Cincinnati Childrens Hosp Med Ctr

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract Somatic FMS-like tyrosine kinase 3 (FLT3) mutations are associated with a poor prognosis and increased rates of relapse in acute myeloid leukemia (AML). There is evolving evidence describing genetic-dependent mechanisms of resistance in FLT3-mutant AML following targeted therapy with FLT3 inhibitors, including the phenylalanine 691 to leucine (F691L) mutation that has been reported in patients treated with current generation of FLT3 inhibitors. While AML cells containing this mutation experience inhibition of kinase activity and canonical FLT3 signaling following exposure to FLT3i treatment, our preliminary data revealed that FLT3-F691L AML cells continued propagating leukemia both in vitro and in vivo. The perplexing finding that kinase activity canonical FLT3 signaling is suppressed, despite ongoing survival of the AML cells, suggested kinase independent signaling resulting from F691L substitution in FLT3. The long-term goal of this project is understanding the mechanism of resistance underlying the FLT3-F691L mutation, which we believe is not fully explained by gatekeeper functionality solely. The specific aims for this project are: (i) determine the effects of kinase-dependent and -independent signaling in FLT3-ITD and FLT3- F691L AML cells on differentiation, survival, and gene expression, (ii) identify the pathways and molecules mediating kinase-independent signaling in FLT3-F691L AML, and (iii) examine novel therapeutic vulnerabilities for FLT3-F691L AML and evaluate prioritized pharmacologic combination therapy to overcome resistance. The impact of this grant application is highly significant, as it will have translational implications, filling an unmet need for relapsed/refractory FLT3 mutant AML. If this mechanism of resistance holds true, this could inform drug development and combinatorial therapies for various pediatric cancer types in the long-term.

NIH Research Projects · FY 2026 · 2023-07

The luminal surface of each epithelial cell is covered by several thousand bristles, known individually as a microvillus and collectively as the brush border. The brush border promotes efficient digestion and absorption of nutrients by increasing intestinal surface area and by acting as a scaffold for enzymes, transporters, and host defense factors. Our proposal is significant because few studies address how microvillus structures are regulated in homeostasis or intestinal disease, despite the critical importance of the intestinal brush border to normal intestinal physiology. Monogenetic diseases with disruption of the brush border present with severe diarrhea and intestinal pathology. We have reported that abnormally short ileal microvillus length is present in a subset of histologically uninflamed Crohn’s disease (CD) ileal tissue samples. This phenotype identifies CD patients at-risk for treatment-refractory disease and future development of strictures, features associated with poor clinical outcome in CD. The major objective of this application is to define mechanisms that regulate microvillus length and brush border maturation. This knowledge could help us understand how to reverse pathological changes that occur at the brush border in Crohn’s disease and other pathologic conditions of the intestine. The key preliminary data supporting this application include 1) high fat diet feeding or acute loss of Ppara in intestinal epithelial cells of mice results in short microvillus length; 2) overexpression of Ppara in intestinal epithelial cells rescues high fat diet-induced microvillus shortening; 3) experimental injury of the duodenum or distal colon of mice cause microvillus length shortening in the ileum; and 4) altered brush border ultrastructure and protein expression are observed in Crohn’s disease vs. control patient ileal biopsy tissues. Our central hypothesis is that microvillus length and brush border maturity are suppressed following high fat diet or intestinal injury and can be restored by PPARα signaling activation within intestinal epithelial cells. In Aim 1, we will determine how high fat diet and intestinal epithelial PPARα signaling alter brush border morphology, ultrastructure, and protein expression using novel conditional mouse models with Ppara gain-of-function or loss-of-function specifically in the intestinal epithelium and air-liquid interface epithelial monolayer cultures. We will also use untargeted and targeted approaches to investigate the cellular mechanisms linking microvillus length changes to PPARα signaling. In Aim 2, we will use Ppara conditional mice to determine if intestinal epithelial PPARα signaling influences inflammatory and tissue injury outcomes and injury-associated ileal brush border changes. We anticipate that completion of the proposed studies will produce the following deliverable: identification of a biological pathway and cellular mechanism that integrates host and environmental influences to modify intestinal brush border morphology and function.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY / ABSTRACT Well controlled antiviral innate immunity is essential for restricting viral pathogens while preventing aberrant inflammation. A key antiviral signaling pathway is initiated by RIG-I-like receptors (RLRs) which sense viral RNA in the cytosol to induce the production of interferons through a higher order signaling platform centered on the adaptor protein MAVS. Although the components of the MAVS signalosome are basally expressed, RLR signaling only proceeds when they coalesce around MAVS at ER-mitochondrial contact sites. Many of the protein-protein interactions and post-translational modifications required for the proper function of the MAVS signalosome are known. While, functional interactions in protein complexes can be mediated by RNA molecules, whether RNA molecules play a role in signaling through the MAVS signalosome remains unexplored. I have found that 1) MAVS is associated with non-viral RNA, that 2) RNA promotes signaling through the MAVS signalosome, and that 3) canonical RNA-binding proteins (RBPs) interact with MAVS. This proposal aims to define the functional roles for RNA and RBPs in regulating antiviral signaling through the MAVS signalosome. During the mentored phase, I will gain new training in the characterization of RNA-protein interactions through biochemical, sequencing-based, and quantitative proteomic approaches, as well as the virological techniques required to study RNA viruses and innate immunity. In Aim 1, I will define the RNA-dependent interactions between components of the MAVS signalosome during the K99 phase, and identify novel proteins that associate with RNA during RLR signaling at the R00 phase. Through Aim 2, I will pinpoint the sequences in MAVS required for RNA-association, and identify a high-confidence set of MAVS-bound RNAs during the K99 phase. During the R00 stage, I will functionally dissect the roles for these RNAs at the MAVS signalosome. In Aim 3, I will screen a shortlist of MAVS-associated RBPs to identify those that influence MAVS signaling during the K99 stage, and determine the molecular functions of three RBPs (STAU1, HNRNPL, and RBM10) in antiviral signaling at the R00 stage. The overall outcome of these experiments will be to define new RNA-centric principles by which the MAVS signalosome is organized. Understanding how RNA molecules influence antiviral signaling could unlock new host-directed therapeutic strategies against viral diseases as well as autoimmune disorders. In addition to my advisor Dr. Savan, I have assembled an Advisory Committee with expertise in the different facets of my research. Together, this excellent training environment at the University of Washington will augment my research during the mentored phase, and equip me with the skills required to transition to an independent academic researcher studying the RNA regulation of innate immune processes.

