University Of California, San Francisco

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Chlamydia trachomatis (Ct) is the leading cause of infectious blindness in the world today. The World Health Organization (WHO) estimates that there are over 142 million people at risk of irreversible loss of sight. This figure likely underestimates the true numbers as it is difficult to account for all communities that are affected globally. The ocular disease caused by Ct is called trachoma and carries an annual price tag of approximately $8 billion from lost productivity. The highest concentrations of this neglected disease include 37 countries in Africa, the Middle East, Asia, and Central and South America along with Australia. Africa has over 89% of the world’s known trachoma cases, reflecting a major region of health disparity. The WHO developed the SAFE strategy to eliminate trachoma as a public health problem by the year 2020. SAFE stands for: Surgery to correct in-turned eyelashes (known as Trachomatous Trichiasis or TT); Antibiotics to treat Ct; Facial cleanliness to improve hygiene, and Environmental improvements to reduce transmission. The A part of SAFE includes Mass Drug Administration (MDA) with azithromycin and has been used in many countries. Ten endemic or hypoendemic countries have been validated by WHO as being trachoma free. However, 6 endemic and 38 hyperendemic countries have not, although they have received 5 to over 10 rounds of MDA. The highest burden of disease is in Ethiopia and South Sudan where ~30-50% of children under 10 years of age have active trachoma, and the reasons for this remain unclear. Trachoma as a public health concern will not be eliminated until we understand why there is ongoing active trachoma following multiple rounds of MDA in hyperendemic countries. Our unifying hypothesis, therefore, is that the natural history of trachoma is defined by the interaction of the ocular microbiome, immune responses and pathogen populations (both Ct and non-Ct) that are influenced by MDA. While there is a growing body of research on the ocular microbiome, few studies have evaluated differences in microbiota composition between healthy and trachomatous eyes and none have looked at the influence of Ct infection. We will employ metagenome shotgun sequencing (MSS) to understand healthy, dysbiotic and chlamydial-associated microbiota in addition to immune responses and pathogen genomic characteristics for a cohort residing in the trachoma hyperendemic Amhara Region of Ethiopia. We aim to: 1) Examine Ct infections based on whole genome sequencing; 2) Identify taxonomic diversity and abundance of ocular microbiota among subjects with and without Chlamydia and their association with trachomatous disease; and 3) Determine host microbiota/immune response profiles associated with and without Chlamydia, and develop models to predict trachoma post MDA. This work will naturally transition to improving chlamydial diagnostics that utilize MSS methods, and developing interventions that, given the ineffective antibiotic regimens to date, may include novel therapeutic strategies and/or revised treatment duration to decrease and eliminate hyperendemic trachoma among at risk populations in Africa that have the greatest need for intervention.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Intra-tumor heterogeneity is a significant barrier to precision oncology. Emerging single-cell and spatial profiling approaches have enabled basic research into tumor heterogeneity. However, the application of these emerging approaches to the clinical decision process is limited. There is a critical need for predictive models that integrate these novel data with existing genomics approaches and histology, to generate actionable clinical recommendations. This proposal builds on my lab’s recent work, using single-cell RNA sequencing (scRNA-seq) to map the cellular hierarchies of complex tumors. Our preliminary data extend these studies to single-cell multi-omics, integrating single-cell assay for transposase-accessible chromatin (scATAC-seq) and spatial transcriptomics (ST). Our long-term goal is to develop models of malignant progression based on sequencing data from patient biopsies and deploy them to support clinical decisions. The overall objective of this project is to develop algorithms to integrate heterogeneous single-cell and imaging data to support therapy selection, trained on data from multiple cancers and broadly applicable pan-cancer. The rationale for this work is that these algorithms will be applied to pre-treatment biopsies to predict progression and to recommend appropriate therapy combinations. In Aim 1 we will develop and validate algorithms to model clonal composition, phylogeny, and evolutionary trajectory. This will be used to rigorously identify combinatorial chemotherapy targets and monitor emerging treatment-resistant clones. In Aim 2, we integrate scRNA-seq with ST as training data to develop a predictive model of gene expression and cellular composition, based on imaging data alone. We validate these algorithms internally, on prospective cohorts, and in situ in adjacent tissue. In Aim 3, we develop predictive models of two clinical problems that are challenging in many cancers: 1) the response to ionizing radiation, 2) the emergence of hypermutation at recurrence. Here, we exploit modern deep-and-wide learning approaches to identify genomic predictors of outcome that are tailored to a patient’s clinical context. We will validate this approach using both internal and external controls. Algorithms will be implemented in clinician dashboards in an existing system and the evaluation of clinical support will take place at two sites: the University of California, San Francisco and the University of Pittsburgh. We anticipate that this project will identify novel prognostic signatures, enable risk stratification, disease monitoring, and the selection of precision therapies. These studies will significantly advance our ability to apply single-cell and spatial profiling in the clinical setting.

