Cincinnati Childrens Hosp Med Ctr

NIH Research Projects · FY 2025 · 2015-07

PROJECT SUMMARY/ABSTRACT A fundamental challenge in developmental biology is delineating hierarchical cellular states including rare intermediates and their underlying gene regulatory networks that mediate cell-type specification. Previously, we utilized scRNA-Seq, developed novel bioinformatics approaches and clonogenic assays to delineate hierarchical genomic and regulatory states culminating in neutrophil or macrophage specification. Our analysis captured prevalent mixed-lineage intermediates (MultiLin*) that manifested coincident expression of hematopoietic stem cell/progenitor (HSPC) and myeloid progenitor genes. Here, we used primary mouse bone marrow progenitors to: 1) rigorously identify stable developmental cell states and rare intermediates, 2) overcome major informatics hurdles to integrate multiomic data sets with flow cytometry to validate hypotheses, and 3) link neural-network-identified TF activity on chromatin to dynamic gene expression; predicting hematopoietic TF cistromes. Our Preliminary Data reveals nascent transcription factor programming within the MultiLin that restricts lineage potentials, yet precurses bipotential progenitors and lineage specification and commitment. We hypothesize that within the homeostatic hematopoietic system, successive myeloid lineage restriction events begin within MultiLin. The proposed work will isolate cluster-defined cell populations and define their developmental potentials, establish a validated network to establish the combinatorial regulatory TFs that program hemopoietic progenitors, then exploit this network to define the genomic and cellular impact of emergency stress adaptations which stimulate erythroid and neutrophil granulocyte production.

NIH Research Projects · FY 2024 · 2015-07

Project Summary/Abstract Despite the importance of myoblast fusion for normal muscle development and physiology, relatively little is known about the molecules that directly function to remodel membranes during the myoblast fusion reaction. Elucidation of fusion mechanisms is critical to fully understand muscle development and to identify novel therapeutic strategies to augment skeletal muscle disease. We previously discovered that myomaker (Mymk) and myomerger (Mymx) are essential for the fusion of skeletal muscle progenitors. Moreover, ectopic expression of these two membrane proteins induces fusion of otherwise non-fusogenic cells (fibroblasts). For the first time, this establishes a cell-based reconstitution system with myoblast fusogens, however many questions exist as to how these two proteins induce fusion. We have recently found that myomaker and myomerger drive fusion through a unique cellular mechanism, by dividing their independent membrane remodeling activities to distinctly impact the fusion process. It stands to reason that the membrane-remodeling activities of myomaker and myomerger must be highly regulated or they could have the potential to compromise cellular integrity. Indeed, our preliminary experiments probing the requirement of myomaker for fusion during dystrophic disease progression unexpectedly revealed that myomaker expression in dystrophic myofibers is deleterious. In this project we will: 1) determine the membrane-remodeling activities of myomaker that control lipid mixing (hemifusion) 2) identify and interrogate the additional factors required for hemifusion 3) elucidate the mechanisms by which myomerger elicits membrane stresses that drive fusion pore formation. Additionally, we will study these fusogens in the context of chronic muscle disease (muscular dystrophy). We will use cell biology, biochemistry, and genetic mouse models to study and define the activities of myomaker and myomaker, thereby elucidating the mechanisms of myoblast fusion. We will also develop a reconstituted proteoliposome system for myoblast fusion. These studies will provide unique insight into the mechanisms of mammalian myoblast fusion. Overall, this work promises to open up a new area of investigation into the cell biology of muscle and positively impact the possibility to harness fusion to improve regenerative medicine.

NIH Research Projects · FY 2025 · 2015-04

PROJECT SUMMARY Our long-term goal is to establish non-invasive approaches to predict drug disposition in children, which includes developing phenotypic biomarkers of drug transporters and drug metabolizing enzymes and pediatric physiologically-based pharmacokinetic (PBPK) models. During the previous funding period, we gained a considerable understanding of age-dependent hepatic metabolism and transport from neonates to adults, which was used to develop PBPK models to predict the disposition of hepatically cleared pediatric drugs. However, ontogeny data remain limited for another key drug elimination organ: the kidney. The main objective of this proposal is to define the ontogeny of drug transport in the kidney. Filling this knowledge gap is critical, as approximately 30% of prescription drugs are cleared predominantly by the kidneys, including several drugs prescribed to children. Further, tubular secretion plays a crucial role in drug detoxification in children, which is often regulated by the rate-limiting organic anion or cation transporters (OATs and OCTs, respectively). Although we and others recently quantified clinically relevant drug transporters in archived kidney tissue samples, these data are highly variable and are from a limited number of subjects, precluding a meaningful interpretation of transporter ontogeny. To address these knowledge gaps, we hypothesize that endogenous substrates of OATs and OCTs in blood and urine can be used as surrogate, non-invasive markers of kidney transporter function in children and adults. We will test this hypothesis via three Specific Aims. Aim 1: Establish robust biomarkers of renal OAT and OCT transport activity in adult humans in a controlled clinical drug-drug interaction study using furosemide and metformin as probe drugs, respectively. The effects of OAT and OCT inhibition by probenecid and cimetidine, respectively, on both exogenous and endogenous probes will be determined. Aim 2a: Confirm the utility of Oat and Oct transporter biomarkers in predicting ontogeny of renal transporter abundance and activity in Sprague-Dawley rats. Aim 2b: Characterize the ontogeny of renal transporters in humans using exogenous (furosemide and metformin) and endogenous (biomarkers) probes of renal transporters. Aim 3a: Characterize the selectivity of renal uptake of the biomarkers in vitro. Aim 3b: Develop and validate biomarker-informed PBPK models for renally secreted anionic and cationic drugs in children and adults. Collectively, this innovative project will establish non-invasive biomarkers that can be used to predict transporter- mediated renal secretion and drug-drug interaction potential in children and adults. Specifically, the effect of inhibition of renal transporters by an investigational drug on xenobiotic or endobiotic toxins (e.g., drugs or uremic toxins) can be predicted in children and adults using these biomarkers. The validated transporter activity biomarkers garnered from this translational project will inform precision dosing of pediatric drugs secreted by kidneys during clinical trials and clinical use.

