OBJECTIVES/GOALS: We report an automated manufacturing system, and a series of cylindrical multi-layer microfluidic artificial lungs manufactured with the system and tested for fluidic fidelity and function. METHODS/STUDY POPULATION: A Roll-to-Roll (R2R) system to engrave multiple-layer devices was assembled. A 100 um-thick silicone sheet passes through an embedded CO2 laser engraver, which creates patterns of any geometry on the surface. The sheet is plasma-activated to create an irreversible bond, and rerolled into a processed device. Unlike typical applications of R2R, this process is synchronized to achieve consistent radial positioning. This allows the fluidics in the device to be accessed without being unwrapped. The result is a cylindrical core surrounded by many layers of microfluidic channels that can be accessed through the side of the device or through fluidic vias. This core is cut to expose the microfluidic layers, and then installed into a housing which routes the fluids into their respective microfluidic flow paths. RESULTS/ANTICIPATED RESULTS: To demonstrate the capabilities of the R2R manufacturing system, this method was used to manufacture multi-layer microfluidic artificial lungs (µALs). Gas and blood flow channels are engraved in alternating layers and routed orthogonally. The close proximity of gas and blood separated by gas-permeable PDMS permits CO2 and O2 exchange. Three µALs were successfully manufactured. Their flow paths were visualized using dyed water and checked for leaks. Then they were evaluated using water for pressure drop and CO2 gas-exchange. The top performing device had 15 alternating blood and gas layers. Test with whole blood demonstrated oxygenation from venous (70%) saturation levels to arterial (95%) saturation levels at a flow rate of 3 ml/min. DISCUSSION/SIGNIFICANCE: The ability to cost-effectively produce high surface area microfluidic devices would bring many small-scale technologies from the realm of research to clinical and commercial applications. In particular, most microfluidic artificial lungs only have small rated flows due to a lack of manufacturing processes able to create high surface area devices.

OBJECTIVES/SPECIFIC AIMS: Background: Children with autism spectrum disorder (ASD) show a broad range of unusual responses to sensory stimuli and experiences. It has been hypothesized that early differences in sensory responsiveness arise from atypical neural function and produce “cascading effects” on development across a number of domains, impacting social and communication skill, as well as broader development in children affected by ASD. A primary challenge to confirming these hypotheses is that ASD cannot be definitely diagnosed in the earliest stages of development (i.e., infancy). A potential solution is to prospectively follow infants at heightened risk for ASD based on their status as infant siblings of children who are diagnosed. We examined the developmental sequelae and possible neurophysiological substrates of three different patterns of sensory responsiveness—hyporesponsiveness (reduced or absent responding to sensory stimuli) and hyperresponsiveness (exaggerated responding to sensory stimuli), as well as sensory seeking (craving of or fascination with certain sensory experiences). Infants at high risk (HR) for ASD were compared with a control group of infants at relatively lower risk for ASD (LR; siblings of children with typical developmental histories). Objectives: Research questions included: (a) Do HR infants differ from LR infants in early sensory responsiveness?, (b) Does sensory responsiveness predict future ASD and related symptomatology? and (c) Is sensory responsiveness predicted by resting brain states? METHODS/STUDY POPULATION: Methods: To answer these questions, we carried out a longitudinal correlational investigation in which 20 HR infants and 20 LR controls matched on sex and chronological age were followed over 18 months. At entry to the study, when infants were 18 months old, sensory responsiveness was measured using the Sensory Processing Assessment and the Sensory Experiences Questionnaire, and a number of putative neural signatures of early sensory differences were measured via resting state EEG. When infants were 24 and 36 months of age, ASD and related symptomatology was evaluated in a comprehensive diagnostic evaluation. RESULTS/ANTICIPATED RESULTS: Results: HR infants trended towards increased hyporesponsiveness and hyperresponsiveness and showed significantly elevated levels of sensory seeking relative to LR controls at 18 months of age. Both groups, furthermore, displayed a high degree of heterogeneity in sensory responsiveness. Atypical sensory responsiveness (increased hyperresponsiveness and/or hyporesponsiveness, as well as sensory seeking behavior) predicted several aspects of ASD and related symptomatology, including social, communication, and play skill, and was associated with differences in resting brain state, including metrics of oscillatory power, complexity, and connectivity, as well as hemispheric asymmetry. Moderation analyses revealed that several relations varied according to risk group, such that associations were stronger in magnitude in the HR Versus LR group. DISCUSSION/SIGNIFICANCE OF IMPACT: Conclusion: Findings provide empirical support for the notion that early sensory responsiveness may produce cascading effects on development in infants at heightened risk for ASD. Differences in resting brain states may underlie atypical behavioral patterns of sensory responsiveness. From a clinical standpoint, results suggest that early sensory differences may be useful for predicting developmental trajectories, and be potentially important targets for early preventive intervention, in infants at risk for autism.

