Cincinnati Childrens Hosp Med Ctr

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY TITLE: Maternal B cells enforce fetal tolerance GOALS/OBJECTIVES: The overall goal of this five-year proposal for a Mentored Clinical Scientist Research Career Development Award (K08) is for me to develop into a productive, independent academic investigator in the field of reproductive immunology. My prior training has provided me expertise regarding T cell and antibody mediated host defense. Through this proposal I will gain additional experience in B cell biology, glycoimmunology and fetal tolerance. The long-term goal of this research is to improve pregnancy outcomes, which will lead to healthier pregnancies and neonates. I graduated from the American Board of Pediatrics Accelerated Research Pathway for Residency in General, and I completed my Fellowship in Neonatal- Perinatal Medicine at Cincinnati Children’s Hospital (CCHMC). I joined the faculty of CCHMC and the University of Cincinnati as an Attending Physician and Research Assistant Professor in the Division of Neonatology. My mentor for this award, Dr. Sing Sing Way, is a physician-scientist with a longstanding track record of scientific innovation and providing exceptional training to mentees at all levels. As an internationally recognized expert in the immunology of pregnancy, microchimerism, commensalism and neonatal sepsis, Dr. Way’s work highly complements my own. My mentorship committee brings needed expertise to the areas of antigen-specific B cell responses (Dr. Justin Taylor), B cell interactions with N-linked glycans (Dr. Shiv Pillai), placental immunology (Dr. Tamara Tilburgs), transgenic lymphocytes (Dr. Koichi Araki) and maternal-fetal immunology (Dr. Hitesh Deshmukh). I am also extremely fortunate to have the unfettered support of CCHMC and the Perinatal Institute, whose combined resources are unmatched. Scientifically, this proposal focuses on deepening our understanding of mechanisms of fetal tolerance. The field currently focuses on immune suppressive CD4+ Foxp3+ regulatory T cells (Treg) as the key mediators of tolerance. This is based on observations that maternal Treg expand throughout pregnancy and are blunted in pregnancy complications associated with fetal intolerance (e.g., preeclampsia, stillbirth). However, there is growing appreciation that Treg do not work in isolation, and often require support from other cell types, including regulatory B cells (Breg). Activation of B cells is tightly regulated by the inhibitory receptor CD22, which binds to sialic acid present at the terminal position of certain glycoproteins. There remain numerous gaps in knowledge regarding the role of maternal B cells in mediating fetal tolerance, factors affecting their activation, and modification of fetal antigens. Additionally, whether these maternal B cells work independently or synergistically with maternal Treg remains to be determined. My central hypothesis is that CD22 impairs maternal B cell immune regulatory functions, preventing B cells from upholding fetal tolerance. This is based on very interesting preliminary data showing that CD22 blockade reverses pregnancy complications and fetal loss when maternal Treg are depleted or functionally impaired by infection. The aims of this proposal will establish: 1) how maternal B cell immune suppression is counter-regulated though antigen recognition and CD22, as well as 2) how maternal B cells enforce fetal tolerance through IL10-mediated inhibition of maternal T cells with fetal-specificity. The scientific rigor of this proposal is supported by numerous career development objectives that will help enable a transition to a successful independent research program. I will work closely with my mentor, Sing Sing Way, and mentoring committee to gain expertise in reproductive immunology, fetal tolerance, spectral flow cytometry, characterization of rare antigen-specific B cells, placental immunology, glycobiology and genetic manipulation of antigen-specific B cells. Coursework, seminars, scientific conferences and hand-on training in laboratories of collaborators/mentors will add additional layers of expertise. Overall, this mentored career development award will broaden my scientific acumen to include reproductive immunology with a focus on B cell biology and glycobiology. As pregnancy complications remain the leading cause of infant and childhood mortality, results of these studies will potentially lead to novel B cell-directed therapies aimed at improving outcomes for pregnant mothers and their children.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY/ABSTRACT Progression of chronic liver disease to fibrosis, cirrhosis, and cancer is the common course of several liver diseases, but treatments for the end-stage liver disease are limited highlighting the critical need to identify a novel therapeutic target. Despite the numerous evidence indicating that accumulation of hepatic progenitor cells (HPCs) - the epithelial compartment of ductular reactions - is associated with fibrosis, definitive evidence supporting the causal role of HPCs in progression of fibrotic liver disease and cancer remains largely uncertain mainly due the lack of a mouse model that allows conditional labeling and tracing of HPCs. Furthermore, although increased angiogenesis in cirrhotic livers is a risk factor for tumorigenesis, whether and how HPCs signal to endothelial cells remains as a knowledge gap. We have previously reported that the forkhead box L1 (Foxl1)- Cre transgenic line can be used for specific labeling and isolation of HPCs. Using this new mouse model, we generated preliminary data indicating that inhibition of Notch signaling in HPCs leads to decreased angiogenesis and fibrosis in mice with chronic liver disease and increased differentiation of HPCs into cells with hepatocytic morphology. Our data also indicate that HPCs secrete several paracrine factors to stimulate proliferation of endothelial cells and angiogenic gene expression. This led to our central hypothesis that Foxl1+ HPCs promote progression of liver disease by crosstalking with endothelial cells and hepatic stellate cells in a paracrine manner, and modulation of the Notch signaling pathway is a valid strategy to promote differentiation of HPCs and inhibit pathogenic mechanisms. We propose HPCs as a novel and unique cellular target that can be modulated to simultaneously inhibit progression of fibrotic liver disease and tumorigenesis and promote liver regeneration. Our overall objective for the proposed study is to establish the causal role of HPCs in pathogenesis using experimental models that recapitulate progression of human fibrotic disease. We will test our hypothesis with the following specific aims: Aim 1 will determine the role of the Notch signaling pathway in reprogramming of Foxl1+ HPCs into mature hepatocytes in vivo. Aim 2 will determine the cellular and molecular mechanism by which Foxl1+ HPCs regulate disease progression. Aim 3 will determine the requirement for Foxl1+ HPCs in multiple stages of tumorigenesis.

NIH Research Projects · FY 2026 · 2023-12

ABSTRACT T cells play important role in cancer cell immunosuppression. Cancer cells can interact with immune checkpoint proteins expressed on effector T cells to cause T cell exhaustion and facilitate regulatory T (Treg) cell suppression of effector T cells. Understanding T cell immunity is an important goal in cancer immunotherapy. Although immune checkpoint inhibitors and CAR-T cell therapies have shown tremendous promise, current immunotherapies only benefit a fraction of cancer patients, and new approaches from mechanism-driven immune modulations are needed to broaden the therapeutic benefits to less responsive patients. Targeting Treg cells to activate effector T cells to combat cancer is an emerging concept in cancer immunotherapy. While systemic depletion of Treg cells can cause excessive T cell activation leading to autoimmunity, proper induction of Treg cell instability without side effects of autoimmune responses may open a new avenue for immune modulation. In preliminary studies, we have found that heterozygous deletion of the Rho GTPase Cdc42 in Treg cells did not affect Treg cell homeostasis nor result in autoimmune response but caused a destabilization of Treg cells that elicited an anti-tumor immunity. Pharmacological targeting of Cdc42 with a small molecule inhibitor, CASIN, mimicked Cdc42 heterozygous deletion in destabilizing Treg cells and in gaining an anti-tumor T cell immunity. CASIN potentiated the effects of immune checkpoint inhibitors in tumor suppression without detectable autoimmunity in mice. This project hypothesizes that the rational designed small molecules targeting Cdc42 activity can destabilize Treg cells and modulate anti-cancer immunotherapy without inflammatory side effects. In Aim 1, to determine the molecular pharmacology of CASIN action we will define the mechanism of action of CASIN and improve CASIN efficacy. We will carry out further medicinal chemistry studies, validate CASIN derivatives for proper target engagement, and examine potential toxicity in T cells and other cell types. In Aim 2 we will demonstrate a proof of concept for CASIN or its derivatives in targeting of Cdc42 in mouse models to trigger anti-tumor T cell immunity. We will determine the toxicity, pharmacokinetics and pharmacodynamics of CASIN and its derivatives, and examine their preclinical efficacies in destabilizing Treg cells to elicit anti-tumor T cell immunity, alone or in combination with immune checkpoint inhibitors. Overall, our work will establish a novel concept and present a useful approach for anti-cancer immunomodulation.