NIH Research Projects · FY 2025 · 2014-12

ABSTRACT This is a second renewal application for a pre- and postdoctoral training program, the title and theme of which is “Understanding Cardiovascular Disease Mechanisms”. The University of Cincinnati and Children’s Hospital has a renowned legacy spanning more than 4 decades of previous NIH T32 support for cardiovascular study, and this current T32 represents the only CV program in Cincinnati. In the past 4 years of this program covered by the renewal application, our 16 trainees published 34 papers and 6 received independent grant funding during their support period, while 5 graduates have matriculated to jobs in scientific careers. The overall scientific emphasis of our training program will continue to build from a basic platform of cardiovascular physiology, cell biology, biochemistry and pharmacology, but will also incorporate the latest approaches in biomedical research, as well as incorporating clinical and translational approaches. The cardiovascular environment at Cincinnati Children’s and the University of Cincinnati is considered one of the very best in the country. Our 20 current training grant faculty are all NIH funded (some 66 NIH funding components amongst them as PI status) with 166 collaborative papers published together, and all are employing the very latest technologies and approaches with outstanding institutional core support. The leadership consists of the co-PIs Drs. Evangelia Kranias and Jeffery Molkentin, both of whom have a long track record of working together (24 years), as well as having excellent mentorship credentials. The Executive Committee (2 members), Internal Advisory Committee (4 members) and External Advisory Committee (3 members) are highly engaged cardiovascular researchers who will continue to help ensure the quality of the training program. The renewal requests continuation of the funding of 3 pre- and 3 postdoctoral trainee positions. Predocs are selected by the Internal Advisory Committee from a vast and outstanding pool of candidates amongst 9 departmental graduate programs, while postdoctoral candidates are selected based on being accepted into a mentor’s laboratory and then passing the screening process by the Internal Advisory Committee and co-PIs. The mentoring program and evaluation process for the program are highly structured and oversight occurs on many levels. Trainees and mentors are evaluated every 6 months with IDPs and committee meetings. The proposed educational training curriculum is highly structured and state-of-the-art.

NIH Research Projects · FY 2026 · 2013-09

SUMMARY: The global obesity epidemic drives dramatic increases in the incidence of metabolic dysfunction- associated steatotic liver disease (MASLD; formerly referred to as non-alcoholic fatty liver disease). A key contributor to MASLD progression/severity is the emergence, accrual, and pathogenic cytokine production of inflammatory CD4+ T cells in the liver. However, cellular mechanisms that govern MASLD-specific, hepatic CD4+ T cell pathogenic functions remain unknown. Emerging evidence links cellular metabolism and post- translational modifications (PTMs)—such as O-GlcNAcylation and neddylation—to MASLD and CD4⁺ T cell- mediated inflammation. We made a striking novel observation that hepatic abundance of amino acid glutamine serves as a critical regulator of hepatic CD4+ T cell functions in MASLD. However, whether glutamine metabolism and PTM work in unison to govern hepatic CD4+ T cell pathogenic functions in MASLD is unknown and represents key knowledge gap. Specifically, using human liver biopsies, human liver organoids, and multiple mouse models of MASLD, we discovered that hepatic glutamine levels are profoundly reduced while O-GlcNAc and neddylation are robustly increased in MASLD. Notably, restoration of glutamine levels through exogenous administration: (i) uniquely reduces pathogenic cytokine production by hepatic CD4⁺ T cells (without affecting non-hepatic CD4⁺ T cells or other hepatic immune cells); (ii) decreases CD4+ T cell O-GlcNAcylation and cullin neddylation; and (iii) diminishes MASLD severity. Unexpectedly, our additional preliminary data show that glutamine utilization pathways act in divergent manners to impact cullin neddylation and hepatic CD4+ T cell pathogenic cytokine production in MASLD. Specifically, T cell-specific glutaminase-mediated glutaminolysis reduces hepatic CD4+ T cell pathogenic cytokine production, hepatic O-GlcNAcylation and cullin neddylation, and MASLD severity. In contrast, whole body O-GlcNAc activity exacerbates these outcomes. Together, our data support the overarching hypothesis that glutaminolysis, via suppression of O-GlcNAc-driven cullin neddylation, dampens hepatic CD4+ T cell pathogenic functions. Here, using samples from our established human cohort, human organoids, and mice with MASLD we aim to: (Aim 1) Determine how CD4⁺ T cell-intrinsic glutaminolysis regulates hepatic CD4⁺ T cell pathogenic functions by suppressing intracellular O-GlcNAc levels in MASLD; and (Aim 2) Determine how glutaminolysis and O-GlcNAcylation influence cullin neddylation to modulate hepatic CD4⁺ T cell function in MASLD. Together, our proposed studies will provide critical knowledge of intracellular processes that uniquely license hepatic CD4+ T cell pathogenic functions in MASLD. Importantly, these cellular processes/pathways are amenable to therapeutic targeting in humans, hence definition of these regulatory mechanisms in various pre-clinical settings and their direct confirmation in clinical samples may lead to discovery of novel predictive, preventive, and therapeutic avenues to MASLD.

NIH Research Projects · FY 2025 · 2013-06

PROJECT SUMMARY/ ABSTRACT: Venous malformations (VM) originate from impaired development of the venous network, resulting in massively dilated and dysfunctional veins. Vascular lesions are usually present at birth, continuing to expand with time and never spontaneously regress. VM result in significant morbidity and pain often leading to serious local and systemic complications. Standard clinical management consists of sclerotherapy and surgical resection. However, because these therapies manage symptoms rather than targeting underlying disease etiology, malformed veins often require repeated interventions. Therefore, novel targeted therapies for VM are of high importance. Gain-of function mutations in the endothelial-specific tyrosine kinase receptor TIE2 have been identified as the leading driver of VM. TIE2 has been shown to regulate both maintenance of vascular quiescence and promotion of angiogenesis, but its role in the vascular lumen expansion has not been explored. Research into the molecular and cellular abnormalities which result from hyperactive TIE2 will provide the necessary groundwork for the development of the targeted molecular treatments for VM. Our results, recently published, show that TIE2 signaling promotes activation of c-ABL (Abelson kinase 1) and that genetic and pharmacological c-ABL targeting significantly reduced vascular lumen size. The mechanisms leading to the pathogenic lumen expansion are still largely unexplored and the role of c-ABL in the pathophysiology of VM and vascular anomalies is unknown. With the identification of novel mediators of the TIE2-c-ABL signaling axis, we can now develop a research program to investigate their role in vascular lumen expansion with the goal of identifying novel targets for VM. To perform these studies, we will utilize our VM xenograft murine models and a recently devised in vitro three- dimensional system to study VM lumen formation and expansion. To advance our understanding of VM, we will employ a rigorous approach based on the complementary use of the well-established HUVEC-TIE2-L914F cell line, VM patient derived EC and patient tissue to confirm the significance of our findings for the pathophysiology of VM. Additionally, our studies on VM will provide cellular and mechanistic insights to advance our understanding of pathological and physiological vessel formation and size maintenance.

