OBJECTIVES/SPECIFIC AIMS: Assess the presence of contact activation at baseline in sera from common variable immunodeficiency (CVID) patients with and without inflammatory complications compared with healthy controls. METHODS/STUDY POPULATION: CVID patients were recruited in the outpatient setting and the measurement of cleaved plasma HK (cHK) levels was determined by Western blot analysis, under reducing conditions, with quantitation of total and cHK bands using an Odyssey imaging system (Licor). One-way ANOVA test for differences among the 3 studied groups will be applied. Biomarkers C3, C4, C1 inhibitor levels and hs-CRP were also measured. RESULTS/ANTICIPATED RESULTS: Participant enrollment continues and to date, 9 CVID patients were studied, 7 with and 2 without inflammatory complications. Repeated determinations of cleaved HK% (cHK%) revealed an average of 1.20% (range: 0.46%–2.66%) in CVID patients with inflammatory complications and those without complications averaged 1.07% (range: 0.79%–1.35%). Healthy controls had an average cHK of 1.15% (range: 0.60%–2.10%). DISCUSSION/SIGNIFICANCE OF IMPACT: Cleaved kininogen detected in the sera of CVID patients was found at similar levels compared with healthy controls (cHK<5%). Findings suggest that systemic activation of the contact system might be absent in CVID, however, future considerations include developing detection methods for local tissue activation.

OBJECTIVES/SPECIFIC AIMS: We sought to investigate the role of the host microbiome during severe, acute respiratory infection (ARI) to understand the drivers of both acute clinical pathogenesis. METHODS/STUDY POPULATION: Nasopharyngeal swabs comprised of mixed cell populations at the active site of infection were collected from 192 hospitalized pediatric patients with ARI. We combined comprehensive respiratory virus detection and virus genome sequencing with 16S rRNA gene sequencing to evaluate the microbial content of the airway during ARI. This data was coupled with 11 clinical parameters, which were compiled to create a clinical severity score. The microbiome profiles were assessed to determine if clinical severity of infection, and/or specific virus was associated with increased clinical severity. RESULTS/ANTICIPATED RESULTS: We identified 8 major microbiome profiles classified by dominant bacterial genus, Moraxella, Corynebacterium, Staphylococcus, Haemophilus, Streptococcus, Alloiococcus, Schlegelella, and Diverse. Increased clinical severity was significantly associated with microbiome profiles dominated by Haemophilus, Streptococcus, and Schlegelella, whereas Corynebacterium and Alloiococcus were more prevalent in children with less severe disease. Independent of the microbial community, more than 60% of patients with the highest clinical severity were infected with either respiratory syncytial virus or rhinovirus. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results indicate that individually and in combination, both virus and microbial composition may drive clinical severity during acute respiratory viral infections. It is still unclear how the complex interplay between virus, bacterial community, and the host response influence long-term respiratory impacts, such as the development of asthma. Nonetheless, during ARIs therapeutic interventions such as antibiotics and probiotics may be warranted in a subset of patients that are identified to have both a virus and microbiome profile that is associated with increased pathogenesis to limit both acute and long-term phenotypes.

OBJECTIVES/SPECIFIC AIMS: Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by progressive fibrosis of the skin and multiple visceral organs. Effective therapies for SSc are needed. Lysyl oxidase (LOX) is a copper-dependent amide oxidase that plays a critical role in the crosslinking of the extracellular matrix (ECM). In this study, we investigated the role of LOX in the pathophysiology of SSc. METHODS/STUDY POPULATION: LOX expression and protein levels were measured in lung tissues and primary fibroblasts from patients with SSc and healthy controls. The effects of recombinant LOX (rLOX) were measured in vitro in primary fibroblasts, ex vivo in human lung tissues and in vivo in mice given bleomycin in combination with rLOX. LOX levels and activity were evaluated in lung fibroblasts treated with an endostatin-derived peptide that ameliorates fibrosis and in mice treated with bleomycin in combination with the peptide. Further, to differentiate the crosslinking activity of LOX from other potential effects, primary human fibroblasts were cultured with rLOX in the presence of the inhibitor, beta-aminopropionitrile. The expression levels of ECM (collagen and fibronectin), pro-fibrotic factors (IL-6 and TGF-beta), and transcription factor (c-Fos) were examined by real-time PCR, ELISA, immunoblotting, or hydroxyproline assay. RESULTS/ANTICIPATED RESULTS: LOX mRNA was increased in lung tissues and matching fibroblasts of SSc patients. rLOX-induced ECM production in vitro and ex vivo in lung fibroblasts and in human lung tissues maintained in organ culture, respectively. Additionally, TGF-beta and bleomycin induced ECM production, LOX mRNA expression and activity. Endostatin peptide abrogated these effects. In vivo, rLOX synergistically exacerbated pulmonary fibrosis in bleomycin-treated mice. The inhibition of LOX catalytic activity by beta-aminopropionitrile failed to abrogate LOX-induced ECM production. LOX increased the production of IL-6. IL-6 neutralization blocked the effects of LOX. Further, LOX induced c-Fos expression and its nuclear localization. DISCUSSION/SIGNIFICANCE OF IMPACT: LOX expression and activity were increased with fibrosis in vitro, ex vivo, and in vivo. LOX induced fibrosis via increasing ECM, IL-6 and c-Fos translocation to the nucleus. These effects were independent of the crosslinking activity of LOX and mediated by IL-6. Our findings suggest that inhibition of LOX may be a viable option for the treatment of lung fibrosis. Further, the use of human lung in organ culture establishes the relevance of our findings to human disease.

