Cincinnati Childrens Hosp Med Ctr

NIH Research Projects · FY 2025 · 2024-07

This project will investigate the neural mechanisms and clinical antecedents underlying listening difficulty (LiD) in school-age children born very preterm using a prospective cohort design. The long-term goal is to enhance auditory and language outcomes for at-risk children. The central hypothesis is that extended high-frequency (EHF) hearing loss stemming from exposures like ototoxic medications disrupts the integration of spatial and talker cues during competing speech processing, thereby being a mechanism underlying LiD in preterm children. The project will leverage an existing cohort of over 300 very preterm infants with prospectively collected perinatal and neonatal clinical data, including audiological, behavioral, and neuroimaging results. Antecedent factors of LiD and EHF hearing loss will be determined by testing associations between neonatal clinical variables (e.g., ototoxic medication exposure, abnormal MRI at term, social risk factors) and later emergence of LiD and EHF hearing loss. Identifying early clinical antecedents will allow targeted monitoring of high-risk infants and guide neuroprotective management in the NICU to mitigate exposures leading to impairments. A subset of 115 preterm children and 35 controls aged 6-8 years will undergo questionnaires, audiological testing (including EHF thresholds), and neuropsychological assessments. EHF hearing loss will be defined as >20 dB HL from 10-16 kHz. Listening difficulty will be quantified using the Evaluation of Children’s Listening and Processing Skills (ECLiPS) caregiver questionnaire. A subset of 35 preterm children and 35 controls will also undergo magnetoencephalography (MEG) during a competing speech paradigm manipulating talker and spatial cues. Neural tracking of the speech envelope will be evaluated using inter-event phase coherence. The specific aims are: 1) Evaluate neural mechanisms underlying LiD in preterm children and 2) Determine clinical antecedents of LiD and EHF hearing loss in preterm children. The hypotheses are: 1a) EHF hearing loss will be associated with greater listening difficulty, 1b) EHF hearing loss will disrupt the integration of spatial and talker cues as evidenced by reduced synergistic effects on neural speech tracking, 2a) Antecedents of LiD will include abnormal MRI at term and high social risk status, and 2b) Antecedents of EHF hearing loss will include ototoxic medication exposure and otitis media history. Linear regression models will evaluate associations between EHF hearing loss and ECLiPS scores. Mixed effects models will test for interactions between EHF hearing loss, spatial cues, and talker cues on neural tracking. Clinical antecedents of EHF hearing loss and LiD will also be determined using regression models. By clarifying the neural mechanisms and clinical antecedents of LiD in preterm children, this project will lay the groundwork for developing targeted therapeutic interventions and early identification of high-risk children.

NIH Research Projects · FY 2025 · 2024-07

Abstract/Project Summary Mice and human physiology are deeply affected by brown fat through its control of metabolic homeostasis and production of signaling molecules. As such, brown fat specific genetic modifications in mice have arisen as a truly relevant tool to understand brown fat role in controlling physiology with applications in an array of organ systems. For this, Cre-recombinase drivers under control of the UCP1 promoter have been used. In this proposal, we show that the most commonly used mouse model of brown fat targeting is the source of some previously unnoticed negative effects. Thus, our preliminary data supports an unmet critical need to generate a new mouse model to target mature brown adipocytes in an efficient and safe manner. For these reasons, the main goals of this application are to generate (Aim 1), validate (Aim 2) and make widely available (Aim 3) a new model of brown fat targeting. For this, we will use an innovative but validated CRISPR/Cas9 targeting technique to insert Cre-recombinase driven by a defined ucp1 promoter into a safe harbor location. Next, we will use Cre-mediated lineage tracing techniques to address its cellular specificity. Due to the broad interest in brown fat biology, this mouse model is anticipated to be broadly used by scientists in an array of fields of interest to multiple NIH institutes. This new mouse model of brown fat targeting is expected to have a positive impact as a step forward towards developing better preclinical mouse models. We will make our new model available to the community through a mouse repository. For these reasons, this proposal is directed to the FOA PAR-21-167, “Development of Animal Models and Related Materials for Research”.

NIH Research Projects · FY 2025 · 2024-06

Project Summary and Abstract The processes underlying human health and disease are highly complex. This necessitates the creation, organization, and dissemination of vast amounts of relevant scientific data. Organizing this information into databases and user-friendly web sites and tools is therefore an integral component of ongoing efforts to understand and ultimately improve human health. Many human diseases are now appreciated to result from genetic and environmental perturbations to gene regulatory mechanisms. As direct readers of genomic regulatory sequences, transcription factors (TFs) and RNA-binding proteins (RBPs) are primary components of gene regulatory mechanisms. Accordingly, the nucleic acid binding specificities of TFs and RBPs (henceforth, “specificities”) provide foundational knowledge for understanding the processes underlying human health and disease states. Comprehensive mapping of TF and RBP specificities is thus regarded as a major goal in the fields of human genetics, functional genomics, and bioinformatics. We aim to ultimately achieve this goal and disseminate the resulting data freely to the research community. To this end, we created the CisBP and CisBP-RNA TF and RBP specificity databases over 10 years ago. Since then, we have improved upon these resources through periodic updates incorporating new experimental data generated in our labs and others. Since their creation, these unique resources have been heavily utilized by the research community, with over 60,000 unique users and over 2,800 citations to date. Our ongoing efforts to maintain and improve CisBP and CisBP-RNA have enabled the comprehensive study of gene regulation in healthy and disease states in humans and other organisms relevant to human health. In this proposal, we seek to build upon these powerful resources by improving the database contents and underlying computational infrastructure (Aim 1), incorporating newly generated specificity data for TFs and RBPs from human viruses and other important human pathogens (Aim 2), and enhancing the user experience through improved web sites, web tools, and community outreach efforts (Aim 3). Implementation of these improvements will directly lead to increased database update frequencies, improved user experiences, a large and more knowledgeable user community, and new data for studying the role of gene regulation in human healthy and disease states.

NIH Research Projects · FY 2026 · 2024-06

Project Summary/Abstract: Daily living skills (DLS), which are the tasks (e.g., hygiene, cooking, laundry, managing money) that individuals do to take care of themselves at home, school, work, and in the community, are impaired in autistic adolescents without an intellectual disability (ID) such that their skills are 6-8 years behind same-aged peers. Age appropriate DLS have been linked to achieving better adult outcomes in employment, college, independent living, and overall quality of life. In the recent Lancet Commission on the future of care and clinical treatment in ASD there was a call to action to address DLS in adolescence as a potential intervention target to increase the likelihood of attaining positive adult outcomes. However, until our team began developing and evaluating the Surviving and Thriving in the Real World (STRW) intervention, there were no known comprehensive, evidence-based DLS interventions for autistic adolescents at this critical developmental period. In two recent pilot randomized clinical trials (RCTs), STRW demonstrated statistically significant and clinically meaningful gains in DLS (i.e., gains of 2-4 years of DLS over the course of a 14-week intervention) compared to a control condition (i.e., PEERS social skills intervention). In our two pilot RCTs, STRW was converted to telehealth due to COVID-19. There were equal DLS gains between in-person STRW and STRW-telehealth (STRW-T) and there were numerous benefits to telehealth delivery. The next step in this line of work is to assess the efficacy of STRW-T in a fully powered Phase 3 RCT compared to an attention control condition (PEERS-telehealth; PEERS-T) and examine the impact of improved DLS on early adult outcomes by following adolescents 6-months after high school graduation. We will enroll 192 autistic teens without ID in the 11th/12th grades and randomize them to receive STRW-T (n = 96) or PEERS-T (n = 96). Caregivers and adolescent participants will complete a comprehensive multi-method DLS battery (i.e., interview, survey, daily phone diaries, goal attainment scaling) at baseline, post-treatment, and 6-month follow- up. Young adult outcomes in work, college, and quality of life will be assessed 6-months post-high school graduation for all participants. The current proposal has the following aims: (1) examine the efficacy of STRW- T on DLS compared to PEERS-T; (2) evaluate whether the improvement in DLS outcomes by STRW-T are sustained at 6-month follow-up; (3) examine the effects of STRW-T intervention on outcomes in college, work, and QoL after high school graduation compared to PEERS. We will also explore the mediating role of improved DLS on young adult outcomes. If the aims of the proposed study are achieved, STRW-T would fill the gap in the current evidence base for treating DLS in autistic adolescents and would be the first study to examine whether a DLS intervention impacts work, college, and QoL outcomes after graduation from high school. Our long term goal is to narrow the gap between age and DLS, and also fill the existing treatment gap by disseminating and implementing this intervention into routine clinical practice for autistic teens.

