OBJECTIVES/GOALS: While mutant TP53 is an attractive therapeutic target in TNBC, attempts to target the mutant p53 protein directly have failed. Thus, we aim to identify pathways critical for the survival of TP53 mutant cells that can be targeted in TNBC. We have identified Kif11 as one such target and aim to further investigate its function in TP53 mutant TNBC. METHODS/STUDY POPULATION: We conducted a dual in silico/in vivo screen that identified Kif11 inhibition as preferentially inhibiting the growth of TP53 mutant TNBC. We obtained data on TP53 mutational status, KIF11 mRNA expression levels, and clinical characteristics from TCGA, METABRIC, and CCLE datasets. We treated breast cancer cell lines with the KIF11 inhibitor SB-743921. Cell counts were obtained through staining with DAPI or Hoechst and imaging on the ImageXPress PICO. We detected cell death by DRAQ7 staining and flow cytometry analysis following Annexin V-PI staining. To investigate mitotic spindle organization, we performed immunofluorescent staining with an anti-tubulin antibody and DAPI co-staining. Cell cycle analysis was performed through flow cytometry. RESULTS/ANTICIPATED RESULTS: KIF11 is highly expressed in TP53 mutant and TNBC clinical samples. High KIF11 expression is associated with poorer clinical outcomes. Kif11 inhibition suppresses growth of both TP53 mutant and wild-type breast cancer cells, but preferentially induces the death of TP53 mutant cells as detected by DRAQ7 and Annexin V/PI staining. Kif11 inhibition induces a G2-M block and growth inhibition in TP53 wild-type cells. On the other hand, following treatment with the Kif11 inhibitor SB-743921, TP53 mutant cells undergo mitotic spindle dysfunction leading to the formation of multinucleated cells and cell death. DISCUSSION/SIGNIFICANCE: These results demonstrate that Kif11 is a promising therapeutic target in aggressive, TP53 mutant TNBCs. Kif11 inhibitors, including SB-743921, have been tested in human trials, and are well tolerated, but it is unclear which patients would most benefit. Our studies show that Kif11 inhibitors may be most useful in patients with TP53 mutant TNBCs.

Effective mentoring is a key mechanism propelling successful research and academic careers, particularly for early career scholars. Most mentoring programs focus on models pairing senior and early career researchers, with limited focus on peer mentoring. Peer mentoring may be especially advantageous within emerging areas such as implementation science (IS) where challenges to traditional mentoring may be more prevalent. This special communication highlights the value of peer mentoring by describing a case study of an early career IS peer mentoring group. We delineate our curriculum and structure; support and processes; and products and outcomes. We highlight important group member characteristics to consider during group formation and continuation. The group’s long-term (6 years) success was attributed to the balance of similarities and differences among group members. Members were in a similar career phase and used similar methodologies but studied different health topics at different institutions. Group members gave and received instrumental and psychosocial support and shared resources and knowledge. Peer mentoring can serve an important function to provide emotional, logistical, and professional development support for early career scholars. Our case study highlights strategies to foster peer mentoring groups that provide a generalizable blueprint and opportunity for improved outcomes for early career professionals.

This in-depth analysis illuminates a translational journey of a community-university research collaboration that examined health disparities among incarcerated pregnant women and spanned the translational spectrum, with the initial collaboration in 2011 paving the way for consequent research grants, publications, practices, programs, and legislation passed years later. The case study utilized data from interviews with research stakeholders, institutional and governmental sources, peer-reviewed publications, and news stories. Identified research and translational challenges included cultural differences between research and prison system; the prison system’s lack of transparency; politics of using and translating research to policy change; and issues of capacity, power, privilege, and opportunity when doing community-engaged research/science. Among the facilitators of translation were the Clinical and Translational Science Award and institutional support; engagement of key stakeholders and influencers; authentic collaboration and team science; researchers as translation catalysts; pragmatic scientific approach; and policies and legislative activities. The research contributed to a variety of community and public health, policy/legislative, clinical/medical, and economic benefits. The case study findings enhance our understanding of translational science principles and processes leading to improved wellbeing and serve as a call for advancing the research agenda addressing health disparities related to criminal and social justice issues.

