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/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/SPECIFIC AIMS: Pelvic organ prolapse, a disorder in which the muscles of the pelvic floor are weakened over time, affects over a million women each year in the United States. A quarter of these women undergo a reconstructive procedure, increasingly using polypropylene mesh as mechanical reinforcement to the pelvic floor. However, the number of complications such as chronic pain and mesh erosion/exposure in women with vaginal mesh implants were reported at rates as high as 10%–20%. This indicates a limited understanding of the host response to mesh in vaginal tissue and strategies to reduce these complications. Utilizing a novel surgical technique in New Zealand white rabbits, we implant mesh analogously to human implantation and evaluate changes in the immunologic response at early (14 d) and tissue remodeling outcomes at late stages (90 and 180 d) of implantation. The mesh-tissue complex was removed from each rabbit and processed for histological staining as well as immunolabeling of immune cells, such as macrophages. Extracellular matrix protease assays and mechanical integrity of the tissue also evaluate the overall inflammatory response associated with each implant. METHODS/STUDY POPULATION: Commercially available polypropylene mesh was used to investigate the modulation of the immune response. An adapted radio frequency glow discharge method is used to create a stable negative charge on the surface of the mesh, followed by the sequential deposition of polycationic and polyanionic polymers to provide a stable, conformal, nanoscale coating. It is well known macrophages are characterized on a spectrum ranging from a proinflammatory M1 phenotype to an M2 anti-inflammatory phenotype. Interleukin-4, an immunomodulatory cytokine known to promote the M2 phenotype, is incorporated into the coating to be released in a controlled manner upon implantation. Utilizing a novel surgical technique in New Zealand white rabbits, we implant mesh using the “gold standard” abdominal sacrocolpopexy procedure and evaluate changes in the immunologic response at early (14 d) and tissue remodeling outcomes at late stages (90 and 180 d) of implantation. The procedure begins with an initial hysterectomy removing the uterus followed by creating space along the vaginal wall on both sides between the bladder and the rectum. Two 3×10 cm2 pieces of mesh are secured along both sides of the vaginal wall. The remaining flaps at the top are then secured to a ligament in the sacral/lumbar space, creating the support to the pelvic organs. Upon closing the incision, a partial thickness defect is made in the abdominal wall, and mesh is implanted inside to repair the abdominal muscle. Both of these implantations of mesh allow for the assessment of the immune response in the pelvic area (relevant for prolapse patients) and in the abdominal area (relevant for translation from hernia repair).The mesh-tissue complex was removed from each rabbit and processed for histological staining as well as immunolabeling of immune cells, such as macrophages. Extracellular matrix protease assays and mechanical integrity of the tissue also evaluate the overall inflammatory response associated with each implant. RESULTS/ANTICIPATED RESULTS: The results of this study show that implants into vaginal tissues elicited an increased host inflammatory response at 14 days as compared with those in the abdominal wall. However, at chronic time points the inflammatory response in the vagina was reduced as compared to that in the abdominal cavity. The present study also demonstrates the scale-up of a previous methodology for a nanoscale coating. We present a nanometer thickness, tunable, and uniform coating capable of releasing bioactive interleukin-4. We evaluated the biological functionality of the coated mesh via bioactivity studies and in vivo implantation. An ideal mesh would provide mechanical support to the pelvic floor while decreasing the inflammatory response and increasing integration with the surrounding native tissue. DISCUSSION/SIGNIFICANCE OF IMPACT: We developed an in vivo model clinically relevant to understanding the early host response to mesh in an anatomically relevant area, the vaginal microenvironment. Previous studies have been conducted in a rodent abdominal defect model while other work has been done in a nonhuman primate vaginal model, but the host response is only observed at later time points (>3 mo). Thus, we developed a rabbit model to investigate early responses and a novel coating to actively working towards improved tissue integration.

OBJECTIVES/SPECIFIC AIMS: To share the experience gained and lessons learned from a cross CTSA collaborative effort to improve the review process for Pilot Studies awards by exchanging external reviewers. METHODS/STUDY POPULATION: The CEREC process is managed by a web-based tracking system that enables all participating members to view at any time the status of reviewer invitations. This online tracking system is supplemented by monthly conference calls during which new calls for proposals are announced and best practices are identified. Each CTSA hub customized the CEREC model based on their individual pilot program needs and review process. Some hubs have supplemented their internal reviews by only posting proposals on CEREC that lack reviewers with significant expertise within their institutions. Other hubs have requested 1–3 external reviewers for each of their proposals or a selection of most promising proposals. In anticipation of potential scoring discrepancies, several hubs added a self-assessment of reviewer expertise and confidence at the end of each review. If a proposal is on the cusp of fundability, then the reviewers’ self-assessment may be taken into account. In addition to the tracking data collected by the online system, a survey of CEREC reviewers was conducted using Qualtrics. RESULTS/ANTICIPATED RESULTS: Across the 144 proposals submitted for reviews, CEREC members issued a total of 396 email invitations to potential reviewers. The number of invitations required to yield a reviewer ranged from 1 to 17. A total of 224 invitations were accepted, for a response rate of 56%. An external reviewer was unable to be located for 5 proposals (3%). Ultimately, 196 completed reviews were submitted, for a completion rate of 87%. The most common reasons for non-completion after acceptance of an invitation included reviewer illness and discovery of a conflict of interest. CEREC members found the process extremely useful for locating qualified reviewers who were not in conflict with the proposal being reviewed and for identifying reviewers for proposals related to highly specialized topics. The survey of CEREC reviewers found that they generally found the process easy to navigate and intellectually rewarding. Most would be willing to review additional CEREC proposals in the future. External reviewer comments and scores were generally in agreement with internal reviewer comments and scores. Thus, hubs could factor in external reviewer scores equally to internal reviewer scores, without feeling compelled to calibrate external reviewer scores. Overall, through CEREC external reviewers, mainly due to the stronger matching of scientific expertise and reduction of potential bias, the quality of reviews appear to be higher and more pertinent. DISCUSSION/SIGNIFICANCE OF IMPACT: Some aspects of the process emerged that will be addressed in the future to make the system more efficient. One issue that arose was the burden on the system during multiple simultaneous calls for proposals. Future plans call for harmonizing review cycles to avoid these overlaps. Efficiency also will be improved by optimizing the timing of reviewer invitations to minimize the probability of obtaining more reviews than requested. In addition to the original objective of CEREC, the collaboration has led to additional exchange of information regarding methods and processes related to running the Pilot Funding programs. For example, one site developed a method using REDCap to manage their reviewer database; an innovation that is being shared with the other CEREC partners. Another site has a well-developed process for integrating community reviewers into their review process and is sharing their training materials with the remaining CEREC partners.