NIH Research Projects · FY 2025 · 2022-07

Despite the overall progress in the U.S. in lowering the prevalence of cigarette smoking and lung cancer incidence rates, the persistent differences in the burden of lung cancer across population groups is a major public health problem. Over decades, African American and Native Hawaiian adults have suffered a disproportionate incidence of lung cancer in comparison to other population groups. The concerning observation that African American and Native Hawaiian adults experience a higher risk, compared to Japanese American, Latino, and White adults, for the same lifetime exposure to smoking has fueled studies to investigate differences in genetic susceptibility, biomarkers of smoking, and epigenetics. While these studies have shed light on the contribution of molecular factors to differences in lung cancer risk, both molecular, genetic and individual-level lifestyle factors are unable to account fully for these population differences in lung cancer risk. Thus, there is a clear need to address how area-level factors such as housing, poverty, tobacco retail outlets, and access to health care influence lung cancer risk across population groups. To address this gap, we will leverage the unique epidemiological resources of the Multiethnic Cohort Study and Southern Community Cohort Study. These two large cohorts include over 272,000 well-characterized adult participants with up to 27 years of follow-up and high-quality cancer surveillance data. Specifically, we will assess the impact of area-level factors on: change in smoking status and internal smoking dose (Aim 1); lung cancer risk across population groups (Aim 2); and DNA methylation in blood leukocytes (Aim 3). In addition, we will evaluate whether known risk factors for lung cancer mediates the relationships between area-level factors and lung cancer risk. We will also assess whether DNA methylated sites and epigenetic age mediate the associations between area-level factors and lung cancer risk (Aim 3). The strengths of this proposal include: 1) the integration of two population-based cohorts, statistically powered to study five population groups from urban and rural settings, ensuring high-risk populations will be studied; 2) the public health significance of understanding risk factors that influence population differences in smoking behaviors and lung cancer risk; 3) the assessment of the biological pathways by which area-level factors lead to lung cancer risk. Findings from this proposal will expand our understanding of the contribution of risk factors to lung cancer development and the underlying biological pathways by which they may operate. This knowledge has translational relevance in providing empirical evidence to support the development of interventions for smoking and lung cancer risk that may have broad health benefits for other chronic diseases.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract The proposal seeks to establish a pathophysiologic link between premature atrial contractions (PACs) and atrial fibrillation (AF). It has been well established that patients with frequent PACs are more likely to develop incident AF. But whether PACs are causative, or an epiphenomenon, is unclear. We hypothesize that PACs from the pulmonary veins or lateral left atrium (LA) lead to more atrial dyssynchrony compared to PACs from other regions, and that this dyssynchrony leads to atrial structural remodeling (via increased wall stress) and fibrosis that serves to facilitate AF maintenance. Our preliminary data in a swine model of chronic PACs demonstrates that chronic PACs lead to atrial electrophysiologic (slow conduction) and structural (fibrosis) remodeling, which is more pronounced for dyssynchronous PACs from the lateral left atrium compared to synchronous PACs from the septum or controls without PACs. In our first Aim, we will explore in the swine model how differences in PAC coupling- interval and atrial rate affect the degree of atrial remodeling and whether PAC cessation leads to complete regression of remodeling. We will test whether an antifibrotic drug, pirfenidone, prevents adverse atrial structural and electrical remodeling in the presence of PACs. We will also assess which molecular changes precede the development of cardiomyopathy to determine the critical molecular pathways leading to PAC mediated atrial remodeling. In our second Aim, we will establish the importance of these findings in humans by performing a longitudinal case-control study of patients with a high burden of atrial ectopy to identify if chronic PAC-induced atrial dyssynchrony leads to echocardiographic atrial remodeling. We will determine whether patients with frequent PACs have more echocardiographic left atrial remodeling and AF over time compared to those without PACs. We will also determine whether specifically dyssynchronous PACs are more likely to lead to atrial remodeling and AF than synchronous PACs. The significance of the proposed work is that if frequent PACs are found to lead to remodeling that leads to the development of incident AF, early intervention in patients with frequent PACs, with medical therapy or catheter ablation, may prevent the later development of atrial remodeling and AF. Successful prevention of AF could mean that millions of individuals could avoid debilitating loss of quality of life and enormous healthcare costs.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY AND ABSTRACT The primary goal of this proposal is to develop novel theranostic agents targeting CD46 for imaging and treatment of multiple myeloma (MM). CD46 is a novel therapeutic target, with a cognate antibody-drug conjugate, FOR46, now in phase 1 clinical trials. Our preliminary data demonstrate high expression of CD46 in MM. In this proposal, we develop and implement [89Zr]DFO-YS5 and [225Ac]DOTA-YS5 as imaging and therapeutic agents targeting MM. The central hypothesis of this proposal is that CD46 is an effective target for molecular imaging and therapy of MM. In this proposal we test this hypothesis in preclinical models, and in a pilot study of patients with MM. In order to test this hypothesis, we have assembled an experienced team of chemists, biologists, imaging scientists, physicists, and physicians to evaluate this method in preclinical models and in patients. In specific aim 1, we will develop [89Zr]DFO-YS5 and [225Ac]DOTA-YS5 as PET imaging and therapeutic agents for MM. In specific aim 2, we will develop new co-treatment approaches to maximize CD46 expression, and use these to augment [89Zr]DFO-YS5 PET and [225Ac]DOTA-YS5 treatment. In aim 3, we perform a pilot [89Zr]DFO-YS5 PET imaging study in patients with MM, and analyze the data to determine its sensitivity for detecting sites of disease. The methods developed in this proposal will ultimately be used to develop novel diagnostic and therapeutic approaches in MM, to help reduce morbidity and mortality for this disease.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Amyotrophic lateral sclerosis (ALS) is a progressive, uniformly fatal neurodegenerative disorder caused by the loss of upper and lower motor neurons. Despite multiple advanced clinical trials, there are currently no therapies that can stabilize or reverse the motor deficits of ALS. My goal in pursuing a K08 Mentored Clinical Scientist Research Career Development Award is to acquire the knowledge and practical training to make major advances in our understanding of the mechanisms underlying neuronal loss in ALS. With my career, I seek to develop novel therapies that will prevent motor neuron death and preserve function. In 2006 Neumann and colleagues discovered that TAR DNA-binding protein 43 (TDP43) accumulated abnormally within diseased neurons and represented a unifying, end-stage neuropathologic hallmark of ALS. With this observation, the field then converged on the cellular process of autophagy — the method by which cells use lysosomes to purge proteins aggregates and maintain homeostasis — as a critical pathway involved in ALS’ pathogenesis. Lysosomes, however, are tremendously intricate and not well understood compartments within neurons. Lysosomes house many complex, hydrolytic enzymes; they assist in adaptive stress responses; and they promote cell survival broadly. While it has been shown that TDP43 can be directed to the lysosome for clearance, past efforts have stopped their investigations at the lysosomal membrane, presuming that presuming that TDP43 degradation happens efficiently by an array of unspecified enzymes thereafter. My preliminary data demonstrates that only a subset of lysosomal enzymes (cathepsins B, D, E, G, L, K, S, and V) can degrade TDP43 in a pH-dependent manner and that ALS-causing TDP43 mutations have the potential to disrupt the functions of these enzymes. Therefore, I hypothesize that cathepsins B, D, E, G, L, K, S, and V are responsible for TDP43 degradation, pathogenic TDP43 mutations are capable of conferring resistance to these cathepsins, and that impaired cathepsin activity (whether due to TDP43 mutations or age- related changes in pH) contributes to TDP43 buildup, TDP43 aggregation, and neurodegeneration over time. This proposal builds upon my solid foundation in neurobiology and neurology that I have cultivated working as a basic research scientist during my undergraduate, medical school, residency, fellowship, and early faculty years. My current research mentor has an established record of impactful discoveries in the field of neurodegeneration. I have also assembled a team of highly accomplished advisors at UCSF to guide me through this next phase of training on my path to becoming an independent investigator. My training plan is specifically designed to provide me with the mentorship, training in advanced experimental skills, and experience required to run a research group. Completing the research and obtaining the skill sets and mentorship outlined in this proposal will prepare me well to obtain R01 or equivalent funding to begin my career as an independent investigator.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Antiretroviral (ARV) agents for treatment and prevention will be key to Ending the HIV Epidemic. Although oral pre-exposure prophylaxis (PrEP) is highly effective, new options less dependent on day-to-day adherence are needed to maximize impact. The era of long-acting PrEP has arrived, with rollout of two new products in 2022. The dapivirine vaginal ring (DVR), self-inserted monthly, is being implemented in sub-Saharan Africa, and injectable cabotegravir (CAB), given every 8 weeks, is now approved by the U.S. FDA. Despite the promise of these modalities to reduce HIV incidence, acute HIV infections (AHI) occur among PrEP users and were seen in DVR and CAB trials. Potential causes of AHI on PrEP modalities include inadequate ARV adherence/ exposure and resistance to the PrEP agent. As oral and long-acting PrEP are scaled up, 300,000 AHI could occur among PrEP users in the next 5 years. DVR showed modest efficacy in trials and high ring adherence will be critical. With high levels of circulating ARV resistance (from ARVs in the same class used for treatment), DVR could fail to block infection with resistant virus. While CAB efficacy was high in trials, AHI occurred despite on-time injections and resulted in resistance. Because the same drug classes are being used for HIV prevention and treatment, resistance from CAB could render U.S. and global first-line antiretroviral therapy (ART) regimens ineffective. Moreover, CAB levels persist for over 1 year in a pharmacokinetic (PK) tail that could further increase resistance risk. As DVR and CAB are scaled up, key knowledge gaps must be addressed: 1) What are causes of AHI on DVR and CAB in rollout settings? 2) What behavioral factors contribute to DVR/CAB non-adherence prior to AHI? 3) What is the risk of DVR/CAB resistance? 4) What are subsequent HIV treatment outcomes? This application proposes intensive studies of causes of AHI on DVR and CAB, leveraging three studies enrolling persons with AHI from large PrEP programs in western Kenya and the U.S. for sample collection, surveys, interviews, and ART outcomes assessment. Innovative methods will be deployed to detect drug resistance (next-generation sequencing), identify novel mutations (full-genome sequencing), retrospectively measure ARV exposure before diagnosis (PK analysis of hair samples cut into segments reflecting exposure over sequential 2-week intervals), and understand DVR/CAB adherence prior to AHI and ART adherence (longitudinal mixed methods). The proposed aims are: 1) To determine the causes of HIV infection among women using DVR in Kenya. 2) To identify the causes of HIV infection among persons using CAB for prevention in the U.S. 3) To evaluate subsequent outcomes on ART after HIV infection among persons using PrEP modalities. Collectively, these aims will provide the first and largest analysis of causes of and outcomes following AHI on DVR and CAB in rollout settings. This study will pave the way to address crucial questions on novel resistance after CAB or DVR use and drug levels at which resistance emerges, and will inform optimal implementation strategies for DVR, CAB, and next-generation PrEP modalities.

