OBJECTIVES/SPECIFIC AIMS: Incomplete spinal cord injury (iSCI) is a life-long disability that typically results in a profound loss of locomotion capability. Current rehabilitation methods rarely restore full community ambulation, which in turn limits quality of life. Most individuals with iSCI exhibit persistent deficits in eccentric muscle control and reach recovery plateaus below the levels necessary for independent community ambulation. Eccentric motor control is essential during the weight acceptance phase of gait, which is emphasized during downhill walking. METHODS/STUDY POPULATION: The overground locomotion of subjects with chronic iSCI was analyzed both prior to and following a 12-week downhill body-weight-supported treadmill training regimen and compared to that of matched healthy controls in terms of kinematics, kinetics, and EMG activation. RESULTS/ANTICIPATED RESULTS: We expect to find significant differences between the controls and subjects with iSCI, with deficits in eccentric motor control accounting for some of these differences. In addition, we expect the downhill training to yield significant improvement in eccentric muscle control that translates into improvements in functional, overground walking for the subjects with iSCI. DISCUSSION/SIGNIFICANCE OF IMPACT: The goal is to determine if downhill training can improve eccentric motor control and extend recovery beyond established plateaus. OpenSim modeling of the experimental data will help quantify changes in eccentric control of individual muscles to clarify where specific gains are made.

OBJECTIVES/SPECIFIC AIMS: Intestinal stem cells (ISC) primarily act in the repair of ulcerated epithelium, and their proliferative capacity relies on Wnt/β-catenin signaling. However, the role of GCs on basal epithelial cell signaling has not been fully characterized. The objective of this study was to interrogate a mechanism by which steroids may limit ISC activation. GCs inhibit NFκB signaling, which has been shown to play a role in nuclear β-catenin activation in epithelial cells. We hypothesized that GCs limit Wnt/β-catenin signaling required for ISC activation and epithelial restitution by inhibiting NFκB activation in epithelial cells. METHODS/STUDY POPULATION: To examine the effects of GCs on intestinal epithelial cells, we treated a nontransformed human colonic epithelial cell line (NCM460) with dexamethasone and observed the effects on NFκB and Wnt/β-catenin signaling events. We isolated mouse epithelial cells from the distal colon for stem cell culture as 3D “organoids.” We obtained pure epithelial cell preparations from mucosal biopsies isolated from patients treated at GI clinics at the University of Kentucky Chandler Hospital and VA Medical Center, Lexington. Steroid treated patients with equivalent levels of inflammation, but no mucosal ulceration were used as controls. RESULTS/ANTICIPATED RESULTS: In steroid-treated NCM460 cells, we saw an increase in steroid-responsive genes GILZ and SGK1. We saw a significant decrease in transcripts for Wnt target genes, including Axin2 and cmyc; NFκB target genes, including IFNG and IL6; and the shared NFκB and Wnt pathway co-activator CREBBP, despite unchanged transcript levels for β-catenin (CTNNB1). This data was corroborated in 3D stem cell cultures from cells isolated from mouse colon tissue, which had significant decreases in transcripts for stem cell markers Lgr5 and Ascl2, proliferative markers KI67 and PCNA, and Wnt target Axin2. NCM460s transfected with a lentivirus carrying a TCF/LEF luciferase construct showed a 2.5-fold decrease in TNF-stimulated luciferase activity with dexamethasone treatment. Interestingly, this effect can be rescued by glucocorticoid receptor (GR) blockade with RU-486. Intestinal epithelial cells from patient biopsies showed significant decreases in colitis-induced Axin2, p-LRP6 (a positive marker of Wnt Signaling) and nuclear β-catenin, which correlated with decreased p-p65 protein levels. DISCUSSION/SIGNIFICANCE OF IMPACT: Together, these data suggest that steroid therapy inhibits Wnt/β-catenin signaling at multiple levels, and effects stem cell proliferation in pure stem cell cultures. Decreases in TCF/LEF transcriptional activation (nuclear β-catenin’s DNA binding target) can be reversed with steroid receptor blockade with RU-486, suggesting that a receptor level interaction may be occurring. Interestingly, the required co-activator CBP, shared between NFκB and Wnt pathways, has decreased transcription following steroid treatment, which may provide a mechanism for limited Wnt activation following steroid therapy. Although steroids play a significant role in regulating the amount of inflammatory damage that occurs during IBD treatment, our data suggest that they may be limiting pathways required for effective healing as well.

OBJECTIVES/SPECIFIC AIMS: Upper-extremity (UE) impairment affects 88% of stroke survivors due to dysfunctional shoulder-hand coordination. Patients may be able to grasp with the arm at rest, but unable to grasp in a functional context (eg, from a high shelf) because shoulder use elicits involuntary hand muscle activity. Further, much rehabilitation research is directed at unsuccessful stroke recovery (patients with persistent UE impairment) but very little towards patients who show successful clinical recovery (such as those with mild UE impairment) even though these patients have attained the desired rehabilitation outcome. We examined the neurophysiological trajectory of successful compared to unsuccessful post-stroke recovery in the context of functional UE movements to clearly identify what factors are necessary for successful recovery of functional UE movements after stroke. METHODS/STUDY POPULATION: We studied 3 populations: (1) mildly-impaired patients, early (at <17 d, 30 d, 90 d, and 180 d) after stroke as a model of successful post-stroke recovery, (2) moderately-impaired, chronic patients (>6-months post stroke) with persistent hand function impairment, as a model of incomplete post-stroke recovery (unsuccessful recovery), and (3) Healthy age-range matched controls. We used transcranial magnetic stimulation (TMS) in all 3 groups at the given time points to measure corticomotor excitability (motor evoked potentials, recruitment curve), corticomotor inhibition (short-interval intracortical inhibition, long-interval intracortical inhibition), and intracortical facilitation of hand muscles with the shoulder positioned in different degrees of flexion or abduction (these shoulder positions are known to elicit involuntary, undesired hand muscle activation, which leads to UE dysfunction and impairment in individuals with stroke). RESULTS/ANTICIPATED RESULTS: Data collection are in process and will be presented. Preliminary data from controls shows that corticomotor excitability of selected hand muscles is affected by changes in shoulder position. Preliminary findings in controls are consistent with clinical findings in stroke that certain shoulder positions elicit involuntary and undesired hand muscle activation, leading to UE dysfunction and disability. Findings from the stroke groups will be presented. DISCUSSION/SIGNIFICANCE OF IMPACT: We hypothesize that this centrally-facilitated coupling between shoulder and hand muscles is disrupted after stroke, which may play a central role in the inability of patients to perform functional UE movements. By comparing the TMS metrics in mildly-impaired Versus moderately-impaired chronic patients, we will be able to identify the longitudinal change in neurophysiology underlying shoulder-hand coordination that is associated with successful or unsuccessful clinical recovery of UE function after stroke. Thus, these findings will help us distinguish between the neurophysiology underlying successful from unsuccessful UE recovery leading to more mechanism-based interventions for UE dysfunction post stroke in the future.

