Washington University

NIH Research Projects · FY 2026 · 2023-05

Project Summary Tumor-stroma/immune cell signaling communications within the tumor microenvironment (TME) play important roles in tumor development and responses to targeted and immunotherapies. However, our knowledge of complex signaling communications within TME, and their roles in tumor development, drug and immunotherapy response is limited. Effective molecular targets are still missing that can inhibit the tumor- stroma signaling communications. Single cell RNA sequencing (scRNA-seq) has been being a powerful technology to capture transcriptional changes in individual tumor, stroma, immune cells within TME. While scRNA-seq datasets of human cancer are rapidly growing in number, which is leading to many basic and translational discoveries, the study of dynamic tumor-stroma signaling communications is limited. Limiting factors include: 1) static and single time-point snapshots of the complex interactions within the TME, and 2) difficulty in perturbing a large number of related signaling targets; and measuring corresponding functional effects to these perturbations in mouse or tumor tissues (to identify novel therapeutic targets and treatments). To resolve these challenges, in this study, we propose to combine the cutting-edge technologies, including novel artificial intelligence (AI) models, scRNA-seq, crispr-based single or double knockouts (CDKOs), 3D tumor-CAF-TAM co-culture assays, and genetic mouse models, in a systems biology manner. Specifically, (in Aim 1), we will develop novel network AI models using valuable large sets of scRNA-seq data of PDAC human tumors at WashU to identify static core tumor-CAF-TAM interaction (TCTi) signaling networks (multi-cell intra- /inter-cellular signaling networks of TCTi); and an initial set of anti-TCTi targets. In Aim 2, we will further develop another network AI model (M-Step) to infer the better anti-TCTi targets using the functional validation feedbacks in Aim 3; and predict synergistic drug combinations (inhibiting multiple key anti-TCTi targets). In Aim 3, the predicted targets and drugs will be efficiently evaluated using scalable 3D Tumor-CAF-TAM co-culture assays and crispr-based knockouts (E-step) with 3 measurable metrics, i.e., tumor proliferation, migration, angiogenesis. The M-step (modeling) and E-step (validation) forms an E-M process to identify key anti- TCTi targets and drugs iteratively. We will apply these AI models in Pancreatic ductal adenocarcinoma (PDAC) because 1) there have been very limited responses to immunotherapy; 2) no effective treatment; 3) nearly all patients will develop chemo-resistant and metastatic tumors within 2 years of diagnosis. Also (feasibility), 4) we have a strong cross-disciplinary team studying the PDAC TME (supported by NCI SPORE and human tumor atlas network (HTAN)), with the valuable state-of-the-art resources. The success of this study will identify novel anti-tumor-TAM-CAF targets and drug cocktails for PDAC treatment. The AI models, supporting the novel E-M systems biology, can be applied to other cancers and diseases.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ABSTRACT Alzheimer disease (AD) is the most common neurodegenerative disorder characterized by neuroinflammation associated with amyloid plaques and tau-containing neurofibrillary tangles in the brain as well as severe neurodegeneration, neuroinflammation and lipid accumulation. The apolipoprotein E (APOE) genotype is by far the most powerful genetic risk factor for late-onset AD and is thought to play an important role in neuroinflammation and lipid metabolism. Pathological activation of microglia and astrocytes contribute substantially to the loss of neurons and synapses and lipid dysfunction in AD and related dementias (ADRD). Despite an important pathogenic role for microglia in tau-mediated neurodegeneration, the specific microglial mediators of neuroinflammation and neurodegeneration are poorly understood. Our lab has recently demonstrated that the microglial immune-oxysterol 25-hydroxycholesterol (25HC) augments the production of the proinflammatory cytokine, IL-1b, in an APOE-isoform dependent manner (E4>E3). Cholesterol 25- hydroxylase (CH25H), the enzyme that synthesizes 25HC is upregulated in AD and PS19 brain tissue as well as in disease-associated microglia (DAM). We have preliminary evidence that 25HC directly contributes to the age-dependent neurodegeneration observed in PS19 mice and regulates cholesterol metabolism in astrocytes. We hypothesize that 25HC synthesized and secreted by activated microglia drives tau-dependent neuroinflammation and neurodegeneration via its effects in regulating cholesterol metabolism. We will test this hypothesis as follows – In Aim 1, we will determine the importance of Ch25h in mediating tau-dependent neuroinflammation and neurodegeneration. In Aim 2, we will determine the role of Ch25h/25HC in mediating the deleterious effects of APOE4 on tau-dependent neuropathology. In Aim 3, we will determine whether and how 25HC alters cholesterol metabolism to reduce neuronal viability. Successful completion of this project may enable the development of novel therapeutic strategies towards ADRD.

NIH Research Projects · FY 2026 · 2023-05

The goal of the proposed 5-year training program is to facilitate Dr. Mai Dang’s full transition to an independent physician scientist. Her work is focused on identifying novel ways to modulate the brain tumor immune microenvironment to improve treatment strategies for pediatric patients. She was recently recruited to Washington University in St. Louis as a tenured-track assistant professor in the Department of Neurology to continue this work with strong institutional support. She will use the next five years of mentored training to acquire additional essential knowledge on immunology and advanced research tools to perform investigations in cancer immunology. While her prior work was specifically on macrophages and microglia, her current proposal will focus on antigen presenting cells and their interaction with the adaptive arm of the immune system. Dr. Dang will be mentored by Dr. Milan Chheda, a physician scientist, whose expertise is on developing new therapies that target glioblastoma brain tumor cancer stem cells and the tumor microenvironment. She will be co-mentored by Dr. David DeNardo, who has expertise in immunology and the tumor microenvironment’s regulation of therapy response in pancreatic and lung cancer. They will be joined by a committee comprised of two highly seasoned mentors, both physician scientists with excellent track record for and commitment to training other physician scientists at WUSTL. Dr. Christine Gurnett is Head of Pediatric neurology, who is highly recognized for her work in the genetics of neurological disorders, and Dr. Joshua Rubin, Professor in Oncology is an expert on pediatric and adult brain tumors. This team is deeply committed to providing Dr. Dang with scientific, technical, and career mentorship to assist her in this launch of her independent research career. The research goal of this proposal is to identify reversible causes of immune suppression in medulloblastomas treated with radiation and drug treatment. The central hypothesis is that effective tumor immunity after radiation in a pediatric brain tumor will require both reducing suppressive myeloid cells and increasing functional antigen presenting cells. She further hypothesizes that reprogramming myeloid cells may allow for more anti-tumor activity during treatment. She will use advanced tools such as inducible depletion of immune cells and single- cell analysis paired with assays to directly measure the function of antigen presenting cells and cytotoxic T cells to study this hypothesis. Findings from this work will uncover ways to effectively use immunotherapy to augment radiation efficacy to improve overall survival and morbidity for pediatric brain tumor patients.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY Our goal is to understand the transcriptional requirements for erythroid-myeloid progenitor (EMP) development and differentiation into monocytes and tissue resident macrophages (TRM). Hematopoiesis is comprised of three waves: primitive hematopoiesis, transient-definitive hematopoiesis and definitive hematopoiesis1–6. Transient-definitive and definitive hematopoiesis require EMPs and hematopoietic stem cell (HSCs), respectively. EMPs are required for embryonic development until HSCs take over as the primary hematopoietic progenitors and thus absence of EMPs results in midgestation lethality7. Despite their importance in fetal development, much remains unknown regarding the transcriptional requirements for EMP development, in part due to shared surface markers and genetic programs between overlapping waves of hematopoiesis and terminally differentiated lineages. Additionally, EMP-derived monocytes are recognized as the main source of majority of the TRM populations in adults4,8,9. TRMs are eventually replaced in the adult by HSC-derived monocytes at tissue-specific rates. The developmental and functional differences between EMP- derived and HSC-derived TRM and the impact on tissue homeostasis are not well-understood. Previous studies and preliminary data indicate that the transcriptional factor Zeb2 is not only required for the maintenance of TRM identity but also required for EMP formation and/or differentiation. We hypothesize that Zeb2 is required for the development and differentiation of primitive hematopoietic progenitors and transient- definitive EMPs. We aim to identify novel regulatory elements and transcription factors regulating Zeb2 expression during primitive and transient-definitive hematopoiesis. I am currently a MD-PhD candidate at Washington University in St. Louis School of Medicine, an institution with a long history of supporting physician-scientists at all stages of their training and is working with a strongly committed mentoring team. The proposed training plan provides new conceptual and technical training, along with scientific, clinical, and career development activities that support a trajectory to become an independent physician-scientist focused on discovering mechanisms of hematopoietic development and lineage differentiation and applying this knowledge toward advancing novel therapeutic strategies.

