OBJECTIVES/SPECIFIC AIMS: Glioblastoma (GBM) carries a prognosis of 14.6 months mean survival despite maximal surgical, chemotherapeutic, and radiation therapy. The pericyte is a recently characterized cell shown to be a critical component of cerebral vessel physiology and pathology. Importantly, alterations in pericyte densities have shown resulting changes in breast and lung tumor growth. We leverage transgenic pericyte-deficient mouse models to evaluate resulting behavior of implanted patient-derived GBM. METHODS/STUDY POPULATION: Patient-derived, green fluorescent protein labeled, GBM will be implanted in right frontal bregma of both 6-month old pericyte-deficient (PDGFR+/−) mice and age-matched wild-type littermate controls (IACUC 20755, IRB 16-00929), which are immunosuppressed via daily intraperitoneal cyclosporine injection. In total, 30 mice of both genders are included in tumor and control cohorts. Fixed cortical sections following 3-week period will be stained for pericytes (NG2), endothelium (CD31), hypoxia (piminidazole), and tumor size. One-way ANOVA with will used to compare groups using SAS software (Cary, NC). RESULTS/ANTICIPATED RESULTS: Feasibility studies show robust in vitro growth of patient-derived GBM cells, showing continued growth over 10 cellular division passages. Lentivirally transduced GFP reveals reliable tumor tracking both in vitro and in vivo. Transgenic mice at 6 months display reproducibly decreased pericyte and microvascular density in triplicate. Wild-type mice tolerate tumor injection up to three weeks with visible tumor growth, peritumoral hypervascularity, and no evidence of mouse neural dysfunction. With current cohorts recently implanted with tumor, we anticipate a significant decrease in tumor size with Cohen’s d effect size of 0.5 in GBM implanted in pericyte-deficient mice when compared to control. Effect sizes are based moderate to large (effect size 0.5–0.8) reductions of in vitro GBM growth in vascular gene (TGF-β knockdown studies). In addition, tagged tumor-derived pericytes should comprise a greater proportion of new vasculature in pericyte-knockdown mice to overcome host pericyte depletion. Finally, tumors in transgenic mice should show increased hypoxia from limitations in angiogenesis. DISCUSSION/SIGNIFICANCE OF IMPACT: Feasibility studies show successful tracking of fluorescently tagged-patient derived GBM samples in transgenic mice with decreased vasculature. GBM grafts show no evidence of immunogenic response with cyclosporine protocol. Successful limitation of tumor size with reduced pericyte density will provide support to increasing study of blood-brain barrier, stem cell activity and inflammatory activity of pericyte microenvironments altering GBM behavior. Furthermore, implementation of known pericyte targeted therapies, such as imantinib, can be evaluated for GBM patient treatment efficacy. Studies with assembled clinical translational research scholar mentorship team will allow the principal investigator to develop an independent career with laboratory focused on contributing to improved patient outcomes, translating successful pericyte-targeted results to patient trials.

OBJECTIVES/GOALS: Following stroke, complement-dependent neuroinflammation exacerbates secondary injury and worsens acute and chronic outcomes. We have shown that an injury site-targeted complement inhibitor (B4Crry), that targets specifically to the ischemic brain, inhibits complement activation leading to improved outcomes. Stroke comorbidities have been shown to promote a pro-inflammatory environment in the brain and systemically, and to exacerbate inflammatory responses after injury. We investigated the impact of age and smoking on acute outcomes after stroke and assessed whether increased complement activation contributes to the worsening outcomes with these stroke comorbidities. METHODS/STUDY POPULATION: Mouse brain endothelial cells (bEnd3) were exposed to hypoxia followed by exposure to serum that was derived from either cigarette smoke (CS)-exposed mice or naïve mice, and IgM and C3d deposition assessed. Adult (12 weeks) and aged (1 year) mice were subjected to 1h transient middle cerebral artery occlusion. Animals were exposed to CS for 3-6 months (5hr/day, 5days/week) by burning 3R4F cigarettes using a smoking machine. Animals were treated with B4Crry or vehicle intravenously 2h post-MCAO. Survival analysis and neurological deficit scores were performed up to 7 days. Brains were extracted for histological and molecular analyses. RESULTS/ANTICIPATED RESULTS: Following hypoxia, bEnd3 cells exposed to serum from CS-exposed mice had higher C3d and IgM deposition compared to naïve serum. Older and CS-exposed mice had significantly worse neurological deficits and mortality compared to younger adults post-MCAO. B4Crry reduced mortality and motor deficits in young, old and old+CS mice with a higher effect size in comorbid animals. Age and/or CS exposure resulted in larger infarct volumes, and increased levels of C3d deposition and microglial activation compared to young adults, but aged/CS animals treated with B4Crry fared comparable to young adults. DISCUSSION/SIGNIFICANCE OF IMPACT: The pro-inflammatory effects of aging and smoking contribute to worse stroke outcomes, and these effects can be successfully mitigated by injury site-targeted complement inhibition.