OBJECTIVES/SPECIFIC AIMS: The goal of this research is to use circulating tumor cells (CTC) enumeration and characterization to monitor anticancer treatment response. Emerging evidence strongly suggests the implications that epithelial-to-mesenchymal transition may have in cancer metastasis. Consequently, we hope to elucidate the significance of mesenchymal and stem-like CTCs in the peripheral blood of metastatic pancreatic cancer patients by analyzing the prevalence and frequency trends of CD133+ CTCs, as they relate to clinical events. We also hope to determine if there is a correlation between EpCAM+ CTCs and CD133+ CTCs numbers with tumor size, disease stage, and patient clinical outcome. METHODS/STUDY POPULATION: Blood samples of patients with metastatic pancreatic cancer (stage IV) were obtained from the University of Florida Health Cancer Center after informed consent through an IRB-approved protocol. CTC capture, characterization, and enumeration was performed on the blood of these cancer patients during their anticancer treatment. Patients had blood drawn for this purpose at time points aligned with clinical phlebotomy (every 2 weeks). CTC capture was performed by introducing treated patient blood samples into antibody-functionalized microdevices. The PDMS devices were functionalized by immobilizing either anti-EpCAM or anti-CD133, through an avidin-biotin complex. After capture, cells were fixated and permeabilized with 4% paraformaldehyde and 0.2% Triton X-100, respectively. Three-color immunocytochemistry (anti-cytokeratin-FITC, anti-CD45-PE, and DAPI) was performed to identify CTCs from nonspecifically captured blood cells. To be counted as a CTC, based on the FDA-approved technical definition, a cell with the appropriate cell size and morphology must be nucleated (DAPI+), express cytokeratin (CK+), and lack the leukocytic CD45 marker (CD45−). RESULTS/ANTICIPATED RESULTS: We tested the clinical utility of the device for monitoring the response of patients with advanced pancreatic cancer to a chemotherapy treatment consisting of anticancer drugs including 5-fluorouracil, leucovorin, oxaliplatin, and dasatinib. We have detected EpCAM+ CTCs in 47/47 (100%) and CD133+ CTCs in 41/47 (87.2%) of blood samples, coming from a cohort of 16 patients. We studied the correlation between the CTC numbers and the clinical result of patients in the study. We found that the size and changes in the size of the primary tumor (confirmed by CT scans) correlated with the frequency and increase/decrease trends in the number of CTCs detected. We expect to find some relationship between the number of detected CD133+ CTCs, or rather stem-like CTCs, and the clinical outcome of patients (eg, disease progression leading to withdrawal from study). DISCUSSION/SIGNIFICANCE OF IMPACT: Enumeration of patient CTCs and stem-like CTCs at different stages of a patient’s treatment may correlate with disease stage and prognosis, and prove useful in monitoring early recurrence, patient-specific treatment response, and newly acquired resistances; all of which would aid in providing guidance for the next step in clinical intervention. This type of liquid biopsy technology has great potential to make an impact in the future of personalized medicine and point-of-care diagnostics, as well as become a sturdy tool for translational research.

