OBJECTIVES/SPECIFIC AIMS: Objectives and goals of this study will be to: (1) compare fecal microbiota and fecal organic acids in irritable bowel syndrome (IBS) patients and controls and (2) investigate the association between colonic transit and fecal microbiota in IBS patients and controls. METHODS/STUDY POPULATION: We propose an investigation of fecal organic acids, colonic transit and fecal microbiota in 36 IBS patients and 18 healthy controls. The target population will be adults ages 18–65 years meeting Rome IV criteria for IBS (both diarrhea- and constipation-predominant, IBS-D and IBS-C) and asymptomatic controls. Exclusion criteria are: (a) history of microscopic colitis, inflammatory bowel disease, celiac disease, visceral cancer, chronic infectious disease, immunodeficiency, uncontrolled thyroid disease, liver disease, or elevated AST/ALT>2.0× the upper limit of normal, (b) prior radiation therapy of the abdomen or abdominal surgeries with the exception of appendectomy or cholecystectomy >6 months before study initiation, (c) ingestion of prescription, over the counter, or herbal medications affecting gastrointestinal transit or study interpretation within 6 months of study initiation for controls or within 2 days before study initiation for IBS patients, (d) pregnant females, (e) antibiotic usage within 3 months before study participation, (f) prebiotic or probiotic usage within the 2 weeks before study initiation, (g) tobacco users. Primary outcomes will be fecal bile acid excretion and profile, short-chain fatty acid excretion and profile, colonic transit, and fecal microbiota. Secondary outcomes will be stool characteristics based on responses to validated bowel diaries. Stool samples will be collected from participants during the last 2 days of a 4-day 100 g fat diet and split into 3 samples for fecal microbiota, SCFA, and bile acid analysis and frozen. Frozen aliquots will be shipped to the Metabolite Profiling Facility at Purdue University and the Mayo Clinic Department of Laboratory Medicine and Pathology for SCFA and bile acid measurements, respectively. Analysis of fecal microbiota will be performed in the research laboratory of Dr David Nelson in collaboration with bioinformatics expertise affiliated with the Nelson lab. Colonic transit time will be measured with the previously validated method using radio-opaque markers. Generalized linear models will be used as the analysis framework for comparing study endpoints among groups. RESULTS/ANTICIPATED RESULTS: This study seeks to examine the innovative concept that specific microbial signatures are associated with increased fecal excretion of organic acids to provide unique insights on a potential mechanistic link between altered intraluminal organic acids and fecal microbiota. DISCUSSION/SIGNIFICANCE OF IMPACT: Results may lead to development of targets for novel therapies and diagnostic biomarkers for IBS, emphasizing the role of the fecal metabolome.

OBJECTIVES/SPECIFIC AIMS: Neonatal hypoxic-ischemic encephalopathy (HIE) is frequently accompanied with physiologic perturbations and organ dysfunction. Markers of these perturbations and their associations with length of stay (LOS) are uncertain. To estimate the association between changes in selected physiologic and/or laboratory values with LOS in newborns with HIE. METHODS/STUDY POPULATION: Using the Children’s Hospitals Neonatal Database (CHND), we identified neonates with HIE at our center born ≥36 weeks’ gestation from 2010 to 2016. Those with major congenital anomalies were omitted. Infants uniformly received therapeutic hypothermia for 72 hours unless death occurred sooner. Inpatient vital signs and selected laboratory markers were collected from our institution’s health informatics, electronic data warehouse (EDW) and then matched to records in CHND. With severity of HIE, gender, and confirmed seizures, each marker’s association with LOS was calculated using multivariable Cox proportional hazards regression equations. These analyses were stratified by mortality. Candidate markers were vital signs, pulse oximetry, creatinine, acidosis (pH), international normalized ratio (INR), and supplemental oxygen (FiO2). RESULTS/ANTICIPATED RESULTS: There were 66 eligible infants (38 males) and 1741 patient-days identified; Severe HIE (48%) and mortality (n=21, 32%) were common. Overall, the median length of stay (mLOS) was 20.5 days (25th–75th centile: 10–31 days), although shorter for nonsurvivors [nonsurvivors mLOS=8 days (5, 20); survivors mLOS=24 days (14, 31), p<0.001). Median birthweight and gestational age were 3.3 kg and 39.4 weeks’ gestation, respectively. In survivors (n=45, 1290 days), regression analyses demonstrated that none of the selected parameters were associated with LOS. Among nonsurvivors (n=21, 451 days), diastolic blood pressure changes [hazard ratio (HR)=0.93, 95% confidence interval (CI)=0.88, 0.97, p=0.04] was related to longer time of survival; conversely, temperature (HR=2.0, 95% CI=1.24, 3.26, p=0.005) was related to shorter survival. Creatinine, pH, INR, FiO2, or other vital signs were unrelated to time-to-death in nonsurvivors. DISCUSSION/SIGNIFICANCE OF IMPACT: In a pilot study of neonatal HIE, changes in physiologic values were related to duration of survival in nonsurvivors, while neither physiologic nor laboratory values were related to survivors’ mLOS. These results both exemplify novel uses for disease-specific, exposure-outcome relationships using EDWs and incorporates required functionalities of required software patches to extract, clean, and report from clinical information captured in electronic health records. We anticipate that text mining with techniques such as natural language processing will augment associations and/or predictions of short-term outcomes.