NIH Research Projects · FY 2025 · 2022-07

The goal of the UCSF Chemistry and Chemical Biology (CCB) predoctoral training program is to foster research in the use of chemical strategies to probe fundamental biological questions. The overall program curriculum focuses on: 1) rigorous didactic and hands-on training courses at the chemistry-biology interface in the first year; 2) research rotations in three different labs during that same year; 3) selection of a thesis advisor and development of an independent research proposal at the end of the first year; 4) an intensive oral qualifying exam in the second year; 5) individual thesis research with a focus on impact, collaboration and publication and finally; 6) a dissertation seminar. Integrated throughout this training program, from the first day to the last, are activities that train and encourage trainees to: i) develop skills in responsible conduct of research (RCR), rigor & reproducibility and chemical and biological safety; ii) openly receive and give feedback through evaluation and assessment tools; iii) foster career interests, including use of Individual Development Plans (IDPs); iv) enhance written and oral communication skills; (v) celebrate a sense of community and vi) gain training in critical analysis via journal clubs, seminars and minicourses. The research in the CCB program is highly inter-disciplinary and broadly categorized as: biological chemistry and synthetic biology, computational chemistry and biology, chemical synthesis and natural products, drug discovery and design, macromolecular structure and function and protein and cellular engineering. To enact this training program, the CCB program is not associated with a specific department; rather, we bring together 48 faculty from nine different academic departments and two research institutes across UCSF’s world-class research enterprise. These faculty are rigorously and holistically evaluated by the CCB Executive Committee, for excellence in research excellence and commitment to mentoring, and these faculty are required to complete annual mentor training. They are among the best scientists in the world (3 HHMI Investigators, 10 NAS members; Lasker, Kavli Prize winners; avg $808,000/yr in funding). Oversight of the CCB program is maintained by the Executive Committee, along with an engaged Dean of the UCSF Graduate School and an expert External Advisory Committee. This CBI proposal builds on 20+ years of educational excellence at the chemistry-biology interface and it has no equivalent elsewhere on the UCSF campus. Approximately 12 students are enrolled annually from a pool of ~220 applicants after a rigorous application process that culminates in personal interviews. Our graduates are highly sought-after in academia, industry and beyond, because of their training, creativity and passion for inter-disciplinary science.

NIH Research Projects · FY 2024 · 2022-07

PROJECT SUMMARY/ABSTRACT The autoimmune regulator (Aire) gene, a key transcriptional regulator expressed in medullary thymic epithelial cells (mTECs), has been shown to be crucial for central tolerance by inducing tissue specific antigen (TSA) expression in mTECs. Interestingly, Aire is also found in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs such as the spleen and lymph nodes. We previously found that eTACs are hematopoietic antigen-presenting cells (APCs) and consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells (AmDCs) and an Aire-high population co-expressing Aire and retinoic acid receptor– related orphan receptor γt (ROR γt) that we termed Janus cells (JCs). Functionally, eTACs are capable of enforcing deletion and anergy on self-reactive T cells, and self-antigen expression in eTACs is sufficient to prevent autoimmunity. The transcriptional, genomic, and functional symmetry between eTACs (both JCs and AmDCs) and mTECs potentially identifies a core program driven by Aire that may influence self-representation and tolerance across the spectrum of immune development. However, the lineage relationship of eTACs, what role Aire plays in these populations, and how extrathymic Aire and eTAC subsets contribute to immune homeostasis are still unknown. This proposal will test the hypothesis that Aire is inducing a tolerogenic phenotype in eTACs and that extrathymic Aire and eTACs are important for enforcing peripheral immune tolerance. Aim 1 of this proposal will define the lineage relationship between eTACs subsets, their antigen processing and presentation functions, and their migratory abilities. Aim 2 will define the cell-intrinsic functions of Aire in eTACs at both the transcriptional and chromatin level. Aim 3 will investigate the contribution of extrathymic Aire and eTACs in maintaining normal immune homeostasis. This proposal will be carried out using a variety of methods including single cell multiomics, flow cytometry, and functional approaches such as ex vivo co-cultures and in vivo monitoring of autoimmunity utilizing novel genetic mouse models. By characterizing eTAC subsets and investigating the functional roles of extrathymic Aire and eTACs, this work will help further elucidate the function of Aire and define basic peripheral tolerance mechanisms. Furthermore, understanding the biology of these tolerogenic populations may have significance for a range of clinical applications from autoimmunity to tumor immunity to maternal-fetal tolerance. This research project and fellowship training will be conducted at a top-funded research institution, the University of California, San Francisco (UCSF), in the laboratories of Dr. James Gardner and Dr. Mark Anderson. Dr. Gardner has expertise in studying peripheral Aire/eTACs and the generation of genetic mouse models. Dr. Anderson is a world expert on Aire biology, thymic selection, and immune tolerance, and a highly respected mentor and leader in the field of immunology. These mentors and institution will provide a rich training environment for completion of this research and development of professional skills necessary for a career in academic research.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Long noncoding RNAs (lncRNAs) have been implicated in a wide range of human neurological disorders including cancer, developmental delay, psychiatric and neurodegenerative disease. While it is known that the brain is enriched in specific lncRNAs, relatively few have been characterized in terms of function and molecular mechanism. Our long-term goal is to understand the function and molecular mechanisms of lncRNAs in neurodevelopment. Such fundamental knowledge is critical to understanding how this large aspect of the noncoding genome regulates brain development and disease. We have taken two approaches for the study of lncRNA function. The first approach is a “traditional” molecular-genetic study of a brain-specific, evolutionarily conserved lncRNA that is a potent regulator of neural stem cells (NSCs). In previous studies, we identified a novel lncRNA transcript that we named Pnky (Pou3f2 intergenic non-koding). In cultured NSCs, either Pnky transcript knockdown or Pnky conditional knockout (Pnky-cKO) increases neuronal production by ~4-fold. Pnky is required for proper cortical neurogenesis in vivo, and the expression of Pnky from a BAC transgene (BAC-Pnky) fully rescues Pnky-deletion – including at the level of the transcriptome – indicating that this lncRNA functions in trans. Pnky interacts with the splicing regulator PTBP1 (Polypyrimidine tract binding protein 1) – a critical regulator of neurogenesis from NSCs – and Pnky appears to function in the same molecular pathway as PTBP1. Preliminary Studies demonstrate that Pnky folds into a compact, monodisperse state that contains intricate structures including a pseudoknot, which is a structural module known to have important function in noncoding RNAs. Given these data, we hypothesize that Pnky contains functional structural modules and regulates the function of PTBP1. Our second approach is to use systematic functional screens to discover key principles of lncRNA genome function. In Preliminary Studies, we used CRISPRi to screen in parallel 10,671 lncRNA and 18,905 mRNA genes for roles in the neural induction of NSCs from human induced pluripotent stem cells (iPSCs). We also performed CRISPRi perturbation coupled with droplet- based single-cell RNA-Seq (Perturb-Seq) for hundreds of screen hits. Based on results from these systematic studies, our working hypothesis is that functional lncRNAs – in comparison to coding genes – are enriched for roles in “focusing” differentiation to specific neural cell types. To further test this hypothesis, we will study lncRNA function in human brain organoids and extend our screens to analyze neurogenesis. Determining the unique functional roles of lncRNAs and coding genes at genome-scale will have important, broad impact on the interpretation of transcriptomic and epigenomic studies of neurodevelopment. Together, by studying lncRNA function at the level of individual transcripts and also at genome scale, we expect to gain fundamental insights into the function of this large aspect of the noncoding genome.