NIH Research Projects · FY 2026 · 2012-06

PROJECT SUMMARY/ABSTRACT There is a significant deficit of well-trained, research-focused physicians equipped to innovate in the prevention, diagnosis, and treatment of childhood respiratory illnesses. This already limited group of established researchers faces further challenges due to a declining number of young physician-scientists opting to specialize in pediatric respiratory fields. At Cincinnati Children’s Hospital Medical Center (CCHMC), training across all levels is a critical priority, with comprehensive programs designed to cultivate MD, PhD, and MD/PhD fellows and faculty into independent investigators in both clinical and basic sciences. The Pediatric Pulmonary Division at CCHMC is particularly well-suited to inspire students to pursue careers dedicated to pediatric respiratory health. The pulmonary research initiatives at CCHMC boast robust and varied clinical and basic science programs, and the Sleep Disorder Center stands out as one of the few pediatric programs in the United States offering a certified pediatric sleep fellowship with a strong emphasis on research. This renewal seeks to sustain a successful 9-week summer research fellowship for medical students after their first year of training. The proposed program will implement a structured approach for selecting trainees and mentors, along with effective methods for evaluating the trainees' progress in both the short and long term. A faculty comprising 37 MDs, PhDs, and MD/PhDs from various departments, all with a proven track record in research and mentorship, will be available to support the trainees. All investigators are well-funded in NHLBI-focused research areas related to pediatric respiratory conditions. The program will also provide opportunities for students to engage in both basic and clinical research projects, featuring weekly meetings that cover responsible research conduct and specific topics in pulmonary and sleep medicine. At the conclusion of the summer, students will present their projects to their peers and mentors during a symposium. The robust capabilities of our research initiatives in pediatric pulmonary and sleep medicine, coupled with a dedicated faculty and a well-structured summer program, create an outstanding opportunity to engage physician trainees during pivotal moments in their careers. This approach significantly enhances the chances that they will choose to continue their journey in research as physician-scientists.

NIH Research Projects · FY 2025 · 2012-05

PROJECT SUMMARY This proposal requests continued support for the “Enhancing Pediatric Treatment Adherence and Health Outcomes” (T32 HD068223) postdoctoral training program, which trains M.D. and Ph.D. researchers to assume leadership roles in developing innovative, high impact research on adherence to medical treatment and chronic illness self-management that will enhance the health outcomes of children with chronic conditions. Modern medical treatments have improved the health outcomes of large numbers of children and adolescents with chronic conditions. However, one of the most critical remaining barriers to improving children’s health outcomes over the course of their lifetime is nonadherence to prescribed medical treatment. As noted by the World Health Organization (WHO), treatment nonadherence is highly prevalent (rates of ≥ 50% in pediatric chronic illness populations) and has a significantly negative impact on children’s health and health care costs. Continued innovative and clinically relevant research is needed to close the gap between the health outcomes that are potentially achievable with more optimal treatment adherence and those achieved in current practice. NIH program priorities and requests for applications, scientific consensus conferences, and scholarly reviews have all identified the importance of research on pediatric adherence to treatment. A critical barrier to scientific advances in pediatric adherence and chronic illness management research is the shortage of talented, well-trained researchers who are devoting their careers to developing innovative, high impact treatment adherence and self-management research with pediatric populations. One of the most promising ways to address this need and improve child health is to develop innovative interdisciplinary research training programs to train leaders in the field of treatment adherence and self-management research. This is the focus of this T32 program. Thus far, we have trained 22 fellows (18 graduates, 4 current trainees) who are developing promising independent research careers in pediatric adherence and self-management. Specific training innovations involve the integration of biomedical, behavioral, biostatistics, and health services/outcomes research and are reflected in a comprehensive training curriculum including experiential research opportunities. Research innovations include novel methods of adherence assessment and intervention involving technology designed to enhance the engagement of children and adolescents, improve the power and duration of intervention effects, and reach diverse populations who cannot easily access traditional clinic-based approaches. Program innovation is also enhanced by novel research training opportunities to evaluate the comparative effectiveness of adherence promotion interventions in pediatric chronic illness management, to integrate objective methods of adherence measurement (e.g., behavioral, pharmacological, technological) and conduct advanced statistical analyses to evaluate the impact of adherence promotion interventions on children’s health outcomes.

NIH Research Projects · FY 2026 · 2011-09

Embryo implantation is a gateway to pregnancy success. A crosstalk between the blastocyst and uterus is vital to implantation. Formation of a healthy implantation chamber (crypt) is critical for normal embryo implantation; an abnormal crypt results in subfertility. Planar cell polarity (PCP) is a classic downstream target of the non-canonical Wnt5a signaling pathway. Uterine deletion of Vang- like protein 2 (Vangl2), a major PCP component, severely derails crypt formation, embryo spacing, and decidualization, ultimately compromising pregnancy outcomes. We have found that crypts invariably originate with preexisting glands conferring direct communication between the implanting blastocyst and glands. Heparin-binding EGF-like growth factor (HB-EGF) is exclusively expressed in the crypt epithelium surrounding the blastocyst, and beads preloaded with HB-EGF, if transferred to day 4 pseudopregnant uteri, evoke implantation-like changes similar to normal pregnancy. However, these responses fail to occur in uteri missing Vangl2, suggesting an intersection of Vangl2 with HB-EGF signaling. HB-EGF executes its function via the EGF family of receptors (Erbbs 1-4) as homodimers or heterodimers and induces tyrosine phosphorylation. This proposal will test a novel hypothesis that HB-EGF signaling intersects with PCP signaling by engaging the ErbBs in implantation. HB-EGF working via ErbBs 1-4 induces tyrosine phosphorylation. Our preliminary results show a physical association of Vangl2 with ErbB2 and ErbB3, suggesting that these receptors, if activated by HB-EGF, transphosphorylate Vangl2. The following two specific aims will study interactions between PCP and HB-EGF signaling in implantation using mouse models: Specific aim 1 will test the hypothesis that HB-EGF signaling intersects with Vangl2 signaling to direct the formation of an appropriate glands-crypt assembly for direct cross-talk between the implanting blastocyst and the glands. This interlocking of two different signaling pathways involves phosphorylation of members of the ErbB family of receptors followed by transphosphorylation of Vangl2. Specific aim 2 will test the hypothesis that the functional coupling of HB-EGF and Vangl2 signaling in implantation requires the presence of ErbB1, ErbB2 and/or ErbB3 in the uterine epithelium and/or stroma. We will use conditional deletion of ErbB1, ErbB2 and/or ErbB3 using Pgr-Cre and Ltf- iCre drivers to explore the Vangl2 phosphorylation status in the uterus, and correlate these results with pregnancy phenotypes. Successful completion of this project will reveal a conceptual attribute to the field of implantation and pregnancy biology that a growth factor signaling pathway intersects with PCP signaling in forming implantation chambers (crypts) for pregnancy success.

