OBJECTIVES/GOALS: Septal cholinergic innervation to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer’s disease. To understand the molecular events underlying this loss, we optimized a primary septal-hippocampal co-culture system that facilitates study of central cholinergic synapses. METHODS/STUDY POPULATION: We developed an optimized in vitro septal-hippocampal co-culture system modified from previous published protocols. Briefly, hippocampal and septal tissue were harvested from embryonic day 19 (E19) Sprague-Dawley rats, digested with 0.1% trypsin, and an equal number of cells from each region plated onto coverslips coated with poly-D-lysine and laminin at a final density of 300 cells/mm2. We use immunostaining with validated primary antibodies and a fluorescent binding assay, together with confocal microscopy, to determine the structure of cholinergic synapses that are 1) native, 2) mammalian, 3) CNS derived, 4) comprised of physiological synaptic partners, and 5) developmentally mature. RESULTS/ANTICIPATED RESULTS: After DIV21, co-cultures maintained a healthy morphology. A subpopulation of neurons strongly expressed the cholinergic markers vesicular ACh transporter (vAChT), choline acetyltransferase (ChAT), and the high-affinity choline transporter (ChT1), whereas most neurons lacked vAChT expression and were presumably glutamatergic or GABAergic. The percentage of cholinergic neurons in the co-culture attained up to ~5-7%, depending on conditions such as embryo age at dissection or ratio of septal to hippocampal cells. We also report on cholinergic synapse structure by examining postsynaptic markers (excitatory and inhibitory) and staining for nicotinic acetylcholine receptor subunits. DISCUSSION/SIGNIFICANCE OF IMPACT: Primary septal-hippocampal co-cultured neurons have not been exploited extensively in the field, perhaps due to the difficulty in maintaining such cultures for extended periods. Here, we optimized an in vitro septal-hippocampal co-culture system, a powerful tool to comprehensively analyze central cholinergic synapse formation and dysfunction.

OBJECTIVES/SPECIFIC AIMS: Angiotensin type 1 receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) are frequently prescribed for hypertension and associated cardiovascular and renal complications. In animal models, these drugs also reduce anxiety and depression. OBJECTIVE—to determine if Veteran Affairs (VA) clinical pharmacy data indicate a protective effect of ARBs and/or ACEIs for major depression. METHODS/STUDY POPULATION: Pharmacy records from nationwide VA electronic medical records (EMR) were extracted for patients prescribed ARBs, ACEIs, α-blockers, β-blockers, calcium channel blockers, or diuretics (n=4,081,359). Patients were excluded if: they had not received medications for 6 months with >70% coverage; were diagnosed with substance/alcohol abuse, dementia, psychosis, schizophrenia, or prescribed insulin. The study population was categorized as “ARB/ACEI” (A/A) or “Never ARB/ACEI” (NA/A). Using the Greedy Matching Algorithm, subjects were matched on a 1:1 ratio for sex and race over a 5 year age range resulting in 2 equal groups of n=1,350,236 each. Subjects were older (M=71.6, SD=12) and mostly men (97%). RESULTS/ANTICIPATED RESULTS: In the A/A Versus NA/A, respectively, the incidence of anti-depressant use was greater during (9.9% vs. 8.9%) but was lower after (11.8% vs. 12.2%) the study period. PHQ-2 scores (Mean±SD) were statistically lower, albeit similar, during (0.79±1.56 vs. 0.85±1.63) and after (1.00±1.73 vs. 1.07±1.79) the study period. DISCUSSION/SIGNIFICANCE OF IMPACT: These preliminary data suggest that inhibiting angiotensin II action does not provide a protective effect on major depression when compared with other classes of antihypertensive drugs. This study illustrates how “Big Data” may inform the design, or obviate the need, for large-scale randomized-controlled trials.