OBJECTIVES/SPECIFIC AIMS: Transforming growth factor-beta (TGFβ) is a genetic modifier of cystic fibrosis (CF) lung disease. TGFβ’s pulmonary levels in young CF patients and its mechanism of action in CF are unknown. We examined TGFβ levels in children with CF and investigated responses of human airway epithelial cells (AECs) and mice to TGFβ. METHODS/STUDY POPULATION: TGFβ levels in bronchoalveolar lavage fluid from CF patients (n=15) and non-CF control patients (n=21)<6 years old were determined by ELISA. CF mice and non-CF mice were intratracheally treated with an adenoviral TGFβ1 vector or PBS; lungs were collected for analysis at day 7. Human CF and non-CF AECs were treated with TGFβ or PBS for 24 hours then collected for analysis. RESULTS/ANTICIPATED RESULTS: Young CF patients had higher bronchoalveolar lavage fluid TGFβ than non-CF controls (p=0.03). Mouse lungs exposed to TGFβ demonstrated inflammation, goblet cell hyperplasia, and decreased CFTR expression. CF mice had greater TGFβ-induced lung mechanics abnormalities than controls; both CF human AECs and CF mice showed higher TGFβ induced MAPK and PI3K signaling compared with controls. DISCUSSION/SIGNIFICANCE OF IMPACT: For the first time, we show increased TGFβ levels very early in CF. TGFβ drives CF lung abnormalities in mouse and human models; CF models are more sensitive to TGFβ’s effects. Understanding the role of TGFβ in promoting CF lung disease is critical to developing patient specific treatments.

OBJECTIVES/SPECIFIC AIMS: The objective of our collaboration is to develop a strong transdisciplinary team consisting of microfluidics engineers, cancer biologists, and clinicians, to identify cell surface markers capable of detecting circulating osteosarcoma cells (COC) using microfluidic devices. Our goals are 3-fold: (1) Identify cell surface markers unique to osteosarcoma (OS) for COC isolation, (2) develop a Geometrically Enhanced Mixing (GEM) device to isolate COCs, and (3) Evaluate the efficacy of GEM device to detect COCs in OS patients under treatment. The long-term goal is to utilize this cell detection approach to correlate the presence of COC with metastatic incidence. METHODS/STUDY POPULATION: To identify a marker to capture COCs we are utilizing flow cytometry and microfluidic capture devices. Flow cytometry will be used to evaluate the relative expression of epithelial cell adhesion molecule (EpCAM), CD45, cell surface vimentin (CSV), insulin-like growth factor 2 (IGF2R), interleukin 11 receptor subunit alpha (IL-11Rɑ), ganglioside 2 (GD2), and receptor activator of nuclear factor κ-B (RANK) on a panel of OS cell lines. These cell surface markers were selected based on an extensive review of OS cell surface markers. OS cell capture efficacy will be assessed by passaging a known concentration of OS cells through a GEM microfluidic device coated with antibodies targeting the selected marker, as indicated by flow cytometry. Once captured, COCs on the device will be analyzed and the capture efficiency for the indicated marker will be measured. ANOVA will be used to determine any significant difference in capture efficiency between marker types. Once an optimal marker or panel of markers has been selected we will conduct capture studies using OS cell spiked blood samples followed by clinical samples obtained from OS patients. In clinical samples, COC detection will be validated using the FDA approved triple immunocytochemistry technical definition of a circulating tumor cell (CTC). This will enable COCs to be differentiated from the normal whole blood cell population by selecting for CD45−, EpCAM+, and cytokeratin+ cells. RESULTS/ANTICIPATED RESULTS: Our preliminary studies have shown that on our microfluidic device, EpCAM, a marker commonly used to identify circulating tumor cells in other cancer settings, has a poor capture efficiency (15.9%+7.7%) for HU09 OS cells while the same setup with EpCAM has a capture efficiency of 56.9%+2.7% for BXPc-3 pancreatic cells. We therefore anticipate our flow cytometry studies to show a low expression of EpCAM and CD45 for OS cell lines, while showing a moderate to high expression of CSV, IGF2R, IL-11Rɑ, GD2, and RANK. We expect to show a 60%–80% capture efficiency for markers selected for COC capture. Currently, CSV and GD2 are particularly promising as markers based on previously published studies. DISCUSSION/SIGNIFICANCE OF IMPACT: OS is the most common primary bone tumor and the third leading cause of pediatric cancer deaths. At diagnosis 80% of patients will present with metastasis, however only 20% of these cases are clinically detectable. Innovative strategies to identify patients at risk of metastasis would allow for stratification of intervention therapies. Currently, tumor recurrence and metastasis are primarily dependent on diagnostic-imaging modalities such as computerized tomography or positron emission tomography scans. Unfortunately, these imaging modalities can only detect tumor masses of significant size (106 tumor cells). Liquid biopsies are a novel alternative to current diagnostic imaging systems to monitor metastatic incidence and treatment efficacy. The detection of CTCs through routine blood sampling has the potential to be used clinically for earlier detection, monitoring the treatment of metastatic cancers and surveying the effect of therapeutic interventions on metastasis. To date, the majority of the studies on CTCs have evaluated their presence in carcinomas. Although sarcomas are rare, they generally have a poor prognosis. This study will address one of the unmet medical needs in the field of CTC detection; the identification of cell surface OS makers to improve binding specificity, increase purity, and maintain a high capture efficiency. This phase of our proposal will evaluate the most abundant and conserved markers across a panel of OS cell lines. Once a marker or panel of markers is selected, we will begin to develop a microfluidic device that can be used clinically to detect CTCs in this disease setting.