NIH Research Projects · FY 2026 · 2023-07

Project summary/abstract Enteric infections remain highly prevalent worldwide with more than two billion cases and over one million deaths per year. Although diet has long been linked to infection susceptibility, the dietary factors that regulate intestinal defense remain poorly understood. In the intestine, there are trillions of microbes, collectively called the microbiota. Different diets can alter the composition and metabolites produced by these resident microbes in the intestine. In addition, recent work has highlighted clear links between microbiota colonization and effective immune responses at mucosal sites. Thus deciphering the interactions between diet, microbiota, and intestinal immunity will enable development of novel and practical dietary approaches to prevent and treat enteric infection. My new data provoke the hypothesis that metabolism of the nutrient phytate by commensal bacteria can decrease intestinal infection and boost host defense mechanisms. Studies outlined in this proposal will directly test this hypothesis by determining (i) how phytate-enriched diet enhances host intestinal defense in mouse and human intestine, and (ii) whether the microbiota is needed for phytate-mediated defense against intestinal infection. Delineating the contribution of diet and microbiota will significantly expand our understanding of mechanisms involved in intestinal immune regulation and inflammation. During my training as a veterinarian, I discovered my strong interest in understanding how the diet-microbiota relationship regulates intestinal infection. For this reason, I initiated the proposed project with Dr. Theresa Alenghat that will build upon my knowledge of mucosal immunology and enable me to transition into the fields of nutrition and microbiota. My thesis and initial postdoctoral work have provided me with an excellent foundation in immunology and epithelial biology, but I still lack technical skills and conceptual expertise in diet, gnotobiotics, metagenomic analyses, and human intestinal organoids. My mentors, Dr. Alenghat and Dr. James Wells, along with an exceptional scientific environment, will enable me to utilize modern, innovative approaches in my research and collaborate with top investigators. My understanding of disease in a broad range of species, coupled with my prior research, have provided me with a strong and unique foundation. Over the next five years, I fully anticipate that this background in conjunction with my current research plan will allow me to successfully carry out the proposed project. The mentoring and training I will receive from Dr. Alenghat and Dr. Wells, and from the wider Cincinnati Children’s community, will enable me to successfully transition into an independent scientist that can address clinically relevant questions at the interface of nutrition, microbiota, and infection.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT: Septic shock impacts 10% of children admitted to the pediatric intensive care unit, 25% of whom die. Outcomes are particularly poor in those who suffer sepsis-associated acute kidney injury (SA-AKI), a heterogeneous syndrome driven by the dysregulated host immune response to infection, and for which limited treatment options exist. Informed by existing adult literature and a strong foundation of preliminary data, Dr. Stanski’s proposal seeks to develop a precision medicine approach to management of these high risk children with septic shock and SA-AKI, by leveraging clinical and biomarker data to identify unique SA-AKI subphenotypes that may inform vasoactive medication selection at the bedside. The results from these studies will guide future prospective work to identify treatment strategies for pediatric SA-AKI, and improve outcomes for children with septic shock as a whole. Candidate: Dr. Natalja Stanski is an Assistant Professor of Pediatric Critical Care Medicine at the University of Cincinnati College of Medicine and Cincinnati Children’s Hospital Medical Center (CCHMC). She has a strong early track record of patient-oriented research in the areas of acute kidney injury and sepsis precision medicine, focusing on identifying strategies to characterize syndrome heterogeneity. Dr. Stanski’s long- term goal is to become an independent physician-scientist leading precision-medicine informed patient-oriented research to improve outcomes for children with sepsis and SA-AKI. As such, her training goals as part of this award include gaining advanced knowledge in the following areas: design and conduct of patient-oriented research (including clinical trials), implementation science and qualitative research methodologies, advanced biostatistics (including techniques for cluster analysis), and content expertise in precision medicine, sepsis and SA-AKI. To accomplish these goals, Dr. Stanski’s career development plan includes formal coursework in advanced biostatistics, clinical trial management, implementation science and qualitative research; individualized expert mentoring; training by expert consultants; and completion of the proposed research aims. Research: The aims of this study are: 1) to use cluster analysis methodology to identify novel pediatric SA-AKI clinical subphenotypes and describe their associations with outcomes and serum biomarker profiles, and 2) to use Implementation Mapping to develop a rigorous, theory-based implementation plan for personalized vasoactive selection in children with septic shock and SA-AKI. This application leverages the outstanding research infrastructure at CCHMC, and the support of a formidable expert mentorship, consultant and collaborator team. Specifically, Dr. Stanski’s access to the existing large patient datasets and biorepositories necessary to conduct this important work makes her uniquely suited to complete her proposed research aims. The support and training provided by this K23 will leave Dr. Stanski well-positioned to achieve her long-term goal of research independence, and the preliminary data generated will provide the foundation for a feasible precision medicine approach to SA-AKI management that is needed to improve outcomes in pediatric septic shock.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory esophageal disorder characterized clinically by esophageal dysfunction (vomiting, pain, dysphagia, and food impaction); histologically by esophageal eosinophilia, epithelial hyperplasia, and dilated intercellular spaces associated with impaired barrier function; and by a high degree of heritability. Most genetic studies have focused on analyzing common genetic variants by genome-wide association studies (GWAS), with evidence implicating the calpain 14 (CAPN14) and thymic stromal lymphopoietin (TSLP), which are notably both expressed by the same relevant cell type, esophageal epithelial cells. Recently, we have performed whole-exome sequencing (WES) on EoE multiplex families and identified a set of rare genetic variants involved in EoE. Though recent research progress provides the evidence for EoE genetic etiology being linked to genetic variants, testing single genetic variants separately does not consider the complex interaction landscape of genes. Our central hypothesis is that a subset of EoE results from the combination of multiple variants in the same biological pathways. This study will statistically and experimentally evaluate combinatory effects and how the genetic variants are contributing to EoE and will develop risk scores based on the biological pathways to predict EoE by using recently developed innovations (e.g., WES, GWAS, RNA-seq, ChIP-seq, and ex vivo disease modeling [esophageal organoids and organotypic culture]). In the attached proposal, we have outlined an integrated set of multidisciplinary studies with the necessary statistical and experimental support to evaluate the impact of the combinatory effects among EoE genetic variants. In Aim 1, we will test the hypothesis that the risk for EoE will be increased by the combinatory rare-rare variants, rare-common variants/SNPs, and biological pathways. To determine the impact on the risk for EoE, we will jointly analyze combinatory effects at variant, gene, and pathway levels. In Aim 2, we will test the hypothesis that DSP and PPL rare variants have combinatory effects on esophageal barrier functions and gene/protein expression. To explore the operational mechanisms, we will examine whether these variants have combinatory effects using ex vivo, 3-dimensional culture models (e.g., esophageal organoids, organotypic culture) of EoE. Finally, in Aim 3, we will test the hypothesis that the synthesis of genetic and genomic data will lead to the ability to predict who is at risk of developing EoE, its disease features, and/or response to therapy. Aim 3 will serve as the foundation for a future R01 application to conduct a mechanistic study to characterize the impact of convergent genes/pathways and a case-control study to further validate and explore the clinical utility of risk scores. The proposed study will address an unmet medical need as outlined by a recent NIH workshop, providing insight into disease genetic mechanisms and thereby potentially contributing to personalized medicine, especially the application of enhanced screening or preventive therapies.