NIH Research Projects · FY 2026 · 2024-06

Abstract. Prematurity remains the leading cause of infant mortality, responsible for well over 1 million deaths each year. Another 1.9 million babies are estimated to die in utero from stillbirth. We do not have effective cures because our understanding of how pregnancy works remains rudimentary. Important clues come from human epidemiological studies highlighting protective benefits of prior pregnancy against complications in future pregnancy, and the partner specificity of these effects. For example, the risk of preeclampsia is sharply reduced in women with prior healthy pregnancy, but rebounds with a change in paternity in subsequent pregnancy. Partner-specific resiliency against pregnancy complications is reproduced in mice using inbred strains expressing defined MHC haplotype or other model antigens for siring first and subsequent pregnancies. These parallels establish our scientific premise that investigating how fetal-expressed antigens are recognized and remembered by mothers will efficiently unveil essential new knowledge on how pregnancy works, and urgently needed strategies for improving pregnancy outcomes. Pregnancy in humans and mice each stimulate expansion of immune suppressive maternal CD4 T cells that express the FOXP3 transcriptional regulator, called regulatory T cells (Tregs). Tracking maternal CD4 cells using antigen-specific tools show selective Treg expansion among cells with fetal-specificity. In turn, resiliency against pregnancy complications is associated with postpartum persistence fetal-specific “memory” Tregs, establishing an instructive framework for investigating how mothers immunologically remember their children. Our recent studies tracking maternal CD4 cells using FOXP3 lineage fate tracking reporter mice further show a large proportion (40-70%) of fetal-specific Tregs loose FOXP3 expression after parturition. These FOXP3-negative “exTregs” are essential for enhanced resiliency against complications in future pregnancy, because their depletion overrides the protective benefits of adoptively transferred postpartum donor splenocytes. At the same time, these findings also highlight exciting new gaps in knowledge regarding the interplay between FOXP3 expression plasticity, fetal tolerance and pregnancy outcomes. These knowledge gaps, directly aligned with the goals of RFA-AI-23-027, Immune mechanisms at the maternal-fetal interface, will be addressed through the following specific aims designed to further investigate our overall hypothesis that mothers remember not only through fetal-specific FOXP3+ Treg memory, but also via exTregs with distinct phenotypic and functional features: Establish the suppressive molecules utilized by exTregs and FOXP3+ Tregs for enhanced resiliency against pregnancy complications (Aim 1), Investigate maternal CD4 cell differentiation after fractured fetal tolerance induced pregnancy complications (Aim 2), and Determine whether pregnancy primed exTregs are committed for FOXP3 re- expression or liable to proinflammatory differentiation with fetal antigen re-stimulation in the physiologically relevant non-tolerogenic context of solid organ transplantation (Aim 3).

NIH Research Projects · FY 2025 · 2024-06

Project Summary/Abstract The sharing of data in biomedical research, including data generated from clinical trials, is paramount for ensuring reproducibility, improving study design, increasing analytic power via concatenation of disparate data sets, and identifying and testing new hypotheses. With more data being made publicly available, there is a critical need to facilitate their re-use. The reusability of publicly available clinical trial datasets is more complex when data are shared in an unstructured format, as compared to in a structured database, due to the need for arduous manual annotation of individual files to permit secondary analyses. One platform to share individual participant level clinical trial data is the Immunology Database and Analysis Portal (ImmPort), a NIH/NIAID-funded public warehouse providing open access to clinical and mechanistic data from immunological and clinical studies. The overall aim of this proposal is to enhance the reusability of clinical trial data shared through ImmPort and to encourage their re-use. Specifically, the goal is to develop an automated tool to simplify the transformation of laboratory test data from clinical trials into the format of the ImmPort submission template. Upload of the data in this template format is necessary for integration into the structured database. Otherwise, the data will be made available to the public as custom formatted study files, complicating their re-use. The complexity in current upload protocols is what prevents many data submitters from fully annotating their clinical data for integration into ImmPort’s structured database. The tool will be an extension of our prototype ImmPort curation approach that facilitates the formatting of assessment data for submission to ImmPort. The tool will rely on a manually curated data dictionary to give context to the data in the study files. With that data dictionary and minimal additional manual input, the approach automates the complex file formatting steps required to transform the data into the laboratory test submission template. The tool will be developed to guide users through the annotation process with a graphical user interface, making full annotation of lab test clinical trial data more accessible to researchers without informatics expertise. Additionally, to increase confidence and proficiency in re-using clinical study data publicly available through ImmPort, a comprehensive tutorial based on a secondary analysis of two food allergy related clinical trials shared through ImmPort will be developed. The tutorial, created in R, will cover various ways of data retrieval, processing, and an in-depth analysis with visualizations. Overall, this proposal aims to enhance the reusability of clinical trial data available through ImmPort, thereby increasing the return on investment for biomedical research and benefiting scientific knowledge and clinical care.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMRAY Eosinophilic esophagitis (EoE) is a chronic allergic disease of the esophagus, partly mediated by type 2 immunity and epithelial dysfunction. Patients with EoE suffer from dysphagia, pain, and the lowest quality of life among children with chronic, pediatric conditions. The epithelium is the first line of defense against potential threats, providing a physical barrier between the host and the external environment. The site where the barrier breach occurs may be a location where normally harmless antigens encounter immune cells and potentially lead to production of danger signals, priming a break in immune tolerance and initiation of allergic responses. Understanding the physiologic factors that preserve/restore esophageal homeostasis will facilitate our long-term goal of developing improved therapies with long-lasting effects and a cure for EoE. The sharp edge between intact barrier versus barrier breach is orchestrated by proteases and protease inhibitors. We demonstrated that losing an epithelial protease inhibitor or altering proteolytic activity provide a paramount signal in the development of EoE. Delivery of the serine protease inhibitor A1AT reversed EoE phenotypes (e.g., barrier function, type 2 mediator production) in vitro and in an EoE mouse model. We have now revealed another variable in the protease-inhibitor equation; the low-density lipoprotein receptor– related protein 1 (LRP1). LRP1 is a scavenger receptor that mediates endocytosis and elimination of ligands, including serine protease inhibitors (serpins) in complex with proteases. Interaction of a serpin with a protease, exposes encrypt motifs in the serpin’s sequence that interact with LRP1 and serve as LRP1 agonists. Following the binding of a serpin-protease complex to LRP1, LRP1 initiates signaling pathways that terminate inflammatory responses and promote epithelial homeostasis. We demonstrated that LRP1 agonists potently reverse EoE disease in vitro and in vivo. In addition, we showed that an intracellular truncated LRP1 fragment appears exclusively in EoE biopsies. The function of the truncated LRP1 fragment in EoE biopsies is unknown. We hypothesize that excessive LRP1 processing during EoE promotes transcriptional changes that propagate EoE. In this study, we will explore the mechanism of action by which serpins and synthetic LRP1 agonists promote inflammatory resolution in vitro and in vivo. We developed an experimental platform to investigate the function of full-length serpins, serpin-derived peptides with LRP1 binding motifs, and small molecule inhibitors that inhibit proteolytic activity but lack LRP1 binding motifs. Our unique experimental framework enables us to test serpin functions that are independent of protease inhibition. We will elucidate the downstream functional consequences of LRP1 processing in esophageal epithelial cells and investigate the enzymes responsible for LRP1 processing. Successful completion of this study will provide fundamental insights into LRP1-serpin-protease crosstalk and facilitate developing drugs to achieve inflammatory resolution and epithelial homeostasis in individuals with EoE.