OBJECTIVES/SPECIFIC AIMS: Precise biomarkers are urgently needed to characterize the tumor immune microenvironment in primary melanoma tumors both for prognostication and to predict the benefit of immuno-therapeutic intervention. The goal of this work is to define spatial relationships between CD8+ T cells, CD68+ macrophages and Sox10+ melanoma cells in order to define features correlating with prolonged survival METHODS/STUDY POPULATION: Five micrometer slides from either the primary biopsy or subsequent wide local excision procedure were stained using Opal multiplex IHC for DAPI, CD3 (LN10, Leica), CD8 (4B11, Leica), CD68 (KP1, Biogenex), SOX10 (BC34, Biocare), HLA-DR (LN-3, Abcam), and Ki67 (MIB1, Abcam). Cell phenotypes within representative fields preselected by a trained dermato-pathologist and were visualized using the Mantra quantitative pathology workstation (PerkinElmer), and analysis of spatial distribution of CD3+ CD8+ cells analyzed using inForm® image analysis software (PerkinElmer), and Spotfire software (TIBCO). In order to test whether mIHC can better characterize the tumor immune microenvironment, we screened databases at the Herbert Irving Cancer Center (HICC) at Columbia University for stage II/III melanoma patients diagnosed between 2000 and 2012, with available FFPE of primary melanoma tissue and documented clinical follow-up. We identified a preliminary population of 57 patients to begin our analysis. Clinical follow-up was available on 35 patients of whom 21 patients were alive with no evidence of recurrence or died with no evidence of recurrence and 14 had died of melanoma. Twenty-four patients had more than 24 months of survival information available but no detailed clinical information to determine cause of death. RESULTS/ANTICIPATED RESULTS: First, we evaluated whether density of immune cells in tumor and stroma predicted prognosis in 35 patients with disease specific survival information. We find that high number of CD3+CD8+ cells in tumor correlates with Disease Specific Survival (DSS) (p=0.0323*) and CD3+CD8+ cells in stroma may also correlate with DSS (p=0.0671). This is consistent with what is known in the literature regarding tumor infiltrating lymphocytes (TILs). We also found that CD68+ cells in stroma predict poor prognosis (0.0259*). This is consist with the proposed deleterious role for macrophages in tumor progression. Next, using nearest neighbor analysis we examined the effect of HLA-DR and Ki67 expression on spatial distribution of CD3+CD8+ T cells. We find that CD8+ T cells are closer to myeloid (CD68+) cells expressing HLA-DR. This is consistent with the potential of HLA-DR expressing cells to present antigens to T cells, and suggests that T cells may preferentially interact with HLA-DR expressing myeloid cells. Conversely, we find that Ki67 expression on tumor (SOX10+) cells correlates with increased distance from CD3+CD8+ T cells relative to SOX10+Ki67-tumor cells. This finding is consistent with the observation that more advanced tumors with higher mitotic rates have decreased T cell infiltrates, and suggests that dividing melanoma cells are less likely to interact with T cells. In addition, we performed analysis to determine whether spatial relationships defined above impact prognosis. Clinical oncology follow-up was available on 35 of the 57 patients evaluated above. We compared proximity of CD3+CD8+ cells to both myeloid (CD68+) and tumor (SOX10+) cells in patients who recurred and those with no evidence of recurrence. We found that CD3+CD8+ cells in patients who had recurrence were closer to CD68+ HLA-DR− cells than in patients who had no recurrence (t-test, p=0.0377), this correlated with DSS (p=0.003). Conversely, distance from CD3+CD8+ to CD68+ HLA-DR+ in relationship to recurrence was not significant with a trend towards CD3+CD8+ T cells being closer in nonrecurrent patients (t-test, p=0.1362). DISCUSSION/SIGNIFICANCE OF IMPACT: Consistent with the literature, we find that densities of CD8+ T cells correlates with favorable outcomes in early stage melanoma. We also find that density of CD68+ macrophages in stroma correlates with poor outcome. If proximity is a surrogate for interaction, these data indicate that dividing, Ki67+, melanoma cells interact less with CD8+ T cells than do Ki67+ melanoma cells. Further, HLA-DR expression on CD68+ infiltrating cells likely enhances their interaction with T cells. Interestingly, on further analysis, CD3+CD8+ cells were significantly closer to CD68+ HLA-DR− cells in patients who recurred, implying that interactions between these cell types may not be favorable. This analysis demonstrates that spatial analysis may be useful in predicting prognosis in early stage melanoma, and this is the first report of this type of analysis predicting outcomes in primary tumor specimens to our knowledge. Further staining and analysis of the complete patient cohort (n=120) is ongoing.

OBJECTIVES/SPECIFIC AIMS: The goal is to understand the underlying mechanism of epicaridial fat biology and its response to cardiometabolic disease by using quantitative multi-echo Dixon (mDixon) of water and lipid sequence, T2* blood-oxygen-level-dependent (BOLD) sequence of iron content, and data analysis methods to determine the quantity of brown Versus white fat. To accomplish this goal, we propose to define the histological, genetic, and metabolite state of epicardial fat and to confirm the relationship between fat phenotype and magnetic resonance (MR) characteristics. We will then investigate whether MR is more effective in identifying patients with lower cardiovascular disease risk than computed tomography (CT). METHODS/STUDY POPULATION: We will recruit 100 patients undergoing open-heart surgery and will quantify mDixon (proton density fat fraction), BOLD (T2*), and T2/T1 maps of epicardial, extrapericardial, and subcutaneous fat before their surgery. We will then (a) validate MR findings by direct depot-specific tissue analysis for lipid content, histological, and genetic markers of inflammation and brown and white fat, (b) develop plasma and fat depot specific metabolite profiling of cardiovascular disease risk and correlate with imaging characteristics. We will categorize cardiovascular risk score (Cardiovascular Health Status) of our 100 patients on quartiles. We will then build models where the categorized cardiovascular risk score are regressed on MR measures (epicardial fat fraction, T2*, and T2/T1 maps) and CT measures (epicardial fat volume and coronary calcium score). RESULTS/ANTICIPATED RESULTS: We anticipate to learn about epicardial fat biology and the role of inflammation in cardiometabolic disease. We will validate proton density fat fraction, T2* and T2 map against histology of epicardial fat for lipid content, established markers of brown and white fat and inflammation, respectively, to help us translate imaging technique to clinical practice. In respect to our second aim we anticipate that MR identifies patients at lower cardiovascular risk quartile than CT. DISCUSSION/SIGNIFICANCE OF IMPACT: Interest in epicardial fat as a visceral fat of the heart and coronary arteries is rapidly growing as the scientific based evidence indicates that the anatomic specificity is an important contributor to the cardiovascular diseases. The transformation of epicardial fat from a cardioprotective phenotype to a pro-inflammatory, atherosclerosis-promoting state triggers inflammation that is coincident with the expansion of epicardial fat volume detected by anatomic imaging. This study will impact the management of patients at risk for cardiovascular disease because it will demonstrate that quantification of epicardial fat status by MR identifies fat tissue changes validated by histology at lower cardiovascular disease risk quartile than CT.

OBJECTIVES/SPECIFIC AIMS: Attention is a cognitive function that binds perception and behavior. Recent evidence suggests that attention involves phase-amplitude coupling (PAC) of neural signals. PAC occurs when the amplitude of one frequency (frequency for amplitude) is maximal at particular phases of another frequency (frequency for phase). However, some studies suggest PAC improves attention, while others maintain that PAC inhibits attention. The present study seeks to determine whether PAC promotes or inhibits neural signals that underlie attention. METHODS/STUDY POPULATION: Six adult epilepsy patients with implanted electrodes participated in a cued attention task. Subjects participated in a cued attention task where they oriented attention to one side of the screen at a time and discriminated between stimuli as fast as possible with mouse clicks. Perception-related electrodes discriminated the location and/or shape of the target. These were determined with a cluster-based permutation test. Behavior-related electrodes predicted reaction time (RT) with neural activity prior to target appearance. These were determined with correlations between PAC and RT. PAC was calculated using the modulation index (MI). RESULTS/ANTICIPATED RESULTS: We found 47 perception-related electrodes that discriminated location and/or shape of target (p<0.05, FDR corrected). We found 27 behavior-related electrodes where PAC prior to the target predicted RT (p<0.05 FDR corrected). There was little overlap between the perception-related and behavior-related electrodes (3%). PAC also did not discriminate left-sided and right-sided cues. In addition, behavior-related electrodes had less local neural activity and higher PAC during the period of cued attention than perception-related electrodes. DISCUSSION/SIGNIFICANCE OF IMPACT: PAC minimally facilitates perceptual aspect of visual attention. However, PAC facilitate response speed. We suggest that PAC might improve response speed by “quieting” task irrelevant neural activity. For the same reason, PAC is absent in electrodes that are actively processing meaningful streams of visual data. These findings highlight separable aspects of the human attention system and how PAC contributes to both. Future directions include determining differences in PAC for attentional disorders like ADHD and neurological neglect.