OBJECTIVES/GOALS: Leigh Syndrome, French Canadian-Type (LSFC) is a neurometabolic disorder caused by mutation of mitochondria-related gene, LRPPRC. White matter lesions and demyelination in central nervous system are common in LSFC. LRPPRC is enriched in myelinating glial cells, yet its role is not known. Our goal is to elucidate its mechanistic role in myelination. METHODS/STUDY POPULATION: We crossed C57BL/6N mice bearing a LRPPRC-loxP allele with mice bearing a Plp-CreERT2 allele. Mice with the Plp-CreERT2 allele expresses a tamoxifen-inducible Cre under the control of the Plp promoter, which drives expression in oligodendrocytes. Using these strains, we can target the deletion of LRPPRC, via tamoxifen injection, in both newly formed myelin and mature myelin. Plp-CreERT2; LRPPRCL/L (LRPPRC-KO) or control littermate mice will be injected for LRPPRC deletion at developmental and maturation stages of myelin. Immunofluorescence and electron microscopy of isolated brain tissues will be used for myelin integrity analysis. Cognitive functions of the mice will be measured via behavioral tests. Lastly, we will submit tissues for lipidomic analyses to observe any lipid metabolite variation. RESULTS/ANTICIPATED RESULTS: Behavioral and motor defects would be expected in LRPPRC-KO mice performing in cognitive function tasks across myelin maturation stages. Electron microscopy-based structure analysis of optic nerve, corpus callosum, and spinal cord should reveal thin or loss of myelin on the axons of LRPPRC-KO compared to control. Immunofluorescence staining of major myelin structural proteins, including myelin proteolipid protein (PLP), myelin basic protein (MBP), and myelin-associated glycoprotein (MAG) would be expected have lower levels in LRPPRC deficient tissues. Since myelin is a lipid-rich species, we would also expect lipid concentrations to be affected. LRPPRC-KO lipidomic analyses of myelin-related lipids should depict lower levels in comparison to control, which would imply dysfunctional lipid metabolism. DISCUSSION/SIGNIFICANCE: There are limited studies in ameliorating neural deficits caused by LS and LSFC. Successful completion of this project would help elucidate the functions of LRPPRC in myelination and lipid metabolism and potentially provide insights for developing novel therapeutic strategies for alleviating the demyelination and neural deficits in LSFC.

OBJECTIVES/GOALS: We use a tissue engineered, biomimetic, 3D model to study the pathogenesis of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) by comparing the effect of silicone implant shell on proliferation of patient-derived BIA-ALCL to its precursor T cells within the breast microenvironment. METHODS/STUDY POPULATION: Patient-derived breast tissue was processed for component adipocytes, ductal organoids, and stromal vascular fraction. These were suspended within 50 µl of 0.3% type I collagen matrix to which was added 200,000 cells/mL of either patient-derived BIA-ALCL cells or T progenitor cells. These were then plated into 6mm wells. As a control, both BIA-ALCL cells and T progenitor cells were suspended within type I collagen alone at the same seeding density without breast components. Before plating, wells were lined circumferentially with either textured, smooth, or no implant shell. These were 1cm by 2cm pieces dissected from the whole implant. Wells were imaged using confocal microscopy over 8 days. RESULTS/ANTICIPATED RESULTS: Unstimulated T progenitor cell count showed no significant increase in any of the conditions tested. The change in cell count over 8 days was 3.85% in each condition (p = 0.3352). A Tukey’s multiple comparison test comparing each condition revealed no significant increase in cell count over 8 days for all six conditions. Notably, our previous studies have shown proliferation of BIA-ALCL cells to be significantly more robust in the biomimetic platform compared to collagen-only groups, regardless of implant shell type (p < 0.01). BIA-ALCL cells grew nearly 30% faster in textured and smooth shell biomimetic groups compared to biomimetic wells lacking implant shell. DISCUSSION/SIGNIFICANCE OF IMPACT: Towards elucidating BIA-ALCL’s etiopathology, we show that silicone implant shell has a significant effect on proliferation of BIA-ALCL cells, but not their precursor T cells. If breast implant silicone shell is not a sufficient stimulus for T cell proliferation, co-stimulatory factors are required.