NIH Research Projects · FY 2026 · 2023-11

Pregnancy induced deacetylation of sialylated glycoproteins Abstract. Pregnancy confers susceptibility to severe infection caused by intracellular pathogens, including the prototypical perinatal bacterial pathogen, Listeria monocytogenes (Lm). Residence inside cells has traditionally been thought to be exploited by microbes to evade antibody-mediated immunity since IgG cannot cross the plasma membrane and directly enter cells. Our ongoing studies challenge this paradigm by showing vertically transferred maternal IgG antibodies efficiently protect neonates against Lm infection. The key molecular change enabling protection is deacetylation of terminal sialic acid residues in glycosylated anti-Lm IgG. Expression of the deacetylating enzyme sialic acid acetyl esterase (SIAE) increases during pregnancy. This enzyme is essential since anti-Lm antibodies in SIAE-deficient pregnant mice remain acetylated, and non- protective upon vertical transfer into neonates. Sialylated glycoproteins normally stimulate host cells by binding CD22, an inhibitory Siglec expressed by B cells. However, acetylation blocks CD22 binding, establishing the framework whereby differential acetylation versus deacetylation of sialylated IgG and other glycoproteins can profoundly impact immunity through CD22 binding, which in turn, controls activation and immune-modulatory function of B cells and other cell types with regulatory potential. Although these results significantly broaden the scope for how antibodies work, they also unveil important new knowledge gaps on whether deacetylated IgG similarly protects against infection outside the neonatal window, including during pregnancy with similarly increased susceptibility to Lm systemic infection, and after parturition when deacylated antibodies are still present at high levels. Other questions include the maternal cell types expressing SIAE responsible for antibody protective conversion, and the larger impacts on fetal tolerance and pregnancy outcomes controlled by deacetylation considering many fetal alloantigenic glycoproteins also contain sialylated N-linked glycans. Our overall hypothesis is that SIAE deacetylation unmasks a variety of sialylated glycoproteins as new CD22 ligands, which differentially modulate the suppressive properties of maternal immune cells, with context- dependent impacts on infection susceptibility and pregnancy outcomes. Shared susceptibility to Lm infection during pregnancy in humans and rodents will be exploited to further investigate how deacetylated antibodies work, in concert with fetal alloantigenic glycoproteins, through the following specific aims: Aim 1, determine whether deacetylated IgG protects against Lm systemic infection during pregnancy and after parturition; Aim 2, establish the SIAE-expressing maternal cell subset(s) responsible for IgG protective conversion; Aim 3, investigate how SIAE exposed CD22 ligands control fetal tolerance and pregnancy outcomes. Completion of these aims will not only unveil important insights as to how antibodies protect against intracellular infection, but also new information on the immune-pathogenesis of fetal wastage that occurs with Lm prenatal infection and exciting ways for boosting antimicrobial immunity in this vulnerable physiological window.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Pediatric in-hospital cardiac arrest occurs almost exclusively in pediatric intensive care units and affects thousands of hospitalized children each year. While cardiac arrest survival outcomes have improved, more than half of these children will die prior to discharge making prevention the best approach to improve pediatric patient safety. In our single center prior work, the use of the SAMURAI PICU (Situation Awareness incorporating MUltidisciplinary Teams Reduce Arrests In the PICU) bundle improved early identification of high-risk patients, increased shared situation awareness, and supported risk mitigation plans leading to a >50% decrease in IHCA events requiring cardiopulmonary resuscitation. Using an innovative process of emphasizing shared situation awareness through automated clinical decision support and mitigation of risk, we will use a user-centered design approach to adapt, implement, and assess the feasibility and effectiveness of SAMURAI PICU at other pediatric institutions. We hypothesize that identification of PICU patients at high risk for in-hospital cardiac arrest through the use of our automated clinical decision support tool and integration of this high-risk status in daily safety huddles will lead to improved shared situation awareness and subsequent reduction in cardiac arrest events. We will utilize a five-center pragmatic prospective Hybrid Type 1 effectiveness-implementation study leveraging the existing infrastructure of Pediatric Resuscitation Quality Collaborative to evaluate the following specific aims: 1) adapt the SAMURAI PICU bundle at each intervention site to fit local context through a user-centered design approach, 2) implement the SAMURAI PICU bundle in a hybrid stepped wedge fashion and evaluate the effectiveness of the bundle to reduce CPR events in the PICU. This proposed research is significant it has the potential to inform and transform the field of cardiac arrest prevention and ultimately prevent death related to in hospital cardiac arrest for thousands of children in the future significantly improving pediatric patient safety.

NIH Research Projects · FY 2025 · 2023-09

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NIH Research Projects · FY 2025 · 2023-09

SUMMARY Microphthalmia, anophthalmia and coloboma (MAC) are a group of clinically and genetically related eye defects that cause significant visual impairment. MAC can be caused by pathogenic variants in transcription factors and other genes involved in eye development and by environmental factors such as maternal vitamin A deficiency (VAD). Vitamin A is critical for retinoic acid (RA) synthesis and pathogenic variants causing loss or gain of function for the genes in the RA pathway, such as STRA6, can result in severe MAC. We hypothesize that genetic variation in the RA pathway genes and genes involved in retinol metabolism can result in a predisposition, or lower threshold, for the deleterious effects of VAD on eye development that contributes to the phenotypic severity of MAC. This gene-environment interaction is highly relevant for countries where VAD is common and remains a public health concern, such as India. In addition, the downstream genes that are dysregulated by lack of vitamin A are poorly understood and are amenable to study in animal models. To identify the full spectrum of genetic variation in the RA pathway and other genes in patients with MAC, our first Aim will recruit patients with MAC and perform detailed phenotyping, environmental screening, and imaging so that we can generate accurate phenotype genotype correlations. We will generate, analyze and compare whole genome sequencing (GS) data from our collaborators, Dr. Tibrewal and Dr. Kumar, in patients of Asian ancestry with our studies using GS in patients with MAC of predominantly European ancestry. We will also prospectively recruit patients with MAC diagnosed in the newborn period from centers in the USA, so that we can obtain retinol levels from umbilical cord blood in babies with MAC and retinol levels in maternal plasma around the time of delivery. With this data, we will compare the severity of MAC with sequence variants that we observe in the RA pathway genes and in other eye developmental genes, for patients of different ancestries, with and without evidence of VAD from symptoms or from low levels of retinol. In our second Aim, we will use gene-editing with Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 to determine the effects of altered function for stra6 and other genes in the RA pathway on ocular morphogenesis in zebrafish. All crispants will have detailed phenotyping at different timepoints during eye development, including gene expression studies with single cell RNA-Seq, to determine the downstream genes that are regulated by RA. We will also investigate if we can rescue, or partially rescue, the deleterious effects of genetic variants in stra6 and other genes causing MAC on eye morphogenesis by titrating levels of RA and retinol during periods of ocular development in Danio rerio. With this Aim, we will use an animal model to generate data regarding downstream genes, tissues and developmental timepoints that are affected by deficiencies of RA and vitamin A. Our overall purpose is to improve our understanding of the pathogenesis of MAC to develop targeted prevention strategies and therapeutics for children affected by these devastating birth defects.

