ABSTRACT IMPACT: Analyzing the types of technical assistance (basic, targeted or intensive) provided by the Opioid Response Network (ORN) to unique and hard-to-reach populations (UHRP) informs addiction health services and translational research by identifying technical assistance needs in these populations which may require a higher level of intensity. OBJECTIVES/GOALS: To improve ORN dissemination and implementation efforts, the project classifies TA requests into one of three categories: basic, targeted, and intensive. This TA Framework assists the ORN project team in understanding the level of TA required in the delivery of evidence-based practices to address opioids with communities with respect to UHRP. METHODS/STUDY POPULATION: TA requests from April 1, 2019, to April 1, 2020, were selected. The ORN classifies TA requests in one of three categories: basic (dissemination & brief consultation), targeted (services to enhance readiness and capacity), and intensive (full incorporation of innovation considering context, culture, and linguistics) (Fixsen, et. al., 2009; Becker, et al., 2020). Unique and hard-to-reach populations (UHRP) are defined based on physical location (i.e., remote or isolated), social position, or other vulnerabilities (i.e. member of an ethnic or racial minority group) (Thurman, & Harrison, 2020). ORN classifies 26 types of UHRP these types are not mutually exclusive. A frequency analysis of the UHRP types was conducted. Bivariate correlations between UHRP types that had a minimum of 30 cases were performed. RESULTS/ANTICIPATED RESULTS: Among 746 TA requests selected, 226 had missing information about UHRP types and 29 had missing information TA levels. These requests were excluded from the frequency analysis. The three most common UHRP types were people living in rural or remote areas (n=262, 50%), people who are uninsured or underinsured (n=162, 31%), and people who inject drugs (n=158, 30%). Most TA requests were targeted (69%), 23% were intensive, and 9% were basic. Bivariate correlations were performed between 21 UHRP types. Moderate (Pearson’s r=0.4-0.6) or strong correlations (r>0.6) were found for 11 occurrences for the UHRP type of ‘LGBT’, 8 for ‘Mental Illness’, and 7 for ‘Veterans’. Strong correlations were found between ‘Justice Involved’ and ‘Incarcerated’ (r=0.645), and between ‘Disabilities’ and ‘Chronic Pain’ (r=0.603). DISCUSSION/SIGNIFICANCE OF FINDINGS: There were more TA requests at targeted and intensive levels than basic levels suggesting the need for services to enhance readiness and build capacity. The moderate/strong correlations indicate that UHRP types were likely to coexist with other types. Future research can explore combining UHRP types that have moderate/strong correlations.

ABSTRACT IMPACT: This public health work contributes to the development and implementation of best practices for working with sex trafficked youth who experience placement instability and justice involvement. OBJECTIVES/GOALS: Youth removed from their home into foster care or a group home (i.e., placement instability) are vulnerable to sex trafficking. This study examines whether placement instability predicts mental health, behavioral and justice-related outcomes among sex-trafficked girls. METHODS/STUDY POPULATION: Placement instability occurs when children are temporarily or permanently removed from their home and placed in foster care or a group home. Domestic minor sex trafficking is exploitation and abuse of children for commercial sexual purposes in exchange for money or other goods/services. We hypothesize that sex trafficked girls who experience placement instability will report more mental health challenges, substance use, abuse history and justice involvement than those without placement instability. Data came from participant files in a specialty court program from 2012-2014 (N=184). Multiple sources contribute to the information contained court files; all data extracted by the research team come solely from the court files. Descriptive, bivariate, and logistic regression analyses were performed. RESULTS/ANTICIPATED RESULTS: All participants were (cis)female, 74% were African American, 96% US citizens, with average age of 16 years. Three-quarters of participants had a documented mental health challenge, such as depression and 88% reported substance use. Eighty-one percent of participants had been in a placement, with a group average of 4.5 placements. Girls with placement instability reported more mental health challenges (p<.001), substance use (p<.001), abuse (p<.001), running away (p<.001) and bench warrants (p<.001) than girls without placement instability. Logistic regression estimated housing instability was positively associated with mental health challenge, substance use, running away, number of bench warrants, and number of citations. DISCUSSION/SIGNIFICANCE OF FINDINGS: Among girls who have been trafficked, placement instability places them at greater risk for personal and behavioral challenges, including increased justice involvement. Comprehensive trauma-informed services should be provided to the family to help mitigate issues in the home.

ABSTRACT IMPACT: Identifying factors associated with opioid overdoses will enable better resource allocation in communities most impacted by the overdose epidemic. OBJECTIVES/GOALS: Opioid overdoses often occur in hotspots identified by geographic and temporal trends. This study uses principles of community engaged research to identify neighborhood and community-level factors associated with opioid overdose within overdose hotspots which can be targets for novel intervention design. METHODS/STUDY POPULATION: We conducted an environmental scan in three overdose hotspots’‘ two in an urban center and one in a small city’‘ identified by the Rhode Island Department of Health as having the highest opioid overdose burden in Rhode Island. We engaged hotspot community stakeholders to identify neighborhood factors to map within each hotspot. Locations of addiction treatment, public transportation, harm reduction programs, public facilities (i.e., libraries, parks), first responders, and social services agencies were converted to latitude and longitude and mapped in ArcGIS. Using Esri Service Areas, we will evaluate the service areas of stationary services. We will overlay overdose events and use logistic regression identify neighborhood factors associated with overdose by comparing hotspot and non-hotspot neighborhoods. RESULTS/ANTICIPATED RESULTS: We anticipate that there will be differing neighborhood characteristics associated with overdose events in the densely populated urban area and those in the smaller city. The urban area hotspots will have overlapping social services, addiction treatment, and transportation service areas, while the small city will have fewer community resources without overlapping service areas and reduced public transportation access. We anticipate that overdoses will occur during times of the day when services are not available. Overall, overdose hotspots will be associated with increased census block level unemployment, homelessness, vacant housing, and low food security. DISCUSSION/SIGNIFICANCE OF FINDINGS: Identifying factors associated with opioid overdoses will enable better resource allocation in communities most impacted by the overdose epidemic. Study results will be used for novel intervention design to prevent opioid overdose deaths in communities with high burden of opioid overdose.

