnatal lung function. While our laboratory and others have identified genes, transcriptional regulators, and gene regulatory networks controlling surfactant homeostasis, how chromatin accessibility is modulated to enable gene transcription necessary for AT1 and AT2 cell maturation and function remains relatively unexplained. Lacking are data regarding the molecular mechanisms controlling chromatin accessibility required for the activation of critical transcription factors and their targets before birth and during alveolar repair. The present application is based on our recent identification of the primary roles of PRDM3 and PRDM16 in regulation of genes critical for alveolar maturation, remodeling, and gene expression required for postnatal survival. PRDM3/16 are zinc finger, proline-rich domain-containing transcription factors with histone modifying activity regulating chromatin accessibility throughout the genome. We have shown that deletion of Prdm3/16 in fetal respiratory epithelial cells caused respiratory failure at birth, dramatically impairing surfactant protein production. We will use ATAC-Seq, Cut and Run, and RNA-Seq to identify gene loci regulated by PRDM3/16 to infer mechanisms by which PRDM proteins interact with AT2 specific genes before birth. In this application, we will test the hypothesis that PRDM3 and PRDM16 interact in transcriptional complexes regulating recruitment of histones and chromatin accessibility controlling AT2 cell differentiation and function in late gestation and during regeneration following viral-induced lung injury in adult mice. We will identify and test the functions of binding partners and transcriptional complexes mediating PRDM3/16 activity. The gene regulatory networks, physiologic and biochemical roles of PRDM3/16 on AT2 cells, alveolar structure and function will be identified in vivo and in vitro. The proposed studies represent conceptual advances regarding the molecular control of alveolar epithelial cell differentiation and function and will provide a framework for the development of future strategies to modify epigenetic landscapes controlling AT2/AT1 cell function in health and disease. Rev: 9-3-21

NIH Research Projects · FY 2025 · 2022-07

SUMMARY Extracorporeal membrane oxygenation (ECMO) and cardiopulmonary bypass (CPB) are associated with a devastating but poorly understood thromboinflammatory state. Standard anticoagulation strategies result in significant bleeding risk, yet are inadequate as life-threatening thromboses remain common. There are no effective strategies to mitigate the inflammatory storm initiated within minutes of starting mechanical circulatory support. Data from the PIs lab suggests that factor XII (FXII) promotes both thrombosis and inflammatory mediated organ damage during ECMO. There is significant interest in FXII as a target in mechanical circulation, but the mechanisms coupling FXII to mechanical circulation associated thromboinflammation are under- studied. FXII is a multifunctional protease that bridges the hemostatic and inflammatory systems. FXII activates factor XI (FXI), leading to thrombin generation. The importance of FXII-mediated FXI activation in the setting of mechanical circulation has never been evaluated. Data from the PIs lab suggests mechanical circulation components can promote thrombin-mediated FXI activation, significantly limiting the relevance of FXII in some contexts. Moreover, targeting FXII appears inadequate during CPB with open heart operations where sternotomy and cardiac manipulation increase circulating tissue factor levels. FXII also activates prekallikrein (PKK) to the active protease kallikrein (Kal). Kal is linked to multiple inflammatory pathways, including complement activation and bradykinin generation. Kal has also been proposed to promote thrombosis independently of FXI. FXII-mediated PKK activation has been proposed to drive inflammatory events during ECMO, but there are no studies directly evaluating the role of PKK in mechanical circulation associated thromboinflammation. Data from the PIs lab suggests that targeting PKK significantly limits thrombosis and organ damage in ECMO. The proposed studies will use novel gene-targeted rats developed specifically for this proposal, and cutting-edge pharmacological agents to test the following hypotheses: 1) FXII promotes ECMO and CPB related thromboinflammatory pathologies by independent mechanisms related to activation of FXI and PKK. 2) FXI is a superior antithrombotic target in mechanical circulation contexts with relatively high circulating TF levels, such as exist during CPB. 3) FXII-mediated PKK activation promotes key inflammatory events that lead to organ damage during ECMO/CPB, as well as thromboembolic complications. 4) Combined strategies targeting FXI and FXII, or FXI and PKK, provide better protection from thrombosis and inflammatory organ damage than targeting one of these factors alone, without incurring a major bleeding risk. The proposed studies will provide needed insights into the mechanisms coupling FXII to mechanical circulation associated thromboinflammatory pathologies. The knowledge gained will critically inform future clinical trials of available and emerging agents targeting FXII and FXI. The proposed studies are also likely to identify additional novel targets (e.g., PKK) to limit thromboinflammatory driven morbidity in mechanical circulation.