OBJECTIVES/SPECIFIC AIMS: In this pilot case-control study, the metabolome was quantified in subjects with previously measured serum and clinical biomarkers. The serum metabolome was then integrated with existing serum and clinical biomarkers of WTC-exposed firefighters to identify pathways significant to loss of lung function following acute PM-exposure. This robust subset of metabolite and serum biomarkers may be clinically relevant to predicting progression to lung disease in a larger cohort. METHODS/STUDY POPULATION: Serum drawn within 6 months of 9/11 was analyzed in this pilot. Clinical measures were obtained from electronic medical records. Never-smoking, male, WTC-exposed firefighters with normal pre-9/11 lung function were segregated based on FEV1 percent predicted (FEV1 %Pred) at symptomatic presentation. Cases of WTC-LI (FEV1 %Pred <LLN, n=15) and controls (n=15) were identified from previous cohorts. Ultrahigh performance liquid chromatography tandem mass spectroscopy quantified the metabolomic fingerprints of a group with previously assessed (by multiplex panels; ELISA and Luminex) serum chemokines and cytokines. High-dimensional data analysis and dimension reduction techniques integrated metabolites, cytokines, chemokines, and clinical data to identify pathways of WTC-LI on curated data. Random Forest (RF) out-of-bag estimated success rates were used to measure classification utility of the refined biomarker profile. Principal components analysis (PCA) was used to visualize class separation produced by the refined profile. RESULTS/ANTICIPATED RESULTS: Of the 765 metabolites detected, 580 metabolites were quantified in more than 80% of subjects/group with relative standard deviation ≥15%. Relevant chemokines, cytokines, and clinical biomarkers were included based on previously established clinical importance. Initial PCA explained 34.7% of the variance in the first 3 components. RF was used to identify the top 5% of biomarkers important to class separation. RF of the refined biomarker profile correctly classified cases and controls with a 96.7% estimated success rate. A PCA of the refined metabolic profile now explained 46.2% of the variance in components 1–3, demonstrating improved class separation. Differentiators between cases of WTC-LI and controls included elevated sphingolipids in cases of WTC-LI. The metabolic-inflammatory serum biomarkers MDC, Apo AI, GM-CSF, and heart rate play an important role in class separation. Phospholipids and lysolipids also appeared to differentiate cases of WTC-LI from controls. Specifically, several glycero-phosphatidylcholines (GPC) were elevated in cases of WTC-LI. DISCUSSION/SIGNIFICANCE OF IMPACT: High-dimensional data analysis on the metabolic fingerprints, serum, and clinical biomarker data of a subset of WTC-exposed 9/11 rescue workers has identified pathways associated with the loss of lung function. Sphingolipids, known to function as inflammatory signaling mediators, are thought to play important roles in lung function under both physiological and pathological conditions. Changes in sphingolipid metabolism have been linked to several pulmonary disorders, including asthma, COPD, and acute lung injury. Interestingly, a relation between sphingolipid metabolism and the metabolic-inflammatory pathway is suggested by similarities observed in PCA. Findings of elevated GPCs are similar to COPD literature. Higher levels of GPCs could correspond to elevated levels of lysophosphatidic acid (LPA), a ligand of RAGE. RAGE is a known proinflammatory mediator; LPA species have well-described roles as lipid signaling molecules, function as synthetic intermediates in other metabolic pathways, and were found to be predictive of WTC-LI. Since metabolites are more proximal markers of disease processes, metabolites could capture the complexity of past exposures and, therefore, may better inform treatment. These pathways warrant further investigation into their mechanisms and therapeutic importance.

OBJECTIVES/SPECIFIC AIMS: Deep brain stimulation is currently being evaluated as an experimental therapy for various psychiatric disorders, as well as being investigated as a method for mapping emotional brain functions. This growing area of research requires sensitive measures to quantify effects of stimulation on emotional processing. The current study examined the effects of acute stimulation to 2 limbic regions—the subcallosal cingulate (SCC) and the amygdala—on bias in the perception and evaluation of emotional facial expressions. We hypothesized that transient electrical stimulation to the limbic system would produce acute reductions in negative bias, consistent with its antidepressant effects in patients with severe depression. METHODS/STUDY POPULATION: The current study uses a novel affective bias task, developed to rapidly and covertly quantify emotional state. Over 4–6 minutes, patients rate the intensity and valence of static images of emotional facial expressions. We examined effects of electrical brain stimulation in 2 groups: patients with treatment-refractory depression undergoing SCC DBS therapy, and epilepsy patients undergoing amygdala stimulation via stereo-EEG electrodes during inpatient intracranial monitoring. DBS patients completed the task under stimulation and sham conditions during monthly visits over the first 6 months of therapy, as well as daily during a 1 week, blinded period of DBS discontinuation at the 6-month time point. Epilepsy patients completed the task under stimulation and sham conditions at a single visit. Mixed linear models and paired-samples t-test were used to investigate effects of stimulation as well as depression scale scores on affective bias ratings. RESULTS/ANTICIPATED RESULTS: Four SCC DBS patients showed significant effects of stimulation (p<0.0001) and depressive state (p<0.0001) on affective bias scores across 6 months of chronic DBS therapy, where emotional faces were perceived as less sad with stimulation ON, as well as during visits in which patients were nondepressed (typically later in the treatment course). Furthermore, 2 DBS patients showed rapid negative shifts in bias following acute blinded discontinuation of chronic stimulation, an effect which persisted over the 1-week period of discontinuation (t29=−2.58, p=0.015), in the absence of any self-reported change in mood. Likewise, 6 epilepsy patients showed significant positive shifts in affective bias with acute amygdala stimulation (t5=−4.75, p=0.005). Current analyses are investigating electrophysiological, autonomic and facial motor correlates to affective bias in these patients. DISCUSSION/SIGNIFICANCE OF IMPACT: Affective bias has revealed rapid, significant changes with stimulation at 2 limbic targets—one a white matter hub and one a nuclear subcortical structure—suggesting the task’s utility as an emotional outcome measure in brain stimulation studies. These stimulation-sensitive measures may provide a new metric to track treatment response to deep brain stimulation therapy for affective disorders. Future studies will determine whether affective bias can predict neuropsychiatric complications in patients undergoing stimulation mapping of brain circuitry ahead of resection surgery for epilepsy.

