OBJECTIVES/SPECIFIC AIMS: This project aims to synthesize an angiogenic decorin mimic (VEGFp-DS-SILY) with varying densities of QK and characterize its angiogenic potential and synergism with VEGF by evaluating (1) endothelial cell (EC) migration and proliferation, (2) EC VEGF receptor activation, (3) EC tubule formation in collagen scaffolds, and (4) angiogenesis from a chick chorioallantoic membrane (CAM assay) growing into the scaffold, reflecting the ability of the collagen scaffold to integrate into existing vasculature. The next main goal is to develop and characterize an MMP-degradable nanoparticle system for controlled release of VEGF. Future work will evaluate in vivo effects of VEGFp-DS-SILY bound to a 3D collagen scaffold on ischemic wound repair in a combined excisional wound/bipedical dorsal skin flap rat model. METHODS/STUDY POPULATION: Peptide hydrazides are conjugated to the free carboxylic acid functional groups on dermatan sulfate using EDC chemistry. We added a 3 amino acid spacer (-Gly-Ser-Gly) to the C-terminus of the established QK sequence before the hydrazide functional group and refer to this modified QK as “VEGFp.” VEGFp, SILY, and N-terminal biotinylated versions were synthesized using standard Fmoc solid-phase peptide synthesis protocols and purified using reverse phase HPLC. Coupling efficiencies of peptides to dermatan sulfate were determined spectroscopically at 280 nm measuring the aromatic residues (Trp or Tyr) using a NanoDrop system. Dermatan sulfate with 1 or 4 VEGFp peptides coupled were termed DSV1 and DSV4, respectively. After further conjugation with SILY, we will blend this VEGFp-DS-SILY with unmodified DS-SILY to a total 10 μM to test increasing densities of VEGFp. To verify that the collagen-binding properties of VEGFp-DS-SILY are not compromised by the addition of VEGFp, we will use a streptavidin-HRP system to detect bound biotinylated VEGFp-DS-SILY on collagen-coated plates by established protocols. DSV1 and DSV4 were tested for their effects on endothelial VEGFR2 phosphorylation using an MSD ELISA-type assay and endothelial proliferation using an MTS assay. Cell migration was monitored using an ORIS assay where cells are grown to confluence around a silicone stopper that is then removed to allow cells to migrate inward. Tubulogenesis was evaluated by examining tubule formation on matrigel. Finally, in vivo angiogenesis will be evaluated using a chorioallantoic membrane assay. For extracellular VEGF release, hollow MMP-degradable thermoresponsive nanoparticles [NIPAM, 5 mol% 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 1% Acrylic Acid (AAc), 2 mol% MMP-degradable peptide diacrylate, and potassium persulfate initiator] will be synthesized around noncross-linked polymer cores. The cores will then be diffused out through the shell by dialysis prior to drug loading. SILY (and some biotinylated SILY for visualization) will be conjugated with EDC chemistry for targeting nanoparticles to collagen. NPs size and zeta-potential will be measured on a Malvern Zetasizer. VEGF will be loaded into NPs by co-incubating a loading solution of 1 µg/mL VEGF with 1 mg of NPs, incubating overnight at 4°C. VEGF loading and release will be measured by ELISA. Biological activity of the released VEGF from particles will be determined on ECs using assays similar to those outlined previously. RESULTS/ANTICIPATED RESULTS: Preliminary data have verified the synthesis and purification of SILY and VEGFp (QK-Gly-Ser-Gly-hydrazide), as well as an N-terminal biotinylated version, through mass spectrometry and reverse-phase HPLC, respectively. For proof-of-concept, we have verified binding of VEGFp to the VEGF receptor 2 using a ForteBio Blitz interferometry instrument. In addition to support based on published reports showing retained bioactivity of QK after conjugation using other spacers, our preliminary data suggests that VEGFp still binds to VEGF receptor 2, albeit with decreased affinity like QK as compared with VEGF. Circular dichroism also shows that VEGFp has retained its α-helical structure necessary for bioactivity; however it appears that it has some uncoiling when conjugated to dermatan sulfate. We hypothesize that varying densities of VEGFp conjugated to the decorin mimetic (DS-SILY) will modulate the degree of angiogenic activity and synergy with VEGF. We determined that we can achieve ~70% VEGFp conjugation completion to dermatan sulfate after 3.5 hours. We have quantified VEGFR2 phosphorylation after 5 minute treatments by using phospho-specific antibodies and an ELISA-type protocol in a mesoscale discovery system. Preliminary data with human umbilical vein endothelial cells shows that VEGFp exhibits synergism with VEGF at levels similar to QK. DSV1 and DSV4 data suggests synergy with VEGF, although free-peptides and engineered compounds alone did not show effects similar to VEGF in the conditions tested. Prelminary data with 30 minute treatments suggests that the peptides and compounds may require longer exposures to induce activation, as they may have slower binding rates. In contrast, prolonged stimulation with VEGF causes a sharp increase in receptor activation, peaking around 10 minutes and decreasing significantly by 30 minutes. Peptides QK and VEGFp both slightly increased proliferation of dermal microvascular endothelial cells (HMVECs) after 60 hours incubation. However, incubation with dermatan sulfate and DSV caused significant cell death after 24 hours in reduced growth factor media, likely due to sequestering of growth factors. It is possible that VEGFp-DS-SILY may better stimulate proliferation since it would be presented as a surface bound proteoglycan mimic, rather than as a soluble factor. HMVECs migrated farther for all treatment groups (10 µM QK, 10 µM VEGFp, 1 µM DSV4, and 10 µM DSV4) than the 10 ng/mL VEGF positive control, although more cells migrated in response to VEGF. This may be accounted at least in part by the more pronounced proliferation induced by VEGF. Migration will also be tested in 3D culture within a collagen gel. We are currently testing a 2D matrigel system for tubulogenesis. We have found that 10 µM DSV4 forms qualitatively more well-defined tubules than the untreated control on reduced growth factor matrigel. However, we were not able to quantify the improved tubule formation and are still troubleshooting the tubule analysis. After seeding ECs and culturing for 4, 8, and 12 hours, cells will be fluorescently stained with anti-CD31 and imaged for 3D tubule formation. CAM assay angiogenesis growing into a collagen scaffold. In brief, fertilized chicken embryos are incubated for 2 days before exposing the CAM. VEGFp-DS-SILY bound to a collagen gel will be placed onto the CAM. Some treatment groups will receive additional VEGF to investigate synergistic effects. Light microscope images of angiogenesis into the collagen gel coated with VEGFp-DS-SILY, taken every day from days 10 to 13, will reflect the ability of the collagen scaffold to integrate into existing vasculature and 3D angiogenic potential of VEGFp-DS-SILY with or without VEGF. We expect that VEGFp-DS-SILY treatment will increase the number of vessels formed on the CAM. Preliminary data using a Fluoraldehyde assay indicates that loading of ~300 ng VEGF per mg of nanoparticles can be achieved. We expect that using an MMP-degradable peptide diacrylate crosslinker will allow nanoparticles to degrade in protease-rich environments like the chronic wound bed and release VEGF. Adjustments to the formulation, such as crosslinker density, may need to be modified to control the rate of VEGF release. DISCUSSION/SIGNIFICANCE OF IMPACT: We expect that our angiogenic decorin mimetic will lead to a novel treatment to accelerate healing of ischemic diabetic foot ulcers, thereby reducing the need for limb amputation and mortality rate of diabetic patients. We anticipate that the diabetes research and regenerative medicine communities will (1) gain a platform for targeted delivery of growth factors, (2) understand the dependence of vascularization within 3D collagen constructs on VEGFp densities and VEGF receptor activation in controlling the degree of angiogenesis, and (3) gain the benefits of controlled angiogenesis in ischemic diabetic wound healing.