OBJECTIVES/SPECIFIC AIMS: The goal for this project is to determine the feasibility of using a novel multi-photon fiber-coupled microscope to aid surgeons in localizing STN during surgeries. In order to accomplish this goal, we needed to identify the source of a strong autofluorescent signal in the STN and determine whether we could use image classification methods to automatically distinguish STN from surrounding brain regions. METHODS/STUDY POPULATION: We acquired 3 cadaveric brains from the University of Colorado Anschutz Medical Campus, Department of Pathology. Two of these brains were non-PD controls whereas 1 was diagnosed with PD. We dissected a 10 square centimeter region of midbrain surrounding STN, then prepared this tissue for slicing on a vibratome or cryostat. Samples were immuno-labeled for various cellular markers for identification, or left unlabeled in order to observe the autofluorescence for image classification. RESULTS/ANTICIPATED RESULTS: The border of STN is clearly visible based on the density of a strong autofluorescent signal. The autofluorescent signal is visible using 2-photon (850–1040 nm excitation) and conventional confocal microscopy (488–647 nm excitation). We were also able to visualize blood vessels with second harmonic generation. The autofluorescent signal is quenched by high concentrations of Sudan-black B (0.5%–5%), and is primarily localized in microtubule-associated protein-2 (MAP2)+ cells, indicating that it is likely lipofuscin accumulation in neurons. Smaller lipofuscin particles also accumulate in microglia, identified based on ionized calcium binding adopter 1 (Iba1)+ labeling. We anticipate that colocalization analysis will confirm these qualitative observations. Using 2-photon images of the endogenous autofluorescent signal in these samples, we trained a logistic regression-based image classifier using features derived from gray-level co-occurrence matrices. Preliminary testing indicates that our classifier performed well, with a mean accuracy of 0.89 (standard deviation of 0.11) and a Cohen’s Kappa value of 0.76 (standard deviation of 0.24). We are currently using coherent anti-Stokes Raman scattering and third harmonic imaging to identify different features of myelin that can be used to distinguish between these regions and expect similar results. DISCUSSION/SIGNIFICANCE OF IMPACT: Traditional methods for localizing STN during DBS surgery include the use of stereotactic coordinates and multi-electrode recording (MER) during implantation. MERs are incredibly useful in DBS surgeries, but require penetration of brain structures in order to infer location. Using multi-photon microscopy techniques to aid identification of STN during DBS surgeries offers a number of advantages over traditional methods. For example, blood vessels can be clearly identified with second harmonic generation, something that is not possible with MER. Multi-photon microscopy also allows visualization deep into tissue without actually penetrating it. This ability to look within a depth of field is useful for detection of STN borders based on autofluorescent cell density. When combined with traditional stereotactic information, our preliminary image classification methods are a fast, reliable way to provide surgeons with extra information concerning their location in the midbrain. We anticipate that future advancements and refinements to our image classifier will only increase accuracy and the potential applications and value. In summary, these preliminary data support the feasibility of multi-photon microscopy to aid in the identification of target brain regions during DBS surgeries. The techniques described here complement and enhance current stereotactic and electrophysiological methods for DBS surgeries.

OBJECTIVES/SPECIFIC AIMS: To develop a robust spatial normalization pipeline for brains of individuals with focal cortical lesions. METHODS/STUDY POPULATION: Individuals with chronic focal cortical lesions from stroke. RESULTS/ANTICIPATED RESULTS: We have developed a robust spatial normalization pipeline for brains of individuals with focal cortical lesions, and demonstrate how the pipeline overcomes obfuscated neuroanatomy to yield both consistent and excellent results. DISCUSSION/SIGNIFICANCE OF IMPACT: Our robust normalization pipeline will enable group analyses of individuals with cortical lesions with greater spatial precision. This greater spatial precision will improve answers to questions about functional localization in the brain, and ultimately allow translation of findings from neuroimaging studies in individuals with cortical lesions to the clinic.

