University Of California, San Francisco

NIH Research Projects · FY 2025 · 2022-09

The emergence of microbes resistant to even the most powerful antibiotics represents a serious threat to global public health. The coordinated action of the bacterial machinery of peptidoglycan (PG) synthesis, a process essential for bacterial viability, represents an obvious target for the development of new antibiotics. In this proposed project, we aim to define the molecular interactions of the components of the PG degradation apparatus, consisting of enzymes involved in glycan chain hydrolysis or modification. Our previous studies in Neisseria meningitidis showed that targeting a hot spot on a single lytic transglycosylase (LgtA) also disables the function of the PG-modifying enzyme, Ape1, leading to a disruption of PG assembly, and results in an aberrant peptidoglycan composition, making this pathogen unable to survive in the host. Our studies will reveal, in molecular detail, how these peptidoglycan degrading enzymes work in concert to assemble the bacterial cell wall. Specifically, we will define, in a comprehensive way, how a network of lytic transglycosylases (LtgA, LtgD, LtgE) and their protein binding partners work to facilitate peptidoglycan degradation and the insertion of organelles into the bacterial cell envelope. In this project, we will utilize biochemical and biological approaches to probe protein-protein and enzyme-substate interactions of the various lytic transglycosylases, combined with determination of the molecular basis of activity of the multienzyme complexes in PG metabolism. Genetic modifications of the components of the PG biosynthetic nanomachine will be used to test the observations from our structural studies. Our approach, utilizing high resolution x-ray crystallographic tools along with cryo-EM single particle analysis, will allow visualization of the action of enzymes in PG assembly and degradation and should provide mechanistic insights into their orchestrated activity during the insertion of new PG during cell wall assembly and bacterial cell division. Our studies will lead the way towards the development of new therapies targeting multiple peptidoglycan metabolic enzymes.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Current proton and ion therapy treatment planning procedures utilize either the physical quantity linear energy transfer (LET) as a surrogate for biological effectiveness or make use of relative biological effectiveness (RBE) models that convert absorbed dose to biologically weighted dose, assumed to be iso- effective to photons. LET is indeed important clinically for planning treatments with charged particles, but there are known problems. Ion beams with the same LET can have different RBE, depending on particle type and energy. Therefore, LET by itself is not an ideal parameter to use in radiation treatment planning (RTP). For clinical application of carbon therapy, RBE-models have been developed. However, comparisons of different RBE models used for carbon therapy have shown that dose prescriptions implemented with the European local effect model or the Japanese National Institute of Radiological Sciences mixed beam model can be up to 15% different. We use the term ionization detail (ID) to mean the detailed distribution of ionizing events along a particle track on the nanometer scale. Our chief hypothesis, which is supported by strong prior evidence, is that ID can predict, better than LET and existing RBE models, the biological effects associated with high-LET radiation. We have previously shown how ID can be used together with these models to improve their performance, providing a path for integrating ID-based RTP into clinical practice. Our approach could lead to a consensus in proton and ion therapy RTP. With four Specific Aims, we have chosen a translational and stepwise approach to build an ID-based prediction model. We will test this model for different endpoints and model systems ranging from in vitro cell and molecular data, obtained by irradiating human cancer cells in flasks and anatomical phantoms, to in vivo mice/human tumor data. We will develop advanced algorithms and computational GPU- based methods and use them for effective inverse treatment planning with actively scanned proton and ion beams. This technology will be applied to demonstrate the practicality and evaluate the clinical efficacy of our approach in prostate and chordoma treatments, first prospectively in human-size pelvis and head phantoms, and finally, retrospectively in patients treated for these diseases. We have assembled a strong team with the complementary expertise needed for this project. Members of our team have all successfully collaborated together. Upon completion, we will provide a rigorously tested and validated approach to ID-based particle RTP that will be available for cross-correlation with existing clinical data and for careful testing in prospective clinical particle therapy trials.