OBJECTIVES/GOALS: The purpose of this study is to compare the mental health of African Americans to Whites during 2019 and 2020 using the a National Survey on Drug Use and Mental Health (NSDUMH). METHODS/STUDY POPULATION: Secondary data analysis from the National Survey on Drug Use and Health. The data consisted of 55,772 observations, 3,090 variables. This study will consist of the United State adult data population from 2019 and 2020 using the National Survey on Drug Use and Mental Health (NSDUMH). RESULTS/ANTICIPATED RESULTS: The results of this research will be produced from the following analysis. The analysis will consist of a secondary data analysis from the National Survey on Drug Use and Mental Health (NSDUMH). The primary independent variable of interest is race. All the indicator (race, sex, insurance, etc). Dependent variable is the mental health of African Americans and Whites. This is the variable in the NSDUMH labeled as the Major Depressive Episodes (MDE). The data analysis will be conducted using univariate analysis describing the study population. Bivariate analysis will be performed using chi-square. Since our dependent variable will be dichotomous we will be using several logistic regressions. DISCUSSION/SIGNIFICANCE: Strengthen mental health and psychosocial support services as part of strengthening preparedness, response and resilience to COVID-19 and future public health emergencies. Also adopt the updated Comprehensive Mental Health Action Plan for the future.

