Saint Louis University

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Candidate: During my training, I have been highly motivated to understand how cytotoxic lymphocytes can be used as an adoptive therapy and how immune cells are regulated during immunotherapy. I have served as first, co-, and corresponding author on numerous high-impact original research articles, representing advances in basic, translational, and clinical immunology. I have successfully competed for career development awards that have allowed me to continue my research interests. My long-term research goals are to understand the mechanisms important for NK cell effector functions, how NK cells contribute to and are impacted by immunomodulation and how host immune responses can promote or limit their NK cells as an effective cellular therapy in human and murine systems. Research Career Development Plan: As I begin my independent research program, I will utilize educational and research resources aimed specifically at junior faculty at Washington University to strengthen my career development. While I have the scientific training and expertise to perform the proposed studies, I will utilize the opportunities at WUSM to prepare for my tenure-track position. Specifically, aspects of management, mentorship, and the learning the administrative responsibilities of running a research laboratory (animal protocols, institutional approvals, budget management). Research Project: The long-term goal of this project is to improve our understanding of mechanisms operative in the successes and failures of innate immune therapy. Memory-like NK cells were first identified in mice over a decade ago and studies examining their persistence, anti-tumor responses, and metabolic adaptations to the tumor microenvironment have laid the groundwork for both the human trials currently open to enrollment and the studies proposed herein. One current barrier in the field is translating these novel immune approaches which have had so much success in treating hematologic malignancies into successes treating solid tumors. We hypothesize that ML NK cell differentiation combined with chimeric antigen receptor (CAR)-targeting will improve NK cell trafficking, effector responses, and fitness in both allogeneic and autologous immunotherapeutic paradigms. We will evaluate this concept against cancer and infection. These studies may reveal novel strategies or therapeutic interventions for NK-cell based therapies that will likely be broadly relevant to other cellular therapies. Completion of these studies will lay the scientific groundwork for both future R01 applications and for a successful career studying the interactions of adoptive cell therapy and the host immune system.

NIH Research Projects · FY 2026 · 2023-12

Project Summary/Abstract: Alzheimer's disease (AD) is a devastating neurodegenerative disease with virtually no therapeutic options to reverse its pathology. Glial cells, including microglia, play critical roles in brain homeostasis and disease progression. GPR56 (also called ADGRG1 ), an adhesion G protein-coupled receptor (aGPCR), is one of the critical genes that define "true" microglia: Gpr56 is only expressed in yolk sac-derived microglia but not in fetal liver- and bone marrow-derived microglia-like cells, even after long-term adaptation in the CNS in vivo. Importantly, a recent study from Mathys et al. showed that GPR56 is one of the five genes upregulated in microglia in individuals dying with early-stage AD compared to those with no pathology or late-stage AD. Importantly, their data was generated from participants in a community-based cohort study, the Religious Order Study (ROS)/Rush Memory and Aging Project (MAP), collectively known as ROSMAP. Of those autopsied, the mean age was 89 years. This observation raises the possibility that individuals with upregulated microglial GPR56 survived to advanced age with mild AD pathology. To investigate the unexplored function of microglial GPR56 in AD progression, we generated a new AD mouse model, 5xFAD;Gpr56""";Cx3cr1-Cre'1-(AD-cKO) and 5xFAD;Gpr56+1';Cx3cr1-Cre+1 - (AD-control). Our preliminary showed: (1) a significant reduction in the number of microglia associated with amyloid plaques, (2) a drastic increase in plaque burden, (3) a reduction in NeuNpositive neurons, and (4) more severe dendritic dystrophy in AD-cKO mouse brains compared to AD-controls. Taken together, I hypothesize that microg/ial GPR56 limits AD pathological progression in both AD mouse models and human patients. To test this hypothesis, I will address the following aims: Aim 1 is to investigate the role of microglial GPR56 in restricting AD progression in the mouse model; Aim 2 is to determine cellular and molecular mechanism(s) underlying microglial GPR56 function in AD pathogenesis; and Aim 3 is to characterize GPR56 expression and GPR56-dependent cellular responses in control, mild cognitive impairment (MCI), and AD human brain tissues. In summary, the proposed research will reveal crucial information about the role of microglial GPR56 in AD pathology. The success of this study will extend our knowledge of both glial cells and aGPCRs, providing a novel therapeutic target for AD treatment

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Sepsis is triggered by bacterial, viral, or fungal infection, and it is characterized by multi-organ failure following an impaired host response. Sepsis ranks highly in both national in-hospital mortality and cost burden in comparison to all diseases. Sepsis treatment over the years has been limited to antibiotics, fluids, and organ support. New treatments are needed, which could potentially target sepsis cellular pathophysiology including excessive oxidative stress, inflammatory overactivation at the blood-endothelium interface, declines in mitochondrial health, and disordered lipid homeostasis. Plasmalogens are a unique class of phospholipids containing a characteristic vinyl ether bond at the sn-1 position, which links the glycerol backbone to the aliphatic chain. The vinyl ether bond is a target of reactive oxygen species (ROS), and thus plasmalogens are antioxidants. My recent studies have shown plasma plasmalogen levels are reduced in human sepsis, which likely reflects sepsis endothelial oxidative stress derived from redox enzymes and electron leakage from the mitochondrial electron transport chain (ETC). Mitochondrial damage by ROS impacts cellular respiration and lipid metabolism which is detrimental to overall cell health. This suggests a protective role for plasmalogens which reside in cell and organellar membranes, including the mitochondria. Lysoplasmalogen (lysoPls), a plasmalogen class lacking an acyl chain at the sn-2 position, is a useful plasmalogen precursor that displays more rapid cell uptake than plasmalogen and still contains the ROS-scavenging vinyl ether bond. Pilot data show that supplementation of lysoPls to human lung microvascular endothelial cells (HLMVECs) reduces cellular oxidative stress and maintains plasmalogen pools in the presence of pathogenic bacteria, and lysoPls supplementation protects HLMVEC barrier integrity in the presence lipopolysaccharide. Taken together, this suggests lysoPls have an ROS-scavenging role and provide critical endothelial protection during septic oxidative stress. Therefore, we hypothesize plasmalogen loss reflects injurious endothelial oxidative stress during sepsis, and plasmalogen replacement may limit oxidative stress, improving outcomes via mitochondrial and endothelial effects. Studies in Aim 1 will include: 1) examining human sepsis plasma plasmalogen levels as outcome predictors in collaboration with Dr. Nuala Meyer (University of Pennsylvania) and 2) testing plasmalogen replacement therapy in the mouse cecal ligation and puncture model of sepsis for protection against mortality and organ failure in collaboration with Dr. Richard Hotchkiss (Washington University). Physician-scientists Drs. Meyer and Hotchkiss will also serve as co-mentors for this training program. Studies in Aim 2 will test mechanisms that plasmalogen augmentation reduces inflammation and oxidative stress in the endothelium, improving endothelial function. Overall, these studies open new research avenues to distinguish sepsis targets and therapeutics and to ultimately improve sepsis patient outcomes, especially given the rise of antibiotic resistance in recent decades.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract The bioactive lipid sphingosine-1-phosphate (S1P) plays a key role in regulating the growth, survival and migration of mammalian cells. S1P is produced intracellularly and then released extracellularly to engage in its (patho)physiological roles. The Spinster (Spns) lipid transporters of the major facilitator superfamily (MFS) are critical for transporting S1P across cellular membranes. Of the three Spns proteins in humans, Spns2 functions as the main S1P transporter, which makes it a potential drug target for modulating S1P export and signaling. An endothelial cell-specific defect in Spns2 results in impaired egress of lymphocytes and prevents tumor metastasis in mice, strongly suggesting that Spns2 could be an effective target for reducing metastases by increasing the efficacy of immunotherapy. Thus, detailed characterization of the Spns2 mechanism is of high significance for the development of novel therapeutic strategies for diseases associated with S1P signaling and to target Spns2 as a potential immunosuppressant. The overall goal of this proposal is to define the functional mechanism of the Spns family of sphingolipid transporters. The mechanism of Spns2-mediated S1P transport across cellular membrane remains poorly understood, mainly due to the lack of structural information (Aims 1 and 2). In addition, the precise mechanism of Spns2 regulation is still unclear (Aim 3). We recently defined the proton- dependent conformational dynamics of a bacterial Spns transporter. Our approach capitalizes on a powerful pulsed EPR technique known as Double Electron Electron Resonance (DEER) spectroscopy, an effective nanometer-scale ruler, in the context of high-resolution structures. It is informed by functional studies and contextualized through collaborative molecular modeling. Using this integrated approach, we conduct a thorough mechanistic comparison between human Spns2 and its homologs. The objectives of this proposal are to define the cation- and substrate-coupled conformational cycle of human Spns2 and its bacterial homologs in lipid bilayers. To determine the conformational states involved in the alternating access mechanism, we will apply DEER spectroscopy under conditions expected to stabilize transport intermediates and combine the results with restraint-assisted molecular dynamics to map ligand-coupled conformational changes. Using a similar integrated approach to define the transport mechanism of other Spns family members and their prokaryotic homologs, we will identify the key commonalities and differences in their mechanisms, highlighting the mechanistic flexibility enabling their diverse function with transformative therapeutic potential.

