Washington University

NIH Research Projects · FY 2026 · 2022-12

ABSTRACT Neutralizing antibodies are critical for protection from infectious diseases. The lymph node (LN) germinal center (GC) is the site where B cells undergo antibody affinity maturation and develop into long-lived plasma cells – key events that are required for the development of highly effective neutralizing antibodies following infection or vaccination. T follicular helper cells (TFH) are the CD4+ T cell subset responsible for providing B cell help during an ongoing GC. TFH are absolutely required for GC formation and maintenance. By extension, TFH are necessary to produce effective neutralizing antibodies following antigen exposure. While many aspects of the TFH response and TFH function have been examined in animal models, human TFH responses in the draining LN have only recently been explored with the novel application of an established technique: serial ultrasound-guided fine needle aspiration of draining LN following vaccination. With this technique, we recently demonstrated that the antigen-specific TFH response to SARS-CoV-2 spike (S) protein mRNA vaccination persists in the GC for more than 4 months following vaccination and correlates with the presence of S-specific GC B cells. Furthermore, we have noted substantial LN TFH transcriptional phenotypic changes suggestive of functional maturation over this prolonged GC time interval using single cell RNA-seq in a small preliminary cohort. In this proposal, we will expand upon these findings to address our primary hypothesis: human TFH phenotypic maturation occurs over time in the draining LN following vaccination and these phenotypic changes are associated with changes in TFH function. To explore this hypothesis, we propose three specific aims: 1) We will first establish that TFH phenotypic maturation occurs over time in multiple antigen-specific TFH populations that we will define and characterize from a cohort of fourteen COVID-19 mRNA vaccine recipients using single cell RNA-seq and ex vivo epitope identification methods. 2) We will determine how these antigen-specific TFH populations change in a tertiary immune response following COVID-19 mRNA vaccine “boost” and continued serial LN sampling of the same cohort participants. We will also ascertain if new antigen-specific TFH populations are recruited to the GC during a recall response. 3) Finally, we will verify that the profound phenotypic changes we observe lead to changes in the functional capacity of antigen-specific TFH to provide help to B cells using an ex vivo system of sorted human LN TFH and an in vivo murine adoptive transfer model. By addressing these aims, we will significantly enhance our understanding of the role that human TFH play in directing GC B cell responses to vaccination.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY The discovery of pattern recognition receptors (PRRs), including toll-like receptors (TLRs) and NOD-like receptors (NLRs) has led to the investigation of molecular agonists of innate immunity in adjuvant formulations. An emerging paradigm is that careful selection of adjuvant combinations can result in complementary and even synergistic enhancement of vaccine-induced immune responses. Most combination adjuvants under investigation are chemically heterogeneous mixtures of depot adjuvants mixed with PRR agonists and suffer from batch-to-batch variability and poor chemical definition making investigation of mechanisms and safety a challenge. Our lab investigates self-assembling peptide nanofibers (PNFs) as vaccine adjuvants. A key advantage of PNFs over emulsion adjuvants is that the primary sequence of the self-associating peptide can be designed to control the physicochemical features of PNFs such as morphology, charge, chirality, or hydrophobicity, which are key contributors to adjuvant activity. Mechanistic insights into the mode of action indicates that unlike PAMPs, PNFs do not cause DC maturation but facilitate the release of DAMPs related to osmotic/oxidative stress. In this application, we propose to develop combination adjuvants composed of chemically defined DAMP-inducing peptide nanofibers (PNFs) and TLR2/NOD2 agonists. Our objectives are to understand how molecular mechanisms of DAMP-inducing PNFs and PRR agonists orchestrate innate immune signaling and induce responses that are complimentary, synergistic, or inhibitory for balancing immunogenicity with safety. In aim 1, we will examine the effect of PNFs with varying physicochemical properties and TLR2 or NOD2 agonist combinations on DC activation, DAMP release, and antigen presentation. In aim 2, using a design of experiments (DOE) approach, we will develop an optimal formulation with precisely controlled PNF-TLR2 and PNF-NOD2 combinations and determine the molecular mechanisms of innate immunity in DCs using various KO mouse models. In aim 3, we will validate the efficacy of PNF-TLR2-NOD2 combination adjuvants and investigate translational potential using human DCs. Outcomes of the proposed studies will advance our understanding of the molecular mechanisms that mediate innate immune responses to PNF-PRR agonist adjuvant combinations and will lead to new combinatorial- adjuvant platforms for combating infectious and non-infectious diseases with high translational potential.

NIH Research Projects · FY 2026 · 2022-12

ABSTRACT Supravalvular aortic stenosis (SVAS) is characterized by focal narrowing of the aorta that increases the risk for sudden cardiac death. SVAS is caused by mutations in the elastin gene that lead to decreased elastin amounts and there are currently no pharmaceutical treatments. The mechanisms by which elastin insufficiency cause SVAS are not well understood. Elastin is a critical mechanical component of the aorta and contributes to the passive stiffness (or modulus) that determines how much the aorta will deform (or strain) under applied hemodynamic stresses. Strain on smooth muscle cells (SMCs) within the aortic wall affects differentiation, proliferation, and migration. Cellular transmembrane channels, including Piezo1/2, are mechanosensitive molecules that transduce mechanical changes (such as strain) into biological effects (such as differentiation). Activation of Piezo channels leads to increases in intracellular calcium that can stimulate nuclear translocation of YAP/TAZ, which are transcriptional regulators of target genes including Ctgf. Ctgf is a known modulator of SMC phenotype that encourages dedifferentiation, migration, and proliferation - all characteristics affected by strain that may contribute to SVAS. Preliminary data in our unique SVAS mouse model (TaglnCre;Elnf/f) show a reduced aortic modulus that may increase SMC strain, increased Piezo2 and Ctgf expression in aortic SMCs, and a dedifferentiated aortic SMC phenotype. We hypothesize that SVAS is caused by altered SMC mechanotransduction when enough elastin is not laid down to stiffen the aortic wall and prevent increased SMC strain as stress increases with blood pressure during development. Increased SMC strain causes overexpression/activation of Piezo2, leading to increased intracellular calcium, nuclear translocation of YAP/TAZ, and increased Ctgf transcription that causes SMC phenotype modulation contributing to stenosis. We will address our hypothesis through three complementary aims using TaglnCre;Elnf/f mice and human SMCs derived from induced pluripotent stem cells from SVAS patients. In Aim 1, we will measure the global and local elastic modulus of TaglnCre;Elnf/f aorta and SMC strain under physiologic loading conditions at different developmental time points (before and after stenosis formation) and correlate these results with changes in SMC phenotype as measured by single cell RNA-Seq. In Aim 2, we will apply strain to mouse aorta and mouse and human SMCs and measure Piezo2 expression and activity. We will chemically and genetically alter Piezo2 expression/activity and determine effects on in vitro calcium signaling and in vivo stenosis severity. In Aim 3, we will chemically and genetically manipulate Piezo2 expression/activity, YAP/TAZ localization, and Ctgf amounts in mouse aorta and mouse and human SMCs and determine the effects on SMC phenotype and stenosis severity. Our results will be important for identifying new pharmaceutical strategies that may prevent SMC phenotype changes in response to elastin insufficiency and treat SVAS. .