NIH Research Projects · FY 2024 · 2022-07

Abstract Tuberculosis (TB) is a major global health threat and the only licensed TB vaccine, Bacille Calmette-Guérin (BCG), is inadequate: despite widespread use of BCG, there were 9.9 million new TB cases globally in 2020. Thus, improved vaccines against Mtb are urgently needed. T cell responses remain the primary goal of TB vaccines, as CD4 T cells are necessary to control Mtb in humans and animal models. T cell vaccines have been thus far unsuccessful in preventing infection, but the recent clinical trial of M72-AS01E demonstrated ~50% efficacy as a Prevention of Disease (POD) vaccine. Since TB is transmitted by those with active disease, which is characterized by inflammation and immunopathology, a vaccine that reduces or prevents active disease and immunopathology can have a large impact on the global problem of TB. However, the most optimal antigen target(s) for TB vaccines are not defined, and the lack of evolutionary variation in the known antigens of Mtb suggests that T cell recognition of those antigens is not detrimental to the pathogen or beneficial to the host. Through a comprehensive analysis of genomes from 216 phylogenetically diverse Mtb, my advisor and his colleagues discovered a distinct subset of Mtb antigens that are sequence variable and exhibit evidence of evolutionary selection pressure. In a screen of DNA vaccines encoding these sequence variable antigens, I discovered one (encoding 4 sequence variable antigens) that alters immunopathology and inflammatory Th1 responses in mice subsequently challenged with Mtb, without reducing lung bacterial burdens. Additional studies in our laboratory have demonstrated distinct CD4 T cell effector responses to these same antigens in humans. From these and other results, we hypothesize that a functionally distinct T cell response to one or more of our vaccine antigens is responsible for altered immunopathology in vaccinated mice after challenge. The objective of this proposal is to characterize the cellular responses in vaccinated mice after challenge and identify the mechanism that accounts for altered immunopathology. I will characterize the cellular response using spectral flow cytometry and immunofluorescence microscopy. I will then characterize the vaccine-specific T cell response using peptide:MHC tetramers and single cell RNA sequencing to identify potential mechanisms mediating the altered immunopathology response. Finally, I will identify the underlying mechanism(s) by performing adoptive transfers of sorted T cells to identify the phenotype of the T cells that alter immunopathology during infection. This work will identify differential T cell responses to distinct Mtb antigens and demonstrate a role of immunoregulation in mediating a vaccine response to Mtb infection. The findings will provide an understanding of T cell responses to Mtb antigens that can modulate immunopathology. This will provide a framework of vaccine-induced immunoregulatory responses to inform development of POD vaccines for Mtb.

NIH Research Projects · FY 2024 · 2022-07

Project Summary/Abstract of Parent Grant Meditation training is a promising technique that can help improve emotional health of adolescents and facilitate treatment of adolescent depression. However, there is a fundamental gap in understanding the neural reorganization that takes place as a result of meditation training. Continued existence of this gap represents an important problem because, until it is filled, design of more effective interventions is highly unlikely. The longterm goal is to establish safe and effective methods of promoting emotional health in adolescents. The objective here is to study adolescents undergoing meditation training by using MRI connectomics to map changes in node strength (integrated connectivity) of the putamen. The putamen is a region previously associated with meditation practice and attenuated shrinkage with age in Zen meditators on the one hand, and with love, compassion, anticipation of pleasure, and responses to increasing intensity of happiness on the other hand. The central hypothesis is that structural connectivity of the putamen with other brain regions will increase in adolescents with meditation training and, in turn, positively affect their emotional health. This innovative model is rooted in preliminary results and previous literature. The rationale for the proposed MRI connectomics approach is that regular engagement of the putamen is expected to increase myelination of the white matter tracks connecting it to other regions, which can be probed by using diffusion MRI. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims, which entail studying changes in the putamen node strength and emotional health measured as internalizing problems and depressive symptoms in 1) a cohort of healthy adolescents with a 12-week meditation training compared to waitlist controls (R61 phase) and 2) a cohort of adolescents with mild to moderate depression with a 12-week meditation training compared to waitlist controls (R33 phase). The “Go/No-Go Criterion” is a medium-large increase of the putamen node strength observed with meditation training in healthy adolescents in the R61 phase (Cohen’s d>0.6). The optimization strategy for the R33 phase is based on the fact that anhedonia (diminished ability to experience pleasure) is a key characteristic of depression and preliminary results show that putamen structural connectivity is lower in adolescent depression. It is therefore expected that the mechanistic effect in the putamen will be amplified in the population of depressed adolescents, reflecting normalization of the putamen function. The proposed research is innovative, because it uses advanced MRI connectomics methods to map changes in brain networks of youth with meditation training and tests a novel mechanistic model. The proposed research is significant, because it is expected to greatly advance our understanding of the neural mechanism by which meditation improves emotional health of adolescents. Ultimately, such knowledge will inform treatment and prevention of adolescent depression.

