OBJECTIVES/GOALS: Develop a strategy to restrict GDNF diffusion at an injected CNS tissue site for dopamine neuron rescue by endowing it with binding affinity for carbohydrates that are abundant on the cell surface and extracellular matrix. METHODS/STUDY POPULATION: GDNF will be fused to galectin-3 (G3), a human protein that binds to β-galactoside residues of cell surface and matrix glycoproteins. We characterized the binding of G3 fusion proteins to various glycoproteins and primary human myeloid cells. We incubated G3 fusions with CNS tissue ex vivo to measure their binding and depth of penetration via diffusion. We next plan to administer GDNF-G3 via CNS intracranial infusion in a murine PD model and then conduct behavioral PD phenotype testing via rotarod and pole descent to compare to non-parkinsonian controls. We will further examine the effects of GDNF-G3 on degeneration using immunohistochemical examination of post-mortem brain tissue. RESULTS/ANTICIPATED RESULTS: Based on results from previous clinical trials of GDNF delivery, we anticipate that a successful intervention using GDNF-G3 will result in rescue of midbrain dopaminergic neurons in a murine PD model. In murine CNS tissue, we observed binding to glycans at the tissue surfaces when incubated with G3 fusion proteins ex vivo, suggesting GDNF-G3 will remain localized to the injection site. Next we will administer GDNF-G3 via CNS intracranial infusion in a murine PD model and assess efficacy by behavior and histopathology. GDNF-G3-mediated dopamine neuron rescue are expected to slow or reverse the progression of PD in these animal models. DISCUSSION/SIGNIFICANCE OF IMPACT: PD treatments focus on symptomatic relief. Standard therapies have not been efficacious in rescuing of dopaminergic neurons. GDNF-G3 administered at the site of neurodegeneration would represent a milestone on the path to treating PD pathology and address limitations of GDNF delivery.

OBJECTIVES/GOALS: Following stroke, complement-dependent neuroinflammation exacerbates secondary injury and worsens acute and chronic outcomes. We have shown that an injury site-targeted complement inhibitor (B4Crry), that targets specifically to the ischemic brain, inhibits complement activation leading to improved outcomes. Stroke comorbidities have been shown to promote a pro-inflammatory environment in the brain and systemically, and to exacerbate inflammatory responses after injury. We investigated the impact of age and smoking on acute outcomes after stroke and assessed whether increased complement activation contributes to the worsening outcomes with these stroke comorbidities. METHODS/STUDY POPULATION: Mouse brain endothelial cells (bEnd3) were exposed to hypoxia followed by exposure to serum that was derived from either cigarette smoke (CS)-exposed mice or naïve mice, and IgM and C3d deposition assessed. Adult (12 weeks) and aged (1 year) mice were subjected to 1h transient middle cerebral artery occlusion. Animals were exposed to CS for 3-6 months (5hr/day, 5days/week) by burning 3R4F cigarettes using a smoking machine. Animals were treated with B4Crry or vehicle intravenously 2h post-MCAO. Survival analysis and neurological deficit scores were performed up to 7 days. Brains were extracted for histological and molecular analyses. RESULTS/ANTICIPATED RESULTS: Following hypoxia, bEnd3 cells exposed to serum from CS-exposed mice had higher C3d and IgM deposition compared to naïve serum. Older and CS-exposed mice had significantly worse neurological deficits and mortality compared to younger adults post-MCAO. B4Crry reduced mortality and motor deficits in young, old and old+CS mice with a higher effect size in comorbid animals. Age and/or CS exposure resulted in larger infarct volumes, and increased levels of C3d deposition and microglial activation compared to young adults, but aged/CS animals treated with B4Crry fared comparable to young adults. DISCUSSION/SIGNIFICANCE OF IMPACT: The pro-inflammatory effects of aging and smoking contribute to worse stroke outcomes, and these effects can be successfully mitigated by injury site-targeted complement inhibition.

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.