NIH Research Projects · FY 2025 · 2022-09

Glioblastoma is the most common primary malignant brain tumor in adults with a median survival of less than 15 months despite aggressive standard of care including surgery, radiation and chemotherapy. Novel therapies are therefore in critical need. Tumor-derived 2′3′-Cyclic GMP-AMP (cGAMP) induced by chemotherapy and radiation activates STING in myeloid cells as an immunotransmitter to elicit anti-tumor response. Macrophages and microglia are both potent responder to cGAMP in non-tumor conditions, however, in GBM, macrophages and microglia, which are one of the most abundant cells in the tumor microenvironment, are profoundly immunosuppressive. It is unclear through what molecular mechanisms and fundamental biology that macrophages and microglia minimize STING activation in GBM. While STING signaling is activated by a series of well-characterized protein phosphorylation, the mechanisms of inactivation/dephosphorylation of STING signaling is unclear. Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase comprised of a catalytic (C), regulatory (B) and scaffolding (A) subunit and accounts for 50-70% of the total serine/threonine phosphatase activity. The specificity of PP2A is determined by its regulatory B subunit. We have found that inhibition of PP2A catalytic subunit (PP2Ac) in both macrophages and microglia enhances STING signaling. Mice with macrophages/microglia specific deletion of PP2Ac have increased tumor T cells infiltration and reduced tumor growth. Moreover, from unbiased screening of all known regulatory B subunits, we find that striatin4, a specific B subunit of PP2A, has a similar role as PP2Ac in suppressing STING signaling in macrophages/microglia but not in glioma cells. In this project, we propose to: Aim 1) Determine the impact of PP2Ac inhibition in macrophages/microglia on STING pathway in glioma microenvironment in vivo. Aim 2) Elucidate the molecular mechanisms underlying STING inhibition by striatin4-PP2Ac complex in macrophages/microglia. We will also use clinically annotated GBM samples to verify the clinical relevance of striatin4-PP2A. These findings will firmly establish the role of the specific striatin4-PP2A complex in regulating macrophage/microglia functions and also provide the mechanistic foundation to target this specific PP2A complex leading to precise targeting of PP2A as a novel therapy for glioma. This proposed study will also address the fundamental biology about how glioma cells and macrophages/microglia communicate through cGAMP and how macrophage/microglia turn off STING activation through PP2A. We appreciate that PP2A complexes play significant roles not only in macrophages/microglia, but also in glioma cells and other cell types. Our long-term goal is to identify the specific PP2A complexes in glioma cells, immune cells, astrocytes and other cell types within the central neural system relevant to the modulation of immune response in order to develop novel therapeutics for GBM. We believe this study fits to the mission of NINDS to seek fundamental knowledge about the brain and nervous system and to use that knowledge to reduce the burden of neurological disease such as brain tumor.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Morphogenesis is the biological process by which cells, tissues, and organs acquire the shape that is critical to their function during embryonic development, and it can be repurposed during regeneration of tissues after damage in a mature organism. Work on embryonic explants has revealed that differences in cellular morphologies and mechanical cell-cell interactions, both controlled by signaling molecules, likely drive tissue- specific shapes in multiple epithelial tissues including the symmetric murine molar. Nevertheless, a deeper understanding of the basic principles and cellular behaviors that regulate morphogenesis is required to leverage these processes for future regenerative therapies that can mitigate the effects of aging and disease. I will use the early developmental stages of the murine incisor to study how cell behaviors drive directional growth and morphogenesis. Murine incisor development is highly asymmetric, and the mechanisms regulating this process have remained elusive. Prior studies have shown that perturbations in Sonic Hedgehog (Shh) signaling result in abnormal incisor morphology, and that Shh-dependent cell movement drives tooth bud invagination in the symmetrical molar. Through this proposal, I will test the hypothesis that modulation of the Shh signaling cascade drives changes in cellular morphology and behavior that determine the asymmetric morphogenic development of the incisor. I will measure and quantify localized cellular and tissue morphological changes such as cell shape, nuclear position, and tooth curvature, as well as dynamic cell behaviors such as differential proliferation, oriented cell division, and cell intercalation, using high resolution live imaging and our novel software program, MARGARITA. This will establish a foundational atlas of cell morphologies and behaviors responsible for the epithelial bending events driving early development of the asymmetric incisor (Aim 1). Next, pharmacological perturbation of Shh signaling in incisor explants and spatiotemporal modulation of Shh expression in genetic mutants will determine to what extent the modulation of this signal transduction pathway affects cellular morphology during incisor development (Aim 2). These findings will provide significant insights into basic tooth and developmental biology, which have the potential to be applied towards future dental regenerative therapies. Current strategies to restore missing dentition (i.e., implants, dentures) can lead to significant bone resorption or may fail due to limited osseointegration. Thus, biologically regenerating teeth using morphogenesis-driven techniques has the potential to significantly improve restorative dentistry. These research goals will be conducted in conjunction with a comprehensive training plan designed to develop my career as a dentist-scientist. Training includes structured mentorship from two highly qualified sponsors, as well as scientific and technical training through meetings, seminars, journal clubs, and classes at UCSF, which offers both an outstanding research environment and an excellent dental school for clinical training.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract The purpose of this K01 Mentored Research Scientist Development Award is to promote the candidate's development as an independent health disparities researcher, with a focus on primary care interventions to address disparities in food insecurity and social isolation among adults with chronic conditions. Dr. Thompson-Lastad will use mixed-methods research to study the effectiveness and implementation of primary care interventions in order to meet her long-term career goal of advancing health equity in diverse communities. Towards this end, the proposed mentored research for this K01 award is to assess an innovative model of care that provides prescriptions for fresh fruit and vegetables with or without group medical visits (GMVs) to ethnically and linguistically diverse, low-income patients. To support this research and Dr. Thompson-Lastad’s goal of independent health equity research, this K01 proposal includes formal, mentored training through coursework and tailored tutorials in quantitative methods for practice-based research, measurement of social needs in health care settings, community-engaged methods, and responsible conduct of research. This training will be applied to research with two specific aims: (1) Assess the implementation of produce prescriptions and group medical visits at nine clinics using a longitudinal process evaluation; and (2) Examine changes in food insecurity and social isolation among 400 adults with diabetes receiving produce prescriptions with and without GMV participation. This research leverages interventions implemented by a USDA-funded partnership which includes a network of Federally Qualified Health Centers, county government, and local farms. The partnership will design and implement the intervention. University of California, San Francisco is an ideal environment for the proposed training because it provides access to experts in health disparities, social needs, and diabetes. Completion of the proposed research and career development activities will inform the development of an R01-level clinical trial focused on how group medical visits with food access components influence social connectedness, health outcomes, and social needs over time among FQHC populations. Completing the K01 will ensure Dr. Thompson-Lastad’s ability to conduct independent research on primary care interventions to advance health equity among ethnically diverse, multilingual populations.