ABSTRACT IMPACT: This qualitative study describes health system barriers to high-quality care for Latino older adults with Alzheimer’s Disease and Related Dementias OBJECTIVES/GOALS: Compared to non-Latino Whites, Latino older adults are more likely to receive low-quality dementia care such as high-risk medications or services. Caregivers play a critical role in managing medical care for persons with dementia (PWD). Yet little is known about the perceptions and knowledge of dementia quality of care among Latino caregivers of PWD. METHODS/STUDY POPULATION: We used a qualitative research design and conducted interviews with Latino caregivers of PWD and caregiver advocates. We recruited both from community organizations, senior centers, and clinics. Our interview guide focused on experiences of caregiving, interactions with medical system, and knowledge and experiences managing behavioral and eating problems. We used Grounded Theory methodology for coding and analysis, focusing on contrasting and comparing experiences within and between caregivers and caregiver advocates. RESULTS/ANTICIPATED RESULTS: Preliminary results from interviews with two caregivers and two caregiver advocates illustrate that caregivers of persons with dementia have a difficult time receiving high quality care from primary care clinicians. All participants noted that many primary care doctors didn’t know how to diagnose ADRD and dismissed critical symptoms as part of old age. Caregivers also reported that they wished they had more information on what to expect with ADRD disease progression, noting they received little information from the formal medical care system. With respect to behavioral problems, caregiver advocates noted that primary care doctors often did not provide non-pharmacological alternatives to behavioral problems. DISCUSSION/SIGNIFICANCE OF FINDINGS: Findings from our pilot study demonstrate that there is a clear need to train primary care physicians who serve Latino older adults on ADRD care. Improved diagnosis and management could improve outcomes among Latino older adults with dementia.

ABSTRACT IMPACT: In alignment with principles of community engaged research, feedback from participants in this research study may influence infertility clinics to offer psychological support for individuals or couples experiencing high levels of psychosocial burden when pursuing fertility procedures, especially during periods of interrupted care or uncertainty. OBJECTIVES/GOALS: 1) To assess the psychosocial impact on patients whose fertility treatments were interrupted during the COVID-19 shutdown. 2) To assess the correlation of patient perceived risk at the time of treatment cessation compared to the resumption of treatment, both during an ongoing pandemic. METHODS/STUDY POPULATION: Female patients with scheduled fertility treatments at Mayo Clinic within 6 months of 3/15/2020, were contacted through the patient portal and invited to participate in this study. Interested patients were contacted by a study staff member to obtain their consent and HIPAA authorization and to schedule a phone or Zoom interview. Semi-structured interviews were conducted individually, or in partner dyads depending upon participant preference, and were recorded with their permission. Audio recordings were professionally transcribed and de-identified. Transcripts were qualitatively analyzed using NVivo 12 based on the principles of grounded theory. RESULTS/ANTICIPATED RESULTS: 26 participants were interviewed; 20 interviews were conducted individually and 3 were conducted in dyads with their partners. Initial themes from the interviews show that COVID-19 compounded existing psychosocial burden on individuals and couples undergoing fertility treatments. Women who were older in age, had prolonged history of infertility, or multiple unsuccessful treatment cycles reported feeling an increased urgency to proceed with fertility treatments due to the time sensitive care, which outweighed the perceived risks of COVID-19 to either themselves or the potential pregnancy. Patients also reported a desire for improved communication regarding their procedures and overall well-being, as well as options for counseling services for individuals or couples undergoing fertility treatments. DISCUSSION/SIGNIFICANCE OF FINDINGS: Participants indicated the need for increased psychological support for patients pursuing fertility treatments, especially during periods of interrupted care or uncertainty, as highlighted during the COVID 19 pandemic. Offering counseling as a routine part of the treatment process may mitigate this burden.

