OBJECTIVES/GOALS: This study investigates whether localization of high mobility group box 1 (HMGB1) controls inflammatory signaling and DNA damage response in human keratinocytes, the cell of origin for squamous cell carcinoma (SCC). SCC is especially metastatic in chronic wounds, burns, and in patients with recessive dystrophic epidermolysis bullosa (RDEB). METHODS/STUDY POPULATION: We used CRISPR/Cas9 gene-editing to knock out HMGB1 in a keratinocyte line, p16INK4a-negative keratinocytes immortalized by ectopic hTERT expression (N/TERT-2G [46,XY]). Following gene editing, clonal keratinocyte populations were screened for knockout by PCR followed by TIDE analysis (Tracking of Indels by Decomposition) to identify indels that would result in a frameshift mutation. Total cell lysates for each clonal population were analyzed by immunoblot and immunofluorescence for HMGB1 protein. These cells will be used to assay for DNA damage sensitivity in the presence of genotoxic agents (etoposide, ultraviolet radiation, γ-irradiation). A lentiviral vector will then be used to express mutant forms of HMGB1 that localize to the nucleus (C23/45S) or cytoplasm (C106S) and DNA damage assays repeated. RESULTS/ANTICIPATED RESULTS: We have confirmed by sequencing, immunoblot, and immunofluorescence that HMGB1 is knocked out in a clonal population of N/TERT-2G human keratinocyte cells. We anticipate that cells with a complete absence of HMGB1 will have high sensitivity to DNA damaging agents, but little change in inflammatory signaling. We also expect that cells expressing mutant HMGB1 that localizes exclusively to the cytoplasm will demonstrate an increased sensitivity to DNA damage relative to wild-type controls, while mutant HMGB1 that localizes exclusively to the nucleus will be protected from DNA damage caused by exposure to genotoxic agents. DISCUSSION/SIGNIFICANCE: HMGB1 is a nuclear protein and damage associated molecular pattern that is elevated systemically in patients with RDEB, many of whom will go on to develop metastatic squamous cell carcinoma. The experiments described here investigate whether HMGB1 plays a mechanistic role in skin carcinogenesis via regulation of the DNA damage response.

The recipients of NIH’s Clinical and Translational Science Awards (CTSA) have worked for over a decade to build informatics infrastructure in support of clinical and translational research. This infrastructure has proved invaluable for supporting responses to the current COVID-19 pandemic through direct patient care, clinical decision support, training researchers and practitioners, as well as public health surveillance and clinical research to levels that could not have been accomplished without the years of ground-laying work by the CTSAs. In this paper, we provide a perspective on our COVID-19 work and present relevant results of a survey of CTSA sites to broaden our understanding of the key features of their informatics programs, the informatics-related challenges they have experienced under COVID-19, and some of the innovations and solutions they developed in response to the pandemic. Responses demonstrated increased reliance by healthcare providers and researchers on access to electronic health record (EHR) data, both for local needs and for sharing with other institutions and national consortia. The initial work of the CTSAs on data capture, standards, interchange, and sharing policies all contributed to solutions, best illustrated by the creation, in record time, of a national clinical data repository in the National COVID-19 Cohort Collaborative (N3C). The survey data support seven recommendations for areas of informatics and public health investment and further study to support clinical and translational research in the post-COVID-19 era.

ABSTRACT IMPACT: This study will provide valuable insight regarding the effectiveness of a top-down approach for team formation. OBJECTIVES/GOALS: Rutgers’ Big Ideas is a philanthropic initiative designed to gather team science ideas and present them to donors. We intend to evaluate this Team Science intervention and determine its feasibility in catalyzing the inception of team formation. We will explore the composition of teams that are formed using this particular method and team outcomes. METHODS/STUDY POPULATION: Our group will first evaluate the themes that were covered by the initial 210 submissions as well as the 40 ideas chosen to be presented at the Big Ideas Symposium. We will also be taking a look at the donor population that these ideas were presented to. Then, we will evaluate the 8-12 winning teams that were chosen to move forward. We will compare various success metrics of the 8-12 teams that were chosen compared to the 40 ideas that had not been chosen. RESULTS/ANTICIPATED RESULTS: Encouraging team science through an initiative such as the Big Ideas forum is not only feasible, but also highly effective in creating resilient teams that show prolonged productivity in fundraising, publications, and other academic metrics. DISCUSSION/SIGNIFICANCE OF FINDINGS: Team Science is an exciting movement with immense potential. To that extent, this study seeks to discuss ways that academic leadership can inspire and foster effective team science collaboration. Concurrently, our case review lays the groundwork for further improvements to Team Science initiatives.