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY / ABSTRACT Cardiovascular disease is the major cause of mortality and morbidity worldwide. Despite significant progress in biomedical imaging, the imaging of heart rhythm disorders remains a major technological and scientific challenge. Consequently, the origins and mechanisms for the onset and progression of cardiac arrhythmias remain largely insufficiently understood. Patients suffering from cardiac arrhythmias have high recurrence rates and often require repeated therapeutic interventions, in part because adequate imaging of the processes underlying heart rhythm disorders has yet to be developed. The state-of-the-art for the diagnosis of heart rhythm disorders, such as atrial fibrillation or ventricular tachycardia, is catheter-based electro-anatomic contact mapping. However, catheter mapping is time-consuming and invasive, involving the insertion of electrodes into the heart’s chambers, where abnormal electrical activity triggering the heart’s irregular contractions is recorded on its surface. Because the measurements are superficial, they do not adequately capture the full, three- dimensional electrophysiological dynamics, which evolve underneath the surface and often have their origin inside the heart muscle. In this project, the applicant aims to develop a novel and radically different approach for the in-depth transmural imaging of heart rhythm disorders based on high-resolution 4D (time-resolved 3D) ultrasound and artificial intelligence (AI). Instead of imaging the heart’s electrical activity, the applicant will image the heart’s 4D deformation and use AI to predict the electrical phenomena from the deformation with the precision of high-resolution measurements. To achieve this ground-breaking goal, the applicant will generate an extensive high-resolution dataset, capturing the 4D electrical and mechanical dynamics of arrhythmic hearts, and train an AI to learn the complex relationship between the heart’s deformations and the electrophysiological wave phenomena that cause these deformations. The AI will become highly specialized in recognizing cardiac deformation mechanics and associating them with the corresponding underlying electrical arrhythmia morphology. The data will be generated in beyond-state-of-the-art voltage-sensitive ex vivo fluorescence imaging experiments with intact, isolated hearts, as well as during clinical imaging and in computer simulations. The high- risk approach, which preliminary data suggests is achievable, will be enabled by the applicant’s unique expertise in ex vivo imaging, which, combined with recent advancements in AI, could lead to a major breakthrough. Ultrasound-based imaging providing transmural 4D visualizations of cardiac arrhythmias in real-time would be transformative in cardiac electrophysiology and provide novel insights into many of the yet unseen processes underlying heart rhythm disorders. If successful, the entirely non-invasive imaging technique could greatly advance the diagnosis of heart rhythm disorders and be used to guide therapeutics, such as catheter ablation, more reliably and effectively.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Mutations in DDX3X are associated with autism spectrum disorder, brain malformations, and epilepsy, and account for up to 3% of cases of females with unexplained intellectual disability. However, little is currently known about the molecular mechanism linking DDX3X mutations to neurodevelopmental disease. DDX3X encodes an RNA helicase of the DEAD-box protein family and has been implicated in many aspects of RNA metabolism, yet we still lack a mechanistic understanding of DDX3X’s role in these processes, as well as how patient mutations in DDX3X affect RNA metabolism. To address this gap in knowledge of understanding of how DDX3X mutations perturb cellular function and contribute to neurodevelopmental disease, we will investigate the mechanism of how DDX3X regulates its target transcripts both at the level of translation initiation and mRNA nuclear export, and study how pathogenic mutations in DDX3X alter these processes. DDX3X is implicated in translation initiation as it binds to 5’UTRs of mRNAs, and is required for the efficient translation of a subset of mRNAs with structured 5’UTRs. However, we still do not know the mechanism of how DDX3X can regulate the translation of these mRNAs. In addition to its role in translation, DDX3X has been implicated in nuclear mRNA processing, and our preliminary data show that the genes in the mRNA export pathway are genetic interaction partners of DDX3X. However, we still do not understand what the function of DDX3X is in the nucleus or mRNA export. We hypothesize that DDX3X regulates its target transcripts through both unwinding secondary structures in their 5’UTRs and chaperoning their nuclear export. To answer this hypothesis, in our first aim we will determine how DDX3X impacts mRNA transcript structure and ribosome engagement by measuring RNA secondary structure through in vivo structure-specific chemical modification and high-throughput sequencing. In our second aim, we will determine the function of DDX3X in the nucleus by examining nuclear export of DDX3X -sensitive mRNAs using RNA FISH staining. Upon successful completion of the proposed experiments, we will have gained a greater understanding the precise mechanism of how DDX3X regulates RNA metabolism both at the level of translation initiation as well as mRNA export. This knowledge is critical for advancing our knowledge of RNA metabolism, as well as understanding and developing treatments for patients with DDX3X syndrome.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT A combined behavioral couples therapy and motivational interviewing intervention to reduce intimate partner violence and alcohol use in South India Globally, an estimated 30% of women have reported physical or sexual violence by an intimate partner in their lifetime. Women who report intimate partner violence (IPV) have worse short- and long-term health outcomes, including increased risk for sexually transmitted infections and HIV, poor maternal health outcomes, and increased risk for suicide attempts. Perpetrator Alcohol Use Disorder (AUD) increases risk taking behaviors, and impairs problem-solving and cognitive processes, which may drive IPV. The current scientific understanding of these urgent issues has following limitations: a) most interventions improve either IPV or AUD but not both outcomes; b) interventions that successfully improve both are delivered by highly trained mental health professionals, limiting access and scalability; and c) most interventions focus on either just the husband or the wife but not both. These limitations have led to a strong scientific and implementation gap of interventions that are feasible, effective, and scalable in low-resource settings to target both IPV and AUD. Our Indo-US collaborative team pilot tested an intervention to deliver behavioral couple’s therapy (BCT), based on principles derived from Social Cognitive Theory (SCT) to enhance couple’s communication, combined with contingency management to reduce alcohol use. This intervention was acceptable, feasible and showed preliminary efficacy of IPV and alcohol use in couples when the husband had AUD. We now propose to build on and extend this intervention to combine BCT with motivational interviewing (MI), delivered by primary care nurses, to reduce alcohol use and IPV among couples in India and to test this in a randomized controlled trial. Our research team has a long history of collaborative research in South Asia. Dr. Ekstrand has a 25 year history of research in India, supported by 11 NIH-funded studies where she was the PI or MPI, six of which were at the proposed site. Drs. Acharya and Ekstrand currently oversee two NIH-funded R34 and R21 studies in South Asia successfully using MI. Dr. Srinivasan has led several studies that examined the relationship between AUD and high-risk behavior, including IPV, and was the senior PI of our pilot intervention on which this proposal is based. Dr Srinivasan has also been MPI on three NIH-funded R01 studies with Dr. Ekstrand. We propose to build on this evidence base and robust research infrastructure at primary health clinics at our South India site. We will conduct a randomized controlled trial (n= 400 couples) and study the impact of BCT and MI in reducing IPV and AUD. The intervention will be delivered by nurses in primary health centers who will be supervised by a clinical psychologist. We will perform intention to treat analyses to compare treatment and control groups on the two primary outcomes at 12-months follow-up: 1) mean scores on the Indian Family Violence and Control Scale and 2) number of days with a negative breathalyzer test over a 1-week period. We will assess secondary outcomes and other measures to conduct mixed-methods analyses to assess the theorized mechanisms of change influencing intervention effectiveness. If successful, our study will provide evidence for a low-cost couples’ intervention for IPV and AUD that can be delivered in primary care settings.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Radiotherapy is essential to achieve durable disease control for breast cancer patients. However, despite several decades of research and development in imaging and radiotherapy techniques, breast radiotherapy remains crude due to the lack of viable methods to accurately isolate, immobilize, localize and target the breast. Subsequently, the success of tumor control is at the cost of both acute and chronic toxicities that adversely affect the patients’ quality of life and potentially introduce life-threatening complications decades after the curative treatment. The challenge in providing accurate breast imaging and therapy is due to the unique biomechanical properties of the breast, which is an external organ with no internal skeletal support. As a result, its shape varies substantially with the patient's posture. In the supine position, which is the most stable and common position for radiotherapy, the breast rests on the chest wall, resulting in its close proximity to the chest wall, lung, heart, and other vital organs, which creates an undesirable geometry for radiotherapy. Yet, existing devices for supine breast setup not only provide poor support and immobilization but also adversely interfere with imaging and therapy X-rays. Patients treated in the prone position experience new problems, including the lower setup reproducibility, increased cardiac dose due to heart descending, the difficulty to tolerate, and incompatibility with nodal treatment. To attain the desirable prone breast geometry and avoid drawbacks associated with this posture, a more effective method to lift the breast from the chest wall and to image the breast in the supine position is urgently needed for precision image-guided breast radiotherapy. To achieve the first goal, a pneumatically powered multi-gait soft robot, BreastBot, will be developed and optimized to support and immobilize the breast in the supine position. The BreastBot will be fabricated in several generic form factors to minimize the cost but personalized for each breast via an individualized actuation sequence. The feasibility of BreastBot has been demonstrated using single gait prototypes on volunteers and phantoms. To achieve the second goal, which is to image the BreastBot immobilized breast for image-guided radiotherapy, avoid imaging dose to the patient's body, and achieve a higher image quality, a novel supine ceiling-mounted breast CT will be developed. The following four aims are proposed for image-guided supine breast radiotherapy. Aim 1: Optimization of a breast setup soft robot (BreastBot) for supine breast setup. Aim 2: Development of a ceiling-mounted CBCT for breast image- guided radiation therapy. Aim 3: Specific Aim 3: End-to-end integration, validation, and observational patient study.