ABSTRACT IMPACT: This research suggests that African American and Latinx smokers who bear a disproportionate burden of tobacco-related harms are able to switch to e-cigarettes that present reduced harm to the user due to their similar reinforcement profile to cigarettes. OBJECTIVES/GOALS: Complete switching from combustible to electronic cigarettes (ECs) reduces harm to the user. For ECs to be a viable substitute, they need to be rewarding enough for regular use, indicated by factors such as craving and dependence (reinforcement value). Little is known about short-term changes in reinforcement value across trajectories of EC use. METHODS/STUDY POPULATION: Participants were randomized 2:1 to switch to a nicotine salt pod system EC or continue smoking (assessment-only control) in a 6-week trial. 114 African American (n=60) and Latinx (n=54) smokers were randomized to receive ECs and are included in the current investigation. At week 6, participants were classified by use trajectory: exclusive smokers (n=16), exclusive EC users (n=32), or dual users (n=66). Participants reported on their EC, cigarette, and total nicotine dependence (cigarette + EC dependence), cigarette and EC use, and nicotine craving and withdrawal at baseline and week 6. Cotinine and exhaled carbon monoxide were assessed at baseline and week 6. RESULTS/ANTICIPATED RESULTS: Participants who completely switched from smoking to ECs (exclusive EC users) and those that partially switched (dual users), maintained cotinine levels (ps>.05) and showed reductions in cigarette dependence and withdrawal (ps<.01). However, exclusive EC users showed no significant changes in total nicotine dependence from baseline to week 6 (p=.123), while dual users showed increased total nicotine dependence (p<.001). Dual users displayed similar levels of EC dependence as exclusive EC users but a lesser reduction in cigarette dependence. Exclusive EC users and dual users showed reductions in craving and withdrawal from baseline to week 6. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study is among the first to prospectively examine changes in dependence, craving, and withdrawal among an understudied sample of smokers making a switch attempt. Smokers who completely switch to ECs maintain nicotine levels and dependence, suggesting that they have a similar reinforcement profile to cigarettes and facilitate switching.

ABSTRACT IMPACT: Understanding how perceived discrimination affects Asian Americans can help stakeholders target subgroups that are at highest risk of discrimination-related behaviors and design culturally appropriate interventions to ensure equitable access to healthcare. OBJECTIVES/GOALS: The COVID-19 pandemic has exposed longstanding anti-Asian racism in the US. Yet, effects of discrimination on Asian American health are unknown, partly because diverse Asian American populations are analyzed in aggregate. We aim to understand how perceived discrimination affects healthcare utilization among different Asian American subgroups. METHODS/STUDY POPULATION: We examine the association of perceived discrimination with healthcare utilization using the California Health Interview Survey (CHIS). In the CHIS, respondents reported whether they would’ve gotten better medical care if they belonged to a different race. We examine the association between these responses and physician visits within the past year, in the survey years 2003, 2004 and 2016-2017. We adjust for covariates based on the Andersen Health Behavior model. Subsequent modeling examines potential mediating and moderating factors such as limited English proficiency, immigration status, income, and survey year. Asian American subgroups analyzed include Asian Indian, Korean, Chinese, Filipino, Vietnamese, Japanese, and other Asian. RESULTS/ANTICIPATED RESULTS: Results will highlight how perceived discrimination incentivizes or disincentivizes certain Asian subgroups to utilize healthcare. Asian American subgroups have differing and diverse experiences with discrimination due to their historical and cultural differences; results will elucidate how discrimination affects these subgroups. Results will be compared to non-Hispanic Whites, who represent the racial group least likely to experience discrimination in the US. Mediation and moderation analysis will help understand how traditionally cited factors for healthcare utilization interact with perceived discrimination on Asian Americans. DISCUSSION/SIGNIFICANCE OF FINDINGS: Asian American subgroups are understudied, despite Asian Americans being one of the fastest growing racial groups in the US. Understanding how perceived discrimination affects Asian Americans can help stakeholders target subgroups that are at highest risk of discrimination-related behaviors and design culturally appropriate interventions.

ABSTRACT IMPACT: This work is intended to improve community engagement salons both virtually and generally in order to maximize the benefit of this vital research tool. OBJECTIVES/GOALS: The Meharry-Vanderbilt Community Engaged Research Core (CERC) provides a protocol to maintain high standards in community engagement studios, even in a virtual setting amidst a pandemic. A virtual community engagement salon was selected as a case study to evaluate outcomes. METHODS/STUDY POPULATION: A virtual salon regarding sun safety in the Latinx community was observed live via Zoom and as a recording afterward. Following dissemination and completion of the post-meeting surveys, authors compiled and reviewed the results. An assessment was developed to determine the salon’s alignment with the Meharry-Vanderbilt CERC guidelines in a virtual setting; this was designated as the primary outcome. Data from the session were compared to the available literature on the topic, which produced three subheadings to the secondary outcome essential to the success of virtual community engagement salons: researcher preparedness, participant selection, and survey importance. RESULTS/ANTICIPATED RESULTS: The CERC guidelines of the community engagement salon were met and were effectively translated into a virtual setting. The presentation given by the researcher followed all technical instructions, yet it was clear that the researcher’s demeanor and conversational soft-skills were lacking. Instead of the recommended bi-directional flow of conversation, the conversation flow shifted to a unidirectional state controlled by the participants. Following the session, only three participants completed the survey along with the researcher. This completion rate of under 50% provides limited feedback on participants’ perspectives on the session’s quality and points to improve future sessions. DISCUSSION/SIGNIFICANCE OF FINDINGS: Pre-meeting researcher preparation is necessary to engage community stakeholders effectively. The lack of completed surveys from participants suggests potential fatigue from leading a majority of the conversation. Results demonstrate that solely meeting the requirements of the CERC does not suffice.