OBJECTIVES/SPECIFIC AIMS: The purpose of this study is to use the baboon as a novel animal model for breath research and to identify and characterize baboon breath metabolites that reflect cardiometabolic function to inform us in the development of a noninvasive, cost-effective, and repeatable point-of-care diagnostic breath test. METHODS/STUDY POPULATION: Blood and urine was collected from control and IUGR at the approximate age of 3.5 years. Both groups were then placed on a high fat, high sugar, high salt diet for 7 weeks, after which blood, urine, and breath were collected. The breath samples were then subjected to comprehensive, 2-dimensional gas chromatography coupled with time-of-flight mass spectrometry. Using ChromaTOF software, breath VOCs were identified with at least an 80% spectral match against the National Institute of Standards and Technology (NIST) chemical reference library. The raw data were then statistically analyzed using MetaboAnalyst. We then interrogated multiple online databases to characterize and identify the role of VOCs that were present in both control and IUGR groups. RESULTS/ANTICIPATED RESULTS: Preliminary analyses of the breath VOCs indicate differences in expression between sexes and in control Versus IUGR groups. These results indicate unique “breath signatures.” Further analysis of the breath VOCs reveals the presence of metabolites that are involved in β-oxidation and oxidative stress pathways. DISCUSSION/SIGNIFICANCE OF IMPACT: This breath study, a first of its kind, will develop the baboon as a superior animal model for breath biomarker research. Our observed unique “breath signatures” indicate changes in lipid metabolism and oxidative stress pathways, which we hypothesize are the early metabolic changes at the cellular level that are not yet reflected in clinical lab measures. Future directions include analyzing breath VOCs that did not meet 80% spectral match, validation using SPME technology and commercial standards, and initiating a human pilot study in clinically obese, at-risk children in collaboration with physicians at the Children’s Hospital of San Antonio to develop a noninvasive, cost-effective, rapid, and repeatable point-of-care diagnostic breath test.

OBJECTIVES/SPECIFIC AIMS: Determine whether children with solid tumors maintain intact protective immunity to live vaccines during cancer therapy and after completing cancer therapy (postTx). METHODS/STUDY POPULATION: We will perform a prospective cohort study of children with solid tumors (Hodgkin lymphoma, brain, Wilms, and germ cell tumors) followed at the Puerto Rico’s University Pediatric Hospital. Protective immunity will be measured with antibody titers against live vaccines (Measles, Mumps, Rubella, and Varicella) at diagnosis, during cancer therapy, upon completion and 3 months postTx. RESULTS/ANTICIPATED RESULTS: We hypothesize that those patients with protective immunity to live vaccines prior to cancer therapy will lose it at the end of therapy. DISCUSSION/SIGNIFICANCE OF IMPACT: Loss of protective immunity to live vaccines has been reported in patients with hematologic malignancies after cancer therapy. This lack of protective immunity, which puts patients at higher risk of acquiring vaccine preventable diseases, has been limited studied in patients with solid tumors. The Center for Diseases Control has been established that it is safe to immunize cancer survivors with live vaccines 3 months post Tx. However, no clear guidelines for revaccination have been provided for this population. Understanding the protective immunity variation against live vaccines in children with solid tumors will allow us to identify the need for revaccination with live vaccines in this vulnerable population.

OBJECTIVES/SPECIFIC AIMS: Tissue engineered tracheal grafts (TETG) could provide a life-saving cure for children with long segment airway defects. Computational fluid dynamics (CFD) is a novel and promising technique used to evaluate TETG performance. This pilot study examines the correlation of objective CFD simulations with subjective respiratory symptoms in a TETG large animal model. METHODS/STUDY POPULATION: Three-dimensional geometries of 1 TETG implanted sheep trachea were reconstructed from serial fluoroscopic images, allowing analysis with CFD simulations. Peak flow velocity (PFV) and peak wall shear stress (PWSS) across the graft as well as changes secondary to stenting were determined. CFD metrics were compared with respiratory symptoms seen on exam. RESULTS/ANTICIPATED RESULTS: Two weeks after implantation, the animal developed respiratory distress, which correlated with PFV and PWSS elevations. Although the intraluminal graft appearance changed minimally after dilation, PFV and PWSS decreased across the graft (4.5–0.8 m/s and 0.9–0.1 Pa, respectively). Long-term TETG stenting with dilation returned PFV and PWSS to baseline (0.8–0.3 m/s and 0.1–0.01 Pa, respectively), which correlated with immediate symptom resolution. DISCUSSION/SIGNIFICANCE OF IMPACT: CFD is a noninvasive modality, which allows the evaluation of airflow metrics of symptomatic TETG recipients. This diagnostic tool will permit planned interventions and graft design optimization.

