Johns Hopkins University

NIH Research Projects · FY 2025 · 2024-05

Project Summary: Non-small cell lung cancer (NSCLC) incidence and mortality rates are higher among African Americans (AA) than other racial or ethnic groups. A lower prevalence of lung-cancer-related genetic mutations in AA indicates that studying transcription factors is a new promising direction. We have identified differentially expressed pfeRNAs by profiling 120 clinical specimens from 72 patients, including 12 specimens from 12 AA and 108 specimens from 60 Caucasians and Asians using small non- coding RNA deep sequencing followed by bioinformatic and biostatistics analyses, and all these patients with either benign or malignant pulmonary nodules. We identified a pfeRNA that was only expressed in AA but undetectable in either Caucasians or Asians, suggesting that this pfeRNA is an AA-specific pfeRNA (AA- pfeRNA). The use of AA, Caucasians, and Asians gives us direct evidence that AA-pfeRNA is a potential factor in health disparities in NSCLC. The use of patients’ benign and malignant pulmonary nodules provides a perfect model for understanding lung preneoplasia malignant progression, and the AA-pfeRNA we identified is specifically induced endogenously once lung preneoplasia progresses to NSCLC in AA, giving direct evidence for AA-pfeRNA as a therapeutic target to inhibit the evolution of lung preneoplasia to NSCLC. Importantly, we identified AA-pfeRNA following the requirements of the clinical laboratory improvement amendments (CLIA) compliant a laboratory developed test (LDT) assay, providing evidence for translational development. Further, its high expression predicted worse overall survival of AA with NSCLC. To prevent AA-pfeRNA function and disrupt NSCLC development, we have designed oligos complementary to AA-pfeRNA (cAA-pfeRNA) and optimized modifications and conjugates to deliver pfeRNA to NSCLC and limit accumulation in normal tissues. Therefore, in this project, we will provide mechanistic rationale and therapeutic insights into the druggable AA-pfeRNA: (1) Identify the target protein in cancerous tissues from AA with stage-I/II NSCLC, confirm their interaction pattern and cellular distribution in both cancerous tissues and cells, and observe its effects on the basic biological activities of cells with and without the target protein; and (2) evaluate the efficacy, safety, and pharmacokinetic/pharmacodynamic relationships of ligated-cAA-pfeRNA-2’-MOE-PTO by systemic delivery in preclinical animal models. Our optimized specific & natural pfeRNA-based targeting system will provide a new treatment opportunity for AA with NSCLC to reduce racial and ethnic health disparities. Also, the system will be of significant interest to researchers in the field of sncRNA-based therapeutics for cancer treatment.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Cellular biology of the human gastroesophageal junction cardia (GEJ-cardia) has been notoriously difficult to study, in particular due to a lack of biologically relevant GEJ-cardia-specific disease models. Based on known early genetic events in GEJ-cardia neoplasia, we generated wild-type (WT) and TP53/CDKN2A dual-knockout (DKO) 3-dimensional (3D) human GEJ-cardia-derived organoids using CRISPR-Cas9 genome editing. Notably, DKO organoids grew faster, became larger, exhibited de novo intestinal, metaplastic and dysplastic morphology, and consistently grew as xenografts in vivo. Interestingly, 2-D MALDI mass spectrometric imaging revealed a markedly abnormal lipidomic profile in DKO organoids, with platelet-activating factor (PTAF) and free fatty acids (FFAs) among the most-upregulated lipids. Intriguingly, several of these FFAs (stearic acid, lauric acid, cyclo acid, oleic acid, and palmitate) function as known ligands that stimulate HNF4A, a centrally important master transcription factor (MTF) implicated during early GEJ-cardia neoplastic evolution. We hypothesize that dysregulated FFA metabolism, acting via a novel lipid-epigenome crosstalk featuring MTF hyperactivation, causes abnormal growth, proliferation and differentiation at the human GEJ-cardia. This hypothesis will be tested by two Specific Aims. In Aim 1, we will first assess the biological effects of PTAF- PTAFR signaling in our early GEJ neoplasia organoid model, using either loss- or gain-of-function approaches, followed by an examination of phenotypes, morphology, and differentiation of GEJ-cardia organoids. Similarly, potential functions of candidate FFAs will be interrogated in GEJ-cardia organoids. Furthermore, we will establish the in vivo etiologic role of PTAF-PTAFR signaling as well as candidate FFAs in early GEJ-cardia neoplasia. In Aim 2, we will explore a novel crosstalk between lipid metabolism and epigenomic reprogramming by investigating the functional contributions of FFAs and PTAF to the epigenomic activity of HNF4A. Direct binding of FFAs and PTAF to HNF4A will be measured by competitive radiometric binding assays. The functional regulation by HNF4A of GEJ-cardia chromatin accessibility and activity will be interrogated using a palette of epigenomic sequencing approaches. Finally, we will perform spatial quantification of the abundance of HNF4A- activating FFAs and PTAF in both GEJ-cardia organoids and primary patient samples by conducting advanced histopathology-guided MALDI imaging analyses. These efforts in toto promise to define biological drivers promoting early GEJ-cardia neoplastic evolution and to elucidate a novel crosstalk between lipid metabolism and epigenomic reprogramming, thereby discovering precise molecular mechanisms underpinning GEJ-cardia dysplasia and early neoplastic transformation. Moreover, this project has the potential to uncover and validate candidate approaches toward prevention and early intervention in GEJ-cardia neoplastic evolution by targeting PTAFR, which may ultimately be effectively blocked by an FDA-approved pharmacologic antagonist in human patients.

NIH Research Projects · FY 2026 · 2024-05

Project Summary Recent HIV outbreaks among rural people who inject drugs (PWID) highlighted significant gaps in HIV prevention science. HIV risk behaviors among PWID are influenced by several factors, including access to sterile injection equipment and pre-exposure prophylaxis (PrEP), law enforcement practices, and utilization of medications for opioid use disorder (MOUD) and harm reduction (e.g., accessing syringe services programs [SSPs]). None of the existing HIV prevention literature has examined the relationships between complex patterns of interactions and engagements among a triad of HIV risk modifiers (MOUD, harm reduction, and law enforcement), high-risk behaviors, and HIV incidence among rural PWID during periods of peak HIV transmission. Social network characteristics are another driver of HIV transmission as PWID networks may be characterized as having specific norms surrounding injection practices and risk reduction strategies. Studying how combinations of factors known to influence HIV risks interact at the individual- and network-levels among PWID in rural communities with HIV outbreaks presents a unique opportunity to inform the design of interventions that can respond to rapidly evolving HIV prevention needs and barriers. Evidence generated from urban studies documents that network-oriented interventions focused on social influence processes are able to shift norms related to high-risk sexual behaviors and injection practices and use of risk reduction strategies, leading to reductions in HIV risk behaviors and HIV transmission. However, there is limited literature describing how to adapt network-oriented HIV prevention interventions for rural PWID. To address these gaps, our study aims: 1. To examine longitudinal changes in the associations between the frequency and types of engagements with a triad of HIV risk modifiers (i.e., MOUD, harm reduction, and law enforcement), HIV risk behaviors, and HIV incidence among a cohort of rural PWID (N=400) during a HIV outbreak over 18 months; 2.To examine how the associations between egocentric network characteristics (e.g., perceived norms, turnover, density) and utilization of three evidence-based approaches to HIV risk reduction (MOUD, PrEP, and SSPs) change over 18 months among a cohort of rural PWID during a HIV outbreak; 3. To engage rural PWID in the adaptation and pilot testing of a network-oriented intervention to reduce high-risk injection practices and sexual behaviors; and 3A. To conduct a pilot randomized controlled trial (RCT) of the adapted intervention among rural PWID (N=60) to assess acceptability and feasibility. The proposed research is directly responsive to the 2022-2026 NIDA Strategic Plan. Aims 1 and 2 map directly to Goal 2.2, “Accelerate the science of harm reduction.” Additionally, this study aligns with Priority Scientific Area #3: Accelerate Research on the Intersection of Substance Use and HIV in that our rural study setting is experiencing a HIV outbreak driven in part by social norms surrounding high-risk behaviors and stimulant use. The results of this study will shift paradigms in how we understand HIV prevention in rural contexts.