Beyond medical schools’ historical focus on pillar missions including clinical care, education, and research, several medical schools now include community engagement (CE) as a mission. However, most academic health systems (AHSs) lack the tools to provide metrics, evaluation, and standardization for quantifying progress and contributions of the CE mission. Several nationwide initiatives, such as that driven by the Institute of Medicine recommending advances in CE metrics at institutions receiving Clinical and Translational Science Awards, have encouraged the research and development of systematic metrics for CE, but more progress is needed. The CE components practical model provides a foundation for analyzing and evaluating different types of CE activities at AHSs through five components: research, education, community outreach and community service, policy and advocacy, and clinical care. At the Medical College of Wisconsin (MCW), an annual survey administered to faculty and staff assessed the types and number of CE activities from the prior year. Survey results were combined to create a CE report for departments across the institution and inform MCW leadership. Insights gathered from the survey have contributed to next steps in CE tracking and evaluation, including the development of a CE dashboard to track CE activities in real time. The dashboard provides resources for how individuals can advance the CE mission through their work and guide CE at the institutional level.

Research education and training in Translational Science develops and sustains a workforce to efficiently advance studies designed to improve human health. We evaluated the effectiveness of a Translational Science Training (TST) TL1 Program. Participants had significantly better publications/year, citations/year, h-index, and m-quotient than nonparticipants. Female and male participants, and participants from underrepresented and well-represented backgrounds, performed similarly on all bibliometric assessments. Finally, TST/TL1 Program participants outperformed students from other PhD programs at our institution. This analysis suggests that the TST/TL1 Program has been effective for participants, including those who are female and from underrepresented backgrounds.

While clinical research intends to improve health outcomes for all, access to research participation is often limited and inequitable. Geographic proximity is a recognized barrier, thus, systemic infrastructure solutions through federal programs including General Clinical Research Centers and Clinical and Translational Science Awards have sought to improve accessibility. Even with such support, academic medical centers often have limited clinical research-dedicated space apart from shared exam rooms in difficult-to-navigate hospitals or clinics. In 2019, the Duke University School of Medicine looked beyond its medical center campus to identify free-standing sites within Durham communities for participant study visits. Catalyzed by the COVID-19 pandemic, Duke Research at Pickett, a 22 000-square-foot building with a laboratory, 30 exam rooms, and on-site parking, opened in October 2020 to support vaccine and treatment trials. Upon the lifting of many COVID-19 restrictions, and in partnership with the Research Equity and Diversity Initiative (READI) Community Advisory Council, the building was transformed to encourage community gatherings, education, and training programs. To date, Duke Research at Pickett has hosted 2692 participants in 78 research trials and 14 community-engaged activities.

