Objectives/Goals: We investigated the risk of trauma in the form of fractures and traumatic brain injuries (TBIs) among Medicare beneficiaries with incident Parkinson’s disease (PD) age ≥67 compared to population-based controls. Secondarily, we examined the risk of death following a fracture in PD cases compared to controls. Methods/Study Population: We identified incident PD cases (N = 94,317) within a population-based sample of 2017 Medicare beneficiaries. Controls (N = 471,585) were matched 5:1 on month and year. We obtained claims data from 2017 to 2019 to follow cases and controls to identify new fractures treated in a hospital. Our primary outcome was any fracture. We also considered fracture type and TBI. We compared frailty level between cases and controls. We used logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between trauma and PD after adjusting for the following covariates: selected medical comorbidities, age, sex, race/ethnicity, smoking, and use of care. We used Cox regression to estimate hazard ratios (HRs) and 95% CI for trauma in cases compared to controls using the same covariates. Results/Anticipated Results: Compared to controls, PD patients who developed a fracture were more likely to have a history of falls (OR = 2.20, 95% CI 2.08–2.34) and difficulties in walking (OR = 2.66, 95% CI 2.50–2.82). Compared to controls with a fracture, PD patients with a fracture were more likely to be moderately frail (OR = 1.43, 95% CI 1.25–1.64). PD cases had a higher risk of all fracture types, including hip (OR = 1.93, 95% CI 1.85, 2.01), spine (OR = 1.90, 95% CI 1.79, 2.02), upper extremity (OR = 1.69, 95% CI 1.58–1.80), and other traumas such as a TBI (OR = 2.14, 95% CI 1.88–2.43). PD patients had greater mortality following a fracture (HR = 1.18, 95% CI 1.13–1.24) than controls. Discussion/Significance of Impact: The burden of trauma in the first two years immediately after PD diagnosis is high and warrants the initiation of early fall and fracture prevention strategies, in addition to aggressive treatment of PD symptoms by all providers caring for patients with PD.

OBJECTIVES/SPECIFIC AIMS: The study aims to track and correlate ocular neuropathic symptoms, corneal sensitivity and dry-eye like pain, after scleral buckle and posterior vitrectomy surgeries. The goal is to identify a population of patients that receive these retinal surgeries that experience ocular neuropathic pain. METHODS/STUDY POPULATION: Methods - Prospective and Retrospective cohort studies were designed with the follow cohorts: scleral buckle, posterior vitrectomy, and control. Typical follow up for SB/PV surgeries are: 1 day, 1 week, 1, 3, 6, 12 months post surgery. CS and DELP metrics are measured at each visit. For study interventions, all subjects (from both arms) will undergo the same series of tests, in the same sequence at each visit. Phase 1 of the visit focuses on CS and phase 2 on DELP. These interventions are as follows: first, subjects will receive Drop A; Drop A will be administered in a randomized, double-blinded manner at each visit to either balanced salt solution (control) or Muro 128 5% hypertonic saline (experimental). Drop A will be administered to both eyes. After receiving the drops, subjects will complete a visual analog scale questionnaire to grade their corneal sensitivity. Next, subjects will undergo a five minute washout. After the washout, subjects will receive Drop B; Drop B will be whichever drop was not administered in the Drop A phase. After Drop B is given, subjects will complete the visual analog scale. To begin phase 2, subjects will be given the Ocular Surface Disease Index to record dry eye signs and symptoms. Finally, tear film parameters will be collected using Schirmer’s tear production test and tear film breakup time. Study Population. - Inclusion criteria: For retrospective cohort studies, subjects who have undergone unilateral SB or PV in the past year. For prospective cohort studies, subjects who will undergo unilateral SB or PV in the near future, and age-matched controls. Exclusion criteria: For both retrospective and prospective arms, the same exclusion criteria apply. They include: a previous diagnosis of dry eye; current use of neuropathic pharmacotherapies (including gabapentin, pregabalin, TCAs, SNRIs, carbamazepine, and opioids). RESULTS/ANTICIPATED RESULTS: As of 11/15/18, only the scleral buckle retrospective study arm had enough subjects for any meaningful preliminary report; the arm currently has 8 subjects. Of these 8 subjects, 5/8 subjects report increased surgical-eye corneal sensitivity and 6/8 show discordant dry eye symptoms and tearfilm parameters. Our power analysis showed that N=16 subjects in a group are required to detect a statistical significant difference in corneal sensitivity response. We expect to see a relapsing and remitting pattern of pain (as measured by corneal sensitivity and dry eye questionnaire), as is typical of neuropathic pain. Regarding dry eye symptoms, we anticipate subjects will have prominent dry eye symptoms (as measured by a validated questionnarie), but show no abnormalities in tearfilm parameters. DISCUSSION/SIGNIFICANCE OF IMPACT: To our knowledge, this is the first observational study of neuropathic pain symptoms of corneal sensitivity and dry-eye like pain, in post retinal surgery patients. We recognize the challenge of diagnosing neuropathic pain; currently the gold standard is clinical. However, symptoms of neuropathic pain are non-specific and subtle. Identification of a population suffering from post-retinal surgery ocular neuropathic pain will provide a foundation to test topical naltrexone as a diagnostic tool. If our hypothesis is correct, topical naltrexone could serve as a cheap, easy, and quick diagnostic test for ocular neuropathic pain. We envision this diagnostic test would allow many misdiagnosed and mistreated post-surgical patients to be treated with appropriate therapies aimed at neuropathic etiologies.

