Browsing by Author "Jones, Harlan"
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Item A Robust Model for A Sustainable Diverse Healthcare Community - The Center for Diversity and International Programs, UNT Health Science Center(2019-03-05) Jones, Harlan; Basha, Riyaz; Vishwanatha, Jamboor; Nair, MayaAbout CDIP The Center for Diversity and International Program’s (CDIP) objectives are to broaden partnerships (local, national and international), unify institutional pipeline programs, innovate education and training, and lead diverse constituencies to opportunities in biomedical/behavioral science research and Health Professional career paths. CDIP’S programs A pipe line of programs from “K-12” to “healthcare professionals” are offered by CDIP K – 12 Outreach Stimulate and broaden student’s awareness of biomedical and health professional careers paths by exposing students to clinical and laboratory research environments at UNTHSC. Undergraduate Summer Research Internships Various undergraduate summer research programs at UNTHSC are funded through numerous sources. Participants are created with minority and non-minority-serving institution partners across USA. Graduate and Health Professional Student Training Programs Supports short term and dual degree research training for underrepresented students in health professions. Faculty Grant Writing and Professional Development Provide research and mentoring to underrepresented graduate and heath professional students, post-docs and junior faculty. CDIP’S structure The main pillars of CDIP are constituted with (1) Texas Center for Health Disparities (TCHD): A National Institute of Minority Health and Health Disparities (specialized Center of Excellence in Health Disparities (U54) was announced in 2017. (1) Research (2) Education and Training and (3) Outreach (2) National Research Mentoring Network (NRMN): Develop a culture of mentoring relationships, and more broadly the research workforce. (3) Diversity Training Programs: The plan includes a variety of programs that reach out to students from K-12, through college, and into graduate school and health care professions. (4) Texas Minority Health, Education Research and Outreach: Supports PhD scholars, junior faculty development and scholarship for senior faculty. Impact of CDIP CDIP has pioneered inter professional collaboration at institutional and national levels. 801 students and 201 faculty members from various colleges/schools at UNT Health Science Center and partnering institutions across the nation participated in various programs offered by CDIP and benefiting their educational or professional career. Such collaboration resulted in more than 200 publications and $24.76 Million in research funding. In summary, CDIP functions are in full alignment with the mission of ‘One University’.Item An updated model for maternal separation with early weaning to study early life stress on immune competency(2021) Choe, Jamie Y.; Jones, HarlanEpidemiological data suggests early life stress (ELS) has negative effects on long-term health and a role in immune-mediated diseases. A significant challenge of studying ELS in the basic sciences has been difficulty developing a reliable mouse model with robust, reproducible stress effects. Although variations of maternal separation in rodents have been published, to date no mouse variant has demonstrated predictable effects on peripheral corticosterone. We describe an updated version of the maternal separation with early weaning (MSEW) paradigm and investigate how psychosocial stress during a defined perinatal window impacts the cytokine profile of select primary and secondary lymphoid tissues. Pups were produced through in-house breeding procedures and subjected to our modified MSEW procedure. Euthanasia occurred at postnatal day (PD) 14 or 21 for blood collection via cardiac puncture. Serum corticosterone and catecholamine levels were detected using commercially available ELISA. Corticosterone was significantly increased in stressed males at PD21 (P< 0.001). Thymocytes of stressed females showed increased secretion of IL-10 (P< 0.001), while stress males exhibited elevated IL-17A (P=0.003) and IL-10 (P< 0.001). Splenocytes of stressed males produced decreased levels of IFN-gamma (P< 0.001) relative to the control. This pilot study demonstrates our updated MSEW model is capable of inducing increased peripheral corticosterone in C57BL/6 pups at PD21 and shows ELS skews cytokine production within select lymphoid tissues at PD21. Preliminary data also suggests sex-dependent effects of ELS on secretion of cytokines critical for T helper 17 and regulatory T cell phenotypes.Item Assessing Changes to the Lower GI Tract Microbiome in Response to Neglect-related Early Life Stress Exposure(2024-03-21) Choe, Jamie; Donkor, Michael; Zhang, Yan; Gorham, Isabelle; Allen, Michael; Phillips, Nicole; Jones, HarlanGrowing