OBJECTIVES/SPECIFIC AIMS: The goal of this study is to evaluate a potential strategy to overcome tamoxifen (tam) resistance by using tam in combination with high-intensity focused ultrasound (HIFU). Tam is the most commonly used anti-cancer therapeutic agent in estrogen receptor positive (ER+) breast cancer (BC) which accounts for ~70% of BC cases. Tam treatment decreases a woman’s risk of recurrence by 50%; however, BC that is initially responsive to tam often develops resistance. METHODS/STUDY POPULATION: HIFU deposits acoustic energy locally to a cancerous region, which induces strong vibrations of molecules inside and outside of the cells. The resulting absorption causes rapid heating and mechanical disruption. This clinically relevant, noninvasive, and nonionizing physical force modality, has been shown to synergistically enhance chemical anticancer therapies. RESULTS/ANTICIPATED RESULTS: In this study we found that treatment of MCF7 cells with HIFU and tam has additive antiproliferative effects and mediates increased cell death. Additionally, we used tam resistant (TR) MCF7 cells that had been exposed to low-dose tam over time until they acquired resistance. When MCF7 TR are treated with tam there is no change in viability; however, treatment with HIFU in combination with tam decreased viability of both MCF7 and MCF7 TR to 19% and the viability of the cell lines was indistinguishable. We next evaluated the effect on MCF7 Y537S mutant ESR1, where ER is mutated to be constitutively active. Treatment of MCF7 Y537S had no significant decrease in viability of combination therapy compared with viability after HIFU alone. Analysis of ERalpha gene expression showed that HIFU treatment increased ERalpha expression in MCF7 TR cells, thus resensitizing these cells to tam and allowing these therapies to work synergistically. Our team developed a system to evaluate the potential of this combination of therapies in a patient-derived xenografts (PDX) model. PDX have emerged as a novel translational tool for cancer research with the potential to more accurately recapitulate the molecular and behavioral aspects of cancer. The WHIM20 PDX is a tamoxifen resistant tumor where the patient developed the Y537S mutation in ESR1. Ex vivo experiments on PDX tumor pieces demonstrated that combination therapy of HIFU and tam work synergistically to increase cell death of these tumors. Further, cryogenic-scanning electron microscopy was utilized to directly demonstrate the physical disruption to both cellular and tumor microenvironment post exposure to combination treatment. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies present a novel translational strategy to overcome tamoxifen resistance in ER+BC.

OBJECTIVES/GOALS: Vascularized composite allotransplantation (VCA) restores devastating soft tissue injuries. However, graft viability may be compromised during ischemia time, thus preservation techniques continue to evolve. Here we summarize our preliminary findings from preservation techniques utilizing hyperosmolar extracellular solution (HES) and electric stimulation in a 6 hour ex-vivo perfusion model. METHODS/STUDY POPULATION: A published, MGH rodent hindlimb ex-vivo perfusion model was utilized for this project. Three baseline control elements were taken to compare our results including: a baseline muscle biopsy, harvested hindlimb preserved on ice(4°C) in static cold storage (SCS), and 6 hour perfusion (SNMP) were used to compare results of our four aims. The four aims are shown in Table 1. HES was composed of muscle media and the addition of mannitol until 3 concentrations were made: 300, 500, and 800 mOsm concentrations. In aim 4, the perfusate composition was changed to test a hyper-oncotic purfusate. After 6 hours a muscle biopsy was taken to analyze energy cofactors via liquid chromatography-mass spectrometry, referred to as energy charge. Weight gain (edema), lactate levels, oxygen consumption and energy charge (EC) were used as markers for muscle tissue viability. RESULTS/ANTICIPATED RESULTS: In Aim 1, the higher osmolarity of HES indirectly reduced weight gain but consequentially reduced the EC below 5% when compared to SCS control group. We next incorporate HES into the perfusion model, Aim 2, and noticed a diminution in weight gain. The 500 mOsm group had substantial improvement in EC, lactate production and improved oxygen exchange when compared to a controls: fresh muscle biopsy and SNMP. In Aim 3, after a 6 hour perfusion with the addition of electric stimulation, graft edema improved by 10%, EC improved by 23% and O2 dissociation was highest of all 4 aims. Consequentially, due to muscular contraction the lactate levels were highest. In Aim 4, using a hyper-oncotic perfusate, edema reduced the most during the 6 hour perfusion but revealed lower EC and similar lactate/O2 results. However when left on for 24 hours, edema was significantly higher with lactate build up, EC improved with time as well as O2 dissociation. DISCUSSION/SIGNIFICANCE OF IMPACT: Here we have shown our preliminary results comparing our known ex-vivo perfusion model to multiple hypothesis to improve VCA graft viability. These preservation techniques demonstrate promising results but further studies are ongoing to confirm this encouraging outcome.