NIH Research Projects · FY 2025 · 2023-09

Project Summary More than a third of pediatric adverse events have been attributed to diagnostic error—the failure to establish an accurate and timely explanation of a child’s health problem. Within the pediatric intensive care unit (PICU), a high-stakes, complex, team-driven environment, diagnostic error rates vary between 8% and 25%. To improve diagnostic accuracy, clinical decision support (CDS) tools, including artificial intelligence and machine learning algorithms, have been developed but have failed to be adopted within the PICU. Implementation of CDS into an active high-risk clinical context without a comprehensive understanding of how providers and staff interface with the system can lead to patient safety consequences. These consequences can include disrupted workflows, alert fatigue, and inaccurate documentation that can lead to direct patient harm. We propose leveraging a currently built highly realistic immersive virtual reality PICU clinical environment to assess implementation strategies on clinician acceptance of CDS tools to improve diagnostic excellence within a patient safety learning lab. We will evaluate three aims using a systems-engineering approach combining the expertise of LiveWell, an award-winning design collaborative with user-centered design and human factors expertise, with the clinical knowledge, implementation science expertise, and world-renowned virtual simulation program of Cincinnati Children’s Hospital Medical Center (CCHMC). We will: redesign how clinicians interface with CDS tools for diagnosis-intensive events within the pediatric critical care environment including admission and clinical deterioration; develop and implement design-informed CDS tools for diagnosis and to evaluate their appropriateness/acceptability and impact on diagnostic uncertainty, accuracy, and timeliness within a digital twin immersive virtual reality simulation; and establish best practice guidance for implementation of CDS tools in the critical care environment to improve diagnostic excellence. Outcomes will include 1) appropriateness, acceptability, and adoption of design informed CDS tools; 2) impact on diagnostic accuracy and timeliness in the simulated clinical environment; and 3) a proactive implementation plan for the integration of design informed CDS tools to improve diagnostic excellence into the critical care environment.

NIH Research Projects · FY 2025 · 2023-09

It has been known for some time that development of the visual system can be an adaptive process in which light exposure or visual experience modifies the final structure. Here we propose to investigate a developmental pathway in which photoreception via OPN4 in intrinsically photosensitive retinal ganglion cells (ipRGCs) regulates rod photoreceptor survival. Preliminary data show that light and OPN4 regulate rod precursor cell death in the neonatal retina and rod number in adult mice. This is mediated by the neuromodulator glutamate. We suggest that ipRGC outer retinal dendrites (ORDs) deliver glutamate to the outer retina and regulate rod survival. ORDs are unique to ipRGCs and are transient, existing only during the first week after birth. Single cell sequencing data reveal that the glutamate receptor Grik3 is expressed in rod precursors transiently in the first week after birth and is thus a good candidate to mediate this response. These data suggest the Central Hypothesis that Perinatal light sensing by ipRGCs regulates rod photoreceptor number via glutamate-induced precursor cell death. We propose three Aims designed to define the mechanisms underlying this proposed adaptive response. We will define the developmental time-course of ORDs and assess markers of glutamate function (Aim 1), assess the proposed relationships in the light-OPN4-Gαq-rod cell death pathway (Aim 2), and assess glutamate delivery by ipRGC ORDs and the response of rod precursors (Aim 3). The finding that early developmental light sensing regulates the number of rod photoreceptors is unexpected and of broad interest because it represents a novel mechanism and has implications for human eye development.