OBJECTIVES/SPECIFIC AIMS: Costs associated with the treatment of skin diseases accounted for greater than 4% of total US healthcare spending in 2013, an increase of $46 billion (170%) since 2004. Considering the increase in novel treatments and spending, cost-utility analyses (CUAs) may provide a better understanding of costs in dermatology. In this study, we conduct a systematic overview of study quality among CUAs related to dermatology. METHODS/STUDY POPULATION: We queried studies from the Tufts Medical Center Cost-Effectiveness Analysis Registry (www.cearegistry.org), a database supplying information on all peer-reviewed cost-effectiveness analysis through 2014. Database methodology was previously discussed here. We queried studies using keywords from the 24 major skin disease categories (e.g., diseases relating to actinic damage were searched by using “actinic,” “actinic keratosis”). We collected data on study design, reporting methods, and analyzed relevant data stratified by 2 time-periods (1976–2008 and 2009–2014) chosen to encompass a comparable number of studies. RESULTS/ANTICIPATED RESULTS: In total, 42 and 50 studies corresponding to the 2 time-periods were retrieved (representing 14/24 disease categories). Based on the recommended data reporting guidelines for CUAs, study quality remained largely unchanged across the 2 phases. Across the 2 time-periods, a societal perspective was used in 19% and 12% of studies, costs and (quality adjusted life-years) QALYs were discounted in 67% and 72% of studies, a correct (incremental cost-effectiveness ratio) ICER was reported in 67% and 72% of studies, and a sensitivity analysis was included in 88% and 84% of studies, respectively. DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest the quality of dermatology-related CUAs, as evaluated by recommended data reporting guidelines, to be generally stable during the analyzed time-periods. However, the quality of our results may be limited by the small number of CUAs within dermatology (10/24 disease categories did not have CUAs across any time-period). Moving forward, we encourage researchers within dermatology to pursue additional investigation towards cost-effective practices while adhering closely to recommended quality reporting guidelines for CUAs.

OBJECTIVES/SPECIFIC AIMS: Focal cartilage injuries of the knee joint are common and present a treatment challenge due to minimal intrinsic repair. Cartilage tissue engineering techniques currently used in clinical practice are expensive, cumbersome, and often ineffective in patients with mechanical or medical comorbidities. To address these issues, we developed an acellular nanofibrous scaffold with encapsulated growth factors designed to enhanced articular cartilage repair. Our goal is to evaluate this technology in vitro and pilot a large animal model for eventual translation into human subjects. METHODS/STUDY POPULATION: Hyaluronic acid (HA, 65 kDa) will be methacrylated (~40% modification, MeHA) and conjugated with cell-adhesive (RGD) groups. A solution of 4% wt/vol MeHA, 2% wt/vol polyethylene oxide (900 kDa), 0.05% wt/vol Irgacure 2959, and 0.005% wt/vol stromal cell-derived factor-1α (SDF-1α) and/or transforming growth factor-β3 (TGF-β3) will be prepared in ddH2O. The solution will be electrospun onto a rotating mandrel to achieve a dry scaffold thickness of 0.5 mm. The scaffold matt will be UV cross-linked and 5 mm-diameter samples will be cut out. Four groups of scaffolds will be prepared: MeHA, MeHA+SDF, MeHA+TGF, MeHA+SDF+TGF. All groups will be evaluated for fiber diameter, swell thickness, equilibrium compressive modulus, degradation rate, and growth factor release rate over 4 weeks (n=10). Scaffolds will also be seeded with juvenile porcine MSCs (5×104) in 200 μL of medium incubated for 24 hours. Seeded scaffolds will be evaluated for equilibrium compressive modulus, cell infiltration, and chondrogenesis at 4 and 8 weeks (n=10). Scaffolds will then be evaluated in a juvenile Yucatan minipig cartilage defect model. In total, 6 animals will undergo bilateral knee surgery to create four 4 mm-diameter full-thickness cartilage defects in each trochlear grove. All defects will receive microfracture to release marrow elements. Each knee will receive 2 scaffolds of the same group (replicates) with paired microfracture controls, resulting in a sample size of 3. Animals will be sacrificed at 12 weeks and defects will be evaluated via non-destructive indentation testing for mechanical properties, microCT for defect fill and subchondral bone morphology, and histology for ICRS II Visual Histological Assessment Scoring. RESULTS/ANTICIPATED RESULTS: Our preliminary studies have shown reliable replication of electrospun MeHA scaffolds. We anticipate cross-linking density to correlate positively with compressive modulus, and negatively with swell thickness, degradation rate, and growth factor release rate. We anticipate the addition of SDF-1α and TGF-β3 to increase cell infiltration and chondrogenesis, respectively, within seeded scaffolds. Similarly, we expect minipig defects treated with growth factor-releasing scaffolds to show greater mechanical properties, defect fill, and ICRS II score compared with MeHA scaffolds without growth factor. DISCUSSION/SIGNIFICANCE OF IMPACT: This study has the potential to show how an HA-based cell-free scaffold can be augmented with 2 growth factors that act synergistically to improve cartilage repair in a large animal model. This technology would improve upon the cell-free scaffolds already used clinically for autologous matrix-induced chondrogenesis and is directly translatable.

OBJECTIVES/SPECIFIC AIMS: The serotonin receptor 6 (5-HT6) is a potential therapeutic target given its distribution in brain regions that are important in depression, anxiety, and cognition. This study sought to investigate the effects of age on 5-HT6 receptor availability using 11C GSK215083, a PET ligand with affinity for 5-HT6 in the striatum and 5-HT2A in the cortex. METHODS/STUDY POPULATION: In total, 28 healthy male subjects (age range: 23–52 years) were scanned with 11C-GSK215083 on the HR+PET scanner. Time-activity curves in regions-of-interest were fitted with multilinear analysis-1 method. Binding potentials (BPND) were calculated using cerebellum as the reference region and corrected for partial volume effects. RESULTS/ANTICIPATED RESULTS: In 5-HT6 rich areas, regional 11C-GSK215083 displayed a negative correlation between BPND and age in the caudate (r=−0.41, p=0.03) (14% change per decade), and putamen (r=−0.30, p=0.04) (11% change per decade), but not in the ventral striatum and pallidum. Negative correlation with age was also seen in cortical regions (r=−0.41, p=0.03) (7% change per decade), consistent with the literature on 5-HT2A availability. DISCUSSION/SIGNIFICANCE OF IMPACT: This is the first in vivo study in humans to examine the effect of age on 5-HT6 receptor availability. The study demonstrated a significant age-related decline in 5-HT6 availability (BPND) in the caudate and putamen.