NIH Research Projects · FY 2025 · 2023-07

Abstract. The human intestine harbors an estimated 100 trillion microbes that are increasingly recognized to promote health through tonic immune stimulation. These include innocuous commensal microbes along with pathobionts - those capable of causing gut dysbiosis or invasive infection. Most of what we currently understand about host-microbe commensalism has been evaluated through the lens of bacteria. However, microbes from other taxonomic domains, including eukaryotes, also ubiquitously colonize mucosal tissues and yet our understanding of how these microbes establish commensalism and drive immunological changes remains rudimentary. This gap in knowledge is especially significant for the most common fungal pathobiont Candida albicans, which can translocate out of the gastrointestinal (GI) tract and cause life-threatening systemic infection, particularly in immunocompromised individuals. To address these fundamental gaps in knowledge, an instructive model of C. albicans intestinal colonization in mice was developed. Recombinant C. albicans cells were engineered to express defined model antigens and used to establish colonization so that T cells with surrogate C. albicans specificity could be identified. Using this model, we show that C. albicans cells colonizing the GI tract result in action at a distance - they drive the systemic accumulation of fungal-specific Th17 CD4+ T cells. These T cells work together with IL-17 and activated neutrophils to provide protection against a systemic infection by C. albicans as well as by extracellular bacterial pathogens. These results highlight the protective benefits of commensal C. albicans cells residing in the GI tract, and suggest that co-evolution with this species has led to a mutually beneficial relationship. However, important questions remain as to how C. albicans cells in the gut prime systemic immune responses, and how Th17 signals can be triggered without excessive inflammation. This line of investigation builds upon exciting preliminary data generated together by the laboratories of Dr. Way and Dr. Bennett, two investigators with complementary expertise in clinical infectious disease/cellular immunology and mycology/fungal pathogenesis, respectively. This proposal will address the molecular and cellular mechanisms by which C. albicans cells interact with mucosal host tissues to drive gut local and systemic immunity through the following specific aims: (1) Define how C. albicans morphological changes drive systemic Th17 immunity, (2) Establish the fungal ligand and host pattern recognition receptor(s) that prime systemic Th17 immunity, and (3) Investigate the role of reactive oxygen species and Duox2 (Dual Oxidase 2) for local and Th17 immunity primed by C. albicans cells. Each of these specific aims is supported by extensive published and unpublished preliminary data. Successful completion of these aims will shed light on the important symbiosis between fungal commensal and mammalian host, and the mechanisms responsible for priming systemic Th17 immunity through intestinal stimulation.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Somatic mutations in DNMT3A and TET2 are common in the hematopoietic lineages of elderly individuals, estimated to affect more than 10% of adults over the age of 65. These mutations increase the risk for age-related comorbidities, including severe infection, atherosclerotic cardiovascular disease, osteoporosis, chronic kidney disease and hematologic malignancies, nearly doubling the mortality rate of affected individuals. DNMT3A and TET2 encode enzymes essential for remodeling DNA methylation during cellular differentiation. Animal studies suggest that mutations in these genes drive aberrant activation of immune cells, such as macrophages, which may underlie the disease associations. We recently developed a human pluripotent stem cell (hPSC)-derived macrophage model, where the differentiation-dependent effects of DNMT3A or TET2 perturbation can be precisely delineated. We discovered that DNMT3A- and TET2- perturbations impaired DNA methylation remodeling at thousands of regulatory loci, altering enhancer activities and expression of genes important for macrophage function. Our study highlighted the need for engineering approaches, and mathematical modeling in particular, to unravel the complex effects of DNMT3A and TET2 perturbations on cellular function and disease risk. Here, we pair novel computational modeling approaches with unique experimental resources to mechanistically connect site-specific changes in DNA methylation to aberrant immune responses and disease risk. Aim 1 builds deep neural network models (and requisite training data resources) to predict the effects of DNA methylation on chromatin binding of 100+ transcription factors (TFs), the “readers” of DNA methylation patterns that ultimately recruit RNA polymerase and co-activators to drive gene transcription. In Aim 2, we predict genome-scale TF-binding patterns from chromatin accessibility, transcriptional activity and DNA methylation data in our contexts of interest: DNMT3A- or TET2-perturbed human macrophages in response to viral and bacterial infection-induced immune activation. To discover links between existing and novel disease associations, we will intersect the TFBS predictions with curated sets of age-related disease risk variants, to nominate TFs and contexts where DNMT3A- or TET2-perturbation and downstream alterations in TF binding might mediate disease risk. In Aim 3, we will construct gene regulatory network (GRN) models of DNMT3A- and TET2-perturbed human macrophage to identify TFs driving differential gene expression responses to infection, hypotheses that (1) we will experimentally test and (2) could eventually lead to therapies that mitigate the negative, pathogenic consequences of common DNMT3A and TET2 mutations. Furthermore, we build significant generalizable resources (models, modeling methodologies and training data) that will enable future discoveries in new cell types and disease contexts where alterations in DNA methylation drive phenotypes.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT A severe asthma endotype involving neutrophils infiltrating the airways with a Th17 dominant milieu, and excessive airway remodeling and resistance to conventional therapies, has emerged in patients in the past years. In a transcriptomic screen, we found that the TNF Superfamily member LIGHT, was highly enriched in a murine model of severe asthma mimicking the human disease, in addition to being enriched in the sputum of patients. This proposal will evaluate the contribution of LIGHT signaling in 1) neutrophils to promote airway neutrophilia, 2) stromal cells to promote pathological remodeling and steroid resistance, and 3) it will evaluate the pathogenic role of Th17 cells as drivers of airway remodeling compared to Th2 cells, through LIGHT production. Furthermore, we have evidence that the TNF Superfamily LIGHT decreases airway remodeling and will address how LIGHT blockade compares to neutrophilic depletion, or IL17 neutralization, in decreasing NA symptoms: airway resistance, epithelial damage, fibroblasts activation, and neutrophil recruitment. Lastly, this project will address whether the combinatorial blockade of LIGHT and steroid treatment, can abrogate airway remodeling in severe asthma, in the mouse model and the human lung-in-a-dish model we established.