OBJECTIVES/SPECIFIC AIMS: Clinical guidelines recommend using predicted atherosclerotic cardiovascular disease (ASCVD) risk to inform treatment decisions. The objective was to compare the contribution of changes in modifiable risk factors Versus aging to the development of high 10-year predicted ASCVD risk. METHODS/STUDY POPULATION: Prospective follow-up of the Jackson Heart Study, an exclusively African-American cohort, at visit 1 (2000–2004) and visit 3 (2009–2012). Analyses included 1115 African-American participants without a high 10-year predicted ASCVD risk (<7.5%), hypertension, diabetes, or ASCVD at visit 1. We used the Pooled Cohort equations to calculate the incidence of high (≥7.5%) 10-year predicted ASCVD risk at visit 3. We recalculated the percentage with a high 10-year predicted ASCVD risk at visit 3 assuming each risk factor [age, systolic blood pressure (SBP), antihypertensive medication use, diabetes, smoking, total and high-density lipoprotein cholesterol], one at a time, did not change from visit 1. RESULTS/ANTICIPATED RESULTS: The mean age at visit 1 was 45.2±9.5 years. Overall, 30.9% (95% CI 28.3%–33.4%) of participants developed high 10-year predicted ASCVD risk. Aging accounted for 59.7% (95% CI 54.2%–65.1%) of the development of high 10-year predicted ASCVD risk compared with 32.8% (95% CI 27.0%–38.2%) for increases in SBP or antihypertensive medication initiation and 12.8% (95% CI 9.6%–16.5%) for incident diabetes. Among participants <50 years, the contribution of increases in SBP or antihypertensive medication initiation was similar to aging. DISCUSSION/SIGNIFICANCE OF IMPACT: Increases in SBP and antihypertensive medication initiation are major contributors to the development of high 10-year predicted ASCVD risk in African Americans, particularly among younger adults.

OBJECTIVES/SPECIFIC AIMS: Research on cancer difference is of significant scientific and practical value. For leukemia, the survival disadvantage of the Blacks has been suggested in multiple studies. However, the existing epidemiologic analysis has multiple technical limitations. The goal of this study is to more accurately quantify so as to better understand different sources of racial differences in leukemia survival. METHODS/STUDY POPULATION: A new statistical method, which is based on robust regression and resampling, is developed. Data are obtained from the SEER (Surveillance, Epidemiology, and End Results) database. Using the “classic” epidemiologic methods as well as the new method, analysis is conducted on the prognosis of 4 leukemia subtypes (ALL, CLL, AML, and CML) for 4 major racial groups (White, non-Hispanic White, Black, and Asian and Pacific Islander). RESULTS/ANTICIPATED RESULTS: After effectively removing differences caused by the observed clinicopathological and demographic factors, the survival disadvantage of the Blacks persists for the following patient groups: ALL and age>14, CLL and age>14, and ALL and age≤14. The quantitative results are significantly different from those from classic epidemiologic analysis. Such observed racial differences are more attributable to the unobserved risk factors and cancer disparity. DISCUSSION/SIGNIFICANCE OF IMPACT: This study provides a more effective and more direct quantification of racial difference in leukemia prognosis. The survival disadvantage of the Blacks which is observed for certain subtypes/age groups deserves further attention but should not be overstated. More data collection and analysis are needed to more accurately decipher racial differences in leukemia and other cancer types.

OBJECTIVES/SPECIFIC AIMS: Approximately 1.6 million Americans suffer from inflammatory bowel diseases (IBD), ulcerative colitis, and Crohn’s disease. It is a challenge for both physicians and patients alike to manage the disease, primarily due to lack of disease specific biomarkers. Endoscopy remains the gold standard to diagnose and evaluate IBD activity. Current biomarkers or their combinations cannot adequately predict IBD progression or relapse, and response to therapy. METHODS/STUDY POPULATION: In total, 97 IBD patients recruited at University of Kentucky undergoing endoscopy. Patients medical information was collected from electronic database including C-reactive protein (CRP), fecal calprotectin (FC), endoscopy/pathology report. RESULTS/ANTICIPATED RESULTS: The mean CRP and FC levels were 1.3 (normal <1) and 679 (normal <162), respectively. FC (sensitivity 74%) was more reliable to predict mucosal inflammation compare to CRP (sensitivity 36%). However, 52% of patients did not have FC performed (vs. CRP only 4%), and 45% of these patients failed to submit stool sample for analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggests FC is the most promising noninvasive marker for disease monitoring in IBD. It correlates well with endoscopic activity and mucosal inflammation. However, further analysis must be done to evaluate barriers to testing and issues with compliance from patients. We feel strongly that a blood biomarker for disease activity is vital for disease monitoring and response to therapy in IBD.