NIH Research Projects · FY 2026 · 2023-05

Project Summary/Abstract Osteoclasts (OCs), the bone resorbing cells, arise from myeloid OC progenitors (OCPs) and are critical for bone remodeling and homeostasis. OC differentiation and activity are tightly regulated by intrinsic negative feedback loops (autoregulatory) and by paracrine factors secreted by other cells, most notably osteoprotegerin. Intrinsic regulators are critically important for calibrating physiologic OCgenesis, disruption of which leads to uncontrolled pathologic OCgenesis and osteolysis. Using proteomic studies in an independent study, we have recently identified a novel autoregulatory role of IFN stimulated gene-15 (ISG15), a ubiquitin-like small molecule, in OCgenesis. Specifically, we found that RANKL induces expression of ISG15 in OCPs and OCs, which binds to NEMO (a.k.a. IKKg) to down regulates NF-kB signaling. We found that stimulation of OCPs with RANKL induces IFNa/b secretion by OCPs, which engages and activates type 1 IFN receptor (IFNAR) signaling in OCPs themselves to trigger ISG15-dependent local autoregulatory negative feedback loop to limit the extent of OCgenesis. This mechanism appears to depend on STimulator of INterferon Genes (STING), which we find to be expressed in response to RANKL and is essential for IFNa/b, and ISG15 expression, and was validated by data showing that blocking either STING or IFNa/b diminishes RANKL-induced ISG15 levels and exacerbates OCgenesis. Collectively, these observations suggest that endogenous ISG15 inhibits OCgenesis through its classical binding to target proteins, which is facilitated by the sequential action of highly specific E1 (Ube1L), E2 (UbcH8), E3 (Herc6) ligases, a post-translational modification termed ISGylation that maintains cellular homeostasis. However, ISGylation is a reversible mechanism, whereby under inflammatory conditions, free ISG15 is generated in abundance by cells, secreted to the extracellular (EC) space by secretory vesicles, and acts as a cytokine by binding LFA1 receptor. In this regard, we show that inflammatory stimulation of OCPs with the bacterial product LPS inhibits expression of Ube1L, the E1 enzyme, and induces secretion of ample ECISG15. More surprisingly, we found that ISG15KO mice responded poorly to LPS and developed negligible osteolysis compared to robust bone loss by LPS-induced WT counterparts. This event was TNF-dependent, evident by low levels of TNFa in ISG15KO serum compared to copious amounts of TNF in serum of WT mice. Hence, ECISG15 appears to exacerbate OCgenesis and depends on intact endogenous ISG15. Based on these observations, our overarching hypothesis states that ISG15 has bi-modal functions: under physiologic conditions, ISG15 is conjugated to OC signaling proteins to limit OCgenesis and maintain homeostasis, whereas inflammatory conditions facilitate secretion of free ECISG15, which then acts as an inflammatory cytokine to exacerbate OCgenesis and osteolysis. To test this hypothesis, we will: (1) Elucidate the mechanism by which intracellular ISG15 inhibits OCs, and (2) Determine the mechanism(s) by which ECISG15 exacerbates basal OCgenesis and inflammatory osteolysis

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY This proposal describes a 3-year plan to prepare Dr. Kim Liss, MD, for independence as a physician scientist, studying the role of lipid metabolism in ischemia reperfusion injury (IRI) in steatotic livers. During her Pediatric Gastroenterology, Hepatology & Nutrition fellowship at Washington University School of Medicine, Dr. Liss undertook research training in Dr. Brian Finck’s laboratory, where she demonstrated that steatosis significantly worsens hepatic IRI in an experimental model. She also found that manipulation of a single lipid synthesis pathway has far-reaching effects on inflammation, cell death, and regeneration in the liver. She extended these findings and found alterations in multiple lipid metabolic pathways and macrophage subpopulations in response to IRI. These findings serve as the basis for her proposal. Washington University School of Medicine is an outstanding environment for Dr. Liss to further her research training, with its longstanding history of NIH-funded research, breadth and depth of resources, and, most importantly, commitment to launching young investigators. There is constant crosstalk between departments and research groups. Dr. Liss’ mentoring committee and collaborators have expertise spanning Pediatrics, Medicine, Surgery, Chemistry, Immunology, and Developmental Biology. Dr. Finck is a well-suited primary mentor because he is a leader in the field of intermediary metabolism and has an established record of training young scientists during their transition to independence. Furthermore, the Finck laboratory is a hub of intellectual energy with its trainees working on a broad range of inter-related projects. Dr. Liss will have abundant opportunities to present her work within Washington University and the wider scientific community. Her mentor team has complementary skills, and her Division and Department are highly supportive of her goals. She will take graduate classes to enhance her knowledge of metabolomics, genomics, and bioinformatics, and develop technical skills with her mentors and collaborators. At award conclusion, she will have acquired the knowledge and skills to become an independent physician scientist with special expertise in hepatic steatosis relevant to liver transplant care. In parallel to pursuing her structured career development plans, Dr. Liss will accomplish her proposed Aims: 1) determine how adipose tissue lipolysis interacts with the liver in the context of hepatic IRI, and 2) define changes to the hepatic lipidome that determine the macrophage landscape in hepatic IRI. Using advanced multi-omics approaches, Dr. Liss strives to develop an integrated understanding of the factors that contribute to IRI and recovery. Her work will fill the void in our understanding of the pathobiologic mechanisms underlying IRI in steatotic livers, a condition that limits organ use, and threatens graft function. The efforts described in this proposal will prompt novel interventions to increase donor organ availability, decrease waitlist mortality, improve patient outcomes, and, in the process, serve as a springboard for Dr. Liss’ research career.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY/ABSTRACT Reconstruction of peripheral nerve gap injuries continues to pose a challenge in the clinic due to their significant long-term functional morbidity. Multiple surgical approaches have been developed to promote nerve regeneration across a damaged nerve with a gap. In the clinic, nerve autografts are considered the standard of care in treating nerve gaps due to their superior capacity in promoting nerve regeneration. However, the use of nerve autografts is limited by their availability, increased surgery time, loss of nerve function from the lost nerve and donor site morbidity. Acellular nerve allografts (ANAs) have been proposed as a clinical alternative to autografts in repairing nerve gaps due to their capability in promoting regeneration across short gaps (< 3 cm). ANAs are allogenic nerves that are chemically processed to remove the cells, reducing immunogenicity, while maintaining the extracellular matrix (ECM) that will allow cell migration into the ANA and subsequent nerve regeneration. However, unlike autografts, ANAs do not support regeneration across long defects (> 3 cm). The reason why long ANAs do not promote regeneration or regenerate poorly is not completely understood. Previous studies have demonstrated the critical role of inflammatory cells, such as macrophages and T cells, in regulating and promoting the early stages of nerve regeneration in ANAs (before 4 weeks post-surgery). Specifically, macrophages will promote angiogenesis and T cells will promote axonal myelination. However, when compared to short ANAs capable of robust regeneration, preliminary data shows an increase in the expression of pro-inflammatory cytokines and changes in the morphology of blood vessels in long ANAs at the later stages of nerve regeneration (between 4- and 8- weeks post-surgery). The increase in the expression of pro-inflammatory cytokines and the changes in the morphology of blood vessels happen after angiogenesis has occurred and axons have started migrating into the ANAs. These changes suggest that a pro-inflammatory environment develops inside long ANAs in the later stages of nerve regeneration, and this correlates with changes in the morphology of blood vessels and a decrease in the number of myelinated axons. We hypothesize the state of chronic inflammation in long ANAs is disrupting the regenerative microenvironment in long ANAs. In brief, the proposed research aims to characterize and compare the inflammatory microenvironment that develops in the later stages of nerve regeneration in long ANAs with the microenvironment in the regenerated short ANAs and autografts. This project also explores the effect of reducing inflammation in long ANAs, using a pharmacological intervention, on axonal regeneration. An integral part of this study is to identify the cell subpopulations promoting the pro- inflammatory microenvironment in long ANAs. An in-depth analysis of these cell subpopulations may provide specific molecular targets used to develop therapies aimed to promote nerve regeneration in long ANAs.