NIH Research Projects · FY 2025 · 2023-09

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Sickle cell disease (SCD), the most common life-shortening genetic disorder, affects 100,000 individuals in the United States. SCD has its first health impact in infancy, but more severe complications (e.g., organ damage, chronic pain, risk for early mortality) emerge in adolescence. Further, these youth face social personal and logistical challenges (e.g., financial hardship) that may impact their access to care, resulting in missed opportunities to prevent complications and a higher risk for disease progression. Effective disease self-management is essential to improving care and outcomes and lowering healthcare costs for adolescents and young adults (AYA) with SCD. However, there is limited research available describing the specific factors that need to be addressed to improve self-management and health outcomes for these youth. Our multidisciplinary team used a socioecological framework to develop a novel ehealth self-management intervention for AYA with SCD called SCThrive. SCThrive combines group sessions with a therapist with a companion mobile app. Our pilot work demonstrated improved patient activation (knowledge, skills, and self-efficacy) and self-management behaviors in AYA with SCD compared to a control condition. In addition, those who use the app more frequently showed more improvements. Older AYA used the app less and reported more SDOH-related barriers to self-management. Thus, we will maximize the clinical benefit of SCThrive by 1) adding app engagement strategies for older AYA, 2) conducting a more systematic assessment of personal and logistical challenges that impact health, and 3) integrating ways to address these barriers into the intervention. The objective of this proposed project is to conduct a randomized controlled trial (RCT) with adolescents with SCD across four SCD centers. Our aims are to examine the impact of SCThrive on patient activation (primary outcome; Aim 1) and self-management behaviors, functional disability, health-related quality of life, and emergency room visits (secondary outcomes) at post-treatment and follow-up (Aim 2). We hypothesize that adolescents randomized to SCThrive will have greater improvement in patient activation (primary outcome) compared to those randomized to standard care (control condition). We will also explore the relationship between individual and logistical challenges (e.g., access to care) and treatment response (i.e., patient activation and self-management behaviors). The team has expertise in SCD, patient activation, behavioral interventions, ehealth/mhealth, adherence and self-management, RCT and statistical analyses and has collaborated on pediatric SCD intervention studies that lay the foundation for this proposal. The study is significant because it addresses the need for personalized interventions to improve self-management in adolescents with SCD. The proposed research is innovative because it integrates a robust assessment and intervention strategy for addressing individual and logistical challenges that impede self-management in SCD.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract Cranial neural crest cells (CNCCs) form most of the craniofacial complex. Despite being derived from an ectodermal population, CNCCs are able to differentiate into both non-ectomesenchymal (e.g., neurons/glia) and ectomesenchymal derivatives (e.g., bone, cartilage, and smooth muscle). This unique ability to form a broad range of cell types from more than one germ layer has led to CNCCs being called “pleistopotent.” However, the signaling mechanisms that convey this pleistopotency and drive differentiation of CNCCs are not well understood. The Sonic hedgehog (Shh) signaling pathway has long been studied in the context of craniofacial development. The Shh signaling pathway is mediated by bimodal Gli transcription factors (Gli TFs), which activate or repress target genes associated with proper craniofacial development. Studies done on CNCC in vitro demonstrated that exposure to recombinant Shh protein increased CNCC potency by increasing the proportion of CNCCs that differentiated into both non-ectomesenchymal and ectomesenchymal derivatives and decreased the proportion of CNCCs that only differentiated into non-ectomesenchymal derivatives. To test the role of Shh signaling in CNCC potency, we generated a conditional mouse line lacking Gli activity in CNCCs (Gli2f/f;Gli3f/f;Wnt1-Cre). Single cell RNA-sequencing analysis showed that E13.5 Gli2f/f;Gli3f/f;Wnt1-Cre CNCCs were not separated into distinct clusters as compared to the wild-type CNCCs. Interestingly, while ectomesenchymal clusters were poorly defined molecularly, non-ectomesenchymal clusters were relatively unaffected. Thus, these data suggested that loss of Gli TFs led correlated with a loss of pleistopotency. Based on these preliminary data I hypothesize that Gli-mediated Shh signaling conveys CNCC potency via activation of pluripotent programs and that loss of Gli2/3 signaling limits potency, thus preventing differentiation into the full range of CNCC derivatives. In Aim 1, I will perform CUT&RUN for Gli2 and Gli3 on embryos during CNCC induction to look for binding of Gli TFs at traditional pluripotency markers. I will perform single cell RNA- sequencing on both wild-type and Gli2f/f;Gli3f/f;Wnt1-Cre embryos before, during, and after CNCC induction and specification and compare gene expression profiles to determine if loss of Gli TFs affects expression of potency factors in CNCC, which I will validate with RNAscope. Finally, I will perform Western blots for Gli2 and Gli3 to determine if Gli TFs are predominantly functioning as activators or repressors during CNCC induction. In Aim 2, I will determine if loss of Gli TFs restricts fate commitment of CNCCs. I will generate Gli2f/f;Gli3f/f;R26R-Confetti+/+; Wnt1-Cre embryos, where Wnt1-Cre drives Cre-recombination before establishment of CNCC pleistopotency, and Gli2f/f;Gli3f/f;R26R-Confetti+/+;Sox10-Cre embryos, where Sox10-Cre drives Cre-recombination after establishment of CNCC pleistopotency. This study is important for advancing our understanding of the unique potency of CNCCs in craniofacial development and the knowledge generated in this proposal may be applied to generate reparative tissues for patients with craniofacial defects or injuries.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT: An in-depth understanding of the mechanisms that regulate alveolar septation and septal wall maturation will be required to develop therapies for premature infants with Bronchopulmonary Dysplasia (BPD) and for developing potential therapies for adult lung regeneration. We and others have begun to define changes in PDGFRa myo, lipo and matrix fibroblast (FB) function during alveolarization, homeostasis, regeneration, and fibrosis. Our long-term goal is elucidation of molecular regulators of FB functions and how diverse FBs support epithelial cells. Our objective herein is to identify molecular mech- anisms that result in functional changes in alveolar fibroblasts that regulate ECM organization and epithelial differentia- tion. Integration of transcriptomic datasets predicted GATA6 as a regulator of PDGFRa-FB differentiation. Our preliminary data show that conditional inactivation of GATA6 in perinatal PDGFRa+ FBs resulted in loss of matrix and gain in lipo fibro- blast function comparable to findings in BPD and animal models of hyperoxia. GATA6 inactivation in PDGFRa+ FBs also resulted in fragmented collagen and detachment of AT1 cells from the basal lamina demonstrating a thus far unidentified role of alveolar FBs in ECM organization. Inactivation of GATA6 resulted in significant increase of the lipo FB transcription factor TCF21 expression and preliminary in vitro data suggest suppression of TCF21 by GATA6. The central hypothesis is that GATA6 and transcriptional cofactors regulate FB function in alveolar FBs by suppressing lipo FB differentiation. The rationale for this research is a new understanding of the regulation of matrix FB function and how these functionally different stages modify extracellular matrix and the FB-epithelial crosstalk. Aim 1 will test the hypothesis that GATA6 suppresses lipo FB function and promotes ECM deposition in the alveolar FBs. This aim will use lineage tracing to deter- mine trans-differentiation of the alveolar FB to a lipo FB. And use in vitro cell derived matrices to identify ECM modifica- tions by lipo and matrix FBs. Aim 2 will test the hypothesis that GATA6 is in a transcriptional network that regulates lipo and matrix FB function. In this aim we will use in vitro gain and loss of function of GATA6 and TCF21 and identify and validate transcriptional networks and partnering transcription factors. Aim 3 will test the hypothesis that GATA6 regulates ECM synthesis and paracrine signaling in PDGFRa+ FBs that direct AT2 to AT1 differentiation. The proposed studies in this application will identify the transcriptional regulation of matrix FB function and their role in extracellular matrix deposition and cell-cell signaling in the alveolar niche. The proposed research is conceptually innovative, because we ask questions regarding the nature of fibroblast plasticity and functional switches. The proposed research is scientifically innovative, because the contractile function of alveolar fibroblasts, has been thoroughly investigated, but little is known about their matrix organizing function or their role in supporting AT2 proliferation and AT1 differentiation This contribution will be significant because it addresses 1) lack of knowledge of matrix and signaling function of the alveolar FB; 2) identifies and validates the transcriptional network around GATA6 that regulates FB function.

NIH Research Projects · FY 2025 · 2023-09

Abstract Mucosal tissues like the intestine harbor trillions of antigenically foreign microbes. Unavoidable approximation with commensal microbes in this context highlights the need for expanded peripheral immune tolerance. However, fundamental gaps in knowledge remain as to how this physiological imperative is achieved. Filling these knowledge gaps have enormous potential to reveal novel insights on the immune- pathogenesis of inflammatory bowel disease and other human autoinflammatory disorders. Our current framework for investigating how tolerance expands to accommodate commensal intestinal microbes is primarily focused on the FOXP3+ suppressive subset of CD4+ T cells called regulatory T cells (Tregs). However, several inconsistencies also highlight the limitations attributing commensal tolerance exclusively to FOXP3+ cells. With these considerations, our preliminary studies pivoted to investigate commensal specific CD4+ T cells, without a FOXP3 bias, using an instructive model whereby CD4+ T cells with commensal specificity can be precisely identified. Using recombinant Candida albicans to establish intestinal colonization and tracking endogenous CD4+ T cells with MHC class II tetramers, our initial analysis of gene expression profiles (single-cell RNA-seq) shows minimal (<5%) Treg differentiation amongst CD4+ cells with commensal specificity. Instead, RNA profiling showed nearly half of peripheral cells that expand in response to commensal stimulation are not classified based on expression of other lineage-defining markers, and unified by expression of the zinc finger transcription factor Kruppel-like factor-2 (KLF2). Antigen-experienced KLF2+ CD4+ T cells are further shown to potently suppress responder T cell proliferation during in vitro co-culture. The necessity for T cell expressed KLF2 is further highlighted by spontaneous intestinal inflammation that develops in mice with conditional KLF2 deficiency in T cells, or the rapid (within 10 days) onset of disease in mice with induced KLF2-deficiency in CD4+ cells. Intestinal inflammation that occurs in the absence of KLF2+ CD4+ T cells is triggered by commensal microbes since their elimination using a cocktail of antimicrobials averts intestinal inflammation, efficiently bypassing the necessity for T cell expressed KLF2. Thus, our overall hypothesis is that KLF2 identifies a FOXP3-negative immune-suppressive subset of CD4+ T cells essential for sustaining tolerance to intestinal microbes. Three inter-related aims designed to further develop this potentially ground- breaking hypothesis are proposed which include establishing the molecular basis for how KLF2+ CD4+ T cells mediate suppression, and whether KLF2 is necessary and/or sufficient to promote functional suppression (Aim 1), the molecular basis for how KLF2+ CD4+ T cells protect against intestinal inflammation in vivo (Aim 2), along with gene expression, chromatin accessibility, and KLF2 DNA binding distinctions between KLF2+ CD4+ T cells compared with FOXP3+ Tregs, and other CD4+ T cell differentiation subsets in intestinal and lymphoid tissue as a basis for their unique biological functional properties (Aim 3).