NIH Research Projects · FY 2025 · 2023-06

Project Summary/Abstract Unprecedented rates of obesity are occurring in childhood and disproportionally affect Black youth, Hispanic/Latinx youth, and youth from economically marginalized backgrounds beginning as early as infancy. Obesity in infancy is not outgrown, tracks into adulthood, and places infants and children with obesity at a higher risk for significant medical comorbidities (e.g. cardiometabolic complications) in adulthood. The healthcare cost of obesity is ~$260 billion annually across the lifespan. Recent evidence suggests that infancy may be a critical period for the development of this high weight trajectory, as 10% of infants meet criteria for high weight-for-length; with the incidence being even higher among infants of color (16.3%), infants of Hispanic/Latinx ethnicity (12.1%), and economically marginalized infants (12.2%). Several modifiable predictors of obesity risk have been identified in infancy, including rapid weight gain in the first year, parental use of food to regulate infant distress, early introduction to solid foods, and insufficient infant total cumulative sleep. These risk factors are higher among Black, Hispanic/Latinx, and economically marginalized infants. Thus, obesity prevention starting in infancy has been advocated. The proposed research project addresses a novel opportunity for prevention in pediatric primary care, by pilot testing a promising, innovative infant obesity prevention intervention that utilizes a responsive parenting paradigm (e.g. promotes healthy infant regulation) to target the development of healthy feeding and sleep behaviors in infancy. The intervention will be delivered within pediatric primary care via an emerging model of integrated behavioral health. A responsive parenting approach to obesity prevention is strengths-based and focuses on accurate caregiver interpretation of infant cues, feeding behaviors, soothing, and sleep, rather than potentially stigmatizing messaging related to obesity, and thus, is more salient to caregivers of infants than an obesity-focused intervention. We will conduct a pilot RCT comparing our responsive parenting intervention to treatment as usual in 138 infants who are from communities of color (e.g., non-White; Hispanic/Latinx) or economically marginalized backgrounds (i.e., publicly insured). Families will receive four prevention sessions with the integrated behavioral health specialist at their routine primary care well-child visits during the first six months of life. The primary outcome is conditional weight gain (an indicator of rapid weight gain) at 9 months of age. The responsive parenting approach, delivered by an integrated behavioral health expert, has been culturally adapted for infants and caregivers who are economically marginalized and/or from communities of color through focus groups. The delivery of this intervention via integrated behavioral health in a pediatric primary care setting has the potential to have a significant public health impact in terms of preventing pediatric obesity later in life; and thus, improving health outcomes and reducing health disparities.

NIH Research Projects · FY 2026 · 2023-05

Abstract: Etv4 and Etv5 (Etv4/5) are two transcription factors expressed in the developing mouse kidney, specifically in the ureteric bud tips, the metanephric mesenchyme and the renal vesicle. Our preliminary data using a mouse model of Etv4/5 deletion in the nephron progenitor cells (NPCs) and their progeny demonstrate a critical role for these transcription factors during nephrogenesis; absence of Etv4/5 cause premature NPC exhaustion, thinning nephrogenic zone and hypoplastic/cystic kidneys at birth. In addition, these mutants present segmentation defects as early as the s-shape body stage. We have generated RNAseq data from NPC mutant and littermate control embryonic kidneys and identified Wnt4 as a downstream target of Etv4/5. Based on these preliminary data we hypothesize that Etv4/5 modulate nephrogenesis, at least in part, by downregulating Wnt signaling in the NPCs and in the developing nephron. We further hypothesize that this downregulation is required to favor both self-renewal of NPCs and differentiation of the early nephron. We will test these hypotheses in two aims. In aim one we will investigate the crosstalk between Fgfs, Etv4/5 and Wnt4 signaling to promote self-renewal/prevent differentiation of the NPCs. In aim two we will investigate how Etv4/5 and Wnt4 signaling affect nephron differentiation. These studies will provide further insight into the signaling network driving progenitor self-renewal and differentiation; they will also expand our knowledge of the cellular readout of Etv4 and Etv5 expression, therefore having a significant impact not only on the field of developmental biology but also in cancer and in vitro organogenesis.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT Premature birth is the leading cause of neonatal intensive care unit admissions and exerts a high burden of disease and health care cost, approaching $13.4 billion annually in the United States. Advances in neonatal medicine such as antenatal steroids and surfactant have improved survival, particularly in infants born extremely premature (before 30 weeks’ gestational age). Consequently, bronchopulmonary dysplasia (BPD) is increasing in prevalence. This often results in lifelong cardiopulmonary sequelae that overlap with chronic diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary hypertension, increasing the risk of premature death. Unfortunately, the impact of extreme prematurity or postnatal steroid treatment on cardiopulmonary structure and function has not been rigorously evaluated during early school age (6-8 years), when early identification could prevent long-term adverse outcomes. Thus, significant changes can occur and remain undetected during this critical age of rapid lung growth and alveolarization, a time during which intervention could be the most advantageous. To address this unmet need, we will capitalize on two outstanding resources already in place at Cincinnati Children’s: 1) cutting-edge proton and hyperpolarized-gas magnetic resonance imaging (MRI) that can precisely characterize phenotypes of BPD in the Cincinnati BPD Center and 2) a well- characterized population-based cohort of preterm infants from the NIH-funded Cincinnati Infant Neurodevelopment Early Prediction Study (CINEPS) that will reach school age during this funding period. We will recruit the CINEPS subgroup of extremely preterm infants (140 with BPD; 68 without BPD) and a new cohort of 50 term-born controls and study them longitudinally at 6 and 8 years of age with proton and hyperpolarized gas MRI, spirometry, plethysmography, diffusion capacity, and a parent questionnaire. The primary goal of this proposal is to define changes in cardiopulmonary structure and function in extremely preterm infants during early school age. We will further evaluate the effects of postnatal steroids therapy on lung structure and function compared to untreated infants matched using propensity score weighting. This novel proposal represents the first application of hyperpolarized gas MRI in a cohort of extremely preterm children. Further, this proposal represents the most detailed evaluation of the trajectory of extreme prematurity on cardiopulmonary structure and function through early school age. This high-impact research will define the progression of lung, heart, and pulmonary vascular disease as well as response to postnatal therapies and provide a unique opportunity for early identification of patients who will develop chronic changes related to extremely preterm birth and BPD.

NIH Research Projects · FY 2026 · 2023-05

Project Summary Neurulation is a crucial stage in human development when the nervous system is patterned, and the neural tube is shaped. A large fraction of severe birth defects as well as post-implantation spontaneous abortions occur at this stage. Despite its importance, this stage of human development is almost completely inaccessible as IVF embryos cannot be cultured until this point, and recovery of tissue samples from these early stages is rare. Even if such methods were possible, they present ethical dilemmas. A powerful emerging alternative is to create self- organizing models of these events starting from human pluripotent stem cells (hPSCs). We have created a two- dimensional system that mimics the early patterning of the ectodermal medial-lateral axis and is ideal for studying self-organization of ectodermal fates which include the nervous system, skin, neural crest, and sensory organs. We have also built upon this system to create a controlled three-dimensional system that undergoes reproducible morphogenesis reminiscent of neural tube closure. Here we propose to refine these models, as well as to create new models of the anterior-posterior axis, and to use these models together with live cell reporters to probe the interplay between morphogen signaling through the Wnt, BMP, and FGF pathways, patterning along the anterior- posterior (AP) and medial-lateral (ML) axes, and neural tube morphogenesis. We will address key questions about how spatial patterns of morphogen signals are organized and how they are interpreted by cells to give rise to patterns of fates and the physical movements that shape tissues. Taken together, these experiments will provide insight into ectodermal patterning and morphogenesis and establish a system for studying birth defects associated with this stage of development.

