University Of Colorado Denver

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY The opioid overdose crisis necessitates adapting and implementing evidence-based interventions for opioid use disorder (OUD) in primary care settings to increase access to evidence-based treatment. Buprenorphine treatment for OUD in primary care is highly effective, patient-centered, and recent changes have removed bar- riers to prescribing, yet retention in these settings remains low. Measurement-based OUD treatment, a guide- line-recommended best practice, involves collecting patient outcome data to monitor treatment response and guide changes, thereby improving patient outcomes and retention. Patient-reported outcome measures (PROMs) are validated instruments that assess patients' response to treatment and focus on their experience, making them suitable for measurement-based OUD treatment. Despite the potential benefits, implementing and maintaining measurement-based OUD treatment using PROMs in primary care settings is lacking. This proposal aims to develop and pilot test the “Patient Reported Outcome Measures to Individualize Treat- ment And Improve ReteNtion” (ReTAIN) program intervention package to capture PROMs in Federally-Quali- fied Health Center (FQHC)-based OUD treatment. FQHCs are increasingly caring for patients with OUD who come from underserved communities that are disproportionality impacted by the overdose crisis. Utilizing the Pragmatic, Robust Implementation and Sustainability Model (PRISM) and user-centered design strategies, we will accomplish the following Aims: Aim 1) describe contextual factors and identify intervention package adap- tations and ideal PROM characteristics; Aim 2) Apply user-centered design strategies to refine the ReTAIN program intervention package adaptations; and Aim 3) Conduct a pilot randomized control trial to assess the ReTAIN program's initial effectiveness over 6-months, feasibility, and acceptability. Dr. Jarratt Pytell's research and training plan aligns with the national Overdose Prevention Strategy and NIDA's strategic plan to study the implementation of evidence-based treatments in real-world settings and bridge the evidence-to-practice gap. Dr. Pytell's long-term career goal is to become an independent clinician- investigator leading addiction medicine implementation research in primary care. As a fellowship-trained addic- tion medicine specialist providing primary care-based addiction treatment in an FQHC, Dr. Pytell seeks to lev- erage the HEAL Initiative K23 award to build on his experience and receive training in implementation science, user-centered design, and pragmatic trials. Dr. Pytell will receive guidance from an internationally recognized team of mentors and advisors with expertise in implementation science, pragmatic trials, user-centered design, and patient-centered decision-making who have a proven track record of independent funding and mentoring. Completion of the research and training aims will result in a definitive trial evaluating the effectiveness of meas- urement-based OUD care to improve OUD-related outcome across diverse FQHCs.

NIH Research Projects · FY 2025 · 2024-07

In South Africa, the country with the world’s largest HIV epidemic, an estimated 15-25% of women are living with HIV. Of these, estimates show that 35.5% are not in treatment. Furthermore, there is low engagement with HIV prevention behaviors, such as ever having an HIV test (~20% of women have never tested for HIV) and pre-exposure prophylaxis (PrEP) use (women’s openness to using PrEP is ~65-80%, only). Data on syphilis outcomes, which significantly increase risk for HIV and viral load if already living with HIV, are unknown in this context; though one estimate of prevalence is ~9%. Dual self-testing for HIV and syphilis may be a promising strategy to improve uptake of regular HIV/syphilis testing and linkage to PrEP and/or HIV/syphilis treatment among women in this context. Specifically, self-testing is overwhelmingly supported by stakeholders in South Africa, the infrastructure to scale-up self-testing is in place and rapidly expanding, analyses show that this strategy is life and cost-saving, and PrEP and HIV/syphilis treatment are widely available. Also, self-testing may address the unique social/structural barriers affecting regular testing and subsequent linkage to services, including health system mistrust, discrimination, stigma, cost, and privacy concerns. Thus, interventions to improve dual self-testing uptake and linkage to PrEP and/or HIV/syphilis treatment following a self-test are urgently needed for South African women. Yet, we must first address 3 critical gaps in our knowledge, including women’s needs/preferences on: self-test access and uptake; logistical support needed to correctly use/interpret self-tests; and linkage to and uptake of PrEP and/or HIV/syphilis treatment following self-tests. Using the INSTI Multiplex, a dual blood-based HIV/syphilis test that delivers results in 1-minute, we use mixed-methods to inform these knowledge gaps and draft a dual self-testing and linkage to care intervention for women. Research takes place in Johannesburg, South Africa, brings together an established research team and community advisory board, and is informed by the Mensch Model; work will occur in 3 stages. In Stage 1, we will conduct in-depth interviews with: N=15 women and N=15 healthcare workers. Women will use INSTI Multiplex in front of a study team member to provide immediate feedback on logistical support needed to test/interpret results; they will also discuss preferences on test access, and linkage. In Stage 2, we will enroll N=80 women to complete self-administered web-based surveys on their mobile phones. Surveys are developed from IDI themes and will identify women’s preferred intervention strategy to address each domain. We will engage a data convergence process to ensure that quantitative/qualitative findings inform one another. We will then draft the intervention using the 4 COM-B Intervention Function Matrix. In Stage 3 we will engage an intervention development workgroup consisting of at least n=5 members from each of the following groups: community advisory board, women, healthcare workers. We will present them draft intervention to further develop/refine in 4 meetings of 90-minutes, each. The product will be tested in a future NIH R01.