OBJECTIVES/SPECIFIC AIMS: The purpose of this case series is to show how helping parents instill a non-restrictive, structure-based (i.e., authoritative) approach to feeding is useful in addressing family food conflicts in a clinical child obesity treatment program. METHODS/STUDY POPULATION: Case reports are presented for 3 young children (two 8-year-old males and one 7-year-old female) with obesity (BMI ≥ 95th percentile for age and sex). Patients underwent family-based treatment at Brenner FIT® (Families In Training), an interdisciplinary tertiary weight management clinic. RESULTS/ANTICIPATED RESULTS: All patients experienced a period of rapid weight gain and/or severe onset obesity. Parents reported a combination of problematic eating behaviors (e.g., sneaking food, frequent complaints of hunger, vomiting from rapid consumption). Families implemented structure-based feeding with a meal-snack schedule and allowed children to eat until they were full from the food provided at meal-snack times. BMI z-score decreased from 2.19 to 2.07 in patient 1 and from 2.43 to 2.09 in patient 2 (follow-up weight was not available for patient 3). DISCUSSION/SIGNIFICANCE OF IMPACT: The improvements observed by our clinical program after families lifted restriction and instituted authoritative feeding is anecdotal evidence for the ecological validity of existing empirical work. Randomized controlled trials are needed to examine causality.

ABSTRACT IMPACT: Evaluate the accuracy of applying a predictive algorithm using clinical measures only in persons with stroke in the US. OBJECTIVES/GOALS: PREP2 is an algorithm, that predicts UL functional capacity at 3 months post stroke from measures taken within the first week.(1, 2) Despite its accuracy and ease of use, challenges arise of applying PREP2 in the US. The objective of this study was to evaluate the accuracy of PREP2 using only clinical measures in persons with stroke in the US. METHODS/STUDY POPULATION: Individuals with first-ever stroke were recruited from a local hospital and followed longitudinally, as part of an ongoing observational cohort. Variables captured within two weeks of stroke and entered into the algorithm were: age, SAFE score(1-3) and NIH Stroke Scale(4) total score. The algorithm classifies individuals into one of four expected categories: excellent, good, limited, or poor. The dependent variable was the predicted category of UL functional capacity as defined by ranges of the 3-month Action Research Arm Test score.(5) Accuracy, specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of the algorithm, were calculated using a 4x4 contingency table. Other statistics analyzed include demographic characteristics and a weighted kappa for the algorithm. RESULTS/ANTICIPATED RESULTS: Data from 49 individuals were analyzed (57% male, 88% ischemic stroke, age = 65±8.56 years). Expected categorization matched observed categorization in 29/49 subjects, with the overall accuracy of the algorithm of 59% (95% CI = 0.44-0.73). The sensitivity of the algorithm was low except for the excellent category (0.95). Specificity was moderate to high for good (0.81), limited (0.98), and poor (0.95) categories. PPV was low for all categories and NPV was high for all categories except the good category. Additional results including weighted kappa and inaccuracy of predictions to be presented. DISCUSSION/SIGNIFICANCE OF FINDINGS: PREP2 algorithm, with clinical measures only, is better than chance (chance = 25% for each of the 4 categories) alone at predicting a category of UL capacity at 3 months post stroke. PREP2 is a simple tool that facilitates evaluation of eventual UL outcome from measures routinely captured after a stroke within most healthcare settings in the US.

OBJECTIVES/GOALS: Traumatic interpersonal experiences are associated with higher rates of chronic pain, increased pain severity and poorer functioning. The objective of this ongoing project is to obtain prevalence rates for various forms of interpersonal trauma among individuals with chronic pain, and to explore the potential mediating effect of heightened sensory and social sensitivity on the experience of pain. METHODS/STUDY POPULATION: Patients at Michigan Medicine between the ages of 18 and 65 complete an online survey. Patients are being recruited through a tertiary-care, outpatient pain clinic, as well as through an online health research portal. We aim to recruit 700 participants; we currently have 59.6% of our goal (n = 417). Participants also have the option to be included in a registry from which we can recruit for future studies. Approximately 85% of our participants have agreed to be in the registry. RESULTS/ANTICIPATED RESULTS: Preliminary data show that, of the 263 (63.4%) participants for whom data on chronic pain is available, 167 (63.5%) report chronic or persistent pain over the previous 3 months. Of these, 54% reported some form of childhood abuse or neglect. Approximately 41% reported four or more adverse childhood experiences. Additionally, of the 122 participants (73%) who were in a current romantic relationship, 20% reported some form of physical violence victimization from their romantic partner. We anticipate that interpersonal trauma will be associated with poorer perceptions of social relationships, higher sensory sensitivity, and higher perceived stress. DISCUSSION/SIGNIFICANCE OF IMPACT: The PASE Project parent study will be used to better understand prevalence rates for various forms of interpersonal trauma in our chronic pain population. Future analyses and studies will explore alternative pathways linking interpersonal trauma to the experience of pain through sensory and social sensitivity, which will inform interventions aimed at reducing pain among patients with a history of trauma.