NIH Research Projects · FY 2026 · 2022-12

ABSTRACT There is an unmet critical need for noninvasive methods to interrogate the genetic and molecular properties of the malignant brain tumor known as glioblastoma (GBM). Our group was the first to introduce the focused ultrasound (FUS)-enabled liquid biopsy technique for noninvasive and spatially targeted molecular diagnosis and characterization of brain tumors, which we term sonobiopsy. The current barrier to the widespread use of sonobiopsy in clinical practice is the lack of human data that rigorously characterizes the safety and feasibility of this technique. Our long-term goal is to transform the clinical management of GBM patients by providing genetic signatures of the disease using sonobiopsy. The overall obiective of this proposal is to perform a randomized, blinded, double-armed, single-center prospective clinical trial to validate the safety and diagnostic feasibility of sonobiopsy. We have strong preliminary data that demonstrated the feasibility and safety of sonobiopsy in rodent and porcine GBM models and also developed a neuronavigation-guided FUS system for performing sonobiopsy in patients. The proposed clinical trial will accomplish two specific aims: 1) Demonstrate enrichment of GBM plasma circulating tumor DNA (ctDNA) to a detectable level with sonobiopsy and 2) Define the safety profile of sonobiopsy in GBM patients. Under the first aim, we will enroll forty presurgical GBM patients who will be randomized (1 :1) for sonobiopsy versus sham. Immediately prior to surgical resection, sonobiopsy or sham will be performed, and plasma will be collected pre- and post-sonication. Genetic sequencing will be performed on plasma samples, as well as surgically resected sonicated tumor samples. We will compare: 1) the frequency of GBM-specific variants in the blood pre- and post-sonobiopsy, 2) the differences in ctDNA enrichment between sonobiopsy and sham, and 3) the concordance in mutation detection between post-sonobiopsy plasma samples and standard sequencing of tumor samples. For the second aim, because the sonobiopsy or sham intervention will be performed in an intraoperative MRI surgical suite prior to resection, we will use MR imaging to define the blood-brain barrier permeability changes and detect tissue injury or edema/hemorrhage at the surrounding healthy tissue and sonicated sites if they occur, Histological analysis of surgically resected sonicated and un-sonicated tumors will be performed to define the safety profile of sonobiopsy by staining for hemorrhage, necrosis, apoptosis, and inflammation markers. This project is innovative because it is a substantial departure from the status quo by using FUS in a novel fashion to substantially increase the presence of tumor biomarkers in the blood. The proposed research is significant because it will establish the foundation of knowledge to enable the translation of this innovative technique and ultimately advance the diagnosis and monitoring of brain cancer patients by identifying genetic signatures of the tumor without surgery. In addition to the standard diagnostics of anatomic imaging and surgical histology, sonobiopsy has the potential to become the third pillar for brain tumor management which will have a dramatic impact on patient survival and quality of life.

NIH Research Projects · FY 2025 · 2022-12

PROJECT SUMMARY CD8 T cells are critical for immune surveillance of both intracellular pathogens and cancer. Chronic and acute antigen exposure entails distinct differentiation trajectories of CD8 T cells. In acute infection, naïve CD8 T cells that recognize antigen undergo clonal expansion and differentiate into either cytotoxic effectors or quiescent central memory cells (Tcm) that mediate rapid responses to subsequent infections. In settings of chronic antigen exposure, such as cancer, CD8 T cells instead differentiate into to terminally exhausted (TEX) cells. TEX have attenuated proliferative, cytokine-secretory, and cytolytic functions relative to the effector cells elicited by acute infection. Although they still exert a significant level of viral control, TEX are unable to sustain durable responses and require constant replenishment from a pool of quiescent, stem-like T progenitor exhausted (TPEX). Importantly, immune checkpoint blockade therapy acts on TPEX to mobilize T cell responses to tumor antigens. Despite the therapeutic imperative to elicit Tcm in response to vaccination and TPEX in responses to cancer, the molecular mechanisms by which these T cell subsets develop are incompletely understood. It is known that the transcription factor (TF) TCF-1 is required for both Tcm and TPEX populations and is downregulated during the differentiation of effector and exhausted CD8 T cells. Although pro-inflammatory cytokines such as IL-2 and IL-12 are known to induce the downregulation of TCF-1 (encoded by Tcf7), it is unclear how these extracellular cues are integrated to regulate TCF-1 expression during the fate decisions of effector and TEX differentiation. It is also incompletely understood how TCF-1 contributes to the establishment of unique transcriptional profiles of TPEX and Tcm despite the dependency of both cell subsets on this TF. The proposed study seeks to address these gaps in knowledge by testing the central hypotheses (1) that self- amplification of IL-2 receptor signaling through upregulation of the IL-2R alpha chain (Il2ra) promotes downregulation of TCF-1 by direct repression of Tcf7 by the IL-2-inducible TFs AP4 and Blimp1 and (2) that differential engagement of TLE family co-repressors and intrinsic HDAC activity by TCF-1 in TPEX and Tcm result in subset-specific transcriptional regulation by TCF-1. The research project will interrogate the necessity of IL- 2R self-amplification in terminal differentiation by ablating IL-2R signaling-responsive cis-regulatory elements in the Il2ra and Tcf7 loci in mouse models of chronic and acute infections. The requirements of TLE co-repressor recruitment and HDAC activity in TPEX and Tcm will be interrogated by complementing Tcf7-deficient CD8 T cells with Tcf7 mutants deficient in either HDAC or TLE co-repressor binding activity and infecting mice with chronic or acute LCMV. The proposed research project is expected to advance the long-term objective of determining the molecular basis by which Tcm and TPEX fate decisions are made, with the ultimate goal of engineering durable CD8 T cell responses to infection and cancer.

NIH Research Projects · FY 2026 · 2022-11

The past decade has seen a significant increase in the incidence of emerging virus infections that transmit directly from animals to humans or are vectored by mosquitos and ticks. Changes in demographics and climatic factors likely contribute to this increase. In the United States, ticks are the dominant vector for transmitting arthropod borne diseases including the Thogotovirus, Bourbon virus (BRBV) which can cause fatal disease in humans. Like other enveloped viruses, entry of BRBV begins with attachment to host-cell receptor molecules with subsequent membrane fusion to deliver the contents of the virion into the host cell. Viral attachment and fusion proteins are targeted by antibodies that contribute to our natural immunity against viruses and are consequently a validated therapeutic target. The single envelope glycoprotein (GP) of BRBV is responsible for both attachment to cellular receptors and catalyzing fusion. BRBV GP is structurally related to the envelope glycoprotein G of vesicular stomatitis virus, gB of herpes simplex virus, and GP64 of baculovirus which collectively are termed class III fusogens. Our understanding of class III fusogens and their inhibition lags behind the class I and II viral fusogens, exemplified by influenza HA, and flavivirus E respectively. We have developed a set of unique tools and reagents that will allow us to characterize existing and newly developed monoclonal antibodies against BRBV GP both structurally and functionally. We will identify those that are potently neutralizing, identify their mechanism of inhibition, test the efficacy of neutralizing and non-neutralizing antibodies in a mouse model of disease, and identify the breadth of related thogotoviruses against which such antibodies function. Using chimeric VSV reporter viruses that depend on the GP of BRBV for infection, we will define the precise step in entry by which specific antibodies impede infection. We will also determine the entry pathway of BRBV into cells and define the host requirements for this process. In preliminary data, we carried out a CRISPR inactivation screen that identified glucosylceramide synthase (UGCG) as an important host factor for entry of BRBV into mammalian cells and demonstrate related thogotoviruses are also dependent upon UGCG. Using a combination of genetic approaches, coupled with pharmacological inhibition and imaging of single virions during entry, we will precisely delineate the requirement for UGCG in entry, and identify and characterize additional host factors coopted during this process. Genetic and structural studies will permit us to map the critical determinants on GP required to coopt host-factors during the entry pathway, which will synergize with our antibody studies to provide a detailed mechanistic picture of entry and its inhibition. Successful completion of the proposed studies will provide new insights into the mechanism and structural requirements for attachment, internalization and membrane fusion driven by a class III fusogen, uncover the mechanism by which the host glucosylceramide synthase functions in entry, and identify antibodies that target GP to block those critical functions.