OBJECTIVES/SPECIFIC AIMS: Aim 1: To determine the true incidence of trauma-related acute respiratory distress syndrome (ARDS) in India. We propose to perform a prospective observational study to determine the incidence of ARDS in India. Aim 2: To perform a preliminary assessment of risk factors for ARDS in the Indian trauma population. We will leverage these findings against the global ARDS data to provide a foundation for further interventional studies. Aim 3: To evaluate the current management strategies and patient outcomes from ARDS in trauma subjects admitted to the Jai Prakash Narayan Apex Trauma Center (JPNATC). These findings will identify areas in need of practice-based performance improvement in ARDS therapies in India. METHODS/STUDY POPULATION: This application proposes an observational study of trauma patients with ARDS, a population that continues to have substantial in-hospital mortality. The approximate number of ICU-admitted trauma cases for the study period is 1700. Specific data elements to be collected include patient demographics, comorbidities, mechanism of injury, Injury Severity Score, risk factors for ARDS, sequential organ failure and assessment scores, vital signs, laboratory values, and evidence-based treatments received, including mechanical ventilation and adjunctive therapies. Outcome data will include discharge location, ICU and hospital length of stay and all-cause mortality. Selection of Subjects: We will include all patients admitted to the JPNATC Trauma and Neurosurgical ICUs intubated and mechanically ventilated and meeting the definition of Berlin definition of ARDS8. We will collect data for a total of 12 months. RESULTS/ANTICIPATED RESULTS: Due to gaps in reporting, the incidence, mortality, and practice-based management algorithms applied in trauma patients suffering from ARDS in India is unknown. We hypothesize that the overall incidence of trauma-related ARDS is higher, and the fraction of patients managed with evidence-based therapies is lower than global reported averages. DISCUSSION/SIGNIFICANCE OF IMPACT: Although the true incidence of ARDS in trauma subjects in India is currently unknown, we suspect that it is much higher than reported. Such data are important in identification of resource allocation including ICU bed and mechanical ventilator availability, particularly in a resource-limited environment. This proposal will aid in the development of research infrastructure at JPNATC, contribute to capacity building, and the establishment of a Clinical Research unit at the Apex Institute. Finally, a provision to develop a consortium and trauma quality improvement program among the existing trauma centers in New Delhi to disseminate important research findings and guidance to the rest of India is a future benefit of the study.

OBJECTIVES/SPECIFIC AIMS: One of the driving mechanisms of cancer progression is the reprogramming of metabolic pathways in intermediary metabolism. Cancers increase their energy expenditure by increasing ATP production for utilization in anabolic pathways to increase production of proteins, nucleic acids and lipids. The Warburg effect, where cancer cells predominantly use aerobic glycolysis rather than oxidative phosphorylation to produce ATP, was long thought to be the main initiating pathway in increasing tumor burden. However, compelling new evidence shows that there exists metabolic heterogeneity among and within tumors. Mitochondrial respiration often plays a major role in tumor progression, as many different cancers contain a subpopulation of slow-cycling tumor-initiating cells that are multidrug-resistant and dependent on oxidative phosphorylation. These cells represent a target for cancer therapy. In this study, we identification a novel endogenous regulator of mitochondrial respiration, retinoic acid receptor responder 1 (RARRES1). METHODS/STUDY POPULATION: We assessed the metabolic phenotype of RARRES1-depleted normal epithelial cells through metabolomics, a flux analyzer and blotting for phosphorylation of AMP kinase, a major regulator of energy homeostasis. We further examined mitochondrial energetics by staining the mitochondria with TMRM and Mito-Tracker. We then analyzed the apoptotic phenotype of epithelial cells with depletion of RARRES1 with fluorescence-activated cell sorting analysis of annexin V-staining. RESULTS/ANTICIPATED RESULTS: Remarkably, fluorescence-activated cell sorting analysis of annexin V-stained epithelial cells with depletion of RARRES1 were resistant to all studied modes of cell death, implying an effect on a fundamental cell process. By using proteomics, metabolomics, cellular and molecular analyses, our data show that RARRES1 regulates mitochondrial membrane potential and subsequently alters 1-carbon metabolism by modulating the function of the mitochondrial voltage-dependent anion channel. We believe this is the first example of a tumor suppressor protein that functions to directly regulate mitochondrial energetics. Using an extracellular flux analyzer, our data also show that depletion of RARRES1 causes an increase in mitochondrial respiration and ATP production, thus enhancing biosynthetic pathways that drive the pathogenicity and survival of cancer. The metabolic and anti-apoptotic phenotype of RARRES1-depleted cells was reversed by treatment of metformin, a mitochondrial inhibitor. DISCUSSION/SIGNIFICANCE OF IMPACT: These data lay the foundation for metabo-therapy of the many tumor types that exhibit RARRES1 depletion and may have the added benefit of targeting drug-resistant tumor-initiating cells.

OBJECTIVES/SPECIFIC AIMS: Triple-negative breast cancer (TNBC) accounts for one-fifth of the breast cancer patient population. The heterogeneous nature of TNBC and lack of options for targeted therapy make its treatment a constant adventure. The deficiency of tumor suppressors p53 and ARF is one of the known genetic signatures enriched in TNBC. Crucial questions remain about how TNBC is regulated by these genetic alterations. METHODS/STUDY POPULATION: In order to address this issue, we established p53/ARF-defective murine embryonic fibroblast and mammary epithelial cell to study the molecular and phenotypic consequences. Moreover, transgenic mice were generated to investigate the effect of p53/ARF deficiency on mammary tumor development in vivo. RESULTS/ANTICIPATED RESULTS: Increased proliferation and transformation capability were observed in p53/ARF-defective cells, and an aggressive form of mammary tumor was also seen in p53−/−ARF−/− mice. Gene expression profiling and knock-down experiments using shRNAs were conducted to identify inflammatory marker ISG15 and RNA-editing enzyme ADAR1 as potential culprits for the elevated oncogenic potential. Interestingly, we found that the overexpression of ISG15 and ADAR1 is also prevalent in human TNBC cell lines. Reducing ADAR1 expression abrogated the oncogenic potential of human TNBC cell lines, while non-TNBC cells are less susceptible. DISCUSSION/SIGNIFICANCE OF IMPACT: These results indicate critical roles played by the tumor suppressors p53 and ARF in the pathogenesis of TNBC, likely through regulating ADAR1-mediated RNA modifications. Further understanding of this pathway promises to shed light on genetics-driven vulnerabilities of TNBC and inform development of more effective therapeutic strategies.