NIH Research Projects · FY 2023 · 2023-09

There will be no changes in the Project Abstract during (or due to) the grant transfer Project Abstract Structural and functional alterations in brain endothelium are observed in ≥90% of Alzheimer Disease (AD) brains in the absence of cerebrovascular disease or amyloid angiopathy. Despite its prevalence, the role of endothelial dysfunction in AD pathogenesis has not been adequately investigated due to the absence of reliable endothelial biomarkers. We have identified a novel panel of endothelial biomarkers whose brain ribonucleic acid (RNA) and cerebrospinal fluid (CSF) protein levels are significantly altered in AD compared to controls. Exciting preliminary data suggest that these proteins offer promise as novel diagnostic and prognostic biomarkers of brain endothelial dysfunction and strongly correlate with cognitive and radiological outcomes in AD. Importantly, our preliminary AD models suggest that endothelial dysfunction is an early pathological substrate which may precede amyloid and tau. We here propose to conduct the first in-depth comprehensive translational study of endothelial dysfunction in AD. In this study, we will utilize proteomics and transcriptomics to measure CSF protein and brain RNA levels of these endothelial markers in 3 large well-characterized longitudinal cohorts of late-onset sporadic AD, healthy controls, and non-AD dementias (combined n>3,400) who have been followed for 20-25 years and for whom we have detailed longitudinal cognitive, CSF, magnetic resonance imaging (MRI including ASL, FLAIR, and DTI), amyloid-positron emission tomography (amyloid-PET), and tau-PET imaging data. In Aim 1, we will examine the diagnostic and prognostic utility of CSF endothelial markers in AD and their ability to predict cognitive decline and brain atrophy over time, independently of amyloid and tau. We will also characterize baseline and longitudinal associations of endothelial dysfunction with CSF and/or imaging markers of amyloid, tau, inflammation, and neuronal/synaptic injury over 20-25 years of follow-up, including 10-15 years prior to symptom onset. In Aim 2, we will leverage event-based modeling (EBM; Aim 2A) in our cross-sectional cohorts and multifactorial data driven analyses (MFDDA; Aim 2B) in our longitudinal cohorts to propose new data-driven predictive models for AD which will elucidate the temporal ordering of fluid and imaging AD markers and the spatiotemporal progression of AD imaging markers (cerebral blood flow, amyloid and tau PET, and brain atrophy) across 37 brain regions and over 10-15 years prior to symptom onset. Functional pathway analyses of proteomic and RNA-seq data from our cohort and others will identify novel mechanisms by which endothelial dysfunction contributes to neurodegeneration, and influences synaptic plasticity, axonal repair, and abnormal protein aggregation in AD (Aim 2C). Through in-depth multi-omics analyses of endothelial dysfunction and the integration of multimodal data from large well-characterized longitudinal multiracial AD cohorts, we aim to propose a new disease model which will be the first to integrate endothelial dysfunction into the AD paradigm and will capture the molecular and pathological heterogeneity of AD beyond amyloid and tau. Findings from this study will provide novel insights into disease mechanisms and new molecular targets for AD drug discovery.

NIH Research Projects · FY 2025 · 2023-08

Exposures to phosgene (COCl2) and phosphorous trichloride (PCl3) are public health threats. COCl2 and PCl3 exposures are the result of industrial accidents or chemical warfare/terrorism acts. Following exposures, acute lung injury presenting as apnea and pulmonary edema are major concerns, which contribute to mortality and morbidity. However, mechanisms responsible for the action of these pulmonary toxic agents have not been elucidated. Both COCl2 and PCl3 rapidly produce HCl in the lung. Plasmalogen phospholipids including plasmenylethanolamine (pPE) in the lung are acid-labile leading to the production of lipidic aldehydes and lysophosphatidylethanolamine containing polyunsaturated fatty acids (PUFA-LPE). In addition to COCl2 and PCl3 reacting with the vinyl ether bond of plasmalogens, other functional groups of lipids may also be targeted. These include primary amines and conjugated dienes suggesting that an array of lipophilic compounds is produced during exposures. Functional groups of proteins and metabolites may also be modified by COCl2 and PCl3 exposures leading to metabolic alterations. Extensive discovery omics analyses following exposures to COCl2 and PCl3 will provide critical insights into mechanisms responsible for their toxicity. We hypothesize that both COCl2 and PCl3 exposures result in lipid and metabolic changes leading to endothelial and epithelial cell dysfunction. Both targeted and untargeted approaches will be performed to identify novel lipidic and metabolic changes following COCl2 and PCl3 exposures. In support of this hypothesis, pilot data show: 1) PUFA-LPE levels are elevated in response to PCl3 exposure to primary human lung microvascular endothelial cells (HLMVEC); 2) PUFA-LPE elicits HLMVEC barrier dysfunction; 3) COCl2 exposure to lung lipids results in the production of a family of lipophilic aldehydes and 4) PCl3 elicits profound changes in HLMVEC metabolites and metabolic pathways following exposure. There are two specific aims for the proposed studies. Specific Aim 1 will identify novel lipid and metabolic products following COCl2 and PCl3 exposures resulting in endothelial and epithelial cell dysfunction. Specific Aim 2 will identify mechanisms by which novel lipid and metabolic species produced following COCl2 and PCl3 exposures elicit endothelial and epithelial cell dysfunction. We will investigate COCl2 and PCl3 exposures to both primary human lung microvascular endothelial cells and primary human small airway epithelial cells. This, together with testing these two distinct toxicants at LD50 levels, meet criteria for this RFA. The proposed studies will examine a potential common mechanism for COCl2 and PCl3 toxicity mediated by PUFA-LPE. Additionally, unique lipid and metabolic changes will be examined following COCl2 and PCl3 exposures using untargeted analytical approaches. Collectively, these studies will provide in- sights into the mechanisms responsible for toxicity and will provide therapeutic targets for the development of countermeasures to COCl2 and PCl3.