OBJECTIVES/SPECIFIC AIMS: The objective of this research was to investigate the effect of alcohol and evocative stimuli on heart rate variability (HRV) in partners with a history of intimate partner violence in a placebo-controlled alcohol administration study with an emotion-regulation task. METHODS/STUDY POPULATION: In total, 17 partners (9 females, 8 males) with a history of partner violence participated in a placebo-controlled alcohol administration study with an emotion-regulation task during which HRV 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 (BAC) 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. RESULTS/ANTICIPATED RESULTS: Controlling for baseline HRV, there was a significant main effect of stimuli (evocative vs. neutral partner stimuli) on HRV in intoxicated partners, F1,16=16.28, p=0.004. There was also a significant main effect of regulation on HRV under conditions acute alcohol intoxication, F1,16=23.55, p=0.001. These effects tell us that intoxicated partners experienced reduced HRV when exposed to evocative stimuli from their partners. These effects also tell us that under acute alcohol intoxication, partners were less able to regulate their emotion when exposed to evocative stimuli than when they consumed a placebo beverage. DISCUSSION/SIGNIFICANCE OF IMPACT: These results suggest that increases in intimate partner violence under acute alcohol intoxication may be the result of reduce HRV. This reduction in HRV would contribute to partners’ inability to response with adaptively in conflict when intoxicated. 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: The specific objectives of this project are (1) identify, test, and validate the parameters for a simplified NLOM imaging probe that will provide specific research and point-of-care information on biofilm presence, therapeutic need and response of individual wounds to treatment. (2) Identify specific proteomic and metabolomic biomarkers of (i) wound susceptibility to infection, (ii) wound response to the most commonly used antibacterial measures in wounds, and (iii) establish criteria for more effective interventions. METHODS/STUDY POPULATION: First, optimal use parameters for NLOM including illumination, field of view, focal length, linear Versus concentric image acquisition, detection and filter wavelengths were identified. Parameters for evaluation included ease and speed of imaging, ability to map diagnostic criteria. Next, using the optimised NLOM imaging modality in bacterial biofilm isolates and subsequently a rabbit ear model of biofilm wound infection, proteomic and metabolomic biomarkers of susceptibility to infection were identified. The effects of 2 standard debridement and anti-infective treatments, polyvidone-iodine solution or cetrimide 15%+ chlorhexidine gluconate 1.5% were mapped in situ for up to 10 days using the NLOM probe. RESULTS/ANTICIPATED RESULTS: Using the novel custom NLOM probe, high resolution mapping of wound biofilm infection, as well as the underlying tissue was performed throughout the onset, development, treatment, and resolution of wound biofilm infection. Specific microbiological, microstructural, oxygenation, and pH parameters were mapped at defined surface and subsurface locations and time-points. Findings included the determination that some standard antimicrobial formulations provide a supportive environment for wound infection, and that micro-channels within the biofilm and their interface with the tissues serve as an important predictor and indicator of wound infection establishment, progression, and response. DISCUSSION/SIGNIFICANCE OF IMPACT: The novel multimodality in vivo NLOM imaging approach establishes an important tool for earlier and more specific diagnosis of wound infection risk, virulence, and invasiveness along with markers of successful treatment, and a simple clinical imaging tool for improving wound infection prevention and treatment.

OBJECTIVES/SPECIFIC AIMS: The goal of this project is to determine the role of ETV6 in early B-cell development and define how germline ETV6 mutations result in predisposition to leukemia. METHODS/STUDY POPULATION: Gene expression commons were queried for expression levels of Etv6 and Pax5 at different stages of hematopoiesis. Mouse bone marrow was isolated and fractioned into cells committed to the B cell lineage via B220+ and CD43+ staining by flow cytometry and then separated into the following fractions: Fraction A (CD24low, CD19−), Fraction B (CD19+, CD24+, BP1−), and Fraction C (CD19+ CD24+ BP1+). Wild-type or germline mutant P214L ETV6 were cloned in an MiG vector and expressed in Ba/F3 cells. ChIP-PCR was performed by cross-linking proteins to DNA with 1% formaldehyde for 10 minute at room temperature, followed by cell lysis with RIPA buffer. Lysates were sonicated to shear DNA to a length of 200–1000 base pairs, then Protein A agarose beads were used to clean and immunoprecipitate chromatin. RESULTS/ANTICIPATED RESULTS: We observed that Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (p<0.0001). Etv6 expression decreases as B cells develop and is negatively correlated with Pax5 expression (r2=0.9993; p=0.0167). We next confirmed the expression patterns of ETV6 and PAX5 during B cell development in human samples. We found that ETV6 expression was higher in the early B cell fraction (CD10+, CD34+, CD19−, and CD20−) compared to the pre-B cell fraction (CD10+, CD34−, CD19+, CD20−). Conversely, we observed that PAX5 expression was higher in the preB cell fraction compared with the early B cell fraction. In Ba/F3 cells expressing ETV6 constructs, ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (p≤0.05). ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We detected association of ETV6, SIN3A, and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results provide a mechanism of interaction for ETV6 and PAX5, 2 genes often disrupted in B-cell leukemia. These findings are significant because PAX5 misregulation results in a B cell development halt, lineage infidelity, and leukemogenesis. In continuing our studies, we have generated a transgenic mouse endogenously expressing the ETV6 P214L mutation by CRISPR/Cas9 editing, and these mice appear to have a thrombocytopenic phenotype similar to that observed in patients carrying the ETV6 P214L mutation. These animals will be the focus of our continued investigation of the mechanism by which ETV6 germline mutation results in a predisposition to leukemia. Our ultimate goal is a comprehensive understanding of how this process may be targeted more efficiently in patients with both heritable and sporadic forms of leukemia involving ETV6.