OBJECTIVES/GOALS: Patients with nsCL/P are prone to infections due to their open like wound cleft and immune system susceptibility. COVID-19 affected nsCL/P patients access to care and treatment delays. This study will determine COVID-19 infection risk between children with nsCL/P compared to those unaffected. METHODS/STUDY POPULATION: Retrospective cohort of children 5 years old and under with nsCL/P (exposed group) and children without nsCL/P (unaffected group); matched by age and gender. Participants will be selected from ongoing case-control study (n=500), School of Dental Medicine, and Pediatric University Hospital. Power analysis will calculate minimum sample size (power=0.80 alpha=0.05). Exclusion criteria: syndromic clefts, patients without diagnosis information and with history of co-morbidity. Other bacterial and viral infections present at the time of COVID-19 diagnosis, sex, age, geographic location, COVID-19 vaccination status and others will be considered as possible cofounders. Descriptive statistics, Chi-square, Odds Ratios at 95% confidence intervals and multiple logistic regression will be estimated. RESULTS/ANTICIPATED RESULTS: We hypothesize that we will identify an increased risk of COVID-19 infection in children with nsCL/P than in those unaffected or children without nsCL/P. Children with nsCL/P will also have an increased risk of symptoms and complications of COVID-19 infection than those unaffected. DISCUSSION/SIGNIFICANCE: Knowing the increased risk of COVID-19 infection in children with nsCL/P will aid to prioritize treatment. If a higher risk of COVID-19 infection is found, it will generate a need to modify elective surgery status to semi-elective; minimizing delayed treatments, unnecessary hospitalizations, complications and increased cost of treatment.

OBJECTIVES/SPECIFIC AIMS: We are exploring the structure of the interaction between an immunogenic neoantigen and a T cell receptor (TCR) that recognizes the neoantigen while tolerating the counterpart self antigen. No structural example exists to date of how a TCR can discriminate between a neoantigen and the self antigen. We aim to determine the structural and biophysical features that underlie the immunogenicity for this neoantigen, and the features we determine are likely to be present in other immunogenic neoantigens. Algorithms to predict the immunogenicity of neoantigens are available, but do not incorporate structural or biophysical factors. We aim to improve these methods for immunogenic neoantigen prediction by determining structural and biophysical factors that result in recognition by the immune system. METHODS/STUDY POPULATION: Recombinant protein expression, production, and purification. Protein x-ray crystallography. Biophysical protein-protein binding experiments RESULTS/ANTICIPATED RESULTS: The T cell receptor (TCR) bound to the neoantigen with an affinity 15-fold higher than the self antigen. The leucine to phenylalanine mutation occurs at position 8 of a 9-amino acid long peptide antigen. This position is typically in the interface bound by the T cell receptor. The structures of the unbound neoantigen and self antigen showed that the mutated residue was in the TCR interface. Additionally we noted a change in the side chain position of a proximal tryptophan, potentially due to clashes with the larger phenylalanine residue. The structure of the TCR bound to the neoantigen showed that the TCR interacted with the tryptophan in the mutation-induced conformation and with the phenylalanine residue. Thus the mutation may be altering TCR binding affinity by interactions of the residue itself with the TCR, and by locking the proximal tryptophan residue in an optimal position to interact with the TCR. We are testing the contributions of each of these factors to the overall affinity change. Hydrophobicity has been linked to immunogenicity, so mutations that increase hydrophobicity compared to the self antigen are likely to be immunogenic. However, leucine and phenylalanine are similar on hydrophobicity scales. On the other hand, a side chain rotation is unlikely to represent a large energy barrier. Therefore, we hypothesize that another property of the phenylalanine, such as size or aromaticity, is driving the affinity difference. DISCUSSION/SIGNIFICANCE OF IMPACT: Traditional forms of cancer therapy do not specifically target cancer cells, and their toxicity to healthy cells limits their effectiveness. Immunotherapy, which involves orchestrating a specific anti-cancer immune response, is now an established cancer therapy. Several forms of immunotherapy target “neoantigens,” which are derived from mutated proteins in cancer, and are therefore are cancer-specific. Neoantigens represent a foothold that can allow the immune system to distinguish between cancer cells and healthy cells, and thus specifically target cancer cells for destruction while imparting no activity toward healthy cells that lack the neoantigen. Most cancer mutations that result in neoantigens arise from random passenger mutations in cancer and will be different among patients. Neoantigen-based cancer therapies are thus a precision medicine technique. The quality of neoantigens to induce an immune response (immunogenicity), which relates to how likely they are to be presented to the immune system and recognized as foreign, has been shown to be a critical factor in predicting the outcome of immunotherapy treatment. We are investigating, on a structural and biophysical level, features that may increase the likelihood of a neoantigen being recognized as foreign by the immune system. The structural insight we gain can be incorporated into algorithms that predict neoantigens from cancer exome sequencing for patient-specific identification of immunogenic neoantigens for immunotherapeutic intervention.