OBJECTIVES/SPECIFIC AIMS: QuantiFERON-TB Gold In-Tube (QFT) conversion from negative to positive, is regarded as a marker of recent latent tuberculosis infection and may be predictive of incident active tuberculosis (TB) disease. However, it remains unclear how conversion is influenced by individual and environmental factors, including the infectiousness of the source case to whom the contact was exposed. We aimed to examine the effect of infectiousness of TB in the source case, as measured by presence of cavitation on chest X-ray, on the incidence of QFT conversion among close contacts of the pulmonary TB index case, after adjusting for potential confounding by contact and source case characteristics. METHODS/STUDY POPULATION: The Regional Prospective Observational Research for Tuberculosis (RePORT)-Brazil is an ongoing prospective cohort study that enrolls close contacts of culture-confirmed pulmonary TB patients and follows them for 24 months for development of active TB. Demographic, clinical, and diagnostic information are obtained at baseline and during follow-up at clinical visits and by telephone. QFT testing is performed at baseline and repeated after 6 months if the baseline QFT is negative. A positive IFN-γ value is defined as >0.35 IU/mL, as recommended by the manufacturer and the CDC, and QFT conversion is defined as a negative QFT at baseline followed by a positive QFT at 6 months. RESULTS/ANTICIPATED RESULTS: Among 260 enrolled contacts with nonpositive baseline QFT results and 6 months of follow-up, 198 (76%) were retested with QFT 6 months after enrollment. Of those retested, 26 (13%) converted to positive. Presence of any cavitation in the source case, based on chest radiography, was significantly associated with QFT-conversion (ORunadjusted=2.4, 95% CI: 1.0–5.7). Additional univariate analyses revealed that QFT conversion was associated with black and brown race (compared with white race) of the contact, current smoking and current alcohol use in the source case. After adjusting for potential confounders (age, sex, and race of the contact and current smoking of the source case), the association between source case cavitation and QFT conversion remained (ORadjusted=2.5 95% CI: 1.0–6.2). As of December 6, 2017, none of the QFT-retested contacts had developed active TB, with a median follow-up of 12.3 months (IQR: 7.1–13.1). We anticipate that ongoing enrollment and follow-up may yield cases of active TB; future analyses will provide greater precision for examining predictors of QFT-conversion and its association with incident TB. DISCUSSION/SIGNIFICANCE OF IMPACT: Our preliminary results agree with published literature suggesting the infectiousness of TB in the index case is a predictor of incident LTBI. Along with recent LTBI, immune suppression, HIV co-infection, and type 2 diabetes are considered risk factors for progression to active TB disease. Because only a small proportion of persons progress from LTBI to active TB disease, it is not appropriate to treat all persons with LTBI. Thus, more research is needed to identify groups at highest risk for QFT-conversion and incident TB disease, so these groups can be targeted for TB prevention, interventions, and facilitate a decline in TB incidence and mortality.

OBJECTIVES/SPECIFIC AIMS: Negative symptoms of schizophrenia, including motivational deficits, social withdrawal, poverty of speech, decreased emotional reactivity, and psychomotor retardation, have been shown to be most predictive of functional impairment and poor outcome in patients with schizophrenia. Furthermore, these symptoms tend not to be responsive to antipsychotic medications. Inflammation could be one mechanism underlying these difficult to treat symptoms. METHODS/STUDY POPULATION: Three cohorts of patients, reflecting different phases of disease, were studied. One cohort was comprised of a sample of patients with deficit schizophrenia (characterized by primary and enduring negative symptoms; n=17), nondeficit patients (n=39), and healthy controls (n=28). ANOVA and multivariate general linear models were used to compare groups, and linear regression models were used to examine relationships between inflammatory cytokines and negative symptoms. The second cohort was comprised of 80 individuals at clinical high risk for psychosis from the North American Prodromal Longitudinal Study. Linear regression models examined the relationship between baseline inflammatory markers and subsequent negative symptoms at follow-up visits up to 2 years. The third cohort consisted of patients with treatment-resistant schizophrenia (TRS) on clozapine (n=10). Correlations were performed to examine relationships between inflammatory markers and negative symptoms. In a subgroup of patients from this third sample, resting state functional connectivity analyses were performed on fMRI data to explore relationships between inflammatory markers and connectivity in brain reward circuitry. RESULTS/ANTICIPATED RESULTS: In a sample of patients with the deficit syndrome of schizophrenia (n=17), a subtype of the disorder characterized by primary and enduring negative Symptoms, tumor necrosis factor (TNF) was significantly increased relative to nondeficit patients (n=39) and healthy controls (n=28; F2,57=3.51, p=0.036), and predicted total negative symptoms (β=0.31, p=0.012), alogia (β=0.30, p=0.024), and blunted affect (β=0.31, p=0.018) items of the Positive and Negative Symptom Scale in linear regression models while controlling for antipsychotics. In another sample of individuals at clinical-high risk for psychosis (n=80), baseline concentrations of TNF significantly predicted negative symptoms, including anhedonia, apathy, and loss of interest in linear regression models, at the 6-month (β=0.25, p=0.011) and 12-month follow-up (β=0.39, p=0.001). Interleukin (IL)-1 receptor antagonist also predicted these symptoms at the 6-month follow-up (β=0.21, p=0.037). In a third sample (n=10) of patients with TRS treated with clozapine, IL-1β was correlated with passive/apathetic social withdrawal (r=0.657, p=0.039) and disturbance of volition (r=0.686, p=0.029) items of the Positive and Negative Symptom Scale and the global avolition-apathy scores of the Scale for the Assessment of Negative Symptoms (r=0.751, p=0.012). Finally, in a small subsample (n=5) of patients from this TRS cohort for whom we collected fMRI data, we found resting-state functional connectivity from a right nucleus accumbens seed to a cluster in medial prefrontal cortex. We found relationships between higher inflammation and decreased connectivity for TNF (r=−0.64) and CRP (r=−0.89). DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, these preliminary data show the predicted relationship between inflammatory markers and negative symptoms and demonstrate the reproducibility of TNF and other monocytic-derived cytokines as reliably elevated in schizophrenia and associated with negative symptoms across samples of patients with schizophrenia and individuals at high risk for psychosis. Cytokines may exert their effects via their impact on brain reward circuitry, and could represent novel treatment targets for motivational deficits and negative symptoms of schizophrenia.