NIH Research Projects · FY 2024 · 2024-07

SUMMARY The Systems Approaches to Cancer Biology (SACB) meeting aims to bring together scientific leaders and early-stage investigators in Cancer Systems Biology to build and foster a thriving and collaborative field through sharing scientific advances. Started as a grassroots effort from early-stage Cancer Systems Biology investigators, the first SACB meeting was held in 2016 and is held every other year. SACB has previously been co-sponsored by the National Cancer Institute and the Association for Cancer Systems Biologists (ACSB). The meeting is solely focused on research topics that address cancer as inherently complex, with spatial, temporal, and interpatient heterogeneity, and with multifactorial causes and consequences that cannot be understood without integrating diverse approaches from a combination of traditional scientific disciplines. The SACB meeting consists of seven plenary sessions, poster sessions, and many opportunities for early- stage investigators to network and gain mentorship from more senior investigators, including “meet the PI” lunches and happy hours. SACB plenary sessions focus on many aspects of cancer biology and cover a diversity of computational/mathematical modeling approaches. This problem-domain-focused programming is particularly conducive to the synthesis of new ideas and collaborations among attendees. Given the history of this meeting, there is a strong emphasis on the development and fostering of early-stage investigators, which includes at least half of the oral presentations being given by trainees and addressing topics of interest to early-stage investigators through special topic sessions. Another focus of the meeting is to grow the Cancer Systems Biology community, which we will accomplish by targeting underserved research communities, reaching out to researchers in complementary topic areas, such as computer science or engineering, as well as funding support for investigators with additional family needs. The SACB Scholars program, initiated in 2022, will support at least 8 undergraduate and/or graduate trainees from under-represented groups in biomedical research to attend the SACB 2024 meeting. This program in 2022 was highly successful and is aimed at providing training and networking opportunities to individuals that might not be able to gain these experiences otherwise. SACB has successfully helped to advance the careers of many early-stage investigators and brings together like-minded investigators to share research findings. SACB 2024 will maintain the hybrid meeting format to both in-person and virtual meeting options. This will provide a venue for SACB to reach colleagues across the world and still be able to provide the intimate environment needed to foster Cancer Systems Biology research into the future.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract Fibrosis is defined as excess deposition of extracellular matrix (ECM), resulting in tissue scarring and organ dysfunction. In the heart, fibrotic remodeling in the context of chronic comorbidities such as hypertension and metabolic disease is associated with increased passive myocardial stiffness and the development of diastolic dysfunction (DD), a contributor to the pathogenesis of heart failure with preserved ejection fraction (HFpEF). The adult heart contains resident cardiac fibroblasts (CFs), which, in response to stress, undergo a cell state transition to become activated fibroblasts, sometimes referred to as myofibroblasts. Activated CFs are characterized by transcriptional reprogramming that results in enhanced production and secretion of fibrotic ECM proteins. Despite the well-recognized roles of CFs in fibrotic remodeling of the heart, there are no targeted therapies to prevent or reverse the phenotypic conversion of these cells into an activated state. Recently, we demonstrated that inhibition of the eicosanoid-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), using SW033291, potently suppresses murine and human CF activation, and blocks cardiac fibrosis and ameliorates DD in vivo in mice. Eicosanoids are 20 carbon-containing lipid signaling molecules that are produced by a variety of cells. Our data support a model in which stress signals trigger 15- PGDH-mediated degradation of eicosanoids in CFs, resulting in reduced ERK1/2 signaling and subsequent activation of the cells to promote fibrosis. SW033291 treatment prevents degradation of eicosanoids, resulting in increased secretion of these fatty acids, which function in an autocrine and paracrine manner to promote antifibrotic ERK signaling in CFs and ameliorate fibrosis of the heart. Three specific aims are designed to extend this new field of cardiac research and test the overall hypothesis that inhibition of 15-PGDH promotes signaling events that prevent and reverse the transcriptional reprogramming that typically culminates in CF activation and pathological fibrosis of the heart.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY The goal of this proposal is to advance our understanding of the mechanism for increased periportal collagen deposition, a sign of developing fibrosis, in the liver of the fetus with fetal growth restriction (FGR) and the impact on future liver metabolic disease. Children born with FGR have a higher risk of developing metabolic dysfunction- associated steatotic liver disease (MASLD) and fibrosis in childhood and adulthood. A distinct feature of MASLD presentation in pediatric patients, and one that is higher in FGR offspring, is localized fibrosis in periportal regions. Previous studies demonstrate that hypoxemia-induced oxidative stress promotes hepatic fibrosis in animal models. Yet, there remains a gap in our understanding of how oxidative stress in the fetal liver may promote a fibrotic niche and future susceptibility to liver disease associated with fibrosis. Our preliminary data indicate that FGR fetal sheep have increased hepatic periportal collagen deposition and increased markers of oxidative stress signaling. In addition, our preliminary data in fetal sheep exposed to long-term hypoxia show an increase in hepatic periportal fibrosis. I hypothesize that hypoxemia-induced oxidative stress in the FGR fetus produces cell-specific and regional effects in the fetal liver that initiate periportal fibrosis. Overall, this proposal will provide critical information for how FGR promotes hepatic fibrosis that persists postnatally. Aim 1 will Identify cell-specific and regional gene expression signatures in the fetal liver in response to FGR. Aim 2 will test if FGR and hypoxemia promote intrinsic fibrogenic capacity in hepatic stellate cells and test whether treatment of an antioxidant, N-acetylcysteine, prevents hypoxia-induced activation. Aim 3 will determine if hepatic fibrosis persists in postnatal lambs born with FGR. Expected Outcomes: This proposal will characterize the impact of FGR on oxidative stress and fibrosis in the fetal liver. Importantly, completion of this proposal will provide me with training in liver physiology and metabolism, bioinformatics, advanced histology, models to study fetal development, hepatic stellate cell culture, and scientific writing to help me achieve my career goal as a tenure-track assistant professor specialized in the fetal developmental programming of immunometabolism. Impact: The aims in this proposal will be used to help understand how fibrosis develops in postnatal liver of FGR offspring and determine future mechanistic experiments used to investigate fetal origins of fibrosis.

NIH Research Projects · FY 2026 · 2024-07

For more than a decade genome-wide association studies (GWAS) have provided insight into the genetic basis of a variety of complex diseases, physiological traits, and molecular phenotypes. Biomedical researchers are increasingly linking genome wide data (e.g., DNA variants, DNA methylation, chromatin accessibility) with other high-throughput molecular data (e.g., transcripts, proteins, metabolites). These additional omics technologies have been useful for extending the biological relevance of GWAS findings by providing functional interpretation of GWAS signals, biomarker identification, disease subtyping, and understanding of molecular processes that underlie disease etiology in relevant tissues. Despite the promise offered by multi-omics data, analysis is often challenging due to multiple large and high-dimensional data sets; heterogeneity across technologies, coverage, range, and signal quality; decisions about how and when to integrate data; and the lack of data from diverse subjects. The multi-omics field is quickly evolving, and we and others have developed methodology regarding multi-omics analyses. In particular, network science and graph analytics has been an intuitive framework for identifying interactions across omics modalities. In response to PAR-22-095 (“National Human Genome Research Institute (NHGRI) Short Courses for Genomics-Related Research Education”), we have developed the Multi-Omics and NETwork analysis workshop (MONET) to provide an interactive experience to learn about multi-omics analysis and the application of network methods. The MONET training would complement the mission of NHGRI as genetic analyses are frequently being integrated with other omics profiles and it is increasingly important to have a trained workforce that has familiarity with multi-omics tools. For MONET, we propose a 7-day immersive experience for ~25 researchers each summer 2024-2028. MONET will consist of ~50 hours of lectures, discussion sessions, computational labs, and tours. Each day will include didactic portions providing overviews of the technologies and methods. The rest of the time will primarily be devoted to hands-on sessions where participants will implement methods using provided sample code and data sets. So that participants also understand how data are generated, we will organize tours of our technology cores, in addition to an outing to an industry partner. We will also include discussion sessions on cross-cutting topics for multi-omics research including omics data preparation, harmonization, and computing, in addition to omics data sharing, privacy and policy issues. Finally, multi-omics research requires a team science approach which will be developed through group exercises. Participants will be post-baccalaureate researchers (e.g., MS/PhD students, research staff, post-doctoral fellows, clinical fellows, junior and senior research investigators) who have interest in analyzing multi-omics data and sufficient programming knowledge to follow data analysis procedures. Instructors will be drawn from experts with experience in genetics, bioinformatics and data science at the University of Colorado Anschutz Medical Campus, affiliated institutions, and external guest lecturers.