OBJECTIVES/GOALS: Interleukin (IL)-6 is produced in excess in Systemic Sclerosis (SSc). Likewise, microarray analysis of Insulin-like Growth Factor (IGF)-II-treated NL fibroblasts revealed increased expression of the basic helix-loop-helix transcription factor, BHLHB2. Our goal is to delineate the role of BHLHB2 in the fibrotic response to IGF-II and IL-6. METHODS/STUDY POPULATION: Primary lung fibroblasts were cultured from human lung tissues at 37oC and 5% CO2. Cell cultures were stimulated with IL-6. Gene expression was measured using quantitative PCR (qPCR). IGF-II mRNA expression levels after IL-6 stimulation were compared with those of the housekeeping gene PPIB (Peptidylprolyl Isomerase B). Western blot was performed on nuclear and chromatin-bound subcellular fractions from treated lung fibroblasts. BHLHB2 protein levels were assayed in response to IGF-II in comparison to PBS as vehicle control. RESULTS/ANTICIPATED RESULTS: Results: Our results show that IL-6 increases IGF-II levels in fibroblasts. In turn, IGF-II increases BHLHB2 nuclear localization. We further show that IL-6 increases BHLHB2 levels and its nuclear localization in lung fibroblasts. Our findings are novel since the role of the transcription factor BHLHB2 in the IL-6 induced/ IGF-II-mediated fibrotic response in SSc lung disease remains unexplored. DISCUSSION/SIGNIFICANCE: Our findings may provide a rationale for combination therapy to block IL-6 and IGF-II function concomitantly and thus halt the progression of SSc pulmonary fibrosis (PF). Our findings may have wide implications for lung fibrosis associated with various diseases, since SSc-PF, is characterized by the activation of common fibrotic pathways.

Objectives/Goals: The standard care for malignant gliomas includes maximal tumor resection, but challenges arise near functional (speech) areas. Direct cortical stimulation (DCS) identifies functional (nonresectable) cortex. We aim to identify electrophysiologic (via subdural electrode recordings [ECOG]) biomarkers of DCS-positive (functional) areas. Methods/Study Population: Our lab maintains one of the largest datasets of electrophysiology analysis of glioma infiltrated brain cortex in the USA. Recordings of intraoperative brain mapping were analyzed to identify cortical sites that were found to be positive (functional) during DCS. DCS positive and negative (nonfunctional) sites were aligned to corresponding subdural electrodes. Future analysis: We plan to compare the temporal and spectral electrophysiologic variations associated with cortical sites found to be DCS positive versus negative during brain mapping. We plan to train machine learning classifiers that utilize these electrophysiologic biomarkers to discriminate between DCS positive and negative sites. Results/Anticipated Results: In total, our database comprised of 110 resections with brain mapping (DCS) and ECOG, including 4 patients who underwent a second procedure for resection. Eight patients were excluded as their resections were for brain metastases, not glioma. Our final cohort was comprised of 98 glioma resections, including 4 patients who underwent surgery twice for recurrence. During these resections, a total of 1393 sites were mapped via DCS for language function (including picture naming, word reading, and sentence syntax tasks). Of these 1393 sites, 100 sites were found to be DCS positive (7.1% positivity rate). (Currently in the process of conducting analysis comparing electrophysiologic features and biomarkers of DCS positive versus negative sites.) Discussion/Significance of Impact: This research is ongoing. Identifying electrophysiologic biomarkers of critical DCS-positive regions may provide a durable alternative to stimulation mapping. Due to its resource intensity, DCS has access barriers. Future neurosurgeons may use biomarkers from subdural electrode recordings to plan safer cortical resections.