NIH Research Projects · FY 2024 · 2022-07

Project Summary/Abstract The Nr2f nuclear receptors are essential for the formation of the facial primordia and for patterning the upper jaw, though their specific role(s) remain incompletely defined. Zebrafish suffer a broad spectrum of phenotypes with nr2f loss of function, ranging from a striking upper-jaw-to-lower-jaw transformation in nr2f2/5 double mutants, to a severe reduction of the pharyngeal arches and an almost total loss of facial skeleton in quadruple nr2f1a/1b/2/5 and nr2f2/5/6a/6b mutants. In mice, preliminary data show that early conditional ablation of Nr2f1/2 in the cranial NC (CNC) results in a similar hypoplasticity of the pharyngeal arches and a severe reduction in the dorsal facial skeleton. The overarching hypothesis of this proposal is that the Nr2fs function in at least two discrete steps of CNC development: First, they are predicted to confer ectomesenchyme fate to a subset of CNC via activation of Twist1. Loss of Nr2f function appears to cause this population of CNC to die instead. To test this model, the fellowship candidate will compare expression of Twist1 and its downstream targets in mouse mutants and controls and attempt rescue of the fish mutant phenotypes with twist1a misexpression in the neural crest. He will perform lineage-tracing experiments in both fish and mice to determine when loss of CNC occurs and use a combination of whole mount in situ and immunofluorescence experiments to examine possible causes. The results of these experiments will be bolstered with RNA-Seq of FACS-sorted mutant mouse CNC. Completing this portion of the proposed study will add the Cre-lox conditional mutation system to his ~3 years of experience in mouse genetics, train him in zebrafish genetics, and provide broad experience in modern imaging techniques. Second, the Nr2fs are proposed to pattern post-migratory CNC ectomesenchyme to make the skeletal structures of the upper jaw distinct from those of the lower jaw. There is evidence for this later patterning role in zebrafish, but early CNC loss in the conditional mouse mutants has confounded attempts to determine conservation of function. To separate this putative patterning role from the earlier NC role, the candidate proposes to use a later-acting Cre driver to ablate Nr2f1/2 and then to examine conditional mutants for homeotic jaw phenotypes. He will also perform RNA-Seq on post-migratory CNC in these mutants to determine whether a different set of targets is dysregulated at this later stage, consistent with these Nr2f roles being discrete. Together, the proposed studies will train the candidate in a wide range of laboratory techniques and analyses, preparing him for future research as an independent PI. Consistent with the stated mission of the NIDCR, this study will add to the body of knowledge on neural crest development and craniofacial anomalies, laying the groundwork for future development of non-surgical therapies to ameliorate patient conditions.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY/ABSTRACT The objective of this multicenter study is to develop and validate a novel, non-invasive biomarker for pediatric NAFLD. The specific aims are to recruit a cohort of 80 youth (6-21 years of age) with NAFLD and 80 controls with obesity and study their skin surface lipids using state of the art lipidomic analyses to: 1. Determine the cutaneous lipids that differentiate youth with NAFLD from controls; 2. Investigate the skin lipidome that detects the presence of advanced disease (non-alcoholic steatohepatitis [NASH], fibrosis) among those with NAFLD; and, 3. Further support the use of skin surface lipids as a biomarker of NAFLD in children by studying its repeatability and variability. Participants will undergo magnetic resonance imaging with proton density fat fraction (MRI-PDFF) at baseline to determine the presence/absence of NAFLD. This methodology has been shown to be accurate in determining the presence of hepatic steatosis. They will then have their cutaneous lipidome sampled using tape stripping. The latter is a rapid (<5 min), painless procedure that samples the lipids of the most superficial layer of the skin (from the stratum corneum and the lipids secreted by the sebaceous glands). All patients with NAFLD will have had prior histologic confirmation of their disease, which will be re-reviewed and scored by a single pathologist, with expertise in pediatric NAFLD, who will be masked to patient clinical and demographic characteristics. Untargeted lipidomic analyses of the collected skin samples will be performed using ultra high-performance liquid chromatography-quadrupole time of flight mass spectrometry to determine the skin surface lipids that alone or in combination predict the presence of NAFLD. The performance of the cutaneous lipidome in predicting the presence of NAFLD in youth with obesity will be compared against that of the currently used screening test, namely serum alanine aminotransferase levels. Among those with NAFLD, the performance of skin surface lipids in predicting the presence of NASH and fibrosis will be assessed. Lastly, a subset of patients with NAFLD will have repeat skin sampling: a. on the same day from the opposite arm, to test the repeatability of cutaneous lipidome, and b. 1 month later, to test the variability of the lipidome as a biomarker. Variables that may affect the skin surface lipids, such as sex, age/pubertal status, change in diet, obesity severity and insulin resistance status, will be assessed and controlled for in these analyses. A non-invasive biomarker for pediatric NAFLD is urgently needed, due to the staggering prevalence of this disease and the fact that the currently available invasive diagnostic modalities (liver biopsy) are not practical and hinder therapeutic discovery and progression.