ABSTRACT IMPACT: This work will inform and improve the way we assess and treat distress in women with endometrial cancer. OBJECTIVES/GOALS: Distress from cancer is associated with worse processes of care. Differences in outcomes by race/ethnicity in endometrial cancer (EC) are well documented, but differences in distress have not been previously explored. Here we characterize the association between race/ethnicity, distress scores, and stressors reported by patients with EC. METHODS/STUDY POPULATION: Patients presenting to a single academic outpatient gynecologic oncology practice for initial evaluation of known EC from January 2013-May 2020 were included. The electronic health record was used to abstract demographics, National Comprehensive Cancer Network Distress Thermometer and Problem List (NCCN DT) scores and stressor categories (physical, emotional, spiritual, practical, and family) from the initial encounter. Referral to support services occurs at NCCN DT score ≥4. We excluded women who received prior cancer-directed therapy and those without an initial NCCN DT score. Summary statistics were tabulated for demographics. Mann-Whitney U tests were used for inter-group difference on continuous variables and 2-sample tests for equality of proportions were used for binary variables. RESULTS/ANTICIPATED RESULTS: 412 non-Hispanic White (NHW, mean age 63) and 149 non-Hispanic Black (NHB, mean age 65) women were included in our analysis. More NHB women presented with high-grade EC (53.7%) vs NHW women (21.9%) and fewer NHB women were privately insured (32% vs 52%). Median distress scores were higher in NHW women compared to their NHB counterparts (4 vs. 2, p<0.001) and NHB women were more likely to report a distress score of 0 compared to their NHW counterparts (32% vs 19%, p=0.001). 50.5% NHW women had a score ≥4 and thus qualified for referral to services compared to 20.7% of NHB women (p=0.02). Of those referred, NHB and NHW women declined referral to support services at similar rates (35.1% vs 34.5%; NS). There was a significant difference in the median number of stressors reported by NHW and NHB women, (4 vs 3 stressors; p=0.02). DISCUSSION/SIGNIFICANCE OF FINDINGS: The NCCN DT, a widely used tool in cancer clinics, may fail to adequately measure distress in NHB women presenting with a diagnosis of EC, despite >30% more high-risk histology cancers in this cohort. This difference leads to disparities in referral to additional support services, which may affect quality of care and quality of life.

ABSTRACT IMPACT: Understanding gene expression changes after viral vaccination and booster may help predict vaccine efficacy. OBJECTIVES/GOALS: Utilize a systems biology approach to identify gene expression changes after administration of Zaire Ebola virus glycoprotein expressed in a Chimp Adeno3 vector (ChAd3-EBOZ) and either boosted ˜7 weeks later with modified vaccinia Ankara MVA expressing Zaire and Marburg GPs plus Tai forest NP (MVA-BN ®Filo) or given saline (placebo). METHODS/STUDY POPULATION: As part of the phase 1b, open-label vaccination trial of ChAd3-EBO-Z in Mali, West Africa, peripheral blood mononuclear cells were isolated from eight volunteers for whole genome transcriptomics analysis. Four subjects received the MVA-BN ®-Filo booster and four received saline. Samples were taken prior to receipt of the booster or placebo, as well as 1, 7, and 14 days afterwards. Significant differentially expressed genes were identified using RNA-seq between baseline and post-MVA-BN ®Filo. Functional enrichment analysis against the GO Ontology Database and the Immune Signatures C7 collection of MSigDB (ImmuneSigDB) was performed. These differentially expressed genes were also examined for associations with Ebola antibody titers and cell-mediated immune responses. RESULTS/ANTICIPATED RESULTS: The majority of gene expression changes occurred on day 1 post-MVA-BN ®-Filo administration. 870 genes had significantly different expression when day 1 samples were compared to pre-booster baseline (791 upregulated/79 downregulated). Those upregulated genes are mainly involved type I interferon and regulation of viral life cycle pathways. The downregulated genes are involved in regulation of cellular defense response, lymphocyte mediated immunity. Comparing to the C7 Immune Signatures collection datasets, we identified more than 100 upregulated genes from 6 studies of yellow fever vaccination that were also significantly upregulated in our study. The top enriched ontological pathway of those genes is cellular response to type I Interferon. DISCUSSION/SIGNIFICANCE OF FINDINGS: The use of a systems biology approach to compare gene expression changes among vaccine studies utilizing whole genome transcriptomics data allows the identification of genes involved in the immune response to vaccination and might aid in predicting vaccine efficacy.

ABSTRACT IMPACT: This project seeks to identify unique host responses that are biomarkers for specific urethral pathogens, and which can be used in the development of point-of-care (POC) STI diagnostics. OBJECTIVES/GOALS: How Chlamydia trachomatis (CT) and other common STIs, e.g. Neisseria gonorrhoeae, evade immunity and elicit pathology in the male urethra is poorly understood. Our objective is to determine how STI-infected urethral epithelial cells, as well as the uninfected ‘bystander’ cells with which infected cells communicate, respond to CT and other STIs. METHODS/STUDY POPULATION: We evaluated how immortalized urethral cell lines - including transduced human urethral epithelial cells (THUECs) - respond to increasing doses of CT infectious particles using in vitro one-step progeny assays performed in the presence or absence of cycloheximide, a drug that inhibits eukaryotic protein synthesis. We will perform concurrent single-cell RNA sequencing (scRNA-seq) and multiplex cytokine analyses to determine how different CT doses impact the transcriptomes of infected and bystander urethral epithelial cells and modulate cytokine production of the overall monolayer. Results of these experiments will inform the feasibility of performing similar analyses in situ using urethral swabs from men with clinically diagnosed urethritis. RESULTS/ANTICIPATED RESULTS: Our results demonstrate that immune-competent urethral cell monolayers strongly resist CT infection, unless most of the cells are simultaneously infected. This suggests that uninfected bystander cells sense CT-infected cells and secrete soluble factors that may act to limit CT proliferation in infected cells and to inform remaining uninfected cells that a potential pathogen is present. We anticipate that our scRNA-seq and cytokine analyses will identify both specific effector pathways that protect against CT and intracellular signals that modulate them. We speculate that these pathways and signals may differ during infection with CT and other STIs. Importantly, we anticipate that our in vitro model of CT infection will be highly representative of in situ immune responses observed in urethras of infected men. DISCUSSION/SIGNIFICANCE OF FINDINGS: In men, common STIs including CT are usually managed syndromically due to a lack of POC diagnostics. By determining how STIs elicit urethral inflammation and identifying countermeasures that STIs use to evade urethral immunity, we can identify host responses that serve as biomarkers for urethritis, generally, and for specific urethral pathogens.

