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

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

OBJECTIVES/GOALS: The goal of this study is to identify if ocular graft versus host disease (oGVHD) patients treated with a systemic JAK inhibitor have a change in their tear cytokine profile (a possible bio-marker) and oGVHD score. oGHVD is a severe inflammatory dry eye disease and major cause of morbidity after a hematopoietic stem cell transplant. METHODS/STUDY POPULATION: Janus Kinase (JAK) is an upstream regulator of cytokine production. A JAK 1/2 inhibitor, Ruxolitinib, was recently FDA approved for the treatment of chronic GVHD. We propose that JAK inhibition results incytokine changes in tears and improvement of oGVHD. To study this, we will quantify tear cytokines in patients with oGVHD, with and without systemic JAK inhibition treatment. Patients with ‘definite’ oGVHD based on the international chronic oGVHD diagnostic criteria (ICOGVHD) whom we have collected tears will be grouped based on JAK inhibition treatment. Tear cytokines are analyzed using Iso spark Meteor bulk quantitative proteomic analysis. RESULTS/ANTICIPATED RESULTS: Seven patients were identified from our patient cohort who met inclusion criteria (oGVHD; tears collected while on Ruxolitinib), five patients were identified whom we have collected tears with oGVHD who have not taken ruxolitinib. The following 10 cytokines will be analyzed in the tears by the Iso spark Meteor bulk quantitative proteomic analysis: GM-CSF, IFN-g, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, TNF-a. The change in cytokine levels will be compared with the ICOGVHD score, corneal fluorescein staining, schirmers test (measurement of tear production), conjunctival injection score, ocular surface disease index score (validated symptomatic score of dry eye disease). DISCUSSION/SIGNIFICANCE: OGVHD is a major cause of morbidity for patients who undergo a hematopoietic stem cell transplant and is the result of a highly complex immune process including dysregulation of pro-inflammatory cytokines. It is critical to understand the effect of cytokine changes on the eyes to potentially identify a biomarker and possible treatment targets.

OBJECTIVES/GOALS: This study seeks to determine the relationship between fear of COVID-19 infection and care-seeking behaviors including provider visit and telemedicine utilization among vulnerable adolescent and young adults in Baltimore, Maryland METHODS/STUDY POPULATION: Participants enrolled in the COVID-19 Youth study were 13-25 years old and recruited from four existing sexual health studies based in Baltimore, MD which focused on 1) PID, 2) HIV, 3) emerging sexually transmitted infections, and 4) a dyadic STI prevention, respectively. Participants agreed to be re-contacted for future studies and completed a telephone survey developed to inquire about the impacts of COVID-19 on care seeking behaviors. Bivariate analyses tested for correlations between fear of COVID-19 infection, demographic variables, and care-seeking behaviors. Multivariable logistic regression modeled associations between fear of COVID-19 infection and care-seeking behaviors. RESULTS/ANTICIPATED RESULTS: Adjusted multivariable logistic regression revealed a statistically significant association between fear of COVID-19 infection and having a provider visit; AYA who feared COVID-19 were at greater than two times increased odds of a provider visit compared to AYA who did not fear COVID-19 infection (OR: 2.37, 95%CI: 1.02, 6.15). Among those with a provider visit, fear of COVID-19 infection was associated with two-fold increased odds of having a telemedicine visit vs. an in-person visit (OR: 2.23, 95%CI: 1.09, 4.51), however this was not statistically significant in the adjusted model. There were however significant associations detected in the adjusted model for HIV status, insurance type and telemedicine utilization respectively. DISCUSSION/SIGNIFICANCE: This study demonstrates the ongoing need for health services during the recent pandemic and overall willingness of AYA to utilize telemedicine. Given the sexual health disparities faced by AYA, who bear more than half of the 50 million STI cases in the US, optimizing services for AYA is essential and consistent with new laws expanding telehealth use.

OBJECTIVES/GOALS: Allopregnanolone (ALLO), a modulator of GABAA receptors, may be useful as a treatment for human and canine benzodiazepine-refractory status epilepticus (SE). Our objective was to develop a phamacokinetic-pharmacodynamic (PKPD) model relating ALLO plasma concentrations to electroencephalographic (EEG) effects in dogs. METHODS/STUDY POPULATION: Four healthy dogs and one dog with epilepsy that had implanted intracranial electrodes were utilized. ALLO doses ranging from 1-6 mg/kg were administered IV over 5 min. EEG data were collected during four IM doses (1-2 mg/kg). Blood samples were collected up to 6 hr following dosing. ALLO concentrations were measured using HPLC-MS/MS. Power density was determined in EEG bands using a custom algorithm. A two-compartment link PKPD model was developed to describe the relation between ALLO plasma concentration and change in EEG power in the alpha, beta, delta and theta bands. RESULTS/ANTICIPATED RESULTS: ALLO caused a rapid increase in absolute power density in all EEG bands measured (1-4, >4 – 8, >8 – 12, >12 – 25, and >25 – 100 Hz). The onset of effect was rapid (1-3 min) and demonstrated by frequency band and dose analysis. Concentration-EEG data were best fit by a two-compartment PK model and sigmoidal Emax PD indirect link model. The beta frequency band was most sensitive, showing increases in power at the lowest ALLO concentrations. The EC50 concentration for the beta frequency was ~270 ng/mL. The EC50 values for effects on the other frequency bands were ~500-700 ng /mL. In conclusion, IV ALLO causes a rapid effect on EEG that can be used to determine minimal plasma concentrations associated with target engagement. DISCUSSION/SIGNIFICANCE OF IMPACT: Dose selection for future clinical trials will use the effective concentrations determined here in conjunction with studies in animal status epilepticus models. Studies are planned in client owned dogs with epilepsy to evaluate clinical efficacy in dogs and as nonclinical proof-of-concept evidence supporting translational studies in people. CONFLICT OF INTEREST DESCRIPTION: Michael Rogawski and Dorota Zolkowska are named as inventors on patent applications claiming use of neuroactive steroids including allopregnanolone and ganaxolone in the treatment of status epilepticus.