OBJECTIVES/GOALS: Develop a strategy to restrict GDNF diffusion at an injected CNS tissue site for dopamine neuron rescue by endowing it with binding affinity for carbohydrates that are abundant on the cell surface and extracellular matrix. METHODS/STUDY POPULATION: GDNF will be fused to galectin-3 (G3), a human protein that binds to β-galactoside residues of cell surface and matrix glycoproteins. We characterized the binding of G3 fusion proteins to various glycoproteins and primary human myeloid cells. We incubated G3 fusions with CNS tissue ex vivo to measure their binding and depth of penetration via diffusion. We next plan to administer GDNF-G3 via CNS intracranial infusion in a murine PD model and then conduct behavioral PD phenotype testing via rotarod and pole descent to compare to non-parkinsonian controls. We will further examine the effects of GDNF-G3 on degeneration using immunohistochemical examination of post-mortem brain tissue. RESULTS/ANTICIPATED RESULTS: Based on results from previous clinical trials of GDNF delivery, we anticipate that a successful intervention using GDNF-G3 will result in rescue of midbrain dopaminergic neurons in a murine PD model. In murine CNS tissue, we observed binding to glycans at the tissue surfaces when incubated with G3 fusion proteins ex vivo, suggesting GDNF-G3 will remain localized to the injection site. Next we will administer GDNF-G3 via CNS intracranial infusion in a murine PD model and assess efficacy by behavior and histopathology. GDNF-G3-mediated dopamine neuron rescue are expected to slow or reverse the progression of PD in these animal models. DISCUSSION/SIGNIFICANCE OF IMPACT: PD treatments focus on symptomatic relief. Standard therapies have not been efficacious in rescuing of dopaminergic neurons. GDNF-G3 administered at the site of neurodegeneration would represent a milestone on the path to treating PD pathology and address limitations of GDNF delivery.

OBJECTIVES/SPECIFIC AIMS: Chemotherapy-related cognitive impairment (CRCI) affects 15-35% of breast cancer survivors and constitutes a significant challenge for survivor quality of life. Among older breast cancer survivors who received chemotherapy treatment, carriers of at least one ɛ4 allele of the APOE gene, which encodes apolipoprotein E, are at higher risk for developing CRCI than non-carriers. APOE4 is well characterized as the strongest genetic risk factor for Alzheimer’s disease, but how it contributes to CRCI is not yet understood, and no animal models of APOE genotype and CRCI have yet been established. To better understand how APOE4 acts as a risk factor for CRCI, we used APOE targeted replacement (TR) mice to develop a model of its effects on cognition following treatment with doxorubicin, a chemotherapy drug commonly used in breast cancer treatment. METHODS/STUDY POPULATION: Twelve-to-thirteen month old APOE3 and APOE4 targeted replacement mice expressing human APOE3 or human APOE4 under control of the endogenous murine promoter were treated with 10 mg/kg doxorubicin or equivolume saline given via two IP injections spaced one week apart. One week post-treatment, mice were tested using Open Field and Elevated Zero apparatuses to assess baseline locomotive activity and anxiety and exploratory behaviors. Five weeks post-treatment, mice were assessed using the Barnes Maze over four days of training trials and one 72 hour memory probe. RESULTS/ANTICIPATED RESULTS: We found no differences in Open Field and Elevated Zero behavior, indicating limited influence of doxorubicin treatment on locomotive and anxiety behaviors in both genotypes. During Barnes Maze training, APOE4 mice treated with doxorubicin showed increased latency compared to untreated APOE4 mice as well as treated and untreated APOE3 mice, indicating deficiencies in spatial learning. In APOE3 mice, no differences in performance were seen between doxorubicin-treated and untreated mice (n = 15-16/group, p <.0001). DISCUSSION/SIGNIFICANCE OF IMPACT: These results indicate that APOE4 targeted replacement mice have specific cognitive vulnerabilities to doxorubicin treatment that can be reliably detected using the Barnes Maze assessment. Future directions include experiments to determine how other chemotherapy drugs or drug combinations impact cognition of APOE4 mice. Ultimately this model may be used to assess preventive measures or therapies for CRCI in the vulnerable APOE4 carrier population with the ability to validate cognitive impacts of these interventions.