OBJECTIVES/GOALS: Can we detect Parkinson’s-disease-related motor impairments using computer vision and machine learning? METHODS/STUDY POPULATION: A sample of 29 people with Parkinson’s disease (PD) and 29 non-Parkinson’s disease (non-PD) controls were recruited from the University of Iowa Movement Disorders Clinic. Videos of 3 motor assessment tasks performed using the hands were recorded and hand location information was abstracted using the computer vision program MediaPipe. Measures from the raw data series and FFT were used as features to train a model using boosted trees to classify each video as PD or non-PD. Model performance was evaluated using leave-one-out cross-validation. Additionally, we used recursive feature elimination to reduce model complexity. RESULTS/ANTICIPATED RESULTS: A model using two features identified by recursive feature elimination yielded a model with an overall accuracy of 81% in cross-validation. In our sample, the model had 86.2% sensitivity, 75.9% specificity, and an AUC of 0.839. Additional improvement may be possible with more data processing, especially in the time-domain. DISCUSSION/SIGNIFICANCE: We built a classifier that was able to reliably and accurately discriminate between videos of motor assessments in people with Parkinson’s and people without. This may provide a low cost screening tool in rural areas or primary care clinics with limited access to neurologist expertise.

OBJECTIVES/GOALS: Determine if exposure to violence at different life stages (childhood versus recent exposure) in postpartum women is associated to increased risk of weight retention more than 1 year and no more than 2 years after delivery, thus increasing health risk in short and long term. METHODS/STUDY POPULATION: Participants will be recruited from a cohort of post-partum women who received services from the Puerto Rico Women and Children Program (PR WIC) during pregnancy and postpartum period as per PR WIC established criteria. Families that have completed their participation in a Lifestyle intervention trial will be paired 2:1 with eligible nonparticipants that fulfill trial enrollment criteria. Language, culture validated instruments will be used to document maternal violence exposure in childhood and recent exposure, defined as within the last 12 months. To asses weight retention, pre-pregnancy weight will be compared to actual weight at the moment of evaluation, anthropometric measurements (weight, length, body composition, and fat mass) will be used to determine the health risk category for each participant. RESULTS/ANTICIPATED RESULTS: Expected results will be that there is an association between violence exposure and weight retention among post-partum women, thus influencing their weight status. The use of the questionnaires as screening tool for history of violence and whether recent or childhood exposure should be considered a health risk during pregnancy and post-partum predisposing women to adipose tissue related disorders. DISCUSSION/SIGNIFICANCE: Findings will reinforce the importance of integrated screening and proactive management of social determinants of health within clinical and social services that target this population. Also, contribute to the development of strategies tailored to break violence cycle as a public health measure.