OBJECTIVES/SPECIFIC AIMS: To identify cardiac structural and function parameters, obtained on usual stroke-care TTE evaluation, associated with cardioembolic stroke (CE) in patients without AF. Hypothesis—left atrial (LA) size and valve dysfunction will be strongly associated with incident CE. METHODS/STUDY POPULATION: Inclusion criteria: July 1, 2013 to July 1, 2015 admission with imaging-confirmed ischemic stroke, no AF, TTE within 1st 7 days. TTE structure/function parameters were recorded. Stroke subtype (CE vs. other) defined using TOAST criteria, blinded to TTE. New AF definition: AF on ECG, telemetry or event monitor. CE/New AF outcome of interest in separate multivariable logistic regression models testing associations with TTE parameters (adjusting for demographics/vascular risk factors). RESULTS/ANTICIPATED RESULTS: Participants (n=332) were ~60 years hypertensive black males with moderate NIHSS and normal ejection fraction. In adjusted models, odds of CE increased with increasing LA systolic diameter (per 0.1 cm), mitral E point velocity(cm/s), mitral valve dysfunction, wall motion abnormality. New AF also associated with increasing LA systolic diameter. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings may suggest cardiac structural changes independent of AF that are on the CE causal pathway. Understanding the relationship between such TTE parameters and stroke subtype would impact clinical practice, as such TTE data is underutilized when considering stroke mechanism and management.

OBJECTIVES/SPECIFIC AIMS: Our main objectives are to study sensory encoding in the adult cortex and quantify rodents’ ability to use intracortical microstimulation to guide behavior. METHODS/STUDY POPULATION: Three rats were implanted with unilateral bipolar stimulating electrodes. The electrodes were connected to a wireless neural stimulator housed in the rat’s backpack. The rat’s swim path was tracked by a video camera above the circular pool, and stimulation parameters were updated in real-time based on distance from the platform. Stimulation was delivered as the distance from the platform increased. Stimulation amplitude was determined through behavioral threshold testing, and parameters ranged from 15–75 μA with 100-Hz pulse trains and 0.2-ms pulses. Rats were first challenged with the 4-platform task in which the submerged platform was randomized across 4 possible locations. This dissociated visual cues from the platform location, as rats had knowledge of the 4 possible locations, but had to use stimulation to guide them efficiently. Next, rats were tasked with the more challenging random-platform task. Visual cues were completely dissociated from the platform location by randomizing the platform location across the entire pool. Performance using the neuroprosthetic device was assessed by comparing trials when the device was on (stimulation trial) Versus off (no-stim trial) for the 2 tasks. RESULTS/ANTICIPATED RESULTS: 4-platform task: Rats visited less potential platform locations when the neuroprosthetic was on Versus off. Rats were also more likely to visit the correct platform location on their first swim trajectory when brain stimulation was delivered. When artificial cues were not available, rats had a greater chance of visiting the platform location from the previous trial. This indicated that rats relied on visuospatial memory without the neuroprosthetic. Random platform task: Performance was measured by taking the ratio of the rat’s actual path length to the optimal path length. When the neuroprosthetic was on, rats demonstrated superior performance through a smaller path to length ratio compared with when the device was off. The platform locations of catch trials were matched to a random subset of stimulation trials, permitting a paired sample t-test. Both rats had significantly shorter path lengths when the device was on. DISCUSSION/SIGNIFICANCE OF IMPACT: Rodents have excellent navigation skills that have been well studied. They have been shown to rely on multimodal sensory information from visual, olfactory, auditory, and idiothetic cues to navigate through their environment. The importance of these cues depends on both their environmental presence and task relevance. In the original Morris water maze experiment, rats use vision to form a visuospatial map of the platform location for allocentric navigation. Here, we have shown that sensory augmented rats can pick up on novel sensory information delivered through ICMS to efficiently find a hidden platform when visual cues are made irrelevant.Our results have implications for the design of the bi-directional sensorimotor neuroprosthetic. We have demonstrated that mammals can interpret artificial sensory information to guide behavior. Future directions include investigating sensory encoding in other primary sensory areas and downstream targets along the somatosensory neuraxis.