NIH Research Projects · FY 2025 · 2024-07

Acute myeloid leukemia (AML) is a devastating malignancy with dismal outcomes for patients, particularly in the elderly where most cases occur. The recent approval of the BCL2 inhibitor venetoclax (Ven) in combination with a hypomethylating agent (HMA; such as azacytidine/Aza) for the treatment of AML in elderly/unfit adults has substantially improved outcomes for these patients. Still, about a third of patients treated with Ven/HMA exhibit poor responses to therapy, and most patients who do respond will eventually relapse with Ven/HMA resistant disease. Moreover, patients whose AML contains mutations in the TP53 gene are refractory to Ven/HMA and all other forms of chemotherapy. The central premise of this project is that intra-patient heterogeneity of the leukemia initiating cell (LIC) population is a key driver of therapy resistance/failure. At least two distinct forms of LSCs can be found in AML patients, one with a more primitive phenotype (p-LIC) and one with a monocytic phenotype (m-LIC). While Ven-based therapies are an effective means to eradicate the p-LSCs in newly diagnosed AML patients, m-LIC are resistant to Ven/Aza and can mediate relapse. The goal of this work is to identify and characterize distinct LIC subtypes and their vulnerabilities to design more effective and more durable therapies. We have further shown that m-LIC exhibit upregulation of Inhibitor of Apoptosis Proteins (IAP) and that IAP inhibition using drugs called SMAC mimetics (SMACm) potently sensitizes AML cells to Ven/Aza- induced apoptosis in vitro and in vivo, particularly for the monocytic subpopulation highly resistant to Ven/Aza alone. Intriguingly, SMACm therapy is also highly effective at eradicating TP53 mutant/deleted AML cells, which are generally refractory to currently available therapies. Thus, varying subtypes of AML LIC are vulnerable to targeting by distinct strategies. Mechanistically, we show that SMACm in combination with Ven/Aza induces noncanonical NF-B signaling and high levels of TNF, which causes cell death by apoptosis. Here, we propose to characterize the molecular phenotypes and therapeutic vulnerabilities of different LIC subpopulations in order to design more effective therapies. In the first aim, we will characterize the pathways and molecular mechanisms that mediate differential vulnerabilities of resistant LIC subtypes, with a focus on monocytic and TP53 mutant subtypes. The second aim will leverage in vitro and in vivo models to gain a thorough understanding of the clonal heterogeneity of different primary AML specimens, the phenotypes of the various subclones, and how distinct LIC subclones differentially respond to Ven/Aza and SMACm therapy. Finally, in Aim 3, a clinical trial will assess the safety and efficacy of combining a SMACm drug, tolinapant, with Ven/Aza in the relapsed/refractory AML setting. Samples from this trial will be used to determine whether and how tolinapant sensitizes LICs to Ven/Aza therapy, thereby inducing deeper remissions. In all, these studies should lead to the development of therapeutic strategies that limit or control the development of drug resistance.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Rural-serving primary care clinics in our Colorado Implementation Science Center in Cancer Control (COISC3) seek to address inequities in behavioral cancer risks and unmet social needs. Clinics need efficient, low-cost tools to screen for and address multiple risks at the same time — and technology can provide important efficiencies. My Own Health Report (MOHR) is a web-based intervention with 3 core functions: 1) Risk Flagging; 2) Goal-setting for patient-prioritized risks; 3) Service Linkage to resources. Our prior trial found that patients receiving MOHR improved physical activity (p < 0.001) and diet (p < 0.001) behaviors that increase the risk for many types of cancer; MOHR also showed improvements in other health behaviors and a signal of reduced tobacco use (with a small n). Thus, MOHR has great potential to prevent cancer. However, we identified two key gaps. First, our COISC3 clinics voiced a need for a more flexible menu of delivery options to feasibly adopt and sustain the intervention. Second, to achieve equitable reach and effectiveness, particularly for those with unmet social needs, clinics need to provide structured follow-up. Such follow-up increases patient access to resources for behavioral risks and/or unmet social needs, thus “leveling the playing field” to address cancer risks. Our new preliminary data show clinics and patients value MOHR with structured follow- up strategies to remind patients of their goals and to provide relevant resources. We developed the infrastructure for and pilot-tested two distinct implementation strategy bundles to provide this follow-up. The first bundle, termed “Reminder-Resource message (R2 message),” involves electronic texts/e-mails sent automatically by MOHR. The second bundle, “R2 Navigation,” trains staff in each clinic to personally deliver these reminders and resources. Each strategy bundle addresses the contextual determinants of successful implementation and sustainment identified in our Pragmatic Robust Implementation and Sustainability Model (PRISM). However, the relative impact of R2 message, R2 Navigation, or their combination is unknown. In Aim 1, we will engage staff, patients, and partners in our COISC3 to finalize the implementation plans to deliver R2 message and R2 Navigation from a menu of options co-developed in preliminary workshops. In Aim 2, we will conduct a randomized comparative effectiveness-implementation trial with 1,400 adult patients with two or more cancer risks (including both insufficient physical activity and fruit/vegetable intake) across 7 COISC3 clinics to evaluate the comparative outcomes of R2 message and R2 Navigation alone or in combination. We will assess multi-level outcomes, including: 1) Effectiveness, 2) Implementation, and 3) Practice Value (including patient experience and costs). In Aim 3, we will use our iterative PRISM process to co-develop an “implementation, adaptation and sustainment guide” for the most cost-effective implementation strategy identified in Aim 2. Overall, we expect to reduce multiple cancer risks equitably, in part by addressing unmet social needs, and to improve outcomes important to patients, primary care, and society.

NIH Research Projects · FY 2025 · 2024-07

Project Abstract: Pediatric HGGs comprise glioblastoma [glioblastoma multiforme (GBM)], anaplastic astrocytoma, and diffuse intrinsic pontine glioma (DIPG) and collectively are the most common malignant brain tumors in children. Patients with HGGs have a very dismal prognosis, with <5% 5-year survival for GBM patients and a median survival of <1 year for patients with DIPG, pointing to an urgent need for alternative therapeutic approaches. Chimeric antigen receptors (CARs) are engineered cell surface molecules that combine the specificity of antibodies with the downstream signaling of effector cells such as T cells. The expression of CARs on T cells has shown enormous promise by effectively treating malignancies by directly killing them. However, CAR-T cell therapy is often hampered by the inability of T cells to penetrate solid tumors and the inhibitory tumor microenvironment (TME). Recently there has been great interest in developing CAR-macrophages against solid tumors to improve phagocytic activity and antigen presentation of macrophages against tumors. Here we hypothesize that this distinctive proficiency of macrophages and microglia to infiltrate malignant brain tumors provides a unique opportunity to exploit their phagocytic properties by engineering the activation of these professional phagocytes using exogenous expression of Chimeric Antigen Receptor (CARs) targeted towards a brain tumor-specific antigen. In Aim 1 we will engineer the most effective CAR-expressing phagocyte for efficient targeting of pHGG and demonstrate the superior efficacy of B7H3 CAR phagocyte therapy in combination with blockage of phagocytosis checkpoint (anti-CD47) immunotherapy in humanized xenograft models of pHGG. In Aim 2 we will Identify the most efficient cell type that can function as a CAR-expressing phagocyte that can most efficiently traffic to the brain and target pHGG tumors in combination with blockage of phagocytosis checkpoint (anti-CD47) immunotherapy and finally in Aim 3 we will study the in vivo biology of CAR-macrophages and its effect on the TME in syngeneic mouse models of high-grade pediatric glioma.