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Sepsis is a dysregulated host response to an infection and can lead to multiple organ dysfunction syndrome (MODS). There are no effective treatments for sepsis-induced MODS most likely because of the heterogeneity of the syndrome. Delineating MODS endotypes and molecular signatures of organ failures may lead to a better understanding of the mechanisms involved in sepsis heterogeneity and allow for personalized treatment strategies. It is difficult, however, to obtain clinical specimens from critically ill patients that would enable investigations into organ-specific mechanistic changes. Extracellular vesicles (EVs) are spherical microparticles enclosed by bilayer phospholipid membranes. Exosomes or small EVs (sEVs) are a subtype of EV formed by endosomal biogenesis. Small EVs can be released from almost any cell type into a variety of bodily fluids and contain many cellular components. The cell-specific cargo can serve as cell-to-cell communicators and be taken up by distant cells which can affect the inflammatory profile. Our preliminary data show that sEVs harvested from serum of pediatric patients with sepsis have a distinct pro-inflammatory trait compared to sEVs of children without sepsis and in vitro sEVs from septic patients can induce atypical inflammatory responses in immune cells. Since circulating sEVs manifest characteristics of the cell of origin, they have been used as liquid biopsy. Thus, sEVs hold potential as useful biomarker for organ-specific changes. There are no available biorepositories of sEVs for pediatric sepsis research, in part, because of the lack of standardized methodology for sEV isolation. The overall goal of our proposal is to establish standardized procedures for reliable biorepositories for sEV biomarker research in critically ill patients with sepsis. This proposal will prove the hypothesis that quality and consistency of isolation and purification protocols of plasma and serum samples enable setting-up reliable biorepositories for future research on sEVs in sepsis. We will take advantage of two large critical care-division based repositories which has biospecimens from pediatric critically ill septic and non-septic studies. The R21 phase Aim 1 is to develop a methodology for sample collection and isolation of sEVs with high yield and purity and Aim 2 is to demonstrate suitability of banked sEVs for high throughput analyses of RNA cargo profile. Once milestones for the R21 phase are met we will proceed to the R33 phase to retrospectively characterize sEV endotypes in critically ill patients with specific organ injuries (Aim 3) and then prospectively determine whether patients can be classified based on their sEV characteristics. Results from these investigations will allow for the novel development of a biorepository of sEVs in pediatric sepsis. This biorepository will enable investigators to explore organ-specific molecular signatures for mechanistic studies of sEVs.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Sepsis is a dysregulated host response to an infection and can lead to multiple organ dysfunction syndrome (MODS). There are no effective treatments for sepsis-induced MODS most likely because of the heterogeneity of the syndrome. Delineating MODS endotypes and molecular signatures of organ failures may lead to a better understanding of the mechanisms involved in sepsis heterogeneity and allow for personalized treatment strategies. It is difficult, however, to obtain clinical specimens from critically ill patients that would enable investigations into organ-specific mechanistic changes. Extracellular vesicles (EVs) are spherical microparticles enclosed by bilayer phospholipid membranes. Exosomes or small EVs (sEVs) are a subtype of EV formed by endosomal biogenesis. Small EVs can be released from almost any cell type into a variety of bodily fluids and contain many cellular components. The cell-specific cargo can serve as cell-to-cell communicators and be taken up by distant cells which can affect the inflammatory profile. Our preliminary data show that sEVs harvested from serum of pediatric patients with sepsis have a distinct pro-inflammatory trait compared to sEVs of children without sepsis and in vitro sEVs from septic patients can induce atypical inflammatory responses in immune cells. Since circulating sEVs manifest characteristics of the cell of origin, they have been used as liquid biopsy. Thus, sEVs hold potential as useful biomarker for organ-specific changes. There are no available biorepositories of sEVs for pediatric sepsis research, in part, because of the lack of standardized methodology for sEV isolation. The overall goal of our proposal is to establish standardized procedures for reliable biorepositories for sEV biomarker research in critically ill patients with sepsis. This proposal will prove the hypothesis that quality and consistency of isolation and purification protocols of plasma and serum samples enable setting-up reliable biorepositories for future research on sEVs in sepsis. We will take advantage of two large critical care-division based repositories which has biospecimens from pediatric critically ill septic and non-septic studies. The R21 phase Aim 1 is to develop a methodology for sample collection and isolation of sEVs with high yield and purity and Aim 2 is to demonstrate suitability of banked sEVs for high throughput analyses of RNA cargo profile. Once milestones for the R21 phase are met we will proceed to the R33 phase to retrospectively characterize sEV endotypes in critically ill patients with specific organ injuries (Aim 3) and then prospectively determine whether patients can be classified based on their sEV characteristics. Results from these investigations will allow for the novel development of a biorepository of sEVs in pediatric sepsis. This biorepository will enable investigators to explore organ-specific molecular signatures for mechanistic studies of sEVs.