NIH Research Projects · FY 2026 · 2022-06

Project Summary/Abstract Crohn's disease is a chronic illness that results in intestinal inflammation and unwanted gastrointestinal symptoms. The only biologic (monoclonal antibody) approved for moderate to severe Crohn's disease in children (<18 years old) are those that antagonize tumor necrosis factor-alpha (anti-TNF). While there is a high initial response rate to labeled infliximab (anti-TNF) dosing, only half of infliximab exposed patients will achieve clinical remission and less than 40% will achieve endoscopic healing after one year on therapy. Several studies have shown that rates of sustained corticosteroid-free remission are improved when patients receive anti-TNF dose optimizations following reactive or proactive therapeutic drug monitoring. Moreover, anti-TNF dose intensification following pharmacodynamic monitoring has led to improved rates of endoscopic (intestinal) healing. Therefore, given the limited therapeutic options for children with active Crohn's disease, there is a critical unmet need for the development of a data-driven, individualized, and scalable anti-TNF dosing intervention used from drug start and continued throughout therapy to optimize drug exposure and ultimately, improve rates of intestinal healing. Our team has developed a precision dosing strategy that uses an innovative physician decision support dashboard that instantaneously applies pharmacokinetic model-informed precision dosing to generate an individual infliximab dosing regimen starting with induction and targeting phase-specific pharmacokinetic and pharmacodynamic endpoints throughout therapy. The central hypothesis is precision dosing (intervention arm) with infliximab during induction and maintenance will improve rates of deep remission vs. conventional care (pragmatic dosing; control arm). The central hypothesis will be tested with two specific aims. Aim1: Conduct a cluster-randomized (by center) clinical trial to assess rates of deep remission at year1 between Crohn's disease patients receiving infliximab with precision dosing vs. conventional care. Aim2: Refine model- informed precision dosing using a continuous learning approach and identify anti-TNF PK/PD targets from induction to maintenance associated with deep remission. Our approach is conceptually innovative with an emphasis on practical implementation. This is the first clinical trial in children to provide anti-TNF dose optimization during induction to target a specific early (week6) trough concentration while the maintenance regimen is selected by specific treat-to-target pharmacokinetic and pharmacodynamic biomarkers. Additionally, precision dosing regimens are produced with a novel web-based decision support dashboard and the study is being performed within the ImproveCareNow Network to streamline clinical trial logistics. In Aim2, a continuous learning approach will be applied to our published pharmacokinetic model to iteratively refine the model by capturing new real-world data to better describe specific patient populations and further reduce prediction error. The long-term goal is for the precision dosing strategy to generate a paradigm shift as the preferred dosing approach to optimize exposure to all biologics and change the natural history of Crohn's disease.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY / ABSTRACT Severe lung disease is an increasingly recognized life-threatening complication of systemic juvenile idiopathic arthritis (SJIA-LD), representing a distinct and highly inflammatory interstitial lung disease that affects as many as 1 in 20 children with SJIA. While the etiology of SJIA-LD is unknown, it is strongly linked to macrophage activation syndrome, (MAS), episodic systemic hyperinflammation with SJIA that is driven by interferon gamma (IFNγ). We recently reported that SJIA-LD and MAS share prominent features of IFNγ activation, supporting a key role for this pathway in the pathogenesis of SJIA-LD. However, the mechanisms by which IFNγ activation drives pulmonary inflammation in SJIA-LD is unexplored. In addition, the widespread use of anti-IL-1 therapy for SJIA has been linked to markedly increased incidence of SJIA-LD including distinct clinical features and development of pulmonary alveolar proteinosis (PAP). Our proposed studies provide a critical step to identify the mechanistic causes of SJIA-LD, a necessary step towards developing targeted treatment strategies for and ultimately to prevent SJIA-LD. The objective of this application is to define the mechanisms by which MAS and persistent IFNγ drive the pathogenesis of SJIA-LD. Accordingly, our central hypothesis to be tested is that persistent IFNγ pathway activation leads to alveolar macrophage dysfunction and lung inflammation in SJIA-LD and is amplified by anti-IL-1 biologic therapy. To study the mechanisms of SJIA-LD, we will utilize overlapping approaches in our established mouse model system to directly test effects of persistent IFNγ on pulmonary inflammation, and the requirement of IL-1 signaling for alveolar macrophage functional phenotypes. In parallel, we will define IFNγ-driven functional polarization phenotypes of alveolar macrophages in children with SJIA-LD. Specific Aim 1 will determine whether persistent IFNγ activation is the key driver of lung inflammation during MAS. We hypothesize that persistent IFNγ activation during chronic/recurrent MAS leads to the development of lung disease in mice. Specific Aim 2 will identify mechanisms of alveolar macrophage reprogramming in experimental MAS. We hypothesize that IL-1 blockade in the setting of persistent IFNγ activation during MAS reprograms alveolar macrophages towards inflammatory phenotypes and inhibits anti-inflammatory/resolution and homeostatic functions. Specific Aim 3 will define IFNγ-driven alveolar macrophage populations in children with SJIA-LD. We hypothesize that alveolar macrophage subsets in SJIA-LD display an IFNγ-driven inflammatory phenotype that prevents normal homeostatic functions. We anticipate that the proposed experiments will define the function of persistent IFNγ activation and IL-1 blockade as drivers of lung inflammation and alveolar macrophage dysfunction in MAS and SJIA-LD. Together these studies will advance our long-term goal of identifying the causes of and developing novel treatment approaches for SJIA-LD.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY Equity gaps in child health outcomes result, at least in part, from insufficiently addressed social determinants of health, defined as “the conditions in which people are born, live, learn, work, play, worship, and age.” Patient and family needs related to determinants like toxic housing, access barriers, and socioeconomic deprivation undermine health and shift attention away from completion of follow-up visits, medication fills, and other ser- vices that promote optimal health and well-being outcomes. Insufficiently addressed social determinants ex- plain much of the persistence in health disparities. As a result, healthcare systems that effectively monitor for risks and then integrate proactive responses to both medical and social needs will be more likely to achieve better, equitable outcomes. Thus, the objective of this project is to co-design and evaluate the “Responding to Identified Sociomedical risks with Effective Unified Purpose” System (RISEUP) to best meet both the medical and social needs of children and their communities. The central hypothesis is that an integrated system co- designed with healthcare-, human service-, payer-based stakeholders, and parents of pediatric patients, will promote better, more equitable outcomes across conditions by enhancing situation awareness and informing cross-sector actions directed at common root causes. This hypothesis will be tested through three specific aims: 1) Identify common root causes of health equity gaps across three chronic pediatric conditions (asthma, type 1 diabetes, and sickle cell disease) using an integrated monitoring system; 2) Co-design a response sys- tem to operationalize shared theory and address medical-social risks; and 3) Evaluate the effects of a medical- social monitoring and response system on patient- and population-level health outcomes, employing RISEUP in the care of children with asthma, diabetes, and sickle cell living in high-morbidity, high-poverty communities. The contribution of the proposed research is to co-design and evaluate RISEUP, this monitoring and response system in partnership with healthcare-, human service-, and payer-based stakeholders, and parents of pediat- ric patients. This research is significant because both medical and social data together would provide AHRQ- prioritized 360-degree views of children and communities, getting us closer to those factors at the root of poor health. This proposal is innovative in its monitoring of disparities’ root causes and the co-design and evaluation of integrated, coordinated response systems. Such data integration, accelerated by human-centered co-design and population-based quality improvement, holds great promise for subsequent scale and spread. Expected outcomes include a potentially transformative template for healthcare and human service systems to collabora- tively monitor and respond to disparities. Indeed, RISEUP will positively affect children by facilitating a preven- tive approach to addressing the social determinants and equitably improving outcomes for children, a vulnera- ble and AHRQ-prioritized population.