NIH Research Projects · FY 2025 · 2011-05

PROJECT SUMMARY This is a competitive renewal application for the continuation of a highly collaborative and successful pediatric clinical pharmacology training program at Cincinnati Children's Hospital Medical Center and the University of Cincinnati. The program will train a new generation of pediatric investigators to assume leadership roles in the application of innovative, high impact quantitative clinical pharmacology approaches to improve the development, rational use and tailoring of new and existing drug therapies for neonates, infants, children, adolescents and young adults. The need for pediatric clinical pharmacology research and training has never been greater. The T32 Training Program in Pediatric Clinical Pharmacology at Cincinnati Children's Hospital Medical Center (CCHMC) is designed specifically to address this critical need. The program is based in the Division of Clinical Pharmacology, which is jointly a unit within the Department of Pediatrics and the University of Cincinnati, College of Medicine (UCCoM). The program outlined in this renewal application draws on strong leadership and a diverse group of well-established faculty mentors actively involved in subspecialty clinical pharmacology research, representing 18 divisions within the Department of Pediatrics, the Departments of Pharmacology & Systems Physiology and Biomedical Informatics at the UCCoM, and the James L. Winkle College of Pharmacy. The program has tremendous institutional support and takes advantage of broad areas of distinction and resources within a uniquely collaborative environment. The T32 training program is innovative and well aligned with the objectives outlined in the program announcement as it: (1) has a focus in early and later phase studies in multiple and diverse pediatric populations through ongoing research collaborations with all major pediatric subspecialties; (2) involves the application and development of innovative quantitative methodologies such as PK/PD modeling and pharmacometrics, quantitative systems pharmacology modeling and simulation and model-informed clinical trial design; (3) is embedded in the institutional and UCCoM pharmacogenetic/genomics research and training endeavors through the Center for Pediatric Genomics; (4) is closely integrated with Bioinformatics and Health Services and Outcomes Research; and (5) can rely on over 10 years of a well-established curriculum after two funding cycles as a successful training program. The program provides a unique training experience to MDs, PharmDs, and PhDs to become the next generation of leaders whose work will advance pediatric clinical pharmacology and to have an extraordinary impact on pediatric therapeutics and health outcomes for children. The program has successfully opened new avenues to enlarge the pool of talented young clinical investigators with a career interest in pediatric therapeutics.

NIH Research Projects · FY 2025 · 2011-04

Project Summary/Abstract Biliary atresia is the most common cause of pediatric end stage liver disease and the number one indication for pediatric liver transplantation. The presence of pathogenic viruses in the liver of afflicted children led to perinatal viral infection as a proposed etiology for biliary atresia as this infection triggers an immune mediated destruction of the biliary epithelium leading to liver fibrosis and ultimately, cirrhosis. The murine model of biliary atresia supports a viral pathogenesis as newborn mice infected with Rhesus rotavirus (RRV) develop portal inflammation extrahepatic bile duct obstruction; however, it is accompanied by high mortality rates precluding the ability to study hepatic fibrosis. We have developed two novel modified virus strains, TR(VP2, VP4) and RRVVP4-K187R, which induce biliary obstruction accompanied by high rates of hepatic fibrosis. We have demonstrated infection with these strains causes bile duct obstruction and hepatic fibrosis that mirrors the human phenotype better than conventional models, including carbon tetrachloride and bile duct ligation. These strains allow us to mechanistically interrogate the post-obstructive fibrotic pathway, a critical next step in the understanding of BA pathogenesis. We will use these strains to determine how infection of cholangiocytes alters the innate immune response leading to the recruitment and activation of monocytes/macrophages. We will also ascertain these modified rotavirus strains’ ability to infect and activate hepatic stellate cells inducing liver fibrosis. These complementary approaches will generate new insight into virus-induced biliary atresia and liver fibrosis.

NIH Research Projects · FY 2026 · 2011-01

Abstract Similar to skeletal muscle myofibers, cardiomyocytes in the heart appear to be particularly susceptible to membrane instability and rupture during disease, in part because of their contractile status that produces ongoing mechanical deformation. Mutations in genes that disrupt or weaken the membrane anchoring proteins of the dystrophin-glycoprotein complex (DGC) or the integrin adhesion network causes a wide range of muscular dystrophies that also cause cardiomyopathy. We have shown that the thrombospondin gene family (Thbs1-5) plays a critical role in membrane stability through both effects on the ER stress response and secretory pathways, as well as controlling the integrin complexes present on the sarcolemma. In our previous cycle of funding, we showed that overexpression of Thbs3 has a remarkable effect of reducing sarcolemma stability in heart (opposite of Thbs4) by removing large arrays of integrin heterodimers from the adhesion complexes, while Thbs3 KO mice have enhanced integrin membrane levels and are protected from insults that would otherwise cause cardiomyopathy, like overexpression of Thbs4 that also increases membrane stability by increasing membrane attachment protein complexes in the sarcolemma. However, several critical mechanistic questions remain to be addressed in attempting to translate our findings into therapeutic approaches. Here we propose the unifying hypothesis that the cardiac expressed Thbs proteins primarily function from within the secretory pathway in mediating the stability, or recycling of membrane attachment complexes, which has profound effects on sarcolemmal stability and healing dynamics within the heart with both acute injury and chronic disease, which will suggest potential novel gene therapeutic ventures for cardiomyopathy in heart failure or with muscular dystrophy. To interrogate this hypothesis, we propose the following 2 Specific Aims: 1) Mechanistically define how Thbs3 and Thbs4 antithetically regulate sarcolemmal stability through integrin processing for gene therapy application in cardiomyopathy and muscular dystrophy. 2) Examine the molecular mechanism of dilated cardiomyopathy in a mouse model for the Human THBS4 D717N variant. The goal will be to better understand human heart disease through the Thbs gene family and how it regulates membrane stability and adhesion complex activity. Translational implications are that a better understanding of these molecular mechanisms will suggest why humans with mutations in Thbs4 show dilated cardiomyopathy and will suggest modified versions of Thbs4 to be used in gene therapy approaches to treat muscular dystrophy, as well as a wide array of heart diseases in which the membrane is weaker.

NIH Research Projects · FY 2026 · 2010-06

Component: OVERALL PROJECT SUMMARY/ABSTRACT Xenbase is the Xenopus model organism knowledgebase (MOK), an online resource that integrates all genomic and biological data from Xenopus research. Our mission is to accelerate the translation of Xenopus research into knowledge that will improve human health. We aim to empower Xenopus research and enhance the impact of Xenopus data in the broader biomedical community, as animal models such as Xenopus are essential for biomedical research and have led to a wealth of discoveries. Xenbase is user-friendly, allowing investigators to quickly find and link different data types in ways that would otherwise be difficult, time consuming, or impossible. It provides high quality curation, data integration, and bioinformatics tools to link Xenopus data to humans and other model organisms, NCBI, UniProt, Ensembl and other resources. In this post genomic era with thousands of scientific publications annually and the exponential growth of “omic” datasets, Xenbase is essential to translate the enormous amount of data generated from research using Xenopus into meaningful connected data. Xenbase thus plays an essential role in maximizing NIH’s >$120 million annual investment in Xenopus research. Xenbase is an essential resource for hundreds of labs around the world. There is a clear need to continue curating Xenopus data, to develop new tools to keep up with technological advances, and link Xenopus data to human biology in novel and insightful ways. Many of the letters of support (more than 110) state that labs could not function without Xenbase. Aim 1 Maintain Xenbase, Curate and Disseminate Xenopus research data. Aim 2 Enhance support for Xenopus models of human diseases Aim 3 Enhance Integrated Omics support