OBJECTIVES/SPECIFIC AIMS: QuantiFERON-TB Gold In-Tube (QFT) conversion from negative to positive, is regarded as a marker of recent latent tuberculosis infection and may be predictive of incident active tuberculosis (TB) disease. However, it remains unclear how conversion is influenced by individual and environmental factors, including the infectiousness of the source case to whom the contact was exposed. We aimed to examine the effect of infectiousness of TB in the source case, as measured by presence of cavitation on chest X-ray, on the incidence of QFT conversion among close contacts of the pulmonary TB index case, after adjusting for potential confounding by contact and source case characteristics. METHODS/STUDY POPULATION: The Regional Prospective Observational Research for Tuberculosis (RePORT)-Brazil is an ongoing prospective cohort study that enrolls close contacts of culture-confirmed pulmonary TB patients and follows them for 24 months for development of active TB. Demographic, clinical, and diagnostic information are obtained at baseline and during follow-up at clinical visits and by telephone. QFT testing is performed at baseline and repeated after 6 months if the baseline QFT is negative. A positive IFN-γ value is defined as >0.35 IU/mL, as recommended by the manufacturer and the CDC, and QFT conversion is defined as a negative QFT at baseline followed by a positive QFT at 6 months. RESULTS/ANTICIPATED RESULTS: Among 260 enrolled contacts with nonpositive baseline QFT results and 6 months of follow-up, 198 (76%) were retested with QFT 6 months after enrollment. Of those retested, 26 (13%) converted to positive. Presence of any cavitation in the source case, based on chest radiography, was significantly associated with QFT-conversion (ORunadjusted=2.4, 95% CI: 1.0–5.7). Additional univariate analyses revealed that QFT conversion was associated with black and brown race (compared with white race) of the contact, current smoking and current alcohol use in the source case. After adjusting for potential confounders (age, sex, and race of the contact and current smoking of the source case), the association between source case cavitation and QFT conversion remained (ORadjusted=2.5 95% CI: 1.0–6.2). As of December 6, 2017, none of the QFT-retested contacts had developed active TB, with a median follow-up of 12.3 months (IQR: 7.1–13.1). We anticipate that ongoing enrollment and follow-up may yield cases of active TB; future analyses will provide greater precision for examining predictors of QFT-conversion and its association with incident TB. DISCUSSION/SIGNIFICANCE OF IMPACT: Our preliminary results agree with published literature suggesting the infectiousness of TB in the index case is a predictor of incident LTBI. Along with recent LTBI, immune suppression, HIV co-infection, and type 2 diabetes are considered risk factors for progression to active TB disease. Because only a small proportion of persons progress from LTBI to active TB disease, it is not appropriate to treat all persons with LTBI. Thus, more research is needed to identify groups at highest risk for QFT-conversion and incident TB disease, so these groups can be targeted for TB prevention, interventions, and facilitate a decline in TB incidence and mortality.

OBJECTIVES/GOALS: In the United States, oropharyngeal cancer (OPC) is the leading human papilloma-virus (HPV) related malignancy, and OPC rates are increasing among the US veteran population. The purpose of this study is to identify demographic and regional factors that may be associated with low HPV-vaccination rates among the US veteran population. METHODS/STUDY POPULATION: This study will use Veterans Health Administration (VHA) administrative data to create a national cross-sectional cohort of veterans ages 18-45 with a VHA primary care visit from 2018-2020. HPV-vaccination status of each subject (initiation of vaccination series, completion of vaccination regimen, and age vaccinated) will be determined. Differences in the prevalence of HPV-vaccination by smoking status, geographic location, socioeconomic status, race/ethnicity, and rural-urban context will be examined in the unadjusted analysis. Factors associated with low HPV-vaccination rates in the VHA will be identified using multivariable logistic regression to model no (vs any) HPV-vaccination, no completion (vs completion) of HPV-vaccination recommendations, and non-routine (vs routine) HPV-vaccination: RESULTS/ANTICIPATED RESULTS: In 2019, HPV-vaccination guidelines were expanded to include some adults between the ages of 26-45, making many young veterans in the VHA eligible for vaccination. From this study’s recently generated dataset, more than 1.2 million subjects (n=1,219,896) met the study inclusion criteria. Extrapolating from trends in the civilian population, it is anticipated that HPV-vaccination rates will be lower among African Americans compared to non-Hispanic Caucasian Americans, within the South Central and Southeastern regions of the US, and in rural communities. This study will define a veteran’s geographic location by their associated Veteran Affairs Integrated Service Network (or VISN), which are groups responsible for healthcare planning and resource allocation in particular regions of the US: DISCUSSION/SIGNIFICANCE: Identifying factors associated with low HPV-vaccination rates within the VHA will be the first step to reducing future incidence of HPV-related cancer burden among US veterans. Assessing geographic variability in vaccination rates by VISNs will create the potential to generate targeted interventions within an existing VHA framework.