NIH Research Projects · FY 2026 · 2024-05

7. PROJECT SUMMARY/ABSTRACT Achieving sustained viral suppression is the pinnacle of HIV management conferring individual-level benefits by improving survival and societal benefits through secondary prevention. While there has been dramatic progress to improve viral suppression among people living with HIV, this progress has been uneven. Globally, 15.2% of the 14.8 million people who inject drugs (PWID) are living with HIV and drug use accounts for 20% of infections outside of Sub-Saharan Africa. PWID lag behind other populations with viral suppression <20% in some settings resulting in high AIDS-related mortality and HIV incidence. Long-acting antiretroviral therapy (LA ART) represents a new way to deliver ART that can improve viral suppression in a population where sociostructural issues such as homelessness and unemployment in addition to substance use complicate adherence to oral ART. Yet, to date, there has been no serious scholarship evaluating the role of LA ART in virologically unsuppressed persons (including PWID) particularly in low- and middle-income countries. Accordingly, our overarching objective is to evaluate the efficacy, safety, acceptability, and cost-effectiveness of LA ART on viral suppression among virally unsuppressed PWID in New Delhi, India. We will utilize a mix of ethnography, a randomized clinical trial and modeling to achieve these objectives. We will first conduct ethnographic work to inform clinical trial design. Ethnography will be conducted among PWID living with HIV, outreach workers and health care personnel. Key topics to be explored during this phase will be attributes of LA-ART including duration of effect (one week vs. six months), mode (oral vs. intramuscular vs. subcutaneous) and site of administration (facility vs. field-based). We anticipate conducting a three arm open-label randomized clinical trial where treatment naïve and treatment experienced unsuppressed PWID will be randomly assigned at an allocation ratio of 1:1:1 across three study arms – Arm 1: TLD (Tenofovir DF [300mg] + Lamivudine [300mg] + Dolutegravir [50mg]); Arm 2: CAB/RPV (Cabotegravir [900mg in 3ml] + Rilpivirine [600mg in 3ml]); two intramuscular injections at Months 0, 1 and every 2 months thereafter); and Arm 3 – CAB/RPV/LEN (Cabotegravir [900mg in 3ml] + Rilpivirine [600mg in 3ml] + Lenacapvir [927mg in 2 x 1.5ml vials]). All participants will be followed for 24 months. The primary short-term and long-term endpoints of this trial will be viral suppression (HIV RNA<50 copies/ml) at Months 6 and 24, respectively. We will then model the impact of Treatment as Prevention on HIV transmission in PWID for each study arm by simulating HIV transmission through injecting and sexual networks based on survey responses incorporating exposure to other interventions (e.g., syringe service programs) and accounting for the cyclical patterns drug use. We will extend transmission models to assess cost savings and cost-effectiveness of each of the intervention arms compared to oral ART. If successful, the findings from this trial can transform the delivery of health care to PWID globally.