NIH Research Projects · FY 2026 · 2022-03

Abstract Clonal hematopoiesis (CH) is an aging associated condition characterized by the clonal outgrowth of mutated pre-leukemic cells. Although individuals with CH are healthy, they are at an increased risk of developing hematopoietic malignancies. To identify cooperating molecular alterations required for malignant transformation of clonal pre-leukemic HSPC, we performed an in vivo shRNA screen and found that shRNAs targeting Traf6 were overwhelmingly enriched in following transformation to overt myeloid leukemias. TRAF6 is an ubiquitin E3 ligase that synthesizes Lysine (K) 63-linked ubiquitin chains on substrates leading to Toll-like receptor (TLR) superfamily pathway activation. In support of our in vivo shRNA screen, promoter hypermethylation and reduced expression of TRAF6 is observed in subsets of myeloid malignancy patients, including ~40-50% of acute myeloid leukemia (AML). Moreover, our preliminary data shows that deletion of Traf6 in pre-leukemic Tet2-deficient HSPC results in an aggressive myeloid neoplasm in part through a novel MYC-dependent mechanism. Based on our findings, we hypothesize that loss of TRAF6 drives subsets of genetically-defined myeloid malignancies, specifically via a novel post-translational modification of MYC resulting in its activation. The objectives of the proposal are to uncover the molecular and cellular basis of TRAF6 deletion on pre-leukemic HSPC function with the long-term goal of uncovering improved therapeutic approaches by investigating the consequences of TRAF6 deletion in models of CH and on leukemia development (Aim 1), identifying the molecular basis of the tumor suppressor-like function of TRAF6 in AML (Aim 2), and evaluating the oncogenic potential of a novel TRAF6-dependent MYC post-translational modification (Aim 3). These studies are highly significant as they will provide critical insight into the progression of pre-leukemic states to overt leukemia as a result of subverting select innate immune pathways, describe a novel disease-modifying role of TLR-TRAF6, and reveal an unreported mechanism of MYC regulation. These studies have direct translational implications and fill an unmet clinical need for genetically- and phenotypically-defined subtypes of AML/MPN.

NIH Research Projects · FY 2026 · 2022-02

This is a new application for a T32 training program that will train the next generation of leaders in vaccine science. The Vaccinology Training Program (VTP) builds on a strong legacy of vaccine development at Cincinnati Children’s Hospital and the University of Cincinnati that includes the oral polio vaccine pioneered by Albert Sabin, the development of one of the world’s leading rotavirus vaccines by Drs. David Bernstein and Richard Ward, and the more recent evaluation of multiple COVID vaccines. Cincinnati Children’s is a longstanding site within the NIH-sponsored Vaccine and Treatment Evaluation Unit (VTEU) network, and features unique strengths in vaccine clinical trials including controlled human infection models (CHIMs) for evaluation of vaccines against Shigella sonnei, norovirus, cholera, and influenza; investigator-initiated trials for novel RSV vaccines, and application of systems vaccinology tools to elucidate the cellular/molecular pathways that lead to successful adaptive immune responses to vaccines in both young and aged individuals. Investigators in our program lead a large U01-funded cohort study of pregnant women and their infants studying the earliest responses to influenza infection and vaccination in order to understand the principles of immunologic imprinting. The VTP features outstanding basic science faculty pursuing a wide variety of projects relevant to vaccine development, including the areas of infectious disease pathogenesis, novel immunogen design, basic aspects of immune development and regulation, computational and functional genomics related to vaccines, and reproductive biology and immunology. The VTP will provide training to M.D., Ph.D., and MD-PhD postdoctoral researchers that will facilitate their development into successful independent investigators devoted to vaccine-relevant research. The program will support 4 trainees in the program per year, each appointed to 2-year terms. 22 expert mentors with strong track records in scientific mentorship and NIH-funded research programs will serve as the VTP mentors and faculty. Formal education in the breadth of vaccine sciences will be provided to all VTP trainees, including a seminar course in vaccinology covering bench-to-bedside aspects of vaccinology, formal education in grant writing, and targeted coursework relevant to the research project and focus (basic or translational) of individual trainees. The intended outcome is to nurture and develop VTP trainees into creative, independent scientists who will form the vaccinology workforce of the future.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY There is a critical need to determine whether premature vascular disease observed in youth with type 2 diabetes (T2D) extends to the brain. This proposal builds on exciting data generated by our team showing differences in brain structure and clinically meaningful declines in neurocognitive function among adolescents with youth-onset T2D compared to their obese and lean peers. Our most prominent findings include lower brain gray matter volume and lower scores on measures of working memory. Importantly, we have also demonstrated some of the first evidence of cerebrovascular impairment in youth with T2D, manifested as reduced gray matter cerebral blood flow. Finally, we have preliminary evidence that cerebral blood flow correlates with reduction in gray matter volume and working memory measures. These findings suggest the breakdown of the neurovascular unit (NVU), a physiological construct describing the interplay between brain vasculature, neurons, and supporting glia and a mechanism for reduced brain volume and cognitive impairment. Our overarching hypothesis that premature vascular disease observed in youth-onset T2D extends to the brain and is associated with structural brain abnormalities and neurocognitive impairment. Importantly, we aim to differentiate the impact of T2D on the brain from the effect of obesity. Thus, we will compare NVU in adolescents with T2D to age, sex and race similar non-diabetic adolescents with obesity (obese controls) and without obesity (lean controls). Aim 1 will comprehensively assess aspects of NVU structure and function and identify risk factors impacting the NVU; 2) Aim 2 will determine the relationships between cerebrovascular, neuroanatomical, and cognitive outcomes cross-sectionally, and 3) Aim 3 will evaluate changes in the NVU, brain structure and cognition over time. Our team is well positioned to conduct this study as we have generated compelling preliminary data in our laboratory (m-PI Shah & DiFrancesco), we have expertise in conducting research studies in youth with T2D with documented ability to recruit and retain participants (m-PI Shah), we developed the state of the art MRI methods to study the vasculature in our laboratory and have applied these to other diseases with vascular sequelae such as systemic lupus erythematous (m-PI DiFrancesco), and we have extensive expertise in neurocognition and brain-behavior research (co-I Beebe & m-PI DiFrancesco). Finally, we have documented collaboration between the members of the study team over the last five years (Shah, DiFrancesco, Beebe & Altaye). The results of this proposal will separate the effects of T2D from obesity, identify noninvasive imaging biomarkers of brain health and treatable risk factors, and explore progression over time, positioning us to test interventions for youth with T2D and obesity as an immediate next step.