OBJECTIVES/GOALS: An increasing number of hospitals and provider groups are consolidating into larger health systems, which hold potential to improve access to and quality of surgical cancer care through clinical integration across sites. In order to study clinical integration, we sought to develop: METHODS/STUDY POPULATION: Hospital data from the American Hospital Association were merged with data from the Agency for Healthcare Research and Quality’s Compendium of United States Health Systems. For each health system with more than one acute care hospital, the hospital with the highest surgical volume (inpatient and outpatient) was categorized as the hub hospital while all other hospitals were categorized as spokes. We evaluated the concentration of case volumes at hub versus spoke hospitals and compared characteristics of these hospitals and their surrounding communities using univariate and multivariable logistic regression analyses. RESULTS/ANTICIPATED RESULTS: Within 624 health systems containing 3,554 hospitals, 355 hospitals were characterized as hub hospitals and had 2,645 affiliated spoke hospitals (median 17 spokes per hub, range 2-151). Hub hospitals performed a median of 68% of all surgical cases (25th-75th percentile 44-87%) and were concentrated in metropolitan (88.5%) and urban areas (11.5%) with none in rural areas; spoke hospitals were located in metropolitan (67%), urban (28%) and rural (5%) areas. On multivariable analysis, spoke hospitals were more often located in rural and small urban counties (OR 9.49, CI 4.57-19.70) and took care of a higher percentage of patients with less than high school education (OR 1.06 for each 1% increase, CI 1.03-1.10) but with lower poverty rates (OR 0.90 for each 1% increase in % poverty, CI 0.86-0.95). DISCUSSION/SIGNIFICANCE: For integrated health systems with multiple acute care hospitals, surgical volume is highest at a single hub hospital, supporting use of a hub-spoke taxonomy. Patient populations in counties with hub versus spoke hospitals differ in urban-rural location, poverty rates, and education level, which may impact access to quality care.

OBJECTIVES/GOALS: The goal of this project is to apply wastewater-based epidemiology to determine the trends of NTS through analysis of influent wastewater, and to isolate and identify circulating serotypes among samples that are representative of the human population in Gainesville, FL. METHODS/STUDY POPULATION: This study is being conducted in Gainesville, FL, a suburban city in North Central Florida with approximately 133,997 residents served by two wastewater reclamation facilities (WRF). Fifty-three weekly influent composite wastewater samples were collected between September 2020 and September 2021 from each WRF. Wastewater samples were processed using an adsorption-based sample concentration method and invA genetic target for NTS DNA detection and quantification via qPCR. The quantified NTS DNA wastewater concentration from each WRF was summed to determine the overall concentration. Bacterial isolation was conducted on stored wastewater of qPCR positive samples using EPA method 1682. Recovered isolate serotypes were identified through whole-genome sequence analysis. RESULTS/ANTICIPATED RESULTS: Overall, NTS was positive in Gainesville wastewater in 48/53 weeks between September 2020 – September 2021. 83/106 (78%) samples tested positive for NTS from both WRFs. The mean wastewater concentration from each WRF was 3.53 (Main St.) and 3.31 Log10 invA Genetic Copies/L. The overall mean city concentration was 6.85 Log10 invA Genetic Copies/L. There were sustained periods of NTS in the wastewater during the months of December-February and May-August. There was no observed seasonal trend in NTS wastewater concentrations over the study period. Culture-based methods will provide a comprehensive review of circulating serotypes that are responsible for both clinical and subclinical infections within the community. DISCUSSION/SIGNIFICANCE: Florida has an incidence of NTS twice the national average. Clinical surveillance is the current standard for community surveillance of NTS. Only 28% of NTS cases are identified in a clinical setting, leaving a major gap in community surveillance. This project has helped fill this gap and provided a comprehensive community-level assessment of NTS.

OBJECTIVES/GOALS: Barriers to childhood vaccine completion include forgeting vaccine appointments, lack of clinic access (distance and funds), and vaccine hesitancy. We tested the impact of automated and real-time appointment reminders, vaccine hesitancy counseling, and targeted vaccine drives on receiving the third dose of the diphtheria vaccine. METHODS/STUDY POPULATION: An implementation study to determine the feasibility and impact of implementing a mobile health-supported intervention bundle. A digital vaccine registry was developed to manage vaccine uptake data. The intervention bundle was applied sequentially: each registered parent received an automated appointment phone reminder (text and voice). If they delayed for >5 days, they received a real time reminder phone call. If during the real time call vaccine hesitancy was deemed to be a barrier, counseling was provided. If access - lack of funds or long distance - to the clinic was the barrier, vaccination was performed at patient's home on the monthly vaccine drives. We compared vaccine completion (all childhood vaccines before 18 months) during the implementation to the preceding three years. RESULTS/ANTICIPATED RESULTS: We anticipate the implementation will be feasible as >90% of all eligible children will be registered. We expect providers will be accepting and would recommend the intervention to other providers. We anticipate the intervention will result in a >10% increase in childhood vaccine completion compared to the average of the past three years. DISCUSSION/SIGNIFICANCE: We anticipate applying a multifaceted intervention will be acceptable to providers, feasible to implement, and significantly improve childhood vaccine completion rates moving Nigeria closer to achieving the global target of >95% childhood vaccine completion rate.