NIH Research Projects · FY 2024 · 2023-08

Project Summary A substantial portion of patients with liver disease, ranging from 5% to 30%, have unknown causes beyond the established etiologies. Unknown etiology is observed across a wide array of clinical phenotypes in liver disease, such as acute liver failure (ALF), hepatitis, cirrhosis, and liver cancer. These are collectively referred to as cryptogenic liver disease (CLD). It has long been hypothesized there exist additional human viruses that cause CLD. In our recent serum virome study, we identified a 387-nt DNA fragment (GenBank MW468091), named Seq260, from 1 of 9 CLD patients. In a series of experiments of gene-walking, enzymatic digestion, and rolling circle amplification and analyses, we have demonstrated that Seq260 is a linear single-stranded DNA. We screened Seq260 in 409 subjects, including healthy blood donors (n=200), hepatitis C virus infection (n=100), Acute liver failure (ALF) patients with indeterminate etiology (n=50), and liver transplantation (LT) patients with (n=45) and without known etiology (n=14). Seq260 was detected in 5 CLD patients (1 ALF and 4 LT) and 1 LT patient with nonalcoholic steatohepatitis (NASH)-associated cirrhosis. One patient had Seq260 quantifiable in liver, showing a titer in the liver 7.74 times higher than that in serum (2.4x106 copies/g vs. 3.1x105 copies/mL). Machine learning analysis reached a high score (likelihood) of Seq260 being a eukaryotic viral sequence. Aggregately, these data lead to our hypothesis that Seq260 represents an unrecognized human virus with liver tropism. To determine if Seq260 represents a novel hepatitis virus, we bring about a research plan in the current proposal that consists of three major experiments. First, we will screen Seq260 in CLD patients as well as the controls. We have been granted access to patient specimens from two NIH- sponsored clinical trials, ALF study group (ALFSG), and the adult-to-adult living donor liver transplantation cohort study (A2ALL). Unknown etiology accounted for 5.5% and 29.5% respectively in the ALFSG and A2ALL. Seq260 copy numbers will be quantitated in both serum and liver in Seq260-positive patients with liver tissue available. Second, we will determine the full genome of the putative virus containing Seq260. Finally, we will evaluate antibody responses in virus-positive patients and the controls. A peptide-based serological test will be developed for the putative virus. Peptides will be individually assessed for their specificity and sensitivity in two virus-positive patients with large volumes of serum available. Selected peptides will then be combined to ELISA tests for the measurement of antibody (IgG and IgM) responses in virus-positive and virus-negative patients. Taken together, the proposed study will characterize a novel human virus and understand its etiological link to liver disease from a clinical aspect. It will expand our knowledge of the human virome as well as the etiology of liver disease without a known cause.

NIH Research Projects · FY 2024 · 2023-06

We discovered that a rabbit polyclonal antibody raised against the second extracellular loop of human G-protein- coupled receptor 160 (hGPR160/ECL2) has two remarkable actions: (1) direct anti-cancer effects on human colon cancer and triple negative breast cancer (TNBC) cell lines that express the receptor without altering the viability of a normal colon cancer cells line, and (2) great potentiation of the cytotoxic effects of the small molecule chemotherapeutic, oxaliplatin, at doses that by themselves have no significant activity. This potentiation indicates that combination with the hGPR160/ECL2 antibody might yield a considerable improvement in the clinical effect with cytotoxin doses that are lower and produce fewer side-effects than those in current use. To translate these observations into a practical therapy, we propose to develop a human monoclonal hGPR160/ECL2 antibody for further characterization of its anticancer effects and as a step towards developing a therapeutically useful human monoclonal hGPR160/ECL2 antibody. The current proposal is in response to PAR-22-216 with aims to develop and test a new biologic agent that both treats cancer and mitigates cancer treatment-related toxicities. We are unaware of any commercially available small molecule ligands for GPR160. Our recent publication1 and preliminary data presented here indicate that an hGPR160/ECL2 monoclonal antibody is a viable chemotherapeutic. However, our preliminary studies used a rabbit polyclonal antibody, which has an irreducible element of uncertainty concerning the locus of binding. Accordingly, a human monoclonal hGPR160/ECL2 antibody will address this issue and permit unambiguous in vitro experiments on anticancer effects in human cancer cell lines and in vivo experiments on human cell line-derived xenografts (CDX) in immunodeficient mice. Success with the work proposed here in three Aims would lead to further work to develop a therapeutic monoclonal antibody. In Aim 1, we will develop human monoclonal antibodies to hGPR160/ECL2 by immunizing transgenic mice expressing human heavy and light genes with the hGPR160’s ECL2 amino acid sequence (or subsequence thereof) conjugated to KLH and developing hybridomas via PEG-fusion. We will screen clonal antibodies by flow cytometry and down-select using a blocking-of-binding assay to a panel of ~10 hGPR160/ECL2 mAbs clones for further in vitro and in vivo studies. We will then test the anti-cancer cell activity of our antibodies with in vitro studies. In Aim 2, the top 2-3 candidates (best binding affinity against the ligand and LC50 in the in vitro cell viability assay) will be advanced for (1) in vivo orthotopic cell line-derived xenograft (CDX) mouse models of colon cancer and TNBC. In Aim 3, the top candidate will be tested in in vitro oxaliplatin and paclitaxel potentiation studies. An hGPR160/ECL2 mAb may prove to be a significant improvement to current anti-cancer treatments that are inadequate for a large percentage of patients. However, small molecule cytotoxins will continue to have an important role and combination therapy with an hGPR160/ECL2 antibody may allow their cytotoxic effects to be maximized while using doses with far fewer side effects.

NIH Research Projects · FY 2026 · 2023-05

Project summary Post-traumatic osteoarthritis (PTOA) is a painful, debilitating and expensive joint disease that conservatively impacts 5.6 million people at any time in the United States. Currently available clinical treatments are mostly palliative and fail to suppress cartilage degeneration or synovial inflammation or to promote tissue regeneration. Consequently, osteoarthritis progresses to an end-stage disease where total joint replacement is the only answer. Our aim is to provide an injectable solution for the treatment of PTOA, through the fabrication of super- lubricious microspheres loaded with platelet-rich plasma (PRP), which is blood plasma that has been spun down to concentrate the platelet component. These platelets contain more than 300 bioactive molecules that play critical roles in controlling and resolving inflammation, while also stimulating cells within the joint to proliferate and regenerate tissues such as articular cartilage. Unfortunately, direct injection of PRP into the knee joint has shown inconsistent efficacy in the clinic, mainly because the harsh environment within the osteoarthritic knee rapidly breaks down and destroys the bioactive molecules rendering them useless for therapeutic effect. Therefore, we propose to fabricate lubricious injectable polyethylene glycol (PEG) microspheres for injection into the intra-articular knee space that cannot be cleared by the body with the synovial fluid turnover. We will evaluate the incorporation and release as well as bioactivity and immunotoxicity of PRP from the hydrogel system, through both quantification of specific proteins released over time and in vitro cellular response studies, respectively. Finally, PRP-loaded microspheres will be used in a mouse model of knee injury and osteoarthritis to determine the ability for our system to i) modify the intracapsular inflammatory state, ii) stop cartilage degeneration, iii) promote new cartilage formation and, iii) restore joint homeostasis.