NIH Research Projects · FY 2026 · 2007-08

PROJECT SUMMARY The overall goal of our Digestive Health Center (DHC): Bench-to-Bedside Research in Pediatric Digestive Disease is to promote research that will yield insights into the fundamental processes and pathogenic mechanisms of digestive disease in children and generate innovative treatment to restore digestive health. The Cores aims are: 1) to strengthen the Research Base and foster collaborations, 2) to catalyze discoveries by giving investigators access to highly innovative Cores, and 3) to develop junior investigators and the future leaders in the field. The last award period of the DHC was very successful, with Core services that evolved to meet the scientific needs of innovative investigators. Our Research Base of 68 investigators attracts $36.5 million of extramural digestive disease-related funds annually; they published 630 articles in the last 4 years. Several Pilot and Feasibility (P/F) Awardees transitioned to R01-funded investigators and the entire research community benefitted from a scientific Enrichment Program presenting the latest advances in digestive disease research. This trajectory of success will be pursued in future years by fostering research and promoting interdivisional and interdepartmental collaboration to maintain a solid critical mass in digestive disease research with a focus on translational research. Specifically, our long-term goals are to improve child health through better diagnosis, treatments and outcomes that will emerge from highly innovative work in our three key focus areas: 1) Mechanisms of Liver Disease modeling, 2) Digestive Disease and Immunity, and 3) Stem Cell and Organoid Modeling of Digestive Diseases. Each focus area brings opportunities for potential impact on the digestive health of children, helps advance the national research agenda, and creates a unique environment to integrate research into patient care. The focus areas are linked by three complementary and uniquely innovative Biomedical Research Cores (Gene Analysis Core, Integrative Morphology Core, and Stem Cell/Organoid Core and Genome Editing Core) and by a Clinical Component of the Administrative Core to facilitate patient-based research. Collectively the Cores and the Clinical Component form a powerful infrastructure that fosters the development of personalized and predictive medical approaches based on the genetics and molecular basis of GI disorders, and of therapies that take into account basic mechanisms of disease. Our working model promotes laboratory discoveries to generate translational research opportunities that lead to validation in patient samples and lead to clinical trials. To strengthen digestive disease research, the DHC will foster collaborations among its investigators and investigators from other disciplines. It will also fund highly promising P/F Projects for junior investigators and will sponsor a dynamic Enrichment Program of scientific seminars, workshops and symposia. With these strategies and an exceptionally supportive institution, the DHC is well positioned to catalyze translational research in pediatric digestive disease.

NIH Research Projects · FY 2025 · 2006-07

Project Summary Epithelial cells are the first line of defense as they interface with the environment and initiate the response to environmental triggers. Our AADCRC is focused on elucidating the mechanisms by which epithelial cells contribute to the pathogenesis of allergic disorders. We found that while some epithelial genes are associated with tissue-specific disease, others are associated with specific combinations of disease and may contribute to disease progression from one end-organ to another. It is known that allergic disorders show substantial lifetime comorbidity. The “atopic march” concept has served as a guiding principle in our field, however, it has become evident that only very small proportion of children follow the traditional atopic march. While early-life AD remains a major risk factor for the development of atopic disease, there is significant heterogeneity in presentation of the atopic march including the timing and organ(s) affected. The atopic march needs to be revised to include this heterogeneity and incorporate the pathogenesis of the various combinations of the atopic march. Our Center is designed to help fill this critical knowledge gap. In the last cycle of funding, we built the first US early-life prospective longitudinal cohort of AD (Mechanisms of Progression of Atopic Dermatitis to Asthma in Children, MPAACH) and conducted mechanistic studies to identify epithelial pathways that promote allergic inflammation and disease. Our collective preliminary data implicate novel epithelial pathways as key drivers of allergic disease persistence in a given tissue as well as progression from one tissue to another. Herein, we will determine how these pathways act to promote the development, persistence, and progression of allergic and inflammatory diseases. Further, the atopic march needs to be revised to include non-White children. The vast majority of studies that served as the foundation for the atopic march principle were conducted in White populations, and our data reveal marked racial differences in the current atopic march concept. MPAACH is comprised of 65% Black children and was designed to help fill this critical need. The overarching hypothesis of this proposal is that homeostatic mechanisms at epithelial surfaces, upon dysregulation, promote allergic inflammation and contribute to the persistence, progression, remission and resolution of allergic disease(s). This hypothesis will be tested by three integrated and synergistic projects focused on epithelial cell biology that combine epidemiologic, basic, and translational research approaches to study multiple end-organs involved in allergic responses. Integration of data across projects by the Data Integration and Analysis Core (DIAC) will provide novel insights into a key unanswered question in the allergy field: Why is allergic inflammation restricted to one tissue in some cases, while it progresses to involve additional tissues in other individuals? Identification of the epithelial genes/pathways that predispose individuals to specific or shared allergic disorders will empower the search for therapeutics aimed specifically at the epithelial surface, and the development of treatment and prevention strategies optimized for allergic disorders alone and/or in combination.

NIH Research Projects · FY 2025 · 2003-07

Our postdoctoral training program at Cincinnati Children’s Hospital Medical Center is a central component of the Center for Child Behavior and Nutrition Research and Training. The Center has a specific research focus on the behavioral, biologic, and nutritional aspects of pediatric chronic illnesses and other nutrition-related health issues. The Center’s T32 training program, funded by NIDDK in 2003, formalized cross-disciplinary training at the postdoctoral level with the aim of training the next generation of academic leaders and interdisciplinary team scientists. The training program integrates the expertise of faculty members across the Divisions of Behavioral Medicine and Clinical Psychology; Endocrinology; Gastroenterology, Hepatology, and Nutrition; Biostatistics and Epidemiology; Neurology; and the Department of Pediatric and Thoracic Surgery. Trainees acquire: (1) expertise in the pathophysiology, diagnosis, and treatment of pediatric chronic medical conditions in which dietary modification is a central component of disease management; (2) knowledge of “state of the art” as well as innovative means of assessment of physical (e.g., body composition, bone mass, disease progression), nutritional (e.g., via electronic data capture), and psychosocial (e.g., quality of life, family functioning, functional behavior) status across the pediatric age range; and (3) knowledge of nutrition, behavioral, and clinical trials science necessary to develop empirically-tested prevention models and clinical interventions that improve dietary adherence, nutritional status, and long-term health and quality of life outcomes for youth. Candidates have backgrounds in clinical psychology or pediatric medical subspecialties (e.g., Endocrinology, Gastroenterology). The focal elements of the training program are mentored experiences within interdisciplinary research teams with our NIH-funded faculty and guidance from a Scholarship Oversight Committee. The mentored research experiences include the fellow’s participation as both an interdisciplinary team member, as well as their initiation of a mentored interdisciplinary independent research project. Based on the fellow’s prior educational pathway (PhD, MD), further training is obtained via practical and applied experiences and engagement in didactics, seminars, and academic coursework tailored to an individual’s training needs and career goals (e.g., behavioral science, nutrition science, clinical trials methodology, advanced biostatistics, grant-writing, team science, responsible conduct of research, diversity, equity, and inclusion, Masters Degree in Clinical and Translational Research). As evidenced by the excellent progress of our program graduates, these training opportunities provide a solid foundation from which young clinical researchers have already secured, and will continue to successfully transition to, faculty positions in the field of academic medicine and emerge as leaders and team scientists involved in NIH-funded clinical research programs that have a significant impact on future pediatric health outcomes. The current renewal application (Years 21-25) for this innovative T32 program requests support for four postdoctoral training positions.