OBJECTIVES/GOALS: Reactive gliosis is a hallmark of neurodegenerative disease and is characterized by the release of pro-inflammatory cytokines and physiologic changes to glial cells. Our work identifies a novel inflammatory glial-glial cell interaction and role for mGluR5 that has the potential to provide novel insight into the mechanisms of neurodegeneration. METHODS/STUDY POPULATION: Cell Culture: Mouse primary astrocytes and microglia were isolated from P0-P3 C57BL/6 or Cx3cr1GFP/+ mice.1Treatment: Glia were treated with oligomeric α-synuclein 1μg/mL or mGluR5 agonist CHPG 100 μM.2,3ELISA: Glia culture media was collected and analyzed according to the manufacturer. qRT-PCR: TaqMan™ probes were used according to manufacturer on extracted glia mRNA. ICC: Microglia were labeled with 1:750 Rb x Iba1 (Wako) and 1:500 Alexa Fluor 488 Gt x Rb. Phagocytosis Assay: Primary glia were treated with α-synuclein or astrocyte-conditioned culture media for 24-48hrs. For treatment of microglia with conditioned media, astrocytes were washed with PBS and fresh media was added to prevent carry over of α-synuclein to microglia. The number of fluorescent microbeads per microglia was quantified. RESULTS/ANTICIPATED RESULTS: Mouse primary cortical astrocytes simulated with α-synuclein aggregates adopt a reactive A1 phenotype independent of microglial stimulation. This A1 phenotype is characterized by release of pro-inflammatory cytokines including Complement Component 3 and the monocyte chemoattractant CCL2. Reactive astrocyte media induces a phagocytic phenotype in primary mouse microglia. Along with this, α-synuclein-directed microglial phagocytosis was attenuated with the addition of the mGluR5 agonist CHPG. DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest that oligomeric α-synuclein is capable of inducing a reactive phenotype in astrocytes independent of microglia and implicate crosstalk between glia as an important mediator of inflammation and microglial phagocytosis in synucleinopathies.

OBJECTIVES/GOALS: Lack of blood vessels remains a major obstacle in tissue regeneration. Angiopoietin 1 and 2 modulate angiogenesis through the Tie2 receptor tyrosine kinase. Ang1 activates pAKT to promote endothelial cell survival while Ang2 antagonizes these effects. We aim to dissect the Ang/Tie2 pathway to uncover the molecular basis for these opposing effects. METHODS/STUDY POPULATION: Ang1 and Ang2 bind Tie2 via nearly identical F-domains (Fd). To investigate the molecular basis regulating the Tie2 pathway, we generated a series of computationally designed self-assembling protein scaffolds presenting F-domains in a wide range of valencies and geometries using Rosette Molecular Modeling Suite. We examined the protein kinase activation, cell migration, and blood vessel formation produced by the designed proteins in human umbilical vein endothelial cells. RESULTS/ANTICIPATED RESULTS: Two phenotypic classes were demonstrated by the number of presented F domains: scaffolds presenting 3 or 4 Fd have Ang2 like activity, upregulating pFAK and pERK but not pAKT and failing to induce cell migration and tube formation; scaffolds presenting more than 6 Fd have Ang1 like activity, upregulating the three signaling branches and enhancing cell migration and tube formation. Scaffolds with 8 or more Fd show superagonist activity, producing significantly stronger phenotypes than Ang1. These results suggest that Fd valency largely determines Ang1 vs Ang2 signaling outcomes, and our designed superagonists can outperform Ang1 in vascularization and wound healing. In in vivo experiments, nanoparticles displaying 60 copies of Fd produce significant revascularization in hemorrhagic brains. DISCUSSION/SIGNIFICANCE OF IMPACT: Targeting the Tie2 pathway is a new paradigm in regenerative medicine. Our designed constructs will enable us to generate high-affinity Tie2 agonists and antagonists as drugs to control angiogenesis, enabling tissue regeneration that recapitulates the biological architecture of the native tissue physiology, improving organ transplant outcome.

OBJECTIVES/GOALS: We investigated the association between gut microbiota features in newly admitted nursing facility (NF) patients and the acquisition of vancomycin-resistant Enterococcus (VRE) and/or resistant Gram-negative bacteria (rGNB) within 14 days. METHODS/STUDY POPULATION: Patients were recruited at 6 Michigan NFs from 09/16-08/18. VRE or rGNB colonization status was determined by culture swabs collected from multiple body sites at enrolment, day 7, and day 14. Our analysis focused on patients with no colonization at baseline, a perirectal swab collected at baseline, and at least one follow-up visit. The V4 hypervariable region of the 16S rRNA gene from bacterial DNA in each sample was PCR-amplified and sequenced on the MiSeq platform. Sequencing results were then processed with the mothur bioinformatics pipeline to classify bacterial taxa present in each sample. Taxa typically associated with the skin microbiota were removed. The primary outcome was acquisition of VRE and/or rGNB within 14 days. Exposures of interest included patient and microbiota characteristics. RESULTS/ANTICIPATED RESULTS: Among 61 patients, 18 (30%) acquired AROs within 14 days of enrolment (3 VRE, 13 rGNB, 2 both) (Table 1). The baseline microbiota features differed significantly in those who acquired a new ARO. Of the major 8 phyla found across samples, patients who acquired an ARO were depleted in the number of phyla present (5.74 ± 1.20 vs 5.06 ± 1.43; p = 0.037) (Fig. 1). The log10-transformed relative abundance of Enterococcus was enriched in patients who acquired an ARO (−0.32 ± 1.47) compared to those who did not (−1.68 ± 1.76; p = 0.021) (Fig. 2). Patients who did not acquire an ARO tended to harbour more butyrate-producing bacterial taxa and strict anaerobes, although the differences were not statistically significant (relative abundance of butyrate producer: 29.49 ± 22.09 vs 22.05 ± 17.76; anaerobes: 64.78 ± 23.54 vs 53.68 ± 27.61). DISCUSSION/SIGNIFICANCE OF IMPACT: Microbiota metrics calculated from perirectal samples are predictive of ARO acquisition. The clinical utility of perirectal samples thus warrants further assessment.