NIH Research Projects · FY 2025 · 2022-07

Metabolism dysfunction associated fatty liver disease (MASLD), with its more severe form, metabolism dysfunction associated steatohepatitis (MASH), is among the most rapidly growing medical burdens in the US. Effective and safe drugs are needed to treat MASH that is often initiated and/or worsened by dysregulation of bile acid (BA) homeostasis. BA homeostasis is tightly regulated by farnesoid X receptor (FXR) that in the gut highly induces the fibroblast growth factor 15 (FGF15) in mice and FGF19 in humans. FGF15/19 are endocrine FGFs that are critical in suppressing BA synthesis and improving energy homeostasis, and under clinical trials aiming to treat BA related disorders. The effects of FGF15/19 on drug metabolism are unknown; however, are critical to ensure safe drug development. The constitutive androstane receptor (CAR; NR1I3), a xenobiotic nuclear receptor, plays a pivotal role in regulating DME gene expression. CAR can be activated directly by ligand binding or indirectly by inhibition of epidermal growth factor receptor (EGFR). We have generated novel genetically modified mouse models with FGF15 gain- or loss-of-function, and showed that overexpression of FGF15 led to induction of the expression of several CAR specific target genes in drug metabolism. Additional evidence suggests that this induction may be from a nutrient restriction and sex specific gene expression pattern switch. We hypothesize that FGF15 overexpression in male mice sends a signal of “nutrient restriction” to the liver, which decreases GH-STAT5b activation and results in a male-to-female switch of DME gene expression. This switch is responsible for CAR activation by decreasing two brakes on CAR: (1) decreasing EGFR activation and (2) reducing endogenous CAR inhibitors. This novel hypothesis will be tested in two independent but related specific aims. 1. Determine CAR activation by FGF15 in vivo and FGF19 in vitro, and determine to what extent CAR activation is responsible for inducing DME genes by FGF15/19. 2. Determine the molecular mechanism of CAR activation in the male Fgf15 Tg mice. Understanding the mechanisms is highly significant to ensure better medicine design and to prevent toxicities and drug-drug interaction.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Our long-term goals are to advance computational protein design to engineer new biological functions and molecular/cellular engineering strategies to uncover principles of biological regulation. This proposal combines our two NIGMS grants in these areas. In our work on computational protein design, we have computationally engineered proteins that sense and respond to new small molecule signals in cells, a capability with important applications in metabolic engineering, diagnostics, bioremediation, and probing fundamental cellular processes. We have also advanced methods to design proteins with precisely tunable shapes entirely de novo. The proposed work builds on our new methods to address a central unsolved challenge, to simultaneously design the geometries of de novo proteins and user- defined functional sites placed into them with atomic accuracy optimized for function. This work should greatly expand the space of new functions that can be designed. We plan to integrate de novo designed proteins into modular systems that can control biological behavior in response to new signals. Our work on natural protein functions seeks to understand how central regulatory proteins operate in interconnected cellular networks, and how these networks are altered upon perturbations such as mutations. We studied a two-state switch (a GTPase) controlled by opposing regulators because this motif is prevalent in biology. Through systematic mutagenesis of the GTPase Gsp1 and integrating measurements at the systems scale (genetic interaction mapping) with biophysics, we uncovered previously unknown allosteric sites on the GTPase central to its function. Our findings moreover suggest a new model how the pleiotropic GTPase Gsp1 differentially regulates distinct cellular functions. Here we will build on these results to investigate the mechanism of allostery in Gsp1 and assess its generality in other GTPases, with implications for understanding mechanisms of disease mutations and for development of modulators. We also plan to test our model of GTPase regulation by determining quantitative cellular consequences of fine-tuned perturbations to GTPases regulators. Future directions include expansion of these perturbation measurements to other central biological switches. The uncovered principles of cellular control can guide cellular engineering, and in conjunction with computationally designed new functions may ultimately lead to new ways to counteract misregulation in disease.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Despite widespread recognition of pervasive patient safety problems in the U.S. health care system, research to develop feasible, scalable solutions has lagged, particularly in the ambulatory setting. The need for new approaches to improve safety is especially pressing in the care of ambulatory patients using high-risk immunosuppressive medications, as use of this category of medications has grown precipitously and reports of preventable adverse events have increased. In the last AHRQ funding period, our research team generated new epidemiological evidence of patient safety risks and adverse events and used this information to develop electronic health record-based quality measures (eMeasures) for the Centers for Medicare and Medicaid Services Quality Payment Program. In addition, we implemented these eMeasures nationally in the American College of Rheumatology's RISE registry, which includes electronic health record data from over one-third of U.S. practicing rheumatologists. In this proposal, we aim to build on this work to improve ambulatory patient safety for individuals using high-risk immunosuppressive drugs. In Aim 1, we will use data from the RISE registry linked to Medicare claims to identify emerging patient safety errors for newly approved immunosuppressive drugs and analyze these errors across race/ethnicity, socioeconomic status, medical complexity, access to care and practice characteristics. In Aim 2, we will perform a comprehensive evaluation of current national patient safety eMeasures related to immunosuppressive drugs, assessing feasibility, reliability, and validity across diverse practice settings, electronic health record systems, and visit types (telemedicine versus face-to-face). In Aim 3, we will use mixed-methods grounded in implementation science theory to determine key drivers of performance on ambulatory patient safety eMeasures. Data from this research will be used to develop and disseminate the first implementation toolkit designed to improve patient safety for high-risk immunosuppressive drugs. The proposed research will contribute significantly to the national infrastructure to improve ambulatory patient safety for the millions of Americans with immune- mediated diseases.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY The adaptive immune system is responsible for the specific recognition and elimination of antigens originating from infection and disease. It recognizes antigens via an immense array of antigen-binding antibodies (B-cell receptors, BCRs) and T-cell receptors (TCRs), the immune repertoire. Because of the enormous breadth of epitopes recognized by immune repertoires, immune repertoires are extremely diverse and dynamic. Advances in immune receptor sequencing (Rep-seq), such as next generation sequencing, have driven the quantitative and molecular-level profiling of immune repertoires, thereby revealing the high-dimensional complexity of the immune receptor sequence landscape. However, the current analysis tools lack the ability to track and examine the dynamic nature of the repertoire across serial time points or correlate with clinical outcomes. We propose to use network analysis and formulate a novel ensemble feature selection approach, along with other advanced machine learning techniques and statistical approaches (e.g., Bayesian nonparametric approach and shrinkage estimation method), to interrogate and measure immune repertoire architecture longitudinally and in a clinical context. Network analysis is a powerful approach that can help us identify TCRs sharing antigen specificity and highly mutable BCR, which can help to develop or improve existing immunotherapeutics and immunodiagnostics. To integrate gene expression data and scRep-seq data in single-cell setting, we propose to apply the multitable mixed-membership approach to construct a network to increase the resolution of T and B cell clusters. In addition, we assess the importance of shared clusters by introducing Bayes factor to incorporate clonal generation probability and real data abundance. B and T cell responses develop in parallel and influence one another, thus we will further study how BCR/TCR network properties interact, in addition to assessing their individual response separately. We will implement the proposed methods on multiple studies to better illustrate the diversity and richness of the data to demonstrate the flexibility and power of the proposed tools. These studies are unique and generalizable, because they include three cancer types spanning from immunogenic to non-immunogenic in both metastatic and localized settings with different immunotherapeutic modalities. In addition, the proposed methods can be used to study immune response to diseases besides cancer, including respiratory coronaviruses, such as SARS-CoV-2. Therefore, first, we will investigate the landscape of bulk Rep-seq changes over serial timepoints for prostate cancer patients who received Sipuleucel-T and COVID-19 patients. We will develop prognostic/prediction model based on network properties with clinical outcome/characteristics for durvalumab-treated lung cancer patients to elucidate the clinically prognostic features of the network as well classify SARS-CoV-2 infected patients from healthy donors. Moreover, based on unique features of single-cell RNA sequencing, we will classify the immune cells and study the T and B cell responses to immunotherapy (CD40 agonist antibody) for esophageal and gastroesophageal junction cancer patients. Furthermore, we will develop bioinformatics software by incorporating the proposed methods and techniques to tackle the complexity of the immunosequencing data in a translational fashion and provide a comprehensive platform with user-friendly visualization tools.