NIH Research Projects · FY 2025 · 2002-09

PROJECT SUMMARY/ABSTRACT We propose to recruit Dr. Cyd Castro-Rojas as Clinical Research Coordinator (CRC) to the multicenter Childhood Liver Disease Research Network (ChiLDReN) to broaden the research workforce for this program and improve enrollment and retention of all populations affected by the cholestatic liver disease, i.e. extrahepatic biliary atresia (EHBA), primary sclerosing cholangitis (PSC), or Alagille syndrome, into the ChiLDReN longitudinal study protocols. Preliminary published data indicate that racial, ethnic, and socioeconomic disparities impact timely diagnosis and access to biliary drainage surgery in EHBA and natural history in PSC. Therefore, it is important that natural history studies on these conditions adequately represent all populations at risk. Using enrollment and retention logs for ChiLDReN studies and electronic health records for patients cared for in Cincinnati, we discovered trends for decreased enrollment of Black and Asian infants with EHBA into the PROBE and BASIC protocols and lower retention rates for Blacks and patients with EHBA of Hispanic ethnicity in the PROBE study. We hypothesize that broadening the research staff for ChiLDReN will allow us to successfully implement strategies to improve recruitment and retention of study participants from a diverse racial, ethnic, and socioeconomic background into ChiLDReN protocols. Dr. Castro-Rojas will broaden the clinical research workforce and improve the enrollment and retention of study participants from diverse backgrounds into the ChiLDReN protocols by 20%. She will lead improvement efforts for the ChiLDReN research program at Cincinnati, including building relations with Black, Hispanic, and socioeconomically disadvantaged communities through the Research Participant Advisory Group. Dr. Castro- Rojas will seek the advisory group’s guidance in editing study documents to make those more approachable for all populations and in identifying strategies to remove barriers to research activities by participants at risk. She will take a 12-credit course at the University of Cincinnati to obtain the Graduate Certificate in Community- Engaged Research for Health and apply the learnings to the improvement efforts for the ChiLDReN network. Additionally, she will take targeted training through the Association of Clinical Research Professionals and the CITI Program to learn about best practices and strategies for recruiting and retaining participants from diverse communities. Furthermore, Dr. Castro-Rojas will disseminate insights into successful research improvement efforts to reduce disparities in observational studies with other ChiLDReN sites and across NIDDK-supported studies at Cincinnati.

NIH Research Projects · FY 2026 · 2002-02

Cincinnati Children’s Hospital Medical Center (CCHMC) is submitting an application for the competitive renewal of its Child Health Research Career Development Award (CHRCDA/K12). This highly successful program at CCHMC has completed 31 years of successful mentoring of physician scientists who leave our program well positioned to catalyze transformational changes in pediatric research. In this, our sixth renewal application, we celebrate the 59 CHCRDA Scholars who have achieved $629M in cumulative NIH grant funding and published more than 4000 research papers. Our Scholars have advanced to become top leaders in pediatric research, many holding major academic positions, with 21 of our Scholars serving as division directors or department chairs in pediatrics. Since our last submission in 2017, the capacity of our research infrastructure at CCHMC has continued to expand, with additions of outstanding new faculty engaged in basic research in genetics, immunology, gene regulation, bioinformatics, rare diseases, and other areas. This expanded faculty base provides outstanding mentor choices for young pediatric physician-scientists. In this renewal, we are proposing 33 accomplished faculty members with mature and well-funded research programs, with a particular focus on strengths in the basic sciences and technological innovation, to serve as primary mentors or co-mentors for the CHRCDA Scholars. We propose four Specific Aims that have been successful for this program through multiple cycles: Identifying top new Scholar candidates (Aim 1); Providing an outstanding mentoring environment and curriculum (Aim 2); Supporting Scholar development through exposure to relevant cutting-edge technologies (Aim 3); and Enhancing the career development opportunities and skills of new faculty entering research-intensive careers (Aim 4). Our program is introducing three new Targeted Innovations this cycle: Formation of a united institutionally-supported career development program (Proctor pre-K Scholar) and CHRCDA Scholar program for physician-scientist early career development; a Technology Innovation Program for our Scholars; and the appointment of a new CHRCDA Director of Academic Affairs and Career Development. The CHRCDA program at CCHMC has matured during its 31 years history into a highly interactive, collaborative, functional community, including the renewed Executive Committee, External Advisors, Internal Advisors, and the CHRCDA Faculty Mentors, all of whom are fully committed to the success of future generations of independent physician-scientists who are leaders in pediatric medicine.

NIH Research Projects · FY 2025 · 2000-12

PROJECT SUMMARY Chronic pain affects approximately 20% of both adults and children in the US and is a source of substantial disability and health care costs. Chronic pain can be challenging to diagnose due to the presence of poorly understood symptoms. When diagnosed, current pharmacologic treatments for pain are remarkably ineffective, while effective non-pharmacologic treatments remain under-utilized. These shortcomings in the diagnosis and treatment of pain arise from tremendous gaps in our knowledge about the basic central nervous system systems that process nociceptive information and instantiate an experience of pain. These gaps are further amplified in the case of pediatric chronic pain due to a lack of basic/translational research. Our team of basic scientists and clinician scientists is uniquely positioned to perform human pediatric studies integrating functional neuroimaging with quantitative sensory testing and psychological assessments to delineate brain systems engaged during chronic pain. We will examine four distinct chronic pain syndromes: migraine, complex regional pain syndrome, functional abdominal pain, and musculoskeletal pain. We seek to 1) Identify shared and distinct brain systems engaged by different forms of pediatric chronic pain, 2) Determine if predictors of recovery differ across different chronic pain conditions, 3) Delineate brain systems associated with the spread of pain. We will follow patients longitudinally for 1 year after initiation of treatment to assess the degree of recovery and spread of pain. This study will provide a critical foundation of basic knowledge for future clinical trials of diagnostic markers for different forms of chronic pain and for the development of new treatments for chronic pain.