OBJECTIVES/SPECIFIC AIMS: Epidemiologic data suggest that BV is sexually transmitted with male partners colonized or infected with the responsible organism(s). The objective of this study was to compare the genital microbiota of women with recurrent BV and their regular male sexual partner using 16S rRNA gene sequencing and quantitative PCR targeting BV-candidate bacteria (Gardnerella vaginalis, Atopobium vaginae, BVAB1-3, Sneathia, Leptotrichia, and Megasphaera type I). METHODS/STUDY POPULATION: Women with recurrent BV (≥3 prior episodes, including a current episode) and their regular male partner participating in a BV treatment trial and providing genital specimens (women: vaginal; men: urethral, coronal sulcus, urine) at enrollment were included. Male specimens for each participant were pooled. 250 bp 16S rRNA V4 region PCR amplicons were sequenced and analyzed using the QIIME pipeline. Taxonomy was assigned using the RDP Classifier against a modified Greengenes database with additional vaginal taxonomies added. An average relative abundance cutoff of 0.5% was used for analysis. qPCR was also performed for specific BV-candidate bacteria. Spearman correlation coefficients were used to investigate associations between all genital bacteria in addition to BV-candidate bacteria between partnerships. To determine positive associations between partnerships, the Wilcoxon signed-rank test was used. RESULTS/ANTICIPATED RESULTS: In total, 45 partnerships were included. Mean partnership age was 31.3 (SD=7.9), 91.1% partnerships were African-American. The majority of partnerships (70.0%) reported condomless sex during the past 3 months. Regarding 16S data, 37 genital bacteria had an average relative abundance of ≥0.5%. The average Spearman correlation across all 45 partnerships was 0.28 (SD=0.27) (median=0.27, minimum=−0.21, maximum=0.84). Overall, a positive association of all genital bacteria existed across the partnerships (p<0.0001). However, regarding specific BV-candidate bacteria, Spearman correlation tests for G. vaginalis, A. vaginae, Prevotella bivia, Megasphaera type I, BVAB1, and BVAB2 were nonsignificant. In contrast, Sneathia spp. were positively correlated between partnerships (r=0.37, p=0.01). With regards to qPCR results, RNA Cq analyses provided significant evidence for a linear association between male and females for only A. vaginae (r=0.52, p=0.006). DISCUSSION/SIGNIFICANCE OF IMPACT: In monogamous heterosexual couples in which the female has BV, the vaginal microbiota of women and the penile/urine microbiota of men were significantly correlated, particularly with regards to Sneathia spp. and A. vaginae, supporting the hypothesis that BV-associated bacteria are exchanged during sex.

OBJECTIVES/SPECIFIC AIMS: Our objective is to create a Resistant Hypertension (RHTN) computable phenotype from electronic health record (EHR)-based data, and to determine the characteristics associated with RHTN within a large, diverse, EHR-based database. METHODS/STUDY POPULATION: The OneFlorida Clinical Research Consortium includes 10 unique health care systems providing care for approximately half of the state (48%, ~10 million). OneFlorida houses a Data Trust which contains longitudinal EHR data and claims data from these providers in a common format, the PCORnet common data model v3.0. For the current project, data from 5 health care systems were considered. All of the adult hypertension (HTN) patients with a HTN diagnosis from an outpatient encounter were extracted from the OneFlorida Data Trust. Additional data such as demographics, prescribing, and vitals information were also extracted. The RHTN computable phenotype was created by constructing a drug exposure variable that took into consideration the number of antihypertensive medications an individual was prescribed at any point in time over the course of the OneFlorida dataset. RHTN was defined as any blood pressure requiring four or more antihypertensive drugs, or uncontrolled blood pressure (≥140/90) on 3 antihypertensive drugs. RHTN cases had to meet the definition criteria twice during the data period, at least 30 days apart. All data extraction, computation phenotype coding, and statistical analyses were conducted using SQL or SAS. RESULTS/ANTICIPATED RESULTS: Our preliminary results show that there were n=342,026 adults with a HTN diagnosis from an outpatient visit in the data set. After the RHTN computable phenotype was constructed, n=11,670 RHTN cases were identified from the n=130,901 HTN individuals with all of the required variables in the data set (8.9% RHTN prevalence). In all, 55% of RHTN cases were Black or African American, compared with the total HTN population (25% Black/African American). RHTN cases also had more prescriptions for loop diuretics, centrally acting agents, α-blockers, and vasodilators compared with the total HTN population. Not surprisingly, the RHTN cases had 26% of the antihypertensive prescriptions in the data set, and the RHTN cases had fewer blood pressure readings that were in control (only 49.4% of readings <140/90). DISCUSSION/SIGNIFICANCE OF IMPACT: Overall, our preliminary data shows that it is possible to create the very complicated computable phenotype of RHTN within the OneFlorida Data Trust. We found that the RHTN prevalence in OneFlorida is 8.9% which is consistent with previous studies from NHANES. Although promising, these results require further validation of the computable phenotype and replication in other similar data sets in order to ascertain their true meaning. Once validated, the experience gained from this computable phenotype can be applied to many other phenotypes.