OBJECTIVES/GOALS: ICD-10 coding inconsistencies hinder timely recognition and treatment of metabolic syndrome (MetS), posing a significant risk for cardiometabolic disease progression. This study employed a digital phenotype for MetS and compared odds for medication and lifestyle intervention compared to those coded for MetS. METHODS/STUDY POPULATION: MetS is a cluster of cardiometabolic risk factors that increase risk for numerous adverse clinical outcomes. Patients with MetS were identified through electronic medical records on TriNetX LLC using the standard ICD-10 code or through a digital phenotype, involving grouping codes for the individual components. Percentage of patients with MetS not captured with the standard code was identified. In addition, disparities in blood pressure, glucose, lipid-lowering medication, and lifestyle intervention between the coding schemas were assessed, shedding light on healthcare inequities and informing targeted interventions. Odds ratios (RR) were presented for all outcomes. RESULTS/ANTICIPATED RESULTS: Patient demographics and lab values were similar between the standard code and digital phenotype cohorts. Of the 4.3 million individuals aged 50 to 80 identified as having MetS using the digital phenotype in the TriNetX research network, only 1.78% of participants shared the standard code. Individuals with the digital phenotype for MetS were at lower odds in receiving glucose lowering medication (OR: 2.11, 95% CI: 1.98–2.13, p <0.001) and exercise or nutrition-based intervention advice (OR: 1.76, 95% CI: 1.55–1.96, p <0.001) after controlling for demographics and lab values for each MetS component. DISCUSSION/SIGNIFICANCE: This project utilized TriNetX to create a digital phenotype for MetS, and suggests most patients are not coded for it using the standard ICD-10 system. This is troublesome given those with the standard code are less likely to receive certain interventions.

OBJECTIVES/SPECIFIC AIMS: Alcohol consumption perturbs the normal intestinal microbial communities (alcohol dysbiosis). To begin to investigate the relationship between alcohol-mediated dysbiosis and host defense we developed an alcohol dysbiosis fecal adoptive transfer model, which allows us to isolate the host immune response to a pathogenic challenge at a distal organ (ie, the lung). This model system allowed us to determine whether the host immune responses to Klebsiella pneumoniae are altered by ethanol-associated dysbiosis, independent of alcohol use. We hypothesized that alcohol-induced changes in intestinal microbial communities would impair pulmonary host defenses against K. pneumoniae. METHODS/STUDY POPULATION: Mice were treated with a cocktail of antibiotics daily for 2 weeks. Microbiota-depleted mice were then recolonized by gavage for 3-days with intestinal microbiota from ethanol-fed or pair-fed animals. Following recolonization groups of mice were sacrificed prior to and 48 hours post respiratory infection with K. pneumoniae. We then assessed susceptibility to Klebsiella infection by determining colony counts for pathogen burden in the lungs. We also determined lung and intestinal immunology, intestinal permeability, as well as, liver damage and inflammation. RESULTS/ANTICIPATED RESULTS: We found that increased susceptibility to K. pneumoniae is, in part, mediated by the intestinal microbiota, as animals recolonized with an alcohol-induced dysbiotic intestinal microbial community have significantly higher lung burdens of K. pneumoniae (5×104 CFU vs. 1×103 CFU) independent of EtOH. We also found that increased susceptibility in alcohol-dysbiosis recolonized animals was associated with a decrease in the recruitment and/or proliferation of CD4+ and CD8+ T-cells (1.5×109 cells vs. 2.5×109 cells) in the lung following Klebsiella infection. However, there were increased numbers of T-cells in the intestinal tract following Klebsiella infection, which may suggest that T cells are being sequestered in the intestinal tract to the detriment of host defense in the lung. Interestingly, mice recolonized with an alcohol-dysbiotic microbiota had increased intestinal permeability as measured by increased levels of serum intestinal fatty acid binding protein (55 vs. 30 ng/mL). Alcohol-dysbiotic microbiota also increased liver steatosis (Oil Red-O staining) and liver inflammation (>2-fold expression of IL-17 and IL-23). DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest that the commensal intestinal microbiota support mucosal host defenses against infectious agents by facilitating normal immune responses to pulmonary pathogens. Our data also suggest that increased intestinal permeability coupled with increased liver inflammation may impair the recruitment/proliferation of immune cells in the respiratory tract following infection. The role of the microbiota during host defense will be important areas of future research directed at understanding the effects of microbial dysbiosis in patients with AUDs.