OBJECTIVES/GOALS: We are launching a multi-center prospective registry for patients with metastatic invasive lobular carcinoma (ILC), the second most common type of breast cancer, to better understand patterns of progression, imaging features of metastatic sites, and if serial cell free DNA measurements can serve as a surrogate marker of disease progression. METHODS/STUDY POPULATION: Patients with biopsy proven metastatic ILC of any receptor subtype will be included in the registry. We will exclude patients with ductal histology only or those with multiple primary malignancies. Patients will be enrolled at four large academic medical centers across the country. Cell free DNA measurements using a tumor informed assay will be obtained every 3 months concurrent with regular clinical imaging. Disease status will be determined by the patient’s medical oncologist by taking into account imaging, tumor markers, symptoms, and cell free DNA measurement. At each time point, patients will be surveyed on their quality of life and their medical oncologists will be asked to rate the clinical utility of the cell free DNA value. Patients will be followed indefinitely. RESULTS/ANTICIPATED RESULTS: We will explore whether the use of serial cell free DNA or a combination of blood-based biomarkers and clinical endpoints can reliably identify treatment response and disease progression in patients with metastatic ILC. Many patients with metastatic ILC have unmeasurable disease on imaging and are thereby excluded from clinical trials. The end goal of this registry is to determine if blood-based biomarkers can be used as a proxy for measurable disease in ILC patients and therefore increase clinical trial enrollment for this subgroup of patients. DISCUSSION/SIGNIFICANCE: The creation of this prospective registry will open the door for future studies of blood-based markers that reflect disease stability and progression, which is an unmet need specifically in ILC. Identification of such markers could lead to a novel treatment response endpoint, changing the way patients are enrolled in trials and managed clinically.

OBJECTIVES/GOALS: During the pandemic, alcohol related deaths increased by 25%. To help understand how we might mitigate this negative outcome, we sought to examine the association of new diagnosis of alcohol use disorder (AUD) with SARS-CoV2 through two years of the pandemic. METHODS/STUDY POPULATION: Using a non-date-shifted TriNetX database, we conducted a retrospective cohort analysis of electronic health records of patients age ≥12 years who had been diagnosed either with COVID-19 (n=1,359,817) or other respiratory infections with no record of COVID-19 (n=2,013,031). Patients were then matched for propensity score risk for AUD, and results were analyzed by three-month intervals from January 2020 through January 2022, in blocks numbered 1-8. Results were expressed as hazard ratios (HR) and 95% confidence intervals (CI) for diagnosis of AUD from two weeks to six months following COVID-19 diagnosis. RESULTS/ANTICIPATED RESULTS: There was significant excess risk compared to control cohorts of AUD following COVID-19 diagnoses made during the first three months of the pandemic (HR (CI)): block 1: 2.41(1.89,3.08); no excess risk was seen for the remainder of 2020 (blocks 2-4) (HR1.01-1.14, NS). The excess risk increased again in 2021 as the delta and omicron variants emerged (HR and 95% CI): block 5 were: 1.26(1.11, 1.43)); block 6: 1.88(1.62-2.18)); block 7: 1.24(1.10,1.41); block 8: 1.12(1.0-1.25). COVID-19 diagnosis was associated with clinically-evident AUD under some pandemic circumstances. DISCUSSION/SIGNIFICANCE: COVID-19 early in the pandemic (block 1) was associated with substantial excess risk for new diagnosis of AUD, with smaller excess risk after COVID-19 during 2021 (blocks 5-7), and no excess risk otherwise. Diagnosis of COVID-19 and pandemic contextual factors are associated with increased risk for AUD.