NIH Research Projects · FY 2025 · 2023-05

ABSTRACT The long-term goal of this project is to advance precision medicine by developing a simple, rapid, and comprehensive approach to molecular diagnostic testing that can be easily performed on any cancer subtype. In lung adenocarcinoma and many other solid tumor types, optimal treatment relies on the identification of specific genomic alterations. These include single nucleotide variants and small insertions and deletions, as well as larger copy number alterations and chromosomal translocations. Currently, clinical testing for these mutations requires multiple assays and significant amounts of tissue so that both DNA and RNA can be obtained for analysis. As a result, many samples fail or simply cannot be tested. At our cancer center 38% of lung cancer patient biopsies sent for comprehensive molecular evaluation either fail or result in incomplete results. More robust and streamlined diagnostic methods are therefore needed to provide truly comprehensive mutational profiling for all cancer patients. We have developed a novel approach for whole-genome sequencing (ChromoSeq) that provides rapid, unbiased evaluation of all mutation types in a single assay. ChromoSeq leverages recent advances in high-throughput sequencing methods to deliver a complete genomic profile in as little as 3 days using minimal DNA input. We have previously shown that ChromoSeq can provide rapid comprehensive genomic profiles from blood or marrow of patients with myeloid malignancies and that ChromoSeq has increased sensitivity to detect clinically significant genomic alterations compared to conventional methods. However, obtaining similar performance from solid tumors with limited amounts of degraded DNA typically obtained from formalin-fixed paraffin-embedded (FFPE) tissue biopsies is challenging. We hypothesize that with substantial methodologic pre-analytic improvements and rigorous clinical validation testing, ChromoSeq can also be used for the comprehensive genomic profiling of solid tumors. In this application, we propose to measure and optimize DNA changes that occur during pre-analytic tissue processing of routine clinical biopsies (Aim 1) and then establish the clinical performance of the assay using retrospectively and prospectively collected patient samples (Aim 2). These Aims will be performed in a CLIA-licensed, CAP- accredited laboratory with the overall objective of producing a CLIA-compliant assay for future use in clinical studies and to improve the diagnosis and treatment of patients with lung cancer. Tools and protocols developed in this application will enable other laboratories to benefit from this simplified approach to cancer genomic profiling.

NIH Research Projects · FY 2025 · 2023-05

SPECIFIC AIMS Ovarian cancer is the most lethal gynecological disease. 70-80% of ovarian cancer patients are diagnosed at stage III or IV when peritoneal metastasis has already occurred. The unique metastasis pattern in ovarian cancer involves the detachment of tumor cells, diffusion through the intraperitoneal space and attachment to the mesothelial layer lining the omentum and abdominal organs. Both increased collagen deposition and extracellular matrix (ECM) stiffening (due to altered collagen architecture) by stromal cells can create a pro- metastatic environment. Paracrine action of tumor growth-inducing molecules like polyamines can also promote metastasis. Identifying mechanisms that underlie collagen deposition and polyamine synthesis will allow better understanding of metastasis and thus further development of therapeutic interventions. One putative candidate for regulating metastasis is the fibrillar collagen-binding receptor tyrosine kinase Discoidin Domain Receptor-2 (DDR2). DDR2 expression is increased in the stroma of high grade serous ovarian cancer (HGSOC) patients and this increase is, alone, associated with shorter survival and worse response to therapy. We found that there were fewer intraperitoneal tumors when DDR2-expressing tumor cells were injected into the peritoneal cavity of syngeneic Ddr2-/- mice vs WT mice. To understand molecular reasons for this difference I performed targeted mRNA sequencing of tumors from Ddr2 WT and Ddr2-/- mice and found that expression of Arginase-1 was highly down-regulated in tumors from Ddr2-/- mice. Moreover, I found that shRNA depletion of DDR2 in WT ovarian omental cancer-associated fibroblasts also significantly decreased Arginase- 1 expression and activity. A major metabolic function of Arginase-I in cells is to promote arginine degradation into ornithine, which is a source of proline for collagen synthesis and polyamines that can impact cellular proliferation. Based upon these, and other, compelling preliminary data I propose to test the hypothesis: DDR2-regulated Arginase-1 expression in ovarian cancer omental CAFs promotes metastasis by impacting collagen production and ECM organization as well as polyamine synthesis. I will test this hypothesis by pursuing two specific aims: Aim 1: Determine the role of intracellular Arginase-1 in omental cancer-associated fibroblasts on collagen synthesis and architecture. Aim 2: Determine the role of intracellular Arginase-1 on polyamine-mediated tumor cell metastasis. These studies will uncover novel mechanisms by which DDR2 regulates arginase-1 in omental CAFs and how arginase-1 promotes metastasis through increased collagen deposition and polyamine synthesis. As DDR2 has been implicated in lung, pancreatic, and breast cancer, my findings may have broad implications in other cancers. This project will encourage my scientific growth as a physician scientist-in-training.