NIH Research Projects · FY 2026 · 2022-11

Project Summary/Abstract Events in early life, particularly those altering the gut microbiota and immune development, can have an important role in determining future risk of immune related disorders. For example, it has been reported that the how substances are encountered by the immune system, the timing of feeding practices, hygiene, and the use of antibiotics, in early life can modify the risk of immune driven disorders later in life. A common theme amongst many of these studies is that these risk factors likely affect the developing microbiota. However, the biologic basis for how early life microbial changes can affect the risk of immune disorders later in life is incompletely understood. Previous data from our laboratories have suggested that during this preweaning period, specific live gut bacteria are selectively delivered to the immune system and that interaction of these live bacteria with cellular immune populations locally in the colonic lamina propria or in distant lymphoid tissues establishes a durable (life- long) and balanced immune system. In preliminary studies, we observed substantial variance in the amount of bacterial translocation in this preweaning period, which was corroborated with studies of antigen-specific T cell responses to a translocating bacterial spp. We therefore hypothesize that the magnitude and quality of the immune response to these translocating bacteria species during the preweaning period is a stochastic event during normal ontogeny which represents an important variable in determining whether an individual establishes a balanced immune system and prevents immune mediated pathologies in later life. However, the nature of these translocating bacteria and methods to quantify the antigen-specific responses to these bacteria remain a gap in our knowledge. We will address this question by identifying and characterizing how the translocating bacteria are encountered by the immune system (Aim 1) and generate reagents to track bacterial antigen presentation via T cell responses in vivo (Aim 2). We will use these reagents to integrate the immune response over the preweaning period to translocating bacterial spp. during physiologic development and correlate it with intestinal immune challenges including infection and experimental colitis in later life (Aim 3). If successful, this project may reveal that preweaning translocation is an important stochastic developmental variable involved in generating intestinal immune health and homeostasis.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Cognitive load during the delivery of clinical care is affected by a number of factors including specialty, practice setting, patient complexity, electronic health record (EHR) use, and clinician expertise. In the United States, clinical care is primarily documented using EHRs: documentation burden, poor usability, and unnecessary navigation contribute to increased cognitive load. Such increases in cognitive load, in turn, contribute to more work hours, dissatisfaction with work, poor patient outcomes (e.g., errors), burnout and poor clinician outcomes. Much of the prior research characterizing clinician cognitive load or its impact on errors has relied on retrospective approaches including self-reports, time-motion studies, and focus groups. Similarly, burnout also has been measured exclusively using surveys. EHR-based audit logs have shown considerable promise as a viable resource for tracking and measuring clinical activities without the incremental survey burden on clinicians. Our research team has demonstrated that workload measures based on audit logs can be used to assess cognitive load, burnout, and errors. Based on this promising pilot work, the primary focus of the IGNITE (Integrating real-time clinical activity and behavioral responses for characterizing cognitive load and errors) study is to utilize EHR-based audit logs and decision support tools to objectively determine the direct relationships between (a) cognitive load and errors, and (b) the mediating role of clinician burnout in explaining the relationship between cognitive load and errors. We will accomplish this through a large-scale multi-site study conducted at three large academic medical centers associated with Washington University/BJC HealthCare, Stanford University, and University of Colorado. For the first aim, we will utilize EHR-based audit logs collected across non-surgical, inpatient settings across three sites over a 3-year period (1/1/2019 to 12/31/2021) to develop measures of cognitive load—both intrinsic and extraneous—and assess the effect of cognitive load on objectively measured wrong-patient errors (using the retract-and-reorder alerts). For the second aim, we will prospectively collect data on a cohort of 300 trainees (residents, fellows) from Medicine and Pediatrics from each study site over a 5-month period. Monthly burnout surveys, along with cognitive load measures from the EHR-based audit logs, and wrong-patient errors during the study period will be used to determine the mediating relationships between cognitive load, burnout and errors. In addition, for both of the proposed aims, we will develop advanced machine learning algorithms to predict errors and burnout from EHR-based activity sequences. Insights from this study will help in designing targeted interventions aligned with the contextual work practices of physicians, designing clinical trials for evaluating such interventions, and in developing informed policy guidelines for the safety and well-being of physicians.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY: The overall goal of this project is to develop and comprehensively validate ultra-low count quantitative SPECT (ULC-QSPECT) methods for alpha particle radiopharmaceutical therapies (αRPTs), including in a first-in-man trial in patients with bone metastatic castration-resistant prostate cancer. αRPTs, such as those based on Actinium-225, Thorium-227 and approved Radium-223 isotopes, are an emerging class of cytotoxic therapies for patients with disseminated metastatic disease using internally administered alpha-particle emitting agents. Despite the great potential of these therapies, current αRPT regimens are not personalized, with administered activity dependent merely on mass, likely leading to non-optimal therapy. To address this challenge, there is a crucial unmet need for methods to measure the isotope uptake, and hence the absorbed radiation dose with these therapies, both at sites of disease and in vital dose-limiting organs. SPECT provides a clinically translatable mechanism to achieve this goal. However, a key challenge to SPECT-based quantification is that the administered activities in αRPTs are orders of magnitude lower than a typical SPECT scan, leading to ultra-low detected count levels. Conventional approaches to quantification that reconstruct the isotope distribution and estimate the regional uptake from reconstructed images are erroneous at these low count levels. To address this issue, we put forwards a novel computational ULC-QSPECT framework for regional activity estimation from αRPTs. These methods quantify regional uptake directly from projection data skipping the reconstruction step, and at the same time, extract the maximal possible information from the acquired projection data. For this purpose, we propose novel methods that accurately model the physics of imaging αRPTs, including stray- radiation-related noise, use data from multiple-energy windows, incorporate scatter-window photons for quantification, and process data in list-mode format. Our extensive preliminary data show that the proposed methods result in nearly unbiased uptake and variances close to the theoretical limit. We propose to further develop and rigorously evaluate these methods. Our proposed evaluations include studies over multiple scanners with different detectors and different collimator configurations. Further, our goal is clinical translation of these methods. Towards this goal, we propose to clinically evaluate these methods for measuring activity concentrations at sites of uptake of [223Ra]RaCl2 in men with castrate resistant prostate cancer. These methods will enable quantification of activity at disease sites in the skeleton as well as clearance through the intestine. The approach has direct relevance to patients as it achieves noninvasive imaging of low-administered activity therapies. Further, this proposal has substantial potential impact to improve both safety and efficacy of drug development efforts in this rapidly evolving space. Further, the methods developed in this proposal will strengthen the clinical utility of SPECT in managing patients with these therapies.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT – COMPONENT A Influenza and SARS-CoV-2 are major causes of morbidity and mortality and constitute the leading causes of vaccine preventable deaths in the United States. A better understanding of vaccine effectiveness for these viral pathogens is critical to drive public health decisions and interventions. We propose utilizing a multidisciplinary approach to conduct a test-negative study to determine influenza and SARS-CoV-2 vaccine effectiveness in ambulatory patients with respiratory tract infections. The team of investigators includes experts in emergency medicine, infectious disease, pediatrics, epidemiology, information technology, molecular microbiology, virology, and genetics. This team has extensive experience in automated electronic medical record alerts, high-volume subject recruitment of ambulatory patients with respiratory tract infections, rapid escalation/de-escalation of recruitment efforts to match viral circulation patterns, respiratory and blood sample processing and shipment, quality data collection and verification, and viral genomic sequencing necessary to ensure the success of this project. The proposed study will encompass the following specific aims: 1)Utilize innovative automated alerting strategies to identify and recruit a diverse population of ambulatory patients with acute respiratory illnesses; 2) Estimate influenza and SARS-CoV-2 vaccine effectiveness using a test- negative study design in the general population as well as different demographic subgroups.; 3) Explore factors that influence influenza and SARS-CoV-2 vaccine effectiveness such as co-morbidities, vaccination type and schedule, and social determinants of health; 4) Determine effect of viral vaccination status on health outcomes in ambulatory patients with influenza and SARS-CoV-2 infection; 5) Contribute biospecimens and viral genomic sequencing data to a national repository of subjects with PCR-confirmed influenza or SARS-CoV-2 infection. To accomplish these goals, we will enroll at least 1000 ambulatory patients/year with acute respiratory tract infections in the proposed study. The subject population will be identified from the emergency departments of 3 large hospitals in the St. Louis area and their associated outpatient clinics. The available patient population at these enrolling sites is diverse with respect to race, ethnicity, age, socioeconomic status, and medical care access which will enhance the generalizability of the study outcomes to the US population.