evidence supports exposure to early life stress (ELS) is associated with alterations in the developing immune system and increases the risk for chronic health conditions. It is widely understood that alterations to the gut microbiome can occur from exposure to various environmental factors, including diet and stress. Early life malnutrition is a form of neglect-related ELS that refers to states of both under- and over-nutrition in which a child may have insufficient intake of one or more nutrients due to an imbalanced diet. Malnutrition during childhood is a public health concern with significant health ramifications. Recent research shows the gut microbiome is intimately involved with immune system development—especially during early life when the immune system is being trained. In the present study, we use a modified version of the maternal separation with early weaning (MSEW) model to study the impact of physical neglect and malnutrition on the gut microbiome in mice. Conditions of neglect-related stress were simulated based on scheduled dam-pup separation (physical neglect) and a high carbohydrate early-wean diet (malnutrition). C57BL/6J mice were bred in-house and ELS pups were subjected to: (1) daily dam-pup separation on postnatal days (PD) 2-13 and/or (2) early weaning (EW) to a high carbohydrate diet on PD14-21. All tissues and stool samples were collected on PD21 for analysis. Pups exposed to MSEW or EW alone were assessed separately. 16S rRNA gene sequencing revealed the neglect-related ELS condition, as described under the present model, led to significant shifts in the predominate species in the lower GI tract microbial community. ELS-mediated shifts included increased Bacteroides and Enterococcus and were accompanied by decreased Lachnospiraceae. RTqPCR of bilateral adrenal glands revealed gene expression changes in key enzymes for stress response pathways, namely those implicated in the synthesis of adrenal glucocorticoids. These results demonstrate ELS-mediated dysbiosis can be observed at PD21 under the present model. Our findings at the PD21 timepoint reveal acute changes to the gut microbiome in the context of ELS and characterizes the baseline microbial community in the lower GI tract.Item Effect of CD44 Expression on T Cell Acute Lymphocytic Leukemia(2014-08-01) Racine, Ronny R.; Mummert, Mark E.; Jones, Harlan; Berg, Rance E.CD44 is a cell surface glycoprotein that serves as a multifunctional receptor aiding in trafficking and adhesion of immune cells. CD44 also serves as a recruitment platform for signaling molecules and has been shown to regulate proliferation. In several types of leukemia the presence or absence of CD44 expression is associated with different clinical outcomes, with patients who have increased expression of CD44 exhibiting a stronger response to conventional chemo- and radiotherapy. By using Jurkat T cells, which do not express CD44, to determine the effects of CD44 expression in a model Acute Lymphocytic Leukemia cell line, we have outlined two major areas of study. Firstly, upon expression of CD44, Jurkat T cells proliferate slowly compared to the control cells. This decrease in proliferation is coupled to an arrest in the cell cycle during the transition from the G1 phase into S phase. The dysregulation of the cell cycle induced by CD44 also leads to the induction of aneuploidy. CD44 expressing Jurkat T cells have reduced mRNA expression for several key regulators of chromosome separation and the mitotic spindle complex. This finding, coupled with decreased EGR-1 expression, which controls the cyclins responsible for transition from G1 into S phase, leads to an unstable cell phenotype which proliferates slowly and accumulates extra chromosomes in daughter cells. The second area of study focuses on the mechanism by which CD44 expression at the cell surface results in the observed decreases in proliferation, Akt activation, and EGR-1 expression. We observed that CD44 expressing Jurkat cells show four to five times higher calcium influx when at rest compared to the vector control cells. This influx in calcium is due to CD44 expression activating a cell surface inducible calcium release activated calcium channel. The excess calcium activates calcium-activated phosphatases and kinases, disrupting EGR-1 expression and inducing a hypophosphorylation of Akt. Together, these findings indicate that CD44 expression can regulate cell proliferation and signal transduction pathways in addition to its role in adhesion. Thus, our data provide a further understanding of how CD44 expression modifies leukemic cells into cells that are favorable for therapeutic intervention.Item Effects of Ionizing Radiation on Human Triple Negative Breast Cancer(2020) Joshi, Rohan; Chaudhary, Pankaj; Jones, Harlan; Choe, Jamie Y.Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype which exhibits high rates of metastasis. Due to lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor-2 receptor (HER2), TNBC is not responsive to hormonal treatment and currently lacks targeted therapies. Radiation is an important component of cancer therapy and used clinically in combination with chemotherapy and/or surgery for TNBC patients. Specialized extracellular vesicles called exosomes are involved in intercellular communication and postulated to play roles in tumor metastasis. In this study, we evaluate effects of single-dose ionizing radiation on the proliferation and release of exosomes from three human TNBC cell lines (MDA-MB-231, CAL-51, HCC-1806). TNBC cells were cultured and irradiated with 8.6 Gy. Exosomes were isolated 72h post-irradiation using differential ultracentrifugation and evaluated for size and purity. Nanoparticle tracking analysis (NTA) quantified exosomes released by tumor cells. Western blot confirmed isolation of exosomes by determining expression of established exosome membrane protein markers. Ionizing radiation suppressed proliferation and migration of MDA-MB-231, CAL-51, and HCC-1806 based on in vitro wound-healing (scratch) assays. Total cell counts for irradiated vs. control conditions at the time of exosome isolation were statistically significant in CAL-51 (P=0.0205) and HCC-1806 (P=0.0261). Exosomes released per cell in response to radiation showed inconclusive trends. Our preliminary data demonstrates radiation is capable of depressing TNBC proliferation and warrants further exploration regarding radiation-induced effects on tumor-derived exosomes.Item Epistatic impact of APOE and ACE2 genetic variants on SARS-CoV-2 and RAS dependent blood-brain barrier dysregulation.(2024-03-21) Tate, Amanda; Barber, Robert; Park, Inwoo; Jones, Harlan; Phillips, NicoleCoronavirus disease 2019 (COVID-19) is associated with respiratory and neurodegenerative symptoms, creating a need to understand the additional impact the pandemic might have on neurodegeneration and risk for neurodegenerative diseases, such as Alzheimer’s disease (AD) in aging populations post 2020. The blood-brain barrier (BBB) is an important interface that connects the periphery to the brain through the vasculature. When this protective barrier becomes dysregulated, the brain is vulnerable to infection, neuroinflammation, and cellular stress, which over time can lead to neurodegeneration and cognitive decline. The renin-angiotensin system (RAS) is an important regulator of vasculature via the activity of the hormone angiotensin II (Ang II). As a mediator of vasoconstrictive, oxidation, and inflammatory responses, its prolonged activity can be damaging and promote neurodegeneration at the BBB. Angiotensin-converting enzyme 2 (ACE2) is highly expressed in endothelial cells which cleaves Ang II into less harmful fragments to offset these potentially toxic effects. Genetic variants of ACE2 are increasingly considered risk factors for the development of vascular disorders (e.g. hypertension) due to their role in RAS-mediated dysregulation of body vasculature. Recent genetic studies have identified a relationship between genetic variants for ACE2, an obligate receptor for the severe acute respiratory syndrome coronavirus 2 (SCoV2), and increased risk and or/severity of COVID-19. Alzheimer’s disease (AD) is the greatest neurological risk among aging individuals and can be caused by both environmental & genetic risk factors. The strongest genetic risk factor for AD is variants in the apolipoprotein E gene (APOE), with the ɛ4 allele being associated with increased levels of amyloid β (Aβ), Tau proteins (p-Tau), neuroinflammation, and BBB permeability. A recent study by Wang et al also suggests a correlation between APOE ɛ4 and increased severity of COVID-19, suggesting some interplay between APOE and host factors related to SCoV2 infection. Furthermore, both ACE2 and APOE genetic variants disproportionately impact minority populations, highlighting a need to understand the health disparity of AD and COVID-19 risk across demographic groups. We hypothesize gene interactions (epistatic interactions) between genetic variants in ACE2 and APOE may exacerbate RAS-mediated BBB dysregulation, leading to increased AD phenotypes and SCoV2 neurological dysregulation. To address this hypothesis, we will create endothelial cells, astrocytes, and neurons containing APOE and ACE2 genetic variants of interest. These epistatic cells will be assessed for expression and functional changes related to BBB integrity. Using Ang II treatments and SCoV2 pseudo-virus models, we will assess the impact of COVID-19 and RAS on BBB integrity and functions. This study will help us understand the mechanisms and interplay of genetic risks for AD and COVID-19 related to RAS-mediated BBB dysregulation, potentially highlighting comorbidities among the aging population. By focusing on