OBJECTIVES/SPECIFIC AIMS: Our aim is to test whether osteocyte-specific CXCL12 expression is critical to exercise-driven bone formation. METHODS/STUDY POPULATION: All procedures were approved by the NEW YORK UNIVERSITY Institutional Animal Care and Use Committee. We generated male and female mice in which CXCL12 was deleted from OCYs (CXCL12ΔOCY) by crossing CXCL12 floxed mice and 10kb DMP1-Cre transgenic mice (gifts from Drs. Geoffrey Gurtner and Lynda Bonewald, respectively). The 10kb DMP1-Cre has been shown to be robustly expressed in odontoblasts and OCYs, with little to no activity in cells from non-mineralized tissues (Lu+ J Dent Res 2007). Growing male and female mice (n=3-8/group) were given fluorochrome labels every two weeks between 4-16 weeks of age, to monitor the role of CXCL12 during development. A second group, of adult 16-week-old mice (n=5/group), were subjected to tibial axial cyclic loading (1200µɛ, 2Hz, 120cycles, 3days/wk for 2 wks) (Liu+ Bone 2018). Basal and load-induced periosteal (Ps) and endosteal (Es) mineralizing surface (MS/BS, %), mineral apposition (MAR, µm/day) and bone formation rates (BFR/BS, µm3/µm2/year) were calculated (Dempster+ JBMR.2013) at mid-length. RESULTS/ANTICIPATED RESULTS: No significant differences were detected in basal bone formation during development. However, relative load-induced Ps MAR (rMAR) was reduced by 50% in female (p=0.02) and 75% in male (p=0.002) CXCL12ΔOCY mice; and similarly, Ps rBFR/BS was reduced by 50% in female (p=0.01) and 70% in male (p=0.001) CXCL12ΔOCY mice (Figure 1). Es bone formation was not affected by CXCL12 deletion. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, osteocyte-specific CXCL12 expression plays a critical role in exercise-driven periosteal new bone formation, suggesting that CXCL12 signaling may positively regulate osteogenic differentiation and/or mature osteoblast function. Further underlying mechanisms are currently being explored. Thus, osteocyte-specific CXCL12 signaling may be a promising target to enhance load-induced bone formation in patients with compromised ability to form new bone.

OBJECTIVES/GOALS: This project will provide novel insights into mechanism(s) by which differences in inflammation develop & resolve, or fail to resolve, in metabolically different groups of bariatric surgery patients determined by Type 2 Diabetes status. My work may uncover unique differences between cohorts, encouraging development of personalized medicine. METHODS/STUDY POPULATION: I analyzed human blood samples collected before and 3, 6, & 12 months after bariatric surgery at the University of Kentucky through an established tissue bank. Subjects hadnormal glucose tolerance, pre-diabetes, or Type 2 Diabetes, based on HbA1c%. Iisolated peripheral blood mono nuclear cells & will comparecytokine profiles among cohorts across all time points. I will define & perturbmetabolic differencesin immune cells among cohorts & across time via isotope tracing, fuel source limitation, and metabolite inhibition. This will determine causal relationships between cytokine profiles & immune cell metabolism. RESULTS/ANTICIPATED RESULTS: I anticipatecytokine profiles, a functional output of immune cells, will differ among cohorts pre-surgery, and that this difference will diminish post-surgery. Differencesmay be insignificant by the 12 month time point. I also anticipate differences in fuel usage and metabolite productionin immune cells among cohorts pre-surgery, and that these differences only partially resolve post-surgery to poise immune cells for continued chronic inflammatory action. I hypothesize that T2D status has a lasting impact on immune cell function and fuel usage patterns, and will continue to supportchronic inflammation following short term T2D remission and longer-term weight loss. DISCUSSION/SIGNIFICANCE: There has been an alarming increase in obesity and its comorbidities over recent decades, and inflammation is a known supporter of T2D. The anticipated rewiring of immune cell metabolism post-surgery, if incomplete, may poise subjects forweight regain and T2D recurrence.