NIH Research Projects · FY 2025 · 2022-01

Kaposiform lymphangiomatosis (KLA) is a devastating congenital lymphatic anomaly with a 51% survival at 5 years, and 34% overall. KLA patients suffer from pleural and cardiac effusions and coagulopathy leading to the high morbidity and mortality. The histopathology of KLA features lesions containing clusters of spindle- shaped endothelial cells accompanying malformed lymphatic vessels typically in the lungs, spleen, abdomen, and/or liver. The role, origin and function of these characteristic spindled endothelial cells is unclear. Definitive diagnosis of KLA is often delayed due to the complex symptoms and the risks of biopsy due to the coagulopathy. As part of a clinical trial using sirolimus (rapamycin; mTOR inhibitor) we were the first to identify a blood biomarker for KLA, angiopoietin-2 (ANG-2), that may provide important insights into the underlying disease mechanisms. ANG-2, which can act as a pro-angiogenic factor, was highly elevated in KLA patients and decreased with sirolimus treatment suggesting that dysregulation of ANG-2 in KLA is dependent on mTOR signaling. Additional possible insights into pathogenesis of KLA have come from the identification of a somatic mutation NRAS Q61R in lesion tissue from patients. Q61R is an NRAS activating mutation in >20% of melanomas and other cancers; however, its role in human endothelial cells and vascular malformations is unclear. Our preliminary studies with human endothelial cells suggest that NRAS Q61R is upstream of ANG- 2 and induces the spindled endothelial cell morphology in KLA lesions. Proposed studies will identify the processes and pathways involved in this regulation and help move the understanding of KLA pathogenesis forward. The goal of this proposal is to test the hypothesis that NRAS Q61R mediates the pathogenesis of KLA by increasing MAPK and PI3K-AKT-mTOR signaling, inducing spindled endothelial cells, upregulating ANG-2 expression, and so driving abnormal lymphangiogenesis. We have developed unique in vitro and in vivo models to test this hypothesis and preliminary data from our laboratory strongly supports this proposed mechanism and demonstrates the feasibility of our approach. These studies will address a critical knowledge gap in KLA. We will test new therapeutic targets since the current treatment, sirolimus, at best only induces a partial clinical response. Our long-term goals are to elucidate the cellular and molecular pathogenesis of KLA and identify new therapeutic targets. We are uniquely positioned having the expertise and experimental models in hand.

NIH Research Projects · FY 2026 · 2022-01

Normal brain function relies on the correct assembly of neural circuits during development. This process starts with the patterning of neural progenitors along the dorsal-ventral and anterior-posterior axes to give rise to distinct subtypes of neurons. A number of key transcription factors (TFs) control the process of neuronal subtype specification. Work in the mouse has shown that the homeodomain (HD) TF Gsx2 plays essential roles in the patterning and differentiation of neuronal cell types that arise from progenitors in the lateral ganglionic eminence (LGE) of the embryonic mouse telencephalon. These progenitors give rise to cell types that include the striatal projection neurons of the basal ganglia and interneurons in the olfactory bulb, both of which are severely reduced in mouse Gsx2 mutants. Accordingly, human patient studies identified 2 pathological GSX2 variant alleles in children with serious neurological symptoms, including dystonia and intellectual disabilities. Consistent with these symptoms, MRI imaging revealed severe basal ganglia agenesis. One GSX2 variant results in a null allele, however, the other is a missense variant (Q251R) that alters a key amino acid in the DNA binding HD. We generated a mouse model of this human variant and our initial studies suggest that the Q>R variant leads to a strong embryonic LGE and basal ganglia phenotype that is morphologically similar to embryos with Gsx2 null alleles. Furthermore, our preliminary data indicate that this human HD variant alters Gsx2 DNA binding specificity, and thereby may account for the observed phenotypes. Moreover, we recently determined that Gsx2 binds and regulates target genes via two mechanisms; as a monomer Gsx2 represses gene expression whereas on a subset of DNA sites cooperative Gsx2 binding to dimer sites appears to facilitate gene expression. Intriguingly, the Dlx HD TFs, which lie downstream of Gsx2 during LGE progenitor maturation, also bind monomer sites but instead of repressing they activate gene expression. In this application, we propose to determine how Gsx2 and the Dlx TFs regulate LGE gene expression during basal ganglia development. To achieve this goal, we will test the following hypotheses in 3 independent specific aims: 1) To test the hypothesis that Gsx2 controls basal ganglia development by mediating distinct gene regulatory outcomes in a DNA binding site dependent manner. 2) To test the hypotheses that Gsx2 and Dlx TFs regulate a common set of LGE genes though direct competition for shared enhancer elements. 3) To test the hypothesis that the GSX2Q251R human variant causes altered DNA binding specificity, and thereby results in the mis-regulation of LGE gene expression and ultimately basal ganglia agenesis. Our approach will combine the use of mouse genetics and human forebrain neural stem cell cultures with molecular, biochemical, and genomic approaches to study transcriptional control of neuronal specification in the developing basal ganglia. The unique expertise of our research team at CCHMC allows us to take this broad approach, and thus increases our chances to gain a deeper understanding of how Gsx factors control basal ganglia development as well as to uncover new gene regulatory mechanisms that underlie dysfunction in certain childhood neurological disorders.