NIH Research Projects · FY 2026 · 2024-05

The major goal of this application is to define viral interactions with host pathways that are essential for the assembly of infectious HIV-1 particles. We previously identified Rab11-FIP1C (FIP1C) as a key adaptor protein required for HIV-1 envelope protein (Env) trafficking and particle incorporation, a finding that established the model stating that host recycling pathways are required for particle incorporation. Recent work from our laboratory has extended this model, defining the role of KIF16B in Env trafficking and identifying discrete motifs within the Env cytoplasmic tail that are essential for recycling and particle incorporation. Preliminary data presented here now identifies the tubular recycling endosome (TRE) as a major new component of the recycling machinery involved in particle incorporation of Env. TRE components including MICAL-L1 and EHD1 were shown to be essential for Env trafficking and incorporation into particles. Remarkably, disruption of TRE trafficking severely depleted Env from particles, but only in those cells that have been shown to be nonpermissive for incorporation of cytoplasmic tail (CT)-depleted Env. Thus, this pathway holds the key to understanding the contribution of the Env CT to the recycling and particle incorporation of Env. Experiments in Aim 1 will define essential components of the TRE that contribute to Env trafficking, examining EHBP1, Rab10, Syndapin2, and MICAL-L1. In Aim 2, the critical role of EHD1-mediated scission from the TRE in Env trafficking to the PM site of assembly will be defined using cellular/biochemical and imaging approaches. A unique FAP-tagged Env construct will be employed to visualize and quantify Env trafficking from the TRE to the PM in live cells. Aim 3 will determine the mechanisms responsible for cell type- dependence of incorporation of Env, using a novel cell-cell fusion approach combined with identification of host factors that interact with the Env CT. Together, the work outlined here will expand upon a productive line of investigation that seeks to comprehensively define the mechanism of incorporation of Env into HIV-1 particles.

NIH Research Projects · FY 2026 · 2024-05

The long-term goal of our research program is to define mechanisms regulating treatment responses and disease complications in Crohn's Disease (CD). Persistent rates of ileal strictures requiring surgery despite widespread use of anti-TNF therapy increase the urgency of this work. Interactions between intestinal epithelial cells (IEC), macrophages, and fibroblasts mediate fibrostenosis in CD. The IEC NADPH oxidase DUOX2 produces reactive oxygen species (ROS) to control mucosal microbes, while also regulating cellular metabolism and repair via redox signaling. The phagocyte NADPH oxidase NOX2 complex produces ROS to kill pathogens, while constraining inflammasome activation and excessive cytokine production. We found that loss-of-function genetic variants in DUOX2, and loss-of-function genetic variants in CYBA, NCF1, NCF2, and NCF4 comprising the NOX2 complex, were associated with higher rates of strictures. This suggested a novel redox signaling mechanism constraining epithelial and macrophage dependent myofibroblast activation and extra-cellular matrix (ECM) production. Our perturbagen bioinformatic analysis of the treatment naïve CD ileal transciptome prioritized small molecules likely to inhibit inflammatory macrophage and fibroblast function and prevent strictures.The overall objective of this project will be to utilize a novel induced pluripotent stem cell (iPSC) derived macrophage:human intestinal organoid co-culture system to test mechanisms by which DUOX2 and NOX2 gene variants and candidate small molecules control intestinal epithelial cell and macrophage mitochondrial function and inflammatory signals regulating tissue fibrosis. We hypothesize that DUOX2 and NOX2 redox signaling regulates mitochondrial complex I assembly and function, thereby constraining mitochondrial ROS dependent cytokines driving tissue fibrosis. Small molecules and monoclonal antibodies will ameliorate pro-fibrotic effects of DUOX2 and NOX2 gene variants. We will test this hypothesis in the following Aims: Aim 1. Define mechanisms of tissue fibrosis in DUOX2var HIO. We will test DUOX2var regulation of epithelial redox signaling, protein kinase A activity, and mitochondrial complex I respiration, and associated mitochondrial ROS dependent TGFB secretion and HIO collagen production. The ability of candidate small molecules and TGFB blockade to prevent pro-fibrotic effects of bacterial products upon DUOX2var HIO will be determined. Aim 2. Define pro-fibrotic mechanisms of NOX2var macrophages. We will test NOX2var regulation of macrophage redox signaling, protein kinase A activity, and mitochondrial complex I respiration, and associated mitochondrial ROS dependent TL1A secretion and HIO collagen production. The ability of small molecules and TL1A blockade to prevent pro-fibrotic effects of NOX2var macrophages upon isogenic HIOs will be determined. These studies will advance precision medicine, by utilizing a platform we have developed to screen candidate small molecules in patient-derived organoids with clinically important genotypes.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY According to the developmental comorbidity framework, listening difficulties in children (despite clinically normal hearing) may occur due to multiple sources of deficits. Hence, in this study, key auditory processing, cognitive, and linguistic factors are conceptually integrated to model how listening ability in noise relates to a crucial language outcome, i.e., sentence comprehension in school-age children. Deficits in speech perception in noise (SPiN) are salient characteristics of children diagnosed with auditory processing disorder and those with a history of recurrent childhood middle ear infection. Furthermore, the majority of children with parental and teacher concern of listening difficulties have coexisting impairments in language, attention, and learning. Existing theories suggest that lexical knowledge and working memory (WM) capacity are crucial for children's listening in adverse situations. However, a direct test of these models within the same study is absent in children. In this study, we will model the relationship between listening in noise ability and sentence comprehension ability in school-age children, controlling for individual differences in attention and with lexical knowledge as a mediator and WM capacity as a moderator. We chose sentence comprehension as the dependent variable because it is a strong predictor of reading comprehension and academic success. Children (7- to 11 years old; N=130) representing a continuum of listening abilities will participate in this prospective cross-sectional study that employs standardized measures with strong psychometric properties. Based on the Lexical Restructuring model and the previous literature, we hypothesize that lexical knowledge as a mediator will explain the causal relationship between SPiN and sentence comprehension (i.e., why better SPiN ability may be positively associated with better sentence comprehension skills). Based on the Ease of Language Understanding model, we predict that WM will moderate the relationship between SPiN and sentence comprehension because this relationship is expected to be stronger for children with high WM capacity than those with low WM capacity. In addition, we predict that WM will moderate the relationship between lexical knowledge and sentence comprehension. This study will fill a significant gap in the developmental literature on the nature of the relationship between listening in noise ability and language comprehension. This is important because many children with listening concerns are reported to have failure in at least one academic area that may not be fully explained by other diagnoses. Study findings will have direct clinical implications to assess listening in noise abilities in a significant number of children. Results will guide long-term intervention objectives on targeting mechanisms such as SPiN and lexical abilities in children taking into consideration WM capacity/cognitive load, to improve their speech perception in realistic situations. This project aligns with NIDCD priority areas across Hearing and Language to understand the influence of auditory perception in realistic listening environments.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ ABSTRACT: Hyperactive, postzygotic mutations in the GNAQ gene (encoding for Gaq proteins) have been identified in vascular anomalies. Patients affected by this class of vascular anomalies experience extensive disfigurement and can develop a life-threatening coagulopathy termed Kasabach-Merritt Phenomenon (KMP). Despite these genetic findings, animal models for GNAQ-related vascular anomalies and KMP do not exist. This has severely hindered the preclinical development of effective targeted therapies for these patients. In our preliminary experiments we generated a transgenic murine model of mutant hyperactive GNAQ-driven vascular anomaly which recapitulates common histopathological findings in patients and a coagulopathy reminiscent of KMP, a potentially lethal disease complication. Furthermore, our studies showed that in patient- derived lesional tissue with GNAQ Q209L mutation, the MAPK/ERK signaling pathway is hyperactive as well as increased EC proliferation. We have exciting data that the MEK/ERK inhibitor Trametinib extended the life span of GNAQ mutant mice suggesting this may be a promising therapy for patients. To date there are no studies examining the role of mutant active GNAQ (p.Q209L) in vascular development and disease. This proposal will culminate with the development of innovative in vivo, in vitro, and Vessel-on-Chip models that we will use for the identification of cellular and molecular mechanisms implicated in vascular disease. Furthermore, with our powerful murine model we are uniquely positioned to develop a comprehensive research program to investigate the GNAQ role in vascular lesion formation and expansion with the goal of identifying highly needed therapeutic targets for vascular anomalies and related complications such as coagulopathies. Additionally, our studies will provide cellular and mechanistic insights to advance our understanding of pathological and physiological vessel formation and size maintenance.