OBJECTIVES/GOALS: There is an excess risk of upper respiratory infection (URI) among Black and Mexican-American children in the US. Factors that underpin these disparities are largely unknown. We evaluated the extent to which socioeconomic status (SES), serum cotinine, obesity, and household size explained the association between race/ethnicity and URI. METHODS/STUDY POPULATION: We studied children, 6-17 years of age, who identified as Black, Mexican-American, or White in the National Health and Nutritional Examination Survey (2007-2012). URI was defined as a self-reported cough, cold, phlegm, runny nose, or other respiratory illness (excluding hay fever and allergies) in the past 7 days. The proportion of the association between race/ethnicity and URI explained by SES, serum cotinine, obesity, and household size was estimated as the average causal mediation effect (i.e., the indirect effect of race/ethnicity via the mediator) divided by the total effect of race/ethnicity. The average causal mediation effect was derived from survey weighted logistic regression models adjusted for age and sex. RESULTS/ANTICIPATED RESULTS: Black children were nearly 40% and Mexican American children were ~60% more likely to report a URI than those who identified as White (OR, 1.37; 95% CI, 1.06-1.77 and OR, 1.61; 95% CI, 1.21-2.13, respectively). Lower SES explained ~25% of the association between Black and Mexican American identity and URI (percent mediated 24.7; 95% CI, 23.0-26.6 and 26.1; 95% CI, 24.2-28.2, respectively). Obesity explained ~7% of the association between Black and Mexican-American identity and URI (percent mediated, 7.6; 95% CI, 7.3-8.0 and percent mediated, 6.7; 95% CI, 6.4-6.9, respectively). Nicotine exposure explained 8% of the association between Black identity and URI (percent mediated, 7.9; 95% CI, 5.6-10.1). DISCUSSION/SIGNIFICANCE: Lower SES explained a quarter of the association between race/ethnicity and URI. Low SES is a broad concept that may work through different mechanisms to lead to disparities in URI by race/ethnicity. Future research is needed to better understand these mechanisms and to identify modifiable aspects that can serve as targets for intervention.

OBJECTIVES/SPECIFIC AIMS: While the role of antiphospholipid antibodies in activating endothelial cells has been extensively studied, the impact of these antibodies on the adhesive potential of leukocytes has received considerably less attention. Mac-1 is a heterodimeric beta-2 integrin primarily expressed by myeloid-lineage cells. In its activated state, Mac-1 mediates cell-cell interactions by engaging a variety of surface molecules, including the endothelium-expressed glycoprotein ICAM-1. Here, our goals were (1) to determine the extent to which APS neutrophils adhere to healthy, resting endothelial cells under physiologic flow conditions, and (2) to identify potential therapeutic targets by elucidating the molecules required for that adhesion. METHODS/STUDY POPULATION: Primary APS patients (meeting Sydney criteria) and non-autoimmune controls were matched for age and gender. Freshly isolated human umbilical vein endothelial cells (HUVECs) were utilized within five passages. Samples were introduced into a flow channel via a programmable syringe pump, and perfused across a resting HUVEC monolayer. After 15 minutes of perfusion, the chamber was flushed, and the remaining adherent cells were quantified. Flow cytometry was used to identify differentially-expressed molecules on the surface of APS neutrophils. Neutrophil extracellular trap (NET) release was assessed in static neutrophil-HUVEC cultures. RESULTS/ANTICIPATED RESULTS: Pre-treating control neutrophils with APS plasma resulted in increased adhesion as compared with control plasma (>2.5-fold for n = 12 plasma samples; p < 0.05). This was true under both venous conditions (low shear) and conditions representative of the microvasculature (pulsatile flow and higher shear). Control neutrophils treated with APS plasma demonstrated upregulation of CD64, CEACAM-1, beta-2 glycoprotein I, and activated Mac-1 on the neutrophil surface, as well as shedding of L-selectin. Upregulation of activated Mac-1 and shedding of L-selectin were also triggered by IgG purified from APS plasma. For these changes to be meaningful clinically, we reasoned that they should be present on neutrophils in the peripheral blood of APS patients. Indeed, perfusion of anticoagulated blood through the flow chamber resulted in increased adhesion of patient neutrophils as compared with controls (>5-fold for n = 18 patients; p < 0.05). Similarly, patient neutrophils demonstrated upregulation of CD64, CEACAM-1, beta-2 glycoprotein I, and activated Mac-1 on the neutrophil surface. A monoclonal antibody specific for activated Mac-1 reduced the adhesion of APS neutrophils to HUVECs in the flow-chamber assay (>2-fold reduction for n = 5 patients; p < 0.05). Importantly, the same monoclonal antibody reduced NET release in neutrophil-HUVEC co-cultures. DISCUSSION/SIGNIFICANCE OF IMPACT: APS neutrophils have an increased adhesive potential, which is dependent upon the activated form of Mac-1. This may lower the threshold for both neutrophil-endothelium engagement and NET release in patients, and thereby have implications for events such as venous thrombosis. Studies are underway to determine the extent to which Mac-1 is a viable therapeutic target in preclinical models of APS.