NIH Research Projects · FY 2025 · 2022-09

Elimination of an infectious disease is often a goal of the public health community. Although that goal is rarely achieved, the tremendous expansion of epidemiological databases provides new opportunities to test hypotheses concerning elimination with mathematical modeling. Besides improving our scientific understanding of disease transmission, hypotheses validated through mathematical modeling provide public health practitioners with a more structured, quantitative assessment of how elimination of specific pathogens can be achieved. This proposal aims to develop an interconnected set of modeling tools to support elimination of communicable diseases. A variety of processes used to achieve disease elimination will be considered including use of mass drug administration to eliminate neglected tropical diseases such as trachoma, vaccination for preventable diseases such as SARS-CoV-2, and antibiotic stewardship efforts to curtail drug resistant infections such as methicillin-resistant Staphylococcus aureus (MRSA). A key theme is the requirement of subcritical transmission for disease elimination, meaning that the average number of new infections each case causes is less than one. A major goal is to elucidate the transmission dynamics of subcritical diseases on the brink of elimination. Transmission heterogeneity may arise from many mechanisms including super-shedding of certain individuals, pockets of susceptibility such as in a community with low vaccine uptake, and contact structure in which some individuals have the potential to infect many others. Simulations of various patterns of disease transmission will be used to develop distinct measurements of transmission heterogeneity. In addition, new techniques to infer and compensate for observation error will be developed that integrate data on the observation process, such as the proportion of cases identified retrospectively via contact tracing programs. Models of transmission dynamics will be used to identify transmission-hotspots and superspreaders that can jeopardize elimination. People, areas, or events that have increased transmission potential can maintain endemic disease transmission even though the population- level average value of R may be less than one. In the first stage of this objective, we will use existing models to construct a suite of in silico simulations to compare the performance of various scan statistics designed to detect disease burden beyond what is expected by chance. In the second stage, we will apply these scan statistics to observational data. Identification of transmission-hotspots and supersreaders permits optimization of disease elimination strategies. To eliminate disease, it is insufficient to merely identify transmission- hotspots or superspreading activity. A strategy is needed for suppressing the sites, events, or people that cause higher levels of transmission. We will use mathematical and computational models for disease elimination to address 1) the impact of control interventions, 2) the optimal distribution of a limited treatment supply, and 3) monitoring of treatment efficacy.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY / ABSTRACT We recently published a study that used genetic encoded voltage indicators to show that gamma-frequency (specifically ~40 Hz) synchronization between parvalbumin (PV) interneurons in the left and right mPFC normally increases during specific cognitive tasks. Furthermore, specifically disrupting this synchrony was sufficient to produce cognitive deficits similar to those observed in schizophrenia. Finally, we have found that transiently increasing or decreasing gamma synchrony using optogenetic manipulations leads to long-lasting changes in both gamma synchrony and cognition. Thus, gamma synchrony is a key mediator of cognition that can undergo bi-directional plasticity thereby correcting or inducing cognitive deficits. This project will now use genetically encoded voltage indicators, optogenetics, chemogenetics, slice electrophysiology and calcium imaging, to identify interventions and cellular/synaptic mechanisms that produce therapeutic increases and deleterious decreases in gamma synchrony, and elucidate exactly how changes in gamma synchrony affect information processing by prefrontal circuits. This will lead to a greater understanding of how gamma synchrony contributes to normal cognition, and reveal specific targets for restoring cognition in conditions such as schizophrenia.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Antitumor immune responses require a functional repertoire of innate and adaptive immune cells. Glioblastoma (GBM), however, harbors a profoundly immunosuppressed microenvironment, particularly its T cell ignorance caused by bone marrow sequestration; T cell exhaustion caused by immune checkpoint molecules on the surface of T cells that suppress T cell function; and impaired memory T-cell responses. Unfortunately, efforts to target the immunosuppressed GBM microenvironment with systemic immunotherapies have not produced meaningful impact in clinical trials. Localized viral treatments have also been investigated for GBM and, while these viruses elicit an anti-tumoral immune response, these treatments have also failed to impact survival in clinical trials. To address these limitations, we have investigated intratumoral delivery of a replicating retrovirus expressing RLI, which encodes an interleukin-15 fusion protein that enhances CD8+ and CD4+ naïve and memory T-cell proliferation, as a therapeutic strategy free of the toxicities of systemic treatments targeting the tumor microenvironment. We demonstrated that replicating retroviral delivery of RLI prolonged survival of immunocompetent mice with intracranial gliomas using multiple different models. Here, we will build upon our data by investigating our central hypothesis that intratumoral RLI immunomodulatory gene therapy can be potentiated by adding other immunomodulatory strategies, incorporating immunogenic cell death, or targeting resistance mechanisms. We will investigate our hypothesis through four specific aims: (1) Potentiate RLI immunomodulatory gene therapy by enhancing T-cell mobilization, co-stimulation, and memory; (2) Determine if targeting checkpoint pathways potentiates retroviral RLI immunomodulatory gene therapy; (3) Enhance RLI immunomodulatory gene therapy by incorporating immunogenic cell death; and (4) Identify and target glioblastoma-expressed proteins that counteract retroviral RLI immunomodulatory gene therapy. Our pursuit of these aims will utilize novel technologies developed by our lab such as our binary retroviral system to deliver a large payload of immunomodulatory genes and our retroviral compact Cas13d RNA-targeting CRISPR to target resistance mechanisms. We will combine these innovative approaches with cutting-edge technologies such as CyTOF to characterize the effects of RLI-based retroviral therapies on the full cohort of innate and adaptive immune responses; customized CRISPRi libraries; paired immunodeficient and immunocompetent mice strains to isolate immunologic resistance mechanisms; and single cell sequencing to profile T-cell subsets altered by these therapies. These studies will develop our novel localized RLI retroviral immunotherapy in a manner that addresses the spectrum of mechanisms creating local and systemic immunodeficiency in GBM by accounting for T-cell ignorance and exhaustion, and identifying and targeting tumor cell and immune cell-driven resistance mechanisms before they evolve. In doing so, we will validate our hypothesis regarding the impact of RLI-based immunomodulatory gene therapy on GBM, a novel strategy with significant translational potential.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Anorexia nervosa (AN) is associated with significant risk for deadly medical complications and an annual cost to the US of around $11.2 billion. Although Family-Based Treatment (FBT) for adolescent AN has demonstrated effectiveness in targeting symptoms of AN, up to 60% of individuals who receive FBT do not remit fully. Notably, no prior work has explored neurocognitive predictors of FBT response, which may help to facilitate the identification of treatment mechanisms and formulation of targeted treatments for non-responders. When considering what neurocognitive processes may be implicated in FBT response, increasing work suggests that adult AN may be characterized by alterations in reinforcement learning. Further, work in other forms of psychopathology suggests that reinforcement learning may predict response to behavioral treatments. However, few studies to date have tested alterations between reinforcement learning and treatment outcome, and none have explored associations between reinforcement learning and FBT outcome. The current investigation will leverage methods from cognitive neuroscience and computational modeling to explore reinforcement learning in adolescents with AN (n = 58) and healthy control subjects (n = 58), as well as its associations with treatment outcome in FBT. I will test the following hypotheses: Aim 1: Consistent with existing data in adults, the AN group will demonstrate poorer performance in the learning task compared to HC, decreased loss learning, and poorer exploitation of prior learned information. Aim 2: Within the AN group, lower rates of learning from loss, as well as lower explore/exploit parameter values will relate to poorer outcomes at 1- and 6-month follow-ups, operationalized as lower body weight and greater eating disorder cognitive symptoms. With the mentorship of five experts across biostatistics, adolescent clinical research, computational modeling, and cognitive neuroscience, the current patient-oriented career development award will allow me access to training that will facilitate unique expertise at the intersection of these fields. Short-term, data from the current investigation will yield insights that can be used to understand the persistence of AN symptoms and identify potential methods to improve treatment outcomes. Long-term, the current project will allow me to launch my career and take the next steps in a programmatic line of research merging complementary expertise in neurocognition, computational methods, and adolescent intervention development.