OBJECTIVES/SPECIFIC AIMS: The aim of this study was to assess the ultra-dose Na18F dPET protocol feasibility for skeleton imaging in a canine model with reduced radiation dose and preserved quantitative characteristics. We hypothesized that administering an ultra-low Na18F dose would provide suitable image quality while reducing subject’s exposure to radiation. METHODS/STUDY POPULATION: In total, 13 adult male beagles [weight (kg) mean±SD; 14.3±2.2] were scanned. The dogs were administered 3 different Na18F doses: 3 (standard dose/SD), 1 (low dose/LD), and 0.05 (ultra-low dose/ULD) mCi. Imaging started ≃45 minutes post injection for ≃ 33 minute total acquisition time. Covering the whole body, 11 bed positions, acquiring 120 (3 mCi) and 180 (1, 0.05 mCi) seconds per bed position. All imaging was performed on a digital photon counting system (Philips Vereos, pre-commercial release). PET list mode data were reconstructed using Time-of-flight with 4, 2, and 1 mm3 voxel volumes. Point spread function, and Gaussian filtering were applied. Two experienced blinded readers evaluated image sets overall quality, tissue characterization, and quality of background in the whole body skeleton. Three-dimensional (3D) regions of interest (ROI) were traced over the distal femur, first lumbar vertebra, and a portion of the liver, recording standard uptake values (SUVmax and SUVmean). RESULTS/ANTICIPATED RESULTS: All the scans and reconstructions were successfully completed in all subjects. Decreasing Na18F dose from the standard dose (3 mCi) to the ultra-low dose/ULDO (0.05 mCi), demonstrated acceptable image quality and quantification. Ultra-low dose Na18F SUVmean values for the 3D ROIs reported (mean±SD) 2.6±0.7, 2.5±1.1, 9±1.6, and 0.6±0.3 from the right and left distal femur, first lumbar vertebra, and a portion of the liver, respectively. When compared the SD with the LD and ULD, dPET demonstrated acceptable image quality and definition for qualitative overall assessment. This was also found for the overall quantitative ROI assessment of the healthy canine skeletons. DISCUSSION/SIGNIFICANCE OF IMPACT: Ultra-low dose Na18F at a level of 50 μCi for a 14 kg canine appears to be diagnostically feasible and a robust option to reduce (60-fold) radiotracer doses in a translational animal model using a dPET system. Furthermore, it allows us to move preclinical nuclear medicine imaging forward with substantial reduced exposure levels while preserving image quality. Both visual and quantitative results indicate that the standard-dose bone Na18F dPET can be decreased with a satisfactory diagnostic image quality. Ultra-low Na18F dose is indeed important for younger populations, control patients, and nononcological diseases/conditions. Favorable pharmacokinetics of Na18F (such as high bone uptake, minimal binding to serum proteins, rapid single-pass extraction, and fast clearance from the soft tissues) in addition to the technological capabilities of dPET/CT demonstrated feasibility enabling dose reduction strategies. Ultra-low dose has diagnostic reproducibility and lower radiation burden compared with higher fixed dose techniques in current available guidelines [Society of Nuclear Medicine and Molecular Imaging; SNMMI (5–10 mCi)]. Na18F dPET/CT provides higher sensitivity and diagnostic accuracy, which enables high-quality images with lower tracer activity in this translational animal model. Future research will apply the same methodology to other anatomical targets as well as to the use of different tracers. Preclinical nuclear medicine imaging using ultra-low tracer doses, demonstrated the potential to obtain reasonable quality images and diminishing radiation surveillance in accordance with as low as reasonably achievable tracer levels.

OBJECTIVES/SPECIFIC AIMS: Background: Renal transplantation (tx) is the optimal treatment for end-stage renal disease (ESRD) in children, but post-tx urinary tract infections (UTIs) may cause morbidity and reduce allograft survival. Objectives: To quantify the number and risk factors for UTIs in pediatric kidney tx recipients in preparation for an analysis of the morbidity and impact of UTIs on allograft survival. METHODS/STUDY POPULATION: Methods: We identified all patients who underwent kidney tx between 2001 and 2016 (n=390) at Children’s Healthcare of Atlanta (CHOA). Patients were included if they had >1 year of follow-up at CHOA. We conducted an IRB-approved, retrospective review of patient demographics, medical history, and tx outcomes in the 5 years following tx. RESULTS/ANTICIPATED RESULTS: Results: Of the 205 records reviewed to date, we identified 176 eligible patients (61.9% male). Mean age at tx was 11.7±5.5 years. In total, 58.5% had a deceased and 41.5% had a living kidney donor. Obstructive uropathy was the etiology of ESRD in 21.0%. Mean UTIs in all patients was 1.1/patient±2.7. On preliminary analysis, patients with a history of obstructive uropathy were more likely to develop a UTI than patients without (45.9% vs. 25.2%, p=0.014). There is a trend to more UTIs in patients with a history of obstructive uropathy compared with patients without (2.1±3.5 vs. 0.9±2.4, p=0.055). In males, there were more UTIs in patients with a history of obstructive uropathy compared to patients without (1.7±2.9 vs. 0.5±1.5, p=0.024). In all, 23.2% of all patients were on UTI prophylaxis post-tx; trimethoprim-sulfamethoxazole was the prophylactic antibiotic in 54.5%. DISCUSSION/SIGNIFICANCE OF IMPACT: Conclusions: UTIs are common post kidney tx in children, especially in those with a history of obstructive uropathy. The associated morbidity and impact on graft survival are unknown.