ABSTRACT IMPACT: We describe a novel methodology that combines CRISPR/Cas9-induced double stranded DNA breaks with homology dependent repair from an adeno associated virus (AAV) encoded template to generate single-allele edited isogenic cell line models of cancer-associated mutations with high efficiency. OBJECTIVES/GOALS: Conventional approaches to creating isogenic knock-ins, to model disease-associated mutations, are limited by poor efficiency and loss of the mutant allele on extended culture. We present an optimized editing approach combining CRISPR/Cas9 with Adeno-associated virus, using mutant SF3B1 as a prototype. METHODS/STUDY POPULATION: Left and right homology arms for SF3B1 were PCR amplified and cloned into pAAV-SEPT-Acceptor plasmid (containing a chimeric intron, neomycin resistance cassette and polyA tail). The disease-associated K700E mutation was introduced by site-directed mutagenesis. Single guide RNA (sgRNA) complexed with recombinant Cas9 along with the AAV donor were delivered into K562 cells, G418 resistant clones selected, and screened for integration by PCR. Confirmed clones were then transduced with a doxycycline-inducible Cre-recombinase containing lentiviral vector. Inducible expression of Cre-recombinase and expression of the mutant allele were confirmed by Western blot and Sanger sequencing respectively. RESULTS/ANTICIPATED RESULTS: Targeted-integration efficiencies among the Neo-resistant clones, generated by AAV-alone and AAV+CRISPR/Cas9, were 16% and 94%, respectively. Single cell cloning after Cre-mediated excision of loxp was unsuccessful presumably due to toxicity of the K700E mutation. To overcome this limitation, clones were transduced with doxycycline-inducible Cre-recombinase lentiviral vector. Doxycycline induction of Cre-recombinase resulted in reliable excision of the loxp cassette and expression of mutant allele at about 50% variable allele frequency (as determined by Sanger sequencing). The approach was validated in additional cell lines and for introduction of N-terminal FLAG tag for SF3B1. DISCUSSION/SIGNIFICANCE OF FINDINGS: Combining AAV and CRISPR/Cas9 can generate scalable single-dominant allele mutants with high precision and efficiency compared to AAV or CRISPR alone. Together with inducible Cre-recombinase, our approach can generate isogenic models where the mutation confers a growth disadvantage.

OBJECTIVES/GOALS: The purpose of this small survey-based study was to characterize the current role of neurocritical care physicians in traumatic brain injury (TBI) systems of care and research. In doing so, we aim to highlight potential roles of neurology providers in the medical management and enhancement of translational science in the field of TBI. METHODS/STUDY POPULATION: Between April and June 2021, a web-based survey was disseminated by email to members of the Neurocritical Care Society. The survey was open to all physician providers. A total of 36 surveys were completed. The survey consisted of 18 questions with pre-defined answer choices. Survey questions aimed to determine areas of practice, primary clinical specialty, hospital practice setting, provider involvement in TBI care, provider involvement in TBI research, and current research roles. RESULTS/ANTICIPATED RESULTS: 92% of survey respondents were in the United States (n=33), representing all national regions. 75% of the physicians were neurocritical care trained (n=27). 69% of providers were practicing in academic institutions while 78% were at sites designated as Level I trauma centers. All respondents managed acute TBI, but 50% served as consultants rather than being the primary service provider. At their sites of practice, 31% of patients were on non-neuroscience services, especially those with non-neurologic traumatic injury. Only 36% reported that TBI protocols were written and adhered to at their site. Only 44% reported that TBI research was performed at their site, while 50% had interest in participating in TBI research. TBI was the primary area of research for 17% of physicians. DISCUSSION/SIGNIFICANCE: This small physician survey highlights heterogeneity in TBI systems-based practice and research roles. Areas of potential improvement include greater involvement of neurocritical care physicians in TBI management, protocol-building and implementation, and TBI research. Reasons for current barriers are multifactorial and will be discussed.