OBJECTIVES/GOALS: Analysis and modeling of large, complex clinical data remain challenging despite modern advances in biomedical informatics. We aim to explore the potential of topological data analysis (TDA) to address such challenges in the context of COVID-19 outcomes using electronic health records (EHRs). METHODS/STUDY POPULATION: In this work, we develop TDA approaches to characterize subtypes and predict outcomes in patients with COVID-19 infection. First, data for >70,000 COVID-19 patients were extracted from the OneFlorida EHR database. Next, enhancements to the TDA algorithm Mapper were designed and implemented to adapt the technique to this type of data. Clinical variables, including patient demographics, vital signs, and lab values, were then used as input to conduct a population-level exploratory analysis with an emphasis on identifying phenotypic subtypes at increased risk of adverse outcomes such as major adverse cardiovascular events (MACE), mechanical ventilation, and death. RESULTS/ANTICIPATED RESULTS: Preliminary Mapper experiments have produced visual representations of the COVID-19 patient population that are well-suited to exploratory analysis. Such visualizations facilitate easy identification of phenotypic subnetworks that differ from the general population in terms of baseline variables or clinical outcomes. In this and subsequent work, we aim to fully characterize and quantify differences between these subnetworks to identify factors that may confer increased risk (or protection from) adverse outcomes. We also plan to validate and rigorously compare the efficacy of this TDA-based approach to common alternatives such as clustering, principal component analysis, and machine learning. DISCUSSION/SIGNIFICANCE: This work demonstrates the potential utility of TDA for the characterization of complex biomedical data. Mapper provides a novel means of exploring EHR data, which are otherwise difficult to visualize and can aid in identifying or characterizing patient subtypes in diseases such as COVID-19.

ABSTRACT IMPACT: This study provides insight into how MED2 impacts the immune cells surrounding glioblastoma that help it to grow and spread; having a more complete understanding of how MED2 works will help us better develop therapies that may one day enter the clinic to improve patient outcomes in glioblastoma. OBJECTIVES/GOALS: The purpose of this study was to determine whether the phosphorylation state of the MED2 peptide impacts its biological activity in GBM and macrophages. MED2 variants include the phosphorylatable wild-type (MED2), pseudo-phosphorylated (MED2-PP), non-phosphorylatable (MED2-NP) and control length (CTL2) peptides. METHODS/STUDY POPULATION: MED2, MED2-NP, MED2-PP, and CTL2 were screened against a panel of molecularly characterized glioblastoma patient derived xenografts and IL4/13 stimulated M2-like THP-1 macrophages. The luminescent cell viability assay, CellTiter-Glo, was used to determine viability. RESULTS/ANTICIPATED RESULTS: The proneural lines XD456 and X1441 were highly sensitive to 5 µM MED2 and 5 µM MED2NP compared to 5 µM MED2PP (p<0.001). There was no statistically significant difference between untreated, 5 µM CTL2, and 5 µM MED2PP groups or between the MED2NP and MED2 treated groups. M2-like THP-1 macrophages were highly sensitive to 10 µM MED2NP compared to 10 µM CTL2 (p<0.01) and 10 µM MED2PP (p<0.01) No statistically significant difference was observed between untreated, 10 µM MED2, 10 µM MED2PP, and 10 µM CTL2 groups. DISCUSSION/SIGNIFICANCE OF FINDINGS: The phosphorylation state of MED2 determines its toxicity. When MED2 is phosphorylated, it is nontoxic to GBM or M2-like macrophages. The non-phosphorylatable version is toxic to both GBM and M2-like macrophages. The wild-type peptide is toxic to GBM but not M2-like macrophages, suggesting that MED2 may be phosphorylated in M2-like macrophages.

ABSTRACT IMPACT: This work should provide further insights to mechanisms of the negative consequences of chemotherapy drugs, specifically in the cardiovascular system. OBJECTIVES/GOALS: Cardiotoxicity remains a safety concern in the development or utilization of chemotherapeutics largely due to the gap in knowledge of the mechanisms of toxicity. The pathophysiology of this cardiotoxicity has not been fully elucidated but data from our lab as well as other recent studies hint toward implications of mitochondrial (mito) biogenesis. METHODS/STUDY POPULATION: Prophylactic use of the beta-blocker carvedilol as well as the ACE inhibitor enalapril have been shown to inhibit the development of anthracycline-induced toxicity, but the mechanism of this cardio-protection remains elusive. To explore this, human stem cell-derived cardiomyocytes and endothelial cells will be either treated with the anthracycline doxorubicin or pretreated with carvedilol or enalapril followed by doxorubicin treatment before cellular lysates are harvested. Western blotting and qPCR will be performed to determine the expression of mito biogenesis markers including Nrf1, TFAM and the master regulator of mito biogenesis, PGC-1α. RESULTS/ANTICIPATED RESULTS: We anticipate that doxorubicin treatment alone will result in decreased expression of the mito biogenesis markers Nrf1, TFAM and PGC-1α and that pretreatment with either carvedilol and/or enalapril prior to doxorubicin treatment will either prevent or reverse this. DISCUSSION/SIGNIFICANCE OF FINDINGS: Doxorubicin’s role in causing mitochondrial dysfunction as well as suppression of biogenesis has already been established. Ideally, generation of new mitochondria would offset the occurrence of dysfunctional mitochondria. Confirming carvedilol/enalapril’s involvement with mito biogenesis would provide a mechanism of cardio-protection.