OBJECTIVES/SPECIFIC AIMS: Our goal is to assess age-related changes in osteogenic stem cell populations of bone tissue. We hypothesize that aging mice have reduced osteogenic capacity in response to physical stimulation due to aging-associated decline in osteoprogenitor cell number and their proliferative capacity. METHODS/STUDY POPULATION: Mechanical loading: The NYU School of Medicine Institutional Animal Care and Use Committee approved all procedures. The response of tibial periosteal cells to physical stimulation or mechanical loading was assessed in 16-week-old adult (n=6) and aged 78-week-old female (n=4) mice subjected to 4 consecutive days of strain-matched axial compressive loading (1400 μm, 120 cycles, 2 Hz). Whole Mount Staining: Baseline periosteal cell numbers and nuclear morphology were assessed by whole bone DAPI staining of the antero-medial region of the tibiae in adult and aged mice (n=6). Immunohistochemistry: Tibiae were fixed in 4% PFA, decalcified in 19% EDTA, OCT-embedded, and thickly sectioned (150 μm) at midshaft. Sca1+, Prrx1+, and Ki67+cell numbers were quantified by simultaneous fluorescent immunohistochemical staining from loaded and nonloaded contralateral tibiae. Nonimmune species specific serum served as negative controls. Imaging: 3D image datasets of the periosteum at the antero-medial region of the tibial midshaft were acquired by multi-photon and confocal microscopy. Quantification of Sca1+, Prrx1+, and Ki67+ cells was carried out using Particle Analysis software (ImageJ) and Imaris 7.4.2 Surface Rendering Statistics functions. Cell number was normalized to periosteal area (~0.04 mm2). A Student t-test determined significance at p<0.05. RESULTS/ANTICIPATED RESULTS: At baseline, aged periosteal cell nuclei (DAPI+) area (14% decrease, p<0.0001), nuclei number, and Prrx1+ cell number (22% decrease) was significantly lower compared with adult mice. In loaded adult mice, Prrx1+but not Sca1+cell number increased significantly (35%, p=0.0115). Proliferating Sca1+(top panel) and Prrx1+(top panel) cells also increased with loading, 62%, p=0.0253 and 115%, p=0.0004, respectively, in adult but not aged mice. The percentage of Prrx1+ cells undergoing proliferation (co-expressing Ki67+) in the total Prrx1+ cell population increased significantly with loading (bottom panel). Aged mice did not exhibit significant differences in loaded versus nonloaded controls for all other outcomes. Our data suggest fundamental changes in periosteal cell morphology, number and response to mechanical loading with aging. The significant increase in total Prrx1+ cell number and the number of Prrx1+ cells undergoing proliferation with loading in adult mice, suggest that the Prrx1+ cell population expands through proliferation. In fact, loading resulted in a 2-fold increase in the percentage of Prrx1+ preosteogenic cells undergoing proliferation. Accordingly, the significant age-related decrease in Prrx1+ cells may explain, in part, the attenuation of load-induced bone formation in aged mice. Loading resulted in greater numbers of proliferating Sca1+ cells (the more primitive cell) in adult mice, though this represented only a small percentage (<10%) of the total Sca1+ population. Mechanical loading expands the Prrx1+ pre-osteogenic cell population, but not the more primitive Sca1+ population. However, this load-induced osteogenic effect in the periosteum is not observed in aged mice, which may explain age-related diminishment of load-induced bone formation. DISCUSSION/SIGNIFICANCE OF IMPACT: Mechanical loading presents an inexpensive treatment for increasing bone mass and bone strength, but may be insufficient to prevent or reverse age-related bone loss due to reduced numbers of osteogenic progenitors in the periosteum. Therapeutic approaches targeting the osteogenic capacity of periosteal cells will be required to address declining mechanoresponsiveness with age.

OBJECTIVES/SPECIFIC AIMS: The purpose of this study was to provide individuals who have experience with cognitive behavioral therapy (CBT) with a demonstration of how using their therapeutic strategies affects their brain activity. Two challenges that face CBT and other cognitive therapies are (1) sustaining the gradual, incremental behavioral changes characteristic of the treatment and (2) measuring associated biological changes. These challenges may impede treatment efficacy and may negatively affect treatment outcomes, including patient discontinuation of CBT. Ideas for addressing these issues include providing patients with (1) a more immediate indicator of therapy effectiveness as well as (2) a biological index of behavioral change. In this study, we aimed to provide participants with an index of biological change based on therapeutic experiences via use of real-time functional magnetic resonance imaging (rtfMRI) neurofeedback. METHODS/STUDY POPULATION: We recruited participants who had already completed cognitive therapy as part of a clinical trial for depression at the University of North Carolina at Greensboro (n=13). In the present experiment, participants were asked to provide a list of negative autobiographical memories or worries as well as cognitive strategies they use to cope with negative moods. The task consisted of COUNT, MEMORY, and STRATEGY trials (30 s each). During baseline COUNT trials, participants counted backwards (e.g., 300–4). During MEMORY trials, they viewed phrases previously developed describing their negative autobiographical memories/worries. During STRATEGY trials participants viewed a strategy they use to help them process the memory/worry. First, a localizer run was completed to determine a unique region of interest for each participant. We identified peak activation within the cingulate cortex to the contrast of MEMORY (STRATEGY+COUNT). Although the task was the same, no neurofeedback was displayed during the localizer run. During the feedback runs, participants were shown neurofeedback from the cingulate cortex following both the MEMORY and STRATEGY trials. This activation was represented on a signal bar display and represented the average cingulate activation during the trial. Unlike many rtfMRI studies, the purpose here was not for participants to interact with the neurofeedback directly. Rather, a feedback summary was shown to participants after each MEMORY and STRATEGY trial as an index of how brain activity changed in response to negative memories/worries and therapeutic strategies. Our goal was not for participants to learn to self-regulate the cingulate cortex, but rather to provide participants with a metacognitive demonstration of strategy efficacy. Participants were given detailed instructions regarding the task design, the role of the cingulate cortex in depression, as well as the hypothesized direction of activation during the MEMORY and STRATEGY phases to help them interpret the neurofeedback. RESULTS/ANTICIPATED RESULTS: Results revealed that “stronger neurofeedback” (defined as the difference between STRATEGY vs. MEMORY trials) correlated with self-reported strategy efficacy ratings immediately following the scan session (p<0.05). More importantly, stronger neurofeedback predicted both self-reported strategy efficacy and frequency of use 1 month following the MRI session (p<0.05). Importantly, this relationship was specific to only those strategies used inside the scanner; and no such relationship was observed at baseline. Neuroimaging results revealed that during the MEMORY phase, activation within inferior frontal gyrus and supramarginal gyrus correlated with baseline BDI score (whole brain, cluster corrected with FSL Flame 1 to p<0.05). During the STRATEGY phase, the periaqueductal gray nucleus, insula, and temporal pole predicted self-reported frequency of strategy use 1 month post-scan session (whole brain, cluster corrected with FSL Flame 1 to p<0.05). DISCUSSION/SIGNIFICANCE OF IMPACT: We believe this study holds promise to provide a powerful demonstration for individuals that strategies used to cope with negative moods can produce significant changes in their brain.