OBJECTIVES/SPECIFIC AIMS: The primary objective of this effort is to develop and distribute an easy to use i2b2 component that is capable of evaluating diverse complex relationships for a wide variety of exposures and outcomes over time. In this manner we are able to leverage the unique design of the i2b2 database to support health services research, comparative effectiveness, and quality improvement using a single tool. Furthermore, our novel database redesign has the potential to provide user-friendly access to individual and group CHC data for CER. METHODS/STUDY POPULATION: For this project we used software experts, clinical informatics specialists, and the existing i2b2 open-source software to convert our legacy HOME Cell into a web-client version. The tool will be used to study health outcomes within a network of Boston based Community Health Centers and the largest safety-net hospital in New England, Boston Medical Center. RESULTS/ANTICIPATED RESULTS: The new web-client HOME Cell will allow i2b2 users to model virtually any exposure (including therapeutic interventions such as medications or tests) in i2b2 against any outcome accounting for complex temporal relationships and other factors. In addition we plan to use our new Community Health Center views to enhance our community engagement activities by allowing direct access to their data for our partners. DISCUSSION/SIGNIFICANCE OF IMPACT: Our project addresses multiple national priorities related to data sharing, clinical research informatics, and comparative effectiveness. The web-client version of the HOME Cell substantially improves our community’s access to HOME Cell functionality and is a novel, sharable resource for use within the CTSA/NCATS community. Our approach provides a new way to perform large-scale collaborative research without the need to actually move patient-level data and has demonstrated that CER, health services research, and quality measurement can share a common framework. In addition, and as demonstrated in our earlier pilot work, the HOME Cell also has the potential to support large-scale multivariate analyses in a distributed manner that does not require sharing of patient-level data. We believe our approach has great promise for supporting the reuse of clinical data for rapid, transparent, health outcome assessments on a national scale. Our efforts support multiple strategic goals including: (1) support for building national clinical and translational research capacity by enhancing a broadly adopted informatics tool (i2b2); (2) enhanced consortium-wide collaborations by offering a tool that can be easily shared within the CTSA network to support multi-institutional collaboration; and (3) improving the health of our communities by offering a tool that has the potential to provide new insights into health care processes and outcomes that could drive innovation and improvement activities.

ABSTRACT IMPACT: We hope to provide a more nuanced understanding of the type-III IFN system, thereby exploring its therapeutic potential in the realm of infectious diseases. OBJECTIVES/GOALS: The role of IFNLR1 receptor dynamics and plasticity in regulating the type-III IFN response is largely unknown. As a specific, powerful component of innate immunity, understanding how the type-III IFN system is regulated could lead to the development of novel therapeutic targets and strategies to face a multitude of viral illnesses. METHODS/STUDY POPULATION: To facilitate our investigation, we will generate doxycycline-inducible FLAG-tagged IFNLR1-expression plasmids representing all known transcriptional variants. These plasmids will allow us to: 1) Evaluate the effect of IFNLR1 surface abundance on the type-III IFN transcriptional profile and 2) Assess the extent of IFNLR1-FLAG co-localization with several notable intracellular structures using immunofluorescence, before and after stimulation with IFNL3. RESULTS/ANTICIPATED RESULTS: We have successfully generated three IFNLR1-FLAG transcriptional variants and confirmed inducible-expression and function in vitro. We are currently assessing the role of surface abundance, internalization, differential isoform expression, and trafficking. DISCUSSION/SIGNIFICANCE OF FINDINGS: By completing this study, we hope to provide a more nuanced understanding of the type-III IFN system, thereby exploring its therapeutic potential in the realm of infectious diseases.