NIH Research Projects · FY 2024 · 2024-07

Abstract The current studies are based on unexpected data from early brain development in mice establishing that reduced neuronal activity results in premature oligodendrocyte progenitor cell differentiation. These data contrast with the extensive data in the literature that axonal activity increases myelination. The fundamental hypothesis under investigation is that during early development, neuronal activity initially allows and/or enhances OPC proliferation but inhibits premature differentiation. The current studies will use Designer Receptors Exclusively Activated by Designer Drugs (DREADD)-expression in cortical neurons to investigate the impact of decreased or increased neuronal activity on oligodendrocyte progenitor cell proliferation, survival and differentiation in corpus callosum and cortex. scRNASeq studies will characterize the transcriptome changes in oligodendrocyte progenitor cells when cortical neuronal activity is reduced or increased. Our data establishing that reduced neuronal activity induces premature oligodendrocyte differentiation in young mice are unexpected given the significant data in the literature establishing that neuronal activity enhances oligodendrocyte progenitor cell differentiation and myelination later in development. We will therefore investigate when during development the impact of neuronal activity shifts from blocking differentiation to enhancing differentiation.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT This proposal describes a rigorous five-year training program leading to the career development of Dr. Lisa L. Korn as an independent physician scientist. The principal investigator is a physician scientist with a PhD in immunology who recently completed clinical fellowship training in rheumatology. Her career goal is to become an independent investigator working at the intersection of mucosal immunology and inflammatory diseases, studying how environmental influences in inflammatory disease are mediated by the intestinal immune system. She proposes to expand her training in gut physiology, the microbiome, and inflammation biology through an intensive training research experience under the mentorship of Dr. Ruslan Medzhitov, a pioneer and world leader in the complex biology of inflammation. In addition to intellectual mentoring and hands-on training in Dr. Medzhitov’s lab, she has designed a series of relevant didactic coursework and has carefully selected an advisory committee with extensive expertise both related to this project and in successfully mentoring physician- scientists. An outstanding scientific environment and extensive resources are provided by Yale University and its Departments of Medicine, Immunobiology, and section of Rheumatology. The research objective of this proposal is to study the interactions between nutrient signals and microbiota on eosinophil residency in the small intestine at steady state and in a mouse model of eosinophilic granulomatosis with polyangiitis (EGPA), an eosinophilic vasculitis that often involves the intestine. We found that small intestine eosinophils undergo a process of tissue adaption at steady state that involved transit of eosinophils up the crypt-villus axis with time, and changes in eosinophil morphology, surface marker expression, and transcriptional profile. The canonical eosinophil survival factor IL-5 was largely dispensable for eosinophil adaptation, while the vitamin A metabolite retinoic acid was required. Because small intestine eosinophils are most abundant in the part of the intestine that is most exposed to luminal nutrients, we asked whether these cells were further regulated by diet. Indeed, a high protein diet caused depletion of the villus-resident subset. Based on these findings, we hypothesize that coordinated environmentally derived signals regulate eosinophil small intestine tissue adaptation, and that they might affect intestinal eosinophilia in EGPA. Therefore, we propose to identify how retinoic acid and dietary protein impact the eosinophil small intestine adaptation process, to determine the impact of the localization of the intestinal microbiota on this process, and to identify mechanisms of intestinal eosinophilia in a mouse model of EGPA.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY AND ABSTRACT The Human Immune Monitoring Shared Resource (HIMSR) at the University of Colorado Anschutz Medical Campus (CUAMC) is applying for funds to add a state-of-the-art high-parameter fluorescence imaging platform, the Lunaphore COMET, to this well-established core facility. HIMSR is substantially supported by the University of Colorado Cancer Center (UCCC, P30 CA046934) and the Colorado Head and Neck Cancer SPORE (P50CA261605). Current multiplex immunohistochemistry (mIHC) tissue imaging instruments include the Akoya Vectra 3.0, the Akoya Vectra Polaris, and the Ionpath Multiplex Ion Beam Imaging (MIBI) system (low- throughput, 40-marker mass spectrometry imaging in regions of interest). HIMSR is the only core lab in the state of Colorado that offers multiplex tissue imaging and specializes in the development of customized high- parameter panels. HIMSR supports a consistently growing number of investigators, approximately 100 per year, 65% of which utilize mIHC instruments. The rationale for adding additional high-parameter imaging capability is that many investigators need affordable hyperplexing options, however the capacity of HIMSR's existing high- parameter MIBI platform is maximized with no available AUT for innovation or new assay development. The Lunaphore COMET instrument utilizes commercially available label-free primary antibodies in a serial staining, imaging, and elution approach that is flexible to each investigator's needs. It maximizes throughput with walk- away automation and preserves tissues and epitopes with unparalleled integrity and stability using innovative microfluidics, making it possible for investigators to perform whole genome RNA-based analyses of the same tissues in downstream transcriptomics assays. The Lunaphore COMET offers whole-tissue imaging with a virtually unlimited hyperplex capability and generates OME-TIFF image files that can be analyzed with either open-source or commercially available image analysis packages. Interest in high parameter tissue imaging and the need for additional instrumentation is driven by a strong group of NIH-funded principal investigators with 23 active NIH-funded grants in the areas of oncology, dermatology, hepatology, nephrology, and hematology. The estimated need for the 12 Major Users is approximately 81% accessible user time. The HIMSR team is highly qualified to develop new assays and operate the Lunaphore COMET instrument, composed of a master's level histologist, a Ph.D. level image analyst, a certified histotech, and three research assistants. The PI (Dr. Kimberly Jordan) has over 7 years of experience in developing, optimizing, and analyzing multiplex IHC tissue imaging data, has collaborated on many NIH-funded R01 projects utilizing these technologies and expertise, and authored 50 peer-reviewed publications in immunology. HIMSR's expertise in developing custom mIHC assays is nationally recognized with a strong track record of providing robust and quantifiable images of tissue microenvironments. There is a well-established financial and administrative infrastructure in place to support the Lunaphore COMET in HIMSR with strong leadership and institutional support.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY GABAergic inhibitory synapses are critical for controlling neuronal excitability, fine-tuning neuronal output, and coordinating neuronal firing. Inhibitory synapses undergo bi-directional, activity-dependent changes that result in both changes to synaptic clustering and synaptic strength. In particular, one form of inhibitory long-term potentiation (iLTP) results in increased post-synaptic clustering of the GABAA receptors (GABAARs) and the inhibitory synaptic scaffolding protein, gephyrin. The clustering of both GABAARs and gephyrin at early stages of iLTP is through trafficking of these proteins to synapses, but later stages of iLTP require de novo protein synthesis in order to sustain this potentiation long-term. We have found that two microRNAs (miRNAs: miR376c and miR153) repress the translation of GABAARs and gephyrin under basal conditions. Following iLTP stimulation, miR376c and miR153 expression is reduced, resulting in increased GABAAR and gephyrin translation required for sustained increases in inhibitory synaptic strength. This decrease in miRNA expression is a result of transcriptional repression, mediated by the transcription factor, nuclear factor of activated T-cells (NFAT), which is regulated upstream by the phosphatase calcineurin (CaN). However, there is still a major gap in our knowledge of signaling mechanisms that control long-term changes in gene expression during inhibitory synaptic plasticity. In this proposal, I will address this gap in knowledge by identifying the upstream signaling cascades responsible for miR376c and miR153 transcriptional repression following iLTP, specifically focusing on the role of both NFAT and the scaffolding protein, A-kinase anchoring protein (AKAP), which mediates the anchoring of CaN in close proximity of calcium influx. I hypothesize NFAT transcription factors regulated by AKAP-anchored CaN signaling are required for long-term strengthening of inhibitory synapses during iLTP. I will utilize live-cell confocal microscopy and whole-cell electrophysiology to identify the requirement for NFAT in increases in inhibitory synapse size, number and strength following iLTP stimulation (Aim 1). Live-cell confocal microscopy, 3D-structured illumination microscopy (SIM), and slice electrophysiology will be performed to characterize the role of AKAP-anchored CaN in regulating NFAT translocation and inhibitory synaptic potentiation following iLTP stimulation (Aim 2). Together, this proposal will help identify the upstream signaling mechanisms that are required to regulate altered gene expression during long-term iLTP and will provide clarity on the mechanisms that regulate inhibitory synaptic plasticity, which are vital for regulating proper circuit function and learning, memory, and cognition.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY The accurate description of cellular states—be it on the genomic, transcriptomic, or proteomic level—has been a major driver in the disentanglement of nature’s mysteries. Mainly supported by technological advances such as next-generation sequencing or single-cell technologies, we now can describe the distinct cellular populations within a tissue or collection of cells in intricate detail. This allows us to understand health and disease, and it often proves instrumental in our ability to develop effective treatments. However, our current technologies are mostly geared toward an understanding of current cellular states. While various technologies can trace the lineage of cells, even the combination with global cellular state readouts does not offer accurate insights into past cellular history. Moreover, current approaches that aim to combine a past transcriptional event with such tracing can only report the historic expression of one or few genes. However, this single moment in time may have comprised many critical developmental or adaptive ‘decisions’ or ‘inputs’ that shape the current function or even location of a cell. Thus, a global recording of a transcriptional state would support a more comprehensive picture of a cell’s history. This proposal’s aim is to provide such a genome-wide record of transcription by building on recently developed technological concepts. For this, we will develop the novel approach of transcriptional endpoint marking (TEM); this technology will introduce a permanent genetic mark at the locus of actively transcribed genes. Expressing the ‘marker’ construct transiently, will inscribe a ‘snapshot’ record of the transcriptomic profile that can later be assessed by targeted genome-wide retrieval of the genomic ‘marks’. Our proposed work will provide proof-of-concept and focus on the development of a genetic toolkit that enables this analysis. In addition, it will provide a computational basis for both the data processing and snapshot analysis. We will initially employ these methods to test cellular state differences that underlie adaptive responses, such as chemotherapy resistance in cell culture methods. We expect that this method will have a broad impact across several fields, which include our laboratory’s interests in vertebrate development. Moving this technology into in vivo systems, such as zebrafish or mouse, will enable the establishment of early developmental markers of defined cellular populations that remained elusive. Alternatively, it will also enable to understand lineage decisions for populations that share developmental history but present with distinct phenotypes. Likewise, snapshot analysis will aid in our understanding of metastasis and resistance in tumour models, resiliency in injury, or response to differentiation factors in vitro. TEM represents a fundamentally distinct approach to dissect development and adaption that complements current efforts in lineage tracing and state analysis. As such, it can serve as a technological driver of novel biological insights.