OBJECTIVES/GOALS: 22q.11 deletion syndrome (22q11DS) is a genomic syndrome that elevates risk for psychosis >20-fold. We used a battery of cognitive and psychophysiological psychosis-risk biomarkers in 22q11DS patients and healthy subjects in order to identify biomarkers of psychosis in 22q11DS that could be used as translational targets in intervention studies. METHODS/STUDY POPULATION: We recruited 15 22q11DS individuals (Mean age=30, M/F=9/6) and 19 healthy controls (HCs; Mean age=34, M/F=5/14). Each individual completed the MATRICS Consensus Cognitive Battery (MCCB), the Wechsler Abbreviated Scale of Intelligence, Second edition (WASI-II) Verbal IQ subtests, and the computerized Wisconsin Card Sorting Task (WCST). To examine auditory EEG responses, each participant completed the 'Double-Deviant' target detection paradigm, which presents a pseudorandom sequence of frequent standard tones and infrequent deviant tones. Mismatch negativity (MMN) metrics were generated from this assessment. Welch's t-tests were completed for neurocognitive variables. One-Way ANOVAs were completed to examine EEG results, with sex entered as a separate factor and age entered as a covariate. RESULTS/ANTICIPATED RESULTS: Significant group differences were found in 8 of the 9 neurocognitive measurements (FDR-adjusted p's< 0.02, average Cohen's d=1.62, average observed power= 0.91) indicating widespread cognitive deficits in 22q11DS subjects across multiple domains. The Double-Deviant MMN ERP response was significantly smaller in absolute magnitude in the 22q11DS group (FDR-adjusted p=0.048, Cohen’s d= -0.864, observed power= 0.58). The MMN ERPs for the frequency and duration deviants were not significantly different (FDR-adjusted p's> 0.33). No group by sex interactions were observed in any of the measures. Neurocognitive variables were associated with psychosis positive, negative, general, and disorganized symptom scales. DISCUSSION/SIGNIFICANCE: Our results identify potential psychosis-risk biomarkers in 22q11DS. If replicated, these biomarkers could provide important translational targets for future clinical trials for individuals with 22q11DS and other individuals at-risk for psychosis syndromes.

OBJECTIVES/GOALS: Ebola virus infection causes severe disease and liver injury in humans. Macrophages contribute to inflammatory signaling and are prevalent in the liver. We assessed the activation status, including therapeutic target expression, of hepatic macrophages. METHODS/STUDY POPULATION: We compared formalin-fixed, paraffin-embedded liver tissue from terminal Ebola virus-infected and uninfected control cynomolgus macaques, a gold-standard model for human disease. We characterized region-specific protein and whole transcriptome expression in these tissues using GeoMx Digital Spatial Profiling. Macrophage (CD68+) and leukocyte (CD45+) accumulation in liver tissue was quantified by immunofluorescence image analysis using digital pathology software. RESULTS/ANTICIPATED RESULTS: Macrophage-specific (CD68+) regions in the liver of Ebola virus-infected macaques demonstrated a shift towards an inflammatory gene expression profile, as compared to those from healthy control tissue. These regions showed differential expression of monocyte/macrophage differentiation, antigen presentation, and T cell activation gene sets, which were associated with decreased MHC-II allele expression. Moreover, macrophage-specific regions in the infected macaques showed enriched expression of genes or proteins associated with known immunomodulatory therapeutics, including S100A9, IDO1, and CTLA-4. DISCUSSION/SIGNIFICANCE: These data demonstrate that hepatic macrophages express an inflammatory phenotype, that their ability to present antigens to the adaptive immune system may be impaired, and that they express therapeutically targetable markers for immunomodulation of these cells during Ebola virus infection.