OBJECTIVES/SPECIFIC AIMS: The objectives of this study were to compare different methods for determining the disciplines involved in a research article. We sought to address the following questions: To what extent does the number of disciplines reported by an article’s corresponding author agree with their description of the article as unidisciplinary or interdisciplinary? (Q1) and To what extent does the corresponding author’s description of the research as unidisciplinary or interdisciplinary agree with its classification as unidisciplinary or interdisciplinary based on the affiliation of its co-authors? (Q2). METHODS/STUDY POPULATION: Using Scopus, we randomly selected 100 articles from 2010 and 2015 from science teams that had at least 1 author affiliated with Johns Hopkins. Author affiliations were grouped into common academic disciplines: Basic Science, Medicine (and all clinical specialties), Public Health, Engineering, Social Science, Computer Science, Pharmacy, Nursing, and Other. Articles with more than 1 discipline were considered, interdisciplinary. We then sent an online Qualtrics survey to the corresponding author of each article and asked them to indicate (1) all of the disciplines that contributed to the research article at hand, and (2) to indicate whether they considered the research to be “unidisciplinary” or “interdisciplinary” based on definitions that we provided. RESULTS/ANTICIPATED RESULTS: For Q1, we asked corresponding authors to indicate the number of disciplines involved in their research and then to choose the definition that best described their research. Among 76 respondents, 42 indicated that their research consisted of 1 discipline, and 34 indicated that their research consisted of more than 1 discipline. Of the 42 respondents who indicated that their research consisted of one discipline, 21 (50%) respondents described their research as “unidisciplinary” and 21 (50%) described their research as “interdisciplinary.” However, of the 34 respondents who indicated that their research consisted of more than 1 discipline, all but 1 (97%) described their research as “interdisciplinary.” For Q2, we assigned a discipline to each co-author based on his/her affiliation and counted the number of disciplines involved. Among 76 respondents, of the 22 who described their research as “unidisciplinary,” 16 (73%) were categorized as “unidisciplinary” and 6 (27%) were categorized as “interdisciplinary,” using this method. Of the 54 respondents who described their research as “interdisciplinary,” 30 (56%) were categorized as “interdisciplinary” and 24 (44%) as “unidisciplinary.” DISCUSSION/SIGNIFICANCE OF IMPACT: Our results highlight that different methods for determining whether a given research article is interdisciplinary are likely to yield different results. Even when researchers indicate that their research is based within one major discipline, they may still consider it interdisciplinary. Likewise, classifying an article as either unidisciplinary or interdisciplinary based on the affiliations of its co-authors, may not be consistent with the way it is viewed by its authors. It is important to acknowledge that assessing the interdisciplinarity of research is complex and that objective and subjective views may differ.

OBJECTIVES/SPECIFIC AIMS: Preliminary animal studies showed that low-intensity ultrasound (US) coincident with intravascularly administered microbubbles locally disrupts tumor vasculature. This study translates the novel therapy of antivascular ultrasound (AVUS) into an autochthonous model of hepatocellular carcinoma (HCC). The differential effects produced by AVUS at low and high doses are evaluated. METHODS/STUDY POPULATION: HCC was induced in 12 Wistar rats by ingestion of 0.01% diethylnitrosamine in drinking water for 12 weeks. Rats received AVUS treatment at low and high doses. Low dose group (n=6) received 1 W/cm2 US for 1 minute with 0.2 mL microbubbles injected IV. High dose group (n=6) received 2 W/cm2 for 2 minute with 0.7 mL microbubbles IV. Perfusion was measured before and after AVUS with contrast-enhanced ultrasound (CE-US) and power Doppler (PD-US). Peak enhancement (PE) and perfusion index (PI) were measured from each US mode. Histology after sacrifice or natural death was compared to pre/post US. Analysis of H&E and trichrome sections was evaluated for percent area of hemorrhage and findings of tissue injury and repair including inflammation, necrosis, and fibrosis. RESULTS/ANTICIPATED RESULTS: After high dose AVUS, PE, and PI of CE-US decreased from baseline by an average of 33.3% and 29.7%, respectively. Histology showed extensive tissue injury (hemorrhage, necrosis, fibrosis) in 58% of tumor cross-sectional area. Conversely, low dose AVUS increased PE and PI of CE-US by an average of 39.3% and 67.8%, respectively. Histology showed smaller areas of microhemorrhage Versus large pools of hemorrhage (only 17% area). PD-US changes were similar to CE-US. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, the opposing effects of AVUS observed at 2 doses allows for multiple roles in tumor therapy. Enhanced perfusion at a low dose may improve drug delivery or radiation therapy. Whereas, vascular disruption at high doses of AVUS may allow noninvasive ischemic therapy. Furthermore, AVUS is ripe for translation given the use its component parts clinically: low-intensity long-tone burst for physiotherapy and microbubbles as an US contrast agent. Thus, AVUS should be evaluated for translation of its differential effects into noninvasive therapies for HCC and other tumors.