NIH Research Projects · FY 2026 · 2024-03

Sialylated antibody defense against intracellular infections Abstract. Division of labor between humoral and cellular adaptive immune components for effective host defense is a foundational immunological tenet. Antibodies are thought to offer limited protection against intracellular pathogens since they cannot efficiently cross the plasma membrane to enter most cells, which may explain the prevalence of intracellular infections in fetuses and newborns given their dependence on vertically transferred maternal IgG for early life immunity. However, we recently demonstrated that pregnancy enables antibody-mediated protection against the prototypical intracellular pathogen Listeria monocytogenes (Lm). The key molecular change during pregnancy is deacetylation of sialic acid (Sia) located in the terminal position of N-glycans on preconceptually primed Lm-specific IgG. Maternal deacetylated IgG then modulates neonatal B cells via the Sia receptor CD22, which can only bind to deacetylated Sia. This establishes a framework whereby differential acetylation versus deacetylation of sialylated IgG serves as a previously unrecognized molecular “switch” that greatly impacts immunity through CD22, which in turn controls responder B cell activation and immunomodulatory functions. While our findings reshape paradigms of antibody-mediated host defense, they also unveil several important new gaps in knowledge, including mechanisms responsible for addition of a six-atom, acetyl group to IgG Sia to purposefully “switch off” CD22-mediated protection. This includes the enzymatic machinery required to add acetylated Sia to IgG N-glycans, as well as factors controlling germinal center responses leading to production of acetylated IgG. Additionally, the subset of neonatal CD22+ B lymphocytes that respond to “switched on” deacetylated IgG and how these B cells modulate other immune cells are unclear. Our overall hypothesis is that overturning maternal B cell Sia acetylation allows deacetylated IgG to bind to CD22 on neonatal Breg, suppressing their IL10 production, and enabling protection against intracellular infection. Shared susceptibility to Lm infection between human and rodent neonates will be exploited to further investigate how deacetylated IgG is formed and mediates protection, through the following specific aims. Aim 1, define cell-specific control of antibody sialic acid acetylation; Aim 2, interrogate how germinal center suppression drives antibody sialic acid acetylation; Aim 3, investigate neonatal B cell IL10 inhibition of antibody-mediated protection. Aims 1 and 2, therefore, will interrogate antibody secreting B cells as producers of “switched off” acetylated IgG, while Aim 3 will investigate “switched on” deacetylated IgG modulation of IL10-poducing Breg. Completion of these aims will expand our understanding of how to control antibody sialylation and acetylation to boost host defense against intracellular infections, informing design of novel vaccine strategies and antibody-based therapeutics given the ubiquity of antibody glycosylation.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Lung diseases impacting the gas exchange alveoli, including COVID-19, are becoming the leading cause of death. A multi-lineage, transcriptional, and epigenetic understanding of alveologenesis and re- alveologenesis upon injury is a timely response to the disease burden and leverages latest single-cell technology. My lab’s track record in studying the lung epithelial, endothelial, and mesenchymal lineages lays the foundation for pursuing a poorly understood process of cellular maturation (Theme 1), a recently identified capillary cell type (Theme 2), and a novel signaling regulation of distinct mesenchymal cell populations (Theme 3). The anticipated knowledge will tackle fundamental questions of cell fate, plasticity, and signaling; shed light on bronchopulmonary dysplasia, pulmonary hypertension, acute lung injury, as well as non-coding variants from genome-wide association studies; and opens the door to single-cell functional genomics applicable to any organ.