NIH Research Projects · FY 2024 · 1999-09

Project Summary/Abstract The long-term goal of this study is to determine the regulation and function of gastrointestinal (GI) eosinophils, a cell associated with multiple inflammatory diseases, especially a new set of emerging chronic allergic diseases referred to as eosinophilic GI diseases (EGID). Whether GI eosinophils exist as heterogeneous cells and subpopulations is currently not known. This is an important subject as distinct cell populations may have different roles in disease and during homeostasis. Understanding this subject has significant clinical implications in view of the emerging class of new drugs that target eosinophils. Perhaps only a specific population of eosinophils should be ablated and only in certain patients. The central hypothesis is that GI eosinophils are dynamic, transcriptionally active cells regulated epigenetically by local microenvironmental cues and that these processes lead to heterogeneous cell populations, under homeostatic and disease conditions. The rationale for this hypothesis is based on the findings that eosinophils express dynamic transcriptomes characterized by distal regulatory elements associated with histone 3 lysine 27 acetylation (H3K27ac) and binding sites for the transcription factor PU.1. We will test this hypothesis using recently developed innovations (e.g., ChIP-seq, ATAC-seq, transcriptomics, ex vivo co-culture) that we have applied to study murine and human eosinophils. In Aim 1, we will examine the epigenetic landscape and function of eosinophils. We will test the hypothesis that eosinophils express dynamic transcriptomes and epigenomes under basal and IL-33–activated conditions. We will mechanistically uncover the involvement of PU.1 as a prototypic transcription factor that regulates eosinophil enhancer function. We will elucidate the epigenetic and transcriptional mechanisms underlying eosinophil responses to a microenvironmental stimulus, IL-33. In Aim 2, we will examine GI eosinophil heterogeneity, aiming to test the hypothesis that murine GI eosinophils exist as heterogeneous populations defined by their epigenomic and transcriptomic landscapes and their proteomic content and functional capacity. In Aim 3, we will examine eosinophil heterogeneity in EGID. We will compare the epigenome, transcriptome, and proteome of human blood and GI tissue eosinophils in healthy and diseased individuals. We will model the interplay of eosinophils with key microenvironmental factors that are likely to influence these processes (e.g., co-culture with epithelial cells and relevant activating cytokines [IL-13]). We are hopeful that the renewal of this grant will allow the elucidation of the fundamental properties of GI eosinophils to continue. We anticipate that the findings from this grant will lay the foundation for defining eosinophil subpopulations in mice and humans. The importance of this undertaking (identifying subtypes and functions of eosinophils unique to different tissues) was highlighted in a recent NIH Taskforce on the Research of Eosinophil-Associated Diseases (RE-TREAD), emphasizing the potential significance of this proposal. In addition, the findings generated have the potential to advance the understanding of EGID and a variety of aspects of mucosal immunology.

NIH Research Projects · FY 2025 · 1995-01

This application requests continued funding for an innovative and highly successful T32 training program in Pediatric Gastroenterology, Hepatology and Nutrition (GI) at Cincinnati Children’s Hospital Medical Center. The training program, funded by the NIH for 30 years, has consistently produced much-needed academic pediatric gastroenterologists. The Aims of our Program are to: (1) Develop young investigators from clinical and research disciplines, and train future leaders in pediatric digestive disease research. (2) Promote interdisciplinary collaboration among scientists with varied expertise that share relevance to pediatric digestive disease to offer a superior training environment. Through a combination of supervised research, graduate level coursework, and dedicated mentorship provided by a consortium of primary research mentors and scholarship oversight committees, six postdoctoral trainees have a unique opportunity to cultivate lifelong scientific proficiency and leadership over a training period of two years. We have chosen to offer this training across three themes which are aligned with the focus groups of our P30 NIDDK-supported Digestive Health Center (DDRCC), including: 1) Mechanisms of Liver Disease; 2) Digestive Disease and Immunity; and 3) Stem Cell and Organoid Disease Modelling. Research training is pursued on either the Clinical or Basic/Translational Research Tracks. Outcomes, health services, or epidemiological research projects are completed on the Clinical Research track and include a formal program of study leading to either a Master of Science degree, or Certificate, in Clinical and Translational research. Laboratory-based research projects are completed on the Basic/Translational Research track and include a formal program of study leading to a Certificate in Clinical and Translational Research. Each of these didactic programs provides instruction in ethics in research, study design, and statistical analysis to provide training in rigor and reproducibility of the research projects. The depth and quality of the research training is monitored by an interdisciplinary team of outstanding scientists with strong training and mentoring track records. The long-term goal of the program remains to foster the development of outstanding physician-investigators and leaders who will meet the pressing academic workforce demands in Pediatric Gastroenterology, Hepatology and Nutrition.

NIH Research Projects · FY 2025 · 1994-07

PROJECT SUMMARY/ABSTRACT Our Pulmonary Development and Disease Pathogenesis T32 Training Program provides advanced research training by supporting stipends for five predoctoral and three post-doctoral candidates within the University of Cincinnati College of Medicine (UCCOM) and Cincinnati Children's Hospital Medical Center (CCHMC) graduate and postgraduate training programs. The training environment draws upon established, integrated, innovative graduate and postgraduate programs focused to molecular, developmental, and cell biology and outstanding clinical translational programs focused to pediatric and adult pulmonary diseases and radiological imaging research. The T32 program benefits from NHLBI-supported solid research programs and brings together 39 distinguished, NIH-funded investigators experienced in molecular and cell biology, physiology, and imaging strategies to study lung development and diseases. Program faculty have shared research interests and active basic and translational research collaborations. Major research themes include the genetic, cellular, and molecular basis of pulmonary disorders across the lifespan, bioinformatic and big data analysis of lung biology and disease, rare lung disease pathogenesis and therapy, and advanced pulmonary imaging. Interactions among pulmonary cells during lung morphogenesis and regeneration, mechanisms underlying branching morphogenesis, alveologenesis, and repair, and the genetic basis of lung disease are areas of expertise. Biomarkers, imaging, and therapeutics for pulmonary parenchymal and vascular diseases are areas of excellence. Meritorious trainees engaged in pre- or post-doctoral training programs are identified and selected on a competitive basis. Ph.D. and MD/PhD predoctoral trainees obtain their PhD degrees in Graduate Programs in Developmental Biology, Immunobiology, Biomedical Informatics, Pathobiology, and Molecular Medicine. Postdoctoral trainees may have an MD or PhD degree, or both, and are recruited to our T32 Mentor laboratories. Special attention is given to recruiting excellent candidates including physician-scientist trainees seeking PhD/MD degrees. Research training includes mentoring in career development, responsible conduct of research, reproducibility and scientific rigor, ethics in research, research presentation skills, and various seminars and courses within the graduate and divisional programs. Trainees attend regular research meetings and pertinent seminar series. Program Administration includes the contact Co-PI and three Co-Principal Investigators, Executive Committee, and External and Internal Advisory Committees. The progress of each trainee, the quality of mentors, and the program's overall effectiveness are critically reviewed. Trainees meet with their Mentor regularly, the T32 Program Director and Co-Directors quarterly, and their Mentoring Committee at least biannually. This T32 renewal application (Years 30-35) will permit us to continue a program with outstanding productivity in preparing new investigators to enhance research training and competence in critical aspects of pulmonary development and disease.