OBJECTIVES/SPECIFIC AIMS: The aims of this study are (1) to develop and characterize a novel nonhuman primate model of pneumococcal pneumonia that mimics human disease; and (2) determine whether Streptococcus pneumoniae can: (a) translocate to the heart, (b) cause adverse cardiac events, (c) induce cardiomyocyte death, and (d) lead to scar formation during severe pneumonia in baboons. METHODS/STUDY POPULATION: Six adult baboons (Papio cynocephalus) were surgically tethered to a monitoring system to continuously assess their heart rate, temperature, and electrocardiogram (ECG). A baseline transthoracic echocardiogram, 12-lead ECG, serum troponin-I levels, brain natriuretic peptide, and heart-type fatty acid binding protein (HFABP) levels were obtained before infection and at the end of the experiment to determine cardiovascular damage during pneumococcal pneumonia. Animals were challenged with 108 colony-forming units of S. pneumoniae in the right middle lobe using flexible bronchoscopy. Three baboons were rescued with ampicillin therapy (80 mg/kg/d) after the development of pneumonia. Cardiac damage was confirmed by examination of tissue sections using immunohistochemistry as well as electron and fluorescence microscopy. Western-blots and tissue staining were used to determine the presence of necroptosis (RIP3 and pMLKL) and apoptosis (Caspase-3) in the cardiac tissue. Cytokine and chemokine levels in the heart tissue were determined using Luminex technology. RESULTS/ANTICIPATED RESULTS: Four males (57%) and three (43%) females were challenged. The median age of all baboons was 11 (IQR, 10-19) years old, which corresponds to a middle-aged human. Infected baboons consistently developed severe pneumonia. All animals developed systemic inflammatory response syndrome with tachycardia, tachypnea, fever, and leukocytosis. Infection was characterized by initial leukocytosis followed by severe leukopenia on day 3 postinoculation. Non-specific ischemic alterations by ECG (ST segment and T-wave flattering) and in the premortem echocardiogram were observed. The median (IQR) levels of troponin I and HFABP at the end of the experiment were 3550 ng/mL (1717–5383) and 916.9 ng/mL (520.8–1323), respectively. Severe cardiomyopathy was observed using TEM and H&E stains in animals with severe pneumonia. Necroptosis was detected in cardiomyocytes of infected animals by the presence of pMLKL and RIP3 in cardiac tissues. Signs of cardiac remodeling indicated by disorganized collagen deposition was present in rescued animals but not in the other animals. DISCUSSION/SIGNIFICANCE OF IMPACT: We confirmed that baboons experience cardiac injury during severe pneumococcal pneumonia that is characterized by myocardial invasion, activation of necroptosis, and tissue remodeling in animals rescued by antimicrobial therapy. Cardiac damage by invading pneumococci may explain why adverse cardiac events that occur during and after pneumococcal pneumonia in adult human patients.

OBJECTIVES/SPECIFIC AIMS: Most incidents of partner violence occur when one or both partners have been drinking, however, the mechanism through which this association exists is unclear. The neural circuits that support self-regulation of emotion and social behavior, as well as autonomic influences on the heart, are co-localized in the brain and represent an integrated bidirectional regulatory system. These physiological regulatory processes are mediated by a neural substrate known as the central autonomic network which includes the peripheral autonomic nervous system. The central autonomic network modulates biobehavioral resources in emotion by flexibly responding to physiological arousal in response to changing situational demands, and serves a fundamental role in emotion regulation and goal-directed motor behavior, and this circuit can be indexed with heart rate variability (HRV). METHODS/STUDY POPULATION: In total, 17 distressed violent (DV) partners (11 females, 6 males) were matched to a sample of distressed nonviolent (DNV) partners (7 female, 6 males) were matched on age, sex, and relationship satisfaction and participated in a placebo-controlled alcohol administration study with an emotion-regulation task during which electroencephalography, HRV, and galvanic skin response (GSR) measures were collected. In the alcohol condition, participants were administered a mixture of 100 proof vodka and cranberry juice calculated to raise their blood alcohol concentration to 0.08%. In the placebo condition, participants consumed a volume of juice equivalent to that consumed in the alcohol condition, but without alcohol. Alcohol and placebo conditions were counter-balanced across participants as were the presentation the blocks of evocative and neutral partner stimuli and emotion-regulation condition (watch vs. do not react). RESULTS/ANTICIPATED RESULTS: Results show that DV partners show greater cortical arousal than DNV partners on measures event-related spectral perturbations, which are mean log event-loced deviations from baseline-mean power at each frequency of the electroencephalography power spectra, when intoxicated and viewing evocative partner stimuli in the “do not react” emotion regulation condition. Results also show a statistically significant 2 (alcohol vs. placebo)×2 (watch vs. do not react)×2 (DV partners vs. DNV partners) interaction of the respiratory sinus arrhythmia measure of HRV when viewing evocative partner behavior (F=7.102, p=0.019, partial η2=0.353). Findings indicate that DV partners have lower HRV than DNV partners across conditions, but particularly when acutely intoxicated and trying not to react to their partners’ evocative behavior. Similarly, results also show a statistically significantly 2 (alcohol vs. placebo)×2 (watch vs. do not react)×2 (DV partners vs. DNV partners) interaction on GSR (F=71.452, p=0.000, partial η2=0.749). GSR findings indicate that DV partners also have lower GSR when acutely intoxicated and trying not to react to their partners’ evocative behavior. DISCUSSION/SIGNIFICANCE OF IMPACT: These results suggest that increases in intimate partner violence under acute alcohol intoxication may be the result of dysfunction of the central autonomic network, especially when DV partners are trying to suppress a behavioral response to their partners’ evocative behavior in conflict. The neurophysiological patterns evidenced by DV partners is consistent with a state of vigilance to threat, and reduced ability inhibit prepotent, but inappropriate responses. They also suggest that HRV may be an important target for intervention with partner with a history of intimate partner violence. One method may be heart rate variability biofeedback which has been shown to increase parasympathetic nervous system functioning, autonomic stability, and emotion regulation.