OBJECTIVES/GOALS: The primary objective of this study was to apply design sprint methodology to develop a proactive outreach tool prototype for patients with chronic obstructive pulmonary disease (COPD). METHODS/STUDY POPULATION: We utilized a 3-day process to align our team and key stakeholders behind answering the following question: “how might we empower COPD patients to understand their healthcare information, make decisions in partnership with their providers, and more easily manage their daily health?” On Day 1, we focused on understanding and defining the problem, and mapping the patient experience. On Day 2, we quickly brainstormed potential solutions, sketched our top ideas, and listed the solutions’ inherent assumptions. On Day 3, we created a prototype of our top solution and storyboarded each step of the prototype experience to review its potential usability and comprehensibility with patients. RESULTS/ANTICIPATED RESULTS: At the end of the design sprint, our team developed a prototype centered around personalized communication between COPD patients and providers. The prototype focuses on augmenting the current transitional care management (TCM) workflow in the post-discharge period. We are working to further develop the prototype prior to formal testing with care coordinators and patients. We anticipate that our prototype will assist in automating the current TCM workflow and facilitate contact with more patients post-discharge. DISCUSSION/SIGNIFICANCE OF IMPACT: Contact with patients is currently challenging due limited resources and the time sensitive nature of the TCM requirements. Automated patient outreach may be especially effective in engaging patients on a large scale, while also minimizing time and resources needed from healthcare staff.

OBJECTIVES/GOALS: The limited treatment options for ischemic stroke patients have resulted in stroke being a leading cause of death and the primary cause of long-term disability in the U.S. Finding effective treatment options requires a better fundamental understanding of the ongoing processes that contribute to poor long-term outcome. METHODS/STUDY POPULATION: Expression of Apolipoprotein E4 predisposes stroke patients to poor long-term outcome. This study aims to test one possible mechanism by which ApoE4 contributes to cognitive decline after stroke. Here, we examine the effect of a major ApoE4 receptor, low-density lipoprotein receptor related protein 1 (LRP1) on sensitivity to stress in astrocytes. LRP1 binds and moves extracellular ligands and plasma membrane proteins into the endocytic system. Others have shown that LRP1 regulates cell-surface TNF receptor (TNFR1) in non-astrocytic cells. We propose That LRP1 similarly regulates TNFR1 in the central nervous system to attenuate inflammatory response after stroke. Studies have shown that ApoE4 slows the recycling of endocytic LDL receptors. We hypothesize that ApoE4 inhibits the ability of LRP1 to remove TNFR1 from the plasma membrane. This is expected to increase cytokine sensitivity, resulting in worse outcome after stroke. We investigated the effect of LRP1 on astrocyte TNFα signaling and response in immortalized ApoE null mouse astrocytes subjected to lentiviral-mediated knockdown of LRP1. The astrocyte response to TNFα stimulation was tested in a time dependent manner using Western blotting of NFkB pathway components, which are the downstream mediators of TNFα signaling. We also tested astrocyte viability after prolonged TNFα stimulation using Alamar Blue reagent. We found that LRP1 deficient cells have increased phosphorylation of NFkB upon TNFα stimulation, and that loss of LRP1 resulted in significant loss of astrocyte viability after prolonged stimulation. RESULTS/ANTICIPATED RESULTS: Altogether, our results indicate that loss of LRP1 renders astrocytes more sensitive to TNFα. Future experiments will focus on testing the influence of LRP1 on recovery after middle cerebral artery occlusion in mice. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will elucidate how astrocyte-LRP1 contributes to outcome after stroke, and helps us to understand one potential way that ApoE4 exerts pathological effects. A better understanding of the long-term processes after stroke will allow identification of therapies which improve the morbidity and mortality associated with stroke. CONFLICT OF INTEREST DESCRIPTION: NA.