NIH Research Projects · FY 2025 · 2023-05

ABSTRACT Stroke is the leading cause of long-term disability, affecting almost 800,000 patients per year in the US. Most stroke survivors have some degree of spontaneous recovery, but this recovery is unpredictable and in many cases incomplete. Successful recovery requires plasticity at the synaptic and cellular level to collectively “rewire” damaged brain networks, in a process called remapping. On a global scale, plasticity in brain networks can be observed in the restoration of functional connectivity (fc) between repaired circuits and distant brain networks. Fc likely contributes to recovery of more complex. However, little is known about the mechanisms underlying network plasticity in remapping and fc. The overarching goal of this proposal is to understand mechanisms of plasticity in brain networks after stroke. Enhancing these mechanisms of repair may be key to designing therapies to improve recovery and attenuate disability after stroke. Many of the processes underlying plasticity in the injured brain mirror those that occur in the developing brain. Most saliently demonstrated in the visual cortex (V1) during development, binocular vision leads to balanced segregation of eye inputs into ocular dominance (OD) columns in V1. Monocular deprivation (MD, suturing one eye shut) during development leads the OD columns of the spared eye to competitively take over the OD columns of the deprived eye, similar to remapping after stroke. This plasticity dissipates in adulthood due to the maturation of inhibitory parvalbumin interneurons (PV-INs) in V1. PV-INs are the most prevalent inhibitory neurons in the brain, and act as ‘brakes’ to close critical periods of developmental plasticity, cementing in place mature spatial/temporal patterns of brain activity. However, recent studies have shown that juvenile-like OD plasticity can be restored in adult mice by selectively reducing firing rates in PV-INs, or by weakening the strength of excitatory synapses onto PV-INs (thus weakening their feed-forward inhibitory activity). PV-INs have been further implicated in restricting plasticity in the hippocampus, striatum, prefrontal cortex, and auditory cortex. Given the prevalence of PV-INs throughout the brain, these findings invite the exciting possibility that PV-INs are “gate-keepers” of neuronal plasticity, and potential targets for therapeutic intervention in the injured brain. The central hypothesis of this grant is that activity in PV-INs regulates network plasticity during sensory deprivation and after stroke. We will employ cutting edge non-invasive optical neuroimaging of cortical calcium dynamics in mice to probe changes in local sensory maps and global fc, in combination with viral gene transfer targeted to PV-INs, to understand the role of activity (Aim 1) and synaptic inputs onto PV-INs (Aim 2) in mediating deprivation-induced cortical plasticity and recovery from stroke. Aim 1: To determine if modulating PV-IN activity can enhance cortical plasticity during whisker sensory deprivation and recovery after ischemic injury. Aim 2: Todetermine the mechanistic role of excitatory synapses onto PV-INs in regulating cortical plasticity during whisker sensory deprivation and recovery after ischemic injury. Aim 3: To identify the translatome of plasticity in PV and Pyramidal neurons during whisker deprivation and after ischemic injury.

NIH Research Projects · FY 2025 · 2023-05

Cochlear implants (Cl) can restore audibility, speech understanding, and communication abilities for adults with hearing loss receiving limited benefit from hearing aids, thereby improving quality of life. Despite the success of Cls, there remains wide variability in performance related to both patient and treatment factors. Biographical/audiological variables such as age, age at onset, duration, etiology, and severity of hearing loss as well as duration of hearing aid and Cl use together with surgical factors have long been used to predict speech perception performance. These variables account for only 10-20% of the variability in speech perception test scores in quiet, implying they are poor biomarkers of auditory system suitability for Cl stimulation. Recent studies demonstrate that Cl performance in background noise is worse in the elderly, in part, related to age-dependent central processing differences. We propose that in adult Cl candidates, accurate assessment of both the cochlear-neural and central auditory substrates can predict performance. Predicting performance has relevance to patient counseling and shared decision making (between clinician and patient), design and recommendations for auditory rehabilitation, consideration for device mapping and troubleshooting, and patient stratification in future clinical trials. Electrocochleography (ECochG) is an acoustically-evoked electrophysiological method to assess the cochlear-neural substrate; responses are present in >95% of Cl recipients, and can account for up to ~50% of open set speech perception scores in quiet. However, the ECochG-total response (ECochG-TR), is necessary but not sufficient to predict speech perception in noise. Adding a preoperative cognitive screener to ECochG-TR improves the prediction model (ΔR2=0.26; R2mode1=0.60). Thus, a good cochlear-neural substrate and cognitive function are both needed for speech understanding in noise. We will use biographical/audiological variables together with preoperative, transtympanic (ttECochG) and/or intraoperative (iECochG) ECochG and surgical factors, to develop clinically useful preoperative and/or postoperative (pre-activation) speech perception prediction models. Aim 1: Determine if preoperative ttECochG-TR is a valid measure of cochlear-neural substrate integrity as it relates to Cl stimulation, compared to the validated iECochG-TR. To this end, we will determine the strength of correlation between ttECochG and: (1a) iECochG-TR and (1 b) implant ear speech perception measures in quiet (6-month). Aim 2: Develop prediction models for implant ear speech perception scores in noise (Primary) and quiet (Secondary) after 6 months of Cl use. Models will include baseline demographic/audiologic variables, cognitive measures plus: (2a) Preoperative ttECochG-TR in the clinic; (2b) Postoperative (pre-activation) iECochG-TR and surgical factors from post-implant CT imaging. Aim 3: Establish the generalizability (external validation) of the prediction models in a geographically separate location (Vanderbilt University) from Washington University in St. Louis as patient and surgical factors can vary across sites.

NIH Research Projects · FY 2024 · 2023-05

Project Abstract Low back pain afflicts up to 80% of Americans and accounts for over $100 billion in healthcare and social costs annually. While intervertebral disc (IVD) degeneration and injury have been strongly associated with low back pain, the causation between pathoanatomical features of the IVD and clinical presentation of low back pain remains poorly understood. Recent rodent work show that axial low back pain symptoms can be recapitulated by the targeted injury of the lumbar intervertebral disc. The injury evokes a degenerative sequala in the IVD and produces neurite infiltration that are associated with chronic behavioral symptoms of axial low back pain. This is corroborated by human studies that observed increased innervation in degenerated IVD from humans. Thus, understanding the molecular events that drives IVD innervation may provide insights to therapeutic opportunities prior to the transition to chronic LBP symptoms. The proliferation of sensory nerves is tightly regulated by Nerve Growth Factor (NGF) and Vascular Endothelial Growth A (VEGFA). NGF serves critical homeostatic functions in neurons, while VEGFA plays a crucial role in spontaneous neurite extension and dendritic outgrowth in connective tissues, in addition to initiating angiogenesis. Degenerate and injured IVD cells express VEGFa as a part of the inflammatory cascade, but the mechanistic effects of VEGFA on neoinnervation in the IVD and subsequent low back pain behavior have not been investigated. The central hypothesis of this proposal is that the ablation of VEGFA attenuates neurite growth and vascularization into the injured IVD and alleviates ensuing low back pain symptoms. We will investigate the hypotheses through the following specific aims: Specific Aim 1: Determine whether the deletion of VEGFA 2-days after injury prevents IVD innervation and vascularization, and low back pain symptoms 3- and 12- weeks following a targeted injury. Specific Aim 2: Determine whether the deletion of VEGFA 6-weeks after injury slows, arrests, or reverses IVD innervation and vascularization, and low back pain symptoms 12- weeks following a targeted injury. The mechanistic understanding of VEGFA’s role in modifying the pathoanatomy will pave the way as a disease modifying therapy for low back pain. The ease for delivery anti-VEGF therapies makes it an attractive candidate for the diseased IVD, and anti-VEGFA has already been approved for the treatment of macular degeneration and cancer in humans. The proof-of-concept work here will also lay the foundation for future studies to investigate the source of VEGFA by utilizing tissue-specific drivers of CreERT2; the VEGF crosstalk between intervertebral disc, endothelial cells, and sensory neurons; and leveraging these chronic low back pain mechanisms as potential therapies.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY The Somatic Mosaicism across Human Tissues (SMaHT) Network aims to propel discovery of new biological processes in human health and disease that are mediated by genomic variation in somatic tissues. We propose to establish the WashU-VAI Somatic Mosaicism across Human Tissues (SMaHT) Program Genome Characterization Center (SMaHT-GCC). A fundamental goal of our proposed GCC is to generate high throughput, high quality, and high consistency genomic DNA and RNA sequencing data, ands to construct a comprehensive catalogue of the human somatic variation together with other members of the Network. First, we will establish and manage a collaborative, efficient, transparent, and flexible GCC as an integral component of the SMaHT Network. We will leverage our experience in participating and leading many past and current genomics consortia to design and implement our GCC. The GCC will manage center administration, maintain high quality, sustainable, and on-time data production, develop and implement data management plans, conduct data quality assurance and integrative data analysis, and fully engage with the entire SMaHT Network. Second, we will generate high quality, high throughput, comprehensive and sustainable genomics datasets for the characterization of human somatic mosaicism, using state-of-the-art technologies. We capitalize on our past experience in generating high quality sequencing-based genomic resources, to design and implement our data production plan. We recommend a collection of human tissue types, and will implement four core genomics assays to profile sample received from SMaHT Tissue Procurement Center. We aim to deliver high throughput, high quality, and high consistency genomic DNA and RNA sequencing data to the SMaHT Data Analysis Center. Third, we will develop and maintain a modern, efficient, scalable, and interactive data management and processing system that seamlessly interfaces with the SMaHT Data Analysis Center. We will leverage our experience in scientific computing, information technology system, large genomic data management and integration, and community engagement to design and implement our data management plan. We will build a streamlined, efficient data infrastructure to manage GCC data and collaborate with SMaHT DAC. Fourth, we will develop and implement data analysis and visualization solutions for the SMaHT-GCC data. We will leverage our extensive experience in genomic data analysis and integration, bioinformatics tool development as well as data visualization in the implementation of our data analysis and visualization plan. We will also bring in unique expertise in analyzing transposable elements and epigenomics data. We will contribute to the Network-wide data analysis, and facilitate the characterization of a full spectrum of the human somatic mosaicism.