NIH Research Projects · FY 2025 · 2023-08

PROJECT ABSTRACT While localized primary pain conditions are prevalent in youth, a significant subset of these patients experience multiple pain conditions and meet the criteria for chronic overlapping pain conditions (COPCs). COPCs have a marked negative impact on daily functioning and quality of life in youth and carry a high risk for continued pain and disability into adulthood. The underlying factors contributing to the development and persistence of COPCs in youth are unknown. The current proposal offers an innovative and previously unexplored approach to determine whether disruptions in spatial (concurrent noxious stimuli across the body) and temporal (noxious stimuli presented over time) filtering of nociceptive processing, reflecting pain amplification (e.g., increased facilitation and/or reduced inhibition), contribute to COPCs. Several quantitative sensory testing methods are uniquely positioned to probe disruptions in nociceptive filtering across spatial (spatial summation, SS; conditioned pain modulation, CPM) and temporal (temporal summation, TS; offset analgesia, OFA) domains. Our recent pilot studies found evidence for greater disruptions in spatial (CPM) and temporal (TS) filtering in youth with COPCs. Our primary objective is to determine if spatial and temporal filtering of nociceptive information differentiates youth with COPCs from those with localized pain and healthy controls and determine whether distinct profiles of disrupted nociceptive processing are associated with the transition of localized pain to COPCs. To accomplish this, the current study will leverage expertise and a vast clinical infrastructure (Migraine, Gastroenterology, Rheumatology and Pain Management clinics) at a large pediatric medical center to enroll 140 youth with a localized pain condition (migraine, abdominal pain, local MSK), and 140 youth with COPC’s. We will also recruit 140 healthy youth to serve as a control group. Following initial phenotyping to delineate disruptions in spatial and temporal dimensions of nociceptive processing (Aim 1), participants will be assessed for changes in pain status (localized to COPCs) every three months for one year (Aim 2). In Aim 1, we hypothesize that youth with COPCs will show disrupted spatial (reflected by reduced CPM and enhanced SS) and temporal (reflected by enhanced TS and reduced OFA) processing compared to youth with localized pain and healthy controls. These findings will delineate specific disruptions of nociceptive processing in patients with COPCs. For Aim 2, we hypothesize that a subset of youth with localized pain and disrupted spatial and temporal filtering will develop COPCs. We will examine the stability of spatial and temporal filtering at clinically relevant time points (3-, 6-, and 12-months). We will also explore whether other factors, including concomitant treatments, influence the disrupted filtering and the transition to COPCs. Our research will provide the first insight into the presence and impact of disrupted nociceptive filtering related to COPCs and its naturalistic progression from localized pain. This information will be critical in identifying risk patterns that can be useful in the prevention of progression to COPCs and mitigating long-term risk.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract Sarcopenia is a devastating skeletal muscle condition that occurs in advanced age and due to various chronic conditions. Despite the widespread prevalence of sarcopenia there are no treatment options and the mechanisms underlying this process are not completely understood. A major hallmark of skeletal muscle aging is a reduction in myofiber size, which can be controlled by the hundreds of myonuclei within a single myofiber. Myonuclei are accrued during development, and new nuclei can also be added in the adult through cellular fusion of muscle stem cells (MuSCs). The presence of hundreds of nuclei and the need to add more has led to questions if the pre-existing myonuclei are at their transcriptional ceiling and thus require the myofiber to add new nuclei for adaptations. To begin to understand the requirement for myonuclei to maintain muscle size, we generated a unique mouse model that allows titration of myonuclear numbers and utilized strategies to track specific myonuclear populations. Our recent studies showed that myonuclear numbers ultimately determine size of myofibers, but that myonuclei possess a transcriptional reserve capacity to increase biosynthetic output and maintain larger cytoplasmic volumes. While the compensatory adaptations in mice with reduced nuclear numbers were advantageous during development, they were associated with evidence of accelerated aging and muscle loss, leading to the hypothesis that loss of functional gene copy numbers is a contributor to sarcopenia. Indeed, by utilizing snRNA-seq technology, we detected altered myonuclear populations during mouse aging suggesting dysregulated transcription, which could be one mechanism to explain a reduction in gene copy numbers. In addition to altered transcription, another mechanism for reductions of gene copy numbers is if myonuclei are lost from the syncytium and not replaced by MuSC fusion, and it is known that MuSCs have reduced activity in aged muscle. Based on these preliminary data, we will utilize unique models and myonuclear tracking systems, to uncover the transcriptional reserve in myonuclei of aging myofibers, elicited either through dysregulated transcriptional profiles or myonuclear loss, and elucidate the link between such myonuclear infidelity and the development of sarcopenia. Specifically, we propose to: 1) understand the molecular and cellular consequences of reductions in myonuclear number during aging 2) molecularly dissect the mechanisms of activation of myonuclear transcriptional reserve during development and aging 3) determine if myonuclei turnover during homeostasis, aging, and atrophy. Successful completion of these studies will provide unique insight into the myonuclear control of sarcopenia and provide new knowledge that will identify new therapeutic strategies to combat muscle loss.

NIH Research Projects · FY 2025 · 2023-08

Contact PI/PD: Pedapati, Ernest PROJECT SUMMARY Fragile X Syndrome (FXS) is an exemplar monogenetic neurodevelopmental disorder (NDD) where a tremendous body of multi-species translational research has elucidated the underlying molecular pathophysiology, and more recently, in-depth electrophysiology of cortical function. Thus far, phenotypic rescue in animal models has not resulted in treatment breakthroughs in humans. Central to this discrepancy is a poor understanding of the constituent neurodynamics of averaged group effects and individual variability in human brain activity as related to higher-level cognitive symptomatology and clinical phenotype. Our large collection of preliminary data demonstrates that individuals with FXS do not mount precise neural responses to the sensory auditory chirp and, instead, have “noisy” asynchronous gamma activity. Furthermore, a marked reduction in alpha power suggests altered thalamocortical function, reducing the ability to detect signal from noise and representing potential tractable targets for “bottom-up” entrainment. Our approach involves three scientific aims, which, if addressed, would ascertain underlying mechanisms that may alleviate sensory and cognitive impairments. First, we will study transient, non-continuous features (neurodynamics) of alpha and gamma oscillations in resting-state EEG and sensory auditory chirp that model patient-level heterogeneity and constitute group effects (Aim 1A). We will also identify what, if any, of these novel features are conserved in the Fmr1-/- KO using preexisting murine EEG data and represent patient subgroups (Aim 1B). Second, we will extend into cognition by studying neurodynamics and circuit modeling associated with statistical learning (SL), which shares similar neural mechanisms to the sensory auditory chirp (Aim 2). Third, we will use individualized closed-loop alpha auditory entrainment (AAE) to attempt the normalization of neural signatures of the sensory auditory chirp and SL tasks (Aim 3). Aim 1 and 2 findings will provide critical data to optimize closed-loop parameters of AAE to serve as a “bottom-up” neural probe to understand the mechanics of disorder-relevant circuit activity through perturbation of thalamocortical drive. Ascertaining the mechanisms underlying these alterations would have a high clinical impact, especially to enhance early intervention to alter the trajectory of intellectual development in which no definitive treatments are available.

NIH Research Projects · FY 2024 · 2023-08

Leveraging multi-omics to maximize the scientific value of pediatric sepsis biorepository and advance patient endotyping. PROJECT SUMMARY: Sepsis is major pediatric health problem and kills more children than cancer in the U.S each year. Primarily driven by a dysfunctional host response to an infection, a subset of patients with persistent or progressive multiple organ dysfunctions disproportionately contribute to sepsis morbidity and mortality. Yet, there are no disease modifying therapies currently available beyond early antibiotics and organ support. Biological heterogeneity among patients has significantly impeded scientific progress and advances in patient care. Although precision medicine approaches have been used to begin to sift through patient-level differences, we fundamentally lack a comprehensive understanding of disease mechanisms. Thus, there is a crucial need to maximize the use of existing pediatric sepsis biorepositories to unravel causal pathways, facilitate rapid identification of biologically relevant patient subclasses more likely to benefit from targeted therapies. To bridge this gap, we seek to utilize state-of-the-art multi-omics approaches to explore the scientific value of our vast collection of biospecimens from critically ill children with sepsis. While gene-expression profiling has been used to identify biologically relevant endotypes, it is increasingly evident that interrogation of single layer of molecular data is likely insufficient. Recent studies suggest that the epigenomic changes, including differential DNA methylation at CpG islands, closely regulate gene-expression in human sepsis. Through this phased innovation award, we seek to determine whether integrated analyses of methylomic and transcriptomic datasets at scale can provide a comprehensive understanding of mechanisms and inform patient endotyping. We further seek to determine whether clinical data linked with patient biospecimens can be used to predict endotype membership, with operational implications for predictive enrichment in future clinical trials. Milestone-driven developmental activities in the R21 phase will focus on stringent quality control of DNA and RNA samples within our biorepository to determine suitability for high throughput methods. We will then generate pilot DNA methylation profiling and RNA sequencing data for study planning and to demonstrate feasibility of integrated analyses. In the R33 phase, we will scale efforts to generate robust methylomic and transcriptomic datasets. We will leverage the bioinformatic capabilities of the investigator team to derive and validate novel multi-omic endotypes and determine their clinical significance. Finally, we will develop a classifier model to predict endotype membership using clinical data within the derivation cohort and test its generalizability in a large electronic health record-based dataset of >15,000 critically ill children with sepsis and multiple organ dysfunctions. Through the successful execution of this proposal, we seek to generate a rich dataset to drive future mechanistic research in human sepsis and develop an actionable framework for rapid and equitable identification of pediatric sepsis subclasses who may benefit from targeted sepsis therapeutics.