NIH Research Projects · FY 2025 · 2022-09

Project Summary HIV incidence remains unacceptably high in sub-Saharan Africa (SSA) due in part to inadequate access, uptake, and retention in biomedical HIV prevention services, including pre- and post-exposure prophylaxis (PrEP/PEP), among persons at increased HIV risk. Alcohol use is a common risk factor for both HIV acquisition and poor HIV prevention uptake and retention in SSA. Interventions that promote biomedical HIV prevention among persons with heavy alcohol use and their sexual partners are urgently needed. Alcohol-serving drinking venues play an important role as sites of HIV transmission in SSA and are ideal sites to engage women and men at increased risk of HIV in biomedical prevention services. However, despite long- standing awareness of drinking venues as transmission “hot spots”, few interventions exist to reach and engage persons in PrEP and PEP from drinking venues in SSA. Major barriers to reaching and engaging persons at high risk of HIV from community settings such as drinking venues in HIV testing – a critical first step to accessing biomedical HIV prevention – include HIV-associated stigma and poor perceptions of risk. To address these barriers, we have developed a mobilization strategy of integrating HIV testing within multi- disease screening to recruit >2,000 people from drinking venues in Kenya and Uganda, reaching >75% of adults recruited for HIV testing. We now need to determine whether multi-disease mobilization can promote uptake of HIV prevention for adults at drinking venues in the context of new biomedical prevention options. Following uptake of biomedical HIV prevention, persons with heavy alcohol use face challenges with retention in care and adherence to PrEP/PEP. We have adapted a brief alcohol counseling intervention (Health Living) to reduce alcohol use and promote antiretroviral therapy (ART) adherence and HIV viral suppression among persons with HIV in Kenya and Uganda. We now need to determine whether this intervention can promote retention in biomedical prevention and PrEP/PEP adherence among adults with heavy alcohol use. The project will rigorously test innovative interventions in Kenya and Uganda to increase uptake and use of biomedical HIV prevention, and assess facilitators, barriers, and cost-effectiveness of these approaches. The project will have the following aims: Aim 1: Compare the effectiveness of two mobilization strategies to increase uptake of biomedical HIV prevention among adults at drinking venues. Aim 2: Determine the efficacy of the Healthy Living Intervention (HLI) to reduce heavy alcohol use vs. standard care (control) on retention in biomedical HIV prevention in a randomized trial among adults with heavy alcohol use. Aim 3: Determine the cost-effectiveness of interventions that increase biomedical HIV prevention uptake (Aim 1) and retention (Aim 2) among adults at high-risk for HIV who attend drinking venues. The proposed research will address the critical intersection of alcohol use and HIV risk in SSA, by promoting reach, uptake and retention in biomedical HIV prevention and exploring associated facilitators and barriers.

NIH Research Projects · FY 2025 · 2022-09

Abstract/Project Summary: In the US, youth and young adults living with HIV (YLWH) have lower rates of antiretroviral therapy (ART) initiation, suboptimal ART adherence and retention in care, and higher rates of virologic failure, compared to older age groups. Additionally, there is an increased risk of substance dependence, psychiatric disorders, and mortality with increased risk of substance use at a younger age. Mental health (MH) and substance use (SU) impact every step of the HIV care continuum from diagnosis to viral suppression and exacerbate socioeconomic challenges of linkage and sustained access to healthcare. Given the strong evidence for the influence of MH and SU on poor HIV health outcomes, there is a clear need for increased access to and provision of these services. Despite the need to address these critical barriers to care in YLWH, there is a severe shortage of MH professionals nationwide and a lack of interventions tailored to this age group. In collaboration with the nonprofit AIDS Healthcare Foundation (the largest provider of HIV care) and a Youth Advisory Panel, the proposed study aims to address these barriers in a tailored manner using a differentiated care approach that is “youth-friendly.” Our goal is to test the effect of a tailored technology-based intervention in a randomized clinical trial (RCT) with an adaptive treatment strategy (ATS) among 200 YLWH (18–29 years old). The intervention includes: (1) brief weekly counseling sessions with a counselor to discuss MH, SU, HIV care engagement, and other barriers to care delivered via a video-chat platform, and (2) a mobile health application designed and developed using Human-Centered Design (HCD) with YLWH to address barriers to engagement in care. Individuals who are not virologically suppressed will be randomized to video- counseling+app or standard of care (SOC). Through this entirely remotely-conducted study, we will be able to: Aim 1: Test the efficacy of video-counseling+app vs SOC on virologic suppression in YLWH. We will compare HIV virologic suppression of those randomized to the intervention vs control arms at 16 weeks via an RCT. Aim 2: Assess the impact of video-counseling+app vs SOC on MH and SU in YLWH. We will evaluate the MH and SU differences between the intervention vs control arms at 16 weeks via an RCT. Aim 3: Explore an ATS to individualize the intervention by assigning the: (a) virologic “non-responders” in the intervention arm to intensified video-counseling+app for 16 more weeks, (b) virologic “responders” in the intervention arm to continue only app use for 16 more weeks. Therefore, in an era of severe shortages of MH providers when MH and SU challenges of YLWH are critical barriers to care, examining an innovative intervention developed using HCD with a differentiated care approach directed specifically to YLWH, grounded in a well-established theoretical model of care and formative research, and with community partnership is necessary for “getting to zero” and ending the HIV epidemic.