NIH Research Projects · FY 2026 · 2024-05

Project Summary Methods to characterize patients who highly benefit on multiple clinical outcomes, from treatments in Alzheimer's disease and related dementias (ADRD), are necessary to treat patients effectively. Treatments may benefit some patients on targeted outcomes, but harm some patients on other, e.g., cognitive, outcomes. So, characterizing patients who highly benefit on multiple outcomes is significant: first it allows these patients to choose a treatment if it is predicted to give them high benefits without the harms; second, accurate characterization methods do not exist. Generally, a characterization method has two stages. One stage “constructs” outcome predictions based on patients' covariates; and another stage “synthesizes” the predictions to estimate the goal - a large high benefit group. As the “construction” stage uses many covariates, it needs methods to estimate predictions from a model, i.e., from a large set of possible distributions (e.g., regression, neural networks). These predictions are then used in the “synthesis” stage for the goal. Such existing methods, however, do not use the clinical goal (to characterize high-benefit patients) as a guide inside the construction stage. For a single outcome, recent work has shown that this lack of linking can produce dramatically inaccurate characterizations, no matter the model. For characterizing patients with multiple high benefits, new methods must explicitly link all multiple clinical goals (i.e. high benefits in all outcomes) in the construction stage. In preparatory work, we showed that existing methods for multiple outcomes, can miss even most of the high benefit patients, and we developed a preliminary better method by establishing the missing links. This new project is motivated by our ongoing work with two studies. The first study tested if citalopram reduces agitation in Alzheimer's patients. Since citalopram may harm cognitive function, we set to characterize patients with high citalopram effect in (a) reducing agitation and (b) maintaining cognitive function. The second study tests the effect of transcranial direct current stimulation on primary progressive aphasia outcomes, with related goals. In preparatory work, we found strong evidence that standard methods miss up to 70% of the patients with multiple high benefits, compared to the new methods. For this project we propose to fully develop methods to characterize patients who highly benefit on multiple outcomes. The methods will be applied to the above studies, and can help more generally in other ADRD studies. Aim 1. Develop methods to characterize patients who highly-benefit in multiple outcomes in randomized trials. These methods are significant because they allow accurate personalized treatment choices. Aim 2. Develop methods to find if a simpler subset of the full multiple outcomes, can have similar patient characterization as the full outcomes. These methods are significant because they can suggest if a high-effect on earlier outcomes is necessary before a high-effect on the later outcomes occurs. Aim 3. Develop methods to characterize patients who highly benefit in multiple outcomes in observational studies. These methods are significant when randomization is infeasible.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY Tau pathology in Alzheimer’s disease (AD) is linked to neuronal activity in multiple ways, including an effect of activity to increase tau phosphorylation [1], tau spread between neurons, and tau shedding into the CSF [2], and activity inhibition reduces tau pathology [3]. NPTX2 (Neuronal Pentraxins 2) is an immediate early gene (IEG) that acts specifically at excitatory synapses on parvalbumin interneurons (PV) to regulate circuit inhibition and is prominently down-regulated in the brain of individuals with AD [4]. During its normal function, a portion of synaptic NPTX2 is shed into the cerebrospinal fluid (CSF) where levels in human subjects inversely correlate with cognitive performance and hippocampal volume in MCI and AD [4-6]. One consistent observation in these studies is that a ratio of CSF NPTX2 and tau or ptau prominently increases diagnostic performance [4-6]. This suggests an important relationship between CSF NPTX2 and tau/ptau in AD. NPTX2 is generated by pyramidal neurons (Py) throughout the forebrain and is trafficked along axons to presynaptic sites where it is exocytosed in response to Py activity and functions specifically at excitatory synapses on PV to strengthen the excitatory drive of PVs, thereby enhancing inhibition within the circuit [7, 8]. Consistent with these synaptic effects, NPTX2 loss of function results in increased Py activity and reduced gamma power, and these effects are amplified by Aß amyloidosis [4]. These observations focus attention on potential interactions between NPTX2 loss of function and tau pathophysiology. To examine the interaction of tau with NPTX2, we will use a newly established rat AD model (TgF344-AD) that expresses human FAD mutations including APP (APPswe) and presenilin 1 (PSΔE9) and mimics human disease by developing tau pathology without requiring mutation of tau [9]. Aim 1 will develop novel tools to image tau accumulation in vivo using 2-photon microscopy. Fluorescent tau indicators, termed Tau1 and Tau 2, will be evaluated for detection of tau accumulations in TgF344-AD rats following either i.v. or local injection of the reporters into the brain using a novel perforated cranial window. In vivo 2-photon images of tau will be correlated with histopathologically imaged tau. Aim 2 will examine the impact of conditional deletion of NPTX2 in TgF344-AD rat neocortex on tau accumulation and cellular localization and correlate these parameters with the activity of Py and PV neurons using cutting-edge multi-color longitudinal 2 photon imaging. The proposed research will develop methods to image pathologic tau accumulation in vivo and test the association of pathological forms of tau with NPTX2 loss of function and resulting changes in brain activity.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Assisted living (AL) is a growing long-term care industry providing housing and supportive services to 800,000+ frail and functionally impaired older adults - over 40% of whom are living with Alzheimer's disease or a related dementia (ADRD). Demographic projections of increased demand for AL, and especially dementia care (also referred to as “memory care”), has attracted notice from private equity (PE) firms. PE ownership advocates argue that PE firms bring much needed capital, allowing providers to update facilities, invest in new technology, and gain economies of scale to enhance efficiency, increase profitability, and improve care quality. However, the growing concern is that some PE firms' focus on short-term profits may compromise the quality of care of AL residents, particularly those living with dementia. Research findings on the effects of PE acquisitions of nursing homes are mixed: while some studies found negative effects (e.g., decreased staffing, increased mortality) others have found no change in staffing or quality outcomes. However, unlike nursing homes, AL is most often paid for privately; and, there is no national framework of regulations, payment policies, or public reporting of care quality to work as “guardrails” against the possible negative impacts of PE acquisitions as there are in nursing homes. Therefore, the objective of this application is to understand the types of AL that PE firms invest in and how PE acquisitions impact the composition of residents with ADRD and residents' outcomes. Our working hypotheses are: 1) ALs that are part of a large chain, located in markets with more demand, and operating under licenses with less regulatory stringency are more likely to be purchased by PE firms; and 2) acquired ALs respond to changes in ownership by restructuring operations in ways that influence the composition of residents and ultimately residents' outcomes. These hypotheses will be tested in an exploratory/explanatory sequential mixed methods study with three specific aims: 1) Explore stakeholders' perceptions of PE firms' motives and the impact of PE investment on AL communities; 2) Compare ALs that were acquired or divested by PE firms between 2010 and 2019 to those that were not; 3) Estimate the causal effect of PE acquisitions on the prevalence of residents with ADRD in AL and residents' outcomes over a 10-year period. Under the first aim, the research team will leverage their existing networks and interview stakeholders to elicit different perspectives on the motives and impacts of PE acquisitions. Under the second aim, the team will apply the methods they developed and data they previously collected to understand which ALs are PE targets. The third aim will examine the welfare effects of PE acquisition on residents with and without ADRD applying an instrumental variable method. The results from this study will be the first to investigate PE acquisitions of AL. As policymakers are urged to consider ownership reporting and monitoring in other sectors, it is important to have insight into the less regulated AL industry that provides supportive services to vulnerable older and disabled adults, almost half of whom are living with dementia.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY The overall long-term objectives for this K01 proposal are to (1) develop and evaluate a nature-based culturally grounded (NBCG) substance misuse intervention tailored to Native American teenage mothers and their young children that live in a tribal community (2) while also advancing my research and professional capacity to become an independent research investigator. Although Native Americans (NA) demonstrate high abstinence rates from alcohol, the generations of trauma and oppression, ongoing discrimination, COVID-19 pandemic-related consequences, and drug companies disproportionately targeting Black, Indigenous, people of color (BIPOC) communities have contributed to NAs experiencing the highest mortality rates from overdose, suicide and alcoholic liver disease (deaths of despair) as of 2020. NA young women experience heightened risk of interpersonal violence that places them at risk for substance use disorder (SUD) and other risk-taking behaviors, which can result in unplanned pregnancies. Their children are then placed in the cycle of addiction as these children are at heightened risk for early problem emotional and behavioral development, partly due to impaired sleeping, which has demonstrated increased risk for later substance misuse. These alarming statistics warrant investigation of a promising culturally grounded substance misuse intervention that targets both young mothers and their children with the goal of breaking the cycle of addiction by way of restoring our traditional and ceremonial knowledges and practices with the land. To do this work, we need Indigenous frameworks. The traditional ecological knowledge (TEK) framework is the theoretical underpinning for the proposed study. Due to the close relational ties NAs and other Indigenous people have to our land and water, TEK posits additional cultural and spiritual mechanisms between the natural environment and human health and wellbeing. The proposed specific aims of this study are to: 1) develop NBCG programming to enhance an evidence- based home-visiting model to fit the needs of the local context; 2) test the NBCG intervention to determine feasibility and acceptability; and, 3) evaluate the NBCG intervention for effectiveness and efficiency and refine modules to maximize fit to the local context to set the stage for a future R01 fully powered Hybrid Type II trial examining implementation and program effectiveness against maternal substance misuse and child negative child emotional and behavioral development, risk factors for later substance misuse. This K01 incorporates a community-engaged exploratory mixed methods sequential study design to inform, evaluate and refine the NBCG intervention. This study will shed light on proposed theoretical mechanisms (e.g., sleep quality, sense of connectedness) between nature-based culturally grounded experiential learning (i.e., NBCG intervention) and resilience and resistance against addiction (NIDA Objective 2.1).