NIH Research Projects · FY 2025 · 2022-01

Abstract Myocardial infarction (MI) due to underlying atherosclerosis is the leading disease sequela that precipitates heart failure in the Western world. Our ability to treat these patients and their heart failure has not progressed beyond a mild 20-30% extension in life span realized some 3 decades ago with neuroendocrine-based management [1]. New therapeutic avenues are needed, the most dramatic of which would be directly generating new cardiomyocyte to regenerate the damaged area of heart tissue. Previous attempts to regenerate the heart through new myocyte production have not been successful despite more than 18 years of research using adult progenitor cells. However, studies with cardiac progenitor cells in rodent models did show a functional benefit to the MI-injured heart, although we now understand that this is not due to significant new myocyte production. Instead we and others have identified a novel mechanism of benefit whereby injected progenitor cells have a rejuvenating effect on the MI-injured heart through refinement of the immune response. Indeed, we have shown that cell therapy injections that flank the recently injured area of the heart from ischemia- reperfusion (7 days later) can optimize healing, reduce infarct area expansion and augment scar borderzone physical properties (Vagnozzi et al., 2020, Nature). These beneficial effects were mediated through selective macrophage subtype activity in the heart, underscoring the importance of the immune response in cardiovascular health and infarct healing and compensation. Here we propose the hypothesis that selective innate immune response signaling pathways, and macrophage subtype polarization can be exploited to help heal the heart. Our more specific hypothesis is that therapy has an underlying protective component through Toll-like receptor (TLR) signaling in both cardiomyocytes and macrophages, and this can be therapeutically exploited to polarize the immune response for better healing. The specific aims are: AIM #1, To examine the mechanism of innate immune signaling in the heart through TLR signaling. AIM #2, To inducibly alter macrophage subtypes in the heart to reprogram the innate immune response and healing dynamics by cell therapy. AIM #3, To determine how fibroblasts communicate with macrophage subtypes in the post-MI injured heart to affect healing dynamics by cell therapy. Such studies will be critical for examining how innate immune signaling at the level of macrophages and cardiomyocytes impacts the heart during an inflammatory injury response with the goal of modifying this response to benefit cardiac healing in patients.

NIH Research Projects · FY 2025 · 2021-12

PROJECT SUMMARY Epilepsy can be a debilitating and sometimes fatal disease for which there are no preventatives, no cure, and for which existing medications fail in one third of patients. Development of preventative treatments for epilepsy is a key NINDS goal (Benchmark II. Prevent epilepsy and its progression; Galanopoulou et al., 2016; Binder et al., 2020). The mechanistic target of rapamycin (mTOR) pathway has emerged as a promising target for epilepsy prevention. mTOR acts as a relatively ubiquitous promotor of cell growth; enhancing neuronal connectivity, excitability and metabolism. Activation of the mTOR pathway occurs during epileptogenesis and appears to regulate pro-excitatory changes implicated in the process. Moreover, blocking mTOR signaling with the antagonist rapamycin mitigates epilepsy development in many epilepsy models. Blocking mTOR signaling, however, does not work in all models, and there is evidence that this pathway may also act to reduce brain excitability. To explain these conflicting effects, we hypothesize that increased mTOR activation among excitatory neurons is pro-epileptogenic, while activation among GABAergic interneurons is anti-epileptogenic. Systemic mTOR antagonists, therefore, block epileptogenic changes among excitatory cells, but also block compensatory protective changes among interneurons. To test this hypothesis, we will use intersectional genetic approaches to selectively delete the obligate mTOR regulatory proteins Raptor or Rictor from vesicular GABA transporter-, parvalbumin-, and somatostatin-expressing interneurons in mice. We will also examine the impact of enhancing mTOR signaling in interneurons by deleting the mTOR negative regulator Tsc2. Studies will be conducted using both acquired and genetic models of epilepsy. We predict that blocking mTOR signaling in interneurons will make epilepsy worse, while enhancing signaling will improve outcomes. Studies will advance understanding of the role of mTOR in epileptogenesis and will have direct implications for ongoing clinical use of mTOR antagonists.