OBJECTIVES/SPECIFIC AIMS: Insufficient endogenous expression of suppressor of cytokine signaling 3 (SOCS3) with subsequent over-activation of its target, the transcription factor STAT3, has been associated with tumorigenesis and cancer development in the lung and other organs. We have observed that a “backup” source of SOCS3 in the lung, namely that secreted in microvesicles (MVs) by alveolar macrophages, is reduced in the bronchoalveolar lavage fluid (BALF) of KRAS mutant mice harboring lung tumors. Here we sought to evaluate levels of SOCS3 in BALF of a cohort of non-small cell lung cancer (NSCLC) patients and to test the effects of vesicular SOCS3 administration on tumor cell transformation and function as potential therapeutic strategy. METHODS/STUDY POPULATION: In total, 22 BALF samples were obtained from healthy volunteers (n=11) as well as patients undergoing diagnostic bronchoscopies for suspected lung cancer (n=11). SOCS3 levels in the BALF were determined by ELISA after brief sonication to disrupt vesicles. In vitro experiments utilized the human adenocarcinoma cell line (A549) or human G12V mutant KRAS-expressing rat lung epithelial cells (RLE-G12V). Proliferation, Fas ligand (FasL)-induced apoptosis, and chemical transformation with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or cigarette smoke extract (CSE) were assessed by CyQuant assay, annexin V staining, and soft agar assay, respectively. For SOCS3 rescue, epithelial cells were treated with natural alveolar macrophages-derived MVs (isolated via ultracentrifugation) or synthetic unilamellar liposomes containing human recombinant SOCS3 for at least 1 hour before assay. RESULTS/ANTICIPATED RESULTS: SOCS3 levels were significantly reduced in BALF samples of patients determined to have NSCLC as compared with healthy volunteers (186.6±26.74 vs. 395.6±74.31 pg/mL, p=0.015, n=11). Addition of exogenous SOCS3-containing liposomes had the capacity to significantly inhibit MNNG and cigarette smoke extract-induced transformation and colony formation in soft agar. Exogenous SOCS3 provided in liposomes or in natural MVs significantly induced apoptosis (both in the presence and absence of FasL) and inhibited basal proliferation of A549 cells. DISCUSSION/SIGNIFICANCE OF IMPACT: These data identified a novel dysregulation of immune surveillance in the form of decreased SOCS3 secretion in the tumor-bearing lung that may contribute to tumorigenesis via sustained STAT3 activation. Future studies will focus on the mechanism underlying this defect and whether rescuing SOCS3 secretion can inhibit cancer progression in vivo.

OBJECTIVES/SPECIFIC AIMS: The food reward circuitry regulates hedonic eating especially in relation to palatable hypercaloric foods, which can lead to chronic overeating and consequent overweight and obesity. Evidence supports that there is considerable overlap within the brain reward circuitry between palatable hypercaloric food intake and substance addiction. The goal of this study was to identify associations between addiction-related genes and body mass index. We hypothesized that addiction-related genes potentially participate in the food reward circuitry if they are associated with obesity traits. METHODS/STUDY POPULATION: A secondary analysis was conducted with 1093 African American adolescents and young adults from the New Mother’s Study. Anthropometric, genetic, demographic and lifestyle measurements were available at the 18-year follow-up assessments. A total of 1350 single nucleotide polymorphisms mapped to 127 addiction-related genes were assessed. A total of 186 ancestry informative markers were used to adjust for population stratification. Generalized estimating equation models were used to identify genetic associations, including additive, dominant, and recessive models, and control for correlations within families. RESULTS/ANTICIPATED RESULTS: The participants ranged from 15 to 23 years of age. Of them, 42.7% were overweight or obese. Significant associations with body mass index were identified for 13 single nucleotide polymorphisms mapped to 11 addiction-related genes, including LEP (p 0.027–<0.001). Most of these genes are involved in dopaminergic, opioidergic, serotonergic pathways, and stress. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results support the role of dopaminergic and opioidergic pathways in the food reward circuitry, and suggest a potential involvement of serotonergic pathways and genes related to stress in the food reward circuitry. Further investigation of the identified genes will facilitate delineation and understanding of the brain food reward system and its relationship with obesity.