OBJECTIVES/GOALS: Our aim is to establish soluble salivary biomarkers indicative of increased risk of oral premalignancy to be used in a point-of-service technology. Our goal is to non-invasively assess risk level for premalignancy by characterizing a molecular signature pattern that can be applied to such a diagnostic tool at routine dental or medical visits. METHODS/STUDY POPULATION: Adult patients 18 years of age and older who are non-smokers and patients of the University of Maryland School of Dentistry Oral Medicine Clinic and have been diagnosed with oral premalignancy (proliferative verrucous leukoplakia) are eligible. Exclusion criteria include history of immunosuppression or immune compromise; use of antifungal, antibiotic, and/or antiviral medications within the past three months; and gross dental disease. Serial unstimulated saliva samples will be collected at baseline or diagnosis of oral premalignancy, 6 months and 12 months. Solubility testing will be completed to determine whether malignant markers such as EGFR/mTOR/PI3K/p53 are soluble in saliva, and patient samples will be analyzed by ELISA and compared to appropriate controls. RESULTS/ANTICIPATED RESULTS: We anticipate demonstrating increased activity of molecular pathways known to be involved in malignant transformation, such as EGFR/mTOR/PI3K/p53, or increased burden of select microbial pathogens to be associated with increased risk of oral premalignancy in the form of proliferative verrucous leukoplakia. Preliminary sensitivity and specificity testing of the identified markers will provide additional insight to the utility of a diagnostic tool with salivary specimen. Therefore, the microbiome and/or molecular profile proposed from these results will serve as a translational application to development of future point-of-service test devices to be used in the prevention and detection of oral premalignant lesions. DISCUSSION/SIGNIFICANCE: Oral cancer is the sixth most common cancer worldwide, and presents challenges in its diagnosis and clinical management. Later diagnosis is associated with poorer patient outcomes—therefore, a molecular and microbiome profile that may be used in a noninvasive diagnostic test technology would prove beneficial to providers and patients.

ABSTRACT IMPACT: Our research identifies key opportunities for increased cross-CTSA collaboration, as a means to improve community-research cooperation and better CBPR practices. OBJECTIVES/GOALS: Currently, team science training prioritizes developing the collaborative competencies of interdisciplinary scientists to work with each other and, more recently, with communities. Community-facing team science resources are scarce but present among some CTSAs, suggesting that capacity gaps might be remedied through cross-hub collaboration. METHODS/STUDY POPULATION: We reviewed online information provided by the 62 current CTSAs to identify: (1) which hubs engage in community research, and (2) what resources the hubs utilize to orient, train, and support community stakeholders as research partners. We then examined the capacities of the collectively available CTSA resources to address needed knowledge, skills, and attitudes that community-engaged researchers have identified as essential for community-based stakeholders to partner equally in research. Finally, we explored practical challenges in team-based dynamics (e.g., interpersonal difficulties, expertise gaps, resource management) that may facilitate or hinder communities’ research endeavors, and suggest resources that CTSAs might implement to facilitate team science dynamics. RESULTS/ANTICIPATED RESULTS: Hubs (n=59) have community engagement programs, 12 of which provide community-based participatory research toolkits. Toolkits vary from basic checklists to fully developed modules. Some hubs also offer consultation services and partner match-making. Learning objectives include: outcome definition, logic models, and goal-setting. Learning resources remain underdeveloped to help communities appreciate the benefits of research engagement and convince academic partners of the value of real-world knowledge and community improvement relative to scientific advancement. Also lacking is easily accessible support to understand the research process, build verifiable trust, maintain bidirectional knowledge and assets, and implement consistent, best practice methodological and reporting protocols. DISCUSSION/SIGNIFICANCE OF FINDINGS: Gaps between current hub offerings and community needs suggest prioritizing creation of resources whose learning objectives highlight the benefits of research engagement for community partners; foster mutual values affirmation between partners; and offer tools that build warranted community-researcher rapport.

OBJECTIVES/SPECIFIC AIMS: Oncostreams represent a novel growth pattern of GBM. In this study we uncovered the cellular and molecular mechanism that regulates the oncostreams function in GBM growth and invasion. METHODS/STUDY POPULATION: We studied oncostreams organization and function using genetically engineered mouse gliomas models (GEMM), mouse primary patient derived GBM model and human glioma biopsies. We evaluated the molecular landscape of oncostreams by laser capture microdissection (LCM) followed by RNA-Sequencing and bioinformatics analysis. RESULTS/ANTICIPATED RESULTS: Oncostreams are multicellular structures of 10-20 cells wide and 2-400 μm long. They are distributed throughout the tumors in mouse and human GBM. Oncostreams are heterogeneous structures positive for GFAP, Nestin, Olig2 and Iba1 cells and negative for Neurofilament. Using GEMM we found a negative correlation between oncostream density and animal survival. Moreover, examination of patient’s glioma biopsies evidenced that oncostreams are present in high grade but no in low grade gliomas. This suggests that oncostreams may play a role in tumor malignancy. Our data also indicated that oncostreams aid local invasion of normal brain. Transcriptome analysis of oncostreams revealed 43 differentially expressed (DE) genes. Functional enrichment analysis of DE genes showed that “collagen catabolic processes”, “positive regulation of cell migration”, and “extracellular matrix organization” were the most over-represented GO biological process. Network analysis indicated that Col1a1, ACTA2, MMP9 and MMP10 are primary target genes. These genes were also overexpressed in more malignant tumors (WT-IDH) compared to the less malignant (IDH1- R132H) tumors. Confocal time lapse imagining of 3D tumor slices demonstrated that oncostreams display a collective motion pattern within gliomas that has not been seen before. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, oncostreams are anatomically and molecularly distinctive, regulate glioma growth and invasion, display collective motion and are regulated by the extracellular matrix. We propose oncostreams as novel pathological markers valuable for diagnosis, prognosis and designing therapeutics for GBM patients.