OBJECTIVES/GOALS: Environmental factors may significantly increase the risk of or buffer against Alzheimer’s disease and related dementias, yet strategies to address cognitive decline and impairment to date largely overlook the role of neighborhoods. This mixed-methods study is the first to examine potential links between access to eateries and cognitive function. The goal is to inform place-specific interventions to help aging individuals reduce risk for cognitive impairment through neighborhood community and design. METHODS/STUDY POPULATION: Following an exploratory sequential mixed-methods design, seated and mobile interviews with 125 adults aged 55-92 (mean age 71) living in the Minneapolis (Minnesota) metropolitan area suggest that eateries, including coffee shops and fast-food restaurants, represent popular neighborhood destinations for older adults and sources of wellbeing. To test the hypothesis that these sites, and the benefits they confer, are associated with cognitive welfare, we analyzed data from urban and suburban dwelling participants in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study, a national racially diverse sample of older Americans followed since 2003 (n = 16,404, average age at assessment 72 years). RESULTS/ANTICIPATED RESULTS: Qualitative thematic analysis of how older adults perceived and utilized local eateries include sites of familiarity and comfort; physical and economic accessibility; sociability with friends, family, staff, and customers; and entertainment (e.g., destinations for outings and walks, free newspapers to read). Quantitative results from multilevel linear regression models demonstrate a positive association between density of eateries and cognitive functioning. Taken together, these results complicate our understanding of fast-food settings as possible sites of wellbeing through social interaction and leisure activities. DISCUSSION/SIGNIFICANCE OF IMPACT: The results contribute new evidence towards an emerging ecological model of cognitive health. Understanding whether and how retail food environments can help buffer against cognitive decline among older adults provides novel opportunities to promote wellbeing in later life through community interventions and neighborhood design.

OBJECTIVES/GOALS: Difficult-to-heal wounds of the skin are a common and costly medical problem. Dermal replacement strategies have emerged as a solution, but a challenge is identification of optimal scaffold parameters. We present a model for assessment of clinical potential of collagen scaffolds for wound healing. METHODS/STUDY POPULATION: In previous animal experiments, we evaluated dermal replacement scaffolds custom-fabricated from fibril-forming collagen oligomer with controlled fibril density (4, 20, 40mg/cm3) and spatial gradients in rat excisional wounds. Wound contraction and cellularization were monitored by gross and histological image analysis for comparison with model outcomes. We now parameterize the scaffold parameters for use in the mathematical model of wound healing with nonlinear curve fitting. A preliminary chemo-bio-mechanical finite element model including collagen, cells, and an inflammatory signal was adapted to simulate wound healing results. RESULTS/ANTICIPATED RESULTS: Collagen oligomer microstructure was quantified from scanning electron micrographs. A constitutive law for collagen mechanics was fit to experimental uniaxial tensile tests. We have conducted preliminary three-dimensional finite element model simulations to be validated against experimental wound contraction, recellularization, and collagen remodeling data collected from each experimental group. We show the effects of collagen density and stiffness on wound contraction by altering early wound mechanical properties. We anticipate future work to further improve the model of mechanotransduction, inflammation, and recellularization. DISCUSSION/SIGNIFICANCE OF IMPACT: This work represents the first step towards a computational model of wounds treated with collagen scaffold dermal replacements. In turn, the model will be used to explore cell-scaffold interactions for purposes of prediction and optimization of tissue regeneration outcomes.