NIH Research Projects · FY 2026 · 2023-05

In 2020, rectal cancer caused over 339,000 deaths globally, and 732,000 new cases were reported. Historically, Stage II and III tumors, also known as locally advanced rectal cancers (LARC), have been treated with surgical resection, radiation, and chemotherapy. However, advances in neoadjuvant (preoperative) treatment now enable up to 35% of patients to achieve complete tumor death, or complete response, with radiation and chemotherapy alone. In these individuals, surgical resection has shown no benefit and carries the significant risks of major complications, prolonged recovery, and reduced quality of life. Unfortunately, standard clinical testing and radiographic and endoscopic imaging modalities poorly differentiate post-treatment scars from the residual tumor. Confounded by post-treatment fibrotic reaction and edema, the poor performance of current technology makes it extremely difficult to identify complete responders before surgery. Due to this technological gap, surgical resection remains the standard of care (SOC) for all patients outside of specialized tertiary care centers. With improved imaging modalities, widespread adoption of nonoperative management would reduce treatment morbidity for thousands of rectal cancer patients annually. One promising modality, photoacoustic imaging, uses hemoglobin as an endogenous contrast agent to map tissue vascular networks. For clinical use, we have developed and tested a new co-registered acoustic resolution photoacoustic microscopy and ultrasound (AR- PAM/US) endoscopy prototype system, together with a deep learning neural network classifier. Initial testing demonstrated a unique marker of complete tumor response – specifically, recovery of normal mucosal vascular architecture within the treated tumor bed. We hypothesize that our co-registered AR-PAM/US system and the neural net classifiers can assist surgeons to examine the residual tumor microvessel network and assess rectal cancer patients’ pathologic complete response after neoadjuvant treatment. We also hypothesize that serial AR- PAM/US studies will perform significantly better than SOC methods in predicting complete response at treatment conclusion and during post-treatment surveillance. We propose to advance and optimize our prototype AR-PAM/US system, probe, and software and to optimize AR-PAM neural network classifiers to accurately differentiate complete responders from those with residual cancer. We will prospectively assess the ability of co-registered AR-PAM/US technology to improve SOC imaging in a cohort of LARC patients on post-treatment risk management and surgery recommendation. We will also monitor a group of LARC patients to determine if the co-registered AR-PAM/US technology can assess changes in tumor vascular and blood oxygen saturation and identify rectal cancer response, both during the course of treatment as well as in post-treatment surveillance. If successful, this technology will directly reduce the number of unnecessary surgeries for rectal cancer and improve quality of life.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ABSTRACT Cervical cancer is one of the most common cancer diagnoses among women, and treatment failure of standard of care (SOC) chemoradiation therapy (CRT) for locally advanced cervical cancer (LACC) is as high as 30-50%. Since recurrent and metastatic diseases are not curable or detected too late to be treatable, there is a pressing need for pre-treatment biomarkers to identify patients at risk of CRT treatment failure and post-treatment biomarkers to detect LACC recurrence and metastasis early. TCGA’s effort to establish pre-treatment biomarkers for cervical cancer by molecular stratification using human genes failed to associate to patient outcomes. On the other hand, we recently demonstrated that HPV genotypes and HPV alternative splicing affect LACC recurrence and survival after CRT. In our preliminary data, we additionally identified a diversity of HPV genomic structures (HPV-GS), including HPV-human gene fusions involving alternative spliced HPV exons, that affect human oncogene expression. We hypothesize that the variance of HPV genomic structural features among LACC patients may represent a valuable clinical sequencing application to develop LACC SOC CRT biomarkers. For post-treatment markers, we currently use F-fluorodeoxyglucose PET/CT (FDG-PET) images at 3–6 months to define metabolic response, which was shown in our previous publications to predict patterns of failure after radiotherapy for cervical cancer. We hypothesize that HPV genomic features can serve as post-treatment biomarkers for LACC recurrence and metastasis detection that are both more accurate and detectable at earlier timepoints. To achieve these goals, we will first test whether variance in HPV-GS can be utilized to develop a clinical pre-treatment biomarker by developing a series of novel HPV-GS analysis tools based on our expertise in both HPV genomics and human structural variants. HPV features will be extracted from matched DNA and RNA sequencing data, and their prognostic values will be tested using samples from our cervical tumor bank of LACC patients uniformly treated with curative-intent CRT. Second, we will examine whether CRT-induced LACC clonal evolution can be used to identify treatment-resistant HPV-GS as on-treatment biomarkers. A novel deep targeted sequencing approach will be used on single-nucleotide variants (SNV) and HPV-GS to identify LACC subclones and fit HPV-GS in the context of clonal evolution. We will also examine the mechanisms of HPV- human gene fusions using clonogenic survival assays and other standard assays. Last, we will use our proven highly-sensitive and flexible CAPP-Seq technology to evaluate whether circulating tumor DNA (ctDNA) can be used to develop HPV-GS tests for early diagnosis and post-CRT recurrence detection. We expect combining both SNVs and HPV-GS will result in an optimized application superior to using single types of features alone. Taken together, we expect our genomic and mechanistic research on HPV-GS biomarkers in the context of CRT- induced LACC evolution will create a series of optimized pre-treatment and recurrence biomarkers that can be applied in the clinic for personalized alternative treatment regimens.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Dominant mutations in the human GRN gene cause haploinsufficiency in Progranulin (PGRN) protein levels and are a leading cause of frontotemporal lobar degeneration with aggregation of RNA binding protein TDP-43 (FTLD-TDP). In addition, single nucleotide polymorphism (SNP) in GRN has been identified as a risk factor for AD and Limbic predominant, Age-related TDP-43 Encephalopathy (LATE), a major cause of memory impairments in patients over 90 years old. Despite the critical role of PGRN in brain aging, however, the exact mechanism by which PGRN haploinsufficiency promotes neurodegeneration remains poorly understood. By analyzing aging cohorts of Grn knockout (Grn-/-) and humanized GrnR493X/R493X knockin mice, we have shown that PGRN-deficient microglia produce more complement proteins and pro-inflammatory cytokines to promote neuronal cell death and TDP-43 proteinopathy during brain aging. Interestingly, our ongoing work shows that blocking complement activation and proinflammatory cytokines TNFa and IL-1a does not completely mitigate neurodegeneration, but rather uncover late-onset microglial pathology characterized by profound accumulation of lipids in Grn-/-, Grn-/-;C1qa-/-;C3-/-, and Grn-/-;C1qa-/-;Tnf-/-;Il1a-/- microglia. Using lipidomics and subcellular fractionation, we further show that loss of PGRN causes severe blockade in the endolysosomal trafficking of lipids in Grn-/- microglia, which interferes with lysosome-mediated degradation of lipid droplets and increased production of proinflammatory oxidized phospholipids. Together, our results broach the hypothesis that PGRN deficiency causes profound endolysosomal trafficking defects of lipids in microglia and astrocytes to promote neuronal vulnerability in neurodegeneration. To test this hypothesis, we propose to (1) delineate the mechanism of endolysosomal trafficking and secretory autophagy that promote lipid-mediated toxicity in Grn-/- microglia, (2) characterize the role of lipid-dependent and -independent mechanisms in promoting neurotoxicity in Grn-/- astrocytes, and (3) delineate the contributions of lipids to glial toxicity in FTLD-GRN using comparative snRNA- seq, IPSC-derived organoids and postmortem tissues. The proposed parallel human-mouse studies will uncover the contributions of lipid-mediated toxicity in highly disease vulnerable regions in Grn-/- mice and FTLD-GRN will uncover critical mechanisms that promote glial pathology and neurodegeneration during brain aging and lead to novel therapeutic targets for ADRD.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY/ABSTRACT The incidence of oropharyngeal squamous cell carcinoma (OPC) driven by high-risk (HR) HPV strains continues to rise. As HPV (+) disease is prognostic for good post-treatment outcomes and arises in relatively young patients with fewer co-morbidities, clinicians now recognize it as a distinct clinical entity from tobacco- associated HPV (-) disease. But despite improved survival following surgery and adjuvant chemoradiation (CRT), many HPV (+) OPC patients suffer prolonged morbidity from severe treatment-associated toxicities. This has led to many treatment de-intensification clinical trials, which seek to reduce toxic side effects while maintaining historic survival rates. Ideally, high-risk patients would remain on standard regimens while low to intermediate-risk patients would receive de-escalated therapy. However, there is a great clinical need for an objective biomarker of risk to aid the subjective clinical assessments: pre-treatment imaging and postoperative pathology. Liquid biopsies can offer such objectivity; they quantify cell-free DNA (cfDNA) shed by cancer cells, called circulating tumor DNA (ctDNA), in biofluids like saliva or plasma. Further, in HPV (+) OPC, cell-free HPV DNA (cf-HPV) parallels ctDNA as a measure of minimal residual disease (MRD). But plasma and saliva assays lack sensitivity to detect this cf-HPV MRD after surgery. To this clinical challenge, we offer our novel liquid biopsy assay of Jackson Pratt (JP) surgical drain fluid (SDF). We believe SDF will be enriched with cf-HPV compared to plasma because it's more proximal to the primary tumor resection site and to the lymph nodes, where locoregional micrometastases are seeded. Additionally, because the JP drains also capture lymph fluid from the lacerated cervical lymphatic system, we also believe the SDF contains informative effector leukocytes that were in transit to the tumor microenvironments (TMEs) of metastatic nodes and the primary tumor. To begin to elucidate the prognostic potential of SDF, we have collected paired SDF, plasma, tumor biopsy, and metastatic lymph node samples. First, using PCR and next-generation sequencing approaches we will quantify the cf-HPV burden in paired plasma and SDF samples. Then we will compare cf-HPV in each sample type individually to histopathological markers of risk (extranodal extension and tumor stage) and recurrences. We will then track tumor-informed variants on ctDNA, isolated from plasma and SDF, to show that ctDNA levels align with cf-HPV and further validate that cf-HPV is a good proxy for MRD. Lastly, we will use digital cytometry tools and mass cytometry to immunophenotype the immune cells within the SDF to determine if they reflect immune response gene expression levels in paired metastatic nodes and tumors. If confirmed, our study has the potential to demonstrate that tri-biomarker analysis (immune cell, cf-HPV, and ctDNA) in a novel liquid analyte (SDF) can provide precision risk-stratification to aid subjective clinical diagnostics.