OBJECTIVES/GOALS: Changes associated with placental vasculature contribute to the progression of gestational hypertensive disease preeclampsia. Caudal-type homeobox-2 (CDX2) regulates trophoblast stem cell differentiation. In this study, we investigate the role of placental CDX2 cells in healthy pregnancy and in conditions of preeclampsia. METHODS/STUDY POPULATION: To understand the role of CDX2 cells, here we collected human placenta samples from the prospectively enrolled cohort and also the de-identified cohort (n=84). We studied CDX2 distribution, and function using a lentivirus-based approach. We studied the CDX2 expression and functional differences using transcriptomics and examined the function in invasion and vasculogenesis in the presence and absence of the new target genes we have discovered in our study. RESULTS/ANTICIPATED RESULTS: Analysis of healthy human placenta samples showed that CDX2-expressing cells were present in fetal chorionic regions and are associated with HLA-G and cytokeratin-7 confirming their trophoblast identity. CDX2 cells demonstrated the potential to form a capillary network akin to endothelial cells. Placental samples from healthy (n=6) and preeclampsia (n=8) patients revealed higher levels of CDX2 expression in preeclampsia. Within preeclampsia CDX2 cells, Natriuretic peptide receptor 1 (NPR1), RET oncogene, and Homeobox D10 (HOXD10) were significantly differentially regulated, including a unique long-non-coding anti-sense RNA (KANSL1-AS1) that affected the function of CDX2 and trophoblast cells in invasion and normal vasculogenesis. DISCUSSION/SIGNIFICANCE: In sum, based on these observations, the present study postulates that CDX2 cells present in a healthy human placenta may serve as a prospective cellular reservoir for angiogenesis. Conversely, altered gene programs within CDX2 cells cause aberrant vascular function that could contribute to the progression of preeclampsia.

ABSTRACT IMPACT: The proposed study has the potential to inform new paradigms of type 1 diabetes prevention and therapy with the overall goal of improving β cell health during autoimmunity. OBJECTIVES/GOALS: Type 1 diabetes (T1D) results from immune-mediated destruction of pancreatic βcells. Recent data suggest that activation of senescence and acquisition of a senescence associated secretory phenotype (SASP) by βcells may contribute to T1D pathogenesis. However, the molecular mechanisms responsible for this phenotype are not well understood. METHODS/STUDY POPULATION: We hypothesize that loss of endoplasmic reticulum (ER) Ca2+ induces βcell senescence, SASP as well as mitochondrial dysfunction which drive T1D development. The current study utilizes SERCA2 KO INS-1 βcells (S2KO) exhibiting loss of ER Ca2+ and a SERCA2 haploinsufficient mice on a non-obese diabetic background (NOD-S2+/-) to test the role of ER Ca2+ loss during T1D development. Senescence associated βgalactosidase staining (SA-βgal), expression of senescence markers (RT-qPCR), mitochondrial function (Seahorse, TMRM) and mitochondrial copy number (qPCR) were all measured in S2KO versus WT βcells and are currently being measured in the NOD-S2+/- mouse model at 6, 8, 12, 14, and 16wks of age. RESULTS/ANTICIPATED RESULTS: RT-qPCR assays detecting senescence markers cdkn1a and cdkn2a and mitochondrial specific genes cox1 and nd1 were developed and validated in both INS-1 βcells and mouse islets. Mitochondrial function assay (Seahorse) was optimized for use in INS-1 βcells and is currently under development for use in intact mouse islets. S2KO βcells displayed increased SA- βgal staining as well as increased mitochondrial coupling efficiency (p=0.0146) and baseline mitochondrial copy number (p=0.0053) compared to WT βcells, suggesting a senescence phenotype and altered mitochondrial function. NOD-S2+/- mice exhibited increased expression of the senescence marker cdkn2a in the islet at 12wks (p=0.0117) compared to control mice, whereas cdkn1a remained unchanged across all timepoints tested. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results suggest that loss of SERCA2 and reduced ER Ca2+ alter βcell mitochondrial function and are associated with features of senescence. Future studies will test whether SERCA2 activation and/or senolytic/senomorphic drugs are able to prevent or delay diabetes onset in NOD-S2+/- mice.