OBJECTIVES/SPECIFIC AIMS: The purpose of this research was to investigate how chronic inflammation promotes the generation of proinflammatory intestinal macrophages and if macrophages contribute to intestinal inflammation through Notch activation. METHODS/STUDY POPULATION: We utilized two animal models of chronic colitis, the chronic DSS-induced colitis mouse model and the spontaneous enterocolitis development in IL-10-deficient mice to investigate the role of chronic inflammation in the generation of proinflammatory intestinal macrophages and its influence in notch signaling. Bone marrow-derived monocytes were collected from each group and differentiated into macrophages (BMM) for gene and protein analysis. Ex vivo phenotypical and functional analysis of colonic macrophages was assessed as was the presence of goblet cells and mucosal T cells. In addition, we analyzed the development of goblet cell differentiation in colonoids in a co-culture system with proinflammatory macrophages. RESULTS/ANTICIPATED RESULTS: Our chronic inflammation models revealed an increase in proinflammatory macrophages present in the lamina propria and that these cells expressed significantly higher levels of notch ligand, Jagged1. Jagged 1 has been shown to enhance TH1 differentiation and T cells isolated from the mucosa of both chronic colitis models display strong TH1 skewing compared to controls. Chronic inflammation also contributes to intestinal barrier defects, enhanced permeability and bacterial translocation. We believe this enhanced intestinal permeability and subsequent bacterial translocation promote Jagged1 expression in intestinal macrophages. To support this concept, we show TLR stimulation induces the upregulation of Jagged1 in BMM. Additionally, the generation of BMM from our chronic DSS-induced colitis mice or age matched controls, revealed BMM derived from a host of chronic inflammation were skewed to a proinflammatory state prior to stimulation showing increased gene expression of several proinflammatory molecules including IL-1α, IL-1β, IL-12 and TNF-α. This would suggest monocytes migrating to the intestinal mucosa have more potential to become proinflammatory instead of traditional anti-inflammatory macrophages. Furthermore, proinflammatory notch ligand-positive macrophages co-cultured with colonoids, derived from unperturbed mice, significantly decreased the number of mucus producing goblet cells. In support of this observation, notch activation in intestinal stem cells promote absorptive (i.e. colonocytes) cell differentiation and prevents secretory cell (i.e. goblet cells) differentiation. DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, our results strongly suggest chronic inflammation modulates macrophages role in maintaining intestinal homeostasis through possible notch activation in both T cells and the intestinal epithelial barrier.

ABSTRACT IMPACT: Characterizing the cellular composition of human adipose tissue may contribute to the prevention and/or treatment of obesity-associated metabolic diseases. OBJECTIVES/GOALS: Our aims in this study were to use single-cell techniques 1. to characterize cell types within the stromavascular fraction of human adipose tissue, 2. to identify subsets of cells within each type (sub-clustering), 3. to identify gene sets and pathways that may provide information on the function and significance of each cell cluster. METHODS/STUDY POPULATION: Abdominal subcutaneous adipose tissue samples from n=6 healthy volunteers (1M, 5F, age 28-38 y, BMI 24.5-63.0 kg/m2) were collected by aspiration or during surgery. In 3 subjects, all females, paired femoral samples were also collected. After collagenase digestion approximately n=10,000 cells/sample were used for single-cell RNA sequencing using the 10X Genomics platform. After QC and downstream analysis, data were analyzed in Seurat v.3.1.5. We identified first different cell types and then subclusters in an unbiased fashion. Gene Set Enrichment Analysis (GSEA) was used for pathway analysis. RESULTS/ANTICIPATED RESULTS: Progenitor markers are related to extracellular matrix, eg DCN (logFC progenitors vs other cells types=3.17, expressed in 99.7% (pct.1) of progenitors, 26.1% (pct.2) of others). Endothelial and pericytes shared markers like RBP7 (logFC=1.67, pct.1 0.88, pct.2 0.39); pericytes also showed unique markers, eg RGS5 (logFC=2.29, pct.1 0.89, pct.2 0.17). Progenitors are further divided into 11 sub-clusters, one of which showed enrichment of CD36 (high proliferation potential), FABP4 (differentiation), and of the novel marker PALMD (logFC=7.13, pct.1 0.94, pct.2 0.48). All p<10E-5. GSEA analysis suggests that inflammatory pathways are downregulated in both adipose progenitors and endothelial/pericyte cells in the femoral compared to the abdominal depot. DISCUSSION/SIGNIFICANCE OF FINDINGS: Single cell RNA sequencing provides unique insights into the molecular profile of cell types and the identification of novel subsets of cells within the human adipose tissue. Such cellular heterogeneity may explain differences in adipose function between individuals and eventually in the risk of obesity-associated metabolic diseases.