NIH Research Projects · FY 2025 · 2022-09

Abstract: Tens of millions of Americans suffer from chronic pain. Opioids represent the main tool for treating pain, but their use in chronic pain conditions suffers from a poor evidence base and the inherent risk of addiction. The current crisis of opioid-related deaths highlights the risk associated with widespread opioid use. The PRECISION Human Pain Network, part of the NIH HEAL Initiative, seeks to provide foundational data on the diversity among cell types that comprise the pain neuraxis. Here, we propose a U19 program entitled “Integrated Research Center for Human Pain Tissues” (INTERCEPT Pain). We will build on prior successes in our human tissue research program, and expand the scope of work to directly address the goals of the PRECISION Human Pain Network. Our program leverages the world-class genomics and computational assets of the McDonnell Genome Institute here at Washington University and substantial institutional strengths in neurobiology of pain, axon degeneration and regeneration, and genetics in a coordinated program to develop foundational new knowledge regarding the transcriptional and functional properties of tissues and cells involved in pain transduction, transmission and modulation in humans. We will provide a comprehensive atlas of cellular gene expression in human peripheral nerve using single nuclei RNAseq, spatial transcriptomics and multiplex proteomics, and leverage this to understand the distribution of genes involved in traumatic (neuromas) and idiopathic (hereditary) painful neuropathies. We will also provide expanded single-nucleus sequencing atlases of dorsal root ganglia, including DRG from donors without and with a recent history of pain. We will generate a spatial atlas of hDRG using imaging mass cytometry, and optimize computational approaches for integrated IMC and single cell transcriptomic analysis of hDRG. Finally, we will combine analysis of electrophysiological, transcriptional, and morphological data from hDRG and human spinal cord dorsal horn neurons. We will work with other centers to optimize protocols and data collection to allow integrated analysis across multiple centers, in collaboration with the U24 DCIC and HEAL data ecosystem. These goals will be accomplished through the coordinated activity of 3 scientific projects, led by international leaders in the fields of pain neurobiology, genetics, and mechanisms of axon degeneration and regeneration. The project titles are as follows: Project 1: Multi-omics peripheral nerve atlas enables fine-mapping of pain molecular phenotypes. Project 2: Characterization of the human dorsal root ganglia at the single cell level via integrated transcriptomics and spatial proteomics Project 3: Functional and genetic characterization of human DRG and spinal cord at single cell resolution The projects are supported by an experienced program leadership team and an administration core, a human tissue procurement and processing core, and a data core.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Within each mammalian cell is a core set of circadian clock proteins that regulate the cellular biology supporting more complex organ physiologies. The central nervous system, entrained by the principle environmental cue – light, synchronizes these peripheral cellular ‘clocks’ across the entire organism, which enables the host organism to anticipate and prepare for the stresses of the active day (e.g., metabolic demands, septic threat). The current evidence, including data derived from our own research program, highlight the profound influence individual clock proteins have on the physiologic capacity with which an organism responds to stress. We first identified that the spectrum of light is a critical determinant of its effects on mammalian biology, and that the short wavelength visible blue spectrum favorably modifies the biology and outcome of sepsis. In murine models of intraabdominal sepsis and Klebsiella pneumoniae (KP) pneumonia, exposure to blue light after sepsis enhanced immune competence, as evidenced by more efficient clearance of bacteria from the septic focus, reduced bacterial dissemination, and attenuated systemic inflammation. The mechanism involved an optic-cholinergic pathway that induced the clock protein Rev-Erba in immune tissues of the spleen and Mj. A Rev-Erba agonist similarly enhance immune function in both in vitro and in vivo studies. Our overarching hypothesis is that the cellular state of the clock protein Rev-Erba is a critical determinant of immune competence and can be modulated to improve the outcome of sepsis. We specifically hypothesize that Rev-Erba regulates the protein machinery supporting the immune phenotype of the mononuclear phagocyte and B cell and is vital to an efficient immune response to microbial threat. In Aim 1 we will study the physiologic and cellular mechanisms by which blue light and the clock protein Rev-Erba regulate monocyte recruitment to the spleen and peripheral tissues and differentiation into a type of monocyte highly efficient in bacterial clearance, using a model of KP pneumonia. In Aim 2, we will explore the mechanisms by which blue light and Rev-Erba modulate B cell PI3K-AKT-mTOR signaling and actin assembly to mediate B cell differentiation, activation, MHC II antigen presentation, and antibody production. As these mechanisms are metabolically demanding and ATP-dependent, we will (Aim 3) determine the mechanisms by which Rev-Erba modulates mitochondrial dynamics to support oxidative metabolism and thereby the phenotype of immune cells during sepsis. The ramifications of light on health and disease remain to be convincingly defined. This proposal will define the biological mechanisms through which circadian clock proteins beneficially alter the host response to acute infectious insult. We will define the dimensions of light and state of clock proteins that are optimally protective and examine their biological relevance and potential therapeutic value in studies of patients with pneumonia.