NIH Research Projects · FY 2026 · 2021-12

Abstract The NF1 gene product, neurofibromin, was identified in 1990, yet fundamental questions remain unanswered concerning its intracellular location(s), binding partners, and signaling properties. It is known that the large (>200kd) tumor suppressor protein contains a small domain that turns of RAS proteins, so that loss of NF1 function leads to RAS activation on cell stimulation. The remaining NF1 sequence can interact with a chaperone, SPRED1, and other proteins in vitro and/or in specific cell types. In Schwan cells, NF1 loss results in formation of neurofibromas, tumors in the peripheral nerves. It is still unknown which interaction partners and RAS proteins are relevant in Schwann cells. Here we propose to identify NF1 interaction partners, including relevant RAS proteins, that contribute to Schwann cell tumorigenesis. This is because in mammals there are six highly homologous NF1-related RAS paralogs, divided into two families. Our studies rely on interdisciplinary approaches, including cell/mouse genetics, and state of the art biochemical and molecular biology techniques, and our expertise in preclinical testing. Our preliminary data, enabled by new reagents and technologies, supports key roles for canonical RAS proteins in NF1 mutant Schwann cell proliferation. We will now assess the specific roles of RAS proteins in primary Schwann cells in vitro, and in neurofibroma in vivo, and test a potential targeted therapy in vivo. Importantly, whether neurofibromin has Schwann cell-specific interaction partners beyond RAS proteins is unknown. Using proximity biotinylation our preliminary studies identify novel potential NF1-interaction partners in Schwann cells. We will identify complexes containing neurofibromin and these proteins and specific RAS paralogs, and additional complexes that may form on cell stimulation, and test if blocking identified pathways is useful therapeutically.

NIH Research Projects · FY 2026 · 2021-12

Abstract Pregnancy is often described as a physiologic “stress test” that may uncover latent risk for hypertension, diabetes and CVD. Even in uncomplicated pregnancies, women experience rapid cardiometabolic changes to support fetal development. While many of these adaptations normalize soon after delivery, some changes persist, including echocardiographic changes in cardiac structure and lower high-density lipoprotein cholesterol (HDL-C). Whether these pregnancy-related changes, or alterations in HDL particle structure or function, contribute to long-term cardiovascular risk is unknown. Parity, or the total number of live births, among post- menopausal women is also associated with greater risk of cardiovascular disease and alterations in HDL-C levels, HDL structure and function, and cardiac and vascular function. However, a significant research gap exists in linking observed cardiovascular changes across pregnancy to the observed associations of parity with cardiovascular health post-menopause. We will address this gap by leveraging existing data and samples from the NHLBI Growth and Health Study (NGHS) and conduct a new in-person visit in 350 participants at median age 46, when the women will be pre- or peri-menopausal. NGHS enrolled 871 girls (50% African- American and 50% white) in 1987 at age 9 or 10 and examined them up to 17 times, to age 27, including 7 echocardiograms between ages 20-27, multiple saved samples and reproductive history questionnaires. This study will conduct detailed lipoprotein sizing and functionality assessments from stored and new samples and conduct cardiovascular imaging and repeat echocardiograms at median age 46 to: 1) Determine the specific lipoprotein particle size distribution and function changes which occur from before to short- and long-term post pregnancy; 2) Determine whether pregnancy-related cardiac adaptations result in long-term alterations in cardiac structure and function, thereby increasing CVD risk for women in their forties; and 3) Test whether parity increases CV risk independently of socioeconomic status (SES) in African-American and white women. With the completion of our aims, we will have determined the prospective pathways between pregnancy-related lipoprotein and cardiovascular changes and pre-menopausal cardiovascular health for women in their forties. Results from our proposed studies will fill a critical gap in our understanding of how such risks may accumulate during a woman's reproductive life, as well as how socioeconomic status contributes to parity- related risk long-term. Women in this study have also been exposed to the obesity epidemic, lending greater understanding to contemporary reproductive development.

NIH Research Projects · FY 2026 · 2021-12

Project Summary Heart failure is a complex clinical syndrome that is driven by impaired myocardial contractile performance. Several metabolic alterations contribute to heart failure, including mitochondrial dysfunction and changes in cardiac substrate utilization, resulting in energy deficiency and reduced cardiomyocyte contractility. Current therapies for heart failure treat the symptoms rather than the mechanisms underlying the etiology of the disease and are unable to reverse the molecular changes that occur in diseased cardiomyocytes. Developing novel approaches to enhance mitochondrial function and modulate cardiac metabolism in heart failure is a promising approach towards correcting myocardial energetics to restore heart function. Despite the central role that mitochondria play in cardiac health and disease, we are still lacking critical insight into how many fundamental mitochondrial processes are regulated at the molecular level. Recent computational and experimental data suggest that the mammalian genome contains thousands of previously overlooked small proteins called microproteins, and hundreds of these have been linked to the mitochondria where they are thought to play important roles as regulatory molecules. Examples of mitochondrial microproteins (MitoMPs) have been shown to regulate essential mitochondrial processes including cellular respiration, substrate utilization, metabolism and stress signaling. MitoMPs typically manifest their functions by binding to and regulating larger protein partners or multiprotein complexes within membrane domains. In line with this, we recently discovered 2 novel MitoMPs named MOXI (micropeptide regulator of b-oxidation) and mitolamban, which each interact with discrete metabolic regulatory complexes to perform distinct functions. MOXI plays a critical role in regulating long chain fatty acid oxidation, likely through a direct interaction with the mitochondrial trifunctional protein (MTP), while mitolamban interacts with complex III of the electron transport chain and contributes to complex assembly and function. Here we propose a comprehensive research plan to dissect the molecular mechanisms of action of MOXI (Aim 1) and mitolamban (Aim 2) in the heart using gain- and loss-of-function mouse models. Additionally, we aim to evaluate their potential as therapeutic targets using experimental models of heart failure and ischemic heart disease. Furthermore, towards the goal of gaining a more complete understanding of mitochondrial biology in the heart, we propose the functional analysis of 3 newly identified MitoMPs (Aim 3). We hypothesize that these microproteins play unique roles in regulating distinct aspects of mitochondrial function and metabolism and that their functional characterization could give rise to novel targets for heart failure therapeutics.