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Cannabis and tobacco are two of the most widely used drugs and are frequently used together. People who use both cannabis and tobacco tend to have higher rates of problematic cannabis use and dependence, sustained long-term tobacco use, and poorer outcomes during treatment for tobacco use disorders. With rates of cannabis use on the rise, understanding the mechanisms driving cannabis and tobacco co-use is imperative for developing effective policy and public health efforts aimed at decreasing co-use rates. Although cannabis and tobacco co- use is common, mechanism(s) underlying co-use remain unclear. Preliminary work by our group shows that delta-9-tetrahydrocannabinol (THC) can alter the subjective experience of commonly used tobacco products including combustible cigarettes and e-cigarettes. Preclinical evidence supports this interaction by documenting increases in motivation to use nicotine, including less price sensitive nicotine demand in THC-exposed rodents. Human laboratory data are needed to extend this work by comprehensively determining effects across the range of commercially-relevant THC and nicotine doses and routes of administration. We will concurrently conduct two full-factorial laboratory studies to determine the impact of acute THC administration on the dose-related motivational, subjective, and physiological effects of two widely used tobacco products – combustible cigarettes (Study 1) and electronic cigarettes (Study 2; “e-cigarettes”). Participants in both studies will receive pretreatment with placebo or active THC. Following THC pre-treatment, participants will self-administer a combustible cigarette containing a randomized nicotine dose (Study 1) or an e-cigarette containing a randomized nicotine dose (Study 2) using a standardized puff procedure. We will use NIDA standardized products (SPECTRUM cigarettes and SREC e-cigarettes) to ensure consistency across study years and applicability to other ongoing research. A battery of craving, mood, withdrawal, and subjective drug effect measures will be collected before and after nicotine product administration. Participants will then complete an incentivized demand task previously validated by the study team to evaluate cigarette and e-cigarette use motivation and increase experimental rigor through experienced consequences of task performance. Participants will be randomized to either smoked or vaporized THC administration to evaluate the effect of congruent versus incongruent routes of THC and tobacco administration on study outcomes. We hypothesize that THC will in a dose-orderly manner increase motivations to use tobacco as well as decrease tobacco craving and increase positive affect with tobacco use. Understanding the influence of cannabis on tobacco use across varied routes of administration and a broad nicotine dose range will inform key regulatory policies within the cannabis and tobacco regulatory fields to include cannabis standard unit dosing and nicotine reduction policies.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Background: Allogeneic hematopoietic cell transplant (allo-HCT) remains the only curative therapy for many aggressive malignancies and is increasingly used for non-malignant disorders. Success is limited by 1) relapse due to insufficient graft-vs-tumor (GVT) activity and 2) graft-vs-host disease (GVHD). Current GVHD therapies predispose to infection and relapse. Understanding the mechanisms that optimize GVT activity and mitigate GVHD remains a critical unmet need. Donor T-cells are essential to the allogeneic immune response. De novo DNA methylation by DNMT3a allows activated T-cells to acquire patterns of gene expression that define alloreactivity. Human donor DNMT3a mutations result in decreased relapse, increased GVHD, and improved survival in HCT recipients. Based on a large body of preliminary data, both GVHD and GVT activity significantly depend upon DNA methylation, the role of which is largely unknown in the HCT context. I hypothesize that targeting epigenetically controlled pathways can skew allogeneic immunity away from host and towards tumor. Design: Mice with cell-lineage specific DNMT3a deletion will be used as donors in well-established, clinically relevant, murine models of HCT. Following HCT, donor cells will be isolated from recipient mice and undergo whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNAseq) in parallel. This novel approach, proven to be feasible and successful in my preliminary work, produces a comprehensive epigenetic and transcriptomic profile of the cells directly involved in the pathogenesis of GVHD and the GVT effect. Importantly, this method readily identifies the pathways that are dysregulated in the absence of DNMT3a. Aim 1: Target epigenetically regulated pathways to mitigate GVHD. Dnmt3a KO donor T-cells accelerate GVHD. WGBS and RNAseq post-HCT identified multiple pathways, normally silenced by DNMT3a, that may contribute to this phenomenon. We will explore these putative novel targets, starting with CCR9, a chemokine receptor that guides T-cells to the intestine, a major GVHD target organ. Aim 2: Determine the contribution of myeloid cell DNMT3a deletion to enhanced GVHD and GVT activity. Myeloid-derived cells hold significant immunoregulatory potential. The impact of myeloid DNMT3a deletion is unknown. We will use donors with myeloid Dnmt3a deletion to test this. Aim 3: Identify genes that distinguish beneficial GVT activity from harmful GVHD via epigenetic and transcriptomic comparative analyses. In GVT models, where allo-HCT is performed in the presence of leukemia, KO T-cells provide superior disease control. We will apply WGBS and RNAseq to interrogate how the presence of tumor alters the epigenome and identify the changes that allow KO T-cells to better eradicate leukemia. My long-term goal is to become an independent laboratory-based clinician-scientist, focusing on complications of HCT and novel therapeutic approaches. My aims and training plan have been specifically and thoughtfully crafted to provide me with the research skills and experience needed to reach this goal.

NIH Research Projects · FY 2026 · 2024-04

General Introduction, Abstract Smell deficits are one of the most reproducible changes in patients with schizophrenia (SZ). They are tightly correlated with specific clinical features, such as negative symptoms and some cognitive deficits (e.g., social cognitive deficits), but rarely with positive symptoms. Many studies have also observed molecular and cellular changes in the olfactory epithelium (OE) in living patients with SZ. Our group has accumulated evidence that the immune/inflammatory changes associated with redox imbalance may be major pathological changes in the OE of SZ patients. Nevertheless, whether and how the OE pathology mechanistically contributes to these specific clinical features has been a major knowledge gap. Using an inducible animal model, we have demonstrated that chronic local OE inflammation elicits a functional alteration in prefrontal cortex (PFC) pyramidal neurons, likely via the olfactory-prefrontal circuits. As a result, the mice showed behavioral deficits in the positive valence systems and social processes. Consistent with this basic science observation, we have recently obtained promising results that specific molecular changes in the inflammatory/redox signaling in the OE are directly correlated with negative symptoms (deficits of the positive valence systems) and smell deficits in SZ patients at the cross-sectional and longitudinal levels. Altogether, we hypothesize that molecular and cellular changes in the OE in SZ patients, or at least in a subset of them, significantly contribute to specific clinical manifestations that correlate with smell deficits. We expect that the OE pathology alters the olfactory- prefrontal circuits and PFC pyramidal neuron functionality, which in turn contributes to negative symptoms and social cognitive deficits in SZ patients. We hypothesize that the impact of OE pathology on the olfactory- prefrontal circuits is more robust in adolescence/young adulthood when the PFC maturation dynamically happens. We propose three Projects and three Cores. Project 1 (P1) will define how OE perturbation (e.g., chronic local OE inflammation) elicits the neuronal alteration in the OE and OB, which results in behavioral deficits; P2 will determine the circuitry mechanism(s) that links OE pathology to the PFC via the olfactory- prefrontal circuits, and identify the critical developmental period vulnerable to the OE influences; P3 will define OE pathology in early-stage SZ patients at the molecular/cellular levels, stratify patient groups depending on the specific mechanisms (e.g., molecular expression levels), and elucidate how the OE pathology influences specific clinical symptoms (e.g., negative symptoms and cognitive deficits) via the olfactory-prefrontal circuits. This molecular/circuitry mechanism cannot solely be causal for these clinical deficits. Nevertheless, we consider this mechanism as a significant contributory factor for them. Given that inflammation/redox-associated pathology in the OE is potentially treatable with local, non-invasive interventions, we believe that this study will provide new translational insights into these treatment-refractory clinical domains in SZ.

NIH Research Projects · FY 2026 · 2024-04

Sudden cardiac arrest (SCA) is a major public health concern, accounting for up to 400,000 deaths each year. While SCA cases are heterogeneous in etiology, the most common underlying pathologic substrate is ischemic heart disease and the most common electrophysiologic mechanism is ventricular fibrillation (VF). Survival following VF-SCA is less than 20% in most communities with mortality due largely to fulminant injury of either the heart or brain. Accordingly, there is a compelling case to identify novel modifiable SCA risk factors, to elucidate its underlying pathophysiology, and to identify novel preventative and therapeutic targets. The mitochondrion has primary function in energetics, oxidative stress, and apoptosis, and serves as both a target and an effector of ischemia-reperfusion injury. Consequently, mitochondria have a fundamental role in arrhythmia risk and in cardiac and brain resuscitation. Indeed, we have previously demonstrated that mitochondrial DNA (mtDNA) copy number (-CN), which reflects the number of MT genomes per cell, is a novel risk factor for SCA, independent of traditional SCA risk factors. MtDNA-CN, however, captures the quantity of mitochondria. It does not capture their quality. We have recently identified a series of mtDNA haplotypes that capture functional genetic variation, and developed a computational tool to accurately assess somatic mutations in mitochondria (heteroplasmy), allowing for a comprehensive survey of mtDNA genetic variation. Thus, we propose to test the hypothesis that mtDNA variation (mtDNA-CN, inherited mtDNA haplotypes and rare mutations, mtDNA heteroplasmy [somatic variants]) will be associated with SCA risk and resuscitation outcomes. We will examine the association of SCA with mtDNA characteristics in 2,600 SCA cases from a large population- based case-control study of SCA and validate our findings in two population-based cohort studies where SCA cases have been identified prospectively and two case-control studies of autopsy confirmed SCA subjects. We will also examine the association of mtDNA variation with cardiac resuscitation (restoration of sustained circulation) and brain recovery among 2,600 VF-SCA cases followed prospectively. Second, we will characterize the consequences of specific mtDNA variation on mitochondrial function and cardiomyocyte (CM) electrical activity. We will assess the impact of mtDNA variants associated with known rare diseases with CM involvement across a range of heteroplasmies using human induced pluripotent stem cell CMs (hiPSC-CMs) to directly test whether deleterious heteroplasmic variants modify electrical excitability, repolarization or conduction, or compromise the development or maturation of CMs. We will also leverage existing mtDNA base editing technologies to assess the impact of variants associated with SCA risk and/or resuscitation outcomes. Together, these aims will determine whether mtDNA genetic variation are associated with SCA risk and resuscitation and will characterize the consequences of mtDNA variation on CM electrical activity. These investigations will lead to innovative approaches to prevent SCA and improve VF-SCA resuscitation outcomes.