NIH Research Projects · FY 2026 · 2023-05

Summary DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. The precise mechanisms of how RPA imparts functional specificity is poorly resolved. Towards addressing this gap in knowledge, our long-term goals are to answer the following questions: a) RPA physically interacts with over three dozen DNA processing enzymes. How are these interactions determined, regulated, and prioritized? b) RPA binds to ssDNA with high affinity (KD <10-10 M). How do DNA metabolic enzymes that bind to ssDNA with hundred-fold lower affinities remove RPA? c) RPA plays a role in positioning the recruited enzymes (with appropriate polarity) onto the DNA. What are the structural, kinetic, and thermodynamic properties that regulate this process? d) How are the DNA and protein interaction activities of RPA tuned by post translational modifications such as phosphorylation? RPA achieves functional dexterity through a multi-domained architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. This dynamic plasticity has hindered structural, biochemical, and biophysical investigations of full-length RPA. Over the past eight years, our group has developed non-canonical amino acid based site-specific fluorescence labeling tools to investigate the dynamics of the individual domains of RPA. While difficult to accomplish, our breakthrough enabled us to reestablish how the individual domains of RPA bound, dissociated, and remodeled during various DNA metabolic processes. The findings were in stark contrast to commonly assumed models for RPA function and has opened numerous avenues to finally investigate and establish how RPA functions in specific DNA metabolic processes. For example, we showed that the commonly assumed high-affinity DNA binding domains of RPA were in fact the most dynamic and not bound to ssDNA in the context of the full-length protein. Utilizing our powerful biochemical, structural, and biophysical toolkit we here seek to resolve how RPA functions in the context of nucleosomes, R-loops, telomere, and in other DNA repair pathways.

NIH Research Projects · FY 2026 · 2023-03

ABSTRACT The knowledge gaps this application addresses are how precancerous lesions (i.e. metaplasia) arise and subsequently fuel adenocarcinoma of the stomach. Infection with Helicobacter pylori (H. pylori) and autoimmune gastritis both cause chronic inflammation and increase the risk of gastric cancer. This application includes numerous mouse models and human tissue samples to investigate the importance of immune cells (mast cells and Th2 T cells) and cytokines (IL4 and IL13) in inducing gastric metaplasia and promoting the development of gastric metaplasia and tumorigenesis. Identifying inflammatory cells and signals that initiate metaplasia and drive tumor development could improve the ability to identify individuals at an increased risk of disease progression, and new immune based strategies to prevent and treat preneoplastic lesions associated with gastric diseases, including gastric cancer.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Chronic gastritis initiates a pathological progression of disease which in some individuals culminates in gastric cancer, the fourth leading cause of cancer related mortality worldwide. Helicobacter pylori infection is the most common cause of gastritis and responsible for most gastric cancer cases worldwide. Autoimmune gastritis is another prominent cause of gastritis increasing the risk of gastric cancer development. Persistent gastric inflammation initiates the transformation of healthy epithelial cells into pre-cancerous metaplastic cells which transform into adenocarcinoma in a subset of individuals. Recent transcriptomic analyses of gastric adenocarcinoma have led to the identification of four distinct molecular subtypes. This discovery has improved targeted treatment and surveillance strategies for each distinct gastric cancer subtype. Currently, there is a need for an in-depth molecular analysis of pre-cancerous gastric metaplasia. We recently discovered that gastric metaplasia can also take on distinct molecular phenotypes in the autoimmune setting. It needs to be determined whether metaplasia arising out of H. pylori infection, known to inject cytotoxins that can interfere with cell- signaling pathways possibly promoting oncogenic transformation, is phenotypically distinct from metaplasia arising out of autoimmune gastritis. If metaplastic subtypes are conserved, then chronic inflammatory signals, independent of etiology, likely contribute to cancer progression; however, if metaplastic subtypes in infection are distinct from autoimmunity, then factors unique to the bacterial infection likely drive an alternative trajectory of oncogenic transformation. Furthermore, how certain inflammatory cytokines (e.g., IL-4/IL-13) impact the development of gastric metaplasia and specific molecular subtypes of metaplasia needs to be established. Identifying molecular phenotypes of pre-cancerous metaplastic cells, in distinct disease settings, that may carry differential oncogenic potentials, will contribute to the discovery of novel screening targets to decrease the gastric cancer burden in highly susceptible individuals. Identifying the inflammatory signals that promote the development of potentially high-risk metaplastic cells will aid in discovery of new therapeutic strategies for inhibiting gastric cancer. In this proposal, metaplasia arising out of two common etiologies of gastritis, H. pylori infection and autoimmune gastritis, will be molecularly defined and compared. Gastric metaplasia from human gastritis patients will be transcriptionally profiled to determine the phenotypes of metaplasia induced by human disease. The impact of inflammatory cytokines, IL-4 and IL-13, on inducing/expanding phenotypically distinct subtypes of gastric metaplasia will also be determined. This work will improve the mechanistic understanding of gastric carcinogenesis in the settings of H. pylori infection and autoimmune gastritis. This knowledge can then be used to improve medical prevention strategies and identify novel targets for therapeutic intervention of gastric cancer.

NIH Research Projects · FY 2025 · 2022-09

Chemotherapy-induced cognitive impairment (chemobrain; CICI) is a common neurotoxicity affecting >50% of patients8 treated with widely used chemotherapeutics, including taxanes (e.g., paclitaxel)9-11 and platinum- based agents (e.g., cisplatin).8,9,12 There are no FDA-approved interventions. Mitochondrial dysfunction and neuroinflammation in the central nervous system (CNS) are mechanisms thought to drive CICI, but how these are triggered remains elusive.6,13-15 Our preliminary studies implicate CNS alterations of sphingolipid metabolism and increased sphingosine-1-phosphate (S1P) formation, offering a novel target for therapeutic intervention with S1P1 receptor subtype (S1PR1) antagonists. In preliminary studies of cisplatin-induced cognitive impairment, S1P increased in the prefrontal cortex (PFC) and hippocampus (key centers of cognition) where S1PR1 was expressed. This was associated with mitochondrial superoxide dismutase (MnSOD) nitration and inactivation and activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. MnSOD tightly regulates peroxynitrite (PN)-mediated nitroxidative stress,16,17 but is also inactivated >80% when nitrated by PN.18-20 This PN-mediated process has been linked to mitochondrial dysfunction in many disease states.17-19,21-23 NLRP3 is critical for interleukin-1β (IL1β) and IL18 maturation,24-26 powerful inflammatory cytokines in cognitive impairment.27-31 Systemic administration of the orally bioavailable, CNS penetrant S1PR1 functional antagonist FTY72032 blocked MnSOD nitration/inactivation, NLRP3 activation and attenuated CICI without adversly affecting locomotor activity (preliminary data). These findings are very exciting, since FTY720 (fingolimod) is FDA-approved for multiple sclerosis with good safety profiles.33-36 How chemotherapy triggers S1P/S1PR1 signaling is not known, but preliminary data suggest glial cell toll-like receptor 4 (TLR4) as a likely link. We hypothesize that chemotherapy engages TLR4 to drive S1P/S1PR1 signaling in the CNS that contributes to CICI through PN-mediated mitochondrial dysfunction and NLRP3-mediated neuroinflammation (Fig. 1). We will test this hypothesis in naïve and tumor-bearing male and female mice. In Aim 1, we will investigate S1P/S1PR1 signaling in the CNS, temporal and cellular distribution of S1PR1 and test whether S1PR1 antagonists prevent and reverse CICI without interfering with antitumor effects. In Aim 2, we will use pharmacological and genetic approaches to define the role of TLR4 in sphingolipid metabolism and S1P formation during CICI and test the impact of S1PR1 inhibition on mitochondrial dysfunction and NLRP3-driven neuroinflammation. S1PR1 antagonists are not expected to interfere with chemotherapy efficacy; we and others have shown that these drugs block tumor cell growth, inflammation and metastasis.37-43 Our studies are anticipated to provide paradigm-shifting insights that establish S1PR1 in CICI and expedite translational investigation of medicines that are already FDA approved (fingolimod/ozanimod) as adjunct to chemotherapy.