NIH Research Projects · FY 2024 · 2022-09

Abstract Chimeric antigen receptor (CAR) T cells targeting CD19 are highly effective in children with refractory/relapsed acute lymphoblastic leukemia (ALL), including those with primary refractory or CNS disease. Current CAR T cell therapies infuse patients with T cells constitutively expressing CARs, which are not susceptible to any controllable regulation. Cytokine release syndrome (CRS) and CAR-associated neurotoxicity (CAN), both of which can be fatal, arise from uncontrolled CAR T cell activation and expansion. While a few pharmacological management approaches have been attempted to overcome this issue, they are often suboptimal. In addition, chronic B-cell aplasia from persistent CD19 CAR T cells requires monthly infusions of immunoglobulin, which is burdensome and expensive, especially for pediatric patients facing potentially a lifetime need. Here, we propose to develop a system for controllable CAR T cells that can be turned on and off as needed. We have previously demonstrated that exogenous expression of the tyrosine phosphatase SHP-1 acts as a negative regulator to dampen T cell activation. Recently, we have developed an inducible and reversible protein degradation system for SHP-1 by adapting the plant Auxin-induced degron (AID) system for T cells. Combining these two tools in Aim 1, we propose to develop CD19 CAR T cells that will be kept basally dormant through overexpression of SHP-1. However, upon administration of Auxin, the CAR T cells can be temporarily and reversibly activated through the degradation of SHP-1. As the doses of Auxin sufficient to activate the AID system had no significant toxicities in humans, we do not foresee a problem translating this system into the clinic. In Aim 2, we will examine the efficacy of this novel CAR T cell system in a murine model of ALL. In Aim 3, we will expand the studies to test whether this regulatable CAR T cells system can control and/or limit CAR T cell-associated toxicities using a muring model of ALL, CRS and neurotoxicity. Such an exogenously regulatable CAR T cell system may provide clinicians a tool to avoid/limit severe CRS and CAN, and allow repopulation of the B-cell compartment after a sufficient treatment course. This approach will greatly enhance the safety of CD19 CAR T cells and is likely applicable to CARs for other malignancies, including solid tumors, where on-target, off-tissue cytotoxicity is more problematic.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY A rotator cuff tear is a common shoulder condition that affects approximately 40% of individuals over the age of 60. This condition is painful, debilitating, and reduces quality of life. Despite their prevalence, the etiology of rotator cuff tears is not fully understood but is generally believed to involve extrinsic factors (i.e. tendon impingement during shoulder motion), intrinsic factors (i.e. tendon degeneration), and/or overuse. These factors have been studied extensively in animal models, which have provided support for each factor contributing to rotator cuff pathology. However, these findings have not yet been confirmed in human studies largely because of the difficulty in accurately and reliably assessing intrinsic factors and overuse in humans. Ultimately, understanding the etiology of rotator cuff pathology in humans will remain difficult without a model that characterizes the role of each factor in rotator cuff pathology. The objectives of the proposed studies are to: 1) develop a preliminary multivariable model classifying the effects of extrinsic, intrinsic, and overuse factors on rotator cuff pathology in asymptomatic individuals (K99); 2) extend the model with additional asymptomatic participants (R00); and 3) expand the model to include symptomatic participants (R00). Our approach will be to quantify shoulder motion and impingement (extrinsic factors) via biplane x-ray imaging, rotator cuff degeneration (intrinsic factors) via shear wave elastography, overuse factors via a novel estimate of lifetime shoulder exposure, and the severity of rotator cuff pathology via diagnostic imaging. We will investigate the relationship between the etiological factors and rotator cuff pathology using classification and regression tree analysis. The proposed studies are the keystone of a career development plan developed to provide the necessary mentorship, coursework, and research training for me to become an independent and impactful researcher. The goals of the K99 phase are to: 1) obtain training in advanced methods of biomechanical data collection and analysis to assess the roles of extrinsic, intrinsic, and overuse factors in the etiology of rotator cuff pathology; 2) develop a preliminary multivariable model describing the role of extrinsic, intrinsic, and overuse factors on rotator cuff pathology; and 3) obtain a tenure track position at a respected research- intensive university. The goals of the R00 phase are to: 1) independently conduct the R00 phase study by implementing the skills learned during the K99 phase; 2) establish multi-disciplinary research collaborations with engineers, orthopaedic surgeons, and physical therapists; 3) lead a well-funded and productive research laboratory; and 4) build upon the K99/R00 research findings to secure independent R01 funding. Together with the rich research environment at Henry Ford Health System, the proposed career development plan will ensure that I have a unique skillset to pursue an independent research career, produce sound and impactful research, and help prepare the next generation of scientists.

NIH Research Projects · FY 2025 · 2022-09

Vaccines, drugs, and modified human cells that activate the immune system against tumor can improve the outcomes and prolong the lives of patients diagnosed with some type of cancers, but have failed to provide survival benefits for patients with glioblastoma (GBM). Activation of the Stimulator of Interferon Genes (STING) pathway represents one of the main innate immune sensing pathway to enable natural killer (NK) and T cell priming against tumor. Intratumoral administration of STING agonists, in particular cyclic dinucleotides (CDNs), was shown to have significant anti-tumor effects in multiple cancer models, including orthotopic GBM models, and is currently being tested in a phase 1 clinical trial in advanced cancer patients (NCT0267754339). Limited bioavailability and stability, however, are limiting factors for clinical CDN development. We have shown that the formulation of oligonucleotides into SNA structures, i.e., the presentation of oligonucleotides at high density on the surface of nanoparticles, leads to biochemical and biological properties that are radically different from those of linear (“free”) oligonucleotides. These include the cellular uptake of SNAs by a wide variety of cells, the gene regulatory activity of SNAs functionalized with siRNA or antisense DNA oligonucleotides, and the TLR-agonistic activity of SNAs conjugated with immunostimulatory oligonucleotides. Importantly, clinical trials with first generation siRNA-based SNAs (NCT03020017; GBM), and toll-like receptor 9 (TLR9)-agonsitic SNAs (NCT03086278; solid cancers) have recently been completed. Our proposed research is to develop a first-in-class immunotherapy by targeting cGAS – the sensor of cytosolic dsDNA upstream of STING – with SNAs presenting interferon-stimulating DNA (ISD) oligonucleotides at high surface density, and to evaluate the potential of SNAcGAS for use in clinical neuro-oncology. This approach is distinct from other current approaches that target the STING pathway with CDNs and small molecules. By targeting cGAS, the strategy of using SNAsISD exploits the ability of cGAS to raise STING responses by delivering dsDNA and inducing the catalytic production of endogenous CDNs. Our use of SNAs addresses the challenges of delivery of therapeutic nucleic acids through the enhanced uptake of nucleic acids formulated as SNAs, and furthermore, exploits the polyvalent presentation of oligonucleotides at high density on a nanoparticle template. Here, the binding of closely-spaced, neighboring dsDNA molecules on the surfaces of SNAs should enhance the formation of 2:2 dimers of cGAS:DNA and thus lead to potent cGAS activation. In three Specific Aims, we will optimize the SNA platform for maximum cGAS-STING pathway activation in vitro and in vivo (Aim 1), assess anti-tumor effect of our lead SNAcGAS architectures together with additional high-activity SNA constructs in vivo (Aim 2), and evaluate treatment regimens combining SNAcGAS with prioritized immunotherapies, including check point blockade and pharmacological strategies to inhibit adenosine signaling (Aim 3).