NIH Research Projects · FY 2025 · 2023-08

Leveraging multi-omics to maximize the scientific value of pediatric sepsis biorepository and advance patient endotyping. PROJECT SUMMARY: Sepsis is major pediatric health problem and kills more children than cancer in the U.S each year. Primarily driven by a dysfunctional host response to an infection, a subset of patients with persistent or progressive multiple organ dysfunctions disproportionately contribute to sepsis morbidity and mortality. Yet, there are no disease modifying therapies currently available beyond early antibiotics and organ support. Biological heterogeneity among patients has significantly impeded scientific progress and advances in patient care. Although precision medicine approaches have been used to begin to sift through patient-level differences, we fundamentally lack a comprehensive understanding of disease mechanisms. Thus, there is a crucial need to maximize the use of existing pediatric sepsis biorepositories to unravel causal pathways, facilitate rapid identification of biologically relevant patient subclasses more likely to benefit from targeted therapies. To bridge this gap, we seek to utilize state-of-the-art multi-omics approaches to explore the scientific value of our vast collection of biospecimens from critically ill children with sepsis. While gene-expression profiling has been used to identify biologically relevant endotypes, it is increasingly evident that interrogation of single layer of molecular data is likely insufficient. Recent studies suggest that the epigenomic changes, including differential DNA methylation at CpG islands, closely regulate gene-expression in human sepsis. Through this phased innovation award, we seek to determine whether integrated analyses of methylomic and transcriptomic datasets at scale can provide a comprehensive understanding of mechanisms and inform patient endotyping. We further seek to determine whether clinical data linked with patient biospecimens can be used to predict endotype membership, with operational implications for predictive enrichment in future clinical trials. Milestone-driven developmental activities in the R21 phase will focus on stringent quality control of DNA and RNA samples within our biorepository to determine suitability for high throughput methods. We will then generate pilot DNA methylation profiling and RNA sequencing data for study planning and to demonstrate feasibility of integrated analyses. In the R33 phase, we will scale efforts to generate robust methylomic and transcriptomic datasets. We will leverage the bioinformatic capabilities of the investigator team to derive and validate novel multi-omic endotypes and determine their clinical significance. Finally, we will develop a classifier model to predict endotype membership using clinical data within the derivation cohort and test its generalizability in a large electronic health record-based dataset of >15,000 critically ill children with sepsis and multiple organ dysfunctions. Through the successful execution of this proposal, we seek to generate a rich dataset to drive future mechanistic research in human sepsis and develop an actionable framework for rapid and equitable identification of pediatric sepsis subclasses who may benefit from targeted sepsis therapeutics.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT During the past 20 years, Congress, the National Institutes of Health (NIH), and the Food and Drug Administration (FDA) have made it clear that safe drug therapy for children is a high priority. With the rapidly changing health care and research environments, it has become essential to focus on the training of the next generation of leaders in Pediatric Clinical Pharmacology who are involved in the design and conduct of hands- on drug studies in children to augment the translation of new and effective therapies for children. The proposed Cincinnati Children’s Pediatric Clinical Pharmacology K12 program aims to provide the resources for a diverse group of junior faculty to become the next leaders in Pediatric Clinical Pharmacology. This includes training in the principles of Pediatric Clinical Pharmacology to those who may never had previous clinical pharmacology experience while also facilitating the transition from fellowship to junior faculty to independent investigator for those who have had previous fellowship training in Pediatric Clinical Pharmacology. The training program we propose in this application for K12 Scholars will leverage the outstanding research infrastructure that exists within Cincinnati Children’s Hospital Medical Center and the University of Cincinnati Academic Health Center, the strong research training activities that we have built within the Cincinnati Children’s Division of Clinical Pharmacology, and the robust collaborations that our Division has developed with other Clinical Pharmacology T32 sites and Clinical Pharmacology programs throughout the nation. Through our rigorous training program, we expect Scholars to achieve the following five core competencies: 1) Foundational Knowledge and Skill in Pharmacometrics, 2) Foundational Knowledge and Skills in Pharmacogenetics, 3) In-depth Understanding of the Impact of Developmental Pharmacology Principles on Drug Action and Toxicity, 4) Ability to Conduct Studies in a Variety of Pediatric Patient Populations, and 5) Collaborative Skills in Interdisciplinary Team Research. The four pillars of training to achieve these core competencies will be focused on 1) research training, 2) responsible conduct of research, 3) teaching/research mentorship and 4) leadership and career development through both experiential learning and didactic courses with knowledge and skills to be attained and expected outcomes in each of these areas. Intended Scholar outcomes include high impact manuscripts describing research in Pediatric Clinical Pharmacology, submission of independent K career development awards or R awards, delivery of lectures and presentations on clinical pharmacology topics in local forums and national conferences, experience in mentoring trainees locally and nationally, and leadership positions in national committees in Clinical Pharmacology societies. We intend to have two junior faculty Scholars appointed at any time with the anticipated appointments being 2 to 3 years, depending on external grant funding. This program will expand the critically small pool of Pediatric Clinical Pharmacology investigators who will advance personalized therapeutics in pediatrics to improve the health outcomes of children.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Diabetic cardiomyopathy is the leading cause of complications in type-2 diabetes. The diabetic heart features metabolic insults that promote its metabolic inflexibility and failure, particularly insulin resistance, impaired glu- cose oxidation and loss of NAD+ biogenesis. The cardiomyocyte-specific glucocorticoid receptor (GR) is im- portant for heart function, but the GR-dependent metabolic mechanisms of glucocorticoids (GCs) in cardiomyo- cytes remain unknown. GR activity is regulated by the circadian clock but the extent to which the GR regulates the cardiomyocyte clock is still unknown. The GR activates its co-factor KLF15, which regulates cardiac metab- olism and promotes oscillating transcriptional programs in heart. However, a direct role for KLF15 in regulating circadian clock genes is still unknown. The circadian clock factor BMAL1 promotes oscillating NAD+ repletion and supports cardiac clock and function in a cardiomyocyte-autonomous fashion. However, the clock response to GC signaling in heart remains unknown. Recently, we have shown two interconnected time dimensions that harness benefits from detriments of GC pharmacology: circadian time-of-intake and chronic frequency-of- intake. Circadian-specific prednisone intermittence has shown positive outcomes of safety and benefits in a pilot clinical trial, underscoring relevance and feasibility in humans. Our preliminary findings with obese diabetic mice suggest that light-phase intermittent prednisone rescues NAD+ biogenesis, glucose metabolism and ceramide lipotoxicity in heart, blunting diastolic dysfunction. This raises the unanticipated idea of repurposing GC drugs to treat diabetic cardiomyopathy, but the mechanism underpinning this action must still be identified. Based on our findings in three cardiomyocyte-restricted inducible KO models, we discovered a new GR-KLF15-BMAL1 mechanism regulating clock and metabolism in cardiomyocytes. We test here the overarching hypothe- sis that this cardiomyocyte-autonomous axis is triggered by specific GC timing and rescues cardiomy- opathy in type-2 diabetes. In Aim 1, we will identify the circadian cue gating the circadian-specific effect of exogenous glucocorticoids in heart. We will test the hypothesis that the rhythmic trough of endogenous GCs is the circadian cue mediating the circadian-specific effects of exogenous GCs in heart. In Aim 2, we will determine the extent to which the cardiomyocyte clock discriminates beneficial versus deleterious outcomes of chronic GC frequency. We will test the hypothesis that cardiomyocyte-specific BMAL1 is the discriminating factor in the dia- betic heart response to chronic GC effects. In Aim 3, we will identify the mechanisms promoting insulin sensitivity and glucose oxidation by the cardiomyocyte-specific GR-KLF15 axis. We will test the hypothesis that the cardi- omyocyte GR-KLF15 axis rescues metabolic flexibility in the diabetic heart through a concerted program of ceramide reduction and pyruvate oxidation, dependent on timing-specific GC exposure. In summary, our study resolves the role of cardiomyocyte GR biology in heart metabolism, while identifying translational cues and drug- gable mechanisms of GR-clock interplay for diabetic cardiomyopathy.