OBJECTIVES/SPECIFIC AIMS: The purposes of this study are to examine the relationships among sleep characteristics (duration, efficiency), stress biomarkers, and child behavior problems among toddlers living in socioeconomically disadvantaged homes and how these characteristics change over time from age of 12 months to 24 months. Aim 1: examine changes in subjective and objective sleep characteristics from 12 to 24 months of age. Aim 2: examine changes in stress biomarkers from 12 to 24 months of age. Aim 3: examine the cross sectional and longitudinal relationships between sleep characteristics and stress response. Aim 4: examine the cross sectional and longitudinal relationships between sleep characteristics and toddlers’ child behavior problems. METHODS/STUDY POPULATION: In this cross-sectional study we are recruiting parents with healthy toddlers from early head start programs and a community clinic to prospectively examine the relationships among sleep characteristics, stress biomarkers, and children’s health. Data on sleep characteristics will include subjective and objective measures of sleep duration and efficiency and parental interactive bedtime behaviors to assist their toddlers’ sleep initiation. Multisystemic biomarkers of stress including cortisol, CRP, IL-6, and BMI, will be measured individually. The associations between sleep characteristics and the biomarkers, considered as a latent variable of the stress response, will be explored. Health measures will include secretory IgA and parent-reported behavioral problems. Generalized linear models will be used in the data analysis. RESULTS/ANTICIPATED RESULTS: To date we have obtained objective (9 days/nights of actigraphy) measures of 33 toddlers’ sleep and subjective measures of parenting interactive behaviors. Using the Parental Interactive Bedtime Behavior (PIBB) Survey and subscales [active physical comforting, encourage autonomy, settle by movement, passive physical comforting (PPC), social comforting], we are currently reporting on the associations between PIBB and toddler’s sleep characteristics. The sample included 33 toddlers (mean age=1.33 years, SD=0.54). The toddlers’ sleep duration averaged 8.22 hours (SD=0.86). There were statistically significant moderate associations between sleep duration and parents’ PPC (r=−0.41, p=0.02). Intra-individual variability in the amount of wake after sleep onset was also significantly associated with total PIBB and PPC (r=0.37, p=0.05; r=0.52, p=0.002, respectively). Intra-individual variability in the amount of sleep fragmentation within toddlers was significantly associated with total PIBB (r=0.36, p=0.05). DISCUSSION/SIGNIFICANCE OF IMPACT: Although active physical comforting (eg, rocking to sleep, patting or rubbing child’s back) is most commonly associated with sleep patterns in infancy and toddlerhood among samples of higher socio-economic status, findings from this study suggest a stronger association between PPC (eg, presence of the parent in the room to fall asleep) and less sleep duration and more individual variability in night wakings. The biomarker data are currently being analyzed and results will be presented within the year. Taken together, these preliminary results and pending results will inform future intervention development that may address the role of parenting behavior in promoting health sleep early in life.

OBJECTIVES/SPECIFIC AIMS: Determine whether GABA-A receptor binding is abnormal and linked to dystonia symptoms in cervical dystonia (CD). METHODS/STUDY POPULATION: There is increasing evidence that a key pathophysiological mechanism in adult-onset focal dystonia is a reduction in inhibitory control over the sensorimotor network. Results from a recent 11C-flumazenil PET imaging study suggest that abnormal inhibitory signaling in genetic and sporadic forms of dystonia may be due to reduced GABA-A binding. It remains unknown whether CD, the most common form of adult-onset focal dystonia, is associated with abnormal GABA-A binding. The goal of this research is to determine if GABA-A receptor binding is abnormal and linked to dystonia symptoms in CD. RESULTS/ANTICIPATED RESULTS: We investigated whole brain GABA-A binding in 15 CD patients (11F; 64±8 y) and 15 healthy controls (10F; 64±9 y) using 60-minute dynamic 11C-flumazenil PET scans. GABA-A receptor binding potential (BP) was estimated using a simplified reference tissue model. A 2-sample t-test was used to identify voxel-wise GABA-A BP differences between groups, and a regression analysis used to test for correlations between GABA-A BP and disease severity as measured with the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS). A conventional region of interest analysis was also conducted to quantify BP changes within the sensorimotor network using the automated anatomical labeling atlas. DISCUSSION/SIGNIFICANCE OF IMPACT: CD patients have reduced GABA-A receptor binding compared with healthy controls, with the greatest reduction seen within the sensorimotor region of the thalamus. Furthermore, reductions in GABA-A binding in brain regions associated with coupling sensory and motor information predict motor severity. These findings support that reduced GABAergic signaling within sensorimotor integration regions is a key mechanism underlying dystonic symptoms in CD and could help inform the development of better, more targeted treatment options.

OBJECTIVES/SPECIFIC AIMS: Mesh properties, such as stiffness, porosity, and weight have been shown to correlate with the degree of mesh integration with vaginal tissue. Previous research in rhesus macaques implanted with polypropylene mesh differing in stiffness, porosity, and weight showed differences in vaginal deterioration following mesh implantation. These differences were correlated with a foreign body response, consisting primarily of activated, proinflammatory M1 macrophages. Previous studies have determined that the early macrophage polarization profile following biomaterial implantation is a strong indicator of overall tissue integration downstream. However, these early responses have not been previously observed in the appropriate surgical models. Prior work from our laboratory in developing a cytokine delivery system has shown that shifting the macrophage response at the host-implant interface from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype in the first 14 days postimplantation resulted in enhanced integration of the mesh with the surrounding tissues. The present study develops an in vivo model clinically relevant surgical model to investigate the modulation of the host response to mesh. Utilizing a moderately-sized animal, we can feasibly implant mesh using the “gold standard” abdominal sacrocolpopexy procedure and evaluate the changes in the host immunologic response at early (14 d) and tissue remodeling outcomes at late stages (90 and 180 d) of implantation. METHODS/STUDY POPULATION: Commercially available heavyweight and lightweight mesh was used to investigate the modulation of the immune response. A custom MTI SILAR Automated Dip Coating machine is used to uniformly coat the mesh in a reproducible manner. 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. Chitosan served as the polycation, chosen because of its known antimicrobial and biocompatibility properties. Dermatan sulfate served as the polyanion, chosen for its important role in regulating extracellular matrix components and enhancing the activity of cytokines. Interleukin-4 (IL-4) is incorporated into the coating to be released in a controlled manner upon implantation. In vitro controlled release profiles were assessed to demonstrate efficient and local release of IL-4. Utilizing a New Zealand white rabbit surgical model, we implant mesh using the “gold standard” abdominal sacrocolpopexy procedure and evaluate the changes in the host 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. Determination of matrix metalloproteinases and fibrotic capsule formation also helps characterize the overall inflammatory response associated with each implant. RESULTS/ANTICIPATED RESULTS: We have developed a clinically relevant rabbit surgical model to implant different conditions of surgical mesh into 2 different sites, including the vagina and the abdomen. 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 nano-scale coating. We present a nanometer thickness, tunable, and uniform coating capable of releasing bioactive IL-4. In vitro assays confirm the bioactivity and the controlled local release allowing for shifts in the immune response to promote implant integration. Improved remodeling has been observed to correlate with a shift in the early host response from an M1 to an M2 phenotype, however, there is limited information on the exact mechanism. Our strategy to achieve enhanced tissue remodeling demonstrate outcomes such as minimal changes to the structural properties of the mesh and a controlled release profile to sufficiently polarize macrophages around the mesh to a pro-remodeling state. DISCUSSION/SIGNIFICANCE OF IMPACT: Pelvic organ prolapse is a condition where the pelvic floor muscles weaken over time resulting in the downward shift of the pelvic organs into the vaginal canal. Moreover, factors such as obesity, age, and vaginal birth increase the susceptibility of being diagnosed with pelvic organ prolapse. Direct costs of reconstructive procedures exceed $1 billion each year in the United States. Synthetic mesh has been used to repair abdominal hernias for over half a century. Biomedical companies, through 510k and the 1976 Medical Device Amendments Act, were able to resell their hernia repair mesh as a treatment for pelvic organ prolapse. However, women who have had vaginal mesh implants have reported an increasing number of complications including chronic pain and mesh erosion/exposure at rates as high as 10%–20%. In fact, in 2008 and 2011, the US Food and Drug Administration issued warnings to doctors and patients about the mesh. In January 2016, the FDA officially had to reclassify surgical mesh for transvaginal repair of pelvic organ prolapse from a class II, moderate risk device, to a class III, high-risk device. Presently, data for the use of synthetic mesh has largely derived from abdominal hernia repair, instead of vaginal repair of prolapse. In the rodent model, the vagina is too small to implant mesh in an analogous manner to human implantation. Instead, implantations are done in the abdomen, a different tissue composition and host response profile than the vagina. Primate models of pelvic organ prolapse have been utilized, but are associated with high costs and investigation of acute immune responses are not considered ethical due to the short time of survival. Thus, our presented work will not only show the development of an improved material for implantation, but also the development of an in vivo model clinically relevant to understanding the early host response to mesh.