NIH Research Projects · FY 2025 · 2022-09

Cervical cancer is a major cause of morbidity and mortality in many sub-Saharan countries, including Nigeria. Expanding human papillomavirus (HPV) vaccination for young girls/women, ages 9-26 years (primary prevention) and HPV self-collection for cervical cancer screening for older women, ages 30-49 (secondary prevention) are both critical to achieving WHO and Nigerian goals for reducing the cervical cancer burden. The Nigerian government now recommends HPV vaccination and self-collection, yet uptake is poor. As such, context-specific, targeted and culturally relevant implementation strategies are needed. We focus on HPV vaccination among girls and self-collection among women because of the substantial burden of cervical cancer. Mother-daughter relationships in Nigeria can be leveraged to increase HPV vaccination uptake among young girls and HPV self-collection among mothers. Mothers (or similar female caregivers) profoundly influence decisions and preferences about young girls’ vaccine uptake in the Nigerian cultural context. At the same time, maternal choices about HPV self-collection can be reinforced in discussions with their daughters. In the proposed study, Actions for Collaborative Community-Engaged Strategies for HPV (ACCESS-HPV), we will use participatory crowdsourcing methods to drive HPV prevention among mother-daughter dyads. Crowdsourcing open calls will allow us to identify locally relevant messages and dissemination techniques to increase uptake of HPV prevention. Then, participatory learning communities will build capacity for community-led implementation of selected strategies. Informed by social learning theory and the PEN-3 cultural model, our multi-disciplinary research team proposes the following specific aims: (1) to develop a new combined campaign to increase HPV vaccination for young girls (ages 9-26) and HPV self-collection for mothers (ages 30-49) using crowdsourcing open calls and participatory learning communities; (2) to determine the effectiveness of a final combined campaign on uptake of HPV vaccination among young girls/women and HPV self-collection among their mothers using a stepped-wedge randomized controlled trial; (3) to estimate the impact and cost-effectiveness of the crowdsourced campaign. Our primary outcome will be vaccine uptake (ascertained by clinic records of vaccine uptake) among young girls and HPV self-collection (ascertained by laboratory receipt of specimens) among their mothers. The strong support of the Nigerian Institute for Medical Research (NIMR) alongside national HPV programs creates a rich research infrastructure and increases the likelihood of successful implementation. Our multi-disciplinary research team has experience organizing implementation research focused on crowdsourcing and community participation in Nigeria. This study will enhance our understanding of HPV prevention in resource-constrained settings. This grant application directly responds to strategic priorities of the United States government, the National Institutes of Health, the National Cancer Institute, and NOT-CA-20-025.

NIH Research Projects · FY 2024 · 2022-09

Project Summary/Abstract The goal of this proposal is to develop expert panels focused on curating evidence for the clinical application of somatic mutations associated with childhood cancers. Tumors in the pediatric population have unique genetic profiles that can affect their diagnosis, prognosis and treatment. There is currently a gap in representation of somatic variants for childhood tumors in public cancer databases and knowledgebases. New approaches for evidence curation are needed to identify important mutations in childhood cancers for both diagnosis and therapy response. To address these gaps, our application builds on two prominent developments in the field led by our team. First, as the ClinGen Somatic Clinical Domain Working Group (CDWG) we developed the Minimal Variant Level Data (MVLD) standard to promote sharing and use of gene variants in precision oncology. Second, we developed the Clinical Interpretation of Variants in Cancer (CIViC) expert crowdsourced platform for somatic curation and clinical interpretation. Our goals are to: (1) systematically catalog the clinical relevance of common, rare, and novel variants identified through gene-specific and genome-scale testing in childhood cancers; (2) partner with ClinGen, ClinVar, guideline-setting professional organizations, and other relevant global efforts to translate and present the knowledge derived from genome researchers and clinical laboratories; and (3) develop informatics support for variant assessment of clinical actionability, information display, interfacing with relevant databases, and dissemination. To accomplish these goals we developed a collaboration over the past three years through the ClinGen Somatic CDWG among researchers at Washington University, Georgetown University, and Children’s Hospital Los Angeles that includes clinical and molecular geneticists, genetic counselors, bioinformaticians, and genomic database experts to advance the use of genomic data in childhood cancers. Specifically, we will establish pilot variant curation expert panels (VCEPs) to assess clinical relevance and actionability of somatic variants in pediatric cancers. Our initial focus will be on two disease areas, with timely relevance, pediatric malignancies with NTRK fusions and BCR-ABL1 (Philadelphia)-like B-lymphoblastic leukemia (Ph-like B-ALL). We will adopt ClinGen’s existing VCEP policies and processes for assessing variants of strong clinical significance, potential clinical significance, unknown clinical significance, and benign or likely benign variants in childhood cancers. We will adapt and enhance the CIViC platform to support these expert panels. The CIViC platform will also provide the curated evidence in standard formats for exchange of data with ClinVar and ClinGen resources. Finally, we will seek FDA recognition for the evidence repository developed and curated through this grant.

NIH Research Projects · FY 2024 · 2022-09

Religion is a primary driver of concerns with genomics and genomic healthcare (GGH). It is vital for research on the ethical, legal, and social implications (ELSI) of genomics to understand the role of religion in shaping public attitudes toward GGH. Ninety percent of the US public believes in some kind of higher power and 55% pray daily. The US public attends religious services at a rate 3 times higher than scientists in the US, who are at risk of misunderstanding the role religion plays in attitudes toward GGH. Further, Black, Hispanic, and rural populations attend religious services more frequently than the general US public. Concerns with GGH activities may lead to lower levels of engagement of GGH, which may in turn contribute to health disparities. This project aims to understand why higher levels of religious practice are associated with greater concerns with GGH activities. Additionally, this project will interview faith leaders to identify how public health genomics might engage faith communities in ways that are respectful and constructive. While some concerns with healthcare technologies may disappear with new information, others stem from worldviews, deep moral commitments, or mistrust of the healthcare system. Engaging religious communities is essential to identify which concerns can be addressed with information, which call for alternative responses, and what alternative responses might look like. Very few models exist for genomic public health engagement with religion, particularly models that directly address value-laden concerns.13,14