ABSTRACT IMPACT: We present preliminary data and an outlined approach to assess peripheral immune changes associated with PTSD in a clinical setting and in a pre-clinical rat model of PTSD. OBJECTIVES/GOALS: We report our methodology and findings indicating a relationship between CNS dopamine signaling and peripheral immune cell populations and propose to extend this methodology to a PTSD patient population to elucidate immune-brain connections in this disorder. METHODS/STUDY POPULATION: Using an IRB-approved protocol in collaboration with a board-certified psychiatrist, we will recruit PTSD patients undergoing treatment, newly diagnosed drug-naiive PTSD patients, and age-matched healthy controls. Flow cytometry will be used for immunophenotyping on blood samples from each group. To complement this data, we will also measure serum cytokine levels in each group. In order to elucidate the connection between the observed immunophenotypes in the PTSD population and CNS neurotransmitters levels, we will employ a rodent model of PTSD and high-pressure liquid chromatography to measure dopamine levels in tandem with peripheral immune changes. RESULTS/ANTICIPATED RESULTS: In both humans and rodents with low CNS dopamine, an expansion of monocyte-derived suppressor cells was observed via flow cytometry. We anticipate that human PTSD patients will exhibit a similar expansion in suppressive immune cells’‘ in agreement with existing literature suggesting a chronic inflammatory state in PTSD. Moreover, in an animal model of PTSD we anticipate an inverse correlation between the CNS dopamine levels and the size of the immune suppressor cell population. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings will indicate whether altered dopamine neurotransmission underlies peripheral immune system changes in the context of PTSD models and human patients. Thus, these findings will provide an alternative avenue for future investigations on the role of the immune system in PTSD.

ABSTRACT IMPACT: Melanoma leptomeningeal disease (LMD) is a devastating subtype of central nervous system (CNS) metastatic disease that is associated with limited treatment options and an extremely poor prognosis, thus requiring the development of preclinical models of LMD for therapeutic development. OBJECTIVES/GOALS:

1. 1. Develop an immunocompetent murine model of melanoma LMD with tumors bearing genetic mutations commonly found in patients, specifically BRAF(V600E)/PTEN-/-

2. 2. Assess the safety of intrathecal (IT) immunotherapy, specifically anti-PD1 antibody (aPD1)

3. 3. Evaluate the therapeutic efficacy of IT aPD1 checkpoint blockade in murine melanoma LMD METHODS/STUDY POPULATION: To develop BRAF(V600E)/PTEN-/- LMD models, we acquired BP, D4M, and D4M-UV2 (irradiated) murine melanoma cell lines and luciferase-tagged them. 1.5x10^4 cells were suspended in 10 uL serum-free media and injected into the cisterna magna of female C57BL/6 mice. Brain and spinal cord were harvested for histologic assessment once mice were moribund. To assess safety of IT aPD1, we injected IT control IgG or IT aPD1 (13 ug, 26 ug, 39 ug) and monitored weights or harvested at days 7 or 14 for IHC staining of inflammation markers. To evaluate therapeutic efficacy of IT aPD1, BP cells were directly injected as above. After 3 days, mice underwent imaging to confirm tumor uptake and randomization to receive 13 ug IT control IgG or aPD1 once + 200 ug systemic (Sys) control IgG or aPD1 (days 0, 3, and 5), and then monitored for survival. RESULTS/ANTICIPATED RESULTS: For LMD development, all mice survived cisternal injection of BP, D4M, and D4M-UV2 cells and median survival was 17, 19, and 30 days, respectively. Presence of leptomeningeal deposits was confirmed for all tumor-bearing mice by IHC for MART1. For safety of IT aPD1, all mice survived the procedure and no mice displayed morbidity or >10% weight loss over 14 days of observation. IHC assessment of brain and spinal cord samples from mice treated with 13 ug aPD1 revealed focal ischemia related to injection site and no other signs of neurological damage or inflammation. IT aPD1 treatment of mice with BP leptomeningeal tumors demonstrated no significant survival advantage, although both IT aPD1 +/- Sys aPD1 had mice live up to days 29 and 26, respectively, compared to both IT control IgG +/- Sys aPD1, for which all mice died by day 22. DISCUSSION/SIGNIFICANCE OF FINDINGS: We demonstrate that cisternal injection of murine BRAF(V600E)/PTEN-/- melanoma cell lines yield LMD with reproducible survival and that treatment with IT aPD1 in this model is feasible and safe. Together these findings establish a new model to facilitate the development of more effective immunotherapy strategies for melanoma patients with LMD.