NIH Research Projects · FY 2026 · 2022-09

PROJECT SUMMARY Alzheimer’s disease (AD) is a large and growing worldwide public health epidemic. Because there is no cure for AD, much clinical effort is focused on interventions targeting potentially modifiable risk factors. Recent epidemiological studies have identified hearing loss (HL) as a major modifiable risk factor for AD. These findings have prompted debate over whether HL specifically accelerates AD pathology, or if the link instead reflects shared risk factors such as age, genetics, or metabolic conditions. Because human studies are necessarily correlational, an animal model is necessary to determine whether HL specifically influences AD pathology. We address this question in Aim 1 using the E4FAD mouse model of AD, which is homozygous for the most prevalent genetic AD risk factor (ApoE4) and co-expresses five additional familial AD mutations. We will determine whether HL induced by cochlear ablation accelerates AD pathology, including hallmark accumulations of amyloid β (Aβ) as plaques and phosphorylated tau (p-tau), as well as increased neuronal hyperactivity and reductions in memory-related sharp wave ripples (SWRs). Because these pathologies emerge first within entorhinal cortex (EC) and hippocampus (HC), we will assess cognitive impairment (CI) reflecting EC-HC dysfunction (e.g., spatial memory). The expected results will be informative for clinical best practices, e.g., by suggesting whether prevention and treatment of HL could itself mitigate AD, or if instead targeting a common cause is needed to address both HL and AD. Independent of these outcomes, recent studies in rodents have shown HL causes long lasting CI reflecting HC dysfunction. Although the underlying mechanisms remain poorly understood, they may include side effects of noise exposure in rodents (e.g., elevated stress hormones in HC), as well as psychosocial factors in humans (e.g., communication difficulty and social withdrawal). However, recent rodent studies have documented CI following HL induced without noise exposure (e.g., cochlear ablation), implicating mechanisms other than psychosocial factors and noise side effects. A leading candidate mechanism in such cases is disrupted neuronal activity in the EC-HC which could result from altered input from the auditory pathway following HL (e.g., decreased sound-related activity and increased spontaneous hyperactivity). We address this possibility in Aim 2 through longitudinal physiology recordings in EC-HC and auditory cortex (ACtx) before and after HL induced by cochlear ablation. We will examine whether hyperactivity emerges in EC-HC in parallel with upstream ACtx, quantify possible reductions in SWRs, and determine whether functional connectivity is altered between EC-HC and ACtx. The expected results will clarify mechanisms of HL-induced CI and may suggest clinical responses to both HL and CI (e.g., pharmacological attenuation of hyperactivity). Collectively, our proposal will resolve outstanding unresolved questions surrounding the role of HL in HC dysfunction including AD-related pathology. These findings will be highly relevant to large clinical populations suffering disability due to AD and HL.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY / ABSTRACT A hallmark goal in human biology is to define the relationship between genes and phenotypes. Mapping the function of every gene in human cells will enable us to begin to define how gene expression programs impart specialized and adaptive human cellular functions required for life. We are especially interested in how transcription factors and epigenetic regulators enact cell type specific gene expression programs to dictate cell function during early development. Elucidating how individual genes function to regulate transcription and thus to program cell phenotypes will transform our understanding of human biology, development and disease. A mechanistic understanding of gene function requires scalable approaches for perturbing gene activity, single cell molecular phenotyping assays and robust models of human multicellular biology. We recently developed CRISPRoff— a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that durably and robustly silences gene expression. Unlike CRISPR mutagenesis approaches, CRISPRoff gene silencing effectively programs null alleles at the level of target gene mRNA and protein in polyclonal cell populations without induction of DNA damage or the unpredictability of DNA repair processes. We are proposing to optimize a generalizable multiomic CRISPRoff platform for molecularly phenotyping null alleles at single-cell resolution in multicellular models of human development. We will then use this CRISPRoff platform to create single-cell molecular multiomic maps of nuclear gene function across space and time. Lastly, we will evaluate genetic compensation and paralog functional redundancy in multicellular models. Our proposed research will serve to demonstrate the utility of this multiomics CRISPRoff platform for characterizing null alleles and motivate extending this approach to functionally map null allele phenotypes for all genes encoded by the human genome. The results of the proposed research will serve as a fundamental resource and roadmap for a broad community of biomedical scientists and greatly inform our understanding of gene function in human biology and disease.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Gliomas account for 80% of all malignant brain tumors and have an extremely poor prognosis, with a 5-year survival of 5.1%. The etiology of glioma remains poorly understood, with few established modifiable risk factors. Multiple studies have implicated infections in the development of glioma, however the underlying mechanisms and putative causal pathogens remain unclear. In addition to risk, there is also accumulating evidence from studies investigating novel therapeutics suggesting that immune response to infection may be prognostic in glioma patients. Previous epidemiologic studies have investigated a limited number of pathogens using serological assays that only allowed detection. We seek to conduct a large serologic study measuring 41 antigens from all 12 infections previously associated with glioma using assays that provide quantitative measures of antibody response. Our study will include 1000 glioma case-control pairs with extensive clinical and epidemiologic data. In Aim 1 we will estimate the effect of each individual infection on glioma risk and survival and also examine grouped patterns of co-infections. In Aim 2, we will employ innovative long read sequencing technology to detail all polymorphisms in human leukocyte antigen (HLA) class I and II genes in the same set of subjects. Genetic variation in the HLA region plays a pivotal role in regulating immune response to viral challenge and has been previously linked to glioma risk and progression. We will investigate a range of functional HLA polymorphisms, including antigen-presenting classical alleles and amino acid residues, with respect to glioma risk and survival. In Aim 3, we will integrate serological and HLA sequencing data to delineate host genetic mechanisms of immune response to infection and subsequent effects on glioma endpoints. This will allow us to develop comprehensive immunogenomic models for predicting glioma risk and survival. Taken together, the proposed study will contribute new, high-quality data that will significantly advance our understanding of glioma pathogenesis, as well as inform avenues for prevention and improvement of outcomes in glioma patients.

NIH Research Projects · FY 2025 · 2022-09

PROJECT ABSTRACT Dementia is the 6th leading cause of death in the U.S. with marked disparities by socioeconomic status, gender, and race/ethnicity. Food insecurity is a common experience in U.S. children and adults, and it likely influences Alzheimer's Disease and Alzheimer's Related Dementias (AD/ADRD) via multiple mechanisms. Yet, there is almost no quantitative research evaluating food insecurity and AD/ADRD. The very limited prior work in this area is cross-sectional which introduces challenges: 1) the temporal ordering of food insecurity and cognitive performance is ambiguous; 2) researchers are unable to disentangle if acute vs. chronic food insecurity differentially impacts dementia risk; and 3) measuring cognitive assessments at a single time point precludes the possibility of evaluating cognitive decline. Through innovative uses of the U.S. Health and Retirement Study (HRS), and the National Longitudinal Survey of Youth, 1979 cohort (NLSY79), the proposed research fills this critical gap. Both data sources collect prospective information on food insecurity and dementia risk among older adults (HRS), and across the lifecourse (NLSY79). We will leverage the complementary strengths of these datasets to evaluate: [1] if food insecurity among older adults predicts dementia risk (Aim 1; HRS); [2] if lifecourse food insecurity (from ages 18 – 48) predicts dementia risk (Aim 2; NLY79); and [3] if Supplemental Nutritional Assistant Program (SNAP) benefit amount patterns (ages 18 – 48) predict dementia risk (Aim 3; NLY79; SNAP is a federal food insecurity alleviation program). This innovative work on lifecourse food insecurity is possible through our novel application of sequence analysis, which will be applied to markers of food insecurity collected over time in both nationally representative data sources. Our research questions focus on food insecurity, which is a modifiable target for AD/ADRD prevention that is biologically plausible, common, and potentially high impact through existing policy interventions, such as SNAP.