OBJECTIVES/SPECIFIC AIMS: Astroglial glutamine synthetase (GS), which metabolizes glutamate and ammonia to glutamine, is critical for the detoxification of brain ammonia, clearance of synaptic glutamate, and production of brain glutamine. Perturbations in the expression and activity of GS are thought to play a causative role in the pathogenesis of several conditions of abnormal neurotransmission. Although the long-term consequences of GS inhibition on amino acid homeostasis in the brain are unknown, it is thought that amino acid influx in the brain is tightly coupled with glutamine efflux via the L-type amino acid transporter. Both glutamine and leucine serve many critical functions in the brain including protein synthesis, gene expression, insulin regulation, and immune signaling. The objective of this study was to determine the effects of chronic GS inhibition with methionine sulfoximine (MSO) on glutamine and leucine homeostasis in the brain. METHODS/STUDY POPULATION: In total, 12 rats were surgically implanted with microdialysis guide cannulas in the bilateral dentate gyrus. Rats were randomly divided for surgical implantation of either a MSO (n=6) or phosphate buffer saline (PBS; n=6) pump in the right dentate gyrus. After 7 days, bilateral microdialysis probes were placed under brief isoflurane anesthesia, and microdialysis flow was established by infusing 0.5 µL/min of artificial extracellular fluid. Dialysate samples were collected every 30 minutes for the duration of the experiment. A 113 mM 15N-Leucine (3.6 mL/h) and 2 M 2–13C-sodium acetate (0.0633 μL/g/min for t=0–5 min, 0.0316 μL/g/min for t=5–10 min, and 0.0253 μL/g/min for t>10 min) solution was infused intravenously for 300 minutes. The EZ:Faast Free Amino Acid analysis kit and ultra-performance liquid chromatography/tandem mass spectrometry was used for quantification of amino acids in the dialysate fluid. RESULTS/ANTICIPATED RESULTS: At baseline (t=0 h), the concentrations of glutamine were significantly lower in MSO-treated rats (p<0.001) in the ipsilateral (GS-inhibited) hippocampus. There were no differences in glutamine concentrations between MSO and PBS-treated rats in the contralateral hippocampus. In PBS-treated rats, there was a significant increase in 15N-leucine between t=0 hour and t=5 hour in the contralateral (p<0.05) and ipsilateral (p<0.05) hippocampus. In MSO-treated rats, there was a significant increase in 15N-leucine between t=0 and t=5 hours in the contralateral (p<0.05) hippocampus, but not in the ipsilateral hippocampus (p=ns.). DISCUSSION/SIGNIFICANCE OF IMPACT: This study demonstrated for the first time that basal glutamine concentrations are low in areas of the brain where GS is acutely inhibited, and that leucine uptake in these brain areas are markedly decreased. Perturbations in glutamine and leucine homeostasis have been implicated in several disease processes including diabetes, obesity, liver disease, immune system dysfunction, epilepsy, and cancer, and the glutamine-dependent leucine influx in the brain may be a novel and important therapeutic target to treat these conditions.

OBJECTIVES/GOALS: The objective of this study is to investigate the neurophysiological mechanism of vision therapy in male and female adolescents with persistent post-concussion convergence insufficiency (PPCS-CI). This study may improve diagnostics and inform more effective personalized point of care treatment strategies to remediate symptoms. METHODS/STUDY POPULATION: Participants (ages 11-25) were diagnosed with PPCS by a physician, CI was diagnosed by an optometrist and OBVAT was performed by certified therapists. Patients with PPCS-CI were randomly assigned to a therapy type (immediate therapy or natural recovery). Hemodynamic measures were examined in patients with PPCS-CI at baseline (1-3 months post-concussion), and post OBVAT to evaluate recovery outcomes. Non-invasive techniques were used to measure middle cerebral artery velocity (MCAv), blood pressure, heart rate, and end-tidal CO2 at rest and during objective symmetrical convergence step eye movements. Functional magnetic resonance imaging (fMRI) was acquired during convergence step eye movement experiments contrasted to sustained fixation and resting state data collection. RESULTS/ANTICIPATED RESULTS: To investigate the neural mechanism of OBVAT, eye movements, fMRI and physiological measures were collected in 8 patients with PPCS-CI (4 men and 4 women). Results show an 10% decrease in the MCA during 4-degree symmetrical convergent eye movement responses in males post-OBVAT and a 19% increase in MCA during convergent eye movement responses in females. Furthermore, there was a group level activation of the frontal eye fields, which improves post-OBVAT. The beta weights in the left frontal eye fields show a trending decrease in male patients post-OBVAT and trending increase in females. Males had a decrease in MCAv post OBVAT (baseline 83.6 ± 7.5 cm/sec & 75.7 ± 12.5 cm/sec post-OBVAT), while females show a significant increase post-OBVAT (baseline 53.77 ± 5.2 cm/sec & post-OBVAT 65.13 cm/sec ± 12.5). DISCUSSION/SIGNIFICANCE: This initial pilot demonstrates there may be different underlying pathophysiological outcomes associated with a concussion dependent on sex. This work may have direct implications on treatment strategies for male and female adolescent patients with PPCS-CI.