NIH Research Projects · FY 2026 · 2024-03

Abstract Submucous cleft palate (SMCP), defined by abnormal attachment of palatal muscles with intact oral and nasal mucosa, is a common craniofacial birth defect that disrupts the essential function of the soft palate for swallowing, causing feeding difficulties, middle ear dysfunction leading to hearing loss, and lifetime speech problems. Humans have five pairs of palatal muscles that all attach to the palatine aponeurosis, a fan-like dense fibrous connective tissue comprising the core of the soft palate that connects to the hard palate bone. Whereas the palatal muscles are derived from embryonic pharyngeal mesoderm, all connective tissues in the soft palate, including the palatine aponeurosis, tendons, and the muscle connective tissues that are embedded within and ensheathing the muscles, develop from cranial neural crest cell (CNCC) derived mesenchyme. Studies of limb muscle development have identified the embryonic muscle connective tissue as an important source of signals for patterning both the nascent muscles and tendons. However, how muscle connective tissue and tendon cells are specified from a common mesenchymal progenitor population and how they regulate muscle and tendon development remain unclear. We found that the Foxf2 and Foxd1 transcription factors exhibit complementary and partly overlapping patterns of expression along the medial- lateral axis of the embryonic soft palate mesenchyme at the onset of palatal myogenesis, with Foxf2 expression enriched in medial mesenchymal progenitors and with Foxd1 strongly expressed in the mesenchyme cells in the muscle-forming lateral region of the embryonic soft palate in mice. We demonstrate that Foxf2-deficient mice exhibit specific soft palate defects, including mispositioned palatal muscles and failure of palatine aponeurosis formation. Expression of Foxd1 and muscle-associating mesenchyme markers are ectopically activated in the medial mesenchymal progenitor cells in Foxf2 mutant embryonic soft palate. Furthermore, Foxd1 heterozygosity substantially rescues palatine aponeurosis formation and integration with the tensor veli palatini (TVP) muscles in Foxf2 mutant mice. On the other hand, homozygous disruption of Foxd1 causes aberrant splitting and abnormal attachment of TVP muscles to the pterygoid plate. Our central hypothesis is that Foxf2 and Foxd1 act antagonistically to regulate the specification and differentiation of distinct connective tissue lineages from the CNCC-derived soft palate mesenchyme to guide the morphogenesis and integration of palatal muscles. We propose comprehensive experimental studies to gain unprecedented understanding of the cellular and molecular mechanisms regulating soft palate connective tissue development and palatal muscle morphogenesis. Data from these studies provide a rich resource for uncovering mechanisms coordinating development and integration of musculoskeletal tissues and will lead to improvement in diagnosis and treatment of SMCP and other musculoskeletal disorders.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY The proposed Consortium of Food Allergy Research Clinical Research Center: Cincinnati Children's Hospital Medical Center (CoFAR-CRC:CCHMC) aims to bring the experience, expertise, and scientific productivity and the diverse patient populations of CCHMC to CoFAR. The patient population seen in the clinics and participating in the research of the Division of Allergy and Immunology at CCHMC encompass varied presentations and classifications of food allergy, including IgE-mediated food allergy (IgE-FA), eosinophilic esophagitis (EoE), and food protein–induced enterocolitis (FPIES). The CoFAR CRC:CCHMC approach to research is multipronged and self-sustaining by attracting patients with excellent care and creating and maintaining databases and registries supported by biospecimen collection and biobanking. Those are utilized in conducting mechanistic research that forms the basis of discoveries and clinical trials and advances knowledge that improves patient care and attracts more patients. The description of the CoFAR CRC:CCHMC will provide a) data on the capacity and readiness of the CRC; b) solid structure and wide range of services available; c) recruitment abilities from the CRC databases and the community, specifically of difficult-to-recruit populations, including minorities, infants, and adults; d) track record of initiation and successful and regulatory- compliant execution of collaborative, multi-center clinical trials; e) support of professional development of faculty and staff; and f) development and inclusion of junior faculty. We propose a network-wide, multicenter clinical trial focused on maintaining acquired tolerance to food allergens and a center-specific research project focused on defining the mechanism by which environmental factors contribute to the pathogenesis of food allergies. For our potential network-wide clinical trial, we hypothesize that the gradual lengthening of the dosing intervals in oral immunotherapy (OIT) in a similar pattern as in allergen immunotherapy protocols or cluster dosing (4 days consecutive dosing + 3-day dosing holiday weekly) will maintain OIT-acquired tolerance as well as daily dosing while providing a more manageable and acceptable dosing schedule for patients and their families. We will conduct a prospective, open, 3-arm, non-inferiority trial of peanut OIT in children 3–8 years of age; the primary outcome is passing an end-of-study (24 months) oral challenge to a serving size of peanut protein. Mechanistic studies of changes in B cell receptors and basophil activation will explore novel biomarkers for assessing long-term tolerance. For our center-specific project, we hypothesize that EoE and IgE-FA are driven by environmental exposures that abrogate the aryl hydrocarbon (AHR) pathway; we propose to investigate the mechanism of AHR function, therapeutic potential of esophageal delivery of AHR ligand- producing bacteria, and metabolic composition of AHR ligands in food allergies, intersecting the AHR ligand composition with cellular and molecular profiles of the epithelium and atopic manifestations.

NIH Research Projects · FY 2026 · 2024-02

Project Summary/Abstract Anaphylaxis can be life-threatening but exacts an outsized toll on patients and the healthcare system. This system’s burden is due in part to one size fits all national guidelines that necessitate emergency department (ED) care for all patients and ≥ 4-6-hour observational periods that are agnostic to reaction severity and patient characteristics. This generic approach is ill-suited to most reactions, thereby contributing to ED overutilization, long ED wait times, ED overcrowding, avoidable hospitalizations, and inflated healthcare costs. The proposed K23 study seeks to deliver personalized anaphylaxis care and will further develop my skillset as an independent translational investigator. I hypothesize that machine learning can be applied to my actively enrolling KL2 cohort to develop prediction models capable of customizing ED observation periods in real-time based on patient- specific risk. The rationale for this hypothesis comes from preliminary KL2 data (n = 91) in which 51.6% of patients had complete symptom resolution within 2 hours from the first epinephrine dose. The next logical step is to expand my prospective cohort and employ machine learning to develop a model capable of predicting time-to-symptom resolution in real-time to reduce observation periods, prevent unnecessary hospitalizations, and identify patients who may not require ED care and can instead be safely managed at home. Additionally, there is an unmet need for clinically actionable biomarkers to enhance patient risk stratification and enable customizable therapeutic approaches; however, there is an incomplete understanding of immunological mechanisms contributing to human anaphylaxis pathogenesis. Most research has focused on the role of IgE- mediated mast cell and basophil activation, yet emerging data support my second hypothesis that neutrophils are key drivers of anaphylaxis that govern divergent progression into either refractory or biphasic courses. The rationale for this hypothesis comes from preliminary KL2 data in which 100% of patients with severe reactions exhibited elevated markers of neutrophil activation and levels were dynamic over time. The specific aims of this application are 1) To derive a dynamic clinical prediction model to customize anaphylaxis observation periods. Simultaneously, I will acquire training in biostatistical and biomedical informatics, machine learning, dynamic prediction modeling, and mediation analysis; and 2) Demonstrate that serum biomarkers of neutrophil activation predict reaction severity and clinical courses. This aim will help me acquire essential training in translational approaches to neutrophil biology and will provide a mechanistic gateway for future research on the immunology of anaphylaxis courses. Findings from this study will result in a novel prediction model to optimize anaphylaxis observation periods and prevent unnecessary hospitalizations. These outcomes will aid my transition to independent investigator status by conducting validation studies of the predictive model in a multicenter cohort. Study findings will also open avenues of investigation on the integration of neutrophil measures into clinical care and the prognostic machine learning tool.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Non-alcoholic fatty liver disease (NAFLD) affects billions of people. NAFLD was first defined in the USA 35 years ago, yet progress toward a true cure has been incremental and limited to lifestyle modification. The long-term goal of our research program is to take a fresh view on human liver metabolism through the lens of precision hepatology to define personalized mechanisms as a fundamental step toward developing evidence-based, rational solutions for mechanism-directed diagnostic and therapeutic investigation. Here, we propose to explore liver effect by integrative pre-clinical research, combining patients’ and engineered human tissues using new technologies such as human organoids, gene editing and comprehensive genomics/transcriptomics. We propose to use our pioneering steatohepatitis organoids in combination with an innovative population-scale pluripotent stem cell (PSC) approach to identifying NASH genotype-phenotype associations under disrupted metabolic states. Our Preliminary Data using this human organoid GWAS system suggests that GCKR rs1260326 confer risk for developing steatosis and inflammation under oleic acid induced insulin resistant conditions, supported by cohort data from thousands of NASH patients. Based on these exciting Preliminary Data, the overall objective in this application is to elucidate variant-dependent effects of common coding single nucleotide polymorphism on liver specific steatosis and inflammation under disrupted metabolic states. Our central hypothesis is that GCKR rs1260326 activates glucokinase to trigger hepatic lipogenesis under an insulin resistant state, thereby exacerbating pathogenic inflammation through mitochondrial oxidative stress. The rationale for the proposed research is that a mechanistic determination of GCKR genetic predisposition in T2D complicated steatohepatitis will provide a strong foundation for identifying susceptible populations and molecular pathway directed therapy development. We will critically test and validate our hypothesis in the following Aims: In Aim1, we will determine the mechanistic role of GCKR rs1260326 in steatohepatitis liver organoids. In Aim2, we will interrogate GCKR rs1260326 mediated mitochondrial dysregulation in organoid and patients. In Aim3, we will redefine GCKR rs1260326 and steatohepatitis associations in diabetic NASH patients. At the completion, we will have determined how common risk variants and metabolic activities in the liver interact to cause aggressive forms of NASH, informing risk stratification strategies, as well as the potentially diverging efficacy of available therapies. Our studies will lead to a better mechanistic understanding of mitochondrial dysregulation associated with GCKR rs1260326 at the level of liver, forming a basis for mitochondrial metabolism-directed therapeutic investigations. Further, by demonstrating the utility of our population-based organoid model system, this investigative strategy may open the door to many other studies of pleiotropic heritable factors, potentially transforming the future of precision hepatology.