NIH Research Projects · FY 2024 · 2022-08

Exposures to chlorine (Cl2) and bromine gases (Br2) are public health threats. Cl2 and Br2 exposures occur as a result of industrial accidents as well as in chemical warfare. Cardiopulmonary failure is a major concern following exposures, which contributes to mortality and morbidity; but the mechanisms underlying end organ injury after exposure to Cl2 or Br2 remain to be determined. We discovered that Cl2 and Br2 gas exposure target host plasmalogen lipids, resulting in high levels of 2-halofatty aldehyde and 2-halofatty acids in the lung and circulation. Recently, we have shown 2-chlorofatty acids, at levels found in the plasma of mice and rats exposed to Cl2, elicit neutrophil extracellular trap (NET) formation. Since NETs are critical and early initiators of coagulopathies that cause end organ injury, the proposed studies will test the hypothesis that Cl2 and Br2 derived 2-halofatty acids elicit NET formation to induce lung injury. Furthermore, our preliminary data show 2- bromofatty acid also causes NET formation, underscoring the potential for NET formation as a unifying mechanism mediating both Cl2 and Br2 gas toxicity that will identify common therapeutic targets and countermeasure development opportunities. Moreover, while Cl2 and Br2 are similar, their unique physicochemical properties endow differences in mechanisms by which each cause injury. Salient to this proposal, we have shown that 2-bromofatty aldehyde reactivity with nucleophiles is 25-fold greater than that of 2-chlorofatty aldehyde. In addition to testing the role of NET formation, proposed studies will also identify protein targets of 2-bromofatty and 2-chlorofatty aldehydes and their respective 2-halofatty acids in mediating NET formation, and we anticipate results from these studies will demonstrate overlapping and unique targets in the pathways by which Cl2 and Br2 mediate circulatory and pulmonary dysfunction. There are two specific aims for the proposed studies. Specific Aim 1 will identify chlorolipids and bromolipids as critical mediators of NET formation and subsequent lung injury following Cl2 and Br2 exposure. Specific Aim 2 will identify mechanisms by which chlorolipids and bromolipids elicit NET formation in human neutrophils. We will employ both mouse and rat models of Cl2 and Br2 gas exposure. This, together with testing two distinct toxicants at LD50 levels, meet criteria for this RFA. Collectively, the proposed studies will delineate a common mechanism for Cl2 and Br2 toxicity mediated by halolipid-stimulated NET formation and organ failure. This mechanism could lead to a common treatment for both of these Chemical Countermeasures Research Program concerns in the future.

NIH Research Projects · FY 2024 · 2022-08

Posttraumatic stress disorder (PTSD) is the 4th most common, non-substance related, psychiatric disorder in the United States. Patients with PTSD vs. those without have a significantly greater risk for type 2 diabetes (T2D) and cardiovascular disease (CVD) and mortality. Determining if PTSD improvement is associated with addresses NHLBI priorities and informs efforts to reduce health disparities related to psychiatric disorders. But even in patients who experience improvement, residual PTSD symptoms may contribute to adverse T2D/CVD outcomes. Thus, it is important to determine which PTSD symptoms or combination of symptoms are most strongly associated with adverse T2D/CVD outcomes in patients with comorbid PTSD and T2D or CVD. better T2D/CVD outcomes and lower mortality This proposal builds on our past 4-years of funded research We now move our focus away from incident T2D/CVD to outcomes in comorbid PTSD and T2D/CVD. There are no studies that have determined if patients with comorbid PTSD and T2D or CVD who do vs. do not have clinically meaningful PTSD improvement are at lower risk for adverse T2D/CVD outcomes and death. There are no studies which have determined if specific PTSD symptoms (e.g., hyperarousal) or combination of symptoms are associated with adverse T2D/CVD outcomes and mortality. Our design determines if PTSD improvement is followed by better T2D/CVD outcomes and whether this is mediated by disease management variables such as medication adherence. focused on PTSD treatment and improved health behaviors, and incident T2D and CVD. We do not study a specific type of PTSD therapy because we want to demonstrate whether PTSD is a modifiable risk factor for T2D/CVD adverse outcomes and mortality. As in the prior funding period, we use Department of Veterans Affairs’ medical record data. This is an ideal ‘laboratory’ to test our hypotheses. In a 10 year observation period (2012-2022) we will sample 15,193 patients with PTSD and comorbid T2D and 17,442 patients with PTSD and comorbid CVD. Our aims are 1) determine if separate PTSD phenotypes (derived from latent class analysis of PTSD symptoms), and symptom change, in patients with comorbid PTSD- T2D/CVD, are differentially associated with an increased risk for adverse T2D and CVD outcomes, including disease related and all-cause mortality, 2) determine whether risks for adverse T2D and CVD outcomes, all- cause and cause specific mortality are lower in patients with vs. without a clinically meaningful PTSD improvement ; 3) determine if measures of T2D/CVD management, such as good glycemic control, statin medication adherence and improved depression, mediate the association between clinically meaningful PTSD improvement and T2D/ CVD outcomes. and 4) conduct planned age, gender, and race sub-group comparisons. Decades of research on depression and T2D/CVD led to guidelines that acknowledge depression as a risk factor for poor CVD outcomes. Positive results and confirmatory studies may eventually lead to similar guidelines that advise providers to screen and treat PTSD to improve T2D/CVD outcomes.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Some critical proteins, with functions both inside and outside of cells, circumvent conventional secretion via the ER and Golgi and are released through Unconventional Protein Secretion (UPS) pathways. These routes are evolved either to spatially and temporally control the function and the triggered release of these UPS cargoes by certain stimuli, or to activate upon impairment of the conventional pathway. Hence, UPS pathways are often triggered by cellular stress, e.g., in hypoxic metastatic tumors and cells under low energy conditions. Some UPS cargos are assisted by chaperones, but many others are released independently. Their release involves self- sustained direct crossing of a membrane, either the cell membrane (Type I UPS) or organelles (Type III UPS). The fundamental question here is how UPS secreted proteins enter organelles and how their essential translocation across membranes is regulated. Defining the molecular regulatory mechanisms is of high significance to drive new therapeutic strategies (e.g., UPS modulators) for diseases associated with their perturbed cellular distributions. We propose a novel hypothesis that explains the regulated and directed release of these key proteins in tumor progression. Hypoxia instigates a transient or enduring cellular acidification. In this model, the interplay between the local acidity and membrane curvature determines the conformational states and membrane-binding mode of these cargoes. In the context of a Type III UPS, this promotes self-sustained protein translocation across endosomal membranes and ultimate secretion. To test this hypothesis, we will determine the extracellular release mechanism of two important UPS cargo proteins, the brain-type creatine kinase and sphingosine kinase isoforms 1 and 2. Extracellular release of these proteins in various cancers contributes substantially to the survival of metastatic cells. This mechanism is mediated by extracellular production of their biologically active products. The subjects of this proposal as potential amphitropic proteins are able to reversibly interact with a membrane. Thus, defining the conformational rearrangements triggering the release of these proteins entails identifying the conformational states that are populated under low energy status of the hypoxic metastatic cells. Testing the involvement of reversible structural refolding and incorporation into the membrane is challenging due to their dynamic states and the difficulties of gaining high-resolution structural information of membrane-bound protein states, particularly the effects of membrane curvature on the protein structure. Thus, we have combined approaches encompassing a range of complementary and cutting-edge methods as well as cell biology studies. Using a similar methodology to study the release mechanism of other key therapeutic targets will test commonalities and differences in their extracellular release mechanism. Ultimately, our research has the potential to define an unconventional protein secretion pathway employed by cancer cells and other pathological conditions. In addition, as a long-term goal, we will bridge our basic research studies that elucidate mechanism with translational research by testing our key conclusions in model organisms.