OBJECTIVES/GOALS: To explore the severity of posttraumatic stress disorder (PTSD) symptoms in association with hippocampal and amygdala volumes in ICU survivors. We hypothesize that the severity of posttraumatic stress symptoms in ICU survivors is associated with lower volumes of both the hippocampus and amygdala. METHODS/STUDY POPULATION: Secondary analysis of the VISIONS study, a prospective sub-study of the BRAIN-ICU cohort, which included survivors of critical illness. Patients were screened for preexisting PTSD before discharge. The PTSD Checklist Specific (PCL-S) was used at 3 and 12 months to evaluate the ICU as a traumatic experience. A score of >30, indicated significant symptoms of PTSD. A Philips Achieva 3T MRI scanner was used to scan patients at both discharge and 3-month follow-up. To compare median brain volumes at discharge and 3 months for those with and without significant PTSD symptomatology (PCL-S ≥30) at 3 and 12 months, we used a Kruskal-Wallis (KW) equality-of-populations rank test. RESULTS/ANTICIPATED RESULTS: The median age for our sample was 58.5 (52.6, 63.7). One-third of the sample was female, and 90% were Caucasian. Fifty-seven percent of individuals (N = 12) had at least one prior mental health diagnosis, with two having a prior history of PTSD. One third of individuals experienced delirium during their critical illness. At 3-month follow up, there were three patients with PTSD symptomatology and one at 12-month follow up. Median brain volumes (hippocampus or amygdala) did not differ between individuals with or without PTSD symptomatology at either 3 or 12 months (p-values for all tests >0.05). DISCUSSION/SIGNIFICANCE OF IMPACT: Although our study did not reveal significant differences in brain volumes between PTSD patients and non-PTSD patients, sample size is a major limitation and larger scale studies should be undertaken to elucidate possible neurobiological markers of PTSD in ICU survivors. CONFLICT OF INTEREST DESCRIPTION: Dr. Wilson would like to acknowledge salary support from the Vanderbilt Faculty Research Scholars Program (1KL2TR002245), HL111111 and GM120484. Drs. Ely and Jackson as well as Mrs. Kiehl all receive funding for their time working on this investigation from AG035117 and HL111111. Dr. Ely would additionally like to acknowledge salary support from the Tennessee Valley Healthcare System Geriatric Research Education and Clinical Center (GRECC). Dr. Ely will also disclose additional funding for his time from AG027472 and having received honoraria from Orion and Hospira for CME activity; he does not hold stock or consultant relationships with those companies. The authors would like to acknowledge the following: this work was conducted in part using the resources of the Center for Computational Imaging at Vanderbilt University Institute of Imaging Science and the Advanced Computing Center for Research and Education at Vanderbilt University, Nashville, TN, and study data were collected and managed using REDCap electronic data capture tools hosted at Vanderbilt University.

OBJECTIVES/GOALS: To reduce hospitalizations, health care systems are studying ways of improving social determinants of health (SDoH) in patients with chronic disease such as diabetes (DM). Our goal was to better characterize the SDoH of a cohort of DM patients by using socio-economic information from census data. METHODS/STUDY POPULATION: Our study population included DM patients seen in primary care practices of a large health care system in 2013-2017. We integrated socio-economic status (SES) information from the American Factfinder to data extracted from the electronic health record (EHR). Addresses for the cohort were geocoded using ArcMap to obtain the census tract information for median income, poverty status, educational level, and supplemental food benefits using American Community Survey 5-Year estimates. We used multivariable logistic regression to calculate odds ratio (OR) and 95% confidence intervals [], with 3+ comorbidities as the dependent variable and demographic and SES variables as independent variables. RESULTS/ANTICIPATED RESULTS: Our study population included 13,782 patients: 53% were female, 65% white, 28% Black, 27% were on Medicare, 3% on Medicaid, median age was 60, 53% had 3+ comorbidities. Median income was $66,243, poverty level 6%, receiving food benefits 8%, no high school degree 8%, and bachelor’s degree or higher 30%. After evaluating collinearity, our multivariable analysis showed that patients with 3+ comorbidities were more likely to have income < $52,000 (lower quartile) versus $84,001 (upper quartile), OR = 1.2 [1.0-1.4]; be female, OR = 1.6 [1.4-1.7]; divorced or widowed versus married, OR = 1.5 [1.3-1.7], 1.4 [1.3-1.6]; and be on Medicare, Medicaid or both, OR = 2.4 [2.2-2.6], 2.2 [1.8-2.6], 6.0 [4.5-8.3]. DISCUSSION/SIGNIFICANCE OF IMPACT: Census tract-based SES could provide invaluable information to health care providers when associated to the EHR. We found that median income, which is not collected in the EHR, was significantly associated with a higher burden of disease. Census tract SES could serve as a proxy for evaluating SDoH.

ABSTRACT IMPACT: Neighborhood disadvantage was significantly associated with brain structure and function in trauma-exposed adults, providing evidence that contextual factors should be assessed in mental health research, particularly in high-risk populations. OBJECTIVES/GOALS: Over 13 percent of Americans live in a socioeconomically disadvantaged neighborhood. Previous work has linked lower individual socioeconomic position to alterations in brain structure and function. However, the neural effects of area-level socioeconomic factors, such as neighborhood disadvantage, are unclear. METHODS/STUDY POPULATION: We recruited two-hundred and fifteen traumatically-injured participants from an Emergency Department in southeastern Wisconsin. An Area Deprivation Index (ADI) score, a national measure of neighborhood socioeconomic disadvantage, was derived from each participant’s home address. Two-weeks post-trauma, participants underwent a battery of self-report measures and functional magnetic resonance imaging (fMRI) scans. Using a multi-modal approach, we investigated the impact of ADI on brain structure as well as neural activation during rest and during an emotional uncertainty task. We sought to disentangle the relationship between neighborhood and individual socioeconomic position and neural activity in the context of trauma. RESULTS/ANTICIPATED RESULTS: We demonstrated that neighborhood disadvantage is associated with decreased volume and alterations of resting state functional connectivity of structures implicated in affect processing, including the hippocampus, amygdala, and ventromedial prefrontal cortex. These results held even after controlling for relevant individual variables, including acute post-traumatic stress symptoms and years of education. Moreover, individuals from disadvantaged neighborhoods exhibited heighted activation of these same structures in response to aversive stimuli. Thus, brain regions critical for recognizing and processing negative stimuli are susceptible to the effects of area-level socioeconomic factors. DISCUSSION/SIGNIFICANCE OF FINDINGS: The results offer additional evidence that neurobiological mechanisms clarify how stress ‘gets under the skin’. Changes to key brain regions may explain why those living in disadvantaged neighborhoods are at a heighted risk of PTSD. Broadly, these findings should inform future policies and community-driven interventions aimed at reducing poverty.