OBJECTIVES/GOALS: Preeclampsia (PE) is a hypertensive disorder of pregnancy, affecting 5 - 7% of pregnancies worldwide. A major cause of morbidity and mortality, PE is also associated with subsequent adverse health outcomes, including long-term increased risk of cardiovascular disease. The genetics conferring increased risk for PE are incompletely understood. METHODS/STUDY POPULATION: We performed a cross-ancestry, fixed-effects meta-analysis, incorporating both published and unpublished genome-wide association study (GWAS) summary statistics. In addition to publicly available summary statistics from two prior studies, we generated GWAS data from three electronic health record biobanks (BioVU, eMERGE, and PMBB). In total, we utilized data from 359,378 individuals (4,411 cases and 354,967 controls). Leveraging this large-scale biobank data importantly allows for detection of complex factors contributing to the diverse etiology of PE. Cases across cohorts were defined using PE-specific ICD-9/ICD-10 codes and phecodes. Cohorts included pregnant individuals of self-identified non-Hispanic Black, non-Hispanic White, and East Asian ancestry. RESULTS/ANTICIPATED RESULTS: 2 of 20,204,625 loci achieved genome-wide significance (p < 5 × 10–8) when minor allele frequency was limited to common variants (>0.01). The most significant locus was rs138180605 (p = 1.77 × 10–8), located in an intergenic region between FGFR2 and ATE1, both previously associated with breast cancer. The other significant locus was rs137895377 (p = 2.33 × 10–8), located in an intronic region of PLEKHO1. Another 225 loci achieved suggestive significance (p < 1 × 10–5). 203 loci could be mapped to 109 unique genes, some previously associated with related phenotypes such as hypertension. Next steps will focus on functional analyses, including genetically predicted gene expression incorporating placental tissue, followed by construction of a PE polygenic risk score to demonstrate predictive utility of results. DISCUSSION/SIGNIFICANCE: This work has contributed to the limited body of knowledge surrounding maternal genetic susceptibility to PE by identifying several loci warranting further investigation. Further work will expand on these results to improve understanding of genetic factors and clarify clinical risk of disease.

OBJECTIVES/GOALS: HIV-specific CD8+ T-cells play a critical role in partially controlling viral replication in infected-individuals, but ultimately fail to eliminate infection. Enhancing these T-cell responses through lymphocyte engineering approaches has the potential as a novel therapy capable of achieving durable control or eradication of infection. METHODS/STUDY POPULATION: IL-15 Superagonist (IL-15SA) potently supports the in vivo persistence and antiviral activity of adoptively transferred CD8+ T-cells. The Deep-PrimingTM technology platform, developed by Torque, allows for loading of immunomodulators onto the surface of T-cells via electrostatic ‘nanogels’, which slowly release to deliver sustained autocrine immune stimulation without the harmful effects of systemic exposure. Here, we investigate the impact of IL-15SA Deep-Priming on HIV-specific CD8+ T-cells in a humanized mouse model of HIV infection. Humanized mice were generated by engrafting NOD-scid-IL2Rgnull mice with memory CD4+ T-cells isolated from an ARV-suppressed HIV+ donor. An autologous HIV-specific Cytotoxic T-Lymphocyte (CTL) clone was isolated, and killing potential confirmed. Four weeks post humanization, mice were infected with HIV and received an infusion of unmodified HIV-Specific CTLs, or IL-15SA Deep-Primed HIV-specific CTLs (CTL-DP). T-cell numbers and plasma viral loads were quantified weekly by flow cytometry and qRT-PCR. RESULTS/ANTICIPATED RESULTS: Mice receiving unmodified CTLs trended toward reduced viral loads compared to the No Treatment condition, while mice receiving CTL-DP saw significant, 2-Log10 reductions in VL (p < 0.01). At 41 days post-infection 100% (5/5) of the No Treatment, 66.7% (4/6) of the CTL treatment, and 16.7% (1/6) of CTL-DP treatment mice had detectable viremia. IL-15SA Deep-Priming increased CTL expansion and persistence in peripheral blood which correlated with improved CD4+T-cell preservation. DISCUSSION/SIGNIFICANCE OF IMPACT: Here we demonstrate the first in vivo analysis of IL-15SA Deep-Priming of HIV-Specific CTLs. These data suggest that Deep-Priming of patient T-cells can enhance in vivo function and persistence, leading to improved viral suppression; a significant advancement in the field of HIV cure research. CONFLICT OF INTEREST DESCRIPTION: Austin Boesch, Thomas Andresen, and Douglas Jones are employees of Torque. Darrell Irvine is a co-founder of Torque and Chairman of Torque’s Scientific Advisory Board.

We analyzed the publication productivity supported by the Puerto Rico Consortium for Clinical and Translational Research (PRCTRC) using the structured process of scientometrics. The objective of this study was to evaluate the impact of the research and collaborations as presented in publications. Manuscripts published from 2010 to 2018 and that had the PRCTRC award number and a PMCID number were retrieved from the Science Citation Index database. Scientometric indicators included h-index (HI), average citation (AC), collaboration coefficient (CC), collaboration index (CI), and degree of collaboration (DC) analysis, and relative citation ratio (RCR) was done with Web of Science Platform, iCite, and Stata software. Joinpoint Trend Analysis Software was used to calculate the annual percent change (APC). From 2010 to 2018, 341 publications were identified with an average of 38 publications per year and a total of 3569 citations excluding self-citations. A significant growth (APC: 17.76%, P < 0.05) of scientific production was observed. The overall HI was 31, and the AC per item was 11.04. The overall CC was 0.82, the CI was 8.59, and the DC was 99.1%. This study demonstrates a statistically significant increase in the PRCTRC scientific production. Results allow for the assessment of the progress resulting from the provided support and to plan further strategies accordingly.