The Clinical and Translational Science Award (CTSA) Consortium and the National Center for Advancing Translational Science (NCATS) undertook a Common Metrics Initiative to improve research processes across the national CTSA Consortium. This was implemented by Tufts Clinical and Translational Science Institute at the 64 CTSA academic medical centers. Three metrics were collaboratively developed by NCATS staff, CTSA Consortium teams, and outside consultants for Institutional Review Board Review Duration, Careers in Clinical and Translational Research, and Pilot Award Publications and Subsequent Funding. The implementation program included training on the metric operational guidelines, data collection, data reporting system, and performance improvement framework. The implementation team provided small-group coaching and technical assistance. Collaborative learning sessions, driver diagrams, and change packages were used to disseminate best and promising practices. After 14 weeks, 84% of hubs had produced a value for one metric and about half had produced an initial improvement plan. Overall, hubs reported that the implementation activities facilitated their Common Metrics performance improvement process. Experiences implementing the first three metrics can inform future directions of the Common Metrics Initiative and other research groups implementing standardized metrics and performance improvement processes, potentially including other National Institutes of Health institutes and centers.

Cardiovascular disease (CVD) is largely preventable, and the leading cause of death for men and women. Though women have increased life expectancy compared to men, there are marked sex disparities in prevalence and risk of CVD-associated mortality and dementia. Yet, the basis for these and female-male differences is not completely understood. It is increasingly recognized that heart and brain health represent a lifetime of exposures to shared risk factors (including obesity, hyperlipidemia, diabetes, and hypertension) that compromise cerebrovascular health. We describe the process and resources for establishing a new research Center for Women’s Cardiovascular and Brain Health at the University of California, Davis as a model for: (1) use of the cy pres principle for funding science to improve health; (2) transdisciplinary collaboration to leapfrog progress in a convergence science approach that acknowledges and addresses social determinants of health; and (3) training the next generation of diverse researchers. This may serve as a blueprint for future Centers in academic health institutions, as the cy pres mechanism for funding research is a unique mechanism to leverage residual legal settlement funds to catalyze the pace of scientific discovery, maximize innovation, and promote health equity in addressing society’s most vexing health problems.

The Harvard Catalyst KL2/CMeRIT program is a 2-year mentored institutional career award that includes KL2 grants funded by National Institutes of Health (NIH) and CMeRIT grants funded by Harvard Catalyst nonfederal funds. The purpose of this study was to compare outcomes for early-stage investigators funded by the KL2/CMeRIT program to a group of applicants who were not chosen for support to assess the potential impact of the program on early career outcomes. Career data, including academic promotions, subsequent grant funding, and publication rates, from both successful and unsuccessful 2008–2018 KL2/CMeRIT applicants were compiled throughout the year 2020. Data were obtained directly through outreach to both groups and through assessment of online resources. The cohort comprised 487 individuals, 109 awardees, and 378 nonawardees. Awardees were more likely to be subsequently involved in clinical and translational research than nonawardees (92% vs 75%, p < 0.001). A higher proportion of awardees also had achieved academic promotion (81% vs 69%, p = 0.016) and subsequent NIH funding (72% vs 58%, p = 0.047), while there was no difference in publication rates (p = 0.555). Participants in the Harvard Catalyst KL2/CMeRIT program demonstrate greater early career success than nonparticipants though the nonparticipants also fared relatively well.