NIH Research Projects · FY 2025 · 2022-09

Abstract/Project Summary Kidney disease is common and deadly with frequent onset in childhood. Kidney and urinary tract congenital anomalies account for most of the renal failure in children while, in addition, secondary acute kidney injury (AKI) occurs in up to 60% of neonatal and pediatric intensive care patients, directly correlating with length of stay, subsequent disability, and with early mortality. Kidney insults in childhood including ischemia, hyperoxia, infection and nephrotoxic drug/environmental exposures impair kidney maturation and function resulting in chronic renal disease (CKD) and with stealthier hypertension, renal stones and proteinuria. The development of effective interventions and methods of early detection and severity measurements of renal disease in children is lagging in part due to a lack of knowledge of physiological and pathological changes that occur as the kidney matures. Molecular blueprints would dramatically enhance our ability to design effective approaches to intervene and prevent kidney dysfunction. This goal cannot be met, however, without having a source of pediatric kidney tissue to begin molecular interrogations to identify the uniquely human and developmental, ’omic instructions required to make and maintain healthy kidneys. The objective of the Washington University Kidney Single Cell Atlas Project (pKidCAP) is to create a highly unique and innovative Pediatric Center of Excellence that delivers novel concepts, knowledge and resources by providing spatially resolved single cell molecular maps of pediatric reference and diseased kidneys at several time points across the pediatric lifespan. The pKidCAP investigators will apply paired snRNAseq, snATACseq technologies for decoding gene regulation and expression from the same cell and use spatial transcriptomics to resolve the cellular diversity with morphology using healthy and disease samples from pediatric kidneys procured from the Biomedical core and in mouse model of glomerular disease. The educational and opportunity pool programs will promote enthusiasm and progress in pediatric kidney disease research by 1) providing human age-specific references for fetal and childhood kidney disease tissues, 2) enabling studies aimed to delineate cellular, morphological, physiological and molecular changes associated with postnatal kidney maturation, 3) accelerating scientific research aimed at ex vivo human kidney organoids, 4) establishing protocols for isolating differentiated kidney cell types at stages consistent with those seen in kidney tissue samples, 5) advancing drug toxicity screening, and by 6) designing validation studies of gene function and kidney engineering. The availability of the tissue and the outstanding data generated from them will attract new expertise outside kidney research developing spatial imaging and analytical technologies and research interested in physiological aging across the lifespan.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Myofascial pain is a type of pain that originates in the muscle and surrounding fascia and is accompanied by a loss of range of motion, weakness, and sometimes autonomic symptoms. Affecting more than 85% of the general population sometime in their lives, myofascial pain has been a leading cause of health-care visits, absenteeism, and invalidity pensions—representing a significant health concern and economic burden. However, despite the high incidence of myofascial pain and substantial financial cost, the underlying pathophysiology remains largely unknown. As a result, clinical diagnosis and management of myofascial pain are largely empirical. Objective and quantitative biomarkers acquired by imaging, electromyography, laboratory tests, or muscle biopsy do not exist in the clinic for the assessment of myofascial pain. To address this unmet challenge, we propose to develop and translate a multi-modal, multi-parametric, multi-scale imaging approach to comprehensively assess the different aspects of the pathophysiology underlying myofascial pain both locally at the trigger point and regionally across the entire myofascial tissue, based on which we will identify candidate biomarker(s) that are most sensitive and specific to distinguish different states of myofascial pain and then test the biomarker’s ability to monitor treatment responses and predict clinical outcomes in a randomized clinical trial. Specifically, in the R61 phase (technology development & observational study), we will develop noninvasive magnetic resonance and electrophysiological imaging biomarkers and minimally invasive fiber-optic imaging biomarkers in parallel (Aim 1), and then identify candidate biomarkers that are best capable of differentiating different states (healthy, latent, and active) related to myofascial pain in an observational clinical study (Aim 2). Our prior research and preliminary studies provide strong premises for the proposed biomarker development and clinical testing. In the R33 Phase (interventional study), our research team—consisting of expertise in imaging, myofascial pain, quantitative analysis, and clinical trial—will examine the ability of the imaging biomarker(s) identified in the R61 phase for monitoring the responses to local chemical injection treatment and predicting clinical outcomes in a randomized clinical trial. The proposed research will create vital knowledge about myofascial pain and contribute to the creation of quantitative imaging biomarkers that may guide our choices of appropriate pain management and reduce opioid addiction.

NIH Research Projects · FY 2025 · 2022-09

Project Summary This K08 proposal will expedite the principal investigator’s progress towards his goal of becoming an independent physician-scientist focused on increasing our understanding of pathogenic mechanisms of pulmonary hypertension (PH) and developing innovative therapies to improve outcomes. Candidate: Dr. Kel Vin Woo is a physician-scientist at Washington University School of Medicine (WUSM). He completed a fellowship in Pediatric Cardiology and is developing expertise at the intersection of Fibroblast Growth Factors (FGF) signaling and vascular biology under the mentorship of Dr. David Ornitz, a world authority on FGF biology. Under Dr. Ornitz’s mentorship, the PI investigated how endothelial FGF signaling modulates hypoxia-induced PH by mitigating endothelial-to-mesenchymal transition. He will leverage the skills gained during his fellowship to further analyze FGF signaling in vascular smooth muscle cells and use adenovirus- delivered endothelial FGF as a potential method of regulating vascular remodeling. Career Development Plan: Dr. Woo will pursue this line of research with primary mentorship from Dr. Ornitz and co-mentorship from Dr. Curiel (an expert in adenovirus vectorology). Additionally, a team of intramural and extramural advisors include experts in PH, and vascular and pulmonary biology, and all have considerable experience in nurturing independent investigators. WUSM provides a highly interactive environment with excellent facilities, resources and opportunities. This 5-year plan builds on the PI’s prior experience and further enriches his training, providing him with the tools needed for independence. It includes the following objectives: (1) Master techniques in advanced mouse hemodynamic phenotyping; (2) Become proficient with adenovirus engineering to improve PH outcomes using preclinical models of hypoxia-induced vascular remodeling; (3) Disseminate research findings in diverse venues and actively establish productive collaborations. Research Plan: The overall hypothesis of the proposal is that FGF signaling in lung endothelial and vascular smooth muscle cells protects against hypoxia-induced PH. Specific Aim 1 will investigate how smooth muscle cell FGF prevents pulmonary vascular remodeling. Aim 2 will interrogate how endothelial targeted adenovirus- delivered FGF reduces hypoxia-induced vascular remodeling and PH severity. Upon completion of the proposed research, Dr. Woo will be proficient in: (1) modulating intracellular pathways important for endothelial and smooth muscle cell remodeling; (2) analyzing newly developed conditional knockout mice to ascertain the hemodynamic effects on the lungs; and (3) developing approaches for gene delivery to lung endothelium as a potential therapy for pulmonary vascular disease. These acquired skills will be readily applicable to other forms pulmonary vascular disease and endothelial-smooth muscle interactions in vascular remodeling. Upon completion of the proposed training, Dr. Woo will have acquired the necessary expertise to become an independent investigator focused on reducing the burden of pulmonary diseases, in alignment with the NHLBI mission.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Lymphocyte development is precisely controlled to enable clonal expansion and expression of a diverse immunoglobulin receptor repertoire, which proceeds through DNA double-stranded breaks (DSBs) generated by the RAG endonuclease. These two dichotomous, but interdependent processes, are managed through the cooperation of diverse cellular signals to prevent cells with DSBs from entering cell cycle where they could be aberrantly repaired as translocations. During early B cell development, the pre-B cell receptor (pre-BCR), through activation of the SYK kinase, coordinates both the proliferative expansion of pre-B cells and the assembly of immunoglobulin receptor genes. Negative regulation of the pre-BCR is required to ensure cell cycle arrest and limit the number of DNA breaks generated during immunoglobulin receptor gene assembly. Indeed, unopposed pre-BCR signaling, particularly increased SYK activity, drives proliferation and leukemic transformation. However, the mechanisms that repress SYK and pre-BCR signaling are not known and remain a critical gap in our understanding of B cell maturation. We have identified a novel cell-type specific program activated by signals from RAG DSBs that suppresses SYK and inhibits pre-BCR signaling. Deficiencies in this DNA damage- mediated feedback circuit result in initiation of pre-B cell leukemia. Surprisingly, this signaling network is not triggered by all DNA injury but, rather, is specific to RAG DSBs generated during immunoglobulin receptor gene assembly. Our goal is to determine how signals from RAG DSBs integrate with developmental programs to coordinate B cell maturation and prevent leukemic transformation. We propose that RAG DSBs suppress SYK to enforce cell cycle arrest and, thereby prevent B cells with DNA breaks from re-entering cell cycle. This DNA damage-mediated checkpoint program would permit iterative attempts at generation of a mature antigen receptor to promote B cell differentiation while preventing leukemic initiation. Further, we propose that these DNA damage signals are activated through distinct domains of the RAG endonuclease that interact with proteins at sites of DSBs to modulate signaling pathways. This RAG-specific mechanism in B cells discriminates between normal and errant DSBs to activate appropriate cellular responses. Utilizing an innovative experimental approach that allows interrogation of DSB signals within the context of B cell developmental programs, our proposed studies will define how RAG DSB signals maintain pre-B cell checkpoint and will resolve the mechanisms that distinguish RAG-mediated from non-RAG-mediated DNA damage. Completion of these studies will delineate pathways critical for dampening proliferative signals in early B cells, establish signals that restrict leukemogenesis, and define novel functions of the RAG endonuclease in regulating DNA damage responses.