NIH Research Projects · FY 2025 · 2021-12

ABSTRACT Alveolar macrophages (AMs) are believed to be a self-renewing cell population without a requirement of replenishment from extra-pulmonary sources in healthy adult mice; however, the mechanism(s) involved and whether replenishment occurs by stimulating the proliferation of progenitors or mature AMs are not known. We reported that lung levels of granulocyte/macrophage-colony stimulating factor (GM-CSF) control long-term maintenance of macrophages trans- planted into the lungs (via a reciprocal feedback loop) as well as endogenous AMs. My preliminary data demonstrate that GM-CSF is a critical regulator of AM mitochondrial turnover, integrity and functions and is required for fatty acid oxidation- derived energy production, processes vital to cell proliferation. Prior studies in a leukemia cell line (TF-1) suggest the pleotropic effects of GM-CSF on macrophages may be mediated by biphasic, ligand concentration-dependent receptor signaling, i.e., low levels of GM-CSF promote survival and differentiation (but not proliferation) while high levels also stim- ulate proliferation. Our long-term goal is to determine mechanisms responsible for AM self-renewal. The objective here is to elucidate the mechanism by which GM-CSF regulation of mitochondrial homeostasis controls AM self-renewal. Cen- tral Hypothesis: AMs are maintained by homeostatic self-renewal driven by GM-CSF threshold-triggered/concentration- dependent, niche-limited proliferation of mature (long-lived) AMs (not progenitors). Rationale: This hypothesis was de- veloped based on my reported and preliminary data demonstrating GM-CSF deficiency results in reduced mitochondrial metabolism and integrity despite increased mitochondrial mass. Approach: I will utilize complementary genetic and phar- macologic tools, in vivo and ex vivo studies (with isolated AMs), and pathway-specific inhibitors to pursue two Specific Aims: 1) Ontogeny and transcriptional control of AM renewal, and 2) Role of GM-CSF regulated mitochondrial metabolism in AM renewal. The expected results will inform cellular and molecular mechanism(s) by which GM-CSF regulates AM population size and will lay the foundation for developing novel therapeutic strategies to modulate AM population size. The proposed research is innovative, in my opinion, because it challenges the previously widely-held concept of AMs as short-lived, non-dividing cells replenished from circulating monocytes (regulated by M-CSF) – and instead posits that AM population size is maintained by homeostatic self-renewal mediated by GM-CSF threshold-triggered/concentration-de- pendent, niche-limited proliferation of mature (long-lived) AMs. My novel preliminary data identified GM-CSF dependent regulation of mitochondrial homeostasis as a molecular mechanism for AM self-renewal. In addition, utilization of novel mouse models and novel methodologies will enable determination of how GM-CSF regulation of mitochondrial homeosta- sis regulates AM proliferation and will identify the regulatory genes and their downstream targets responsible for long- term maintenance of AMs. The proposed research is significant because it will advance our knowledge of a critical pulmo- nary hormone (GM-CSF) by informing the mechanism(s) by which GM-CSF regulates the long-term maintenance of endog- enous and transplanted AMs and inform us on the therapeutic mechanism of action of PMT, a novel cell therapy in devel- opment to treat patients with hereditary pulmonary alveolar proteinosis and potentially other lung diseases.

NIH Research Projects · FY 2025 · 2021-11

Project Summary/Abstract The candidate is currently an MD-PhD student at the CCHMC (University of Cincinnati) in the laboratory of Dr. Artem Barski. The proposal describes a combined research and training program leading to independent carreer in biomedical research. The research goal of this project is to identify functional silencers in human T cells and to understand their mechanism of action. Over 98% of the human genome is non-coding. It is believed that many non-coding sequences are regulatory, and are dynamically utilized in a cell type specific manner to dictate programs of gene expression. While enhancers have widely been studied in health and disease including cancers like T-ALL, the location and biological function of silencers is largely unknown. Like enhancers, silencers are believed contribute to cell type specific patterns of gene expression, and thus hold unique requirements for regulation and function in different cell types. Particularly, the contribution of silencers to transcriptome maintenance in human T cells, and the mechanism for silencer activation by canonical repressors (PRC2) is unknown. There is a critical need to directly interrogate functional silencers in CD4+ T cells. I have constructed a silencer screen using a negative selection method to identify the sequences of functional (active) silencers from a genome-wide library of open chromatin in T cells. Preliminary results from my screen in Jurkat cells suggests that functional silencers are largely in unique locations compared to recently published silencer assays. Functional silencers in Jurkat cells also enrich for T cell specific transcription factor motifs, and exist nearest to genes involved in T cell homeostasis, activation, and Th differentiation. I hypothesize that a T cell specific repertoire of silencers actively maintain homeostasis of resting T cells cell by active suppression of aberrant gene expression. In Aim I, I seek to identify functional silencer elements in human CD4+ T cells using the novel assay which I have developed. This work will contribute an atlas of functional silencer elements, including patterns of histone marks and repressor transcription factors enriched at these elements, and elucidate a silencer controlled regulatory network to stimulate hypothesis generation and the research of others. In Aim II, I will investigate a mechanism of silencer activation by canonical repressor PRC2, and determine if some functional silencer sequences also dictate H3K27me3 mediated gene repression. Collectively this work is expected to provide new insights into T cell biology with novel functional data of gene regulation by silencer elements. I expect this work to generate a highly relevant atlas of silencer elements in CD4+ T cells, experimental validation of transcription factors which regulate these elements, and support for a hypothesis that PRC2 coordinates silencer function by mediating both transcriptional repression and H3K27me3 directed transcriptional regulation. Guided by my supportive and experienced mentorship team, I hope to continue developing the scientific and clinical skills necessary to make meaningful contributions to patient outcomes throughout my independent research career.