OBJECTIVES/GOALS: Susceptible mucocutaneous membranes of the eye and nasal cavity are easily infected by viruses leading to pink eye or respiratory infections whose direct cost has been estimated as $16 billion annually in the United States. We have developed a novel and effective barrier that will be agnostic to variants enveloped viruses like coronaviruses. METHODS/STUDY POPULATION: We evaluated the efficacy of hydroxypropyl cyclodextrin barrier in preventing respiratory coronavirus infections using 25 humanized angiotensin converting enzyme-2 receptor (hACE-2) mice under a BSL3 laboratory setting. We have shown the barrier is safe and efficacious in preventing coronavirus infections in in vitro respiratory cell lines. We instilled 10 uL aliquot of the barrier into the nostril of the mouse 30 minutes before exposing them to a 10uL titer containing 10,000 plaque forming units of the SARS-CoV-2 delta variant. The control mice received the SARS-CoV-2 infection but not the barrier. The mice were observed for 5 days after which they were sacrificed. We analyzed the lungs and nasal palates for viral load using reverse transcription-polymerase chain reaction. RESULTS/ANTICIPATED RESULTS: We observed our barrier to be effective in preventing SARS-CoV-2 delta variant infection in hACE2 mice models. The lungs and nasal secretions of treated mice were less infectious with lower viral load than the control mice. The lungs of treated mice showed decrease in IFN gene expression and many cytokines and chemokines that regulate virally induced inflammatory responses such as IL-1b, IL-8, CXCL9, CXCL10, and the CCLs. We observed the plasma Angiotensin I and Angiotensin II decreased with barrier treatment, correlating with the viral load observed in the lungs. These peptides may be useful biomarkers for monitoring viral load within the lungs of virally infected individuals. DISCUSSION/SIGNIFICANCE: This supports the barrier’s efficacy to reduce transmission and prevent infections of SARS-CoV-2. This easy to use barrier can augment the mucocutaneous layers of the eye and nasal cavity. Our agnostic barrier will reduce the economic and public health burden of seasonal respiratory and eye viral infections and their related deaths amongst the public.

Objectives/Goals: Understanding the interconnections among over 20,000 human diseases spanning organ systems could inform more precise diagnosis and treatment of diseases. Here, we examine whether the ability of large language models (LLMs) to learn universal representations of concepts can be leveraged to discover complex relationships across human diseases. Methods/Study Population: To address the challenge of computationally representing thousands diseases spanning multiple organ systems, we used internal representations of concepts by LLMs to encode diseases based on their descriptions from standard disease ontologies (ICD10 and Phecodes). To do this, we leveraged application programming interfaces (APIs) of three LLMs-GPT3.5, Mistral and Voyage to encode disease relationships. We then performed unsupervised clustering of the diseases using their encodings (embeddings) from each LLM to determine whether the resulting clusters reflect disease relationships. To enable deeper exploration of disease relationships, we developed interactive plots that provide a system level view of the relationships between thousands of diseases and their association with specific organ systems. Results/Anticipated Results: We found that unsupervised analysis of disease relationships using the LLM encodings reveal high similarities among diseases based on organ systems they affect. All the LLMs clustered diseases into groups largely defined by the organ systems they affect without being trained to specifically classify diseases into their corresponding organ system classification. An exception to this was tumors in which we observed that most tumors cluster together as a group irrespective of the organs they affect. Interestingly, we found that tumors affecting anatomically related organs show higher similarity to each other than to those affecting distantly related organs. In addition to anatomical relationships between diseases, we found that the LLM embeddings capture genetic relationships between diseases. Discussion/Significance of Impact: Overall, we found that the LLM-derived encodings uphold biologically and clinically significant relationships across organ systems and disease types. These results suggest that LLM encodings could provide a universal framework for representing diseases as computable phenotypes and enable the discovery of complex disease relationships.