OBJECTIVES/GOALS: HIV-specific CD8+ T-cells play a critical role in partially controlling viral replication in infected-individuals, but ultimately fail to eliminate infection. Enhancing these T-cell responses through lymphocyte engineering approaches has the potential as a novel therapy capable of achieving durable control or eradication of infection. METHODS/STUDY POPULATION: IL-15 Superagonist (IL-15SA) potently supports the in vivo persistence and antiviral activity of adoptively transferred CD8+ T-cells. The Deep-PrimingTM technology platform, developed by Torque, allows for loading of immunomodulators onto the surface of T-cells via electrostatic ‘nanogels’, which slowly release to deliver sustained autocrine immune stimulation without the harmful effects of systemic exposure. Here, we investigate the impact of IL-15SA Deep-Priming on HIV-specific CD8+ T-cells in a humanized mouse model of HIV infection. Humanized mice were generated by engrafting NOD-scid-IL2Rgnull mice with memory CD4+ T-cells isolated from an ARV-suppressed HIV+ donor. An autologous HIV-specific Cytotoxic T-Lymphocyte (CTL) clone was isolated, and killing potential confirmed. Four weeks post humanization, mice were infected with HIV and received an infusion of unmodified HIV-Specific CTLs, or IL-15SA Deep-Primed HIV-specific CTLs (CTL-DP). T-cell numbers and plasma viral loads were quantified weekly by flow cytometry and qRT-PCR. RESULTS/ANTICIPATED RESULTS: Mice receiving unmodified CTLs trended toward reduced viral loads compared to the No Treatment condition, while mice receiving CTL-DP saw significant, 2-Log10 reductions in VL (p < 0.01). At 41 days post-infection 100% (5/5) of the No Treatment, 66.7% (4/6) of the CTL treatment, and 16.7% (1/6) of CTL-DP treatment mice had detectable viremia. IL-15SA Deep-Priming increased CTL expansion and persistence in peripheral blood which correlated with improved CD4+T-cell preservation. DISCUSSION/SIGNIFICANCE OF IMPACT: Here we demonstrate the first in vivo analysis of IL-15SA Deep-Priming of HIV-Specific CTLs. These data suggest that Deep-Priming of patient T-cells can enhance in vivo function and persistence, leading to improved viral suppression; a significant advancement in the field of HIV cure research. CONFLICT OF INTEREST DESCRIPTION: Austin Boesch, Thomas Andresen, and Douglas Jones are employees of Torque. Darrell Irvine is a co-founder of Torque and Chairman of Torque’s Scientific Advisory Board.

OBJECTIVES/GOALS: To characterize the oncogenic potential of HNSCC cell lines harboring 17 non-canonical PIK3CA mutations. METHODS/STUDY POPULATION: Non-canonical PIK3CA mutant constructs generated via site-directed mutagenesis are subcloned into doxycycline-inducible vector pLVX-Puro. Serum-dependent HNSCC cell line (PCI-52-SD1) is then stably transfected with vectors and undergo doxycycline-induction. Cell survival is determined by depriving cells of fetal bovine serum for 72 hours and quantifying remaining cells with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell proliferation and migration is evaluated with colony formation assays and transwell assays respectively. RESULTS/ANTICIPATED RESULTS: To date, the survival behavior of eight non-canonical mutants was assessed. Three mutants – Q75E, V71I, and E970K – exhibited 18.7-26.7% greater survival rate relative to cells transfected with wild-type. Five mutants – R519G, Y606C, W328S, C905S, and M1040I – demonstrated survival rates that differed only by −4.3% to +6.6% relative to wild-type. We hypothesize the three activating mutants that exhibited increased survival will also demonstrate increased cell proliferation and migratory behavior whereas the three neutral mutants will not differ from control. DISCUSSION/SIGNIFICANCE OF IMPACT: Ongoing HNSCC PI3K inhibitor trials could be more effective if all PIK3CA hyperactivation mutations are known. Identifying non-canonical mutation effects could result in greater efficacy if drugs are restricted only to those with activating mutations. CONFLICT OF INTEREST DESCRIPTION: JRG and DEJ are co-inventors of cyclic STAT3 decoy and have financial interests in STAT3 Therapeutics, Inc. STAT3 Therapeutics, Inc. holds an interest in a cyclic STAT3 decoy oligonucleotide. The remaining authors declare no conflicts.

OBJECTIVES/GOALS: Individuals with a family history of alcoholism (FH+) are more likely to develop an alcohol use disorder than those with no such history. Early life adversity has a high coincidence with FH+ making pathogenic studies difficult in clinical studies. Here, we developed a mouse model to study pathogenic mechanisms underlying these risk factors. METHODS/STUDY POPULATION: Male and female C57BL6/J mice were exposed to increasing concentrations of ethanol (3-21%) or water for 15 days prior to breeding. Ethanol was not present during gestation. Offspring were either removed from the home cage and isolated for 3 hours or left undisturbed from postnatal days 1-21. Beginning on PND 56 offspring mice were assessed for clinically relevant behavioral disruptions in social behavior, cognitive working memory, locomotor activity, anxiety-like phenotypes, ethanol preference and binge drinking behavior. In a separate experiment, brains of Cx3cr2+/GFPxCcr2+/RFP mice from ELA or control conditions were collected every 7 days after birth for assessment of neuroinflammation and central immune cell morphology and density. RESULTS/ANTICIPATED RESULTS: Mice with a family history of ethanol exposure and ELA are predicted to exhibit behavioral changes (impaired working memory, reduced social behavior, increased anxiety-like behaviors, increased ethanol consumption) to a greater extent than mice with a family history of ethanol exposure or ELA alone. We expect markers of neuroinflammation (cytokine expression, immune cell activation) to predict the behavioral changes in these mice. DISCUSSION/SIGNIFICANCE OF IMPACT: Alcohol consumption and stressful life events are known environmental precipitants to neuroinflammation, which in turns may predispose individuals to anti-social and risky behavior. A mouse model of these early postnatal conditions will allow basic scientists to unravel the biological underpinnings of the behaviors driven by these factors.