NIH Research Projects · FY 2026 · 2024-04

CRC Abstract / Summary FA (FA) is very common, affecting up to 32 million Americans, including up to 8% of children and 10% of adults, and its prevalence appears to have increased significantly over the past 20 to 25 years.4,10 The past decade has seen tremendous progress in the investigation of FA, with significant advances in both prevention and treatment, as well as in the understanding of the immunologic basis of FA. However, at this time there is still only a single FDA-approved therapy for FA, and given its limitations most patients are still left with the age-old option of strict avoidance and the use of emergency medications upon accidental exposure. Given these facts, safe and effective therapies, or even better preventative approaches that can be widely applied, are highly desirable. Johns Hopkins University (JHU) has been a leader in the study of FA for over 30 years, and a CoFAR Clinical Research Center (CRC) since its inception. With its large referral population, the center has been a world leader in defining the natural history of FA and in the study of FA treatments. In addition, JHU has a remarkable track record of training the next generation of leaders in FA research, and most important to this application, the entire Hopkins team of physicians, nurses, research assistants, and laboratory personnel remains intact, poised to move forward with this next 7 years of CoFAR. From the scientific perspective, this application puts forward three outstanding protocols. The first is a cutting edge, network wide clinical trial on the treatment of peanut allergy using a novel approach to immunotherapy utilizing a peanut peptide vaccine. The second and third protocols are site-specific, led by outstanding young investigators, the first focusing on the mechanisms of food-induced anaphylaxis and the second on patient-centered outcomes in FA treatments. Given these facts and the details to follow, it is clear that JHU is exceptionally qualified to remain a CRC for this next iteration of CoFAR. The site brings to CoFAR an unbeatable combination of highly experienced investigators and staff, available patients, and a track record of innovation, leadership, and performance, as well as an unparalleled record in the development of the next generation of clinician scientists focused on the study of FA.

NIH Research Projects · FY 2026 · 2024-04

Leadership Center Summary/Abstract Food allergy research has increased dramatically over the past 20 years, in large part to the efforts of CoFAR. New approaches for both treatment and prevention have been introduced, as have the understanding of the immunologic basis of food allergy. The overarching goal of the research agenda proposed in this application is to maintain CoFAR's position as an international leader in the study of food allergy, including optimal characterization of the disease and development of the next generation of treatments ad prevention. The research agenda will be based first and foremost on the best possible science, but to maximize productivity we will also need to plan judiciously so that all available resources can be used to their utmost capacity. Therefore, the research agenda will need to carefully select and stage protocols based not just on their potential to advance the field, but also with careful consideration regarding other novel treatments that may be under development, and how one study may inform the next. Finally, the overall research strategy will need to work in unison with the NIAID, the SACCC and each CRC in the consortium. This overall agenda will be accomplished through the following major components: 1) To implement a leadership team capable of providing optimal administration of the Consortium, including financial, operational, and managerial aspects, as well as protocol development and implementation, data collection and analysis, and publications and presentations, with judicious financial management. Dr. Robert Wood will lead this team, with co-PIs Drs. Scott Sicherer and Supinda Bunyavanich. The team will include experts in clinical trials as well as the laboratory study of food allergy. The team will also include a financial administrator as well as a highly experienced grants management group at Johns Hopkins. The leadership team will work closely with the NIAID and SACCC, together providing day-to-day management of all clinical operations within the consortium. 2) To develop the necessary committee structure to guide and implement the Consortium's administrative and clinical operations. 3) To develop a structure of financial management that will make optimal use of all CoFAR funds and resources. 4) To integrate the Biomarker Facility into each clinical protocol. It is anticipated that a minimum of 2 major protocols, including both clinical trials and non-interventional studies, will be initiated and completed over the seven-year funding period, and this could rise to 4 or even 5 protocols depending on the size, duration, and complexity of each study. If successful, this research agenda will define not just the next 7 years of CoFAR, but will also establish the platform for the next decade(s) of food allergy research.