NIH Research Projects · FY 2026 · 1991-04

PROJECT SUMMARY The NICHD Neonatal Research Network (NRN) was established to conduct rigorous research in neonatal medicine, facilitating collaboration among centers to complete studies more rapidly than investigators acting alone. Cincinnati Children’s Hospital Medical Center has been one of the most productive sites in the NRN for over 30 years. Our site’s strong performance is due to our large, diverse patient population, outstanding research infrastructure and clinical facilities, strong faculty investigators with expertise in a wide variety of neonatal research, and experienced research support staff. Our leadership understands the importance of large, multicenter trials to answer clinical questions in neonatology. Dr. Stephanie Merhar, Principal Investigator for the Cincinnati site, has led NRN studies and other multicenter trials in Cincinnati for over 7 years. She is joined for this renewal by Dr. Tanya Cahill as Follow-up Principal Investigator and Drs. Vivek Narendran and Jae Kim as alternate PIs. During this next cycle, Cincinnati will work collaboratively with other Clinical Centers, outside investigators, and the Data Coordinating Center to develop and implement rigorous and reproducible common protocols. Due to our exceptional model of regional, population-based care with more than 1900 annual NICU admissions in our 3 recruiting NICUs, we are able to contribute a large number of subjects to NRN observational studies and clinical trials. Our well-established regional follow up infrastructure allows us to maximize retention rates for both 22-26 month and school age follow up. We are committed to conducting definitive multi-site clinical research through the NRN to improve outcomes for newborns born preterm and with other serious conditions.

NIH Research Projects · FY 2026 · 1990-08

Abstract. We study neurofibromas, benign peripheral nerve tumors characteristic of patients with the common dominantly inherited disease neurofibromatosis type 1 (NF1). In the 30-year history of this project we have developed cell and animal models and studied many of the cell types that are recruited to neurofibromas: neurofibroma cells include Schwann cells (SC), which we now know are outnumbered by a combination of fibroblasts, macrophages, and other immune cells. We use a neurofibroma mouse model in which, in the peripheral nervous system, biallelic Nf1 mutations are present only in Schwann cell precursors (SCP) and Schwann cells; other wild type stromal and immune cells are recruited to neurofibromas as they form. We showed that neurofibroma SC and SCP express CXCL10, whose receptor, CXCR3, is expressed on immune cells-- T cells and dendritic cells (DC)-- and that CXCR3 is required for neurofibroma formation. Further, we observed a strong interferon signature in SC and neurofibroma macrophages; interferons are known to stimulate the production of CXCL10 and of CXCL9, chemokines that signal through CXCR3. Our striking Preliminary Data show that neurofibromas require T cells to form, that transfer of T cells back to mice lacking them is sufficient to promote neurofibromas. Mice deficient in a key type of APC, dendritic cells (DCs), also show reduced neurofibroma numbers. Finally, immune cells are known to secrete factors that influence appear to promote Nf1-/- SCP self-renewal and neurofibroma formation. Based on these observations we propose that: (i) Schwann cells/SCP require type I IFN signaling to produce CXCL10 to recruit DCs and T cells. (ii) subset(s) of DCs stimulate tumor-specific T cells to produce IFN-g, which induces Cxcl9 expression by macrophages. (iii) Cxcl9/10 act redundantly to sustain T cell recruitment; and (iv) cytokines produced by immune cells themselves promote SC de-differentiation and neurofibroma development, in a feed-forward loop.

NIH Research Projects · FY 2025 · 1987-07

Project Abstract There are 124 publications that establish 185 disease risk loci for the genetics of systemic lupus erythematosus (SLE) with robust supporting data. This is a completely different situation than when AI0274717 started 33 years ago as a search for genetic linkage. Now, in addition to the 185 risk loci we have strong circumstantial evidence for an etiologic role for Epstein-Barr virus (EBV) at the origin of SLE, especially considering the recently published powerful association between the location in the genome of transcription complexes containing the Latency III gene product, Epstein Barr Nuclear Antigen 2 (EBNA2) and the genetic risk loci of SLE. This association has been confirmed and extended to the at present known SLE risk loci. In addition, new data show that EBNA3C and EBNALP, both also EBV Latency III gene products, are also powerfully concentrated at the SLE risk loci, all three, EBNA2, -3C, & -LP, clustering together and in aggregate binding indirectly to more than half the known SLE risk loci. A set of human transcription factors and co-factors (TFs) tend to bind DNA at these same SLE risk loci. Our hypothesis is that many genetic mechanisms that cause SLE operate in the EBV transformed B cell, because of the EBV Latency III gene expression. Since ~90% of the plausibly causal variants in the 185 risk loci are in genomic regions, predicted to have regulatory function; therefore, the role of TFs promises to be important in the genetic mechanisms of SLE. What is missing, a serious gap in our knowledge, is the identify of the Target genes regulated by these risk variants. Given the unexpected, but dominating influence of the Latency III gene products in associations with the SLE risk loci, we conclude that the cell type in which to begin the systematic search for the disease relevant Target genes is the EBV-infected and transformed B cell. In Aim 1 we will focus on identifying Target genes in these cells for as many of the 185 SLE risk loci as possible with special attention focused on the involvement of EBNA2. In Aim 2 we will concentrate on the allelic differences in Target gene expression induced by the risk and non-risk alleles of SLE risk locus variants, again with special attention to the possible role of EBNA2. We have preliminary data that support our technical capacity to perform the experiments proposed and have constructed many of the reagents needed to perform the proposed experiments, which have been initiated. We will adapt high throughput systems biology methods to screen and explore the gene regulation of the 185 loci to build a foundation to evaluate the role EBNA2 has in SLE etiology relevant regulation. Extraordinary new commercially available methods will make the screening procedures proposed practical, feasible and affordable. If our hypothesis is correct and we demonstrate EBNA2-dependent mechanisms of gene regulation at the risk loci, then these would be nominated as potential causal mechanisms for the genesis of SLE. These results will lay the foundation for the important next step, to perturb the identified mechanism in ways that change the risk of developing SLE or that change the capacity of the illness to persist.