OBJECTIVES/GOALS: We aimed to conduct an updated genome-wide meta-analysis of keloids in expanded populations, including those most afflicted by keloids. Our overall objective was to improve understanding of keloid development though the identification and further characterization of keloid-associated genes with genetically predicted gene expression (GPGE). METHODS/STUDY POPULATION: We used publicly available summary statistics from several large-scale DNA biobanks, including the UK Biobank, FinnGen, and Biobank Japan. We also leveraged data from the Million Veterans Program and performed genome-wide association studies of keloids in BioVU and eMERGE. For each of these datasets, cases were determined from ICD-9/ICD-10 codes and phecodes. With these data we conducted fixed effects meta-analysis, both across ancestries and stratified by broad ancestry groups. This approach allowed us to consider cumulative evidence for genetic risk factors for keloids and explore potential ancestry-specific components of risk. We used FUMA for functional annotation of results and LDSC to estimate ancestry-specific heritability. We performed GPGE analysis using S-PrediXcan with GTEx v8 tissues. RESULTS/ANTICIPATED RESULTS: We detected 30 (23 novel) genomic risk loci in the cross-ancestry analysis. Major risk loci were broadly consistent between ancestries, with variable effects. Keloid heritability estimates from LDSC were 6%, 21%, and 34% for European, East Asian, and African ancestry, respectively. The top hit (P = 1.7e-77) in the cross-ancestry analysis was at a replicated variant (rs10863683) located downstream of LINC01705. GPGE analysis identified an association between decreased risk of keloids and increased expression of LINC01705 in fibroblasts (P = 3.6e10-20), which are important in wound healing. The top hit in the African-ancestry analysis (P = 5.5e-31) was a novel variant (rs34647667) in a conserved region downstream of ITGA11. ITGA11 encodes a collagen receptor and was previously associated with uterine fibroids. DISCUSSION/SIGNIFICANCE: This work significantly increases the yield of discoveries from keloid genetic association studies, describing both common and ancestry-specific effects. Stark differences in heritability support a potential adaptive origin for keloid disparities. Further work will continue to examine keloids in the broader context of other fibrotic diseases.

ABSTRACT IMPACT: The knowledge acquired from my research can inform the development of early diagnostic methods for HIV-associated neurocognitive disorders. OBJECTIVES/GOALS: In the era of combination antiretroviral therapy (cART), the prevalence of HIV-associated neurocognitive disorders (HAND) remains high but the neural mechanisms are unclear. We examined whether older people with HIV (PWH) with minimal cognitive impairment have reduced functional connectivity in frontostriatal circuits compared to controls. METHODS/STUDY POPULATION: 99 PWH (mean age 56.6 years, 75% male, 62% Black, mean duration of HIV-infection 26.2 years ±9.3, 90% viral load <50 copies, 98% on stable cART) and 38 demographically-comparable controls (mean age 54.5 years, 71% male, 58% Black) participated in a cross-sectional study. A 7-domain neuropsychological battery and an Activities of Daily Living index were used to determine HAND diagnoses: 32 PWH met criteria for asymptomatic to mild HAND. Motor skill was assessed using the Grooved Pegboard Test by measuring performance speed. Structural MRI and resting-state functional MRI were collected. Seed-to-voxel analyses were conducted using 4 distinct regions in the striatum as seed regions. We used a voxel threshold of p<0.001 and cluster threshold of p<0.05 (FDR-corrected) after controlling for demographic variables. RESULTS/ANTICIPATED RESULTS: Compared to controls, PWH had lower resting state functional connectivity between the default mode region of the striatum (i.e., medial caudate) and bilateral superior frontal gyrus, supplementary motor cortex and paracingulate gyrus (p<0.05; cluster size: 567 voxels). Also, compared to controls, PWH had reduced resting state functional connectivity between the motor division of the striatum (i.e., posterior putamen) and anterior cingulate cortex and left supplementary motor cortex (p<0.05, cluster size: 405 voxels). Performance speed on the Grooved Pegboard motor test negatively correlated with functional connectivity between the motor region of the striatum and supplementary motor frontal regions in all participants (Spearman’s rho=-0.18, p=0.04). DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results support the hypothesis that frontostriatal abnormalities are widely present in PWH and might play a key role in HAND development. Our data suggest that dysfunction within the frontostriatal circuits may be involved in motor impairment in PWH, and ongoing inflammation may contribute to motor impairment and frontostriatal injury.