NIH Research Projects · FY 2024 · 2024-07

PROJECT ABSTRACT Since 2008, the Pathology Shared Resource (PSR) has been providing Colorado researchers with access to a full spectrum of expertise in biobanking, pathology, histology, molecular pathology, and cytogenetics, leveraging the clinical infrastructure and research enterprise at the University of Colorado Anschutz Medical Campus (CU AMC). The PSR is a regional resource and receives funding and other support from the CU AMC, the University of Colorado Cancer Center, and the Department of Pathology. Services provided by the PSR include archival and real-time biospecimen collection and management, clinical trial support, and access to annotated biospecimens. The PSR provides histology and immunohistochemistry services on animal and human tissue specimens, CLIA-level molecular testing, and analysis for cancer genomic aberrations at chromosomal and whole genome levels to support basic, translational, and clinical research projects. The PSR, overseen by a strong collaborative team of researchers and clinical pathologists, is fully equipped to support the logistics and management of institutional and multi-institutional clinical trials including investigator-initiated trials. The PSR also manages the large Surgical Pathology archive that contains formalin-fixed paraffin-embedded (FFPE) biospecimens obtained from diagnostic and surgical procedures performed at the University of Colorado Hospital (UCH). Many research studies utilize specimens from the Surgical Pathology Archive. Most projects require analysis of a prepared tissue section on a glass slide to be analyzed by immunohistochemistry or other methods. Analyzing large tissue cohorts using individual sections on individual glass slides is labor intensive, costly, and can introduce experimental variation. Tissue microarrays (TMA) are created by taking small tissue areas (cores) from a donor FFPE block and combining it with tissue areas (cores) from other donor blocks in a single recipient block to make it possible to analyze many different tissue specimens (up to hundreds) simultaneously on a single glass slide cut from the TMA block, reducing labor time, cost, and experimental variability. The PSR has manual TMA building capabilities, but manual construction has its limitations as it is labor intensive. Currently we cannot provide the level of support needed for TMA design and construction to the research community. This proposal request funding for the purchase of the TMA Grand Master instrument which is capable of fully automated TMA construction that will greatly enhance our TMA building capabilities and decreasing our workflow time. In addition, the TMA instrument has excellent data and image tracking features which would greatly improve our study design, rigor, and quality of obtained data, and data management capabilities. An added benefit is that many tissue sections (100-200+) can be derived from a single TMA block that can be utilized in different research projects as an off-the-shelf solution and thereby expediting research and reducing analysis costs. Modernization of our TMA building capabilities by implementing the automated TMA Grand Master instrument in the CU PSR will greatly improve, streamline, and modernize our workflows and expedite services to the research community.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY Schizophrenia (SZ), and related psychotic disorders, affects about 1% of the global population and is associated with highly increased rates of cardiovascular disease, diabetes, and cancer compared to the general population. Greater obesity severity and prevalence in this population are the primary contributors to these increased risks. While antipsychotic (AP) treatment is a known contributor to weight gain, the neurobiology underlying this and other contributing factors are largely unknown. Without an improved understanding of the neurobiology of obesity in SZ, developing targeted treatments remains challenging. A framework that may be useful in understanding this neurobiology is the "Triple Network Theory" of SZ, which postulates that disruptions in three brain networks (default mode, executive control, and salience networks) heavily contribute to SZ pathophysiology. Disruption of these networks are similarly strongly associated with obesity severity in the general population. This proposal, therefore, aims to determine how disruptions in the “triple network” in SZ contributes to the observed increased obesity risk. Associations between triple-network disruptions and obesity in SZ will be assessed with functional magnetic resonance imaging (fMRI) measurements of brain connectivity within the “triple network” in individuals with and without SZ. Connectivity will be measured at rest and during visual food cues, both before and after consuming a meal. Machine learning analyses will measure how AP-related weight gain correlates with disruptions in the triple network in SZ, how obesity relates to these disruptions in individuals with and without SZ, and how other factors (i.e., appetite-related hormones, eating behaviors, and mood ratings) may influence these relationships. Mr. Dodd’s training plan focuses on the neurobiology of obesity and psychiatric disorders, advanced neuroimaging analysis, and professional development (see Training Plan). He will be supported by mentors with relevant expertise, as well as state-of-the-art resources in obesity, neuroimaging, and psychiatry research at the University of Colorado Anschutz Medical Campus. This training will support both this proposal and his career goal to become a neuroimaging physician-scientist investigating the neurobiology of psychiatric conditions and their comorbidities. The research conducted during this training has the potential for high clinical impact. Determining how the Triple Network Theory relates to obesity in SZ, and related psychotic disorders, may lead to improved treatment development and targeting for obesity and obesity-related comorbidities for this population.