ACE2 variants disproportionately found in minority populations, we will provide knowledge surrounding two co-morbidities for neurodegeneration and elevate those at the highest risk for developing AD and/or COVID-19. Funding: This work is supported by the Neurobiology of Aging and Alzheimer’s Disease T32 Training Fellowship, and the IMSD Fellowship, Grant # 5 R25 GM125587-05 from the National Institutes of General Medical Sciences (NIGMS).Item EXOSOME PROFILING OF BRONCHIAL LAVAGE FLUID IN A MOUSE MODEL OF SURGERY RESECTION OF BREAST CANCER WITH LUNG METASTASIS(2024-03-21) Marikh, Morad; Brown, Ainsley; Hall, Courtney; Donkor, Michael; Garlotte, Isabelle; Subasinghe, Kumudu; Elkassih, Omar; Jones, Harlan; Phillips, NicoleThe lung serves as a primary site for breast cancer metastasis, carrying profound implications for patient prognoses. About 60% of people diagnosed with metastatic breast cancer have lesions in either the lungs or the bones, with triple-negative breast cancer (TNBC) more likely than other types of breast cancers to metastasize to the lungs. Although current targeted chemo-radiotherapy and surgery result in higher survivorship, studies have documented that such curative treatments may also increase risk of lung metastasis. To date, the causal factors that mediate metastasis in the context of cancer treatments remain elusive. Our long-term goal is that a deeper understanding of the mechanisms that mediate relocation of breast tumor cells from its primary origin to its distal site (e.g., lung) will reveal novel complementary diagnostic and preventative treatments to improve TNBC survivorship. Exosomes, serving as tiny extracellular vesicles within tumor cells and other cells (e.g., immune cells) release diverse biomolecules have been implicated in tumor pathogenesis. Specifically, miRNAs as cargo within exosomes are known to regulate cellular function. miRNAs are small RNA molecules that can bind to messenger RNA (mRNA) and inhibit protein synthesis or promote mRNA degradation. This regulatory function allows miRNAs to modulate the expression of multiple genes involved in various cellular processes and their dysregulation has been implicated in various diseases, including cancer. The objective of this study was to determine the expression of miRNA-200b-3p and miRNA-141-5p as known regulators of lung cancer are influenced by surgical removal of a primary breast cancer. We hypothesized that miRNA-200b-3p and miRNA-141-5p mRNA expression is increased in response to surgery. Using an established model of breast cancer metastasis, exosomes were isolated from the bronchiole alveolar lavage fluid (BALF) of tumor bearing mice and mice in which primary tumors were resected compared to tumor-free mice. Results demonstrated that miRNA-200b-3p was present in both tumor-bearing and non-tumor-bearing mice. In contrast, miRNA-141-5p was not expressed in tumor-bearing, non-tumor-bearing mice, and naïve mice determined by quantitative reverse transcriptase polymerase chain reaction (qrtPCR). In conclusion, as we navigate the intricacies of miRNA dynamics in the lung microenvironment, future studies will involve broadening the miRNA panel and refining exosome recovery techniques. This strategic evolution aims to enhance sensitivity, facilitating the detection of elusive, tumor-derived exosome miRNAs. All studies have been approved by UNTHSC IACUC, approval number #2018-0031. Acknowledgement: This research is partially supported by a grant from the Cancer Prevention and Research Institute of Texas (Award#: RP210046) to Dr. Jamboor K. Vishwanatha and National Institute of Cancer Research of the Health under Award 1 P20 CA233355-01 (Vishwanatha, Jones-Project 1).Item Health Advocates Addressing Vaccine Hesitancy in Minority Communities: A Program of the Texas Community Engagement Alliance (CEAL) of Fort Worth, Texas(2022) Nguyen, Helen; Scott, Gabrielle; Davis-Lydia, Elizabeth; Nava, Marcela; Bunnell, Bruce; Jones, HarlanRacial and ethnic minority groups have experienced worse health outcomes related to the COVID-19 pandemic, including higher rates of infection, hospitalization and death when compared to White people. Community health outreach offers a tailored approach to delivering health information, taking into account the cultural differences among ethnic minority groups. While health profession schools often incorporate cultural competency training in the curriculum there is often little time for students to work with the community in a partnership. Through support provided by the Texas Community Engagement Alliance (CEAL) Against COVID-19 Disparities program, we recruited health profession students from the UNTHSC campus as community health advocates to provide a service learning experience. Students were provided a structured course providing contextual information on health disparities and participated in building a