ABSTRACT IMPACT: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and benefit to patients; our work will identify novel mediators of this process for both therapeutic potential and underlying mechanism, allowing for both potential immediate clinical utility and identification of future targets based on new mechanistic insights. OBJECTIVES/GOALS: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and clinical benefit to the majority of patients. We aim to interrogate a novel mediator of dysfunction identified from transcriptome analyses, pyruvate kinase muscle isozyme isoform 2 (PKM2), for therapeutic utility and underlying mechanism. METHODS/STUDY POPULATION: Transcriptome analyses of CD8+ lymphocytes from tumor-bearing lungs from both murine KrasG12D p53-/- and human non-small cell lung cancer (NSCLC) patients were performed, and differentially expressed genes identified. Flow cytometric analyses for PKM isoform expression and effects of target knockdown on accumulation of dysfunctional characteristics, including checkpoint and transcription factor expression, proliferation, and cytokine production, were performed using an in vitro co-culture of murine antigen-specific T (OT-I) cells and antigen-expressing NSCLC (HKP1-ova) cells. In vivo examination of the same was performed using adoptive transfer of OT-I cells into immunocompetent recipient mice with engraftment of HKP1-ova cells, and subsequent evaluation of mouse survival and T cell phenotypes. RESULTS/ANTICIPATED RESULTS: Transcriptome analyses demonstrated that PKM expression was upregulated in dysfunctional T cells from both murine and human samples. This was confirmed both in vitro with co-culture and in vivo with adoptive transfer approaches, with both activated and dysfunctional OT-I cells expressing higher levels of isoform 2 of PKM than naive OT-I cells. Expression of PKM2 mimicked the kinetics of the transcription factor Tox, a known driver of dysfunction, and knockdown of PKM2 resulted in reduced granzyme B expression, and increased proportions of progenitors with fewer terminally differentiated dysfunctional cells. Knockdown of PKM2 in adoptively-transferred OT-I cells led to enhanced tumor control; results are being extended to other tumor models, and T cells metabolically profiled with PKM2 manipulation. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work identified a novel mediator of dysfunction whose targeting has the potential to enhance anti-tumor immunity. Mechanistically, targeting PKM2 led to altered T cell differentiation to a dysfunctional state, linking metabolic phenotypes to these traits and underlining the importance and therapeutic potential of T cell metabolic pathways.

ABSTRACT IMPACT: Our goal is to identify bacterial biomarkers of adverse Clostridioides difficile infection outcomes OBJECTIVES/GOALS: We characterized microbiota features of Clostridioides difficile infections (CDIs) and will investigate the association between bacterial taxa and adverse outcomes, which includes severe and recurrent CDIs. METHODS/STUDY POPULATION: 1,517 stool samples were collected from patients diagnosed with a CDI at the University of Michigan along with 1,516 unformed and 910 formed stool control samples. We characterized the microbiota of the 3,943 stool samples by sequencing the V4 region of the 16S rRNA gene and used the Dirichlet Multinomial Mixtures method to cluster samples into community types. Severe CDI cases were defined using the Infectious Diseases Society of America criteria and recurrent CDIs were defined as CDIs that occurred within 2-12 weeks of the primary CDI. We will use machine learning to examine whether specific bacterial taxa can predict severe or recurrent CDIs. We will test 5 machine learning models with 80% training and 20% testing data split. RESULTS/ANTICIPATED RESULTS: Similar to findings from a previous study with 338 samples, we found there was no difference in diversity between CDI cases and unformed controls (Inverse Simpson index, p > 0.5) and samples from the 3 groups (CDIs, unformed controls, and formed controls) clustered into 12 community types. To investigate the bacterial taxa that are important for predicting adverse CDI outcomes, we will select the best machine learning model based on performance and training time and examine how much each feature contributes to performance. We anticipate the large number of CDI cases in our cohort and robust machine learning approaches will enable us to identify more bacteria associated with adverse outcomes compared to other studies that have attempted to predict CDI recurrence with fewer CDI cases. DISCUSSION/SIGNIFICANCE OF FINDINGS: Adverse CDI outcomes are a significant source of the morbidities, mortalities, and healthcare costs associated with CDIs. Identifying bacterial biomarkers of severe and recurrent CDIs could enhance our ability to stratify patients into risk groups and may lead to the development of more targeted therapeutics.