ABSTRACT IMPACT: This study assesses patient and volumetric risk factors for distant recurrence within 6 months of completion of curative chemoradiation with brachytherapy in locally advanced cervical cancer. OBJECTIVES/GOALS: Initial tumor volume and tumor shrinkage velocity are prognostic of cure and survival after curative chemoradiation (CRT) for cervical cancer. We explored whether local tumor volumetric changes influence time to distant recurrences outside the radiation field. METHODS/STUDY POPULATION: We performed a retrospective cohort study of patients with FIGO Stage IB-IVA cervical cancer treated with curative CRT and brachytherapy at a tertiary academic center with minimum 3 months follow up and standard post-treatment FDG-PET. Patients received 6 weekly fractions of brachytherapy interdigitated with external beam radiation and cisplatin. Tumor volumes were assessed by MRI at brachytherapy planning. Patients who developed distant metastasis were classified as earliest (3-6 months), early (6-24 months) or late (>24 months) following completion of CRT. Absolute and percent decrease in tumor volume for each fraction were calculated with respect to first brachytherapy volume. Fisher’s exact and Mann Whitney-U tests were used for comparison of categorical and continuous variables. RESULTS/ANTICIPATED RESULTS: 143 of 574 (25%) patients developed distant metastasis. Distribution of age, histology, FIGO 2018 stage, primary tumor SUVmax, treatment length, and pre/post treatment squamous cell carcinoma antigen levels were not associated in each group. Para-aortic lymph metastases were more common in patients with earliest distant recurrence (33% earliest, 26% early, 12% late, p=0.03). Median initial tumor volume in the earliest (n=24), early (n =29) and late (n=9) groups was 57, 28 and 40 mL, respectively (p=0.08); 57 (earliest) vs 30mL (early+late groups), p=0.04. Average mid treatment (fraction 4) and end of treatment (fraction 6) percent shrinkage was 80 (earliest) vs 73 (early+late), p=0.84 and 94 vs 92, p=0.95, respectively. Neither absolute nor percent tumor shrinkage differed between early vs. late groups. DISCUSSION/SIGNIFICANCE OF FINDINGS: Tumor volumetric changes during definitive chemoradiation were not associated with the timing of developing distant metastasis, which is linked to presence of lymph node metastasis and tumor volume at diagnosis.

ABSTRACT IMPACT: Urine tumor DNA non-invasively detects minimal residual disease and infers tumor mutational burden in locally advanced bladder cancer prior to radical cystectomy, which may potentially enable the selection of patients for bladder-sparing treatment or facilitate personalized adjuvant immunotherapy. OBJECTIVES/GOALS: Standard-of-care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy. The inability to assess minimal residual disease (MRD) non-invasively limits our ability to offer bladder-sparing treatment. We sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis. METHODS/STUDY POPULATION: We applied uCAPP-Seq, a targeted sequencing method for detecting utDNA, to urine cell-free DNA samples acquired on the day of radical cystectomy from 42 patients with bladder cancer. utDNA variant-calling was performed non-invasively without prior tumor mutational knowledge. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. Tumor mutational burden (TMB) was inferred from the number of non-silent mutations detected in urine cell-free DNA by applying a linear relationship derived from TCGA whole exome sequencing of 409 MIBC tumors. RESULTS/ANTICIPATED RESULTS: utDNA levels were significantly higher in patients with residual disease detected in their surgical pathology compared to those who achieved a pathologic complete response (P = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD significantly predicted the absence of pathologic complete response with a sensitivity of 81% and specificity of 81%. Positive utDNA MRD also portended significantly worse progression-free survival (HR = 7.4; P = 0.03) compared to negative utDNA MRD. Furthermore, we applied a linear relationship (Pearson r = 0.84; P < 0.0001) to identify patients with high inferred TMB who may have been candidates for early immune checkpoint blockade. DISCUSSION/SIGNIFICANCE OF FINDINGS: utDNA MRD analysis prior to surgery correlated significantly with pathologic response and progression-free survival, which may help select patients for bladder-sparing treatment. utDNA can also non-invasively infer TMB, which could facilitate personalized adjuvant therapy for patients in the future.

OBJECTIVES/GOALS: Congenital cytomegalovirus (cCMV) remains to be the leading infectious cause of fetal anomalies. The role of fetal natural killer (NK) cells during cCMV remains largely unknown. The objective of this study is to define the transcriptomes of fetal NK cells exposed to human cytomegalovirus (HCMV) infection during gestation. METHODS/STUDY POPULATION: Four sets of umbilical cord blood and matching umbilical cord tissues were collected from two HCMV seropositive (HCMV+) and two HCMV seronegative (HCMV-) fetuses that did not experience any complications during gestation. These samples were provided by the Medical College of Wisconsin Tissue Bank and were processed within 24 hours following live birth. CD7+ CD3e-CD14-CD19-CD20- fetal NK cells were isolated, using the BD FACSAria sorter. Following cell sorting, single-cell RNA sequencing (scRNA-seq) was performed, and cDNA libraries were constructed and sequenced via NextSeq 550. Cell Ranger was then used to algin the cDNA reads and the Seurat R package was used to analyze the transcriptional data. Cells were filtered and clustered based on the number of uniquely expressed genes. RESULTS/ANTICIPATED RESULTS: Four sets of umbilical cord blood and matching umbilical cord tissues were collected from two HCMV+ and two HCMV- fetuses. We were able to successfully sort and capture fetal NK cells and perform scRNA-seq on these samples. Following unbiased clustering, we observed and characterized five distinct fetal NK cell subsets in the umbilical cord blood and four fetal NK cell subsets in the corresponding umbilical cord tissue. Our findings revealed that HCMV+ fetal NK cells primarily consisted of mature NK cell subsets, while HCMV- fetal NK cells constituted the majority of the immature subsets. Importantly, we identified a unique subset of NKG2CHi fetal NK cells that were significantly elevated in the HCMV+ fetuses. Finally, we defined a group of transcription factors involved in the formation of antiviral fetal NK. DISCUSSION/SIGNIFICANCE: Here, we demonstrate that HCMV infection can induce the formation of distinct NK cell subsets and drive their unique transcriptional profiles. These findings have the potential to guide the development of an innovative NK cell immunotherapy that could help prevent fetuses from developing symptomatic cCMV.