NIH Research Projects · FY 2026 · 2023-04

Project Summary: Adolescents and young adults (AYA) are disproportionately affected by HIV in the US. Despite adolescents accounting for over 20% of new infections, this age group is the least likely to be tested for HIV, linked to care, and achieve viral suppression when compared to their adult counterparts. Further, AYA also have low rates of HIV awareness and initiation of HIV Pre-Exposure Prophylaxis (PrEP). Therefore, there is an urgent need to expand HIV screening and prevention strategies to nontraditional healthcare settings such as emergency departments (ED) to reach those adolescents who would otherwise not receive preventive healthcare. The goal of this application is to leverage our recent insights obtained from a multi-center, ED-based, adolescent gonorrhea and chlamydia screening study and apply them across a national pediatric ED research network by (1) adapting, refining, and testing this process to increase universally offered, opt-out HIV screening among adolescents in the pediatric ED and (2) link at-risk adolescents to PrEP services and preventive care. This will be accomplished through a network of children’s hospital EDs with a track record of robust research collaboration (Pediatric Emergency Care Applied Research Network or PECARN). This research will contribute to the evidence base for creating clinically effective and sustainable HIV screening programs that can be successfully implemented into the clinical workflow of the ED. It will also improve identification and linkage to PrEP care for at risk adolescents using mHealth strategies by first identifying AYA who are PrEP candidates based on their responses to a computerized sexual health screen (cSHS) and subsequently (1) providing clinical decision support to providers via the electronic health record and (2) direct text messaging from the cSHS to PrEP candidates providing educational content and connecting youth to a PrEP navigator. This intervention will rely on an innovative approach that electronically integrates patient-reported data to guide clinical decision support. This work is significant because it has the potential to shift current ED clinical practice paradigms from only acute health encounters to participation in the broader management of public health and prevention, and it will fill gaps in the literature needed to provide evidence for the best method of HIV screening in a pediatric ED setting. Using a previously developed tablet-based, broad scale GC/CT screening process, we will adapt, refine, and test this process with the aim of increasing universally offered, opt-out HIV screening in the pediatric ED through electronic integration of patient reported data for provision of clinical decision support for HIV screening and identification of PrEP candidacy, and then use mHealth to link patients to PrEP services. This research is novel in that it shifts the usual clinical practice paradigm of HIV screening and prevention in the pediatric ED from a scattered approach to a consistent and sustainable approach that is critical to addressing the HIV epidemic among adolescents.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract Appropriate differentiation and maintenance of cellular identity are required for normal development of all organs. In the heart, mutations in genes that are necessary to maintain cardiomyocyte identity are associated with structural congenital heart defects, which are the most common malformations found in newborns. Despite their frequency, the etiology of most congenital heart defects remains poorly understood. Furthermore, numerous structural congenital heart defects are associated with arrythmias. Although advances in surgical techniques have been successful in allowing patients to survive to adulthood, the surgeries do not repair arrythmias associated with the structural defects. Thus, it is essential to understand fundamental mechanisms directing normal vertebrate heart development, in order to inform us of the etiology of congenital heart defects and their associated arrythmias. A long-term goal of our lab is to understand the conserved molecular and genetic mechanisms that direct cardiac chamber size during early vertebrate development. Nr2f transcriptions factors have highly conserved requirements in vertebrate heart development. Furthermore, mutations in Nr2f genes in humans are associated with a spectrum of congenital heart defects, including atrial septal defects. This proposal will investigate fundamental mechanisms determining atrial chamber size through investigating Nr2f-dependent mechanisms controlling atrial cardiomyocyte differentiation and the maintenance of atrial cardiomyocyte identity. While requirements for Nr2f factors are well-established in atrial development, the mechanisms controlling Nr2f gene expression in atrial cardiomyocytes and by which Nr2f transcription factors direct atrial cardiomyocyte development remain poorly understood. Our work has shown that zebrafish Nr2f1a is the functional equivalent of Nr2f2 in atrial development. Our preliminary data has identified a conserved enhancer that that is sufficient to promote Nr2f1a expression in atrial cardiomyocytes zebrafish and that Nr2f1a has a previously unrecognized requirement concurrently maintaining atrial cardiomyocyte and inhibiting the acquisition of pacemaker cardiomyocyte identity. In Aim 1, we will interrogate the signals that regulate the conserved nr2f1a cis-regulatory enhancer that promotes atrial cardiomyocyte expression. In Aim 2, we will determine the temporal requirements of nr2f1a and the differentiation state of cardiomyocytes within the atria of nr2f1a mutants. In Aim 3, we will elucidate the Nr2f-dependent gene regulatory networks that repress pacemaker cardiomyocyte identity in venous atria. Our studies may provide a foundation of information that may inform us of the etiology of congenital heart defects and their associated arrythmias, which ultimately may lead to novel therapies that can prevent or ameliorate congenital heart defects and associated arrythmias in humans.

NIH Research Projects · FY 2026 · 2023-04

Abstract This multiple-PI application proposes investigations of cellular and molecular mechanisms of Fibrolamellar Hepatocellular Carcinoma, FLC. FLC is a disease that occurs in children, adolescents and in young adults without a history of other liver disorders such as fibrosis or cirrhosis. Numerous studies in FLC patients and in animal models clearly showed that FLC is caused by a fusion protein kinase DNAJB1-PKAc which is sufficient to induce liver cancer resembling FLC in mouse models. We found that the fusion kinase DNAJB1-PKAc phosphorylates b-catenin at Ser675 in FLC patients and in FLC-derived spheroid cells. This phosphorylation leads to elevation of the b-catenin-TCF4 complex which might activate multiple cancer genes via two pathways: interaction with and activation of unique chromosomal regions (Cancer-Enhancing Genomic Regions or Aggressive Liver Cancer Domains, CEGRs/ALCDs) and via direct activation of the promoters of genes involved in metabolic rewiring. The main hypothesis of this application is that the DNAJB1-PKAc-b-catenin axis is a critical mediator of FLC pathology via activation of CEGRs/ALCDs-dependent neuronal genes, oncogenes and collagens as well as genes involved in pathological metabolic changes. Three Specific Aims are designed to test this hypothesis. Specific Aim 1 will examine the role of the DNAJB1-PKAc-b-catenin pathway in activation of CEGRs/ALCDs-dependent cancer genes in FLC. The focus of these studies is on the FLC patients who have relapsed tumor and lung metastases which are potentially caused by activation of DNAJB1-PKAc-b-catenin- dependent neuronal pathways. Specific Aim 2 will investigate the role of the DNAJB1-PKAc-b-catenin-TCF4 axis in FLC using a mouse model of FLC. We have generated an FLC mouse model, in which DNAJB1-PKAc protein is created and expressed at high levels at 4 months after injections of gRNAs. We will examine development of pathological changes in the liver and investigate the DNAJB1-PKAc-b-catenin-TCF4 pathway at different stages of FLC. We will inhibit b-catenin by PRI-724 in the FLC mouse model, examine development of FLC, and determine downstream molecular mediators. Specific Am 3 will determine the role of the DNAJB1- PKAc-b-catenin-TCF4 axis in rewiring of metabolic pathways in FLC. Our preliminary data suggest that the urea cycle and associated metabolic processes are rewired toward increased collagen synthesis and maturation in FLC, and the treatment of FLC spheroids with PRI-724 reverts these changes. Therefore, we will test the hypothesis that the DNAJB1-PKAc-β-catenin-TCF4 axis is a critical event for metabolic rewiring and will also determine the impact of metabolic rewiring on FLC proliferation and survival. Thus, this project will elucidate mechanisms of FLC and develop a background for b-catenin-based therapy.