OBJECTIVES/SPECIFIC AIMS: This study aims to first describe the unique cytokine profile and TGFbeta levels of young children with CF, then understand the pathologic effects of TGFbeta on lung function in a CF animal model. These powerful translational studies linking observations in clinical disease with a transgenic mouse model allow us a unique opportunity to investigate the role of TGFbeta in early CF lung disease. METHODS/STUDY POPULATION: Cytokine levels (TGFbeta, TNFalpha, IL-8, IL-6, HNE, and IL-1beta) in bronchoalveolar lavage fluid (BALF) from CF patients (n = 15) and non-CF control patients (n = 21) under 6 years old were determined by ELISA and Luminex assay. Tracheotomized patients without significant underlying lung disease were chosen as non-CF inflamed control patients, as they had similar levels of neutrophilic inflammation and infection as CF patients. The percentage of BAL neutrophils (% PMNs) in each sample was assessed. The relationships between cytokines were analyzed using linear regression and principal components analysis. In animal studies, CF and non-CF mice (n = 4-5 per group) were treated with intratracheal adenoviral TGFbeta1 vector, an empty vector control, or PBS. After one week, animals were collected; lung function, response to the bronchoconstrictor methacholine, and rescue with albuterol were measured utilizing a FlexiVent machine. Lungs were collected for histology. Immunohistochemistry for alpha-SMA was performed and pictures of all cross-sectional airways were obtained. Burden of ASM was assessed by dividing the square root of alpha-SMA stained airway smooth muscle by the basement membrane perimeter length of each airway. RESULTS/ANTICIPATED RESULTS: Patient characteristics of CF and non-CF inflamed control patients were similar in terms of age (3.6 yrs vs 3.3 yrs respectively, p = 0.49), positive BAL culture (13% vs 14%, p = 0.94), and % PMNs (65% vs 56%, p = 0.64). Despite these similarities, TGFbeta levels were 2-fold higher in CF versus non-CF BAL (p = 0.034). Analysis of BAL cytokines from both patient groups showed that three principal components describe 86% of total variance across the cytokine variables. These components represent different contributions from the cytokines, with TGFbeta, IL6, and % PMNs comprising one component of similarly regulated inflammatory markers. These components can distinguish CF versus non-CF patients with 77% accuracy (area under the curve: 0.77). TGFbeta concentrations were uniquely associated with increased IL-6 in CF samples (r = 0.74; p = 0.0015) but did not demonstrate association with other cytokines. After TGFbeta exposure, CF mice demonstrated greater abnormalities in airway resistance than non-CF mice, with heightened response to methacholine. Importantly, this increase in airway obstruction in CF mice was reversible with albuterol treatment, indicating airway smooth muscle dysfunction as a principal driver of lung function abnormalities. Furthermore, TGFbeta induced an increased ASM burden on lung histology in both CF and non-CF mice (p<0.05). IL-6 levels in the BAL of CF mice showed greater increases after TGFbeta treatment compared to non-CF mice (p<0.05). Empty vector control treatment did not cause lung pathology. DISCUSSION/SIGNIFICANCE OF IMPACT: Young children with CF have a unique pattern of pulmonary inflammation compared to inflamed non-CF control patients. In CF, TGFbeta pulmonary levels are uniquely associated with IL-6, a driver of ASM dysfunction in other pulmonary diseases. We followed up this clinical observation study by investigating the effect of TGFbeta on pulmonary disease in a mouse model. CF mice demonstrate increased pulmonary IL-6, airway obstruction, and ASM dysfunction after TGFbeta exposure. This study provides evidence that TGFbeta is associated with a distinct cytokine pattern that may promote ASM dysfunction in early CF lung disease. Understanding the mechanism of early CF pathophysiology will be critical in developing targeted therapeutics that can prevent early lung damage.