OBJECTIVES/GOALS: Primary graft dysfunction (PGD) is acute lung injury in the first three days after lung transplant. Patients that experience PGD have increased mortality and an increased risk of chronic lung allograft dysfunction. The pathogenesis is thought to be an ischemia-reperfusion injury but is incompletely understood and there are no specific therapies. We investigated the role of the microbiome in PGD and associations with inflammation and markers of aspiration. METHODS/STUDY POPULATION: We collected airway lavage samples from lung transplant donors before procurement and recipients after reperfusion. We extracted DNA, amplified the bacterial 16S rRNA gene, and sequenced on the Illumina MiSeq platform. QIIME2 and Deblur were used for bioinformatic analysis. R packages were used for downstream analysis and visualizations. The host response was quantified using the Milipore 41-plex Luminex and an ELISA for pepsin. Clinical data was collected by the Penn Lung Transplant Outcomes Group. PGD was assessed by degree of hypoxemia and chest X-ray findings in the 72 hours after transplant. RESULTS/ANTICIPATED RESULTS: There was no significant difference in alpha diversity (Shannon index, p = 0.51), biomass (via comparison of 16S amplicon PicoGreen, p = 0.6), or beta diversity (Weighted UniFrac, p = 0.472, PERMANOVA) between subjects with PGD grade 3 (n = 36) and those that did not (n = 96). On taxonomic analysis, we found an enrichment of Prevotella in donor and recipient lungs that went on to develop PGD (p = 0.05). To follow up this finding we measured immune response and pepsin concentrations in recipient lungs. We found elevated levels in 35/41 cytokines measured in subjects that developed PGD as well as an elevation in pepsin and a correlation between pepsin concentration and Prevotella relative abundance (Figure 1). Additionally, Prevotella relative abundance had statistically significant positive correlations with multiple cytokines such as IL-6 (Pearson’s = 0.26, p = 0.009) and eotaxin (Pearson’s = 0.24, p = 0.016). DISCUSSION/SIGNIFICANCE OF IMPACT: There is an enrichment of oral anerobes in lung allografts that eventually develop PGD. This is associated with elevated levels of pepsin and markers of inflammation. These lines of evidence suggest aspiration contributes to priming the allograft for PGD.

ABSTRACT IMPACT: Understanding how spinal cord stimulation works and who it works best for will improve clinical trial efficacy and prevent unnecessary surgeries. OBJECTIVES/GOALS: Spinal cord stimulation (SCS) is an intervention for chronic low back pain where standard interventions fail to provide relief. However, estimates suggest only 58% of patients achieve at least 50% reduction in their pain. There is no non-invasive method for predicting relief provided by SCS. We hypothesize neural activity in the brain can fill this gap. METHODS/STUDY POPULATION: We tested SCS patients at 3 times points: baseline (pre-surgery), at day 7 during the trial period (post-trial), and 6 months after a permanent system had been implanted. At each time point participants completed 10 minutes of eyes closed, resting electroencephalography (EEG) and self-reported their pain. EEG was collected with the ActiveTwo system and a 128-electrode cap. Patients were grouped based on the percentage change of their pain from baseline to the final visit using a median split (super responders > average responders). Spectral density powerbands were extracted from resting EEG to use as input features for machine learning analyses. We used support vector machines to predict response to SCS. RESULTS/ANTICIPATED RESULTS: Baseline and post-trial EEG data predicted SCS response at 6-months with 95.56% and 100% accuracy, respectively. The gamma band had the highest performance in differentiating responders. Post-trial EEG data best differentiated the groups with feature weighted dipoles being more highly localized in sensorimotor cortex. DISCUSSION/SIGNIFICANCE OF FINDINGS: Understanding how SCS works and who it works best for is the long-term objective of our collaborative research program. These data provide an important first step towards this goal.