community project to increase COVID-19 vaccine uptake. Pending IRB approval, we will evaluate student's cultural competency and efficacy in participating as student health advocates. This research was, in part, funded by the National Institutes of Health (NIH) Agreement 1OT2HL156812-01 as part of the NIH Community Engagement Alliance (CEAL)Item High expression of N-acetyl transferase 9 in cholangiocarcinoma and its possible role in tumor progression(2024-03-21) Hoteit, Tamara; Jones, Harlan; Chaudhary, PankajCholangiocarcinoma, CCA, is an aggressive type of liver cancer due to the scarce number of biomarkers and its resistance to anticancer drugs, leading to difficulty in its early detection. Its 5-year survival rate ranges from 2% to 24%, depending on severity and metastasis. Currently, surgery is the most effective treatment option, but only under certain criteria, such as the cancer being caught early and having not metastasized. CCA incidence is the highest in Asian countries because of the presence of a carcinogenic liver fluke. N-acetyl transferase, NAT, is an enzyme with many functions, including regulating protein stability, membrane targeting, gene silencing, and drug resistance. Different subsets of NATs are also known to be biomarkers for different types of cancer, such as colorectal, prostate, and breast cancers. According to The Cancer Genome Atlas database, NAT9, a subset of the NAT family, is overexpressed in patients with CCA; however, no studies have demonstrated its role in CCA, indicating the need for more research. In this study, we aim to assess the expression of NAT9 in CCA using cell lines and patient tissue samples through RT-qPCR, western blotting, and immunohistochemistry. This data will help us shed light on NAT9’s role in the tumorigenesis of CCA and could be a promising biomarker and therapeutic target for CCA.Item NASAL NANO-VACCINE PREVENTS PRIMARY BREAST TUMOR FROM MAKING THE LUNG ITS NEW HOME(2022) Donkor, Michael; Jones, HarlanDespite medical advances in the diagnosis and treatment of cancer, metastatic breast cancers remain a leading cause of death in the U.S. Increasingly, novel immune-based treatments which harness the patient's immune system have promise for improving survivorship in metastatic breast cancer patients. Such therapies take advantage of the immune system's natural defense mechanisms to halt progression of breast cancer. This is mainly through the early activation of innate immune cells such as natural killer cells and the subsequent activation of the adaptive immune responses such as T and B lymphocytes which elicits a tumor-specific cytolytic and humoral antibody response, respectively. Researchers have taken advantage of these immune mechanisms of tumor defense as a complementary approach to current radio-chemo treatments, which have shown to be limited by adverse off-target effects on patients. This is particularly problematic for recurrent highly metastatic lung, brain, and bone disease, where the physiological function is a premium. Ongoing research in our laboratory is focused on using nanotechnology to develop immune-based vaccines to target local immune protection against metastatic lung disease. Because the lung is naturally tolerogenic, making it easy for disseminated tumor cells to grow, the expectation is that boosting immune responses at the lungs before seeding tumors from primary organs would mitigate metastasis and reduce mortality risks. Using an experimental murine breast cancer model of metastasis, we sought to examine the effect of intranasal vaccination to induce local and systemic adaptive immune responses as a first step in conceptualizing an immune-based nano-vaccine. We hypothesized that an intranasal vaccine protocol would induce protective lung mucosal immune protection against secondary lung metastasis. Our results demonstrated that intranasal vaccination provides protection against secondary lung metastasis using murine model of experimental lung metastasis. This protection was due to increased accumulation of both CD4+ and CD8+ T cells in the lungs that produced IFN-gamma as shown by flow cytometry and ELISA techniques. Again, our results show that intranasal vaccination produces higher tumor-specific IgG responses across respiratory tissues. These results provide initial findings suggesting the potential for targeted tumor vaccines to produce a local tumor-specific T-cell and antibody response with the potential to prevent tumor metastasis. Future challenge studies using spontaneous model of lung metastasis will test our working hypothesis that intranasal tumor vaccination protects the lung from tumor development in the presence of a primary breast tumor.Item OCIMUM TENUIFLORUM DECREASES THE RATE OF GROWTH AND METASTATIC POTENTIAL OF MURINE 4T1 MAMMARY CARCINOMA CELLS(2021) Donkor, Michael; Jones, HarlanCombination therapies involving chemotherapy and radiation, aimed at reducing metastasis and mortality have faced challenges, including limiting the effectiveness of immunotherapy. Ocimum tenuiflorum (O. tenuiflorum) has been used traditionally in Indian culture for the treatment of disease and proven scientifically to have immunomodulatory effects. The purpose of this study was to determine the anti-tumor effect of the natural plant O. tenuiflorum on 4T1 murine mammary carcinoma cells (4T1). Hypothesis: O. tenuiflorum decreases the rate of growth and metastatic potential of 4T1 tumor cells. 