OBJECTIVES/SPECIFIC AIMS: Bladder cancer patients being considered for immune checkpoint blockade are often judged on immunohistochemical staining for the checkpoint target protein PD-L1 in the original surgery or biopsy sample. However, sampling error or the clinical evolution of most patients’ cancer can render the original PD-L1 assessment no longer accurate. In contrast, circulating tumor cells (CTCs) allow serial noninvasive sampling of the current tumor status throughout a patient’s clinical course including those with the highest metastatic potential. We therefore sought to develop a method for quantifying PD-L1 expression in CTCs towards addressing inherent limitations of current UC management. METHODS/STUDY POPULATION: This work utilizes both cancer cell lines as well as patient samples. Positive and negative control cancer cell lines were assessed via “industry standard” antibodies for PD-L1 expression via Western blots and immunofluorescence, and a threshold-based method was developed for reliable quantification. PDL-1 expression was additionally verified via interferon-mediated up-regulation. CTCs isolated from bladder cancer patient samples via a density centrifugation method were then assessed for PD-L1 via the same antibodies. RESULTS/ANTICIPATED RESULTS: We will show preliminary preclinical and clinical data that validates the sensitivity and specificity of our assay. A case study will be presented that illustrate the potential useful of the novel approach we describe and which should be complementary to current clinical practices. In a patient with metastatic bladder cancer, this method effectively detected the PD-L1 expression in CTCs taken at a time coincident to when the patient derived an excellent response to the PD-L1 checkpoint inhibitor Pembrolizumab. DISCUSSION/SIGNIFICANCE OF IMPACT: This work highlights the potential utility of CTCs in the management of bladder cancer. It may be the case that this assay in conjunction with current methods of patient selection for immunotherapy may allow for better response prediction than either method alone.

OBJECTIVES/SPECIFIC AIMS: We set out to describe important associations and outcomes among those requiring maintenance renal replacement therapy with the patient volume per provider. METHODS/STUDY POPULATION: Through the combination of several large administrative datasets, including the United States Renal Data System (n=237,485), the American Medical Association Master file (n=6249), and Medicare data limited to 2012, we compared characteristics of patients, by quintile of patient/provider volume. χ2 and logistic regression, adjusted for various patient and provider factors for categorical and continuous variables, was used for baseline comparisons, respectively. Cox regression, adjusted for patient, provider, and socioeconomic variables, was used to calculate risks for important clinical outcomes such as kidney transplant listing, transplant receipt, and all-cause mortality. RESULTS/ANTICIPATED RESULTS: There is a threshold patient volume at which important clinical outcomes, including kidney transplantation and all-cause mortality, may be influenced. Higher patient volume is associated with adverse patient outcome. Those receiving care from providers with the highest patient volumes are less likely to receive kidney transplantation, live in a more rural area, and be non-White. DISCUSSION/SIGNIFICANCE OF IMPACT: There is a need to identify novel and potentially modifiable factors associated with patient outcome among those with end-stage kidney disease on maintenance renal replacement therapy. Provider level variables, such as patient volume, is one such variable. As nephrologists are often tasked with the care of variable numbers of patients on dialysis, a better understanding of this association is an unmet need.

OBJECTIVES/SPECIFIC AIMS: Current practice frequently dictates hospitalization for TIA and minor stroke (TIAMS) in order to obtain comprehensive evaluation of stroke risk factors and mechanism. Inpatient hospitalization is often done to expedite workup and to coordinate care although may be associated with nosocomial risks and increased healthcare cost. However, a subset of these patients who do not have debilitating deficits may not require inpatient hospitalization. We conducted a pilot study to assess the feasibility of conducting rapid outpatient stroke evaluations in low risk patients with TIAMS without disabling deficits. METHODS/STUDY POPULATION: The rapid access clinic was initiated at a single-site urban tertiary care facility for outpatient evaluation of TIAMS within 24 hours of emergency department (ED) evaluation. Patients were selected using a decision tool identifying presumed low-risk TIAMS seen in the ED. Criteria included medical (e.g., no disabling deficit, no thrombolytic agent given, negative CT for hemorrhagic stroke) as well as social criteria (e.g., patient ability to follow-up as an outpatient). We evaluated rates of noncompliance with post-ED follow-up, need for hospitalization from clinic, and 90 day stroke and health outcome data. RESULTS/ANTICIPATED RESULTS: Between December 2016 and December 2017 a total of 93 TIAMS patients seen in the ED were recommended for the rapid access clinic utilizing the decision tool. Of these patients, 94.5% (86) were evaluated within 24 hours of ED discharge. Only 2 patients (2.4%) who received outpatient evaluation required hospitalization; 61 (71.8%) patients had TIAMS on final evaluation in clinic. DISCUSSION/SIGNIFICANCE OF IMPACT: Our pilot data suggests that for a subset of patients, rapid outpatient evaluation may be a feasible and safe strategy for TIAMS management. Future work exploring such strategies may help improve TIAMS outcomes and reduce ED crowding and unnecessary hospital admissions.