OBJECTIVES/SPECIFIC AIMS: Specialized pro-resolving lipid mediators (SPM) actively counter proinflammatory cascades. A deficit of SPMs is one possible mechanism through which inflammation leads to the development of atherosclerotic disease. The purpose of this study is to characterize the profiles of intermediates of SPM synthesis pathways and end-product SPMs in the plasma of patients with peripheral artery disease (PAD). METHODS/STUDY POPULATION: A cross-sectional sample of 52 patients with PAD was recruited at the San Francisco Veterans Affairs Medical Center. PAD was defined as the presence of claudication symptoms and an ankle-brachial index <0.9, or a history of revascularization for claudication. Patients were excluded if they were taking immunosuppressive medications, had a severe acute illness (infection, surgery, illness, critical limb ischemia) within the last 30 days, or had severe hepatic, renal, or nonvascular inflammatory disease. Intermediates of SPM synthesis pathways and end-product SPMs were measured in plasma samples of patients by liquid chromatography-tandem mass spectrometry. RESULTS/ANTICIPATED RESULTS: The average age of the cohort was 69±6.3 and patient comorbidities reflected common comorbidities associated with PAD (hypertension 96%, hyperlipidemia 87%, diabetes mellitus 42%, coronary artery disease 34%). Rutherford categories, measurements of PAD symptom severity, ranged from 0 to III (0 10%, I 40%, II 27%, III 23%). Three EPA products were measured: 18-hydroxyeicosapentaenoic acid (18-HEPE), resolvin E1 (RvE1), and resolvin E2 (RvE2). 18-HEPE, an intermediate of SPM synthesis, was detectable in the plasma of every patient (median: 105 pg/mL, IQR: 54.9–195), whereas the SPM end-products, RvE1 and RvE2, were only detectable in 6 and 10 patients, respectively. In total, 7 DHA products were measured: 14-hydroxydocosahexaenoic acid (14-HDHA), 17-HDHA, resolvin D1 (RvD1), resolvin D2 (RvD2), protectin D1, protectin DX, and maresin 1. The intermediates 14-HDHA (median: 6546 pg/mL, IQR: 3329–12061) and 17-HDHA (median: 644 pg/mL, IQR: 340–1056) were detectable in the plasma of every patient. However, the end-products RvD1, RvD2, protectin D1, protecin DX, and maresin 1 were identified in less than half of the cohort. DISCUSSION/SIGNIFICANCE OF IMPACT: We report the presence of several intermediates of SPM synthesis pathways (18-HEPE, 14-HDHA, and 17-HDHA) in every patient but the presence of SPM end-products in only a limited portion of the cohort. These results suggest that some patients with PAD may have a deficit of SPMs. Further investigation is required to better understand the role of SPMs and mediators of resolution of inflammation in PAD.

OBJECTIVES/SPECIFIC AIMS: Characterize the expression kinetics of HIV-1 Envelope and their relationship to virus production at the cellular level. METHODS/STUDY POPULATION: In vitro and ex vivo laboratory analyses. RESULTS/ANTICIPATED RESULTS: Initial studies addressing the kinetics of cell surface. Envelope (Env) expression reveal that Env expression to peaks on day 2 post infection. Next steps include a series of experiments to compare the kinetics of Env cell surface expression with broadly neutralizing antibody (bNAb)-mediated ADCC and the characterization of virus production kinetics in this same context. To be maximally effective, ADCC elimination of infected cells should occur before peak Env expression. DISCUSSION/SIGNIFICANCE OF IMPACT: Potent bNAbs to HIV-1 recognize vulnerable sites on the HIV-1 Envelope (Env) protein and are of great clinical interest due to their potential use in the prevention and treatment of HIV-1 infection. Their effectiveness depends not only on the neutralization of viral infectivity, but also on the elimination of productively infected cells via antibody-dependent cellular cytotoxicity (ADCC). On a cellular level, ADCC dynamics are determined by the timing and level of Env expression on the surface of HIV-infected cells. This study aims to delineate the expression kinetics of HIV-1 Envelope and their relationship to virus production. We expect that it will provide new insights into the utility of bNAb-mediated ADCC in treating and possibly curing HIV-1 infection; therefore results might have substantial impact on future HIV treatment strategies.

OBJECTIVES/SPECIFIC AIMS: Cancer clinical trials (CCTs) are vital tools in the advancement of cancer prevention and treatment. Yet, only 3%–5% of eligible patients enroll in CCTs. Low participation can be attributed, in part, to poor communication as well as a lack of awareness and understanding about CCTs. In order to increase participation in trials, interventions should foster meaningful communication about cancer prevention and CCTs, especially between medical professionals and members of the community. Community forums provide a channel to communicate about cancer with members public and to educate prospective participants about CCTs. Thus, our goal was to evaluate the efficacy of hosting community forums about cancer in order to educate the public and influence perceptions of CCT participation. METHODS/STUDY POPULATION: During the Spring of 2016, participants (n=51) who attended a community forum about CCTs completed a pretest and post-test survey assessing their understanding and perceptions of CCTs. RESULTS/ANTICIPATED RESULTS: Results from the pretest to post-test survey revealed a significant positive increase (p=0.01) in participants’ attitudes toward cancer clinical research as well as marginally significant increases in participants’ perceived subjective norms (p=0.06) about participating in CCTs and the perceived personal relevance (p=0.06) of clinical research participation pretest and post-test. DISCUSSION/SIGNIFICANCE OF IMPACT: Findings suggest that community forums about cancer and CCTs could lead to an increased awareness and understanding of CCTs among members of the population and could be useful channels for cancer interventions.