NIH Research Projects · FY 2025 · 2022-07

Chronic neuropathic pain afflicts 15-20 million people in the U.S.1 Defining the molecular mechanisms involved is key to developing novel non-opioid analgesics.2 We recently discovered that the Gαi protein-coupled receptor (GPCR) Gpr183 transcript increases in glial cells of the dorsal horn of the spinal cord (DH-SC) following nerve injury and contributes to ensuing neuropathic pain.3 The primary endogenous ligand for GPR183 is the potent signaling oxysterol 7α,25-dihydroxycholesterol (7α,25-OHC).4-6 However, little is known about the roles of 7α,25- OHC/GPR183 in pain beyond our recent publication.3 Moreover, commercial GPR183 antagonists are very limited and have chemical properties that raise questions about their in vivo utility.3,7 To address this, we initiated an in silico drug discovery approach that screened 5.5 million commercial compounds with similarities to a GPR183 pharmacophore model, then docked and ranked the highest similar docking scores.3 The top 16 compounds were tested in calcium mobilization (FLIPR) assays to identify several small-molecule selective GPR183 antagonists with IC50 values in the nM range, exemplified by SAE-14.3 SAE-14 was counter-screened against the GPCRome at NIMH PDSP8 with no significant activity for over 300 receptors. Systemic SAE-14 administration reversed mechano-and cold allodynia (behavioral hypersensitivities) induced by nerve injury in male and female rodents with no observable side effects or engaging the endogenous opioid system.3 These effects were mimicked by intrathecal (i.th.) injection of SAE-14, identifying the spinal cord as a potential site of GPR183 antagonist action.3 Moreover, pain behavioral phenotypes were recapitulated by i.th. injection of 7α,25- OHC or a fluorinated analog that activated Gαi/o-linked (pertussis toxin), GPR183-dependent mitogen-activated protein kinase (MAPK) signaling (preliminary data). MAPK signaling in the DH-SC is crucial in regulating neuroinflammatory/pro-nociceptive genes and maintaining persistent pain sensitization.9,10 GPR183-dependent MAPK signaling was supported by unbiased transcriptomic analysis of the DH-SC from SAE-14-treated rats with neuropathic pain (preliminary data). SAE-14 lacks sufficient metabolic stability and solubility/absorption (preliminary data) and structure-activity relationship studies on SAE-14 are necessary for optimization and discovery of druggable GPR183 antagonists. Based on our published work3 and preliminary data, we hypothesize that 7α,25-OHC/GPR183 signaling in the DH-SC contributes to the development and maintenance of neuropathic pain states. Proposed studies in Aim 1 will investigate the role of 7α,25-OHC/GPR183 on MAPK activation and neuroinflammation in the DH-SC. Then in Aim 2, we will develop GPR183 antagonists based on SAE-14 and use a testing funnel to screen to find those with optimal stability and bioavailability for in vivo pharmacological profiling in rodent neuropathic pain models in Aim 3. Our findings will further our understanding of GPR183 signaling in neuropathic pain and identify the first small molecule GPR183 antagonists with properties suitable for non-addictive analgesics development addressing a key mission of the HEAL initiative.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY Ribosomes are large ribonucleoprotein complexes that are integral to translational control. The assembly and maturation of ribosomal subunits involves a multitude of trans-acting factors that render the subunits translationally competent. Maturation of the 60S ribosomal subunit is accomplished by the release of eukaryotic initiation factor-6 (eIF6) with the help of the maturation factors-SBDS and EFL1-GTPase. eIF6 sterically hinders association of the 60S and 40S subunits and therefore, its release from 60S is critical to permit interactions between 60S and the mRNA-bound 40S subunits. In addition, eIF6 is essential for rRNA processing in the nucleolus and is associated with the translationally stalled 60S-ribosome quality control complex. Given its essential roles, the spatial and temporal aspects of eIF6 activities and its release from 60S must be tightly regulated to ensure successful initiation of translation. Impaired release of eIF6 is the defining hallmark of certain ribosomopathies: Shwachman-Diamond syndrome and RPL10 mutations-driven pediatric leukemias. eIF6 levels are also deregulated in several cancers and its enhanced expression is associated with a poor prognosis. Remarkably, restricting eIF6 levels inhibits growth of certain cancers without affecting normal growth. Therefore, targeting eIF6 and its release from 60S has been proposed to be a desirable therapeutic strategy for cancers and ribosomopathies. However, we are yet to understand the role of eIF6 in modulating the distinct steps of 60S assembly and maturation. Also, the molecular mechanisms that regulate eIF6 interactions with the 60S are not completely understood. Towards understanding these mechanisms, our recent work has identified key residues in critical interfaces of eIF6 that modulate its interaction with 60S, and our preliminary data provide direct evidence that the disruption of this interaction is detrimental to cancer cell viability. We have also identified novel sites of regulation in the C-terminus of eIF6 and have uncovered its importance for controlling translational rates. Building on these discoveries, in the current proposal we aim to define the mechanistic steps that promote the release of eIF6 by SBDS, and EFL1 from distinct functional states of 60S and to uncover the role of GTPase activity of EFL1 using a rigorous set of biochemical, biophysical, and single molecule approaches. In addition, we aim to uncover the mechanism of allosteric regulation by the C-terminus of eIF6 and elucidate the functional and phenotypic effects of disease-specific mutations of eIF6 using cellular and in vivo approaches. These studies will provide crucial mechanistic insight into 60S dynamics and will enable the development of therapeutics focused on eIF6 and its regulators.

NIH Research Projects · FY 2025 · 2022-07

This application requests 5-years of financial support for training in the pharmacological sciences for six outstanding predoctoral fellows at Saint Louis University. The leadership team includes three experienced research educators who will guide the efforts of 30 well-funded preceptors from eight departments in the Colleges of Medicine, Arts & Sciences, and Biomedical Engineering. Our training program is based on the premise that pharmacology is a broadly-based subject requiring a multidisciplinary and integrative approach. We have two goals. The first and most immediate goal is to ensure competency in modern principles of drug action and related disciplines. Our second and long-term goal is to develop a diverse pool of capable scientists trained to address the challenges presented by barriers to successful treatment of human disease. To achieve these objectives, we designed a training program that includes didactic and interactive instruction in the principles of drug action, grant construction, responsible conduct of research, and methods to enhance reproducibility; seminar and journal club series specifically targeted to drug discovery and medicinal chemistry; career development seminars that include presentations by scientists pursuing alternative career paths; instruction in presentation and teaching skills including an active role as teacher in an undergraduate course completely administered and taught by pharmacology graduate students; and, competitive student presentations at a yearly retreat. Past graduates of our training program now pursue successful careers in research, academics, and industry, including several trainees who hold significant leadership positions in universities and businesses.