OBJECTIVES/SPECIFIC AIMS: To investigate the performance of a metric for passive localization of central sulcus. METHODS/STUDY POPULATION: We studied 7 patients with intractable epilepsy undergoing intra-cranial EEG (icEEG) monitoring at Yale, in whom central sulcus (CS) localization was obtained by standard methods. Our method takes advantage of inherent properties of the primary motor cortex (MC), which exhibits enhanced icEEG band-power and coherence across the CS. For each contact x we calculated the z-score of a composite power and synchrony value log10(px)x;cx, where px is sum of the root mean square of the icEEG in the high gamma band (80–115 Hz) for contact x over the 6–10 minutes of NREM sleep studied, and cx is the mean magnitude squared coherence in the same band using a 500-ms Hamming window between contact x and all other contacts. z-score values lower than threshold (th) were set to 0. Finally, we calculated a metric m=z/d, where d is the mean Euclidian distance of each contact from contacts with z scores greater than 0. The last step was implemented to emphasize local network activity. RESULTS/ANTICIPATED RESULTS: We report the results of a pilot study to test the performance of a new operator independent method for passive identification of CS with intractable epilepsy undergoing icEEG monitoring at Yale, in whom CS localization was obtained by standard methods. The sensori-motor (SM) cortex exhibited higher EEG-gamma power compared with non-SM cortex (p<0.0002). There was no significant difference between the motor/premotor and sensory cortex (p<0.47). CS was successfully localized in all patients with thresholds between 0.4 and 0.6. In 2 patients, knowledge of anatomy was needed to distinguish the MC from adjacent epileptic foci. The primary hand and leg motor areas exhibited the highest metric values consistently followed by the tongue motor area. Higher threshold values were very specific (94%) for the anterior bank of the CS but not sensitive. Intermediate threshold values achieved a reasonable trade-off (0.4: 89% specific and 70% sensitive). DISCUSSION/SIGNIFICANCE OF IMPACT: We present and successfully implement a rapid procedure for task-free and stimulation free localization of the central sulcus during sleep based on intrinsic electrophysiological properties of the primary motor strip which exhibits increased power and enhanced local connectivity. We successfully localized the central sulcus in all patients. When implementing appropriate thresholds, our proposed metric M is very specific for the anterior lip of the central sulcus which may make it ideal to identify this important anatomical landmark. Our method is sensitive for epileptogenic regions as well, therefore basic knowledge about central sulcus anatomy may be needed in cases where there is an epileptogenic lesion in the vicinity of the central suclus. Our method makes a few a priori assumptions: The regions around the central sulcus are adequately sampled and the occipital or parieto-occipital regions are not included in the analysis. In order for the method to function properly, nonsensori-MC should be sampled adequately as well. In the future, normative data could be generated for the composite product of connectivity×power which may replace within-patient z-scoring. Our method is rapid and can be implemented on short segments of ECoG data. The proposed method may be potentially used for identification of seeds in the motor cortex for subsequent network analysis and further studies may delineate its potential use in the operating room.

OBJECTIVES/SPECIFIC AIMS: Determine PTL’s mechanism(s) of action in a panel of canine hematopoietic cell lines; this will enable us to 1) verify that PTL is working as expected and 2) rationally select combination therapeutics. Characterize the in vitro sensitivity of canine hematopoietic cell lines to PTL in combination with other chemotherapeutic agents. Determine immunohistochemical NFκB expression in tissue microarrays of spontaneous canine neoplasms and correlate with outcome-linked data. Characterize the in vivo sensitivity of canine hematopoietic cell lines to PTL using a murine xenograft model. METHODS/STUDY POPULATION: Growth inhibition assays were performed using a panel of canine mast cell, histiocytic sarcoma, lymphoma, and leukemia cell lines, with PTL alone or in combination with redox-perturbing standard-of-care therapeutics. Cell death was assessed using flow cytometry. Immunofluorescence and immunoblotting were used to assess NFκB localization and phosphorylation of NFκB p65 (transcriptional activation), respectively. Intracellular glutathione with and without PTL and combination chemotherapeutics will be assessed spectrophotometrically. Archived spontaneous canine tumors will be evaluated immunohistochemically (IHC) for increased NFκB pathway activation relative to normal control tissues. Nude mice will receive intravenous, intraperitoneal, or subcutaneous injections of canine HS cells and will be treated with PTL or with PTL in combination with standard-of-care chemotherapeutics. RESULTS/ANTICIPATED RESULTS: Results: All immortalized canine cell lines evaluated are sensitive to PTL therapy and undergo dose-dependent apoptosis following exposure to drug. PTL exposure leads to inhibition of NFκB, as evidenced by immunofluorescent nuclear exclusion and decreased p65 phosphorylation. Some chemotherapeutics appear to synergize with PTL in vitro. Anticipated results: We expect to find increased IHC NFκB pathway activation in malignantly transformed tissues relative to controls. We expect standard-of-care therapeutics to synergize with PTL in vivo based on preliminary in vitro data. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will determine whether PTL therapy may be beneficial in dogs with a variety of hematopoietic neoplasms, either alone or in combination with other therapeutics that are currently in clinical use. Dogs with mast cell or histiocytic neoplasia are an excellent model for rare and deadly human diseases, which may also benefit from PTL therapy.