OBJECTIVES/GOALS: Breast cancer metastases are stochastic and difficult to detect. Therapy is often ineffective due to phenotypic changes of tumor cells at these sites. We engineered a synthetic metastatic niche to study the role of phenotypic transitions in the microenvironment on tumor cell phenotype. METHODS/STUDY POPULATION: The engineered metastatic niche is composed of a porous polycaprolactone scaffold implanted subcutaneously in Balb/c mice. The mice received an orthotopic inoculation of 4T1 cells (murine triple negative breast cancer) in the fourth right mammary fat pad and the disease was allowed to progress for 7-21 days (pre-metastatic to overt metastatic disease). The scaffolds and lungs (native metastatic site) were explanted and analyzed by single cell RNA-seq via Drop-seq. Cell phenotypes were identified and tracked over time with the Seurat and Monocle3 pipelines. Assessment of the impact of these cell populations on tumor cell phenotype was conducted through Transwell co-cultures. RESULTS/ANTICIPATED RESULTS: Healthy scaffolds are primarily composed of macrophages, dendritic cells, and fibroblasts – consistent with a foreign body response. Despite differences in the lung and scaffold prior to tumor inoculation, both tissues were marked by >5-fold increase in neutrophils/MDSCs. Additionally, 79% of genes at the scaffold that significantly changed over time were also identified in the lung, indicating key similarities in niche maturation. However, many immune cells at the scaffold had distinct phenotypes, with pro-inflammatory/cytotoxic characteristics. These changes clearly impacted tumor cell phenotype, as cells from the scaffold increased tumor cell migration and apoptosis in vitro. DISCUSSION/SIGNIFICANCE OF IMPACT: Early phenotypic changes at the engineered metastatic niche can identify signs of metastasis prior to colonization of tumor cells. Furthermore, dynamics of immune and stromal cells change throughout niche maturation, influencing tumor cell phenotype and may suggest targeted therapies. CONFLICT OF INTEREST DESCRIPTION: Lonnie Shea, Jacqueline Jeruss, and Grace Bushnell are named inventors on patents or patent applications.

OBJECTIVES/GOALS: Aging is the biggest risk factor for cancer, yet little is known about cancer immunotherapy effects. Here we investigate melanoma response to αPD-1, αPD-L1 and αPD-L2 in young vs. aged hosts. We look at different immune outcomes as possible mechanism. METHODS/STUDY POPULATION: We tested αPD-1 (100 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18-24 months) and young (3-8 months) mice challenged orthotopically with B16. Tumors and draining lymph nodes (TDLN) were analyzed by flow. Bone marrow-derived DC were generated with GM-CSF. RESULTS/ANTICIPATED RESULTS: We reported that αPD-1 treats young and aged with B16 and αPD-L1 only treats young. aPD-L2 treated B16 in aged but, remarkably, not young, the first anti-cancer single agent immunotherapy exhibiting this property. Efficacy in young (aPD-1, aPD-L1) and aged (aPD-1, aPD-L2) correlated with increased T cell stem cells (TCSC) and total tumor-infiltrating lymphocytes (TIL), but TCSC differed by age and treatment (e.g., distinct CCR2, CXCR5, CXCR3, PD-1 and TIM- expression). Aged expressed significantly more T-cell PD-1 and up to 40-fold more PD-L2 versus young in myeloid and NK cells, and TCSC. Bone marrow-derived DC experiments suggest aged DC are destined for high PD-L2 versus young. DISCUSSION/SIGNIFICANCE OF IMPACT: Treatment differences in aged vs. young could depend on immune checkpoint or TCSC differences. We are now identifying mechanisms for increased PD-L2 and contributions to aPD-L2 efficacy in aged, and testing TCSC effects. Our work can improve cancer immunotherapy in aged hosts and further provide important insights even in young hosts.