OBJECTIVES/GOALS: To describe a study to develop, test, and collect implementation data on a youth-led hypertension (HTN) education digital intervention that acts as an electronic tool to guide youth through learning and then teaching adults on how to achieve better HTN control. Adults with uncontrolled HTN are recruited from a New Jersey emergency department (ED). METHODS/STUDY POPULATION: Adults with HTN and youth (15-18 years) participate in a remote user-centered design session focus group to provide input in the development of the youth-led HTN education digital intervention. 100 adult ED patients with uncontrolled HTN (blood pressure (BP) ≥130/80 mm Hg) who live with a youth (15-18 years) and the youth themselves are recruited for a randomized control trial (RCT). The adult-youth dyad is randomized to one of two arms, each a 6-week program with youth earning a digital badge: 1) intervention- youth-led HTN education with the adult, or 2) control- youth learn life skills (such as job readiness/resume building). Implementation metrics are collected through a post-intervention survey and qualitative interviews on the digital badge intervention including acceptability, feasibility, and fidelity. RESULTS/ANTICIPATED RESULTS: We completed two youth focus groups (total of 8 participants) and data collection is ongoing. Youth have shown great interest in the intervention prototype and thought their peers would find it acceptable. They suggested additions to nutrition education activities, such as adding a sodium tracker and examples of high sodium foods. For the RCT, the primary study outcome is adult BP change (from baseline to 1 week and 2-months post-intervention), with secondary outcomes of HTN knowledge and youth self-efficacy. We anticipate that intervention arm adults will have a more significant decrease in BP than control arm adults. We also expect that HTN knowledge and youth self-efficacy will be higher for the intervention arm. Implementation data collected will allow for improvements to future renditions of the intervention. DISCUSSION/SIGNIFICANCE: Bringing health education home while simultaneously empowering youth is an innovative technology-driven model for improving BP for patients with uncontrolled HTN who may lack access to care. Outcomes of this project will result in a scalable and easily adoptable model to reach an otherwise difficult to reach adult population.

OBJECTIVES/GOALS: To quantify changing trends in hepatocellular carcinoma (HCC) etiologies, mainly hepatitis C related HCC (HCV-HCC), nonalcoholic fatty liver disease related HCC (NAFLD-HCC), and alcoholic liver disease related HCC (ALD-HCC), at a single center as well as compared to large national databases. METHODS/STUDY POPULATION: This is a retrospective longitudinal study using a single-center database of patients presenting with HCC from January 1995 to September 2023. Etiologies were confirmed through patient history, clinical exam, and viral serologies. Trends in rate of etiology were analyzed using linear regression. Further investigation will include survival analysis. To improve generalizability, the single-center data were supplemented with national cross-sectional data from the NHANES database on liver disease prevalence from March 1999 to August 2023. Data were provided through questionnaire, clinical exam, and viral serologies. Trends in rates will be analyzed using linear regression. RESULTS/ANTICIPATED RESULTS: Among the single center cohort, NAFLD-HCC increased at an average rate of 1.3% per year (95% Confidence Interval (CI) = 1.1% to 1.4%) and HCV-HCC decreased at an average rate of -0.56% per year (95% CI = -0.83% to -0.29%). Projecting the linear models for the past ten years forward, HCV-HCC is predicted to take up a lower proportion than NASH-HCC by 2026 and lower proportion than ALD-HCC by 2028. Future results will include analysis of the changing proportions of etiologies for liver transplant and survival analysis for HCC by etiology from the single center cohort. Additionally, national trends in HCC etiologies will be provided from the NHANES database. The trends from liver transplant etiology and NHANES are expected to parallel the preliminary results. DISCUSSION/SIGNIFICANCE: As the prevalence of NAFLD increases in the general population, more cases of NAFLD-HCC will be seen in the future. Understanding the changing trends can guide surveillance recommendations, shape treatment algorithms, and frame research priorities.