OBJECTIVES/GOALS: The Colorado Community Engagement Alliance Against COVID-19 Disparities (CO-CEAL) Team has worked with 5 racial/ethnic communities in Colorado to understand COVID-19 vaccine hesitancy and develop messages, materials and dissemination strategies to impact vaccine hesitancy. Relationships have been key facilitators of this work. METHODS/STUDY POPULATION: CO-CEAL was funded for one year as a part of the national CEAL Teams effort. CO-CEAL engaged urban Hispanic/LatinX, rural Hispanic/LatinX, urban African/American, rural African Immigrant and urban American Indian/Alaska Native communities through Community Connectors in each community. Our Community Connectors led recruitment of community members for our community survey data collection (goal of 200 surveys from each community) and participation in our 5 Community (Boot Camp) Translations. Where possible we worked with Community Connectors and leveraged community relationships with whom we had worked over multiple years and projects. In two communities we worked to quickly build and establish new relationships with Community Connectors and their communities. RESULTS/ANTICIPATED RESULTS: The rapid nature of the CO-CEAL project, its timeline and the unusual nature of the funding mechanism required adaptations to usual ways of working on both the academic and community side.In our CO-CEAL partner communities where there was a history of working together, concerns, possible barriers and other issues tended to be rapidly addressed. The prior existing, long-standing relationships fostered trust that enabled open airing of concerns and an ability to address issues as they arose. In CO-CEAL partner communities where new relationships were being forged, trust needed to be build from the ground up. This made adaptations more challenging and the ability to address concerns and issues as they arose more problematic. Although the work has moved forward in all communities, extra care has been required in some. DISCUSSION/SIGNIFICANCE: When crises necessitate rapid action in communities, the depth of prior investments in building and maintaining relationships makes a difference in how rapidly and successfully partners can work together to address a crisis like the COVID-19 pandemic. Funders and Academic researchers should invest broadly in communities to enable rapid responses.

OBJECTIVES/GOALS: More young adults (age <50 years) are diagnosed with metastatic gastric cancer (mGC) every year. We will evaluate the association between environmental risk factors (including historical racial residential segregation) clinical, pathologic, molecular features and H.pylori associated early-onset mGC (mE-GC). METHODS/STUDY POPULATION: This retrospective matched case-control study of patients (1:2 by diagnosis year) with mGC (early-onset [E-GC; <50 years]; vs older-onset [O-GC; >50 years]) from 2000-2022 from the Los Angeles Cancer Surveillance Program (LA-CSP) will be enriched by a chart-abstracted cohort from USC Norris Comprehensive Cancer (NCCC). This annotated database captures sociodemographic, medical, and pathologic features of patients treated for mGC at NCCC. It will link to LA-CSP data exploring neighborhood features (obesity rate, poverty, insurance, access to care, ethnic enclave, historical segregation, etc.) of cases and controls to identify, characterize, and compare geographic “hotspot” neighborhoods of mE-GC. We hypothesize younger Hispanic and Asian patients are at higher risk of H.pylori associated mGC. RESULTS/ANTICIPATED RESULTS: From 2000-2022, 339 patients (mE-GC n = 113; mO-GC n=226) were treated at NCCC. We will have characterized clinical and pathological features of mE-GC vs mO-GC. We determined the proportion of H. pylori associated mE-GC vs mO-GC. We will have established the geographical distribution of patients with mE-GC vs mO-GC to identify high-risk neighborhoods. We will link neighborhood risk factors such as food scarcity, poverty, health care access, ethnic enclaves, to the distinct clinical and pathological features of mE-GC, including H. pylori status. Descriptive statistics, chi-square, t-tests, and multivariable regression will be used to compare mE-GC to mO-GC. After controlling for underlying demographics and tumor features, we anticipate clusters of mE-GC and mO-GC in areas of historical racial segregation. DISCUSSION/SIGNIFICANCE: Linking neighborhood and individual risk factors for mE-GC will inform early detection and prevention efforts for vulnerable individuals in high-risk neighborhoods. Building community partnerships within these neighborhoods is essential for developing interventions targeting H. pylori treatment to reduce health disparities in mE-GC.