OBJECTIVES/GOALS: The objective of this research was to learn how the oncogenic transcription factor, ERG, is regulated in prostate cancer. If we could learn how ERG is regulated and which genes are important for its oncogenic phenotype in prostate cells, we could design new therapeutic strategies against ERG, which has proven to be difficult to target. METHODS/STUDY POPULATION: We conducted an shRNA screen in prostate cells to determine candidate genes and pathways that are important for ERG function. To validate the findings of the screen, we performed a variety of cell-based functional assays, including trans-well migration, wound healing, and clonogenic survival assays. To further investigate the mechanism between ERG and the genes revealed by the screen, we performed biochemical and molecular biology experiments such as Western blotting and qRT-PCR for protein and mRNA expression, co-immunoprecipitation assays to determine protein-protein interactions, and chromatin immunoprecipitation (ChIP-qPCR) to determine transcription factor binding to DNA sites. RESULTS/ANTICIPATED RESULTS: The screen revealed that genes involved in the toll-like receptor 4 (TLR4) pathway are important for ERG-mediated migration. We tested the effect of a TLR4 inhibitor on ERG function and observed decreased migration and clonogenic survival exclusively in ERG-positive cells. Expression of pMEK and pERG was reduced when TLR4 was inhibited, which suggests a mechanism in which TLR4 upregulates pMEK, leading to the phosphorylation and activation of ERG. This is supported by functional assays in which cells expressing a phosphomimetic ERG are resistant to the TLR4 inhibitor. We demonstrated that ERG drives the transcription of TLR4 and its endogenous ligands HSPA8 and BGN. Therefore, ERG can sensitize the cell to TLR4 activation by increasing the number of receptors as well as providing the ligands needed for stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: This research provides a new therapeutic pathway for treating ERG-positive patients through TLR4 inhibition. This can be beneficial because many patients become resistant to the standard therapy, leaving very few treatment options. TLR4-based therapies could provide an alternative for patients who have developed resistance.

OBJECTIVES/GOALS: My long-term career objective is to become an established independent researcher focused on understanding and modulating immune responses to biologics in order to enhance their efficacy and understand the underlying mechanisms by which these interact with the immune system. METHODS/STUDY POPULATION: In this study we will evaluate the utility of cyanuric chloride based synthetic lipids in the presentation of peptide epitopes of the gene delivery vector, adeno-associated virus (AAV). The lipopeptide conjugates will be administered to mice via liposomal formulations to assess their ability to induce immune responses by ELISA as compared to mice treated with the AAV. The three-dimensional conformation of the peptides will be evaluated using nuclear magnetic resonance to determine their similarity with the natural conformation that these peptides adapt on the viral surface. Additionally, to assess the translatability of this conjugation strategy, we will test the ability of our lipopeptides to bind to serum antibodies from human subjects. RESULTS/ANTICIPATED RESULTS: We anticipate that peptide presentation using cyanuric chloride lipids will achieve a robust response in mice following immunization. Immunizations with our lipids should induce the production of antibodies targeting AAV, albeit a milder response that the virus itself, given the complexity of viral epitopes. Nuclear magnetic resonance will inform us on how to improve the synthetic lipids to optimize peptide presentation by altering the characteristics of the lipid anchors. Finally, by using human serum to test for the ability of our lipopeptides to bind to antibodies in serum from patients positive for AAV antibodies, we can become informed on whether our strategy has utility in human studies or whether our method is limited to animal models of human disease. DISCUSSION/SIGNIFICANCE OF IMPACT: The current work seeks to develop a strategy to present peptides from a well characterized biologic, AAV, on a liposome surface. Our ultimate purpose is to employ liposomal formulations as decoys that target AAV-specific lymphocytes to improve the in vivo efficacy of AAV.

OBJECTIVES/GOALS: Our objective is to address the gaps in our epidemiologic understanding of Echinococcosis, a neglected and emergent zoonotic parasite in the US. Comparing regional differences in human cases with known Echinococcus genotypes in canine reservoirs will reveal landscape-level transmission risks. METHODS/STUDY POPULATION: Retrospective analysis of de-identified diagnosis codes for human echinococcosis in the US from 2002-2022 in a federal multi-healthcare network database (TriNetX). Analyses included Kaplan-Meier survival and specific parasite species cohort comparisons with descriptive summaries of demography and co-morbidities, and odds ratios for interventions (surgery, antiparasitics, cyst aspiration) and complications (anaphylaxis, sepsis, etc.) Mitochondrial genomes of Echinococcus spp. from next-generation sequencing ova-positive archival animal scat (National Park Service), along with sequences from published sources in the US, will be used to compare regional genotypic and reservoir host differences. RESULTS/ANTICIPATED RESULTS: Over 36,000 US patients were diagnosed with Echinococcosis in the past twenty years. Most cases had an unspecified parasite species, primarily affecting pediatrics (average age 16y +/- 10y) in the South/Southeast US, with very few interventions or medical complications reported. Patients with specific parasite species diagnoses (E. granulosus or E. multilocularis, N=500) had higher rates of mortality (11.7% at 10y), surgical (8.8%) and medical (18.2%) interventions, and complications (2%-17.2%), with demographic differences noted. Sequencing data and analysis pending for 47 Echinococcus positive wolf samples, with 78 additional sequences available for two types of Echinococcus spp. in wildlife, and lacking data for domestic dog-adapted strains in the US. DISCUSSION/SIGNIFICANCE: This study revealed a higher-than-expected burden of Echinococcosis in the US, confirming it is an under-recognized neglected and emergent disease. Future studies will address gaps regarding the origin of unspecified infections, with targeted surveillance of domestic dogs in high-risk areas.