NIH Research Projects · FY 2025 · 2022-05

Abstract Neuropathic pain is common neurological disorder affecting between 5-10% of the general population and predicted to become more common in the future because of the aging population. Although analgesics are available, this type of pain is particularly resistant to our current treatment strategies leaving patients with few options. In addition, these drugs cause severe side effects. This is of course hugely debilitating for individuals, negatively affecting their quality of life. Furthermore, it has significant economic ramifications (treatment costs, time spent off work) and is a burden on healthcare services that needs to be addressed. As a result, there is a pressing need to develop new better-targeted therapies for the treatment of neuropathic pain. One obstacle has been the lack of translation from basic science findings into the clinic. Here, we aim to address this by using patient samples to enhance the relevance of our basic research. It is now well accepted that neuroimmune interactions contribute to abnormal pain states and one relatively under studied aspect of is through the action of human autoantibodies. The anti-CV2 autoantibodies target a protein called collapsin response mediator protein 5 (CRMP5) and are associated with neuropathic pain in patients. CRMP5 is an understudied onconeural protein highly expressed in the developing brain. In adults, CRMP5 expression is absent from the brain but retained in sensory neurons, nerves and synapses present in the spinal cord. In neuropathic pain, CRMP5 expression is decreased at post-synaptic sites in the spinal cord, while the levels of GluN2B, a subunit of the NMDA receptor, and a novel CRMP5 binding protein, is increased. Using patients' autoantibodies and a rat model of neuropathic pain, we will test the hypothesis that disruption of CRMP5 functions underlies allodynia in anti-CV2 autoimmune neuropathy and in neuropathic pain. Aim 1 will investigate the alterations of sensory neurotransmission elicited by anti-CV2 autoantibodies. This will include a functional profiling of sensory neurons using whole cell and slice electrophysiology. With this we will decipher the site of action of anti-CV2 autoantibodies produce to allodynia. Aim 2 will focus on elucidating the pre- and post-synaptic function of CRMP5 in spinal neurotransmission and neuropathic pain. We will explore the novel idea that loss of CRMP5 regulation of GluN2B underlies allodynia in neuropathic pain. This proposal uses a combination of novel biochemical and functional methods to reverse-translate the clinical findings of CRMP5 (anti-CV2) auto-immunity causing allodynia to interrogate how CRMP5 can contribute to neuropathic pain. The identification of a novel therapeutic target for chronic pain is an exciting outcome with far reaching applications for future therapeutic development.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY/ABSTRACT The lifetime prevalence of voice disorders in the adult United States population is 30% with point prevalence rates of 6.6% to 7.5%1,2. Point prevalence and census estimates suggest that nearly 20-23 million adults may experience dysphonia annually, with the cost of treatment and lost wages approaching $13 billion dollars3. These annual direct costs are comparable to those associated with chronic obstructive pulmonary disease, asthma, diabetes, and allergic rhinitis3. Thus, improving the care of patients with voice disorders remains a significant public health need. To address this need, over the last 17 years, our research program has initiated systematic studies in the understanding of the cellular and molecular pathophysiology underlying vocal fold tissue changes. Our studies to date have provided critical new insights into the cellular and molecular sequalae regulating vocal fold permeability. Over the last 5 years, we have focused our studies on the safety and efficacy of pharmacological treatments for voice disorders. Our preliminary data have revealed mechanisms regulating permeability of the vocal fold epithelial barrier from a class of steroid hormones ubiquitously found in most cells in the human body. Beyond the anti-inflammatory actions of these glucocorticoids, emerging evidence in our laboratory supports a role for glucocorticoids in the regulation of the vocal fold paracellular pathway. These preliminary data have led to an overarching hypothesis that vocal fold epithelial permeability can be selectively regulated using pharmacological approaches that target the paracellular pathway. This novel treatment concept provides exciting new possibilities in the management of vocal fold disease by providing pharmacologic access to the subepithelial space–and a means for transepithelial delivery of commonly available fillers and biomaterials to the vocal folds. Over the next five years, we will converge our next series of studies on the selective regulation of vocal fold epithelial permeability using a combination of in vitro and in vivo experiments. The current R01 builds on a programmatic series of investigations which have provided the necessary preliminary data to support selective permeability of the vocal fold paracellular pathway. The goal of this R01 proposal is to empirically quantify the effects of methylprednisolone on selective regulation of the vocal fold paracellular pathway. These pre-clinical studies are necessary to provide indications for use, safety, and the demonstration of therapeutic efficacy prior to human trials. The specific deliverable upon project completion will be the preliminary studies necessary for rigorous testing in phase I/II/III human trials.

NIH Research Projects · FY 2026 · 2022-04

Knee osteoarthritis (OA) is a costly and worsening problem in the United States with nearly 14 million adults suffering with symptoms of pain and stiffness. Although many treatments exist, they often lack evidence for efficacy, bare significant risk, or are exercise programs that are difficult to maintain. Walking in one’s neighborhood provides a low-cost solution that has been shown to control OA symptoms. However, most Americans, and further, most OA patients do not engage in enough physical activity. Barriers to walking are built and social environment in their neighborhood, such as walkability and fear of crime. Although walkability and social environment have been associated with walking and physical activity, no studies have yet bridged the gap to prevalent knee OA. Investigating and identifying walkability and social environmental components that most influence prevalence of knee OA and trajectory of knee OA to end stage total knee arthroplasty (TKA) can offer targets for intervention. This 5-year K23 award and strong mentorship in “Big Data”, Geospatial Information Science (GISc) and qualitative methods will help Dr. Gebauer become a leader in integrating Electronic Medical Record Data (EMR) and GISc and gain skills in qualitative methodology to facilitate mixed methods studies to explore the influence of neighborhood on walking and painful conditions. Training in spatial statistics and “Big Data” management will facilitate Aims 1 & 2, exploring the associations between neighborhood factors, such as walkability, social capital, and violent crime rates and prevalent cases of knee OA, as well as TKA across the United States utilizing the rich Veterans Affairs (VA) EMR Data set. Innovative GISc modeling with time-varying measures of neighborhood walkability and social characteristics will be used to follow veterans across the country in a dynamic retrospective cohort for 11 years, providing much needed longitudinal data to offer evidence to inform public health action. Training in qualitative methods and triangulation will facilitate Aim 3, using qualitative interviews with primary care providers (PCPs) and OA patients to help understand how patient or PCP knowledge of neighborhood barriers and resources can be leveraged to enhance shared decision making between PCPs and patients and establish concordance around walking for knee OA treatment. Through the completion of these aims and additional training in longitudinal/multilevel modeling strategies, grantsmanship, and networking with experts in neighborhoods and physical activity, Dr. Gebauer will provide novel risk factors for knee OA and time to TKA, introduce innovative spatial survival models to chronic disease epidemiology, as well as a new understanding of shared decision making for patients and their PCPs, providing data for follow up R01 studies examining integration of neighborhood characteristics to increase walking in knee OA patients, replicating this study in civilian populations and other metropolitan areas, and begin to explore other musculoskeletal conditions that benefit from walking in the neighborhood.