NIH Research Projects · FY 2024 · 2024-07

SUMMARY The achievements obtained with different types of cancer immunotherapy are clear, with durable long-term survival in some cases. The most frequently used immunotherapeutic approach is the treatment with immunocheckpoint inhibitors (ICI). However, the major limitation of these immunotherapies is the relative high frequency of long-lasting immuno-related adverse events (irAEs) associated with the treatments. irAEs triggered by ICI can affect most any tissue. Thus, given the increasing burden of irAEs, determining a) the mechanisms that drive the irAEs, b) those patients who are at a higher risk to develop irAEs, and c) novel strategies to minimize irAEs while sustaining the ICI anti-tumor efficacy are unmet needs. The irAEs caused with ICI treatments are mainly due to an overresponse of CD4 and CD8 cells, and T cell-produced cytokines are a major mediator of irAEs. Until recently, minimal attention was given to IL-6 as a mediator of the irAEs from ICI therapy, despite the well-established role of IL-6 in the pathogenesis of a number of inflammatory diseases. This is in part due to the fact that IL-6 is considered a cytokine produced by the innate immune system. We have recently shown that human CD4 cells (but not mouse CD4 cells) from healthy volunteers produce high levels of IL-6 quickly after activation. The levels of IL-6 produced by human CD4 cells after activation are heterogenous among healthy volunteers, suggesting a potential genetic influence. Although IL-6R expression is restricted to leukocytes and hepatocytes, IL-6 can also trigger signals in cells that do not express IL-6R (e.g. endothelial cells, neurons, cardiomyocytes) and cause pathology when soluble IL-6R (sIL-6R) is present, binds to IL-6 and associates with ubiquitously expressed gp130. We have shown that human CD4 cells produce high levels of sIL- 6R upon activation. There is a high-frequency polymorphism in the promoter of human IL-6 gene (-174G/C, rs1800795) that can affect the production of IL-6. Similarly, a polymorphism (rs2228145) in the coding region of the IL-6R gene can lead to enhanced levels of sIL-6R. We propose that the IL-6 and/or sIL-6R produced by activated CD4 cells after treatment of cancer patients with ICI are major mediators of the irAE caused by this therapy, and that the genetic polymorphisms in IL-6 and/or IL-6R genes could determine the risk of patients to develop irAE. We will investigate: 1) the effect of genetic polymorphisms on the production of IL-6 and sIL-6R by human CD4 cells from healthy volunteers; 2) whether polymorphisms in IL-6 and/or IL-6R genes are associated with the development of irAE in ICI-treated cancer patients.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Commensal pathogenic Group B Streptococcus (GBS) is a Gram-positive pathobiont that asymptomatically colonizes mucosal sites including the gastrointestinal tract and the female reproductive tract (FRT), however microorganisms contribute significantly to the mucosal environment and host defense against bacteria. (GI) the interactions with GBS and native microbes in these niches is largely unstudied. During pregnancy GBS vaginal colonization and ascending infection is associated with adverse pregnancy outcomes and fetal/neonatal infection. The to establishment of GBS intestinal colonization in newborns i s also believed to be a critical precursor Late-Onset Disease (LOD) which typically presents as meningitis and results in long-term neurological sequalae in survivors. The bacterial and host determinants that promote GBS mucosal colonization and systemic infection, as well as the role of native microbiota are essentially unknown and represent significant knowledge gaps in the field. Previous microbiome studies have demonstrated that GBS colonization impacts vaginal microbial diversity. Using a mouse model of GBS vaginal colonization, we found that GBS vaginal persistence is associated with the presence of Akkermansia muciniphila (AM), a Gram-negative intestinal commensal that degrades mucin; however, the specific mechanism(s) for this synergy is unknown. Our preliminary data show that the presence of AM dramatically increases GBS adherence to human vaginal epithelial cells (hVEC). Using triple RNA-sequencing we identified numerous GBS and hVEC genes that are significantly altered in the presence of AM, including increased bacterial surface adhesins, such as pili, and decreased transcription of hVEC genes involved in neutrophil signaling and chemotaxis. I hypothesize that AM may promote the observed increase in GBS vaginal persistence by increasing GBS attachment and modulating host immune responses that dampen host defense against GBS. I further hypothesize that AM will similarly promote GBS intestinal colonization which could impact LOD. To examine these hypotheses, we propose the following aims: 1) Determine the mechanism by which AM promotes GBS attachment to epithelium, 2) Characterize the impact of AM on mucosal immunity in the FRT, and 3) Examine the effect of AM on GBS intestinal These mucosal colonization and LOD. studies will greatly increase our knowledge of GBS – commensal interactions which may impact GBS colonization and neonatal disease.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Varicella zoster virus (VZV) is an exclusively human, double-stranded DNA alphaherpesvirus that produces varicella (chickenpox) on primary infection then establishes life-long latency in ganglionic sensory neurons in >95% of adults. With aging and immunosuppression, VZV reactivates in at least 1 in 3 individuals to produce zoster rash (shingles) that can be further complicated by vasculitis (inflammation of the vasculature), myelitis (inflammation of the spinal cord), or chronic pain (post-herpetic neuralgia, PHN). Diagnosing VZV as a causative agent of disease is typically restricted to the detection of viral antigens and/or nucleic acids, however, the disproportionate and sometimes lack of viral nucleic acid/antigen detection in tissues where inflammation persists following acute infection has puzzled clinicians and researchers. Therefore, we suggest a viral-induced, non-infectious “soluble factor” can drive pathology in tissues distant from viral replication in the ganglionic sensory neurons. Exosomes are small extracellular vesicles (~40-200 nm in diameter) of endosomal origin that carry cargo (proteins, nucleic acids) to cells for communication during normal and pathological states, regulating biological processes and response to disease. Using mass spectrometry (MS) and next- generation sequencing (NGS), our preliminary data shows that VZV-infected human sensory neurons (huSNs) release non-infectious exosomes containing unique proteins and miRNAs as well as a single VZV protein (immediate early 62) compared to uninfected neuronal exosomes. These proteins and miRNAs have been shown to suppress the host’s innate antiviral pathway. Indeed, when applied to naïve vascular cells, VZV exosomes induced robust transcriptional changes indicative of suppression of antiviral responses and increased permissiveness to subsequent VZV infection. Compared to mock, VZV exosomes also induced elevated proinflammatory cytokine release in these cells. Given the extensive sensory innervation of multiple organs throughout the body, how these exosomes alter multiple disease-relevant central nervous system (CNS), and non-nervous system cell types remains an important and unexplored area of research. We hypothesize that VZV infected huSNs release non-infectious pathogenic exosomes that contribute to the suppression of innate antiviral responses in cells, resulting in the enhanced permissibility to direct infection, as well as induce a proinflammatory environment. To test this hypothesis, we will: (Aim 1) Determine the content of pathogenic exosomes from VZV-infected huSNs throughout the course of infection and if antiviral treatment alters the pathogenic contents; and (Aim 2) Determine the altered transcriptional and proteomic profile of exosome-exposed primary human spinal cord astrocytes and brain vascular adventitial fibroblasts in the context of innate antiviral immune evasion and inflammation. Understanding how virus-associated, non- infectious exosomes reprogram cells provides a novel mechanism by which virus infection causes pathologies distal from the original site of infection and expands the spectrum of virus-associated diseases.