NIH Research Projects · FY 2024 · 2022-09

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by beta-amyloid (AP) deposition, neurofibrillary tangles, neuronal loss, and gliosis. AD is the 6th leading cause of death in the United States and more than 6.2 million Americans suffer from this disease. This year the estimated cost of AD and other dementias is projected to reach $355 billion and can rise to $1 .1 trillion by 2050. The cause of AD remains elusive, and it is likely multifactorial. The greatest risk factors are known to be age, genetics, and inheritance. Despite monumental efforts and vast funding for research, only one therapy has been approved since 2003 and no medication can prevent acquisition of the disease or halt progression. In the last decade more than 200 research projects have not been completed or have failed. More than 15 clinical trials have attempted to promote AP clearance but only one has received controversial approval from the Food and Drug Administration. Most of these studies have treated patients with mild-moderate symptomatic AD, phases by which irreversible damage have already occurred. Therefore, it has been hypothesized that intervention preceding permanent pathological changes could provide better outcomes. However, it is estimated that pathological changes start 15-20 years before the onset of clinical symptoms. Given the extraordinary cost of antibody-based AP, and prolonged treatment periods both primary prevention and tertiary prevention become cost-prohibitive and unsustainable. To develop novel and cost-effective approaches, we have developed phagocytic chimeric antigen receptors (CARs) that can promote stable AP clearance with minimal treatment frequency. Our initial CAR designs promote remarkable induction of potent phagocytosis of oligomeric AP in human microglial cell lines. Using this technology, we propose to design CAR-based cellular therapies to stably promote clearance of AP plaques and prevent further AP deposition. The approach described in this proposal has the potential to be transformative in the treatment of AD and can likely be developed into successful treatments of other protein aggregation diseases of the central nervous system.

NIH Research Projects · FY 2025 · 2022-09

Abstract More than 80% of the 3 million women who labor and deliver each year in the United States undergo continuous electronic fetal monitoring (EFM) during labor. The goal of such monitoring is to identify fetal hypoxia and prevent the transition to acidemia, expedited operative delivery, and/or neonatal morbidity. Category II EFM, the most commonly observed group of fetal heart rate features, is associated with variable risks for acidemia. One common response to Category II EFM is maternal oxygen (O2) supplementation. The theoretic rationale for O2 administration is that it increases O2 transfer to a hypoxic fetus, thereby reversing hypoxia and preventing acidemia. This practice is so commonly used that 2 out of 3 patients receive supplemental O2 at some point during labor. However, despite the theoretic rationale, there is no rigorous data to support its widespread use. Our pilot randomized noninferiority trial, published in JAMA Pediatrics, demonstrated that room air was noninferior to O2 for improving umbilical artery lactate, a surrogate marker of fetal acidemia and neonatal morbidity. Our subsequent meta-analysis of randomized trials investigating O2 versus room air at time of cesarean section or vaginal delivery found no differences in umbilical artery pH, rates of acidemia, and neonatal intensive care unit admissions between room air and O2 groups. Importantly, none of the trials studied important clinical measures of neonatal or maternal morbidity and only one trial studied O2 in the setting of Category II EFM. The current state of evidence is limited in several ways. First, the existing studies of O2 in labor vary in the dose, duration, and timing of O2 administration. Second, only one pilot trial investigated the utility of O2 supplementation for the ACOG-recommended indication of Category II EFM. Third, all of the studies have used surrogate measures of neonatal morbidity such as cord gases or neonatal intensive care unit admission. Finally, none of the studies were powered to assess the impact of O2 administration on neonatal clinical sequelae of in utero hypoxia or operative delivery. Without data on these definitive outcomes, evidence-based recommendations for managing Category II EFM cannot be made. To fill this important knowledge gap, we propose a large, multicenter, randomized noninferiority trial of O2 supplementation versus room air in patients with Category II EFM in labor. Our central hypothesis is that clinically relevant maternal and neonatal outcomes will not significantly differ between the two arms. We will pursue the following specific aims: 1) Determine the effect of room air, compared with supplemental O2, on neonatal morbidity among patients with Category II EFM and 2) Determine the effect of room air, compared with supplemental O2, on rates of operative delivery. A total of 2124 patients will be randomized to provide adequate power to detect clinically meaningful noninferiority margins for the above stated outcomes.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY (See instructions): Postpartum hemorrhage (PPH), defined as the loss of 1 L of blood or more within 24 hours after birth, is the leading cause of maternal mortality worldwide. Importantly, PPH is the most preventable cause of maternal mortality, and the leading factors causing preventable PPH are delays in diagnosis and treatment. Thus, there is an urgent need for an early and accurate alert system that can facilitate prompt treatment to prevent PPH-related morbidity and mortality. During hemorrhage, the body tries to compensate for blood loss by shunting blood from the periphery to vital organs and replenishing reduced blood volume with water from the interstitium. These compensation mechanisms help stabilize the patient, delaying the time until global vascular indicators such as blood pressure and heart rate are affected, the current measurements used to predict PPH. Thus, monitoring of peripheral blood flow and blood content can yield early indicators of hemorrhage. Optical technologies are well suited to noninvasively measure blood flow and blood content. Preliminary experiments demonstrated sensitivity to reduced perfusion in vivo and measured significant differences between blood samples diluted to physiologic levels seen during PPH using complimentary optical approaches. We hypothesize that early changes in peripheral blood flow and water transfer from interstitium to the vasculature will be detected using wearable optical techniques, and such changes can signal early stages of PPH. We will develop a multifunctional wearable optical device and test it in pregnant patients at high and low risk of PPH. These studies will create a database of PPH signatures that will help inform diagnostic algorithms that will provide an early warning system for improved PPH management.