NIH Research Projects · FY 2026 · 2024-02

Project Summary/Abstract Congenital valve defects (CVDs) constitute some of the most common human congenital heart defects (CHDs). CVDs that lead to valve disease later in life, which can result in heart failure, stroke, blood clots, and even sudden cardiac arrest. The most common therapy for valve diseases is invasive valve replacement surgery, which is one of the most common procedures in the cardiac surgery market at nearly 182,000 per year in the USA, making up nearly a quarter of the total procedural volume. Therefore, elucidating the early developmental and patterning mechanisms driving normal cardiac valve development will give us novel insight into the etiology of CVDs. Retinoic acid (RA) is the most active metabolic product of Vitamin A (retinol) within vertebrate embryos and is reiteratively required for normal vertebrate heart development. Perturbations of RA signaling have been associated with multiple types of CHDs, including CVDs, as well as valve diseases at later stages of development. However, embryos devoid of early RA exhibit enlarged hearts due to the increased specification and differentiation of cardiomyocytes. Despite the known requirements in heart development and previous studies implying RA signaling is required for cardiac valve formation and maintenance, we still do not have clear understanding of the requirements of early RA signaling in vertebrate valve development. Our preliminary work using zebrafish shows that early inhibition of RA signaling results in an enlarged atrioventricular canal (AVC), characterized by an expansion of myocardial AVC markers, which is consistent with the enlarged hearts in RA deficient embryos. Surprisingly, despite the enlargement of the myocardial AVC region in RA deficient embryos and expectation that this would result in typically larger valves, we find that the overlying atrioventricular valves (AVVs) do not form, suggesting that a factor signaling from the myocardium to the overlying endocardium necessary to promote AVV induction is lost. Our preliminary data show that canonical Wnt signaling is lost within AVC cardiomyocytes following early inhibition of RA, suggesting that we have identified a new signaling node necessary to promote normal AVV development. In Aim 1, we will test the hypothesis that early RA signaling directly establishes cardiac progenitors sufficient to turn on Wnt signaling later in the AVC myocardium required for AVV development. In Aim 2, we will test the hypothesis that Wnt signaling functions downstream of early RA signaling in the induction of AVVs. Ultimately, the proposed studies could be used to identify and develop new therapeutic approaches for treatment to prevent or ameliorate CVDs and their resulting valve diseases.

NIH Research Projects · FY 2025 · 2024-02

Project Summary/Abstract Critical maturation steps of mammalian heart development take place during the first few weeks after birth. During this time, cardiomyocytes undergo critical changes in cell structure, metabolism and gene expression, all which are essential for the proper function of the heart. CM are in contact with different cell types including fibroblasts, which have been identified as key central regulators of ventricular maturation and remodeling during both development and injury. However, the pathways utilized by fibroblasts and cardiomyocytes cells to communicate during postnatal heart development remain unknown. Recently RNA sequencing identified Growth differentiation factor 10 (GDF10), also known as Bmp3b, as an upregulated ligand in postnatal cardiac fibroblasts compared to mature quiescent fibroblasts which suggest, GDF10 plays a role in the crosstalk between cardiomyocyte and fibroblasts during neonatal development. Interestingly, in skeletal muscle, GDF10 is essential for maintaining myofiber mass maintenance and adult Gdf10 null mice develop left ventricle hypertrophy. Further, single-cell sequencing of fat cells found GDF10 to directly modulate PPAR-gamma nuclear abundance and transcriptional activity, which is known to be important for cardiac energy shift in cardiomyocyte, suggesting that GDF10 might be important for mitochondrial maturation during heart development. However, the exact role of GDF10 in the proper maturation of the heart remains to be defined. Our preliminary studies indicate that in vitro treatment with GDF10 induces size increase and a switch towards mature myosin isoform expression in neonatal cardiomyocyte which suggests that GDF10 action during cardiac development might be important for proper maturation. Further, gene expression profiling of neonatal cardiomyocyte and fibroblasts revealed fibroblast expressed GDF10 to strongly correlate with Activin receptor expression on cardiomyocytes, which have been implicated in GDF family signaling in cardiomyocytes. Finally, our preliminary studies show that gdf10 null hearts at postnatal day 7 have myocardial growth defects similar to hypoplastic left heart syndrome. The central hypothesis of this proposal is that cardiac fibroblast secreted GDF10, signaling through activin receptors, is essential for cardiomyocyte hypertrophy and mitochondrial maturation during development. Aim 1 will determine if GDF10, through Smad 1/5/8 signaling induces cardiomyocytes hypertrophy and mitochondrial maturation. In parallel, bulk RNA-sequencing of GDF10, activin receptor inhibitor ACE031 and GDF10+ACE031 treated cardiomyocyte cells will give us an in-depth understanding of direct GDF10 targets that induce CM maturation. In Aim 2, we will determine whether GDF10 signaling is required for CM hypertrophy and metabolic maturation in vivo by extensively examining gdf10 null hearts and measuring changes in cardiomyocyte cell size and sarcomeric assembly. Additionally, we will evaluate fetal and adult metabolic and contractile protein gene expression of Gdf10 null mice compared to controls. Our long-term goal is to determine GDF10 as a key crosstalk ligand between fibroblasts and cardiomyocytes in the mammalian postnatal cardiac development and if the loss of this communication impairs maturation of cardiomyocytes by decreasing cardiomyocyte hypertrophy.