OBJECTIVES/GOALS: Nicotinamide adenine dinucleotide (NAD) plays essential roles in energy metabolism and cell signaling pathways. NAD functions as a coenzyme by accepting electrons during glycolysis and the TCA cycle and subsequently donates them to complex I of the electron transport chain providing the driving force for ATP production. NAD also acts as a co-substrate for several classes of enzymes, including sirtuin deacetylases. Both NAD and the enzyme that is rate limiting for synthesis, Nicotinamide phosphoribosyltransferase (Nampt), are depleted in the failing heart, concurrent with hyperacetylation and mitochondrial dysfunction. Moreover, treatment with NAD precursors reduced cardiac injury in several heart failure models. However, NAD precursors may have systemic effects, and it remains unproven whether depletion of myocardial NAD is causative or merely correlative for the onset and progression of heart failure. METHODS/STUDY POPULATION: To test this, we generated a cardiac-specific tamoxifen-inducible (αMHC-MerCreMer) model for deletion of Nampt (Nampt cKO) in cardiomyocytes. Adult mice were administered tamoxifen for 5 days leading to deletion of Nampt, resulting in a 72% reduction in myocardial NAD after two-weeks. RESULTS/ANTICIPATED RESULTS: Echocardiography revealed that Nampt cKO mice displayed a significant reduction in left ventricular (LV) contractility as well as cardiac hypertrophy. Despite the further loss of NAD, the majority of animals survived to 8 weeks of age before experiencing sudden deaths resulting in significant mortality over the next several weeks. Remarkably, we observed only a slight increase in acetylation of mitochondrial proteins, and cardiac mitochondria isolated from Nampt-null mice even at 8 weeks displayed a normal or higher oxygen consumption rate. We found that mitochondrial NAD levels were preferentially maintained and depleted at a slower rate compared to those in bulk tissue. DISCUSSION/SIGNIFICANCE OF IMPACT: While mild depletion of cardiac NAD has been reported in heart failure, our data indicate that the heart can adapt to much more severe loss of NAD prior to the loss of viability.

OBJECTIVES/GOALS: The primary objective of this study is to evaluate the risk of an early syphilis diagnosis following a chlamydia, gonorrhea, or HIV diagnosis, and to determine differences by race, repeat infection, diagnosing provider and STI/HIV risk behavior among cisgender women in Baltimore, MD. METHODS/STUDY POPULATION: Public health surveillance data from 2009-2021 was used to examine the overall incidence of syphilis infections among cisgender women ages 13-50 diagnosed with a reportable STI (chlamydia, gonorrhea, or HIV) and the percentage of total infections that were early infections (primary, secondary, or early latent syphilis) in Baltimore City. Data were collected on age, race, diagnosing location (i.e., STI clinic, private provider, etc.), preceding STI diagnoses, and sexual risk behaviors. STI-specific cumulative incidence and incidence rate ratios were used to compare syphilis diagnoses among Black vs. white women, women with repeat STI diagnoses vs. one STI diagnosis, women diagnosed at a public vs. private clinic, and commercial sex workers and substance users vs. those not reporting these risk behaviors. RESULTS/ANTICIPATED RESULTS: Based on recent surveillance data, we expect approximately 79,000 chlamydia, gonorrhea, and HIV diagnoses among cisgender women between 2009-2021. We hypothesize that 3% of chlamydia, gonorrhea, and HIV diagnoses among cisgender women will be followed by a syphilis diagnosis within the study period. Extrapolating from previous studies of early syphilis in men who have sex with men in Baltimore, we expect the rate of syphilis diagnosis following STI diagnosis will be higher in Black vs. white women, women with a prior gonorrhea or HIV diagnosis vs. chlamydia diagnosis, women with repeat STI diagnoses vs. one STI diagnosis, women diagnosed at public STI clinics vs. those diagnosed by private providers, and women reporting commercial sex work and/or substance use vs. those not reporting these risk behaviors. DISCUSSION/SIGNIFICANCE: Local healthcare providers should offer syphilis screening to any woman diagnosed with a chlamydia, gonorrhea, or HIV infection. The higher rates of early syphilis diagnosis among women with repeat STI diagnoses or a prior gonorrhea or HIV diagnosis suggests regular screening is critical in these populations.