NIH Research Projects · FY 2025 · 2024-04

The primary function of the lung is gas exchange. Immune cells and airway nerves orchestrate interoceptive defensive responses and adjustments to homeostatic function that preserve airway patency, eliminate noxious irritants and pathogens from air spaces, clear airway mucus, and optimize the work of breathing. Dysfunction or dysregulation of these systems directly contribute to the emergence of the physiological and pathophysiological attributes of respiratory diseases and their associated symptoms. Sensory nerves often rely on specialized chemosensory signaling mechanisms at their nerve terminals to transduce mucosal irritation. In the airways, we have described the chemosensory functions of brush cells, their association with the peripheral terminals of vagal afferent nerves and the reflex effects resulting from their activation. The central hypothesis of this research proposal is that brush cells orchestrate both immune and reflex responses in the airways and lungs. We further hypothesize that brush cell dysfunction may contribute to the emergence of symptoms associated with both acute and chronic diseases of the airways and lungs. Studies proposed herein aim to: 1) characterize the mechanisms by which brush cells are activated, how they transmit their activation to adjacent sensory nerves, and the reflexes initiated upon their activation; and 2) determine the role of ATP and the unique expression of carbonic anhydrase by airway brush cells in transducing airway mucosal irritation, acidification of airway surface liquid and the accumulation of carbon dioxide in the airway lumen. We will utilize the innovative techniques that are unique to our laboratories, including transgenic approaches enabling optical recordings of afferent neurons and brush cells, reflex physiological recordings, single afferent neuron recording and our molecular approaches to studying both neurons and brush cells. Our focus on brush cell interactions with afferent nerves is a logical direction for our group, and our plans for hypothesis testing will be enabled by intriguing recent discoveries summarized below in our Research Strategy. We anticipate that the results of these proposed studies will reveal novel roles for brush cells and ATP in transducing reflexes resulting from mucosal irritation in the airways of patients with chronic diseases of the airways and lungs.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT The objective of this project is to elucidate mechanisms underlying HMGA1 proteins in leukemogenesis and therapy resistance in KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML). KMT2A-r AML is an aggressive form of leukemia that is resistant to current therapies and therefore highly lethal. KMT2A-r leukemia is caused by rearrangements of the KMT2A gene (formerly MLL1), which encode abnormal fusion proteins that drive pro-leukemogenic gene expression, including HOX genes (HOXA9, HOXA10, MEIS1). Despite advances in our knowledge of the fusion partners and proteins that form complexes with KMT2A-r proteins, inhibitors developed to target these fusion proteins or their complexes show only modest efficacy in clinical trials thus far. Moreover, mechanisms underlying KMT2A-r leukemogenesis remain incompletely understood. Thus, there is a dire need for further research and new therapeutic strategies. Here, I propose a novel approach by focusing on the High Mobility Group A1 (HMGA1) chromatin regulator as a fundamental molecular switch required for leukemogenesis and therapy resistance in KMT2A-r AML. Our scientific premise that HMGA1 drives leukemogenesis and therapy resistance in KMT2A-r AML is based on the following preliminary results: 1) HMGA1 is upregulated in KMT2A-r AML with the highest levels after blasts become resistant to therapy, 2) HMGA1 is required for proliferation and clonogenicity in KMT2A-r AML cell lines, 3) HMGA1 binds to the promoter regions and induces expression of leukemogenic HOX genes in KMT2A-r AML cell lines, 4) HMGA1 drives proliferation and represses the CDKN1A tumor suppressor locus in KMT2A-r AML. 5) A recent publication identified CDKN1A repression as a driver of chemoresistance in KMT2A- r AML, although mechanisms underlying its repression were unknown, 6) Prior studies show that treating refractory AML patients with a cyclin-CDK inhibitor (flavopiridol/alvocidib) represses HMGA1 and downstream genes in the patient blasts, suggesting it could target HMGA1 pathways in KMT2A-r AML. Based on these exciting preliminary data, we hypothesize that: 1) HMGA1 drives leukemogenesis and chemoresistance in KMT2A-r AML by directly inducing HOX genes and repressing CDKN1A, and, 2) Targeting HMGA1 will enhance responses to cytotoxic therapy. To test this, we propose the following Specific Aims: 1) To define the role HMGA1 in KMT2A-r leukemogenesis and chemoresistance, and, 2) To determine how HMGA1 induces HOX genes and represses CDKN1A in KMT2A-r AML. Impact: This study will shed light on mechanisms underlying leukemogenesis in KMT2A-r AML and could lead to novel therapeutic strategies. The JHU School of Medicine provides an excellent environment for the PI, Bailey West, to complete the proposed studies. Ms. West will learn cutting-edge techniques, produce first-author manuscripts, and present her work; this training will rigorously prepare her for an academic career.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Osteoarthritis (OA) is the most common form of arthritis, affecting 1 in 8 adults in the United States. It is characterized by pain, stiffness, and swelling in the hands, hips, or knees, significantly impacting daily functioning and overall quality of life. Current treatment approaches primarily focus on pain management rather than addressing the underlying cause of OA: the degradation of articular cartilage (AC). AC is a smooth and protective layer of hyaline cartilage that covers the ends of bones in synovial joints that is actively maintained by long-lived articular cartilage chondrocytes (ACC). However, ACCs have limited regenerative capacity and fail to repair AC after damage. Ultimately, AC damage leads to improper joint loading and further pathological remodeling of the synovial joint, including AC ossification. AC ossification occurs through the inappropriate activation of endochondral ossification-like programs, similar to the development of growth plate chondrocytes (GPC) during embryogenesis. Interestingly, both ACCs and GPCs originate from chondrocyte progenitors (CPs) in the developing limb. As skeletal elements mature, CPs can differentiate into hypertrophic chondrocytes, which eventually form bone. In contrast, at the prospective synovial joint site, CPs undergo a process of "dedifferentiation," downregulating Sox9 and Col2a1 to form a structure called the interzone (IZ). Cells within and around the IZ later develop into ACCs and other components of synovial joints. To investigate the critical decision between GPC and IZ/ACC fate, we employed gene regulatory network (GRN) analysis and identified the transcription factors PITX1, ATF4, and SOX4 as regulators of CP cell fate before interzone development. Notably, PITX1 and ATF4 have each been shown to bind the Sox9 promoter, but PITX1 and ATF4 are associated with different lineage biases (ACC and GPC, respectively). The mechanisms underlying the maintenance of CP multipotency and the reprogramming of CPs to commit to ACC differentiation remain unclear. In our proposed project, we will utilize murine Sox9 lineage tracing models to characterize changes in cell identity during differentiation. The objectives of the study are to determine: 1) the extent to which candidate CP transcription factors, such as PITX1 and ATF4 exhibit mutually antagonistic effects and promote the expression of ACC and GPC genes and 2) the requirement of CP reprogramming during IZ development for ACC fate. We foresee that the completion of this project will reveal novel properties of synovial joint development for potential therapeutic targeting.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Obesity risk shows individual variation such that some children are more likely than others to gain excess weight. One potential reason is that, due to genetic and environmental factors, individuals vary in appetitive behaviors that drive food intake and weight. However, the neurodevelopmental mechanisms underpinning variation in appetite and weight, and effects of risk and protective factors on those outcomes, are not understood. Preliminary data from RESONANCE, our large MRI cohort, suggests obesity risk factors (maternal pre- pregnancy obesity, obesity-associated genetic variants) are associated with not just heightened parent-reported child appetite and BMIz, but with slower global myelination, decreased gray matter volume within fronto-cingulate control circuits, and increased volume in striatal reward regions, from infancy through early childhood. However the relevance of these findings to appetitive behaviors and development of obesity in middle childhood is unknown. This is important because obesity rates and metabolic complications increase through development, adiposity and eating habits measured in later childhood track into adulthood, and obesity is harder to treat later in development, making middle childhood a key stage for capturing outcomes with relevance for lifetime metabolic health. Further, although task fMRI studies have identified altered patterns of activation in brain appetite circuits in association with pediatric obesity and early risk factors for obesity, the predictors of altered functioning of brain appetite circuits in middle childhood are unknown. Identifying the patterns of brain development that predict obesity-promoting behaviors and brain functioning in middle childhood is essential to understand the neural mechanisms by which early obesity risk factors drive excess intake and obesity, and may help pinpoint neurobehavioral targets for early obesity prevention. Finally, although preclinical research and MRI studies of children >9y support that hypothalamic gliosis, a cellular inflammatory response, plays a role in obesity pathogenesis, it is unclear whether it occurs or impacts appetite in earlier life. For the proposed study, RESONATE, we will address the above research gaps by extending the RESONANCE study to administer meal tests, behavioral and fMRI tasks assessing food and non-food reward and cognitive control, and weight/adiposity measures in middle childhood, and examining hypothalamic gliosis, in a sub-sample of RESONANCE children. By combining this data with extant MRI data and extant or newly-collected data on obesity risk and protective factors, we will test a multi-faceted hypothesis that prenatal, genetic and postnatal factors lead to differential early development of brain appetite circuits, which in turn gives rise to variation in appetitive behaviors and behaviors involving reward processing and cognitive control as well as altered function of brain appetite circuits, that act to influence the development of obesity into middle childhood. Our long-term goal is to lay foundations for developmentally-appropriate, neurobehaviorally-informed interventions to address child obesity.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY / ABSTRACT Identification of targeted therapies for chronic obstructive pulmonary disease (COPD), a leading cause of morbidity and mortality in the United States and globally, has lagged other lung diseases. Eosinophil count, which has been a good biomarker for identifying type-2 inflammation and response to monoclonal therapies in asthma has proven to be less beneficial in COPD with unclear molecular mechanism. Since COPD is a heterogenous disease marked by systemic inflammation and high comorbidity burden it is critical that the impact of dysfunctional platelets, which act as immune and inflammatory cells, is considered in the mechanistic pathway of type-2 inflammation and eosinophil activation. Mounting evidence suggests that the magnitude of platelet activation, independent of cardiovascular comorbidity, is associated with respiratory outcomes in COPD. Activated platelets conjugate to eosinophils which leads to eosinophil activation and translocation into the lung tissue. Understanding the role of platelet-eosinophil conjugates in COPD morbidity and associations between platelet activation and eosinophil activity would enhance understanding of underlying mechanisms for type-2 inflammation in COPD and lead to novel therapeutic options. To elucidate the association of activated platelets with eosinophils in COPD we will directly measure platelet-eosinophil conjugates and eosinophil activation using flow cytometry in 50 individuals with COPD without overt or subclinical cardiovascular disease co-enrolled in the ongoing study "Platelet Activation Pathways and Respiratory Morbidity in COPD" (NHLBI K23HL151758). We hypothesize that a higher proportion of platelet-eosinophil conjugates and activated platelets as measured by flow cytometry will be associated with disease severity, worse respiratory symptoms, higher proportion of circulating activated eosinophils, and more type-2 inflammation. Platelet-eosinophil conjugates will be identified in whole blood as cells simultaneously expressing eosinophil (CCR3) and platelet (CD42a or P-selectin) surface markers in unstimulated and agonist-stimulated samples while activated eosinophils will be distinguished as those expressing CD69, CD63, CD29, or CD18. Proportion of activated platelets will be identified in platelet rich plasma before and after in vitro stimulation as part of the K23 study as those expressing P-selectin, CD63, CD40L, or PAC1. Completion of the proposed aims will elucidate the role of platelet-eosinophil conjugates and associations of activated platelets with eosinophil activation which will inform future pathway-specific mechanistic and interventional studies toward the ultimate goal of personalized therapy for COPD. The proposed study uses the infrastructure and refined laboratory protocols created as part of the K23 study to investigate the role of activated platelets with eosinophils. A more refined understanding of the mechanisms through which eosinophils are associated with COPD morbidity could potentially reveal more broadly applicable endotypes for targeted therapy.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Injuries of the growth plate account for nearly 20% of all fractures in children and often result in impaired bone growth. Once damaged, cartilage tissue of the growth plate is often replaced by unwanted bone tissue. This “bony bar” can cause angular deformities of the bone or stunt longitudinal bone growth. Angiogenesis is known to precede bony bar formation after growth plate injury, yet the mechanistic underpinnings of this (and by extension the means to prevent it) are poorly understood. Recent studies from our laboratory have demonstrated an essential role for skeletal sensory nerves in the regulation of skeletal cells and tissues. Most skeletal neurons are NGF (Nerve growth factor) responsive TrkA (Tropomyosin receptor kinase A) fibers, which our group has shown to interact with skeletal tissues to influence skeletal development and repair. Most recently, we reported that TrkA+ neurons regulate vascular proliferation and angiogenesis. This work has led us to the entirely new hypothesis that skeletal sensory neurons may regulate response to growth plate injury, potentially via regulation of angiogenesis, and that this could be targeted therapeutically. To assess this hypothesis, our studies are divided into two Specific Aims: Specific Aim 1. Identify molecular determinants of sensory neural regulation of growth plate injury response. We will utilize multi-tissue single cell RNA sequencing (scRNA-Seq) to map the neuroskeletal interactions after growth plate injury. Analysis of both the injured physis and neural soma in the corresponding lumbar dorsal root ganglia (DRG) neurons will recreate the neuro-skeletal interactome before and after injury. Next, experiments will be performed within TrkAF592A transgenic animals, in which injury site innervation and bony bar formation are attenuated. Our Aim 1 hypotheses are that: (1) chemical-genetic neuronal inhibition may disrupt pathologic bony bar formation, and growth plate angiogenesis, and (2) single cell sequencing will map the neuro-skeletal interactome and predict neural regulatory mechanisms. Specific Aim 2. Target sensory neurons via a small molecule inhibitor to prevent growth plate innervation and bony bar formation. In Aim 2, we will utilize a small molecule inhibitor of TrkA (AR786) in order to pharmacologically target TrkA signaling so as to inhibit growth plate injury site innervation, vascularity and bony bar formation. Studies will be performed in Thy1-YFP reporter animals which permit visualization of nerve ingrowth during growth plate repair. Our Aim 2 hypothesis is that small molecule TrkA inhibition during growth plate injury will prevent injury site innervation and pathologic bony bar formation.