NIH Research Projects · FY 2025 · 2024-01

Asthma is an inflammatory lung disease that affects >300 million people worldwide. Obesity/metabolic dysfunction, which affect 40% of adults in the US, is associated with a more severe asthma endotype. Unlike mild asthma which is driven by a Th2 immune response, the “obese asthma” endotype is associated with frequent asthma exacerbations, a shift away from Th2-dominated inflammation towards a Th17-dominated profile, and a marked female bias. A major gap in our understanding of mechanisms driving the “obese asthma” endotype is due to deficiencies in common mouse models of obesity. Standard mouse models of high-fat diet (HFD) driven obesity are unable to induce extensive weight gain and metabolic dysfunction in female mice - a major shortcoming given the female bias of the “obese asthma“ endotype. Our group has uncovered that HFD feeding of C57BL/6 mice housed at a temperature in which they are at metabolic homeostasis (thermoneutral temperature (TN); 30-33°C) promotes severe obesity and metabolic dysfunction in female mice. Preliminary data using this model demonstrate that allergen-challenged obese female mice exhibit: (1) worse asthma outcomes compared to lean controls; (2) reduced markers of type 2 immunity (eosinophilia, Th2 cytokine production); and (3) a selective increase in the IL-17A production by innate lymphocytes, particularly gd T cells and Mucosal Associated Invariant T cells (MAIT cells). Critically, none of these changes are observed in obese asthmatic male mice. Although both gd T cells and MAIT cells have been ascribed protective roles in the context of asthma and metabolic homeostasis, in obesity their proinflammatory vigor increases, and they become important mediators of glucose intolerance, insulin resistance, and tissue inflammation invoking extreme functional plasticity. Importantly, no studies have examined the role of these cells in obese asthma to date. Based on our novel preliminary data, we hypothesize that, innate lymphoid populations (gd T cells, MAIT cells) acquire pathogenic functions in obese female mice and contribute to the development of the “obese asthma” endotype. This hypothesis will be tested in two aims. Aim 1: To determine if gd T cells or MAIT are necessary and sufficient to trigger more severe asthma in obese female mice. Using gd T cell or MAIT cell blocking agents, and adoptive transfer of purified gd T cell/MAIT populations from lean or obese asthmatic females to lean asthmatic male and female mice, followed by assessment of asthma and metabolic endpoints, we will determine the necessity and sufficiency of these cells in obese asthma. Aim 2: To compare the proinflammatory and metabolic landscapes of innate lymphocytes from lean and obese asthmatic female mice. Transcriptional landscape of purified lung innate lymphocyte populations (gd T cells, MAIT cells, ILCs, iNKT T cells) from lean and obese asthmatic female mice will be analyzed. Changes in inflammatory and metabolic pathway gene expression will be correlated to asthma and obesity-related sequelae severity. Notably, completion of these studies will advance understanding of the mechanisms underlying “obese asthma” endotype.

NIH Research Projects · FY 2025 · 2024-01

This application represents a new Clinical Research Center consortium that will strengthen the Pediatric Heart Network. Over the last seven years, Cincinnati Children’s (CCHMC) and the University of Kentucky (UK) have built The Joint Pediatric and Adult Congenital Heart Program, a collaborative “one program, two site” model of care within our distinctive region of the United States. The clinical program spans areas of Ohio, Kentucky, Indiana, and West Virginia which includes a large portion of Appalachia. Our population experiences significant health outcome disparities with the rural and remote members of our communities often most affected. Such disparities are seen daily in our clinics and inpatient units, motivating this application. During the time our clinical program has matured, CCHMC has been a highly productive member of the Pediatric Heart Network and the National Heart Lung and Blood Institute’s Bench-to-Bassinet program, bringing experience that will facilitate the integration of this consortium into the PHN. The Joint Heart Program’s large, combined clinical volume of patients will strengthen the previously outstanding enrollment of the CCHMC site. Our current role as vascular and MRI core lab, and single IRB for the Network, bring added value. Important to the next iteration of the PHN, the Joint Heart Program has prioritized data science and data integration, partnering with broader institutional resources to enable transdisciplinary research that generates new understanding of health and disease while informing novel therapies that improve clinical outcomes for all affected patients and families. Furthermore, the Joint Heart Program will bring diversity to the research through the following: 1) program structure with institutional partners of different size and perspective, 2) a clinical population that is includes a rural and remote population often not involved in such research, 3) institutional commitment, existing infrastructure, and local expertise to develop partnerships with underrepresented and special populations in our region. The latter is enabled by extensive community engagement resources and research outreach capacity supported by two CTSAs with an established collaborative regional relationship. In Aim 1, we will accelerate scientific discovery and improve clinical outcomes by leveraging our proven expertise in multicenter research, data science, and data integration including pediatric learning health networks, clinical registries, and large translational datasets. In Aim 2, we will reduce inequity in outcomes for CHD and childhood acquired heart diseases by fostering diversity of both research teams and participants. In Aim 3, we will cultivate a new generation of pediatric and adult congenital researchers through a strong transdisciplinary training platform that emphasizes diversity and inclusion within mentors and mentees. We are eager to collaborate with the clinical centers, data coordinating center, NHLBI staff, industry partners, and broader CHD and acquired pediatric heart disease communities to address the most important problems facing affected children and their families.

NIH Research Projects · FY 2026 · 2024-01

ABSTRACT Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease that affects 15-30% of children worldwide and is characterized by dry, pruritic, and eczematous skin lesions that diminish quality of life. In addition, AD predisposes to other allergic comorbidities including asthma and food allergy that impose considerable morbidity and a significant public health burden. AD is associated with a dysfunctional skin barrier with reduced skin structural protein filaggrin (FLG) expression, Th2 immune dysregulation and microbial dysbiosis including increased Staphylococcus aureus (SA) prevalence. However, current standards of care aimed at reducing SA colonization on AD skin have exhibited minimal benefits and various unintended side effects. Therefore, there is a critical need to elucidate microbial colonization patterns and drivers of SA persistence in early life to guide the development of novel targeted therapies aimed at regulating the microbiome. We are uniquely equipped to address this need by utilizing our established MPAACH (The Mechanisms of Progression of Atopic Dermatitis to Asthma in Children) cohort, the first US-based early-life cohort of children with AD, which includes contact plates and tape strips sampling for microbial data and extensive clinical data at each annual visit. Our novel preliminary studies on the non-lesional skin of MPAACH participants demonstrate: (a) vast microbial diversity on the AD skin surface; (b) an association of persistent SA colonization with increased AD severity, allergen sensitization and low FLG expression; (c) an association of specific SA genes, including lipoprotein-like lipoprotein (lpl) cluster genes, with low FLG expression; and (d) decreased adhesion to keratinocytes in SA lacking the lpl gene cluster, especially in the context of low FLG expression and Th2 cytokines. Together, our findings and existing literature inform our central hypothesis that severe AD will be associated with decreased global microbial diversity, evenness, and richness over time and that persistently SA colonized AD children harbor SA with strain- specific genes that induce increased keratinocyte adhesion, inflammation, and barrier dysfunction. Using additional samples from non-lesional skin over multiple annual visits, we propose to test this hypothesis by (i) identifying longitudinal microbial and SA colonization patterns on non-invasive skin tapes (Aim 1), (ii) identifying SA genes involved in binding to AD skin (Aim 2) and (iii) identifying the mechanisms of SA adhesion and invasion on WT and FLG deficient primary human keratinocytes (Aim 3). Our studies will enhance our understanding of microbial patterns and identify mechanisms of SA adhesion, invasion and persistence over time in early life. More broadly, these studies will give crucial insight into novel therapeutic targets to mitigate skin dysbiosis and attenuate disease severity, which may have biological and clinical significance that extends far beyond AD.