NIH Research Projects · FY 2026 · 2022-09

Extreme heat, wildfires, and other natural disasters have led to acute effects on morbidity and mortality; yet, the long-term consequences of extreme heat and wildfires have been sparsely studied. This is due in part to the paucity of studies with longitudinal information on individual- and area-level factors along with fine-scale weather and wildfire data. There is an urgent need to investigate the long-term consequences of extreme heat and wildfires in well-designed studies that include a comprehensive assessment of these exposures and their independent and joint effects on life expectancy, coupled with a critical evaluation of possible mediators (ambient particulate matter) and moderators (sociodemographics, comorbidities, health behaviors, area-level factors, and geography). Furthermore, the biological processes by which extreme heat and wildfire affect mortality are not well understood. While evidence of the impact of particulate matter on DNA methylation as a measure of epigenetic regulation is accumulating, studies of extreme heat and DNA methylation are sparse. Elucidation of the underlying pathways and the identification of interventions for high-risk groups are needed. To address these gaps, we will conduct a rigorous investigation of the impacts of extreme heat and wildfire smoke on adult life expectancy, leveraging the unique epidemiological resources of the Multiethnic Cohort Study, a large population-based study that includes 112,000 adult men and women from California, who were ages 45-75 at enrollment in 1993-1996 and currently ages 72-103. Specifically, we will generate and characterize extreme heat and wildfire smoke for California Multiethnic Cohort participants spanning a 24-year period (Aim 1); assess the impacts of long-term exposures of extreme heat and wildfire smoke on life expectancy (Aim 2); and DNA methylation and epigenetic age (Aim 3). The strengths of this proposal include: 1) the use of state-of-the-art exposure assessment methods to characterize extreme heat, wildfire smoke, and particulate matter; 2) a large population-based sample with detailed individual- and area-level data with sufficient power to detect modest effects that are broadly generalizable to similar population groups in the US; 3) the assessment of the role of biological pathways (DNA methylation) by which extreme heat and wildfire smoke may operate; and 4) a thorough investigation of effect modification by a variety of factors such as greenspace and cooling centers as well as the mediation of effects by particulate matter. Findings from this proposal will expand our understanding of the contribution of long-term extreme heat and wildfire smoke on life expectancy. This knowledge has translational relevance in providing empirical evidence for implementation scientists to develop strategic interventions and response plans to combat the health effects of extreme heat and wildfire smoke.

NIH Research Projects · FY 2024 · 2022-09

Project Abstract/Summary People with HIV (PWH) have a high burden of respiratory symptoms due to chronic lung disease, of which COPD, diagnosed by spirometric obstruction on pulmonary function testing (PFT), is best studied. The most common finding on PFTs, however, is an abnormal diffusing capacity for carbon monoxide (DLco) with normal spirometry, or iso↓DLco. The clinical relevance of the iso↓DLco PFT phenotype is not known. Iso↓DLco is more common in PWH than in the general population, and HIV is an independent risk factor for reduced DLco. Preliminary work from our lab has shown that PWH with iso↓DLco have an increased respiratory symptom burden compared to PWH with normal PFTs. Iso↓DLco is also associated with a unique set of plasma inflammatory/immune biomarkers compared to other PFT phenotypes like spirometric obstruction, suggesting that the iso↓DLco PFT and biomarker pattern has a unique clinical correlate. The pathophysiology underlying this finding is not known but may be related to early structural lung disease (emphysema or interstitial lung disease) or pulmonary hypertension. Alternatively, iso↓DLco may be a sequela of chronic inflammation in the setting of long-standing HIV infection and possibly co-infection with other viruses like cytomegalovirus (CMV), which affect HIV persistence and immune activation. The central hypothesis for this study is that iso↓DLco is a unique HIV phenotype, possibly mediated by CMV-induced vasculopathy. The study will be nested within I AM OLD-DA, an established longitudinal cohort of PWH in San Francisco, USA and Kampala, Uganda, and will leverage the existing research infrastructure. In San Francisco we will use advanced imaging analyses of chest CTs to understand the etiology and potential causes of iso↓DLco. In Aim 1, we will evaluate CTs for emphysema, interstitial lung disease, pulmonary hypertension and air trapping; based on our pilot study, we expect that in about half of the PWH with iso↓DLco, imaging analysis will not identify a reason for the PFT finding. In Aim 3, we will test for association between iso↓DLco, CMV and distal pulmonary vascular remodeling (‘vascular pruning’) using quantitative CT methods with a working hypothesis that CMV-mediated vascular pruning is associated with iso↓DLco. In Kampala, Uganda, we will study a demographically and clinically distinct cohort or PWH and HIV-negative controls to determine the prevalence of iso↓DLco and its associated respiratory symptom burden (Aim 2). Altogether, the results from this study will help generate a deeper understanding of this PFT phenotype and determine if CMV is a modifiable risk factor for iso↓DLco and a target for therapeutic intervention. Completion of this project will also provide a platform for training Dr. Katerina Byanova, a pulmonary and critical care fellow at the University of California San Francisco, in the conduct of high-quality, patient-oriented clinical research. This grant will provide Dr. Byanova with the support necessary to acquire the knowledge and skills to become an independent clinical investigator and a leader in HIV-related lung disease. .