ABSTRACT IMPACT: Identification of microRNA (miRNA) associated with neuropathic pain after spinal cord injury (SCI) will elucidate underlying epigenetic mechanisms contributing to its development and identify targets for intervention to optimize treatment strategies and outcomes. OBJECTIVES/GOALS: Approximately 70% of individuals with SCI experience neuropathic pain, which is refractory to pharmacologic intervention, and can reduce overall health and wellbeing. This study aims to identify predictive miRNA biomarkers of neuropathic pain after SCI to identify targets for the development of efficacious interventions. METHODS/STUDY POPULATION: Blood samples and clinical outcome measures were collected from adult participants with SCI with neuropathic pain (n = 28) and without neuropathic pain (n = 15). The sample population consisted of a mean age of 39 (SD = 12.12), 8 female (20%), with 13 classified as acute SCI (within 3 months post injury) and 30 as chronic SCI (> 3 years post injury). Pain presence, type, and intensity were assessed with the International Spinal Cord Injury Basic Pain Dataset (ISCIBPDS). Serum miRNA sequencing counts were produced from blood samples. Fold change and independent t-tests assessed differential expression between those with and without neuropathic pain, and those with chronic or acute SCI. Linear regression was performed to explore the relationship between miRNA expression and ISCIBPDS pain intensity ratings. RESULTS/ANTICIPATED RESULTS: In individuals with SCI, significant downregulation of expression of miR-338-5p was present in those with neuropathic pain compared to those without neuropathic pain (fold change = 0.81, p = 0.04). A significant relationship between expression of miR-338-5p and highest reported neuropathic pain intensity on the ISCIBPDS was identified (R2 = 0.15, F = 7.32, p < 0.01). Covariates of sex, age, and years post injury were not found to significantly influence the relationship between miRNA expression and ISCIBPDS intensity ratings. No significant differences in miR-338-5p expression were identified between participants with acute and chronic SCI, or with nociceptive pain ratings, demonstrating specificity of the relationship between miR-338-5p differential expression with pain of a neuropathic nature. DISCUSSION/SIGNIFICANCE OF FINDINGS: These findings, along with validated targets of miR-338-5p in the NF-KB neuroinflammatory signaling pathway, suggest that miR-338-5p may serve a neuroprotective role in modulating neuroinflammation, and that its downregulation may result in maladaptive neuroplastic mechanisms contributing to the development of neuropathic pain after SCI.

ABSTRACT IMPACT: Extrapolating from mouse data we explored eosinophil content in human adipose tissue and its effect on adipocyte biology potentially leading to the discovery of novel therapeutic targets for treatment of obesity and insulin resistance. OBJECTIVES/GOALS: The interaction between immune cells and adipose tissue (AT) in obesity has not been fully elicited. Mouse models of diet-induced-obesity show AT resident eosinophils (EOS) help preserve insulin sensitivity (IS). As data in human obesity are lacking, here we explored AT-EOS content and their role in AT metabolism in subjects with and without obesity. METHODS/STUDY POPULATION: We recruited lean (L) subjects and patients with obesity (Ob) to undergo abdominal subcutaneous AT biopsy and evaluation of insulin resistance (IR) by determination of HOMA-IR. Circulating EOS were isolated from all participants under fasting conditions and exposed to high glucose (HG) or high lipids (HL) for 4 hrs. AT EOS number was assessed via FACS analysis. Circulating EOS and AT mRNA was assessed by qPCR for multiple genes involved in inflammation and cell migration. To evaluate the effect of EOS on primary human adipocytes, in vitro cultures were exposed for 4 days to either interleukin-4 (IL-4), interleukin-13 (IL-13) or to human EOS. Adipocytes mRNA levels were evaluated for genes involved in adipogenesis and lipid metabolism. RESULTS/ANTICIPATED RESULTS: 16 lean, IS subjects (BMI 22.5+ 0.4kg/m2) and 22 age-matched IR patients with obesity (BMI: 38.9 + 1.0kg/m2) participated. We observed a ratio of 2:1 in AT EOS content of L vs Ob subjects (P<0.03). To assess the reduced AT-EOS content in obesity, we evaluated expression of Chemokine-C receptor 3 (CCR3) in circulating EOS. We show decreased CCR3 mRNA levels in Ob vs L subjects (P=0.006). We expect HL in vitro experiments on peripheral EOS of L subjects to affect CCR3 mRNA levels. In AT of Ob subjects, we found a significant decreased expression of Eotaxin 2, the main EOS chemokine binding CCR3 expressed on EOS. Preliminary data from in vitro primary adipocytes culture suggest for IL-4 and IL-13 to increase mRNA level of Peroxisome Proliferator Activated Receptor Gamma (PPARG), the master regulator of adipogenesis. DISCUSSION/SIGNIFICANCE OF FINDINGS: Comparable to animal studies, we found a decrease of AT-EOS content in patients with obesity. Alterations in CCR3/Eotaxin 2 signaling may be involved. IL-4 &IL-13 are secreted predominantly by EOS and appear to directly regulate gene expression in human adipocytes. These data represent the first evidence for a novel role of EOS in human AT biology.

ABSTRACT IMPACT: Use of this novel murine model of inflammatory bowel disease (IBD) and C. difficile infection (CDI) will aid in developing new clinical approaches to predict, diagnose, and treat CDI in the IBD population. OBJECTIVES/GOALS: IBD is associated with intestinal inflammation and alterations of the gut microbiota, both of which can diminish colonization resistance to C. difficile. Here, we sought to determine if IBD is sufficient to render mice susceptible to C. difficile colonization and infection in the absence of other perturbations, such as antibiotic treatment. METHODS/STUDY POPULATION: C57BL/6 IL-10-/- mice were colonized with Helicobacter hepaticus to trigger colonic inflammation akin to human IBD. Control mice, not colonized with H. hepaticus, were pretreated with the antibiotic cefoperazone to render the gut microbiota susceptible to CDI. Mice were then gavaged with spores of the toxigenic C. difficile strain VPI 10463 and monitored for C. difficile colonization and disease. The fecal microbiota at the time of C. difficile exposure was profiled by 16S rRNA gene sequencing and analyzed using mothur. Statistical analyses were performed using R. RESULTS/ANTICIPATED RESULTS: Mice with IBD harbored significantly distinct intestinal microbial communities compared to non-IBD controls at the time of C. difficile spore exposure (14 days post-IBD trigger). Mice with IBD were susceptible to persistent C. difficile colonization, while genetically identical non-IBD controls were resistant to C. difficile colonization. Concomitant IBD and CDI was associated with significantly worse clinical and intestinal disease than unaccompanied IBD. DISCUSSION/SIGNIFICANCE OF FINDINGS: Patients with IBD who develop concurrent CDI experience increased morbidity and mortality. These studies in a novel mouse model of IBD and CDI emphasize the dual importance of host responses and alterations of the gut microbiota in susceptibility to C. difficile colonization and infection in the setting of IBD.