OBJECTIVES/GOALS: This project seeks to understand how personalized medicine can optimize care for patients with colorectal cancer. It identifies opportunities for personalized medicine to improve clinical outcomes, and uses cost-effectiveness analysis to assess the clinical and financial impact of this approach. METHODS/STUDY POPULATION: This project uses two methods to understand the impact of personalized medicine. First, this project has used SEER-Medicare data in conjunction with Clinical Pharmacogenetics Implementation Consortium guidelines to identify medications used by patients with colorectal cancer that can be impacted by genetic variants. This data will then be combined with population genetic variant rates to understand the likely impact screening for a given variant will have on medication response and adverse events. Medication use frequencies and genetic variant rates are then used to populate cost-effectiveness models that simulate the clinical and financial outcomes, identifying optimal genes to screen. RESULTS/ANTICIPATED RESULTS: The first result will be a comprehensive overview of treatment patterns for patients with colorectal cancer in the United States, as well as the treatments used for disease-induced comorbidities. The second result will be the identification of genetic variants based on population rates and medication utilization that should be screened in this patient population. The final result will be a breakdown of the clinical and financial outcomes associated with implementing screening for the identified genes. Preliminary results from a two-gene cost-effectiveness analysis demonstrates that screening for variants in those genes improves both clinical and financial outcomes. DISCUSSION/SIGNIFICANCE OF IMPACT: This project demonstrates how current treatment approaches can be optimized via personalized medicine. It uses epidemiological methods to identify opportunities to integrate genetic findings from other diseases, and uses cost-effectiveness analysis to understand the impact of transforming care. CONFLICT OF INTEREST DESCRIPTION: Stocks-Aurinia, Syndax, Adaptimmune, Rigel pharma

OBJECTIVES/SPECIFIC AIMS: Previously, we showed decreased development of endometriotic lesions in CD44 knockout mice compared to control.(1) CD44 has 10 different variants and a standard form. Menstrual endometrial cells (MECs) from women with endometriosis have increased adhesion and also express higher levels of CD44 variant 6 (v6) than v3, compared to MECs from women without endometriosis. (2) Here, we assessed the effects of CD44 standard (CD44s), CD44v3 and CD44v6 overexpression (OE) on immortalized human endometrial epithelial (iEECs) and stroma cells (hESCs) in vivo attachment in a nude mouse xenograft model. 1. Knudtson JF, Tekmal RR, Santos MT, et al. Impaired Development of Early Endometriotic Lesions in CD44 Knockout Mice. Reproductive sciences (Thousand Oaks, Calif.). 2016;23(1):87-91. 2. Griffith JS, Liu YG, Tekmal RR, Binkley PA, Holden AE, Schenken RS. Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants. Fertility and sterility. 2010;93(6):1745-1749. METHODS/STUDY POPULATION: Overexpression of CD44s, CD44v3 and CD44v6 was carried out using lipofectamine and their expression verified with qRT-PCR in iEEC and hESCs. Nude mice, 8-10 week old, were injected with estrogen 1 week prior to injection of iEECs and hESCs (n=7 per group). The cells were counted after transfection and at least 300,000 iEECs and 300,000 hESCs were injected per mouse. The transfected cells were tagged with cell tracker red (iEECs) and green (hESCs). Forty-eight hours after injection into the xenograft, the mice were sacrificed. The cells were counted using fluorescent stereo microscopy (FSM). Percent attachment was calculated based on the number of cells visualized by FSM divided by the number of transfected cells injected. Unpaired student t-test was performed to analyze differences in the percent attachment of the cells. RESULTS/ANTICIPATED RESULTS: The majority of cells were attached to the peritoneum. There was increased attachment of hESCs with OE of CD44v6 compared to control (p=0.03). CD44v6 OE did not change attachment of iEECs. There was no difference in attachment in iEECs or hESCs with OE of CD44s or CD44v3. DISCUSSION/SIGNIFICANCE OF IMPACT: Overexpression of CD44v6 increases attachment of ESCs to PMCs in an in vivo xenograft model. Menstrual endometrial cell type and CD44 variants play a complex role in the development of the early endometriotic lesion.