NIH Research Projects · FY 2025 · 2023-05

Post-traumatic osteoarthritis (PTOA) is a degenerative joint disease that arises after injury and affects millions worldwide. There are currently no disease-modifying treatments. PTOA is a complex, multi-tissue joint disease characterized by pain, cartilage degradation, synovial inflammation and fibrosis, and formation of ectopic bone growths called osteophytes. The inherent complexity of this disease is a barrier to developing effective treatments, as little is known about the intricate tissue crosstalk that underlies PTOA progression. Our long- term goal is to uncover and comprehensively characterize cellular and molecular mechanisms central to key pathological sequalae of PTOA: synovial fibrosis, inflammation, and osteophyte formation. We will focus on canonical Wnt/β-catenin (cWnt) signaling. cWnt overactivation has recently been implicated as a driving factor of arthritis. Our data show that the cWnt signaling agonist R-spondin 2 (Rspo2) is strongly induced in multiple joint tissues during PTOA, and that Rspo2 alone is sufficient to induce pathological features characteristic of PTOA. Using single-cell RNA-seq, we profiled synovium of mice with PTOA and found that Rspo2 is produced by synovial lining fibroblasts. We identified a novel population of pro-fibrotic cells that arise after injury and express Lgr cell surface receptors for Rspo2. We showed that synovial fibroblasts respond to Rspo2 by secreting cytokines that in turn activate pro-inflammatory macrophages (known to drive synovial pathology in PTOA). Single-cell profiling also revealed a novel subset of injury-induced, Lgr-expressing osteochondral progenitors in synovium, which we propose give rise to osteophytes. We hypothesize that Rspo2-driven cWnt signaling mediates pathological crosstalk between joint-resident cell types to potentiate PTOA. To test this, our aims in the K99 phase are to: 1) determine the role of Rspo2-driven cWnt signaling in the emergence and function of pro-fibrotic synovial cells during PTOA using transgenic reporter mice, multi-omic analyses, and in vitro differentiation assays, and 2) characterize crosstalk between cWnt-active synovial fibroblasts and pro- inflammatory macrophages, using knockout mice and crosstalk assays. To extend upon my molecular biology and immunology expertise, I will receive rigorous technical and conceptual training from my diverse mentorship committee during the K99 phase, and valuable career guidance. This expert training in bioinformatics; cWnt signaling; bone, cartilage, and synovial biology; and multi-modal imaging, will be crucial for carrying out my K99 aims and especially critical for successfully launching my independent career. These skills will be utilized in my R00 phase to: 3) determine how Rspo2/Lgr signaling promotes osteophyte formation in PTOA, using tissue-specific deletion and reporter mice, and in vitro differentiation assays. This work will significantly extend our understanding of cellular and molecular mechanisms that underpin synovial fibrosis, inflammation, and osteophyte formation in PTOA. These insights will have meaningful, tangible outcomes for human health, by accelerating development of effective disease-modifying treatments for PTOA sufferers.