OBJECTIVES/GOALS: Since 2013, managers of community engagement (CE) programs across the Clinical and Translational Science Award (CTSA) consortium funded by the National Center for Advancing Translational Sciences (NCATS) have convened monthly to build connections, share knowledge, and enable collaboration. METHODS/STUDY POPULATION: Notable for focus on staff leadership, the CE Brokers group has been central to the ongoing success of CTSA community engagement partnerships and approaches to research. In early 2022, a survey of the 139-member group asked about their roles and responsibilities, the ways the CE Brokers network has contributed to their hubs adoption and development of best practices and innovations, resources and lessons learned, and the creation of opportunities for members to collaboratively conduct and disseminate original research, and research on the science of community engagement. The survey also asked CE Brokers if they or their community partners are part of an underrepresented community. RESULTS/ANTICIPATED RESULTS: These demographic data will be shared, along with analyses of data on growth of the group over time, evolving themes, and a SWOT analysis completed in 2021. DISCUSSION/SIGNIFICANCE: This will provide a platform to explore new avenues for the CE Brokers and their impact within the NCATS CTSA consortium, in line with the evolving direction of the clinical and translational research enterprise.

OBJECTIVES/GOALS: The prevalence of patients with end-stage kidney disease (ESKD) in PR has increased in the last decade along with an aging population, while waitlisted patients have decreased. Our objective is to compare clinical and sociodemographic characteristics of patients with ESKD on dialysis who are waitlisted from those who are not waitlisted. METHODS/STUDY POPULATION: Retrospective study of all patients with ESKD who received dialysis in PR from 2013-2019, based on the United States Renal Data System data. Variables: (1)waitlisting status (yes or no; waitlisting date); (2)sociodemographics (age at dialysis initiation, sex, zip-code area, health insurance type, and employment status); and (3)clinical characteristics (dialysis initiation date, primary diagnosis of renal disease, comorbidities including obesity, diabetes, peripherovascular/cardiovascular disease, cancer, etc.). Statistics: (1)descriptive statistics (continuous - central tendency [mean, median] and dispersion [standard deviation, interquartile range]; and categorical [frequencies and percentages]); (2)Chi-square test, Students t-test, and Mann Whitney U test to evaluate differences. RESULTS/ANTICIPATED RESULTS: We expect patients on dialysis not waitlisted will be older and have more comorbidities than those who are on dialysis and waitlisted. DISCUSSION/SIGNIFICANCE: Kidney transplant is the choice of therapy for ESKD and its benefits are better quality of life, improved survival, and best long term-cost effectiveness. If our hypothesis is true, findings will highlight the importance of characterizing which patients with ESKD on dialysis are kidney transplant candidates when considering barriers to waitlisting.