NIH Research Projects · FY 2024 · 2022-07

PROJECT SUMMARY/ABSTRACT The goal of this proposal is to curate clinically relevant variants in genes associated with the inherited monogenic diseases autosomal recessive Leber congenital amaurosis (LCA)/early-onset Retinal Degeneration (eoRD) that cause lifelong blindness beginning in infancy or childhood. More than 30 genes associated with these phenotypes have been identified and the first gene replacement therapy was approved for LCA/eoRD associated with RPE65 variants, while clinical trials are currently underway to treat disease caused by 3 other genes (AIPL1, GUCY2D, and CEP290). Despite these advances, it is still challenging to make accurate clinical diagnoses and decisions based on current knowledge of variants in LCA/eoRD- associated genes. A major limitation is the lack of uniform classification criteria optimized for gene-disease specific features that enable accurate and consistent interpretation of the clinical relevance of variants. To address this, we have assembled a variant curation expert panel (VCEP) comprised of an international group of experts with in-depth knowledge in LCA/eoRD genetics and clinical care. Further, we established collaborative relationships with the clinical domain working group (CDWG) oversight committee and the Ocular CDWG of the NIH-sponsored Clinical Genome Resource (ClinGen) for advice and guidance. With this leadership team, we propose to curate variants in genes associated with LCA/eoRD phenotypes for which gene therapies are available, or clinical or advanced pre-clinical studies are underway. The proposed project involves 2 Specific Aims: 1. Complete the approval process for the LCA/eoRD VCEP through 4 steps (assembling a group of experts for LCA/eoRD variant curation, rule specification for the classification of variants in LCA/eoRD genes using disease-gene specific characteristic features, pilot testing rules specified for the curation of variants in LCA/eoRD associated genes, and submitting the rules and pilot results to the ClinGen Sequence Variant Interpretation Working Group for approval), and 2. Curation of variants in selected LCA/eoRD genes by implementing the specified rules and submission to ClinVar. All steps will be carried out with the approval of the ClinGen CDWG oversight committee utilizing a suite of variant curation tools and protocols developed by ClinGen. The proposed project will lead to the development of variant interpretation criteria that are in harmony with rules established for other diseases and optimized for LCA/eoRD genes, and generate a comprehensive resource of LCA/eoRD gene variants with FDA-designated expert level variant classifications in the ClinVar public database. This information will advance research on LCA/eoRD and enable accurate, consistent, high quality interpretation of genetic test results, and improve patient care. Further, the rules specified by the LCA/eoRD VCEP will advance development of rules for other IRDs and other hereditary diseases.

NIH Research Projects · FY 2026 · 2022-07

ABSTRACT / PROJECT SUMMARY More than 6 million people live with neurodegenerative diseases in the United States, receiving care and support from both unpaid care partners and paid workers. The paid workforce is large and diverse, employing millions of people, most of whom are in occupations that do not require a college degree. The largest occupation by far is the “direct care” workforce, which consists of 2.4 million personal care aides, 1.1 million nursing assistants, and 800,000 home health aides. These workers are affected by a range of policies and programs that vary across states, payers, and healthcare organizations, including differing training requirements, state- level regulations regarding what services direct care workers are allowed to provide, and reimbursement strategies that may affect demand for and wages of direct care workers. We have little evidence to guide evaluation and refinement of policies even as the importance of ensuring an adequately-sized and well-prepared direct care workforce is growing. There also is an urgent need to identify effective approaches to support direct care workers in their interface with an increasingly diverse population of people living with dementia and with other healthcare professionals. To advance research on the direct care workforce that serves people living with dementia, we will establish the AWARD (Advancing Workforce Analysis and Research for Dementia) Network. Workforce researchers come from a range of academic disciplines (e.g. public health, economics, sociology, medicine, nursing) and are spread throughout the nation with few centered at any one institution. They often are in separate departments or research units from scholars with expertise in dementia care, limiting opportunities for interdisciplinary research collaborations and mentoring of early-stage researchers in the theories and practicalities of conducting studies of the healthcare workforce. The AWARD Network will offer programs and activities to create a strong community of researchers engaged in research on the direct care workforce and its role in care for people living with dementia. Activities of the AWARD Network will include: (1) hosting an annual meeting, (2) holding monthly virtual trainings, webinars, and workshops, (3) organizing two Summer Training Institutes, (4) supporting competitive research internships, (5) directly funding pilot research, and (6) developing resources and data to harmonize and accelerate research. The AWARD Network will develop infrastructure and relationships to generate research that supports evidence-based policy and practice to advance the capacity of the direct care workforce in serving PLWD. This work will ultimately inform policy leaders and healthcare organizations striving to ensure appropriate care and reduce health disparities for this population.

NIH Research Projects · FY 2025 · 2022-06

ABSTRACT We understand very little of how invasive fungal pathogens overcome host blocks to infection, yet such infections are among the most challenging to diagnose and treat. Our focus is the most common cause of fungal meningitis, Cryptococcus neoformans. Macrophages and other myeloid cells are critical for killing pathogens and promoting immune responses. Within the initial site of Cryptococcal infection, the lung, multiple populations of myeloid cells exist, including alveolar macrophages (AMs), interstitial macrophages (IMs), monocytes, and neutrophils. Importantly, a type 2 immune response is triggered by C. neoformans infection and mediates increased susceptibility to Cryptococcal infection. We observed that incubation of C. neoformans with murine bone marrow- derived macrophages (BMDMs) results in induction of arginase-1 (Arg1) a marker for type 2 responses. Through forward genetics, we identified a C. neoformans secreted protein, CPL1, required for Arg1 induction and virulence in vivo. Remarkably, recombinant CPL1 protein is sufficient to induce Arg1 expression in BMDMs in vitro and to boost the bona fide M2 response to the type 2 cytokine IL-4. Correspondingly, CPL1 is critical for C. neoformans to activate Arg1 in interstitial macrophages in vivo. TLR4, known best as an LPS receptor but which is also a poorly-understood mediator of type 2 responses, is required for CPL1 to act on BMDMs. As TLR4 does not directly couple to the key type 2 transcription factor STAT6, we sought indirect mechanisms of action. Salmonella promotes M2 polarization via a secreted effector, steE, that activates STAT3 by mimicking cytokine signaling. Indeed, we found STAT3 to also be required for Arg1 induction by CPL1. These data lead to the following working model: 1) CPL1 promotes macrophage polarization indirectly by weakly activating TLR4 to produce cytokines that activate STAT3, and 2) cooperation between STAT3 and the key type 2 regulator STAT6 leads to enhanced macrophage polarization to drive virulence. To test this model, we have assembled a collaborative team with expertise in fungal pathogenesis and immunology. Through Aim 1, we will elucidate mechanisms by which CPL1 promotes macrophage polarization in vitro. We will test the model that CPL1 boosts the M2 response via STAT3-STAT6 cooperation, and we will investigate how CPL1 functions through TLR4. Through Aim 2, we will test the hypothesis that CPL1 polarizes lung interstitial macrophages in vivo, thereby producing a replicative niche. We will investigate the role of TLR4 in the macrophage/monocyte compartment, and we will use thick section imaging and other modalities to test predictions of the model that CPL1 acts locally to promote macrophage polarization to enable pathogen replication. These studies have the potential to transform our understanding how an invasive fungal pathogen reprogram the innate immune system to its advantage.