OBJECTIVES/SPECIFIC AIMS: Objective: apply checkpoint inhibitors that are specific to the exhaustive markers expressed on tumor CD8+ T-cells ex vivo in order to improve cytokine release and cytotoxic function in comparison to two control groups: (1.) T-cells that receive no antibodies; (2.) T-cells that receive standard inhibition with PD-1 and CTLA-4 antibodies only. Long-term objective: provide personalized medicine in the treatment of HCC by using checkpoint inhibitors that are specific to the receptors expressed by an individual tumor. METHODS/STUDY POPULATION: The study population includes patients undergoing liver transplantation or surgical resection for HCC. Two grams of tumor, two grams of healthy liver tissue at least one centimeter from the tumor margin, and 50 milliliters of blood will be obtained. Solid tissue will be mechanically and enzymatically disrupted and CD8+ T-cells will be isolated from all sites. Using flow cytometry, the expression of surface receptors PD-1, CTLA-4, LAG-3, TIM-3, BTLA, CD244, and CD160 will be categorized in each tissue to identify which receptors are upregulated in the tumor microenvironment. Up to three antibodies specific to the upregulated receptor(s) on the tumor T-cells will be applied per specimen. The experimental arm will receive these antibodies and co-stimulation with CD3/CD28 and will be compared to two controls. One control will receive only CD3/CD28, and the other will receive CD3/CD28 in addition to the standard combination of PD-1 and CTLA-4 inhibitors. From each condition, flow cytometry will be used to assess the mean production of interleukin-2, tumor necrosis factor-α, interferon-γ, granzyme B, and perforin expression as an assessment of T-cell function. RESULTS/ANTICIPATED RESULTS: Preliminary data from the peripheral blood of healthy controls confirms that the developed flow cytometry panels effectively identify the surface receptors and cytokine production of CD8+ T-cells. Two patients have successfully been enrolled in this study. It is predicted that T-cells extracted from the tumor will express more inhibitory receptors than normal liver or peripheral blood and will have increased function after they are targeted with checkpoint inhibitors that are specific to the inhibitory surface receptors they express. DISCUSSION/SIGNIFICANCE OF IMPACT: HCC is the second leading cause of cancer-related death worldwide and therapeutic options are limited for patients who are not surgical candidates. T-cells are a critical component of the anti-tumor response to HCC. However, T-cells can develop an exhausted phenotype characterized by up-regulated inhibitory receptors (PD-1, CTLA-4, LAG-3, TIM-3, CD-244, CD-160, BTLA) and decreased function, allowing for immune escape. Clinical trials using combined checkpoint inhibition with PD-L1 and CTLA-4 antibodies have been considered a breakthrough for patients with advanced HCC, as up to 25% show an objective tumor response. The explanation for the varied susceptibility to checkpoint inhibition remains unknown and is hypothesized to be secondary to inconsistencies in the expression of surface inhibitory receptors. Although inhibitory receptor expression has been shown to be upregulated under conditions of hepatitis and/or HCC, there has been no single study to effectively investigate the expression of all known inhibitors in order to better explore the interplay between them. It will be of great academic interest and clinical purpose to evaluate individual receptor expression and engage the correlating antibodies given the possibility of synergism between receptors and the need for a more profound anti-tumor T-cell response in HCC.

OBJECTIVES/SPECIFIC AIMS: This abstract describes a recently-funded 2 year study that aims to: (1) explore the community advisors’ perspectives of the safe sleep intervention’s acceptability, feasibility, and adaptability using focus groups and key informant interviews. (2) Adapt the selected safe sleep interventions (SSI) and identify promising implementation strategies to support it through an evidence-based quality improvement process with a multistakeholder group. METHODS/STUDY POPULATION: Background sudden unexpected infant death (SUID) is the leading cause of post-neonatal infant death in the United States. Sudden infant death syndrome (SIDS), accidental suffocation and strangulation in bed account for over 50% of SUID, leading to recommendations for supine sleep position and safer sleep environments for infants. However, despite significant reductions in SIDS after “back to sleep” and “safe to sleep” campaigns, significant racial and urban-rural disparities persist. In 2015, the rural-urban crude death rate ratio was 4:1 and Black infants are twice as likely to die from SUID as White infants. Adherence to safe sleep recommendations is highly variable and a number of hospital and community-based interventions have been suggested to improve knowledge and change parent behavior. Hospital programs to promote safe sleep education and policies may serve to educate families about safe sleep, but may not be uniformly available in rural and underserved areas. The AAP evidence-based safe sleep guidelines have demonstrated reductions in SIDS and SUID when child caregivers adhere to them. Community-based SSI, including safety baby showers, promote safe sleep practices, but barriers may exist for participation, especially in rural areas. Partnering with community groups serving a high risk area, we will explore the barriers and facilitators to more widespread safety baby shower (SBS) delivery/adoption in rural underserved communities (RUC). Observation of the evidence-based SBS as it is currently delivered, focus groups and key informant interviews will be conducted with program leaders and participants. Based on this knowledge and using an evidence-based development process, we will adapt the SBS and identify implementation strategies to support its uptake in RUC. RESULTS/ANTICIPATED RESULTS: We expect to develop a modified safe sleep intervention that reaches more expectant and new mothers is more efficient at delivering safe sleep guidelines to rural community members and can be more readily adopted and implemented by RUC. Supporting implementation strategies will be identified during the formative evaluation. DISCUSSION/SIGNIFICANCE OF IMPACT: Developing a safe sleep intervention adapted for the local context through a collective decision-making process between intervention experts and local community advisors will potentially improve safe sleep guideline delivery and adherence in RUC. The next study will pilot test the effectiveness of the adapted safe sleep intervention with identified supporting implementation strategies.