OBJECTIVES/GOALS: Our overall objective is to develop a directly observable and reproducible method of enhanced blood flow through tumor vessels (i.e. dynamic control) at the time of systemic treatment delivery. Our central hypothesis is that the dynamic control of tumor vessels will improve (1) systemic drug delivery and (2) effector cell trafficking to target tumor. METHODS/STUDY POPULATION: B16 melanoma cells were inoculated into C57BL/6 (B6) mice (male and female) in both regional (hind leg) and systemic (flank) models. Dynamic control consisted of an IV saline bolus (500 ul) and phenylephrine (10 ug). Tumor vessel response was observed in real-time through window chambers using intravital microscopy (IVM). Dynamic control was combined with melphalan (20 mg/ml) either regionally (isolated limb perfusion) or systemically. Outcomes included tumor growth, survival, IHC, and toxicity. Dynamic control will be combined with adoptive transfer of effector T cells. B6 mice will be inoculated with B16/OVA (flank with window chamber) and treated with fluorescently labeled (calcein), OVA-specific CD8+ T cells from OT-1 transgenic mice. IVM, IHC, and flow cytometry will be used to measure T cell trafficking. RESULTS/ANTICIPATED RESULTS: Dynamic control (1) restored blood flow in non-functional tumor vessels and (2) increased and then transiently reversed blood flow in functional vessels. Vessel diameters did not change, suggesting that shunting of systemic blood to the tumor vasculature accounted for the observed changes. Dynamic control augmented tumor responses in our regional therapy model of melanoma. Increases in DNA adduct formation (melphalan mechanism of action) detected by IHC, decreased tumor growth, and increased survival were observed with dynamic control. There was no increased limb toxicity. Similarly, dynamic control augmented responses in our systemic therapy model (decreased tumor growth and improved survival). We anticipate that dynamic control will improve trafficking of effector T cells in the next set of experiments. DISCUSSION/SIGNIFICANCE OF IMPACT: Heterogeneous responses to systemic therapies represent a major gap in current cancer treatment. An essential requirement for any effective therapy is its ability to reach tumor via the tumor-associated vasculature. We have therefore developed an approach to enhance drug delivery (dynamic control), which we also plan to test in clinical trials.

OBJECTIVES/GOALS: The overall goal of this study was to determine the effect of early life stress (ELS) on the intestinal CD4+ T cell immune compartment, at homeostasis and after induction of experimental Inflammatory Bowel Disease (IBD). METHODS/STUDY POPULATION: We used a mouse model of ELS, maternal separation with early weaning (MSEW). We used IL-10 reporter mice to enable analysis of IL-10-producing cells. Mice were examined on postnatal day 28 to determine the impact of ELS on gut regulatory T cells. Plasma levels of corticosterone (rodent stress response hormone) was determined by ELISA. Colitis was induced in MSEW and normal rear (NR) mice via intraperitoneal injection of α-IL-10R every 5 days until day 15. Mice were euthanized on days 20 and 30. Colonic tissue sections were stained for histological analysis. Remaining tissue was further processed for flow cytometric analysis of CD4+ T cells and innate lymphoid cells. RESULTS/ANTICIPATED RESULTS: Plasma corticosterone was elevated in MSEW mice compared to their NR counterparts at 4 weeks of age. We observed that the MSEW stress protocol does not affect the baseline colonic CD4+ T cell or innate lymphoid cell populations. There was a reduction in the intestinal CD4+ T cells and regulatory T cells on day 20 in α-IL-10R MSEW mice compared to NR counterparts. This difference disappeared by day 30. Histological scoring showed no difference in disease severity between α-IL-10R treated MSEW and NR mice on day 20. However, on day 30, when α-IL-10R NR mice are recovering from colitis, MSEW mice showed persistent histological inflammation, mainly attributable to sustained epithelial damage. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that ELS prolongs intestinal inflammation and impairs epithelial repair. Future studies will focus on elucidating the mechanisms responsible for ELS-dependent impairment of mucosal repair in experimental colitis.

OBJECTIVES/GOALS: High aortic stiffness is associated with increased cardiovascular morbidity and mortality. The purpose of this work is to demonstrate the potential of our compliant stent-graft design to therapeutically increase aortic compliance over a standard aortic stent-graft. METHODS/STUDY POPULATION: The aorta from a human cadaver will be excised and placed into a pulse duplicator circuit. The stiffness of the system will be estimated using the pulse wave velocity (PWV), which will be calculated using the time delay between pressure measurements at proximal and distal locations in the system. Baseline measurements with the unstented aorta will be compared to two cases: (1) with a standard stent-graft placed, and (2) with our compliant stent-graft prototype in the descending thoracic aorta. PWV is calculated as the distance between the pressure sensors divided by the time delay. Faster PWV is associated with a stiffer vessel, or lower aortic compliance. RESULTS/ANTICIPATED RESULTS: Prior work in vitro showed that the compliant stent-graft reduced peak and pulse pressures compared a standard, rigid stent-graft. We also expect the compliant device to exhibit lower PWV compared to a rigid stent-graft. Depending on the aortic tissue stiffness, the compliant stent-graft could raise or lower PWV compared to no stent. Mean pressure in the compliant case is likely to be slightly higher than the other two cases because the compliant stent-graft’s narrower lumen increases flow resistance. Although mean pressure will be higher, peak pressure should be lower than in the standard stent-graft because the added compliance decreases overall pressure swing between systole and diastole. DISCUSSION/SIGNIFICANCE OF IMPACT: Lower PWV in the compliant stent-graft over the standard stent-graft will indicate its potential to therapeutically lower aortic stiffness in patients needing aortic stenting. Positive outcomes from this study will be a step toward the eventual translation of a compliant stent-graft to clinical use.