NIH Research Projects · FY 2025 · 2022-06

The cancer research community is on the verge of a major leap in our understanding of the factors that contribute to human cancer risk. While it is clear that mutations in DNA, either spontaneous or environmentally induced, are essential for cancer development, recent advances have highlighted the importance of non-mutagenic factors as rate-limiting determinants of cancer risk in human populations and in mouse cancer models. The root causes of human cancer have been widely debated, but most of the emphasis has been on the origins of the “driver” mutations that are ubiquitous in human tumours. Although epidemiology studies have highlighted the possible roles of lifestyle factors such as obesity, alcohol consumption, inflammation and poor diet in cancer risk, it has generally been assumed that these factors act directly or indirectly to cause mutations in DNA, thus contributing to tumour mutational burden and resulting in increased cancer risk. In contrast, recent sequencing studies have uncovered abundant mutations in normal human tissues, suggesting that even strong cancer driver mutations are not sufficient for cancer formation. These results were presaged by studies of mouse tumour models, some carried out more than 50 years ago, showing that promotion is the rate-limiting step in tumour development. To identify the mechanisms that control mutated normal cells, and to elucidate the precise mechanisms by which promoting factors stimulate the conversion of these cells to neoplastic growth, we have assembled a multidisciplinary team of investigators with wide-ranging experience in epidemiology, genetics, computational network analysis and machine learning, tissue imaging of gene expression, single cell transcriptomics, and genome-wide CRISPR functional screens. We will focus human analysis on a unique collection of several thousand human normal and matched tumour samples from >20 countries, including regions of both high and low cancer risk. Detailed risk factor information and whole genome sequence data is available from all these samples as part of the Grand Challenge Mutographs study. Analysis of these samples, together with detailed intervention studies in human populations, mouse models and human organoids, will allow us to develop a roadmap of tumour promotion from single normal cells carrying driver mutations, through to malignant progression. Our findings will facilitate identification of the causative environmental factors that promote cancer and provide routes to new methods and approaches to cancer prevention based on a deeper understanding of the process of initiated cell selection by tumour promoting agents.

NIH Research Projects · FY 2025 · 2022-06

Project Summary/Abstract……………………………………………………………………………………………… This is an application for a K23 award for Dr. Tasce Bongiovanni, an acute care, trauma and surgical critical care physician at University of California, San Francisco (UCSF). Dr. Bongiovanni is establishing herself as an investigator in patient-oriented research using implementation science to prevent the prolonged use of pain medications in older adults in the postoperative period. Although short-term use of opioid-sparing medications may be appropriate for postoperative pain control, long-term use in older adults can lead to adverse events including increased risk of hospitalization and death, and should be avoided. However, a shift towards multimodal pain regimens driven by the opioid epidemic has taken place without attention to ensuring that opioid-sparing medications are discontinued appropriately. Since older adults account for roughly half of all surgeries in the United States, a proportion expected to increase as the population rapidly ages, a deeper understanding of prolonged use of opioid-sparing medication is an urgent public health concern. The objective of this K23 proposal is to better understand and address, via a targeted, evidence-based intervention, prolonged used of postoperative pain medication in older adults. My central hypothesis is that continuation of pain medication postoperatively is common in older adults, has worsened as surgeons shift to multimodal regimens, and that this phenomenon is largely due to lack of communication and coordination between care teams and patients. The aims of this proposal are: 1. In a nationally representative Medicare population, define the epidemiology of prolonged use of pain medications prescribed postoperatively, including patient, clinician and health system risk factors; 2. Conduct qualitative interviews with clinicians and older adult patients and their caregivers to document experiences of prolonged use of pain medication in the postoperative period and obtain feedback about a planned pilot intervention to address these issues and 3. Pilot an intervention to prevent the prolonged use of postoperative pain medication for older adults after surgery. The aims of this proposal are developed to directly support career development activities with a focus on training in 1. Advanced statistical analysis, specifically of Medicare data; 2. Robust qualitative methodology; 3. Design and evaluate effective implementation strategies for older adults and 4. Career development and leadership, focused on surgery in older adults, with the long-term goal career goal to combine her clinical and research interests to improve postoperative care and medication use in older adults. Dr. Bongiovanni will conduct this work with an exceptional mentoring team, led by Dr. Steinman and embedded in the UCSF Pepper Center. This K23 proposal will advance our knowledge of the risk factors and drivers of prolonged use of pain medication in the postoperative period, and use this knowledge to design, refine and test the feasibility and acceptability of an intervention to prevent prolonged use for older adults. It will also provide advanced research skills and valuable data to launch Dr. Bongiovanni’s career as an independent investigator at the intersection of surgery and aging. Together, the data and training plan will form the basis for a compelling R01 proposal to improve postoperative care for older adults.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY/ABSTRACT The ability to enhance cognition by targeting circulating factors in the systemic milieu provides a unique and underexplored therapeutic approach to brain aging. We and others have shown that systemic manipulations, including exposure to young blood through heterochronic parabiosis (in which the circulatory system of a young and old animal are joined) and young blood plasma administration, revitalize the aged hippocampus and ameliorate cognitive decline in aged mice. The rejuvenating effects of young blood mirror those observed with exercise, positing common bloodborne mechanisms of action by which broad systemic interventions exert their beneficial effects. Preliminary data from our labs indicate that systemic administration of platelet factors derived from either naïve young mice or exercised mice reverses age-related impairments in adult neurogenesis, deceases neuroinflammation and restores cognitive function in aged mice. Moreover, using proteomics, our two groups have independently identified CXCL4/platelet factor 4 (PF4)—a chemokine released from platelets, involved in coagulation and a variety of immunomodulatory functions—as a potential young blood-derived and exercise-induced anti-geronic circulating factor. The proposed study will investigate the rejuvenating and therapeutic effects of platelet factors on the aged brain. Specifically, our hypothesis is that systemic exposure to platelet factors rejuvenates adult neurogenesis, attenuates neuroinflammation and improves cognition, while ameliorating neurodegenerative phenotypes. We will test this theory with three specific aims: 1: Determine mechanisms downstream of PF4 underlying cognitive and regenerative rejuvenation in the aged hippocampus. 2: Examine the rejuvenating effects of PF4 on neuroinflammation in the aged hippocampus. 3: Investigate the beneficial effects of PF4 in a mouse model of Alzheimer’s disease. Successful completion of these studies will have significant translational potential, identifying platelet-derived circulating factors as candidate therapeutic targets to restore age-related cognitive dysfunction and potentially treat dementia-related neurodegenerative disorders such as Alzheimer’s disease.