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Candidate: Patricia Vega-Fernandez, MD MSc RhMSUS is a pediatric rheumatologist whose overarching career goal is to improve the outcomes of children affected by autoimmune rheumatologic diseases through a career as an independent clinician scientist focused on biology-based imaging and clinical research to support the development of personalized treatment approaches. Dr. Vega-Fernandez, an Assistant Professor of Pediatrics at Cincinnati Children’s Hospital Medical Center (CCHMC), has conducted multiple studies focused on addressing the feasibility and reliability of musculoskeletal ultrasound (MSUS) in Juvenile Idiopathic Arthritis (JIA). The proposed K23 career development plan will focus on identified gaps in Dr. Vega-Fernandez’s knowledge and training that are essential to advance her career towards an independent investigator: 1) Critical appraisal of the biological signatures in JIA; 2) Performance of US-guided synovial biopsy in children with JIA; 3) Validation of instruments and longitudinal studies; 4) Strengthened personal, academic, and professional skills to become an effective independently funded clinical-scientist. Mentors/Environment: Dr. Vega-Fernandez and her mentors, Hermine Brunner MD MSc MBA, Sherry Thornton PhD, Jonathan Dillman MD MSc, and Jareen Meinzen-Derr PhD MPH, have assembled a strong team of advisors and collaborators from inside and outside of CCHMC to guide the proposed training and research activities. The career development plan utilizes intellectual resources at CCHMC and the University of Cincinnati. Research: JIA, the most common chronic rheumatic disease in childhood, is characterized by joint inflammation (arthritis). Diagnosis of arthritis in children is based on the presence of joint swelling and limited range of motion and/or tenderness on palpation. Given the young age of children at diagnosis, the report of joint pain or limitation of motion can often times be inaccurate. In addition, clinical assessment of active arthritis in the developing skeleton is subjective and has low inter-rater reliability. MSUS can objectively inform presence and severity of joint inflammation. There is a knowledge gap on the clinical significance of MSUS findings in children. The central hypothesis of this study is that a 10-joint focused MSUS score can serve as a diagnostic imaging tool to provide an accurate assessment of both clinical and biologic activity of inflammation over time in children with JIA. Guided by strong preliminary data, this hypothesis will be tested with three specific aims: 1) Determine the convergent validity of the MSUS-10 score across the spectrum of JIA disease activity; 2) Determine the ability of the MSUS- 10 score to capture clinically relevant change of JIA; 3) Evaluate the relationship of the MSUS-10 score with biologic markers of JIA inflammation. The proposed experiments are significant because the MSUS-10 examination and score may support safer and more appropriate choices for the treatment of arthritis, which in turn will improve the outcomes of children with JIA. This project is innovative as this novel approach of using MSUS scoring may lead to a paradigm shift in the treatment and care that JIA patients receive.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Oxylipins are oxygenated bioactive lipids derived from polyunsaturated fatty acids that have diverse and integral functions in health and disease, including inflammation, cancer, and cardiovascular diseases. Oxylipins are short-lived, locally acting signaling molecules that are synthesized on demand by cyclooxygenases (COX), lipoxygenases (LOX), or cytochrome P450 monooxygenases. Advances in lipidomics have led to the detection of disease-specific changes in oxylipins. Although the identification of disease-specific changes in oxylipins has the power to be used for disease diagnosis, prognosis, or treatment, the translation of lipidomic studies into the clinic remains challenging due to a lack of biological understanding of oxylipins. To better understand the clinical relevance of disease-specific changes, we identified critical gaps in our knowledge that need to be addressed, including 1) what mechanisms regulate the coordinated synthesis of multiple oxylipins leading to cell-specific oxylipin patterns; and 2) how the signals elicited from individuals oxylipins are integrated into biological functions. To address these gaps in our knowledge, the long-term goal of our research program is to decipher the signaling mechanism responsible for the synthesis and function of individual oxylipins to understand the functional consequence of their alterations in diseases. Without further mechanistic insights into disease-specific changes in oxylipins, it is unlikely novel oxylipins will be effectively targeted for clinical purposes. Platelets are the ideal model system to study oxylipin biology because they produce nanomolar levels of approximately 15 oxylipins from COX and 12(S)-lipoxygenase (12-LOX) and offer a simplified model to study the biological consequences of oxylipin dysregulation. In this proposal, we will focus on the function of 12-LOX and its arachidonic acid (AA)-derived metabolite, 12-HETE, which have broad clinical and biological significance. However, due to the lack of consensus on the function of 12-HETE, the mechanism by which 12- LOX contributes to inflammation, cancer progression, and clotting is controversial and represents a substantial knowledge gap. This proposal will study 12-LOX and 12-HETE as a prototypical examples to address its role in disease, and develop tools to characterize the function of oxylipins by using gene-edited human megakaryocytes, which have been shown to faithfully recapitulate the donor-derived platelets. Our short-term goals are to 1) determine the intracellular mechanisms used to release and deliver substrate to 12-LOX and 2) identify the downstream signaling pathway(s) activated by 12-HETE in platelets. Our studies will provide valuable insight into the mechanistic understanding of oxylipin synthesis and function that could ultimately aid in developing new therapeutic approaches for a broad range of diseases.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting 1 in 3500 individuals worldwide due to inactivating mutations in the NF1 gene. Cognitive symptoms in NF1 include impaired executive functioning, autistic features, speech and language delays, attention deficits, hyperactivity, and impulsivity. Disease manifestations are due to reduced expression of functional neurofibromin, the protein product of NF1 that inhibits Ras-MAPK (Ras-Raf-MEK-ERK) signaling cascades by accelerating Ras-GTP hydrolysis. Using a heterozygous knockout (Nf1+/-) mouse model of NF1, we previously discovered that Nf1 haploinsufficiency causes hypersensitivity to salient visual stimuli, such as a looming disc that promotes rapid escape to an available shelter by simulating predator approach from above. These phenotypes are likely controlled by the superior colliculus (SC), which receives direct visual input from retinal ganglion cells (RGCs) and coordinates behavioral responses to looming visual threats. Recently, it has been shown that cultured Nf1+/- RGCs are more excitable in vitro. These results raise the possibility that Nf1 haploinsufficiency enhances the sensitivity of SC-projecting RGCs to light; however, no study to date has examined RGC firing in an intact Nf1+/- retina or recorded neuronal responses in the superior colliculus in Nf1+/- mice. In this proposal we will test the hypothesis that a MAPK-dependent increase in the sensitivity of Nf1+/- RGCs to light produces visual hypersensitivity phenotypes via the downstream superior colliculus. In Aim 1, we will directly measure RGC responses to visual stimuli ex vivo and in vivo in NF1 model mice. In Aim 2, we will use calcium imaging and optogenetics to define the contribution of ventral SC glutamatergic neurons to phenotypic expression. Finally, in Specific Aim 3, we will test the role of activated MAPK signaling in NF1-associated phenotypes by determining if visual hypersensitivity is recapitulated in a novel knock-in mouse model of Noonan syndrome. If successful, these experiments will identify a new role for retinotectal visual processing circuits in NF1 and may paradigmatically shift how attentional and visuospatial deficits are conceptualized in this disease.