NIH Research Projects · FY 2026 · 2024-04

The overall goal of this proposal is to characterize senescent cells (SnCs) and their communication networks during fibrosis induced by the foreign body response (FBR), specifically with respect to biomaterials, across the lifespan. SnCs are rare but impactful cells that contribute to multiple biological processes; to date, their identity and function in vivo, including interactions with the immune system, remain elusive. We recently discovered that SnCs contribute to fibrosis induced by the FBR in young animals. In preliminary studies, we found senescent pericytes and fibroblasts in the FBR that each had a unique senescence-associated secretory phenotype (SASP) and that communicated with different macrophage subpopulations. We also found differences in SnC induction and phenotype in older animals, and sex differences in the immune response to implants in both young and aged mice. In older animals, for example, SnC communication shifted toward increased communication with T cells. We developed an approach that defines senescence signatures (SenSigs) from transgenic p16-Cre reporter mice that can be used in combination with transfer learning methods to predict which cells are senescent in single-cell datasets. Using these transgenic tools and computational approaches, the proposed research will address long -standing questions of SnC phenotype, heterogeneity, function, and immunological interactions in the context of fibrosis in the foreign body response. We anticipate that this will enable development of more sophisticated and targeted senolytic therapies for the FBR and for fibrosis in general. Our Aims are: Specific Aim 1. Define senescent cell types and their senescence signature in the FBR across lifespan in male and female p16-reporter mice. Specific Aim 2. Define SnCs and their communication patterns in the FBR. Specific Aim 3. Determine the functional role of SnCs and SnC-subtypes on immune response and tissue structure.

NIH Research Projects · FY 2025 · 2024-04

Mitochondria play an essential role in cellular function and impact human health through varied mechanisms, including energy metabolism, cell signaling, and apoptosis. Many aging-related diseases have a mitochondrial (MT) component, with studies highlighting associations with cardiovascular disease, frailty, and overall mortality. In addition to inherited variation, mitochondria experience somatic mutations at much higher rates than the nuclear genome, introducing a state of heteroplasmy, defined as having more than 1 mtDNA allele within a cell. Using whole-genome sequence data from ~200,000 samples in the UK Biobank (UKB), we found that increased levels of MT heteroplasmy are associated with all-cause mortality, with individuals harboring a heteroplasmic nonsense mutation having a 1.7-fold increased risk of death, even after adjusting for the total number of heteroplasmies. These data indicate that the functional nature of the heteroplasmic mutation is a key driver of increased mortality risk. We propose to directly test the impact of heteroplasmic nonsense mutations across a range of variant allele fractions (VAF) on MT function through base editing of the MT genome followed by a comprehensive suite of MT functional assays. Given the strong link between primary MT disorders and cardiac function, we further propose to test the impact of these variants on cardiomyocyte electrical activity using human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs). In the ~200,000 UKB participants, we have identified 47 ultra-rare heteroplasmic nonsense single base-pair changes. Nineteen of the variants are targetable by existing MT base editing technologies. We propose to optimize constructs to target all 19 bases in HEK293T cell lines, which have served as the model system for MT genome editing, and then select up to 2 constructs per gene for downstream functional analyses in additional cell lines. To functionally characterize heteroplasmic nonsense mutations, we will first expand the range of VAF through a combination of single-cell expansion and the use of mitoTALENs and test the hypothesis of a dose-response relationship between VAF and MT function. We will then use a series of high-throughput assays to comprehensively characterize the mutant cell lines by assessing MT function (e.g., cellular respiration, glycolytic flux) and quantity (mtDNA-CN, nucleoid density, mass). We will then use hiPSC-CMs to test whether deleterious heteroplasmic variants across a range of VAFs modify electrical excitability, repolarization, or conduction, or compromise the development and maturation of cardiomyocytes. The increasing recognition of the role of MT heteroplasmic mutation in aging-related disease has created an urgent need to functionally characterize these mutations. Indeed, while this proposal focuses on heteroplasmic nonsense mutations, we have already identified ~1000 ultra-rare heteroplasmic missense and non-coding mutations at highly constrained sites that likewise increase risk for all-cause mortality. Thus, the proposed experiments will provide the foundational framework for future studies to distinguish benign from deleterious MT heteroplasmic mutations at these 1000s of potentially deleterious heteroplasmic sites.