OBJECTIVES/GOALS: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths by 2030. Though many other cancers have seen improvements in patient survival rates, patients diagnosed with PDAC have a 5-year survival rate of only ~9%. A major contributor to decreased survival is late-stage diagnosis of the disease. New methods of early detection are urgently needed. Extracellular vesicles (EVs) are secreted from cells of all tissue types into the circulation. EVs play important roles in a variety of diseases. They have shown to promote cancer progression and they are being studied as potential biomarkers for disease diagnosis. The purpose of this study was to perform qualitative and quantitative characterization of small-molecule profiles of EVs derived from various pancreatic cancer (PC) and normal pancreas cell lines, to provide proof-of-concept for evaluating the efficacy of leveraging EVs as potential biomarkers of PDAC. METHODS/STUDY POPULATION: EVs were isolated from the conditioned media of six PC and two normal pancreas cell lines using differential ultracentrifugation with filtration. EV enrichment was validated using quantitative ELISA, immunoblot and transmission electron microscopy. Targeted liquid chromatography coupled to mass spectrometry (LC-MS/MS) and untargeted (UPLC-QTOF-MS) metabolomics were used to analyze the biochemical composition of EVs. RESULTS/ANTICIPATED RESULTS: The biochemical profile of PC EVs was found to be significantly different from the profiles of normal cell EVs. Interestingly, amino acids were downregulated in PC EVs as compared to normal cell EVs. However, PC EVs were enriched in lactate and malate. PC EVs also had significant upregulation in other small molecules such as xanthosine, guanosine diphosphate and nicotinamide. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results indicate that the biochemical characterization of EVs using metabolomics has the potential to yield biomarkers which can delineate cancer cell-derived EVs from normal cell-derived EVs. Further work will test the clinical significance of these findings by similar analyses of plasma of PDAC patients. Furthermore, these profiles may be detectable before progression of the disease to late-stage PDAC, leading to the development of assays for earlier diagnosis in patients.

OBJECTIVES/GOALS: The glucocorticoid receptor (GR) is a ubiquitous steroid hormone receptor that is emerging as a mediator of breast cancer metastasis. We aim to better understand the biology associated with phospho-GR species in TNBC and their contribution to tumor progression. METHODS/STUDY POPULATION: To better understand how p-S134 GR may impact TNBC cell biology, we probed GR regulation by soluble factors that are rich within the tumor microenvironment (TME), such as TGFβ. TNBC cells harboring endogenous wild-type or S134A-GR species were created by CRISPR/Cas knock-in and subjected to in vitro assays of advanced cancer behavior. RNA-Seq was employed to identify pS134-GR target genes that are uniquely regulated by TGFβ in the absence of exogenously added GR ligands. Direct regulation of selected TGFβ-induced pS134-GR target genes was validated accordingly. Bioinformatics tools were used to probe publicly available TNBC patient data sets for expression of a pS134-GR 24-gene signature. RESULTS/ANTICIPATED RESULTS: In the absence of GR ligands, GR is transcriptionally activated via p38-MAPK-dependent phosphorylation of Ser134 upon exposure of TNBC cells to TME-derived agents (TGFβ, HGF). The ligand-independent pS134-GR transcriptome primarily encompasses gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TGFβ-induced TNBC cell migration, anchorage-independent growth in soft-agar, and tumorsphere formation, an in vitro readout of breast cancer stemness properties. Finally, a 24-gene pSer134-GR-dependent signature induced by TGFβ1 predicts shortened survival in breast cancer. We expect to find similar results using an in-house tissue microarray. DISCUSSION/SIGNIFICANCE OF IMPACT: Phospho-S134-GR is a critical downstream mediator of p38 MAPK signaling and TNBC migration, survival, and stemness properties. Our studies define GR as a required effector of TGFβ1 signaling and nominate pS134-GR as a biomarker of elevated risk of breast cancer dissemination.