NIH Research Projects · FY 2026 · 2023-04

Helminth infections and allergic diseases impact billions of individuals worldwide. While induction of type 2 immune responses are necessary for combatting helminth pathogens, inappropriate type 2 immunity triggers inflammatory and allergic conditions. Therefore, improved understanding of mechanisms that control type 2 immune responses are needed. The intestinal microbiota continually influence immune responses, and several studies have demonstrated that microbiota-derived factors dampen type 2 immunity. Intestinal tuft cells are specialized epithelial cells that are essential for sensing luminal signals and initiating downstream type 2 immune responses. My preliminary data indicate that the microbiota regulate tuft cell homeostasis and tuft cell-dependent type 2 immune responses in the intestine. Studies outlined in this proposal will directly test (i) how intestinal stem cells instruct tuft cell differentiation, and (ii) how commensal bacteria impact tuft cell-dependent type 2 immunity. Collectively, these studies will provide new insights into how the microbiota direct intestinal epithelial differentiation to instruct intestinal immune responses, and will guide novel microbiota-based approaches for investigating and treating type 2-driven intestinal diseases. My career goal is to establish myself as a successful investigator studying microbiota-regulated intestinal diseases. To progress towards this goal, I propose in this application to dissect interactions between the microbiota, epithelial cell differentiation, and mucosal immune responses. I will specifically concentrate on developing expertise in four new areas that will facilitate my transition to independence: 1) gnotobiotics and metagenomics, 2) type 2-driven murine disease models, 3) epithelial development and human intestinal organoids, and 4) chromatin and single cell analyses. My mentors, Dr. Alenghat and Dr. Wells, along with the exceptional scientific and intellectual environment at Cincinnati Children’s Hospital will enable me to utilize modern, innovative approaches in my research and collaborate with top investigators. Over the next five years, I fully anticipate that my background, in conjunction with the career development plan outlined in my application, will allow me to successfully carry out the proposed project. The mentoring and training I will receive will enable me to successfully transition to an independent research career directed towards fundamental advances in intestinal immunity, as well as innovative and targeted strategies for investigating microbiota-sensitive diseases.

NIH Research Projects · FY 2026 · 2023-04

Project Summary Functional abdominal pain disorders (FAPD) constitute one of the most common pediatric chronic pain conditions. FAPD is associated with significant disability, school absenteeism and suboptimal treatment outcomes. Treatment is hampered by an incomplete understanding of contributing factors like sleep disturbances. Subjective sleep disturbances have been reported in FAPD but the role of sleep architecture and its relationship to gastrointestinal (GI) symptoms has not been established. Preliminary data by the candidate has shown decreased slow wave sleep (%NREM3) measured via polysomnography (PSG) in FAPD patients vs. controls. Percutaneous Electrical Nerve Field Stimulation (PENFS) is an FDA cleared therapy for pediatric FAPD. Preliminary data indicates that PENFS improves sleep in addition to symptoms of FAPD. However, there is limited data on factors predicting response, effects on sleep architecture and underlying mechanisms. The primary aims of this K23 training application are to: (1) Determine the relationship of sleep architecture (NREM3 and other variables measured via PSG) with abdominal pain and other patient-reported outcomes in a case-control study of adolescents with FAPD. (2) Compare changes in sleep architecture before and after PENFS, associate with patient reported outcomes. This is the first study to objectively characterize sleep architecture in pediatric FAPD and changes with PENFS. Comprehensive measures of sleep physiology such as sleep questionnaires, daily diaries and actigraphy will be employed in addition to PSG to associate with GI measures. The primary objective is for the candidate to acquire advanced research training through a Master of Science in Clinical and Translational Research, learn sleep (PSG and actigraphy) and pain assessments, and advanced statistical methods. The long-term goal is to build an independent research program to study risk factors in pediatric FAPD and identify novel treatment targets to improve outcomes. The K23 candidate is an Assistant Professor of Pediatrics, and a pediatric gastroenterologist at Cincinnati Children's Hospital Medical Center (CCHMC). This application integrates a thoughtfully designed research and training plan developed in conjunction with mentors and collaborators who are leading scientists and clinical experts in pain, GI, sleep and behavioral medicine. CCHMC provides a rich collaborative research environment and is deeply dedicated to the development of young investigators with significant institutional funding opportunities, support in developing clinical trials, study monitoring, and statistical analysis through core resources at the Digestive Health Center, and Clinical Translation Research Center. This environment will be conducive to the candidate to complete the proposed research and training to achieve full research independence. Results will build the foundation for a subsequent R01 trial to further optimize PENFS outcomes and determine the relationship of central pain processing with NREM3 sleep.

NIH Research Projects · FY 2026 · 2023-03

PROJECT DESCRIPTION One in six pregnancies is affected by some form of gestational diabetes (GD), a prevalence that is steadily rising in the context of the worldwide epidemics of obesity and early diabetic onset. Early diagnosis and advances in maternal glucose control have greatly mitigated the perinatal consequences of gestational diabetes on the mothers and their offspring. However, these advances have only marginally impacted its long-term consequences. As such, exposure to hyperglycemia in utero remains associated with increased long-term morbidities in offspring. The mechanisms driving this pathological transmission across generations have not been established. The overarching hypothesis of this project is that the hematopoietic stem cells (HSCs) of the offspring are stably altered by gestational diabetes and are essential “effectors” of the long-term pathological effects of gestational diabetes in offspring. Our preliminary data established two reliable mouse models of gestational diabetes that reproduce not only the perinatal adverse features of the human pathology, but also its long-term consequence in adult offspring. In these models, we show that gestational diabetes alters the hematopoiesis of the offspring and that this effect persists to adulthood, even in absence of overt diabetes. This phenotype indicates the acquisition of a long-lasting memory of metabolic events by the most upstream hematopoietic stem cell (HSC) compartments. Importantly, our results also indicate that hematopoietic alterations present in offspring can contribute to pathologies, such as atherosclerosis. Here we propose to investigate the interplay between gestational diabetes and the hematopoietic system. Based on our preliminary data, we will determine in aim 1 how signaling through the receptor of advanced glycation end products (RAGE) contributes to the acquisition of a diabetic hematopoietic memory in GD offspring. In aim 2, we will establish the epigenetic modifications that underlie the long-term maintenance of this hematopoietic memory in adult GD offspring. Our work will decipher the mechanisms underlying the hematopoietic memory associated with gestational diabetes. Defining these mechanisms will establish potential biomarkers for diabetic hematopoietic memory. it will also reveal new therapeutic targets to alter the trajectory of the hematopoietic memory and prevent its long-term pathological consequences.