NIH Research Projects · FY 2026 · 2023-12

Project Summary Obesity has solid inflammatory underpinnings, which are risk factors for various chronic inflammatory and metabolic diseases such as type 2 diabetes (T2D). However, how obesity provokes aberrant inflammation remains to be defined. Recent studies indicate that small extracellular vesicles (sEVs, a.k.a., exosomes) carrying RNA, including microRNA (miRNA), play a critical role in inducing inflammation in obesity. In this study, we aim to demonstrate that RNA silencing machinery plays a critical role in modulating RNA cargo profiles in sEV-secreting cells and also in responding to exposure of RNA cargos of sEVs in sEV-receiving cells. Our overarching goal is to reveal how RNA silencing machinery-mediated, sEV-induced events are critical for controlling obesity-associated inflammation, impacting the development of chronic inflammatory and metabolic diseases. Small EVs are released from many cell types into the extracellular space and are distributed in body fluids. These sEVs, taken up by neighboring and distant cells, subsequently modulate the functions of recipient cells. Hence sEVs have emerged as important transducers of intercellular communication, with their cargos, including miRNAs, playing critical roles in modulating sEV-recipient cellular activities. Our Preliminary data show that circulating EVs from obese adolescents are enriched with specific RNA cargo and exhibit higher pro-inflammatory traits, than those from lean subjects. Further, our newly established mouse model, which enables us to monitor specific cell type-derived sEVs in vivo, indicates that in obesity, sEVs become pro- inflammatory and when engulfed by macrophages, these pro-inflammatory sEVs induce inflammatory responses in macrophages. These intriguing Preliminary data suggest novel mechanisms for EV-induced inflammation in obesity. Studies in this proposal will: (1) assess the role of RNA silencing machinery in sEV-secreting cells in generating pro-inflammatory sEVs, and (2) evaluate the role of RNA silencing machinery in sEV-receiving cells in mediating sEV-induced inflammation. By utilizing our novel mouse models coupled with access to human samples and models, our systematic approaches will reveal novel mechanisms of how the pathogenicity of sEVs is critical in the development of inflammation in obesity-associated pathophysiology.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY Calcium is an essential regulator of muscle function and its dysregulation contributes significantly to the common pathogenic signaling cascades that drive multiple striated muscle diseases including heart failure and muscular dystrophy. The sarcoplasmic reticulum (SR) is the major calcium storage site in muscle and SR calcium loading is achieved by the SR calcium-ATPase (SERCA). Cardiomyocytes from human heart failure patients show robust calcium defects including decreased systolic calcium transients, increased diastolic calcium, and SR calcium leak, which contributes to impaired contraction/relaxation and can induce arrhythmias. Muscular dystrophy is a family of genetic disorders caused by mutations in genes that serve a structural role in stabilizing the membrane of skeletal muscle myofibers and cardiomyocytes. These mutations result in membrane instability, rupture, and unregulated calcium influx leading to mitochondrial calcium overload and myocyte necrosis. Numerous studies have shown that in heart failure and muscular dystrophy, calcium homeostasis can be restored by enhancing SERCA activity or expression, thereby reducing cytosolic calcium and increasing SR calcium stores, resulting in significant protection from disease. We recently discovered a novel SR membrane microprotein, DWORF, that binds to SERCA and potently activates its calcium transport activity. In mice, DWORF overexpression in the heart leads to robust SERCA activation, increased SR calcium uptake and SR calcium stores, faster cytosolic calcium clearance, and increased cardiomyocyte contractility. Additionally, DWORF overexpression by cardiac- specific transgene or adeno-associated virus (AAV9)-mediated gene delivery has proven to be cardioprotective in experimental and genetic mouse models of heart failure. Notably, a significant limitation to previous AAV9- DWORF gene therapy studies in mice has been the reliance on gene delivery in neonates to achieve meaningful cardiomyocyte infectivity, as adult cardiac and skeletal muscle are not efficiently targeted by AAV9. A recent breakthrough in the field was achieved with the development of the muscle-tropic MyoAAV capsid, which is significantly more potent than any current AAV in transducing cardiac and skeletal muscle. Here we will leverage the optimized MyoAAV system to comprehensively assess the therapeutic potential of DWORF gene therapy in mouse models of striated muscle disease with diverse etiologies where calcium dysregulation plays a primary role in disease pathogenesis. Aim 1 will use two distinct mouse models of heart failure, myocardial infarction and pressure overload, which mimic different clinically relevant scenarios. Aim 2 will employ a robust model of muscular dystrophy caused by combined loss of dystrophin and utrophin, and analysis will focus on both cardiac and skeletal muscle. Aim 3 will utilize a newly developed humanized mouse model of monogenic phospholamban disease, which presents with calcium defects that lead to ventricular arrhythmias. In all 3 Aims, we expect that MyoAAV-DWORF gene therapy will protect from cardiac and skeletal muscle disease pathogenesis and these findings will inform on future translational studies in large animals and human gene therapy trials.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY/ABSTRACT Thrombotic microangiopathy (TMA) occurs in 20% of children after hematopoietic stem cell transplant (HCT) and can cause multi-organ dysfunction and death. Over the last decade, our research team has advanced understanding of the diagnosis and management of TMA. Standardized diagnostic criteria for TMA, developed by our group, were recently adopted by international consensus (Transplantation and Cellular Therapy, 2022). Our published data demonstrate that dysregulation of complement and interferon are associated with endothelial injury and a high-risk TMA phenotype. Complement blockade has been associated with improved outcomes in children with TMA (70% versus 16% historical survival), but is not effective in all patients. Furthermore, we still do not fully understand the specific initiators or triggers for TMA. Our preliminary proteomic data demonstrate increased phosphatidylinositol 4-kinase (PI4K) expression – which regulates viral replication – in HCT recipients. In our published prospective natural history study of BK polyomavirus (BKPyV), we observed that ~20% of children had BKPyV in their blood within the first few months after HCT and BKPyV viremia was associated with a seven-fold increased risk of TMA, making BKPyV a good model of a viral trigger in TMA. Finally, we have sequenced over 150 clinical BKPyV samples and have identified high levels of genomic diversity after HCT. Our established collaborations across Cincinnati Children’s Hospital, the Children’s Hospital of Philadelphia, and the University of Cincinnati make us uniquely qualified to perform the proposed studies. The objective of the current proposal is to examine initiators and mechanisms of TMA in an established cohort of children and young adults (N=300) undergoing HCT. We hypothesize that exogenous and endogenous mechanisms both initiate TMA, mediated by complement and interferon. Our hypothesis focuses on complement and interferon because this hypothesis is supported by our published and preliminary data, and because these initiators can be targeted in future interventional studies. Specifically, available therapeutic options now include inhibitors of the complement and interferon systems and virus-specific T cells to eradicate BKPyV. To test our hypothesis, we propose two Specific Aims. Specific Aim 1 will systematically evaluate complement and interferon as triggers for TMA in children undergoing allogeneic HCT. We will test for complement and interferon activation pre-HCT and then weekly after HCT. Aim 2 will evaluate host susceptibility and viral exposure as novel triggers for TMA in children undergoing allogeneic HCT. We will evaluate susceptibility to TMA in children with BKPyV viremia by testing samples for BKPyV diversity and PI4K after HCT. Finally, the knowledge gained from Aim 1 and Aim 2 will be applied to a new performance cohort (N=100), to be enrolled in the final years of the award period. The performance cohort will test, during clinical care, how the identified triggers can predict TMA and its severity after HCT. This approach will inform the design of future interventional trials to prevent, treat, or decrease the severity of TMA after HCT and improve patient survival for this high-risk population.