OBJECTIVES/SPECIFIC AIMS: Chlamydia trachomatis (CT) infection can lead to reproductive morbidity in women. Animal models suggest that protection against CT is mediated through the cytokine interferon-gamma (IFN-γ), produced by CD4+ T-cells, which clears CT through intracellular tryptophan depletion. In humans, correlates of protection remain to be elucidated, which hinders chlamydia vaccine development. Natural clearance of CT infection (e.g., clearance before antibiotics) may be an immunological correlate of protection, evidenced by (1) CT clearance without antibiotics; and (2) a 4-fold reduced risk of CT reinfection within 6 months. We have identified women with and without natural clearance of CT infection. By comparing these two groups of women, the role of IFN-γ-mediated natural clearance of CT infection will be investigated. METHODS/STUDY POPULATION: Through collaboration with a cohort study of CT-infected women, we have access to stored specimens from women who naturally cleared CT or had persisting CT infection. Using peripheral blood mononuclear cell (PBMC), we will assess whether natural clearance of CT infection is associated with IFN-γ-producing CD4+ T-cells by stimulating PBMC ex vivo with CT antigens using intracellular cytokine staining. We will also use cervicovaginal lavage (CVL) and untargeted High-Performance Liquid Chromatography-Mass Spectrometry to assess for tryptophan-dependent and -independent metabolic pathways associated with natural clearance of CT infection. RESULTS/ANTICIPATED RESULTS:: To date, IFN-γ has been measured in 10 women who did not clear CT infection, demonstrating that <20% of these women produced significant levels of IFN-γ. Women who naturally cleared CT have yet to be studied. Untargeted HPLC-MS has been performed on 6 women (3 who cleared matched to 3 with persisting CT infection). To date, 11 pathways that are significantly associated with natural clearance have been identified. DISCUSSION/SIGNIFICANCE OF IMPACT: The outcome of natural clearance of CT infection is distinct from women with persisting chlamydia. These studies may inform whether IFN-γ, produced by CD4+ T-cells, or tryptophan-dependent or -independent metabolic pathways are associated with natural clearance, which may advance chlamydia vaccine development.

Objectives/Goals: Neurodegenerative diseases involve progressive neuronal loss or dysfunction, often due to accumulated damage and impaired repair mechanisms. Our research evaluates the role of innate immune recognition proteins to provide insights into age-related neurodegeneration and cognitive decline. Methods/Study Population: We will utilize transcriptomic data from the Long-Life Family Study (LLFS), a cohort rich in genetic and phenotypic data related to aging and longevity. Our approach includes assessing a set of innate immune recognition proteins, also known as pattern recognition receptors (PRRs) expression across various age groups, focusing on potential correlations with cognitive performance. By analyzing serum transcriptomic profiles, we aim to map changes in expression and DNA repair genes over time, evaluating their connection to cognitive health and neurodegeneration in aging populations. Results/Anticipated Results: We anticipate that the expression of some PRRs will increase with age and correlate with cognitive decline, suggesting a role in age-related neurodegeneration. We also expect a decrease in DNA repair pathway gene expression in older age groups, contrasting with an increase in genes involved in endogenous DNA detection. These results will reveal how PRRs may function as neuroprotective factors and how their expression changes may relate to the decline in DNA repair processes with age, providing a better understanding of innate immune recognition in cognitive health. Discussion/Significance of Impact: This study will reveal the role of PRRs in aging and neurodegeneration, potentially establishing them as a key player in neuronal protection. Findings may guide future research into therapeutic strategies targeting them for Alzheimer’s and other age-related neurodegenerative diseases.