NIH Research Projects · FY 2025 · 2023-04

The goal of this proposal is to develop a comprehensive approach for evaluating the efficiency and specificity of genome-edited human cells using whole-genome sequencing. Genome editing has enormous therapeutic potential by making it possible to restore genetic defects, inactivate deleterious mutant alleles, and augment the function of cellular therapies. Although genome editing technologies are designed for optimal efficiency and specificity, on-target editing can be variable, and unwanted mutations in edited cells can result in unintended functional consequences, including disruption of genes due to off-target mutations, transgene insertions, or deletions, duplications, or structural rearrangements. As a result, current draft guidance from the Food and Drug Administration (FDA) recommends that genome-edited cellular therapies be evaluated for both on- and off-target mutations. However, existing approaches for performing these analyses are logistically complicated and either use antiquated methods or involve modifications to the editing process that cannot be applied to cellular drug products that will be used in patients. We hypothesize that whole-genome sequencing (WGS) is an ideal platform to address FDA guidelines for genomic analysis of genome-edited cellular products because it detects the full spectrum of mutation types and can be used to evaluate fully manufactured ‘patient-ready’ cellular therapies. Here we propose to develop a comprehensive WGS assay specifically designed to characterize mutations in genome-edited human cells. In Aim 1, we will modify our recently developed clinical WGS assay for somatic mutations (ChromoSeq) to measure the efficiency and specificity of genome editing in human cells. We will use high coverage (>250x) WGS of paired edited and unedited control cells and joint somatic variant calling methods to quantify on-target editing efficiency and detect transgene integration sites and unintended single nucleotide variants, insertions/deletions (indels), and chromosomal rearrangements. We will then qualify this WGS approach using a dataset of high confidence mutations generated in three human cell lines with CRISPR/Cas9 and multiplex pools of guide RNAs (gRNA), which will be identified via iGUIDE and targeted, error-corrected deep sequencing. In Aim 2, we will use our WGS assay to define the landscape of mutations in genome-edited human CAR-T cells. These will include 5 replicate experiments with reagents to common CAR-T targets, and 15 existing primary human CAR-T products edited at a range of therapeutically relevant genes that have already been generated in our labs. We will use these data to generate a benchmark dataset of on-target editing efficiency measurements, CAR integration sites, and unintended mutations in human CAR-T cells that will provide valuable data for future clinical trials. Finally, we will analyze up to 20 additional genome-edited cellular products from the Somatic Cell Genome Editing Consortium to further establish the performance and utility of WGS for evaluating the safety and efficacy of genome-edited cellular therapies that will enable future investigational clinical studies.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY The Somatic Mosaicism across Human Tissues (SMaHT) Network aims to propel discovery of new biological processes in human health and disease that are mediated by genomic variation in somatic tissues. Efficient and effective logistical and scientific coordination is vital to maximizing value of SMaHT Network data. We propose to establish the WashU SMaHT Program Organizational Center (SMaHT-OC). Our proposal combines three key strengths: 1) broad and deep expertise in genomics, bioinformatics and genome variation; 2) strong collaborative ties among the WashU management team; and 3) longstanding experience as leaders of consortia building genomic resources, including Roadmap, ENCODE, 4DN, TaRGET, CCDG, Human Pangenome Project, and IGVF. The WashU SMaHT-OC will serve as the nucleus for facilitating all activities of the SMaHT Network. We will define rules and mechanisms of communication, and will empower researchers to engage with multimodal "big bio-data". We will develop and organize management, networking, reporting methods, and regular meetings for establishing efficient and synergistic Network operations. We will provide administrative support to ensure that SMaHT Network-related resources, data and other products are available to the community. We will coordinate all Network meetings and working groups, including the Kick-Off meeting and annual meetings. We will build and manage both internal and external SMaHT Network websites, which will be designed to evolve with community needs. We will integrate and infuse the SMaHT Network with information and data from other large-scale resources and projects by navigating data sharing requirements and coordinating collaborations with other consortia. We will extend the successful IT infrastructure of the WashU Epigenome Browser to coordinate data visualization efforts to enable the scientific research community to take full advantage of the SMaHT Network. We will provide administrative and budget support for developing and managing engagement and training, including coordinating opportunities for researchers within and outside the Network. The WashU SMaHT-OC will integrate the efforts of all the funded initiatives of the diverse SMaHT Network, and, through innovative methodology, establish a research environment that promotes collaboration, coordination, and communication among Network participants and the scientific community. .

NIH Research Projects · FY 2026 · 2023-04

Project Summary Pulmonary arterial hypertension (PAH) is characterized by a progressive increase of pulmonary vascular resistance and obliterative pulmonary vascular remodeling that result in right heart hypertrophy, failure, and premature death. The underlying mechanisms of vascular remodeling and obliterative vascular lesion formation remain unclear. Fatty acid metabolism dysfunction is linked to PAH. However, the mechanistic role of fatty acid metabolism in regulating pulmonary vascular remodeling in the pathogenesis of PAH has not been reported. We hypothesize that endothelial fatty acid-binding proteins 4 and 5 (FABP4-5) regulate endothelial glycolysis and arterial programming through HIF-2a/SOX17 signaling which contributes to severe vascular remodeling in the pathogenesis of PAH. We will 1) define the novel role of endothelial FABP4-5 in the pathogenesis of PAH using multiple transgenic animal models. 2) delineate the cellular and molecular mechanisms that FABP4-5 induces arterial programming and pathogenesis of PAH. Completing our proposed study will provide a novel therapeutic strategy for the effective treatment of PAH in patients.

NIH Research Projects · FY 2026 · 2023-04

Project Summary Non-specific low back pain (LBP) is a highly prevalent and costly health condition characterized for many by recurrent, fluctuating or persistent pain and limitations in function over time. The overall objective of this project is to curtail the costly long-term course of LBP. The goal of the current project is to understand the role of specific spinal movement impairments to the course of recovery of people presenting with acute LBP, and to examine the immediate and short-term effects of treating the impairments in people with acute LBP. Spinal movement impairments in people with acute LBP are of particular interest because the impairments have been found to be highly relevant to the clinical course in people with chronic LBP. Our central hypothesis is that the spinal movement impairments will 1) be prevalent in patients with acute LBP, 2) persist to varying degrees over time, 3) be related to the course of recovery of acute LBP and function, and 4) be a viable target for treatment in the acute stage. We will conduct a prospective, observational cohort study of 212 people (ages 18-60) who have acute LBP but do not have a history of chronic LBP. We will collect 1) measures of spinal movement impairments, 2) self-report surveys of patient characteristics, LBP history, psychosocial behavior, comorbidities, treatment use, imaging, LBP intensity and functional limitations, and 3) findings from our clinical exam. The measures of movement, self-report surveys and exam data will be collected at baseline of the acute episode, and at 2 and 6 months thereafter. We will collect a subset of the self-report surveys electronically weekly from baseline-to-8 weeks and monthly from 3-12 months. At baseline and 2 and 6 months after the acute episode we will 1) examine the prevalence of the impairments, 2) test if the degree of the impairments at baseline, as well as change over time in the degree is related to LBP and functional limitations, and 3) test if the degree of impairments at baseline and change in impairments over the 6 months after the acute episode will predict transition to chronic LBP. To test the effect of treating the impairments in the acute stage, we will conduct an early phase learning trial (Phase 2A) in a 2nd sample of people (n=68) who present to an Emergency Department (ED). We will randomize people to 1) motor skill training in functional activities (MST) + usual care (UC) or 2) UC. MST will be provided in the ED and twice in the following 2 weeks. In the ED and 2 weeks after the ED visit we will collect self-report surveys and movement data. Then for each of 10 weeks, we will collect a subset of the self-report surveys electronically. We will test the immediate (within-session) and short-term (2 and 12 week) effect of MST+UC to UC. Expected outcomes will be specific information about the 1) change in prevalence of the impairments over the course of recovery, 2) relevance of change in the impairments to change in the person’s pain and function over time, including the transition to chronicity, and 3) effects of treating the impairments. Successful completion of this project will have an immediate, high impact on research and evaluation and treatment of people with acute LBP that could improve the costly course of LBP for people who have incomplete recovery from an acute episode.