NIH Research Projects · FY 2026 · 2022-03

-γ-producing Th1 cells have been extensively studied for development of novel vaccines and therapeutics against Mycobacterium tuberculosis (Mtb) infection. However, recent studies demonstrate that Mtb-specific Th1 cells alone do not predict protection against tuberculosis (TB). Therefore, although Th1 cells are required for controlling Mtb replication, other types of immune cells may be needed to prevent infection and eradicate the bacteria. We recently found that a subset of IL-9-producing CD4+ T cells (Th9) can mediate protective immunity against Mtb infection. Th9 cells can be differentiated from naïve CD4+ T cells with TGF-β and IL-4, and play important roles in antitumor immunity and immunity related to allergic reactions. The role of Th9 cells in Mtb infection, however, remain largely unknown. We discovered a role for Th9 cells in TB immunity when we analyzed the transcriptomes in CD4+ T cells from healthy volunteers vaccinated with BCG and from people with latent TB infection (LTBI). IL-9 mRNA in CD4+ T cells topped the list of genes increased after BCG vaccination and LTBI. A significant increase in IL-9 production by bronchoalveolar lavage (BAL) cells after stimulation with infectious Mtb was observed only in LTBI patients and people vaccinated with oral (PO) BCG, indicating induction of Mtb-specific Th9 memory responses. To test whether Th9 cells mediate intracellular Mtb killing, we differentiated Mtb-specific Th9 cells from ESAT6-TCR transgenic mice and co-cultured these Th9 cells with Mtb-infected murine macrophages. Intracellular mycobacterial growth was significantly reduced in macrophages after interacting with Th9 cells. Moreover, neutralizing IL-9 abolished the inhibitory effects of Th9 cells, while addition of recombinant IL-9 alone inhibited intracellular bacterial growth. Furthermore, adoptive transfer of Mtb-specific Th9 cells into syngeneic RAG1/2-/- mice suppresses Mtb growth in vivo. In addition, we found that IL-9 was increased and Mtb growth decreased in mice deficient in MCPIP1, a new RNA-binding protein affecting T cell activation. Deletion of MCPIP1 in Th9 cells resulted in a significantly enhanced IL-9 production upon restimulation. Based on our preliminary findings, we hypothesize that Mtb-specific Th9 cells represent a distinct subset of CD4+ T cells important for protective TB immunity. Strategies inducing Mtb- specific Th9 cells could be used to develop more effective TB vaccines and/or immunotherapies. We propose three aims to test this novel hypothesis: Aim 1 to determine the effects of Mtb-specific Th9 cells on protective TB immunity; Aim 2 to define the roles of IL-9 in vaccine-induced protection against Mtb infection using genetically engineered mice, and Aim 3 to identify the source for IL-9 in PBMC and BAL cells from BCG vaccinated individuals, and the mechanisms of Mtb-specific Th9 cell-mediated bacterial killing in human macrophages. These studies will have a significant impact on improving our understanding of host protective immune mechanisms against Mtb infection, and provide the rationale for targeting Mtb-specific Th9 cells as a new strategy for more effective TB vaccines and/or immunotherapies against Mtb infection.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY The devastating effects of illicit substance use during pregnancy on mothers and children is widespread, affecting more than 500,000 births between 2017 and 2018. Adverse birth outcomes can include fetal growth restriction, neonatal abstinence syndrome, prematurity, and death. Illicit drug use during pregnancy puts women at risk of activities that expose them to sexually transmitted infections and legal consequences including loss of child custody and incarceration. Long-term negative consequences for children exposed to substances in-utero range from growing up in an environment where one or both parents have substance use disorders (SUD) to increased risk of depression, developing SUD themselves, and suffering from abuse. While the American Society of Addiction Medicine recommends reproductive planning education be incorporated into SUD treatment programs, interventions have primarily focused on increasing access to birth control and treatment strategies for women already pregnant. A proactive approach to address substance use prior to pregnancy, in the preconception period, is needed. Preconception interventions that address alcohol and tobacco cessation have been found effective. However, to our knowledge, there are no interventions to address preconception illicit substance use. The prevalence of SUD among women in jail is as high as 63% and this population is at risk of both drug relapse and pregnancy after release. There is an optimal period of time (prior to release) when preconception SUD interventions are feasible and ideal for this population. Using a proactive approach for women in jail and before pregnancy will reduce the risk of substance exposed pregnancies (SEPs) and mitigate the numerous negative consequences for mothers, newborns, and families. The proposed study will be the first to adapt an evidence-based preconception intervention, CHOICES, for use among incarcerated women with illicit SUD. CHOICES was developed to reduce the risk of alcohol- exposed pregnancies by targeting both risky drinking and effective contraceptive use.1 The proposed study will adapt CHOICES for use among incarcerated women with illicit SUD who are at risk for an SEP when they are released. The specific aims of this project are to: (1) adapt CHOICES for use among incarcerated women with illicit drug-related SUD to create a manualized intervention, CHOICES-PLEAS (Pregnancy Liberated from Exposure to Alcohol and Substances) and (2) evaluate the feasibility, acceptability, and efficacy of CHOICES- PLEAS at reducing the risk of SEPs (continued drug abstinence and/or reduction in risky sexual behavior) among women in a court-mandated SUD treatment program. Through completion of these aims and additional training in behavioral intervention design, advanced statistical methods, and SUD treatment for reproductive-age women, the trainee, Dr. Bello, will gain the expertise necessary to be an independent investigator. Her long-term goal is to establish herself as an independent investigator with expertise in addressing the reproductive health needs of women with SUD.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY / ABSTRACT Bowel resection leads to the devastating condition of Short Bowel Syndrome (SBS). SBS patients cannot maintain nutrition through regular enteral nutrition (EN) due to insufficient intestines. Such patients, in the absence of EN, require intravenous nutrition via a process called Total Parenteral nutrition (TPN) for survival. Worldwide, tens of thousands of patients require TPN. Despite the lifesaving TPN, side effects in SBS include potentially fatal liver and gut injury. Although, many researchers have focused on the detrimental effects of the constituents of TPN, results from our published studies highlight our novel hypothesis that the state of luminal content deprivation in SBS, disrupts the normal gut derived signals and drives injury mechanisms in SBS. Our published data shows that during EN, as part of normal enterohepatic circulation of bile acids (BA), activation of gut Farnesoid X Receptor (FXR) by BA results in release of Fibroblast Growth Factor 19 (FGF19). FGF19 regulates hepatic BA, cholestasis, lipid, and glucose homeostasis. Due to a lack of gut FXR activation in SBS we hypothesize that the FXR-FGF19 signaling axis is impaired. We have also published that BA prevent gut atrophy by enhancing Glucagon Like Peptides (GLPs). GLPs are regulated via BA activated gut receptor TGR5. While GLP-2 is a gut trophic factor; GLP-1 modulates hepatic steatosis, insulin, and glucose. We hypothesize that inadequate gut TGR5 activation in SBS additionally drives liver and gut injury. Novel Model: We have established a novel untethered ambulatory SBS piglet model using miniature pumps, jugular and duodenal catheters, and surgical bowel resection (SLU#2346,43-R-011) to closely recapitulate human SBS. Proof of Concept: We have published that in animals on TPN without bowel resection (and not receiving EN), treatment with gut FXR agonist, Chenodeoxycholic acid (CDCA) or gut TGR5 agonist Oleanolic Acid (OA), as well as intravenous FGF19 and GLP-1/GLP-2 can prevent liver and gut injury. Importantly, we have shown inadequate gut FXR and gut TGR5 activation and decreased FGF19, GLP-1/GLP-2 in SBS animals. In pilot studies we have noted hepatic and gut protection with CDCA and OA treatment in SBS. Thus, our central premise is to critically understand alteration in gut FXR and gut TGR5 driven signaling in SBS and to test if its restoration in SBS animals mitigates injury. As detailed in the research plan, we will test our hypothesis under the following aims. With Aim 1 we will critically test the roles of intravenous FGF19 and the gut FXR agonist, CDCA on liver injury in SBS. We will analyze serum, histology, key receptors, and transporters along the FXR-FGF19 axis to understand mechanistic links. With Aim 2 we will deliver the gut TGR5 agonist, OA as well as GLP-1/GLP-2 in SBS animals and explore TGR5-GLP axis driven protective mechanisms in SBS, assessing serology, histology, gut trophic factors, morphometrics and gene expression. This project, using a highly translatable SBS model will help advance strategies to mitigate serious complications and provide key insights into drivers of injury in SBS.