NIH Research Projects · FY 2025 · 2024-06

ABSTRACT. The Down syndrome (DS)-Connect® registry is an important resource that enables collaboration between the NIH, individuals with DS and their families, advocacy and professional organizations, and a multidisciplinary community of professionals involved in medical care and scientific research serving people with DS. Through collection of information on demographics, health histories, medications, and other self- reported information from self-advocates and their families, DS-Connect® provides an unprecedented view of this genetic condition that can elicit new research and clinical trials tailored to this population. Additionally, self-advocates and their families enrolled in DS-Connect® are served in several ways, including access to important information and resources, as well as opportunities for research participation. Furthermore, the DS-Connect® datasets inform NIH and researchers to ensure that scientific research in this field is attuned to the needs of the community. Our team brings multidisciplinary expertise in development of patient registries and online research portals, cohort studies of the population with DS, clinical trials in DS, open science approaches, and communication and outreach campaigns in the DS community. We look forward to applying this expertise to transition the DS-Connect® registry to a modernized, secure, and sustainable platform that can serve the diverse stakeholders involved in this important resource.

NIH Research Projects · FY 2026 · 2024-06

Project Abstract Eosinophilic Esophagitis (EoE) is a chronic immune mediated inflammatory disease of the esophagus that has emerged as a common cause of swallowing difficulty in children and adults leading to increased health care utilization and negatively impacting quality of life. With rapidly increasing prevalence, clinical phenotypes have emerged including a fibrostenotic phenotype (FS-EoE) defined by characteristic endoscopic appearance, significant esophageal stiffness and narrowing. This phenotype has had lower treatment response and increased symptom burden. Management guidelines do not yet provide a paradigm that consider symptom severity or the presence of fibrostenosis and do not yet incorporate newly approved biologics for EoE. With the recent approval of biologics targeting pathways implicated in the propagation of inflammation and fibrotic remodeling, comparative studies that inform patients and providers alike on the effectiveness of alternate treatment strategies are needed. The purpose of this small R01 proposal is to establish pilot data on the comparative effectiveness and feasibility of a randomized clinical trial of dupilumab versus swallowed topical corticosteroid, fluticasone, in the treatment of FS-EoE. This trial proposes the use of a novel and innovative outcome measure of treatment response, distensibility as measured by EndoFLIP, for its ability to measure changes in esophageal narrowing and association with patient reported symptoms. Biospecimens collected during the course of this trial, including esophageal mucosal biopsies and a state-of-the-art Esophageal String Test, will be used to establish a biorepository linked to trial outcome data to explore next step mechanistic questions related to treatment response and esophageal remodeling in EoE. Results from this proposed study will lay the foundation for a definitive multicenter comparative therapeutic trial for the management of FS-EoE.