OBJECTIVES/SPECIFIC AIMS: The objective of this research is to determine under what conditions endpoints based on estimated glomerular filtration rate (eGFR) slope or on relatively small declines in eGFR provide valid and useful surrogate endpoints for pivotal clinical trials in chronic kidney disease (CKD) patients. METHODS/STUDY POPULATION: We consider 2 classes of surrogate endpoints. The first class includes endpoints defined by the average rate of change in eGFR during defined portions of the follow-up period of the trial, following initiation of the randomized treatment interventions. The second class includes composite endpoints defined by the time from randomization until the occurrence of a designated decline in eGFR or kidney failure. The true clinical endpoint is considered to be the time from randomization until kidney failure, irrespective of the trajectory in eGFR measurements prior to kidney failure. We apply statistical simulation to determine conditions under which alternative endpoints within the 2 classes are (1) valid surrogate endpoints, in the sense of preserving a low probability of rejecting the null hypothesis of no treatment effect on the surrogate endpoint when there is no treatment effect on the clinical endpoints and are also (2) useful surrogate endpoints, in the sense of providing increased statistical power that allows significant reductions in sample size and/or duration of follow-up. Input parameters for the simulations include (a) characteristics of the joint distribution of the longitudinal eGFR measurements and the time to occurrence of renal failure, (b) characteristics of the short-term and long-term effects of the treatment, and (c) design parameters, including the duration of accrual and follow-up and the spacing of eGFR measurements during the follow-up period. We use joint analyses of 19 treatment comparisons across 13 previous clinical trials of CKD patients to guide the selection of input parameters for the simulations. We apply longitudinal mixed effects models for analysis of endpoints based on eGFR slope, and Cox regression for analyses of the composite time-to-event endpoints. RESULTS/ANTICIPATED RESULTS: We have previously shown that surrogate endpoints defined by eGFR declines of 30% or 40% can provide valid and useful alternative endpoints in CKD clinical trials for interventions that do not produce short-term effects on eGFR which differ from the longer-term effects of the interventions. Other factors influencing the validity and utility of these endpoints include the average baseline eGFR, the mean rate of change in eGFR, and the extent to which the size of the treatment effect depends on the patient’s underling rate of eGFR decline. We will extend these results by presenting preliminary results describing conditions under which outcomes based on eGFR slope provide valid and useful alternatives to the clinical endpoint of time until occurrence of kidney failure. DISCUSSION/SIGNIFICANCE OF IMPACT: The statistical simulation strategy described in this research can be used during the design of clinical trials of chronic kidney disease to assist in the selection of endpoints that maximize savings in sample size and duration of follow-up while retaining a low risk of producing a false positive conclusion in the absence of a true effect of the treatment on the time until kidney failure.

OBJECTIVES/GOALS: Recent NCATS funding announcements emphasize pursuing domain-agnostic translational science projects that seek to transform the system of science. We aimed to articulate the social responsibility of translational science, defined as prioritizing improved health outcomes and decreased disparities. METHODS/STUDY POPULATION: We focused on the framing of social responsibilities of translational science and distinctions between (a) domain-agnostic translational science that aims to transform the system of science and (b) translational research that takes place within a specific therapeutic area. We reviewed CTSA funding calls, translational research ethics papers, and statements by leaders in the field of translational science. We integrated the social responsibilities of improving health outcomes and decreasing disparities with the values of translational science, which prioritize the relevance, usability, and sustainability of translational interventions. RESULTS/ANTICIPATED RESULTS: We drew on our review of the literature and case studies to offer guidance aimed at helping to ensure that differently positioned actors and entities within the translational ecology can advance the values of translational science while also fulfilling the social responsibilities of translational science. We specify how (a) Funders and policymaking institutions, (b) Organizations such as research universities and CTSA institutes, (c) Translational health science teams working on innovative translational science projects, and (d) Individual translational scientists can all contribute to ensuring that translational science fulfills its ethical obligations and social responsibilities. DISCUSSION/SIGNIFICANCE: The social responsibility of translational science can be fulfilled by centering its efforts to develop useful, sustainable, and relevant innovations. These criteria clarify how social responsibilities manifest in practice and can help funders shape and guide the next era of translational discovery.

OBJECTIVES/GOALS: Last year, we reported on storytelling events highlighting the experiences of Black biomedical professionals. The goal of this continuation study was to put Black Voices into action through a companion curriculum, instructional materials, and facilitated meaningful discussions around racial justice. METHODS/STUDY POPULATION: We developed instructional materials and pilot-implemented the curriculum in a 250-student Healthcare Leadership class. Prior to viewing the video, participants were asked to provide a definition of diversity, inclusion, and equity. Afterwards, the students reflected and suggested changes to the definition. Throughout the class, instructors facilitated group discussions about the role of diversity. As a culminating activity, students submitted a written reflection summarizing their understanding of the role of diversity, inclusion, and equity in healthcare. RESULTS/ANTICIPATED RESULTS: Instructional materials included videos, a facilitators guide, a reflection worksheet, and online course modules. The facilitators guide included an overview of best practices in the facilitation of group discussions, common rules groups can establish for open dialogue, and discussion questions specific to the Black Voices videos. In-class and lab discussions brought up several prominent metaphors for diversity and inclusion. For example, students first defined diversity as pieces of the puzzle that somehow fit together, but later surmised that the pieces do not need to fit, or be from the same puzzle. DISCUSSION/SIGNIFICANCE: Our pilot showed that participants experienced the power of storytelling, particularly for amplifying Black voices, gained a vehicle to expand awareness and consciousness of diversity, equity, and inclusion, and identified plans to promote and facilitate stories of race, equity, and diversity.