NIH Research Projects · FY 2024 · 2022-06

PROJECT SUMMARY/ABSTRACT Dementia causes substantial burdens for patients and caregivers, which have been exacerbated by the COVID- 19 pandemic. The current state of dementia care is inadequate to meet the needs of this growing, vulnerable population. Scalable, effective, and person-centered dementia care models that are aligned with value-based healthcare reforms are needed now. The Care Ecosystem is an accessible, remotely delivered team-based dementia care model, designed to add value for patients, providers and payers in complex organizational and reimbursement structures. Care is delivered via the phone and web by unlicensed Care Team Navigators, who are trained and supervised by a team of dementia specialists with nursing, social work, and pharmacy expertise. Care Protocols guide proactive, quality care that is documented in the electronic health record. The evidence base to date suggests that the Care Ecosystem improves outcomes important to people with dementia, caregivers, and payers when delivered in a controlled research environment, including reduced emergency department visits, higher quality of life for patients and lower caregiver depression. We propose a rapid pragmatic trial in 6 health systems serving geographically and culturally diverse populations. We will leverage technology, delivering care via the phone and web and using electronic health records to monitor quality improvements and evaluate outcomes while maximizing external validity. In Aim 1, we will use implementation science to identify the model adaptations, facilitators, and barriers to implementing and sustaining the Care Ecosystem during the COVID-19 pandemic. In Aim 2 we will use mixed methods to rigorously evaluate the effectiveness of the Care Ecosystem on outcomes important to patients, caregivers, healthcare providers, and health systems during the pandemic. In Aim 3, we will characterize the patient and caregiver factors associated with treatment benefit. This will include investigating effectiveness in underrepresented groups and elucidating unmet needs that will guide future development work. By simultaneously evaluating the real-world effectiveness and implementation strategies in diverse health systems, this project will bridge the science-practice gap in dementia care during an unprecedented time of heightened strain on family caregivers, healthcare providers and health systems. Furthermore, this work will pave the way for expanding access to high quality dementia care in the future, mitigating the negative impact of dementia on patients and their families across the nation.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY/ABSTRACT Oral and oropharyngeal cancers result in over 10,000 deaths each year in the United States. Although oral squamous cell carcinoma (OSCC) patients with localized disease have survival rates of up to 80%, about two-thirds present clinically with regional and distant metastases associated with five-year survival rates of 50% and 35%, respectively. Despite advances in immunotherapy, the five-year mortality rate for OSCC has remained constant over the last several decades, underscoring the importance of early detection and intervention. The majority of OSCCs arise from pre-cancerous lesions called oral epithelial dysplasias (OED), only some of which will progress to invasive cancers. Patients with OEDs that will progress would likely benefit from more aggressive treatment early on; however, the morbidities associated with aggressive treatment are significant, preventing their broad use in all patients. While this general outlook is similar across many cancers, the accessibility of oral cavity lesions also provides a unique opportunity for detailed analysis to understand the biological processes that contribute to or protect against progression into invasive and malignant cancer. We will test the hypothesis that the immune response to OED regulates the risk of progression. The immune system responds to disruptions and danger in tissues. Significant evidence supports the important role of the immune system in responding to early lesions in the oral cavity, including an abundance of immune cells infiltrating these tissues, elevated risk in immunosuppressed individuals, and loss of MHC class I antigen presentation machinery in many OSCC tumors. However, features of the immune response are not currently utilized to define treatment strategies or to stratify risk in OED or OSCC patients, presenting an unmet opportunity. The recent development of multiplexed ion beam imaging (MIBI) enables unprecedented detailed analysis of archival pathological tissues. This technology, which we recently implemented with the help of an NIH Instrumentation Grant, uses antibodies conjugated to heavy-metal reporter ions to quantify up to 50 proteins simultaneously at subcellular (400nm) resolution in formalin-fixed paraffin-embedded tissues. We have collated a substantial number of archival tissues from OED patients with detailed clinical and follow up data, including progression to OSCC. Here, we will leverage MIBI to conduct a detailed analysis of immune responses in these tumors, providing new insight into the immunological mechanisms and cellular interactions in these microenvironments. In Aim 1, we will test the hypothesis that the types of immune cells present and their activation states are distinct between OEDs that went on to progress versus those that have not. In Aim 2, we will test the hypothesis that the architecture and cellular neighborhoods within the tissue are distinct in OEDs that progressed to OSCC. In Aim 3, we will use these data to identify immune features associated with and predictive of risk of progression. These studies will harness a new imaging technique to answer fundamental questions about the immune response and to guide precise treatment decisions for patients with OED.

NIH Research Projects · FY 2025 · 2022-06

Project Summary Sepsis is a dysregulated host response to infection that culminates in organ failure leading to millions of deaths worldwide each year with an increasing incidence as the population ages. There is a fundamental lack of understanding of the complex host immune response in sepsis that has limited the development of targeted therapeutics for which there are none beyond antibiotics and supportive care measures. At its core, there is substantial immunopathology in sepsis with contributions from an overly exuberant immune response and ineffective pathogen clearance. We and others have studied the critical role of platelets in immune responses during acute infections, including the role of the lung in extramedullary platelet biogenesis. In this application, we will explore the role of the spleen, a central immune organ, in extramedullary megakaryopoiesis and platelet production in sepsis. Based on preliminary data, we hypothesize that the spleen co-opts a significant role in platelet biogenesis during sepsis and that the platelets produced from the spleen are immunomodulatory and important in host defense. In Aim 1, we will utilize a mouse model of peritonitis and polymicrobial sepsis resulting in thrombocytopenia to understand the mechanics of ‘stressed’ platelet biogenesis in this setting. We will study the role of adrenergic-dependent hematopoietic progenitor mobilization from the bone marrow during sepsis and the niche-promoting factors that regulate this process. In Aim 2, we will interrogate the engraftment of circulating hematopoietic progenitors in the spleen, their maturation into megakaryocytes, and the mediators (SCF, CXCL12, IL-3) regulating this process. Using state-of-the-art techniques such as intravital imaging and lineage tracing enabled by splenic transplantation, we will test the hypothesis that the spleen significantly contributes to platelet biogenesis during sepsis. In Aim 3, we will use novel methods of single-cell RNA sequencing of platelets to test for platelet heterogeneity during homeostasis and sepsis in mice and humans. Within this aim, we will test a novel cellular therapy for sepsis by transfusing immune-skewed platelets into septic mice and testing for therapeutic benefit. In summary, these studies will produce paradigm-shifting knowledge on the role of the spleen in extramedullary megakaryopoiesis and platelet production and the importance of platelet driven immunity, which will be foundational in the design of new therapeutic approaches to treat sepsis.