NIH Research Projects · FY 2024 · 2024-06

PROJECT SUMMARY The University of Colorado Anschutz medical campus requests funds to purchase a Zeiss LSM980 confocal/2- photon microscope system equipped with fluorescence lifetime imaging (FLIM). This is to replace an equivalent LSM780 confocal/2-photon FLIM system that was purchased in 2013 but is now aging and will very soon no longer be supported by the manufacturer. The LSM780 system we seek to replace represents the only inverted 2-photon microscope system available for use on campus. This system has received consistently high usage since its purchase (>185h/month in 2023, close to maximum capacity) by 149 principal investigators over the past 5 years, and it is critical to support NIH funded research across a wide range of disciplines. The proposed system consists of a Zeiss LSM980 confocal microscope system with spectral detector unit that is equipped for 2-photon excitation via coupling of a Ti;sapphire fs infra-red laser, and inclusion of non-descanned detectors. This system also includes a specialized Picoquant multiharp FLIM module that consists of fast detectors and high-speed photon counting card. This Zeiss LSM980 confocal/2- photon FLIM system will be housed within the institutional Advanced Light Microscopy Core (ALMC). The ALMC is well equipped to house the instrument, which includes in depth expertise with the technologies included via PhD-level staff. The ALMC also receives strong institutional support from the school of medicine via the Neurotechnology center (NTC). This includes -2000sq ft of space that houses 10 instruments, as well as salaries for 3 full-time professional staff and support to backstop any budget shortage to cover instrument service contracts. For the proposed system, institutional support in the shape of $155,000 is provided, as well as a recent purchase of a new Ti:saphhire laser. In choosing the proposed Zeiss LSM980 system, we tested 3 other microscope systems and quantified multiple aspects of the system performance (resolution, detection sensitivity, live sample handling), finding the Zeiss system showed superior performance. Similarly, we determined the proposed Picoquant FLIM module provided the most accurate lifetime measurements. Every component of the system is required by all major users (5) and either the majority of or all minor users (8). These users represent those performing the most cutting-edge microscopy in their NIH-funded research that includes 2-photon microscopy and FLIM and requiring the highest level of usage. These users demonstrate the broad range of microscopy use among the 149 principal investigators who are using the current aging system. Thus, replacement of the current Zeiss LSM780 confocal/2-photon FLIM system with the proposed Zeiss LSM980 confocal/2-photon FLIM system is urgently needed in order to maintain ongoing access to required multi-spectral confocal microscopy, 2-photon microscopy and FLIM, for NIH funded research.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Diabetic wound infections are a major public health burden, with approximately 25% of diabetic individuals developing a wound in their lifetime, 25% of these wounds not healing and 28% requiring surgical amputation. The diabetic wound environment is complex, with hyperglycemia driving immune dysfunction and bacterial pathogenesis leading to poor infection outcomes. One of the most frequently isolated bacterial species from diabetic wound infections is Group B Streptococcus (GBS). Interestingly, GBS is notoriously absent from non- diabetic wounds, suggesting that components of the diabetic environment specifically influence GBS success in this niche. Previously, I have developed two murine models of GBS diabetic wound infection and determined that GBS promotes hyper-inflammation in vivo. Dual RNA-sequencing of the murine and GBS transcriptome has identified multiple host and bacterial targets with altered transcription during diabetic infection that have informed the hypothesis herein. This proposal therefore seeks to elucidate the environmental factors that promote GBS infection of diabetic wounds. I hypothesize that multiple facets of the diabetic wound environment including neutrophil function, hyperglycemia and polymicrobial interactions contribute to GBS success. This hypothesis will be addressed via the following specific aims: Aim 1: Evaluate the consequences of altered neutrophil function to GBS pathogenesis during diabetic wound infection. Aim 2: Determine how hyperglycemia contributes to GBS pathogenesis in the diabetic wound. Aim 3: Examine the contribution of Staphylococcus aureus to GBS diabetic wound persistence during polymicrobial infection. These studies will increase our understanding of how the diabetic wound differs from the non-diabetic wound environment, and the consequences of that altered environment on pathogen success.

NIH Research Projects · FY 2025 · 2024-06

Project Summary/Abstract The COVID-19 pandemic has affected over 690 million people worldwide and caused 6.9 million deaths. Although the recent variants are less infectious and cause lower mortality than earlier ones, diagnosed active cases are as high as 20 million worldwide. SARS-CoV-2 spike protein is continuously accumulating mutations. Our earlier published studies on its receptor-binding domain (RBD) showed that increased receptor binding and escape from neutralizing antibodies direct the natural selection of mutations. However, mutations have also been found in other regions of the spike protein, and the role of these non-RBD mutations is not well understood. Computational models propose a long-range allosteric communication between different parts of the spike protein. During this 2-year proposal period, we will design experiments to test the role of long-range communication by probing the effect of distant mutations on RBD binding to ACE2 and to neutralizing antibodies (Aim 1). Spike protein is a trimer and exists in multiple conformations with individual RBDs either accessible or inaccessible to ACE2 and antibodies. We will probe the relative populations of the four conformational states of the spike protein and the effect of mutations. In addition to SARS-CoV-2 emerged in 2019, two other coronaviruses (CoVs) have caused severe disease: SARS-CoV emerged in 2003 and MERS- CoV in 2012. Though based on only three data points, the data suggests another COVID outbreak might be imminent within the next ten years. Human coronaviruses (CoVs) are widely considered to have originated from bats, and hence, multiple efforts worldwide have been focused on identifying bat CoVs capable of infecting humans. So far, about 700 bat CoVs with unique spike proteins have been identified, and it is crucial to determine which receptor pathways they might use to infect humans in order to develop effective therapies. To begin with, we will examine which of these bat CoV spike proteins might use the ACE2 (SARS-CoV/SARS- CoV-2) and DPP4 (MERS-CoV) pathways by combining bioinformatic sequence analysis and determining the relative binding affinities (Aim 2). These assays will be further applied to determine which other receptor pathways bat CoVs might use to infect the human respiratory system. The molecular knowledge gained during this proposal will help us in predicting the evolution of the current SARS-CoV-2 virus and any future COVID outbreaks, which will finally lead to developing effective therapeutics that can neutralize multiple CoVs.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT The purpose of this K99/R00 application is to provide support for Dr. Zachary Clayton, a promising post- doctoral fellow in the laboratory of Dr. Douglas Seals. This additional research and training will allow him to successfully transition into an independent investigator in the field of translational cardiovascular (CV) physiology. As part of his proposed K99 training plan, he will learn new technical skills, enhance his intellectual and professional skills and participate in various career development activities, including those that will help establish him as a leader in the fields of cardio-oncology and cellular senescence. His proposed project seeks to investigate the role of cellular senescence in regulating arterial dysfunction (i.e. the primary risk factor for CV diseases [CVD]) with the common chemotherapeutic agent Doxorubicin (DOXO), first in mice (K99) and later translating to mice and humans (R00). Senescent cells accumulate in the CV system following DOXO and cellular senescence may exacerbate upstream regulators of arterial dysfunction (i.e. inflammation and oxidative stress). Guided by strong preliminary data, Dr. Clayton will first (Aim 1) confirm that DOXO causes arterial dysfunction via cellular senescence by utilizing complementary mouse models in which he can systemically clear senescent cells. With guidance and training in technical skills from an internationally recognized expert in cellular senescence, he will then (Aim 2) conduct innovative ex vivo cell culture experiments in vascular cells to determine the role of cellular senescence in mediating key phenotypes underlying arterial dysfunction, evoked by plasma from DOXO-treated mice. After transitioning to a faculty position, Dr. Clayton will next (Aim 3; R00) translate his findings first to mice, by performing mouse-to-mouse plasma exchange experiments to determine whether DOXO-mediated arterial dysfunction can be transferred via the circulation and whether this response is dependent upon cellular senescence activation. Next, he will extend his findings to humans by determining the role of plasma from DOXO-treated cancer patients in facilitating arterial dysfunction-related phenotypes in cultured vascular cells. Overall, the proposed research has the potential to address 2 strategic research priorities of NHLBI: 1) investigate new pathobiological mechanisms important to the onset of CVD; 2) determine strategies for reducing vascular morbidity in cancer survivors. The proposed projects will provide opportunities for future fundable research, culminating in submission of a novel R01 during years 4-5 of this award. Dr. Seals (primary mentor) is an internationally recognized and NIH funded scientist with a strong history of successful mentoring in translational CV research. With his guidance and the guidance of co-mentor Dr. Judith Campisi, advisory team members Drs. Youngho Bae, Thomas LaRocca, Saul Villeda, Lavanya Kondapalli, Kamali Kimdar and biostatistician Dr. Zhiying You, Dr. Clayton will be able to successfully complete the proposed research and training plan and transition to an independent, extramurally-funded tenure-track position at a top-tier (R1) research institution.