ABSTRACT IMPACT: The ability to restore mitochondrial health in neurons derived from FXTAS patient-induced pluripotent stem cells by novel natural compounds is critically important to the management of patients experiencing this syndrome and other Fragile X associated disorders. OBJECTIVES/GOALS: The goal of this research is to assess the biological potency of MAM, NADA and MAM analogues’ neuroprotective capacity with respect to mitochondrial damage, and antioxidant properties that can restore mitochondrial health in patients with FXTAS. METHODS/STUDY POPULATION: To establish mitochondrial dysfunction, normal human cell lines and human induced pluripotent cells will be exposed to multiple concentrations of glucose/ glucose oxidase (GluOx) at several time points to induce varying intensities of oxidative stress. The degrees of oxidative stress will be measured by apoptosis and mitochondrial reactive oxygen species (ROS) production. N-arachidonoyldopamine (NADA), macamides (MAM) and its analogue compounds, effective against oxidative damage in mitochondria, will be used to rescue glucose oxidase induced oxidative damage in both cell lines. To test the ability of these drugs to restore mitochondrial health, cell viability and cellular superoxide production will be assessed by propidium iodide and the MitoSox fluorescence assay, respectively. RESULTS/ANTICIPATED RESULTS: We anticipate that GluOx at varying concentrations and time points will proportionally increase levels of apoptosis and mitochondrial ROS, reflective of mitochondrial damage, with the most severe dysfunction occurring at the maximum dose of 40µM and the longest duration of 72-hr exposure. Moreover, administration of NADA, MAM, and MAM analogues at seven concentrations, ranging from 10-8 to 10-5 M in half-log increments, will successfully treat the oxidative defects induced in the cell lines by decreasing apoptosis, and superoxide production, and increasing cell viability. DISCUSSION/SIGNIFICANCE OF FINDINGS: This research allows for the development of an in vitro neuronal model of FXTAS, lends flexibility to testing therapeutics, and expands the discovery of mitochondrial biomedical markers for the syndrome. Data generated should inform mechanistic studies of the relationship between mitochondrial damage and FXTAS-induce neurodegeneration.

ABSTRACT IMPACT: This unique approach has the capability to elucidate the pathological mechanisms underlying traumatic brain injuries and neurodegeneration, both separately and in concert, while simultaneously providing a semi-high throughput model for investigating potential pharmaceutical interventions: discoveries that would have major translational implications and a significant impact worldwide. OBJECTIVES/GOALS: We aim to improve our understanding of the mechanisms behind the development of neurodegenerative diseases by utilizing the link between traumatic brain injuries and demonstrated biomarkers with our innovative TBI on a chip model. With this tool, we hope to provide new pathological insights and explore potential pharmaceutical interventions. METHODS/STUDY POPULATION: E16 murine cortical networks were cultured onto reusable, optically transparent MEAs (fabricated in-house), and subjected to a clinically-relevant range (30-300g) of impact g-forces, utilizing our exciting new in vitro model of trauma (TBI on a chip) with real-time electrophysiological and morphological access. Impacts were systematically applied at varying intensities, repetitions, and time points, and fixed 24-hours post. Basic immunocytochemical techniques were used to investigate post-impact levels of acrolein, an established biomarker of both post-TBI oxidative stress and neurodegeneration (ND), and compared to procedurally and age-matched non-impact control networks. In addition, several other TBI/ND biomarker investigations are in progress (βA42, α-synuclein, and phosphorylated tau). RESULTS/ANTICIPATED RESULTS: Impact experiments revealed significant, force-dependent increases of acrolein (acrolein-lysine adducts) at 24hrs post impact, indicative of impact-linked neuronal degeneration. These changes were amplified by the following manipulations: increasing g-force exposure (30-250 g); the rapid (4-6 sec interval) application of multiple impacts (1, 3, 5 and 10x); and exposure to 40 mM EtOH (10 min duration immediately following impact). Further, we demonstrate the enhancement of injury recovery as a function of: increasing time intervals between repeated hits; Hydralazine exposure. In addition, conditioned media from maximally-impacted cultures can cause acrolein elevation when introduced to non-impact, control networks, indicating acrolein’s role as a diffusive-factor in post-TBI secondary injuries. DISCUSSION/SIGNIFICANCE OF FINDINGS: This novel approach provides unprecedented resolution, and is improving our understanding of the pathological mechanisms underlying both TBI and ND. Combined with our established in vivo models of trauma and computer modeling, we hope to better guide our translational laboratory endeavors and help improve clinical diagnoses and treatments.