OBJECTIVES/SPECIFIC AIMS: Diabetic retinopathy is an increasingly prevalent disease, difficult to screen for across the globe. We have developed and began optimizing an innovative technique to visualize and quantify retinal blood flow, to elucidate the role of the choroid in retinal pathologies such as diabetic retinopathy or choroidopathy. METHODS/STUDY POPULATION: Preliminary retinal was obtained from a surgical retina video library (Truvision, Goleta, CA, USA). Videos of different organs were recorded while vessels were occluded via a blood pressure cuff, using consumer-grade digital video cameras (NEX-5T, a7sii; Sony, New York, NY, USA). All other retinal videos were taken using a fundus camera (50×; Topcon, Oxland, NJ, USA) modified to support the above digital video cameras. All videos were processed using experimental software (MATLAB, Mathworks, Natick, MA, USA). RESULTS/ANTICIPATED RESULTS: Video imaging of the retina was optimized for lighting conditions and software requirements. Parameters were defined for the software imaging pipeline, such as frequency range of interest, sampling rate, and noise minimization. Software was developed to stabilize frames, accounting for eye saccades. Use of a biosensor enabled accurate measurement of pulse waveform, increasing signal-to-noise ratio. The optimal light requirements were determined such that adequate exposure of the retina is reproducible yet still comfortable for use in human subjects. DISCUSSION/SIGNIFICANCE OF IMPACT: This novel technique allows for an inexpensive, noninvasive, and reproducible ocular blood flow imaging platform. By optimizing this technique, we can proceed with our future plans for a pilot study to compare our imaging technique with the current standard, paving the way for future clinical studies.

OBJECTIVES/GOALS: Developing pharmacokinetic (PK) models to guide selective serotonin reuptake inhibitor (SSRI) dosing in youth is costly, time-intensive, and requires large numbers of participants. We evaluated the use of remnant blood samples from SSRI-treated youth and developed precision PK dosing strategies. METHODS/STUDY POPULATION: Following IRB approval, we used a clinical surveillance platform to identify patients with routine phlebotomy within 24 hours of escitalopram or sertraline dosing. Remnant blood samples were obtained from youth aged 5–18 years, escitalopram and sertraline concentrations were determined, and clinical characteristics (e.g., age, sex, weight, concomitant medications that inhibit sertraline or escitalopram metabolism) and phenotypes for CYP2C19, the predominant enzyme that metabolizes these SSRIs, were extracted from the electronic medical record (EMR). A population PK analysis of escitalopram and sertraline was performed using NONMEM. The influence of clinical variables, CYP2C19, and dosing was evaluated from simulated concentration-time curves. RESULTS/ANTICIPATED RESULTS: Over 21 months, we collected315 samples from escitalopram-treated patients (N=288) and 265 samples from sertraline-treated patients (N=255). In youth, escitalopram and sertraline exposure (concentrations over time) and specific pharmacokinetic parameters (e.g., clearance) were influenced by CYP2C19 phenotype, concomitant CYP2C19 inhibitors, and patient-specific characteristics. Escitalopram and sertraline concentrations from remnant blood samples were 3.98-fold higher and 3.23-fold higher, respectively, in poor metabolizers compared to normal metabolizers (escitalopram, p<0.001) and compared to normal, rapid, and ultrarapid metabolizers combined (sertraline, p<0.001). DISCUSSION/SIGNIFICANCE: Combining remnant blood sampling with pharmacogenetic-integrated EMR data can facilitate large-scale population PK analyses of escitalopram and sertraline in youth. This real-world approach can be used to rapidly develop precision SSRI dosing strategies, including slower titration and reduced target doses in CYP2C19 poor metabolizers.

Effectively addressing public health crises requires dynamic and nimble interdisciplinary collaborations across the translational spectrum, from bench to clinic to community. The Clinical and Translational Science Award (CTSA) Program hubs are uniquely suited to facilitate interdisciplinary collaborations across universities and academic medical centers. This paper describes the activities at the Columbia University CTSA Program hub to address a current public health crisis, the opioid epidemic. Columbia’s CTSA Program hub led a three-phase approach, based on the Conceptual Model of Transdisciplinary Scientific Collaboration as described by Stokols et al.: (1) a university-wide planning and brainstorming phase to identify key leaders across many domains who are influential in addressing the opioid epidemic, (2) a campus-wide and community outreach to identify all interested parties, and (3) ongoing targeted support for collaboration development. Preliminary metrics of success are interdisciplinary collaborations and grant funding. We describe recent examples of how interdisciplinary collaboration, academic-community partnership, and pilot funding contributed to the development and funding of innovative interdisciplinary research, including the New York site of the HEALing Communities initiative. The processes are now being used to support interdisciplinary approaches for other translational public health issues.