OBJECTIVES/SPECIFIC AIMS: The overall objective of this proposal is to establish and modulate the inflammatory profile of individuals across the spectrum of multiple sclerosis (MS), with a focus on determining the potential of interleukin 4-induced protein 1 (IL4I1) as a possible marker of progression and modulator of inflammation in human blood samples. METHODS/STUDY POPULATION: The proposed experimental approach involves isolating plasma and peripheral blood mononuclear cells (PBMCs) from individuals across the spectrum of MS phenotypes, and analyzing these samples primarily by quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) methods. Specifically, study groups include: (1) actively relapsing-remitting MS (a-RRMS), (2) non-actively relapsing-remitting MS (n-RRMS), (3) non-active secondary-progressive MS (SPMS), (4) other autoimmune diseases (OAD), (5) healthy controls (HC). RESULTS/ANTICIPATED RESULTS: We expect that IL4I1 treatment increases regulatory cytokine (eg, IL10, TGFb) expression while decreasing Th1 and Th17-derived cytokines (IFNg, IL17), as well as increasing relative composition of regulatory cells (Th2, Treg, M2) as compared with Th1, Th17, M1 (aim 1). Preliminary data on healthy control cells support this prediction. Our central hypothesis is that IL4I1 level indicates the body’s ability to repair itself. As such, we anticipate that all MS groups are deficient in IL4I1, to varying degrees, such that HC>n-RRMS>a-RRMS>SPMS. HC have full repair capacity. RRMS>SPMS as remission indicates existent repair capacity, which is lost in SPMS. n-RRMS>a-RRMS since both, as RRMS, capable of repair response, but a-RRMS triggered this response more recently in response to more recent relapse. In all groups, we expect IL4I-treatment to mitigate inflammation (aim 2). Finally, we expect that H2O2 production by IL4I1 is a key player in IL4I1 function, and that H2O2 will preferentially induce oxidative stress to pro-inflammatory subsets of PBCMs (aim 3). DISCUSSION/SIGNIFICANCE OF IMPACT: MS is a chronic inflammatory neurodegenerative disease of the central nervous system that, with an average age of onset of 34, afflicts over 2.3 million individuals worldwide during many of the most productive years of their lives. The pathogenesis of MS, which involves autoimmune destruction of myelin, is poorly understood. Accurate biomarkers, which could predict disease progression, are yet to be identified and would provide valuable information to patients and their treating clinicians. Likewise, effective treatments are few and in high demand. IL4I1 is a promising candidate for both roles.

OBJECTIVES/GOALS: Biliary tract cancer is an uncommon, aggressive malignancy. Incidence varies geographically and is highest in East Asia and South America and lowest in Western countries. Previous dietary risk evaluations have primarily been case-control studies. We evaluated associations of dietary intakes with BTC risk in three cohort studies in two countries. METHODS/STUDY POPULATION: We evaluated 638,860 adults in China and the United Kingdom enrolled in the Shanghai Women’s Health Study (SWHS), Shanghai Men’s Health Study (SMHS), or UK Biobank (UKB). Dietary intake information was obtained from study participants at baseline using food frequency questionnaires previously validated for these studies. Dietary intakes of major food groups and macronutrients were divided into low, middle, and high intake tertiles. Cox regression was used to estimate hazard ratios and 95% CIs for biliary tract cancer risk associated with major food groups in all three cohorts and macronutrients in the SWHS and SMHS. Participants were excluded if, at enrollment, they had a history of cancer, CHD, stroke, a total daily Kcal count below 500 or exceeding 3500, or developed cancer or died within one year after enrollment. RESULTS/ANTICIPATED RESULTS: The analyzed cohort includes 558,372 participants: 66,945 SWHS, 55,750 SMHS, and 435,677 UKB participants with median enrollment ages of 49, 52, and 57 years old and median follow-up times of 18.1, 12.3, and 10.3 years, respectively. The SWHS observed 205 eligible BTC cases, SMHS 97, and UKB 366. SWHS and SMHS participants were combined for dietary evaluation and the highest tertile of fruit intake showed an inverse association with biliary tract cancer risk when compared to the lowest tertile: HR 0.74 (95% CI, 0.55-0.99); p-trend 0.044. In UKB, the highest tertile of fish intake was associated with a reduced risk when compared to the lowest tertile: HR 0.76 (95% CI, 0.59-0.98); p-trend 0.034. DISCUSSION/SIGNIFICANCE: High dietary fruit intake was associated with a reduced risk of biliary tract cancer only in SWHS and SMHS. High fish intake was associated with a reduced risk of biliary tract cancer only in UKB. Our findings reflect geographic-based BTC risk variation which we will further explore in our next model accounting for population-specific risk factors.