NIH Research Projects · FY 2026 · 2023-03

SUMMARY Immunization represents one of the most successful public health interventions in human history, preventing more than 2 million deaths each year. Vaccine success depends on a variable combination of antibodies that can neutralize the invading pathogen and virus-specific T cells that kill infected targets. However, the induction of neutralizing antibodies and antiviral T cells that are sufficiently functional and broadly targeted to thwart a highly mutable pathogen like HIV has proven exceptionally difficult in both humans and animal models. Thus, there is currently no efficacious vaccine to prevent the nearly 5,000 new infections with HIV that occur each day. This shortcoming in vaccine success is likely due to intrinsic immune regulatory mechanisms that limit the quantity and quality of HIV-specific immune responses. Development of translational means to overcome these immunological roadblocks holds great promise for advancement of next-generation vaccines to prevent HIV infection and improve global health. Our research focuses on the remarkable capacity of natural killer (NK) cells to suppress the magnitude and quality of antiviral T and B cell responses triggered after immunization. NK cells impair the generation of protective neutralizing antibody responses by inhibiting follicular helper T cell responses and restricting affinity maturation of antibodies within germinal centers. This NK-cell immunosuppression also limits the quantity and quality of antiviral memory T cell responses. NK cells achieve this suppressive effect via perforin-dependent killing of activated T cells, although the specific receptors used to recognize target T cells and perforin-delivered granzymes involved in triggering cell death remain incompletely defined. Whereas inhibition of perforin could curtail NK cell-mediated immune suppression, this broad of an approach could temporarily undermine immunity against pathogens and tumors, and thus a more refined approach targeting granzymes is proposed. Therefore, the goal of this proposal is to advance an innovative high risk, high impact approach to foster HIV vaccine efficacy through selective inhibition of granzymes involved in the immunosuppressive activity of NK cells. Initial experiments in mice will use small molecule inhibitors and CRISPR to define the utility of targeting a specific granzyme to limit NK-cell killing of T cells and suppression of vaccine-elicited adaptive immunity. Select inhibitors will be validated in Rhesus macaques. Based on quantitatively defined go/no-go criteria establishing the success of granzyme targeting to enhance vaccine efficacy, we will proceed to evaluation of this approach in vaccine- mediated prevention of SIV infection in non-human primates. These experiments will also open impactful avenues of investigation into the molecular features of both the immunosuppressive subset of NK cells and targeted subpopulation of T cells. Thus, the proposed work will facilitate subsequent development and deployment of innovative strategies to enhance HIV vaccine efficacy.