NIH Research Projects · FY 2026 · 2024-04

Peripheral nerve injury can result in loss of cutaneous sensory function, with associated defects in protective reflexes and tactile function, as well as neuropathic pain. While regeneration from injured nerve is the best- studied mechanism of reinnervation following injury, it is inherently slow even with surgical augmentation. Collateral sprouting from neighboring uninjured neurons represents an additional mechanism for recovery. Although axonal regeneration has been studied extensively, much less is known about the mechanistic basis or functional consequences of collateral sprouting. After peripheral nerve injury, macrophages are recruited into or proliferate within the injured nerve stump and areas undergoing Wallerian degeneration, where they play pivotal roles in phagocytic removal of neuronal and myelin debris and facilitate the regeneration of injured axons. However, changes and potential functions of macrophages in denervated skin after nerve injury remain less studied. In this project, we will explore the hypothesis that macrophages promote skin reinnervation by collateral sprouting of uninjured nociceptors after peripheral nerve injury. In Aim 1, we will characterize changes in the abundance, localization, morphology, and phenotype of macrophages in denervated skin over time and define the anatomical relationship of these cells to sprouting nociceptive fibers. In Aim 2, we will test the hypothesis that macrophages are essential for normal nociceptor sprouting and functional sensory recovery by quantifying sprouting-mediated skin reinnervation and behavioral sensory recovery after SNI surgery in mice in which macrophages have been globally or locally depleted. In Aim 3, to define the mechanisms by which macrophages promote collateral sprouting after nerve injury, we will assess whether macrophage depletion alters peripheral nerve injury-induced upregulation of sprouting-associated genes (SAGs) in spared nociceptive neurons. We also will use in vitro DRG neuron culture to examine the impact of prior in vivo injury, with or without macrophage depletion, on neurite outgrowth and branching. Finally, we will ask whether in vitro exposure of DRG neurons to either paw skin or macrophages isolated after nerve injury alters axon outgrowth or branching. Success in this proposal will allow us to elucidate the importance of cutaneous macrophages in anatomical and functional sensory recovery by collateral sprouting. It will also help to identify candidate transcriptional and cell biological changes driving these effects and establish a system with which to subsequently identify the responsible secreted macrophages effectors. Based on our findings, targeting of the recruitment, differentiation, survival, or effector function of macrophages might be explored as therapeutic strategies to promote the progression of sensory recovery and/or limit unwanted events such as chronic pain after peripheral nerve injury.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY Endemic Burkitt lymphoma (eBL) outcomes in children with advanced stage disease are markedly inferior to those in children with early-stage disease. eBL is one of the most rapidly growing tumors. As a result, diagnostic delays of days-to-weeks contribute to late presentations, and directly affect stage and curability of disease. BL is the most common childhood cancer in Uganda. A child with a large jaw mass will be readily recognized as having eBL, and will typically be treated before histologic confirmation. However, a child with a lesser jaw swelling, or an abdominal mass, may be treated with empiric antimicrobials or observed for weeks. Even after procedures are performed for suspected BL, final histopathology is typically not available for another two weeks. EBV is consistently associated with eBL, and is detectable in blood and saliva of eBL patients. Although EBV DNA holds promise as an early detection marker for eBL, diagnostic utility is limited by poor specificity. EBV DNA not related to tumor is present in blood in a small fraction of latently infected normal lymphocytes and EBV virions may be present in plasma. EBV virions are also frequently detectable in saliva. We hypothesize that the specificity for malignancy would be markedly enhanced by enriching specimens for circulating tumor-derived EBV DNA. This may be accomplished by modifying preanalytical variables in specimen preparation, including upfront cell stabilization to limit viral DNA leakage from latently infected cells, elimination of normal lymphocytes (and epithelial cells in saliva) that may harbor latent EBV DNA, and enrichment for CpG methylated DNA, which effectively reduces virion DNA since that is never CpG methylated. Enrichment for methylated EBV DNA is a particularly innovative approach to improve assay specificity. We will test these hypotheses in two aims. In Aim 1 (blood), PBMC-derived viral DNA will be reduced by analyzing plasma, as opposed to whole blood or buffy coat cells, collected in cell-stabilizing tubes to minimize ex vivo cell lysis. Virion EBV DNA will be excluded via CpG methylated DNA separation. The effect of specimen transport temperature, time, and other preanalytical variables on cell-free DNA (cfDNA) recovery and EBV DNA measurements will be evaluated. In Aim 2 (saliva), collection techniques that minimize oral epithelial cells and lymphocytes and minimize ex vivo cell lysis along with exclusion of virion EBV DNA will be assessed. The effect of specimen transport temperature, time, and additive on cell-free DNA (cfDNA) recovery and EBV DNA measurements will be also be evaluated. These studies aim to define the optimal specimen preparation methods that will be feasible in African settings, and elsewhere, and can serve as the basis for future standards for the biospecimen science community at large. Improved diagnostic specificity of EBV DNA measurements achieved by optimizing preanalytical procedures should translate directly into earlier diagnoses and higher cure rates for eBL. Improved specimen handling also has the potential to improve eBL treatment monitoring, and could extrapolate to the diagnosis/monitoring of other EBV-associated malignancies.

NIH Research Projects · FY 2025 · 2024-04

Project Summary/Abstract Alternative splicing (AS) is the fundamental mechanism of generating isoform diversity in eukaryotic cells. AS occurs at an especially high frequency in the retina and other nervous system tissues, contributing to many important cellular and physiological functions within the cells, including tightly regulated and complex processes like neuronal development. Moreover, dysregulation of AS can have a substantial impact on retinal survival and function. Retinitis pigmentosa (RP) is a group of inherited retinal diseases that cause dysfunction and degeneration of the light-sensitive photoreceptor layer of the eye, resulting in irreversible vision loss and blindness in over 1.5 million people worldwide. Yet, therapeutic options for these patients remain limited. Many mutations that cause mis-splicing of a gene cause RP. Furthermore, defects in the regulation of AS, including mutations in spliceosome components and other associated splicing factors, also cause disease. It is unknown why retinal cells are so particularly, and often exclusively, susceptible to aberrant splicing, despite these defects often occurring in either ubiquitously expressed genes or from mutated splicing factors found in all tissues of the body. By combining novel long-read RNA-sequencing technology with CRISPR engineering and 3D human stem cell organoid models, we can acquire a detailed understanding of AS and learn how dysregulated AS contributes to retina-specific disease. To further investigate the AS landscape in human retinal development and disease, this proposal aims to 1) comprehensively examine the AS events in rod and cone photoreceptors that occur during differentiation of human stem cell-derived retinal organoids, 2) investigate how splicing factor mutations alter normal AS in the retina compared to other neurons, and 3) understand how dysregulated gene expression that results from aberrant splicing causes retina-specific degeneration. During the mentored phase of this proposal, I will take advantage of the many strengths of my multidisciplinary team of mentor/co-mentors, advisors, and collaborators to perform and analyze the single cell long-read transcriptomic experiments with the developing retinal organoids and acquire the training needed regarding brain organoid differentiation and CRISPR methodologies to successfully transition myself to the independent research phase. In the independent phase, I will use the splicing factor mutant cell lines generated in the Zack lab to further understand the mechanism(s) of retinal-specific degeneration caused by dysregulated AS events. The experiments proposed will not only provide the retinal field with a more complete understanding of AS in the retina, knowledge which can possibly be harnessed to design new treatment options for RP, but the training plan we have developed will also provide a robust pathway to establishing my successful and productive independent research career that extends well beyond the aims of this grant.