OBJECTIVES/GOALS: The increasing frequency and severity of heat waves due to climate change present unique risks to children. We aim to assess how various heat wave definitions impact pediatric acute care utilization across California. We also hope to examine heat waves’localized effects at the zip code level and how contextual factors modulate these effects. METHODS/STUDY POPULATION: A time-stratified case crossover will evaluate the association between different heat wave definitions and pediatric acute care utilization throughout California. A within-community matched design analysis coupled with a bayesian model will examine heat waves’ effects at the zip code level. A random effect meta-regression will determine which contextual factors modulate heat waves’ impact on different zip codes. Temperature data will be pulled from Cal-Adapt and interpolated to each zip code population centroid. Data for all unscheduled pediatric hospitalizations and ED visits for selected ICD codes in California from 2000–2019 will be obtained from the California Department of Health Care Access and Information. Contextual factors will be sourced from the US Census and the Healthy Places Index. RESULTS/ANTICIPATED RESULTS: We anticipate that heat waves will be associated with increased pediatric acute care utilization throughout California for select ICD codes. At the zip code level, we anticipate that there will be considerable spatial variation in the association between heat waves and care utilization based on region and zip code characteristics. Furthermore, we expect to see significant variation in the association between heat waves and hospitalizations based on the selected heat wave definition. We predict that zip codes with the highest increases in care utilization will have higher percentages of non-white residents, lower socioeconomic status, and fewer heat protective factors like park density and tree coverage. DISCUSSION/SIGNIFICANCE: As global temperatures continue to rise, children will be increasingly susceptible to health consequences associated with heat exposure. Understanding which pediatric populations are most vulnerable during heat waves is critical for designing policies and interventions that protect the most vulnerable communities.

OBJECTIVES/SPECIFIC AIMS: The central goal of this proposal is to characterize the mechanisms that mediate success or failure of immature intestinal barrier in necrotizing enterocilitis. METHODS/STUDY POPULATION: To do this, I will utilize stem cell derived human intestinal organoids (HIOs), an innovative model of the immature intestine, and a cohort of bacterial isolates collected from premature infants who developed NEC to interrogate the cause-effect relationship of these strains on maintenance of the intestinal barrier. I hypothesize that the epithelial response to bacterial colonization is strain-dependent and results in differences in inflammatory signaling that shape epithelial barrier function in the immature intestine. RESULTS/ANTICIPATED RESULTS: Preliminary data shows that colonization of HIOs with different bacteria leads to species-specific changes in barrier function, and some species selectively damage the epithelial barrier while others enhance epithelial barrier function. I have identified key inflammatory signals that serve as central drivers of intestinal barrier function. DISCUSSION/SIGNIFICANCE OF IMPACT: Characterization of this process is expected to substantially advance scientific understanding of early events in NEC pathogenesis and lead to new opportunities for targeted therapeutic intervention to accelerate barrier maturation or prevent hyperinflammatory reactivity in the neonatal intestine. The research proposed in this application represents an entirely novel approach to studying host-microbial interactions in the immature. Conceptually, this novel translational approach will help to define the pivotal role of colonizing bacteria in initiating epithelial inflammation in NEC patients.