Expanded access (EA) provides a pathway for the clinical use of investigational products (drugs, biologics, and medical devices) for patients who are without satisfactory therapeutic options and for whom a clinical trial is not available. Academic medical centers (AMCs) are likely to encounter EA requests, but it is unknown what support is available at these institutions for physicians seeking EA for patients. Methods: A landscape assessment was conducted at AMCs, focused on those within the Clinical and Translational Science Awards (CTSA) consortium. Results: Forty-seven responses were evaluated including 42 CTSA hubs. The large majority (43 of 47 respondents) reported using single-patient EA, while 37 reported multi-patient industry sponsored EA and 37 reported multi-patient investigator-initiated EA. Only half reported central tracking of EA requests. Support was available at 89% of sites for single-patient EA but less often for multi-patient EA. Types of support varied and were focused largely on the initial submission to the FDA. Conclusion: Use of and support for EA is widespread at AMCs, with support focused on single-patient requests. Gaps in support are common for activities after initial submission, such as FDA reporting and data collection.

OBJECTIVES/SPECIFIC AIMS: To identify cardiac structural and function parameters, obtained on usual stroke-care TTE evaluation, associated with cardioembolic stroke (CE) in patients without AF. Hypothesis—left atrial (LA) size and valve dysfunction will be strongly associated with incident CE. METHODS/STUDY POPULATION: Inclusion criteria: July 1, 2013 to July 1, 2015 admission with imaging-confirmed ischemic stroke, no AF, TTE within 1st 7 days. TTE structure/function parameters were recorded. Stroke subtype (CE vs. other) defined using TOAST criteria, blinded to TTE. New AF definition: AF on ECG, telemetry or event monitor. CE/New AF outcome of interest in separate multivariable logistic regression models testing associations with TTE parameters (adjusting for demographics/vascular risk factors). RESULTS/ANTICIPATED RESULTS: Participants (n=332) were ~60 years hypertensive black males with moderate NIHSS and normal ejection fraction. In adjusted models, odds of CE increased with increasing LA systolic diameter (per 0.1 cm), mitral E point velocity(cm/s), mitral valve dysfunction, wall motion abnormality. New AF also associated with increasing LA systolic diameter. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings may suggest cardiac structural changes independent of AF that are on the CE causal pathway. Understanding the relationship between such TTE parameters and stroke subtype would impact clinical practice, as such TTE data is underutilized when considering stroke mechanism and management.

OBJECTIVES/GOALS: The goal of this study is to leverage a national database to see if autologous reconstruction rates differ in patient and clinical characteristics, readmission rates, and overall survival (OS) compared to other forms of reconstruction. Autologous reconstruction has not been looked at in this way before. METHODS/STUDY POPULATION: • Aim 1: Use the National Cancer Data Base to construct three patient cohorts for women under 70 and above 18 treated surgically for breast cancer with A) mastectomy only, B) implant-based reconstruction, and C) autologous breast reconstruction. • Aim 2: Examine receipt rates of surgical intervention in Cohorts A vs. B vs. C based on clinical and patient demographic/socioeconomic characteristics. • Aim 3: Compare readmission and overall survival (OS) rates for Cohorts A vs. B vs. C while controlling for age and other key variables. RESULTS/ANTICIPATED RESULTS: Based on the literature, we expect rates of autologous reconstruction (Cohort C) to be lower for patients of minority backgrounds compared to white individuals. In addition, we do not expect overall survival to differ between implant-based (Cohort B) and Cohort C reconstruction. Still, we expect mastectomy-only (Cohort A) survival to vary from the two cohorts even when adjusting for different clinical factors, as similar but smaller studies have shown. Finally, we expect readmission rates to be higher for Cohort C, compared to Cohorts A & B, as it is a more complicated procedure typically done in academic institutions with skilled surgeons. DISCUSSION/SIGNIFICANCE: Autologous reconstruction is now considered the gold standard due to its ability to restore the breast shape with higher patient satisfaction and superior long-term outcomes. Multiple studies have documented ongoing racial disparities in post-mastectomy breast reconstruction and autologous reconstruction, with lower rates and referrals.

OBJECTIVES/GOALS: A UCLA Clinical and Translational Science Institute (CTSI) science team partnered with the second largest US school district, with over 500,000 K-12 students, to design and implement a statistical process control dashboard to guide COVID-19 response, including mitigation and vaccination outreach. METHODS/STUDY POPULATION: District data for students, teachers, and staff are updated daily and include COVID-19 test results, counts of quarantine after positive tests, and COVID-19 vaccination rates. Displays used a new hybrid Shewhart control chart to detect changes in test positivity rates and distinguish meaningful signals from noise (random day-to-day variation). The dashboard uses the Shiny and plotly packages in R to display interactive graphs of each data stream (cases, tests, and vaccinations) charted at multiple levels (districtwide, subdistricts, schools). Displays of variation over time show policy impacts and inequities. Selected displays use municipal COVID-19 data to complement district data. RESULTS/ANTICIPATED RESULTS: The district has used the displays to assess the impact of their COVID-19 response and to identify variation in close to real-time to suggest areas with need for additional resources for mitigation or vaccination. The CTSI team has continued to edit and add displays in response to the district’s changing operational needs and questions. DISCUSSION/SIGNIFICANCE: The UCLA CTSI team developed and implemented a robust data visualization dashboard to monitor COVID-19 case rates and plan vaccination outreach efforts. Control charts enabled the district to distinguish noise from signal, thereby rapidly identifying when specific parts of the district needed targeted support to achieve equity goals.