OBJECTIVES/SPECIFIC AIMS: Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a curative procedure for hematological malignancies. Chronic graft Versus host disease (cGVHD) is a lethal complication that often develops after allo-HCT. Fli-1 is an aberrantly expressed protein in cancers including erythroleukemia and melanoma, while being implicated in pathogenesis of systemic lupus in mice and humans, a disease with marked similarity to cGVHD. METHODS/STUDY POPULATION: cGVHD was induced using hematopoietic cells from conditional knock-out mice deficient for the fli-1 gene specifically on T cells and progression of cGVHD in murine allo-HCT recipients was monitored using a clinical scoring system, and changes in activation status of hematopoietic cell populations were quantified using flow cytometry. RESULTS/ANTICIPATED RESULTS: Recipients transplanted with fli-1 deficient T cells exhibited reduced cGVHD clinical scores compared with littermate wild-type controls. Donor-grafts containing fli-1 deficient T cells were associated with restrained T-cell responses including reduced Interferon-y cytokine production, PD-1 expression, and differentiation into follicular helper T cells. fli-1 T-cell deficient donor-grafts also improved donor B-cell reconstitution and reduced plasma cells in allo-HCT recipients relative to littermate wild-type control donor-graft recipients. DISCUSSION/SIGNIFICANCE OF IMPACT: Thus, inhibiting Fli-1 represents a promising therapeutic strategy for the goal of preventing cGVHD after allo-HCT while also directly targeting cancers which aberrantly express Fli-1.

OBJECTIVES/GOALS: The Diabetes Prevention Program (DPP) has been shown to reduce diabetes risk by 58%. Men, particularly men of color, are underrepresented in DPP, while they experience higher diabetes-related morbidity. We examine whether race, ethnicity, and gender disparities in engagement are associated with the risk of referral to DPP in primary care METHODS/STUDY POPULATION: Using electronic health record (EHR) from a large urban health system in the Bronx, NY, with an in-house DPP, we examined patient, visit, referral data for DPP-eligible, adult patients with a primary care visit between July 2015 and December 2017. Eligibility included: hemoglobin A1c between 5.7-6.4%; a body mass index (BMI)≥24 kg/m2 (≥22 if Asian); and having no prior diagnosis of diabetes. A total of 26987 patients were included in this study. We examined patient race, ethnicity, preferred language, visit and prescription history, and health payer, among other characteristics. SPSS was used for univariate and bivariate analyses to examine associations between patient characteristics and referral followed by a logistic regression to examine the multivariate association between predictors and referrals. RESULTS/ANTICIPATED RESULTS: Of all DPP-eligible patients, 49% were Hispanic/Latino, and 39% were non-Hispanic Black. Around one-third (34%) of all eligible patients were men. Among all eligible patients in the sample, only 10% were referred to DPP. There were significant differences in the proportion of eligible patients who were referred versus those who were not referred. Women were referred at more than twice the prevalence of men (8.26% to 2.41%), with Hispanic women being referred most frequently (3.59%), and non-Hispanic white men being referred least frequently (.07%) Ethnicity, race, sex, age, number of provider visits, and number of chronic conditions all impacted healthcare provider referral rates to DPP. The health system’s unique free-of-charge DPP likely influenced the lack of significance of patient health insurance. DISCUSSION/SIGNIFICANCE: Given the implementation of DPP at-scale there is an urgent need to understand the patient and systems-level factors that are associated with referring individuals in the DPP. By detecting characteristics of health systems and patients that warrant special attention, we can improve equitable access to evidence-based diabetes prevention.

A crucial reckoning was initiated when the COVID-19 pandemic began to expose and intensify long-standing racial/ethnic health inequities, all while various sectors of society pursued racial justice reform. As a result, there has been a contextual shift towards broader recognition of systemic racism, and not race, as the shared foundational driver of both societal maladies. This confluence of issues is of particular relevance to Black populations disproportionately affected by the pandemic and racial injustice. In response, institutions have initiated diversity, equity, and inclusion (DEI) efforts as a way forward. This article considers how the dual pandemic climate of COVID-19-related health inequities and the racial justice movement could exacerbate the “time and effort tax” on Black faculty to engage in DEI efforts in academia and biomedicine. We discuss the impact of this “tax” on career advancement and well-being, and introduce an operational framework for considering the interconnected influence of systemic racism, the dual pandemics, and DEI work on the experience of Black faculty. If not meaningfully addressed, the “time and effort tax” could contribute to Black and other underrepresented minority faculty leaving academia and biomedicine – consequently, the very diversity, equity, and inclusion work meant to increase representation could decrease it.