ABSTRACT IMPACT: This work may provide new targets for vaccine and immunotherapeutic development against MRSA infections. OBJECTIVES/GOALS: Staphylococcus aureus is the leading cause of skin and skin structure infection (SSSI), a primary portal of entry for invasive infection. Patients with SA SSSI have a high 1-year recurrence. We have shown innate memory protects mice against SA SSSI. The goal of this project is to determine epigenetic mechanisms of protective memory against SA SSSI. METHODS/STUDY POPULATION: We have shown macrophages (Mf) afford protective memory against recurrent SA SSSI in mice. Priming by prior infection reduced skin lesion size and MRSA burden, which correlated with increased Mf in abscesses and lymph nodes. Priming potentiated the opsonophagocytic killing of SA by bone-marrow derived Mf (BMDM) in vitro, and their adoptive transfer into naive skin afforded protective efficacy in vivo. Here, we investigated epigenetic mechanisms of anti-SA efficacy in BMDMs. BMDM from naive (uninfected) or primed (SA SSSI) wild-type C57Bl/6 mice were cultured ex vivo. DNA from BMDM groups were isolated and analyzed for methylation changes using reduced representation bisulfite sequencing (RRBS). Pathway analyses of methylation changes were determined with Panther. RESULTS/ANTICIPATED RESULTS: Present findings indicate the protective memory afforded by BMDM was mediated by epigenetic modifications of the DNA. Using RRBS, we profiled differentially methylated regions (DMR) in DNA from naive vs. primed BMDM. Primed BMDM exhibited significantly different DMRs as compared to naive BMDM. Proximity to known genes were mapped using GREAT. Pathway analyses revealed DMRs predominant in genes integral to immune modulation, such as integrin signaling, cytokine/chemokine networks, and growth regulation. For example, SA-primed BMDM were hypermethylated proximate to GIMAP8 versus naive BMDM, suggesting repression of this protein. Gimap family ligands are small GTPase immune-associated proteins expressed in immune cells known to regulate macrophage lysosomal fusion during parasite infection. DISCUSSION/SIGNIFICANCE OF FINDINGS: These findings reveal epigenetic mechanisms of macrophage innate memory against recurrent MRSA infection. Functional testing of these genes in response to SA infection is needed to confirm their protective role. These insights may provide new targets for vaccine and immunotherapeutic development against MRSA.

Objectives/Goals: We describe the prevalence of individuals with household exposure to SARS-CoV-2, who subsequently report symptoms consistent with COVID-19, while having PCR results persistently negative for SARS-CoV-2 (S[+]/P[-]). We assess whether paired serology can assist in identifying the true infection status of such individuals. Methods/Study Population: In a multicenter household transmission study, index patients with SARS-CoV-2 were identified and enrolled together with their household contacts within 1 week of index’s illness onset. For 10 consecutive days, enrolled individuals provided daily symptom diaries and nasal specimens for polymerase chain reaction (PCR). Contacts were categorized into 4 groups based on presence of symptoms (S[+/-]) and PCR positivity (P[+/-]). Acute and convalescent blood specimens from these individuals (30 days apart) were subjected to quantitative serologic analysis for SARS-CoV-2 anti-nucleocapsid, spike, and receptor-binding domain antibodies. The antibody change in S[+]/P[-] individuals was assessed by thresholds derived from receiver operating characteristic (ROC) analysis of S[+]/P[+] (infected) versusS[-]/P[-] (uninfected). Results/Anticipated Results: Among 1,433 contacts, 67% had ≥1 SARS-CoV-2 PCR[+] result, while 33% remained PCR[-]. Among the latter, 55% (n = 263) reported symptoms for at least 1 day, most commonly congestion (63%), fatigue (63%), headache (62%), cough (59%), and sore throat (50%). A history of both previous infection and vaccination was present in 37% of S[+]/P[-] individuals, 38% of S[-]/P[-], and 21% of S[+]/P[+] (P<0.05). Vaccination alone was present in 37%, 41%, and 52%, respectively. ROC analyses of paired serologic testing of S[+]/P[+] (n = 354) vs. S[-]/P[-] (n = 103) individuals found anti-nucleocapsid data had the highest area under the curve (0.87). Based on the 30-day antibody change, 6.9% of S[+]/P[-] individuals demonstrated an increased convalescent antibody signal, although a similar seroresponse in 7.8% of the S[-]/P[-] group was observed. Discussion/Significance of Impact: Reporting respiratory symptoms was common among household contacts with persistent PCR[-] results. Paired serology analyses found similar seroresponses between S[+]/P[-] and S[-]/P[-] individuals. The symptomatic-but-PCR-negative phenomenon, while frequent, is unlikely attributable to true SARS-CoV-2 infections that go missed by PCR.