OBJECTIVES/SPECIFIC AIMS: Low cancer clinical trial (CCTs) enrollment may contribute to the poor survival improvement for adolescents and young adults (AYAs, aged 15–39 years) with cancer. Treatment site is thought to exacerbate this problem. This study evaluated whether differences in CCT availability explain lower CCT enrollment depending on treatment site for AYAs. METHODS/STUDY POPULATION: This prospective, observational cohort study was conducted at an academic children’s hospital and an adult cancer hospital, 2 affiliated sites within a NCI-designated Comprehensive Cancer Center over 13 months. In consecutive AYA patients newly diagnosed with cancer at both site, it was determined whether an appropriate CCT existed nationally, was available locally, and if enrollment occurred. The proportions of AYAs in these categories were compared by site using the χ2 test. RESULTS/ANTICIPATED RESULTS: Among 152 consecutive AYA patients, 68 and 84 were treated at the children’s hospital and adult cancer hospital, respectively. AYAs treated at the children’s hospital had similar CCT existence nationally compared with AYAs treated at the adult hospital [36/68 (52.9%) vs. 45/84 (53.6%), p=0.938]. However, a significantly higher percentage of children’s hospital treated AYAs than adult hospital treated AYAs had an available CCT [30/68 (44.1%) vs. 14/84 (16.7%), p<0.001]. Enrollment percentages were similarly low in both groups [8/68 (11.8%) vs. 6/84 (7.1%), p=0.327]. DISCUSSION/SIGNIFICANCE OF IMPACT: Significantly fewer AYAs treated at the adult hospital had a CCT available, but national existence was similar at both sites. This suggests that institutional barriers to opening CCT have more importance at adult centers.

OBJECTIVES/SPECIFIC AIMS: Missing data is a common problem in research studies that may lead to inconclusive or inaccurate results. It may even lead to harm secondary to wrong research conclusions. The purpose of this ancillary study is to measure the differences in missing data following implementation of a variety of mechanisms to improve data quality and documentation in a global quality improvement study. Many of the sites involved in the study were in low-income or middle-income countries with minimal research infrastructure. Missing data is defined as “values that are not available that would be meaningful for analysis if they were observed” (The prevention and treatment of missing data, New Engl J Med 367; 14, nejm.org, October 4, 2012). METHODS/STUDY POPULATION: All study sites used REDCap software to enter various data points including hospital and ICU admission and discharge dates as well as whether items on a Checklist relevant to processes of care in the ICU were reviewed. After initial general data collection phase, we categorized data as “must have” and “good to have.” “Must have” variables were defined as data variables that were essential for the study outcomes. “Good to have” variables would not affect the main outcomes of the study if missing. We measured completeness of data using the in-built REDCap data quality check feature. We used several strategies to encourage reduction of missing data. We initially did random data checks but noted that the amount of missing data was substantial and could not be adequately addressed this way. Second, we created excel sheets highlighting missing data for each site and notified sites. This proved onerous to create and made it burdensome for sites to identify easily where data was missing. Third, we built a custom report form in REDCap specifically able to identify which “must have” data points were missing. This could be easily accessed by the principal investigator at each site and made completing the data forms more straightforward. We encouraged all sites to complete their data collection by sending weekly data reports to each site highlighting the patients with missing data. An instructional YouTube tutorial was also created and the link was shared with all sites to demonstrate how to use the custom built report form in REDCap and how to appropriately fill in the missing data. Since this was a global study, we communicated with sites using a variety of locally favored mechanisms including Zoom, FaceTime, WeChat, WhatsApp as well as email. By harnessing the buy-in of local champions our approach was successful. RESULTS/ANTICIPATED RESULTS: The total number of patients recruited for the CERTAIN study is 4843. The rate of all missing variables improved with the efforts described above. Hospital admission dates were missing in 8.4% pre efforts and 4.2% post efforts (p<0.01). ICU admission dates were missing in 5.5% pre and 2.0% post (p<0.01). Documentation of completion of processes of care (including central line review, urinary catheter review, consideration for blood transfusion) improved significantly from pre to post (p<0.01). DISCUSSION/SIGNIFICANCE OF IMPACT: Missing data can be a problem in all types of research studies. This study provides some preliminary evidence for effective approaches that can reduce the problem of missing data when conducting a global study at sites with limited research infrastructure in place. By addressing the concern about missing data, we can be more confident that our results can be accurately analyzed and interpreted, improving the quality of the research.

OBJECTIVES/SPECIFIC AIMS: Smoking during pregnancy (SDP) is associated with negative health outcomes, both proximal (e.g., preterm labor, cardiovascular changes, low birth weight) and distal (e.g., increased child externalizing behaviors and attention deficit/hyperactivity disorder (ADHD) symptoms, increased risk of child smoking). As pregnancy provides a unique, strong incentive to quit smoking, investigating SDP allows analysis of individual predictive factors of recalcitrant smoking behaviors. Utilizing a female twin-pair cohort provides a model system for characterizing genotype×environment interactions using statistical approaches. METHODS/STUDY POPULATION: Using women from the Missouri Adolescent Female Twin Study, parental report of twin ADHD inattentive and hyperactive symptoms at twin median age 15, and twin report of DSM-IV lifetime diagnosis of major depressive disorder, trauma exposure (physical assault and childhood sexual abuse), collected at median age 22, were merged with Missouri birth record data for enrolled twins, leading to 1553 individuals of European ancestry and 163 individuals of African-American ancestry included in final analyses. A SDP propensity score was calculated from sociodemographic variables (maternal age, marital status, educational attainment, first born child) and used as a 6-level ordinal covariate in subsequent logistic regressions. RESULTS/ANTICIPATED RESULTS: For European ancestry individuals, parental report of hyperactive ADHD symptoms and exposure to childhood sexual abuse were predictive of SDP, while a lifetime diagnosis of major depressive disorder, parental report of inattentive ADHD symptoms, and exposure to assaultive trauma were all not significantly predictive of future SDP. For African-American individuals, none of these variables were significant in predicting future SDP. DISCUSSION/SIGNIFICANCE OF IMPACT: Understanding this relationship of risk-mechanisms is important for clinical understanding of early predictors of SDP and tailoring interventions to at risk individuals. Ultimately, the focus of this research is to mitigate risk to pregnant smokers and their children. Additionally, the cohort-ecological approach informs how well research and administrative (vital record) data agree. This allows for evaluation of whether administrative data improve prediction in research cohorts, and conversely if research data improve prediction over standard sociodemographic variables available in administrative data.