NIH Research Projects · FY 2026 · 2023-04

ABSTRACT Atopic dermatitis (AD) is an increasingly common relapsing-remitting skin disease characterized by chronic allergic inflammation and itch. The etiology of AD is unknown, but type 2 cytokines, particularly interleukin (IL)- 4 and IL-13, are involved in driving AD and AD-like pathology, as demonstrated by the efficacy of therapies targeting these cytokines in human AD. Among type 2 cytokine producers, basophils and group 2 innate lymphoid cells (ILC2s) are dysregulated in human AD and are major sources of type 2 cytokines in mouse models of allergic inflammation, yet how these cells are triggered in AD and AD-like disease remain unresolved. Our recent single-cell RNA sequencing (scRNAseq) analysis identified high expression of IL-18 receptor (IL-18R, Il18r1) among skin ILC2s in comparison to ILC2s isolated from other tissues, and we further observed robust production of type 2 cytokines from IL-18-stimulated skin ILC2s, indicating that IL-18 is a candidate driver of type 2 cytokine production in AD-associated allergic skin inflammation. Basophils are also potently activated by IL-18 to produce type 2 cytokines, and our studies have demonstrated that basophils mediate acute itch flares in the context of AD-like disease, suggesting that IL-18 may mediate aspects of AD- related itch. Additionally, we find that IL-18 is elevated in both the skin and plasma of patients with moderate- to-severe AD, and that ILC2s and basophils exhibit unique activation profiles in human AD. Together, these data suggest that IL-18 mediates ILC2 and basophil functions to influence itch and AD pathogenesis. Thus, we hypothesize that IL-18 is a key regulator and potential therapeutic target in AD. In this project, we will determine how IL-18 influences basophil and ILC2 effector functions in the context of AD-associated inflammation and itch behavior. To do this, we will test several novel mouse strains in models of AD-like skin inflammation and translate these findings to human AD settings using innovative spatial transcriptomics approaches. We plan to test our hypothesis and accomplish our overall objective by pursuing three specific aims: 1. Test the contribution of IL-18 to skin ILC2 responses and AD-like disease. 2. Determine whether IL-18 activates basophils to trigger acute itch flares. 3. Examine the relationships between IL-18, ILC2s, and basophils in human AD by spatial transcriptomics. Understanding of how IL-18 contributes to the initiation and propagation of AD in mouse models and in human disease may advance new therapeutic approaches designed to ameliorate inflammation and itch in AD.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ ABSTRACT The purpose of an internal circadian clock is to generate a series of phases - time markers across the day and night by which different aspects of physiology and behavior (e.g., sleep, hormone release, temperature elevations) may be aligned to local time for optimal efficiency. We know a great deal about the molecular mechanisms of the clock (the timekeeping system) and how it is sensitive to local time. We know much less about circadian output, and specifically how the clock generates multiple phases across the entire solar day. Such phases are used by other clock cells, and by non-clock bearing downstream cells and circuits. Our laboratory studies circadian neurophysiology and the overall goal of this project is to understand the generation and usage of different circadian phasic outputs. The work is performed in the model system Drosophila. It builds on observations and a model we created from our past studies of the neural circuit in the Drosophila brain that controls daily locomotor behavior. In the fly brain, ~150 dedicated circadian pacemaker neurons direct daily rhythmic physiology and behavior. These 150 pacemakers are highly synchronized: they all tell the same time. Our model features the cell-intrinsic molecular clock in all pacemakers directing a morning phase of heightened neuronal activity. Yet, different subsets of pacemakers are not all active in the morning, but at different and stereotyped times of the day and night. The diversity of active periods (circadian phases) is generated primarily by cell interactions (especially neuropeptide modulation) and together these activity periods represent the multi-phasic outputs of the pacemaker system. Overall, this research program aims to extend and test this model by providing a cell- and molecular-level understanding for how circadian phase information is transmitted beyond the pacemaker system and received by downstream target circuits. Our work in Drosophila will likely inform our understanding of circadian output in the mammalian brain, and will also be relevant more generally to the mechanisms of neural circuit modulation by neuropeptides.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Peripheral artery disease (PAD) affects 8-10 million people in the US. Clinical trials evaluating stem cell, growth factor, or gene therapy systems for the treatment of PAD have shown some promising results. Use of biomaterial matrices either to enhance therapies or as a standalone treatment are just beginning to be explored in small animal models of PAD, with promising findings indicating that a biomaterial strategy can enhance the efficacy of intramuscular cell therapies in treating the effects of leg ischemia. There are important requirements for optimal delivery, retention, and performance of a bioengineered composite in the mechanically, histologically, and biochemically dynamic intramuscular environment of the PAD leg. The material should: (a) undergo minimal swelling once inside the target tissue; (b) have proper mechanical properties with high resilience to tolerate repeated compressive strain during muscle contraction for its long-term intramuscular retention; (c) be porous enough to facilitate the exchange of trophic factors with the surrounding environment and to permit recruitment of host progenitor and endothelial cells; and (d) have antioxidative and angiogenic properties that can be beneficial to the management of the myopathy of PAD. The objective of the current proposal is to characterize and optimize a biomaterial-based treatment for PAD. We have recently developed an injectable, angiogenic, nanofiber-hydrogel composite with unique interfacial bonding between the hydrogel matrices and the fibers, and successfully applied the composite for the regeneration of soft tissue defects in a rabbit model. We have further modified the hydrogel to have antioxidant properties with minimal swelling and optimized mechanical characteristics to mimic skeletal muscle. Testing in a rat model of PAD, the hydrogel reduced lipid oxidation, enhanced local blood flow in the muscle, and improved running capacity of the treated rats. In addition, we have developed and validated a porcine model of hindlimb ischemia (iliofemoral artery ligation/excision), which recapitulates key aspects of the pathophysiology of human PAD/claudication and can be a platform for the development of therapies for PAD. We are now primed to develop and test our novel therapies for PAD in our porcine model. We have all of the tools in place to address the central hypothesis that a nanofiber-hydrogel composite with optimized mechanical, angiogenic, and antioxidative characteristics will improve hemodynamic, histologic, and physiological endpoints of the ischemic hindlimb in rat and porcine models of PAD. Successful completion of this project will deliver the first off-the-shelf synthetic composite matrix for the treatment of PAD patients. As providing local therapy for the ischemic leg is critical to prevent myopathy and to improve the performance of the affected lower limbs in PAD patients, this study will provide an important advancement over other currently available treatments for PAD. The composite developed in this project can also be readily applied to treat other disease entities, including skeletal and possibly heart muscle pathologies related to ischemia/reperfusion, trauma, infection, and inflammation.