OBJECTIVES/SPECIFIC AIMS: This research seeks to create a next generation documentation system that exists independent of but is complimentary to the packaging system in R. The new documentation can be manipulated programmatically as with all R objects. It also implements multiple translators for creating documentation from different sources, including documentation pages written in latex and code comments. METHODS/STUDY POPULATION: This work is based on input from the R Documentation Task Force, which is a working group, supported by the R Consortium and the University of Utah Center for Clinical and Translational Science, consisting of R Core developers, representatives from the R Consortium member companies and community developers with relevant interest in documentation. An abstraction of the documentation currently in use was created and extended. This abstraction was translated to a class system in R so that documentation can be stored and manipulated in R. RESULTS/ANTICIPATED RESULTS: The class system representing the documentation and the tools for creating the translators are currently being implemented in R. A preview of the system is scheduled to be available at the time of the conference. DISCUSSION/SIGNIFICANCE OF IMPACT: Good documentation is critical for researchers to disseminate computational research methods, either internally or externally to their organization. This work will facilitate the creation of documentation by making documentation immediately accessible and promote documentation consumption through multiple outputs which can be implemented by developers.

ABSTRACT IMPACT: Identifying the causative antigen in membranous glomerulopathy cohorts enables the development of serum assays to detect and monitor disease progression without the need for invasive kidney biopsies. OBJECTIVES/GOALS: Primary membranous glomerulopathy is caused by the formation of autoantibody immune complexes which deposit in the glomerulus and obstruct kidney function. Causative antigens remain to be identified in roughly 20% of cases. Our goal is to identify the antigen in these cohorts, so that non-invasive assays can be developed for disease monitoring. METHODS/STUDY POPULATION: Renal biopsy tissue from known antigen cases (PLA2R, THSD7A), and unknown cases were included in the analysis. Renal biopsy tissue from formalin fixed paraffin embedded tissue was cut at a thickness of 10 µm onto Leica PET-membrane frame slides. These slides were then stained with hematoxylin. The glomeruli were microdissected into microcentrifuge tubes using a Leica DM6000B microscope. The microdissected glomeruli were lysed in 2% SDS and 0.1M DTT at 99 degrees Celsius for 1 hour and processed by filter assisted sample preparation (FASP). Digested peptides were analyzed by liquid chromatography-mass spectrometry using an Orbitrap Fusion Lumos using data-dependent acquisition. RESULTS/ANTICIPATED RESULTS: Mass spectrometry data collected from the laser captured glomeruli was searched against the human proteome fasta database from Uniprot using MaxQuant. IBAQ values were used for quantitation and statistical analysis. Null hypothesis significance testing was performed for each protein by comparing each sample group to the rest of the samples in the data set. In the control groups, the causative antigens PLA2R and THSD7A were detected and quantified with the largest magnitude fold change in their respective category, validating the experimental design. Using this approach, the proteins SAP, NELL1, and NCAM1 were identified and subsequently validated as causative antigens in distinct patient cohorts. DISCUSSION/SIGNIFICANCE OF FINDINGS: Here, we share the results of our efforts to comprehensively identify the spectrum of causative antigens in membranous glomerulopathy. In this context, antigen discovery is an essential first step for the development of non-invasive assays to inform prognosis, monitor response to treatment, and better understand disease etiology.