NIH Research Projects · FY 2026 · 2023-03

(<30 lines) Youth-onset type 2 diabetes (YO-T2D) is increasingly prevalent in parallel with the obesity epidemic, yet effective treatment and prevention strategies are limited. The physiologic increase in insulin resistance occurring during puberty, in combination with obesity-related insulin resistance, enhances the risk of T2D. Yet, it remains unclear why some youth progress through puberty with intact β-cell function, while others do not, despite similar phenotypic and metabolic characteristics. More information is needed regarding the unique events during puberty to better understand 1) the basic pathophysiology of glucose control, insulin sensitivity, β-cell function, and T2D risk in youth, 2) differences among girls and boys, populations at highest risk, and urban and rural geographies, and 3) the potential contribution of other risk factors including psychological, behavioral, and social and external contexts. Importantly, this research needs to address the timeline of pathophysiology and progression from normoglycemia or prediabetes to YO-T2D. The DISCOVERY of Risk Factors for Type 2 Diabetes in Youth (DISCOVERY) study provides a unique opportunity to characterize the risk progression profile and mechanisms underlying the development of YO-T2D, and evaluate the effects of modifiable and non-modifiable risk factors. Ultimately, the results of this study will establish a basic pathophysiology to inform future studies aimed at achieving target glycemia, improving insulin sensitivity, preserving β-cell function, and/or preventing YO-T2D. To address this goal, DISCOVERY will recruit, enroll, and follow a nationally-representative cohort of 3,600 at-risk obese youth in early puberty; extensively phenotype them as they transition through puberty; and characterize the course of decline and dysfunction in pathophysiological indicators that lead to YO-T2D. The expected duration of the DISCOVERY is 5 years, including planning, recruitment, follow-up, analysis, and reporting. In addition, DISCOVERY will store longitudinal biospecimens and genetic material with the intention of acquiring additional ancillary funding to pursue analysis of emerging indicators. Cincinnati Children’s Hospital Medical Center has experience in multicenter and diabetes-related investigations and will contribute to DISCOVERY through the recruitment of approximately 240 at-risk youth, implementation of the IRB-approved consensus protocol, participation on DISCOVERY committees, and collaboration on the analyses and dissemination of the findings from DISCOVERY.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Pediatric chronic non-infectious uveitis (NIU) is an inflammatory ocular disease that has a substantial risk for sight-threatening complications. Methotrexate (MTX) is the preferred first line systemic treatment for all subtypes of NIU given its overall safety and affordability. However, MTX failure is as high as 50%. Biologic drugs are reserved for those who fail MTX. The long delays waiting for therapeutic effect leads to prolonged glucocorticoid use and continued accrual of ocular damage. As MTX may not be the appropriate first line treatment for all subtypes of NIU, identifying predictors of MTX responsiveness will allow expeditious initiation of biologic therapies. Our long-term goal is to prevent sight-threatening damage in children with NIU by initiating early effective treatment that controls inflammation quickly. This investigation is a longitudinal translational study that is a collaborative effort consisting of experts in rheumatology, ophthalmology, ocular imaging, immunology and biostatistics. The objectives of this study are: 1) To identify baseline demographic, clinical, laboratory, and imaging features in children with NIU that correlate with response to MTX; 2) To assess the value of adding quantitative imaging modalities to monitor response to MTX in children with NIU; and 3) To discover gene expression signatures that predict response and non- response to MTX in children with NIU. A total of 120 children who are starting MTX for NIU will be enrolled in all aims and followed prospectively at 3 and 6 months. Children will undergo serial clinical ophthalmic examinations and imaging by anterior segment optical coherence tomography (AS-OCT), ultrawide field fluorescein angiography (UWFFA), and OCT to assess MTX response. Aim 1 will identify the combination of variables at baseline that predict a patient’s risk of response by 6 months. Aim 2 will assess the use of quantitative imaging to evaluate and monitor MTX response over 6 months. These modalities will also be compared to the clinical examination. In Aim 3, 20 pediatric non-uveitic controls will also be included. This aim is designed to discover gene expression signatures that are associated with clinical and imaging based MTX response and non- response. The success of this study will 1) optimize treatment of children with NIU by allowing earlier initiation of biologics in those unlikely to respond to MTX, 2) demonstrate the clinical usefulness of quantitative imaging in therapeutic decision-making, and 3) eventually shift the current treatment standard of NIU leading to improved ocular health of children.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY/ABSTRACT Late-onset, non-infectious pulmonary complications following hematopoietic stem-cell transplant (HSCT) occur in up to half of all patients, are heterogeneous in etiology and in clinical course, and are deadly. Spirometry is the cornerstone of surveillance, yet half of the pediatric HSCT population are unable to perform spirometry. As a result, post-HSCT lung injury in children often is diagnosed in an advanced stage, where therapeutic options are limited, damage is irreversible, and mortality is high. Early detection of lung injury after HSCT is our best opportunity to intervene, stabilize lung-function decline, and improve outcomes, and there is an urgent need for better diagnostics for young HSCT patients—especially for those who are unable to perform spirometry. To this end, we have developed inhaled hyperpolarized xenon-129 (129Xe) ventilation and gas-exchange magnetic resonance imaging (MRI) techniques to spatially resolve, differentiate, and quantify small airway, interstitial, and microvascular abnormalities. The long-term goal of this project is to lower pulmonary-related morbidity in the pediatric HSCT population via the clinical application of 129Xe MRI as an imaging-based, lung-function diagnostic. The central hypothesis of this proposal is that 129Xe MRI is feasible in very young children who are unable to perform PFTs and will detect early lung involvement post-HSCT. Our approach is based on three Specific Aims: (1) develop 129Xe gas-exchange MRI in children by imaging healthy-control volunteers and patients with diagnosed gas-exchange impairment ages 6-18 years old; (2) phenotype post-HSCT pulmonary involvement using 129Xe ventilation and gas-exchange MRI in children ages 6-18 years old who have received HSCT, with longitudinal MRI and PFTs at ~100 days and 1-year post-HSCT; (3) optimize strategies for rapid 129Xe MRI in HSCT patients ages 2-5 years old. This approach is supported by our previous work and experience showing 129Xe MRI is feasible in neonates, in young children who are unable to perform spirometry, and in sedated patients. This approach is innovative because 129Xe gas-exchange MRI is novel to any pediatric population, and imaging children with diagnosed gas-exchange impairment will build a conceptual bridge between better-understood disease pathophysiology and 129Xe gas-exchange MRI findings, which will enhance the clinical interpretation of 129Xe MRI and diagnosis of gas-exchange impairment in the HSCT population. Preliminary data in pediatric HSCT patients revealed diffusion-barrier and RBC-transfer abnormalities, suggesting a clinically-under-recognized, non-obstructive phenotype. This project will revolutionize clinical care for young HSCT patients with pulmonary complications. Longitudinal trajectories of ventilation, interstitial, and microvascular changes from 129Xe MRI post-HSCT will help in clinical risk stratification. 129Xe MRI will enable surveillance and early detection in asymptomatic patients, phenotyping to personalize clinical management and therapeutic approach, and robust assessment of lung function in very young children.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY / ABSTRACT Title: Mechanisms of cardiomyocyte dysfunction in pediatric septic shock Despite decades of research in pediatric sepsis, mortality remains at approximately 25% for children with septic shock. Sepsis-associated myocardial dysfunction (SAMD) is common in children and has an association with mortality that is not simply a reflection of the severity of illness. As no disease-modifying therapies exist for SAMD, there is a critical need to understand the biologic basis of cardiomyocyte dysfunction in sepsis. Furthermore, there is a need for novel human modeling with patient-derived materials given the failure to translate molecular discoveries in murine models of sepsis to improvements in human organ injury. Our objectives are to establish human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) as a model for cardiomyocyte dysfunction in pediatric sepsis and to examine the roles of host genetic background and serum factors in the pathogenesis of SAMD. We have built upon our large, established biobank of serum from children with sepsis to study the cardiomyocyte response to septic serum banked from children who did and who did not have SAMD. We have found that the contractility of hiPSC-CMs is depressed by serum banked from children with SAMD but not by control septic serum from children without SAMD. This depressant effect was reversible after removal of the serum, suggesting this was not reflective of cell death. Furthermore, we identified a significant association of interleukin-8 (IL-8) with SAMD in children with septic shock and found that recombinant human IL-8 depresses hiPSC-CM contractility. Our proposal will address three important questions as specific aims: First, we will determine whether hiPSC-CMs and cardiomyocytes derived from the same children share common functional and transcriptional patterns of responses when exposed to banked septic serum. We will provide a comprehensive comparison of hiPSC-CMs to ex vivo cardiomyocytes by isolating cardiomyocytes from discarded surgical tissue from children undergoing cardiac surgery and by generating hiPSC-CMs from these same patients. Second, we will determine the degree to which host genetic background contributes to cardiomyocyte dysfunction in SAMD. We will develop hiPSC- CMs from pediatric patients with and without SAMD to determine responses to septic serum to identify patterns of functional and transcriptional responses associated with susceptibility to SAMD. Third, we will dissect the role of IL-8 signaling in cardiomyocyte dysfunction in sepsis. We will employ a combination of IL-8 modulation in serum and IL-8 receptor (CXCR1 and CXCR2) knockouts in hiPSC-CMs, providing evidence for IL-8 blockade as a potential therapeutic target in SAMD. This career development proposal will build on my background in translational research in myocardial dysfunction to gain new expertise in cardiomyocyte functional analysis, hiPSC generation and differentiation, and next-generation RNA sequencing technology to facilitate my transition to independence as a physician-scientist focused on elucidating and targeting mechanisms of pediatric SAMD.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY ABSTRACT Rheumatic heart disease (RHD) remains a high prevalence condition in low-and-middle-income countries, currently affecting at least 40 million people, many of whom suffer premature death. Most patients with RHD present late, missing the opportunity to benefit from secondary antibiotic prophylaxis. Screening echocardiography in RHD endemic settings identifies many children with early, latent RHD, but until recently the effectiveness of prophylaxis to protect children with latent RHD was not known. The GOAL Trial (conducted in Uganda by this investigative team) found that children with latent RHD who receive prophylaxis with intramuscular penicillin are less likely to progress at two-years (0.8% penicillin vs. 8.3% no penicillin, p<0.001). However, despite these results, scale-up of echocardiographic screening and early initiation of prophylaxis with intramuscular (IM) penicillin for RHD has a myriad of challenges. Among the most critical are substantial patient (including pain and missed work/school) and health system-level barriers (including cost, time, and training) to delivering prolonged courses of IM injections in low-resource settings. Intramuscular vs. Enteral Penicillin Prophylaxis to Prevent Progression of Latent Rheumatic Heart Disease (GOALIE) will determine if a less burdensome form of prophylaxis, oral penicillin, is non- inferior to IM penicillin in preventing latent RHD progression. GOALIE is a randomized non-inferiority trial comparing the efficacy of Intramuscular to Enteral (oral) penicillin prophylaxis to prevent progression of latent RHD at two years. Based on our strong history of recruitment and retention, we will conduct school- based echocardiographic screening of ~100,000 children and enroll 1004 children into GOALIE, which will provide 90% power to determine if oral penicillin prophylaxis is non-inferior to IM penicillin prophylaxis. GOALIE will also examine economic equivalence and cost-effectiveness of these two prophylaxis strategies (Aim 2) and the patient reported outcomes between these two strategies (Aim 3), providing critical data to inform the integration of prophylaxis for latent RHD into clinical practice. GOALIE builds off our decade long collaboration, including strong Ministry of Health and community support. GOALIE will leverage the structure of the GOAL Trial which developed streamlined protocols for echocardiographic screening (>102,000 screened) and highly successful recruitment (>99% eligible children), retention (97% completion), and adherence support (99% adherence) strategies. The results of our study will have high clinical and public health impact, immediately informing international policy on the standard of care for children diagnosed with latent RHD.