OBJECTIVES/SPECIFIC AIMS: To establish an in vitro quantitative method for the evaluation of polymeric film disintegration that can be applied to predict in vivo behavior. METHODS/STUDY POPULATION: Two clinically advanced vaginal microbicide film products containing tenofovir and dapivirine were used as model films throughout this work. Films were made using the solvent cast manufacturing method in which polymers, excipients, plasticizer, and APIs were either dissolved or dispersed in water, mixed, and cast on a heated substrate. The novel, quantitative method was developed using a TA.XT Plus Texture Analyzer® (Texture Technologies) in combination with a TA-108S5 fixture and the TA-8A: 1/8″ diameter rounded end ball probe. Exponent® was used as the data analysis software. In this method, the film was placed and secured in the fixture, the probe applied a constant force to the film product, and a biologically relevant amount of fluid was applied to the film. The probe was able to penetrate the film upon disintegration resulting in an applied force of zero at that point. A curve of force Versus time was plotted, and disintegration time was defined as the time between fluid addition until the probe force reached zero. Test parameters were optimized in order to reduce error. Visual observation of film disintegration was conducted in the in vivo macaque model using films that included a water-soluble blue dye for film visualization. Colpophotography was also used to confirm film disintegration. In vitro results were compared with in vivo findings. RESULTS/ANTICIPATED RESULTS: The Texture Analyzer disintegration method developed provided quantitative disintegration times and did not rely on user defined endpoints which is common in many visual disintegration tests. The disintegration method was able to distinguish differences between the 2 clinical film products and produced reproducible disintegration times for the tenofovir and dapivirine films. The tenofovir film had a shorter disintegration time (41.28±2.85 s) compared with that of the dapivirine film (88.3 6±9.82 s). This method was also able to distinguish changes made to these 2 clinical film products in terms of volume and formulation alterations. In vitro and in vivo disintegration times differed by orders of magnitude, with in vitro time being measured in seconds and in vivo time being measured in days, for a variety of factors, mainly the application of constant force to the film product. Regardless of these differences, the rank order of film disintegration remained constant for in vitro and in vivo disintegration and an In Vitro In Vivo Correlation (IVIVC) trend could be seen. DISCUSSION/SIGNIFICANCE OF IMPACT: Standardization of preclinical in vitro assessments which minimize user bias are crucial to the field of pharmaceutical film development. As this field continues to develop and more products advance for pharmaceutical application, this method has the potential to become a standard assessment of film functionality. This study represents a first step in the process of developing an IVIVC. More films will need to be tested using both in vitro and visual methods in order to establish and accurate factor to predict in vivo behavior.

OBJECTIVES/SPECIFIC AIMS: To determine the association between clinical characteristics and platelet reactivity in Hispanic patients on clopidogrel therapy. METHODS/STUDY POPULATION: A cross-sectional pilot study was performed in 58 Puerto Rican patients diagnosed with any type of vascular disease and actively receiving a maintenance dose of clopidogrel for at least 7 days. The study population was divided into 2 groups: Group I with non-high on-treatment platelet reactivity (TPR); Group II with high TPR. To determine the platelet function, P2Y12 reaction units (PRU) were obtained by VerifyNow® P2Y12 assay (Accumetrics, USA). RESULTS/ANTICIPATED RESULTS: We studied a heterogeneous cohort of patients with coronary artery disease (57%), peripheral artery disease (30%), carotid artery stenosis (7%), cerebral artery aneurysm (3%), and stroke (3%) on clopidogrel therapy for secondary prevention of thromboembolic events. The mean TPR was 205±49 PRU (range: 61–304), with a prevalence of 28% patients with high TPR (PRU≥230). No significant clinical differences were found between the non-high TPR and high-TPR groups (p>0.05). However, multivariable logistic regression analysis showed that both diabetes mellitus (OR=7.5; CI: 1.01–51.9) and proton-pump inhibitors (OR=13.6; CI: 1.3–142.0) were independently correlated with high TPR (p<0.05) after adjusting for other clinical variables. DISCUSSION/SIGNIFICANCE OF IMPACT: These results provide new insight into the importance of clinical characteristics on platelet reactivity in this Caribbean population. Further studies are warranted to determine whether important clopidogrel pharmacogenes are related with platelet function in Hispanics, as well as the role of TPR in guiding antiplatelet therapy and predicting future adverse cardiovascular events in this population.