OBJECTIVES/SPECIFIC AIMS: Hematopoietic stem and progenitor cells (HSPCs) function to maintain steady state production of new blood cells and to rapidly respond to blood cell loss. Little is known regarding how HSPCs develop the ability to sense and respond to blood cell loss during embryogenesis. Gaining insight into the robust ability of HSPCs to regenerate blood during early development may allow us to develop therapies to rejuvenate this capacity at any stage. METHODS/STUDY POPULATION: We generated a new hematopoietic-specific and inducible cell ablation zebrafish model to uncover the origins of regenerative capacity in HSPCs during development. These transgenic zebrafish express a cyan fluorescent protein (CFP)-nitroreductase (NTR) fusion construct under the control of the draculin (drl) promoter (drl:CFP-NTR), which restricts NTR expression to blood cells. Co-expression analyses of drl:CFP-NTR with known markers of other blood types including HSPCs (runx1+23:mCherry), erythroid cells (gata1:dsRed), and lymphoid cells (rag2:RFP), revealed drl:CFP-NTR was restricted to HSPCs and erythrocytes. To delineate the regeneration potential of embryonic HSPCs, we exposed drl:CFP-NTR transgenic zebrafish embryos to Metronidazole (MTZ), which results in selective ablation of only NTR-expressing blood cells. Embryos were treated from 2 to 3 days postfertilization and recovery of drl+ and gata1+ cells was evaluated over a 7-day recovery period. RESULTS/ANTICIPATED RESULTS: Following MTZ exposure, the nadir of drl+ cell ablation occurs at 2 days post MTZ (dpM) treatment. During the renewal phase of blood regeneration, we first observe recovery of drl+ cells by about 4 dpM, while more mature blood cells like gata1+ erythrocytes show a delayed recovery at about 6 dpM. Our results suggest that HSPCs can respond to injury as early as 2 days of life and that the HSPC-driven regeneration of embryonic blood cells occurs in a hierarchical fashion, similar to regeneration of the adult blood system. DISCUSSION/SIGNIFICANCE OF IMPACT: We have established a quantitative method for in vivo real-time monitoring of embryonic and larval blood regeneration. A significant advantage of our system is that we can use these insights to guide an in-vivo drug screen for factors that accelerate HSPC-driven blood regeneration in a complex biological environment.

OBJECTIVES/GOALS: For shoulder osteoarthritis (OA), the understanding of the patient-specific factors that determine success of both non-operative and operative treatment options is limited. This study aims to identify key factors associated with the response and the heterogeneity of outcomes for both types of treatment. METHODS/STUDY POPULATION: Patients diagnosed with shoulder OA and treated with either reverse/anatomic total shoulder arthroplasty (rTSA/TSA) or non-operative management at the University of California, San Francisco were enrolled in this study. They were followed for a year to ascertain phenotypic traits and patient-reported outcomes (PROs). Magnetic Resonance Imaging (MRI) was used to calculate the Shoulder Osteoarthritis Severity (SOAS) score, a semi-quantitative global assessment of shoulder OA, and to measure fat fractions of rotator cuff muscles. A Microsoft Kinect camera was used to determine the Reachable Workspace (RWS). Linear regression models were used to assess the associations between baseline demographic and radiographic factors on outcomes related to shoulder function. RESULTS/ANTICIPATED RESULTS: It is anticipated that the pre-operative MRI-based SOAS score will be inversely correlated with the magnitude of improvement in PROs 1 year after rTSA/TSA and non-operative management of shoulder OA for the surgical replacement and non-operative cohorts, respectively. Additionally, the non-operative patients who convert to rTSA/TSA within 1 year of observation will have higher SOAS scores compared to patients who continue with non-operative management. The surgical replacement patients with an infraspinatus fat fraction of more than 5% will have worse shoulder function, as measured by RWS, compared to patients with an infraspinatus fraction less than 5%. DISCUSSION/SIGNIFICANCE: MRI may be a novel technique to better predict prognosis of shoulder OA management. This will allow for the development of appropriate algorithms in the prescription of treatments and may be used to counsel patients regarding their expected outcomes or to recommend alternative treatments.