OBJECTIVES/SPECIFIC AIMS: To demonstrate that olanzapine recapitulates the effect of increased lateral hypothalamic (LH) GABAergic activity in the DRN and the DBB. This will provide a potential neural substrate for the observed increase in consumption of food and weight gain. METHODS/STUDY POPULATION: (1) We will examine electrophysiological activity of the DRN and the DBB in response to optogenetic stimulation of LH fibers to these nuclei. (2) We will identify the behavioral phenotype of stimulating these same projections using optogenetic techniques. (3a) Identify the behavioral phenotype of mice possessing cre-loxp-dependent knockout (KO) of LH GABAergic activity, DRN serotonergic activity, and inhibition of DBB cholinergic activity. (3b) Using these mice, we will establish behavioral response to olanzapine in ad libitum feeding and fast-refeeding condition. (4) Using baseline and post-treatment body mass index (BMI), PANSS, and side effect profile scores from a recently completed prospective cohort study of treatment-naive schizophrenic patients receiving atypical antipsychotics for 1 year, we will sequence multiple single nucleotide polymorphisms and explore the correlation of serotonergic, dopaminergic, and cholinergic receptor mutations with the increase in BMI and changes in PANSS score and side effect scores. RESULTS/ANTICIPATED RESULTS: (1) Our preliminary data indicates that the LH exclusively sends GABAergic input to the DBB, and the large majority of its projections to the DRN are GABAergic. (2) We have identified that stimulating LH–>DBB projections produces intense feeding and drinking behavior, a real-time place preference for laser stimulation, and a conditioned place preference for laser stimulation. Preliminary data shows that the LH->DRN also produces feeding behavior. (3a) Our lab has demonstrated that transgenic mice with LH-specific GABA release KO are smaller, have increased anxiety-like behaviors such a repetitive grooming and open field aversion, and have reduced feeding after fasting conditions. We expect the DRN serotonergic KO mice to have increased body weight and reduced anxiety-like behaviors. (3b) Our pilot study demonstrated that the LH GABA KO mice administered olanzapine have a greater consumption of food over 1 hour than controls (n=7, 5, respectively; p=0.08). DRN serotonergic KO mice and mice with inhibition of choline will have an increased baseline feeding behavior, but will not be affected by olanzapine. (4) We believe that SNPs in serotonergic receptors such as 5HT2C, and those affecting dopaminergic and cholinergic receptors, will be more common in schizophrenic patients with increased BMI than those without. Further, we believe that a reduction in the PANSS items reflecting anxiety and aversiveness will correlate with increased BMI, since we postulate that mimicking LH GABAergic activity will produce its previously demonstrated anxiolytic effects. DISCUSSION/SIGNIFICANCE OF IMPACT: Identifying the important role for a reward-oriented feeding center in the brain in producing antipsychotic weight gain will allow a more comprehensive, ethologically sound approach to behavioral modification therapy in these patients. It will lend mechanistic credence to weight control therapies which have used token economy, opioid antagonism, and other inhibition-promoting therapies. This study will also increase the validity for testing further the use of selective serotonin agonists which prevent weight gain such as lorcaserin.

OBJECTIVES/SPECIFIC AIMS: Despite advancements in therapies, such as surgery, irradiation (IR) and chemotherapy, outcome for patients suffering from glioblastoma remains fatal; the median survival rate is only about 15 months. Even with novel therapeutic targets, networks and signaling pathways being discovered, monotherapy with such agents targeting such pathways has been disappointing in clinical trials. Poor prognosis for GBM can be attributed to several factors, including failure of drugs to cross the blood-brain-barrier (BBB), tumor heterogeneity, metastasis and angiogenesis. Development of tumor resistance, particularly to temozolomide (TMZ), creates a substantial clinical challenge.The primary focus of our work is to rationally develop novel combination therapies and dose regimens that mitigate resistance development. Specifically, our aim is to combine TMZ with small molecule inhibitors that are either currently in clinical trials or are approved drugs for other cancer types, and which target the disease at various resistance signaling pathways that are induced in response to TMZ monotherapy. METHODS/STUDY POPULATION: To accomplish this objective, an integrated PKPD modeling approach is used. The approach is largely based on the work of Cardilin, et al, 2018. A PK model for each drug is first defined. This is subsequently linked to a PD model description of tumor growth dynamics in the presence of a single drug or combinations of drugs. A key outcome of these combined PKPD models are tumor static concentration (TSC) curves of dual or triple combination drug regimens that identify combination drug exposures predicted to arrest tumor growth. This approach has been applied to TMZ in combination with abemaciclib (a dual CDK4/6 small molecule inhibitor) based on data from a published study evaluating abemaciclib efficacy in combination with TMZ in a glioblastoma xenograft model (Raub, et al, 2015). RESULTS/ANTICIPATED RESULTS: A PKPD model was developed to predict tumor growth kinetics for TMZ and abemaciclib monotherapy, as well as combination therapy. Population PK models in immune deficient NSG mice for temozolomide and abemaciclib were developed based on data obtained from original and published studies. Subsequently, the PK model was linked to tumor volume data obtained from U87-MG GBM subcutaneous xenografts, again using both original data as well as data from the Raub, et al, 2015 study. Model parameters quantifying tumor volume dynamics were precisely estimated (coefficient of variation < 30%). The developed PKPD model was used to calculate plasma concentrations of TMZ and abemaciclib that would arrest tumor growth, as well as combinations of concentrations of the two drugs that would accomplish the same endpoint. This so-called TSC curve for the TMZ and abemaciclib combination pair evidenced an additive effect of the two agents when administered together. These results will be presented. In addition, results from on-going PKPD studies of TMZ in combination with two other small molecule inhibitors, RG7388, an MDM2 inhibitor, and GDC0068, an AKT inhibitor, will also be presented. DISCUSSION/SIGNIFICANCE OF IMPACT: Our long-term goals are to further elucidate SOC-induced responses in GBM and establish combination treatment regimens that are safe and significantly improve therapeutic efficacy. Collectively, our studies will broadly influence chemotherapy of GBM by establishing a process to rationally design combination approaches that mitigate resistance development. These studies will ultimately provide opportunities to study other targeted agents tailored to individual molecular signatures of GBM, as well as other tumor types.