NIH Research Projects · FY 2024 · 2022-09

Project Summary Lung diseases such as acute lung injuries (ALI) and acute respiratory distress syndrome (ARDS) are leading causes of morbidity and mortality worldwide. Regulatory T cells (Tregs) are traditionally thought of as critical negative regulators of systemic immune responses; however, their local roles in tissues such as the lung are being increasingly appreciated, where they can promote lung epithelial regeneration in both ALI and ARDS. Subsets of tissue-resident Tregs (tTregs) are found to express transcription factors T-bet, Gata3, RORt, and Bcl6, and have enhanced suppression towards their Th1, Th2, Th3/17, and Tfh immune ‘flavors,’ respectively. Although tTreg function are beginning to be understood, how lung tTregs are regulated, positioned, and maintained within their respective tissue niches remains unknown. Mesenchymal stromal cells (MSCs) are immune regulators and have been highlighted to play a role in tTreg biology. Production of IL-33 by a subset of MSCs promotes the expansion and maintenance of visceral adipose tissue tTregs and human MSCs can induce Tregs from conventional CD4+ T cells in vitro. Our group identified a stromal cell niche within the lung where adventitial fibroblasts (AFs), an MSC subset, regulate type 2 effector lymphocytes (e.g. ILC2s and Th2 cells), in part via the secretion of IL-33 and thymic stromal lymphopoietin (TSLP). Using 3D thick section imaging, I have shown that lung type 2-like tTregs (i.e. Gata3hi ST2+, KLRG1+) also localize to this niche, indicating AFs may regulate lung tTregs and their subsets. When co-cultured with lung AFs, lymphoid Tregs significantly increased proliferation, survival, and expression of type 2-like Treg markers ST2 and KLRG1 in a contact-dependent manner. Additionally, AFs preferentially support ST2hi Tregs, as evidenced by higher proliferation, survivability, and ST2 and KLRG1 expression. Using CellphoneDB V2.0, I identified extracellular matrix (ECM)–integrin ligand–receptor pairings, such as ICAM, VCAM, and CD49d, that may mediate interactions between AFs and Tregs. Upon blocking all three in a co-culture system, I found a significant decrease in Treg proliferation and ST2 and KLRG1 expression. I hypothesize that AFs regulate the maintenance and differentiation of lung Treg subsets, preferentially supporting type 2-like lung Tregs, which play critical roles in post-injury lung repair. This proposal will define the topography of type 2 lung tTregs and their role in naïve and inflammatory settings (Aim 1) and determine the role of adventitial fibroblasts in the regulation of lung Treg subsets (Aim 2). This work utilizes murine models of inflammation, as well as genetic ablations to dissect the local type 2 tTreg response in the lung. High-dimensional flow cytometry, advanced imaging, and lung function measurements will be used to quantify impacts on immune cells and functional lung recovery. Completion of these aims will elucidate the role of AFs in the function and regulation of lung type 2 tTregs, providing novel mechanistic insight into the role MSCs play in regulating immune subsets. Completion of this study provides a foundation for the development of precision therapeutics to selectively regulate lung tissue Tregs subsets to impact the outcome of diverse lung diseases.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY AND ABSTRACT Traumatic brain injury (TBI) impacts 69 million individuals worldwide every year. Despite its prevalence, TBI continues to be undertreated, so much so that the Centers for Disease Control and Prevention has dubbed it a “silent epidemic”. TBI boasts no viable treatments, yet the long-term consequences can be profoundly disabling. For example, up to 50% of TBI patients will develop chronic post-traumatic epilepsy (PTE), and a majority will report sleep disturbances like sleep fragmentation and insomnia. A leading candidate for the origin of these post-traumatic deficits is thalamic inflammation. Deep-brain thalamic structures often show signs of robust gliosis and inflammation after TBI, even if the primary injury acutely affects the cortex. The thalamus is a nexus for sleep, and its dense reciprocal connectivity with the cortex render it highly epileptogenic. Studies in the Paz laboratory have shown that aberrant complement activation in the thalamus, and in particular high levels of complement factor C1q, can lead to the development of post-traumatic epileptic activity and a reduction in sleep spindles, electrophysiological signatures of non-REM sleep thought to be crucial for memory consolidation. The question now remains how C1q exerts its maladaptive effects. The proposed research will investigate the cellular source of maladaptive C1q through inducible genetic deletion, EEG, and immunohistochemistry, and will determine the role that microglia—cells whose function in synaptic pruning is known to be modulated by C1q—may play in mediating electrophysiological abnormalities due to C1q (Aim 1). The proposed research will also explore the extent of the classical complement pathway’s participation in the rise of epileptic events and sleep spindle loss by applying a highly novel nanobody against C4b, C1q’s immediate downstream effector (Aim 2), coupled with many of the EEG and molecular techniques described in Aim 1. These studies will be conducted with the ultimate goal of identifying avenues for potential therapeutic treatment for TBI and its resultant deficits, including PTE and sleep disruption. All research will be conducted at UCSF and at Gladstone Institutes and will benefit from the wealth of resources and training available at both institutions. They will be enriched by in-house data science and statistics tools and courses, and profit from internal and external collaborations. All experiments will be guided by the mentorship of supervising sponsor Dr. Jeanne Paz, and made available for feedback and presentation at conferences and meetings.

NIH Research Projects · FY 2025 · 2022-09

Abstract Adult intestinal tissues are maintained in a normal functional state despite rapidly proliferating and differentiating intestinal epithelial cell populations that replenish the epithelium layer every 6-7 days. Intestinal epithelial cells reside in close association with both adaptive and innate immune cells of the gut. A reciprocal and dynamic dialogue among these components maintains intestinal homeostasis and is mediated in part by paracrine cytokine and interleukin networks. Intestinal inflammation predisposes intestinal epithelial cells (IEC) to malignant transformation via incompletely understood mechanisms. We have generated a novel knock-in mouse line that spontaneously develops perturbed IEC differentiation, gut elongation, and have a high susceptibility to colon cancer. These mice provide us with a unique opportunity to discover the key inflammatory mediators that regulate intestinal homeostasis and drive IEC transformation. Using transcriptomic, proteomic, genetic epistasis and cellular approaches, our preliminary data implicate selected immune cytokines in these processes.

NIH Research Projects · FY 2025 · 2022-09

Enteric pathogen infections cause an immense disease burden among children in low-resource settings. Understanding pathogen-specific transmission in high burden populations, and whether transmission is reduced through environmental intervention (reduced exposure) or improved nutrition (reduced susceptibility) is crucially important for informing global public health programs. In preliminary studies, our team has developed multiplex bead assays that measure immunoglobulin G (IgG) response to diverse enteric pathogens, along with seroepidemiologic methods to measure changes in transmission based on enteric pathogen antibody response. Antibody-based measures should complement stool-based PCR measures of infection in studies with infrequent measurement (rather than continuous monitoring) because antibody response integrates exposure over time, thus providing additional information about infections that begin and resolve between measurements. Our overall objective is to use seroepidemiologic methods to measure intervention effects on enteric pathogen transmission, and to leverage large-scale trials to develop new methods that combine multiplex testing with spatial epidemiology to locate communities with highest multi-pathogen burdens. Our team recently completed cluster randomized trials in Kenya and Bangladesh that delivered water, sanitation, handwashing (WASH), and nutritional interventions to pregnant mothers, and measured primary endpoints (growth, diarrhea) among their newborn children through age 24 months. Each trial enrolled and randomized >700 communities. Blood samples were collected among longitudinal substudies of ≈1,500 children in each country at ages 6, 12, and 24 months across factorial arms (Control, WSH, Nutrition, Nutrition + WSH), and among ~4,000 children per country at age 24 months. We propose to test for IgG responses using a multiplex bead assay that includes antigens to nine enteric pathogens: Giardia sp., Cryptosporidium sp., Entamoeba histolytica, Strongyloides sp., Ascaris sp., Campylobacter sp., enterotoxigenic Escherichia coli, Salmonella sp., and norovirus. In Aim 1, we will use the factorial trial design to measure the effects of WASH and nutrition interventions on antibody-based measures of enteric pathogen transmission in each country, including mean IgG response, seroprevalence, and force of infection. We hypothesize that the interventions reduced transmission of enteric pathogens through reduced exposure (WASH) and reduced susceptibility to infection (nutrition). In Aim 2, we will combine multiplex antibody data with spatial models to map landscapes of enteric pathogen exposure and develop generalizable methods to identify communities with highest, multi-pathogen burdens. We hypothesize that antibody-based measures will align with other measures of infection, and that there will be geographic overlap in hotspots, identifying communities with the highest multi-pathogen burdens. Richly characterized trials in two countries ensure results will be rigorous and policy relevant. Completion of these aims will significantly advance the use of serological assays in enteric pathogen intervention studies and population surveillance.