4T1 cells were grown in culture medium and exposed to increasing concentrations of O. tenuiflorum. The metastatic potential was determined using the scratch assay technique. We were also interested in the mechanism by which O. tenuiflorum decreased the rate of growth and metastatic potential of 4T1 cells. We determined the changes in the mRNA expression of IL-4R, previously reported to drive breast cancer metastasis following exposure of 4T1 tumor cells O. tenuiflorum. Results: Exposing 4T1 cells to various concentrations of O. tenuiflorum decreased the rate of growth and metastatic potential of 4T1 tumor cells. Also, O. tenuiflorum downregulated the expression of IL-4R by 4T1 tumor cells with increasing concentration. We conclude that O. tenuiflorum has the potential to be used as adjunct treatment in management of breast cancer. Further studies will investigate in-depth the mechanism of O. tenuiflorum's on 4T1 including apoptosis, migration and other molecular mechanism on tumor evasiveness.Item OCIMUM TENUIFLORUM DECREASES THE RATE OF GROWTH AND METASTATIC POTENTIAL OF MURINE 4T1 MAMMARY CARCINOMA CELLS(2020) Moore, Cayla; Quinn, Bryon; Jones, Harlan; Donkor, MichaelCombination therapies involving chemotherapy and radiation, aimed at reducing metastasis and mortality have faced challenges, including limiting the effectiveness of immunotherapy. Ocimum tenuiflorum (O. tenuiflorum) has been used traditionally in Indian culture for the treatment of disease and proven scientifically to have immunomodulatory effects. The purpose of this study was to determine the anti-tumor effect of the natural plant O. tenuiflorum on 4T1 murine mammary carcinoma cells (4T1). Hypothesis: O. tenuiflorum decreases the rate of growth and metastatic potential of 4T1 tumor cells. Method: 4T1 cells were grown in culture medium and exposed to increasing concentrations of O. tenuiflorum. The metastatic potential was determined using the scratch assay technique. We were also interested in the mechanism by which O. tenuiflorum decreased the rate of growth and metastatic potential of 4T1 cells. Therefore, we determined the changes in the mRNA expression of IL-4R, previously reported to drive breast cancer metastasis following exposure of 4T1 tumor cells to O. tenuiflorum. Results: Exposing 4T1 cells to various concentrations of O. tenuiflorum decreased the rate of growth and metastatic potential of 4T1 tumor cells. Also, O. tenuiflorum downregulated the expression of IL-4R by 4T1 tumor cells with increasing concentration. We conclude that O. tenuiflorum has the potential to be used as adjunct treatment in management of breast cancer. Further studies will investigate in-depth the mechanism of O. tenuiflorum on 4T1 including apoptosis, migration and other molecular mechanism on tumor evasiveness.Item Phosphorylated Annexin A2 at Tyrosine 23 Regulates Exosome Release and Biogenesis in Triple Negative Breast Cancer(2022) Desai, Priyanka P.; Tripathi, Amit K.; Donkor, Michael; Thyagarajan, Srikantha; Jones, Harlan; Van Treuren, Timothy; Lampe, Jana B.; Chaudhary, Panka J.; Vishwanatha, JamboorPurpose: Exosomes are highly involved in the progression of diverse diseases. Targeting exosome biogenesis and release is a potential strategy for the treatment of the disease like cancer which urges an improved understanding of the process. During the exosomes biogenesis, invagination of the plasma membranes forms early endosomes which mature into late endosomes and multivesicular bodies. Annexin A2 (AnxA2), a calcium dependent phospholipid binding protein, is one of the cargo proteins which gets uploading into the exosomes and impart aggressive phenotype in triple negative breast cancer (TNBC). The mechanism how AnxA2 uploads the exosomal cargo into the exosomes and releases exosomes in the tumor microenvironment remains to be unidentified. In this study, we have explored the potential mechanism for exosome biogenesis and release to target it in TNBC, which lacks the targeted based therapies. Methods: Plasmids expressing constitutive phosphomimetic (AnxA2-Y23E) and non-phosphomimetic AnxA2 (AnxA2-Y23F) mutant gene were transfected in MDA-MB-231 cells. Exosomes isolated from AnxA2-Y23E and AnxA2-Y23F mutant cells were analyzed for expression of the exosomal cargo proteins and RNAs by Western blot and RT-PCR. The number of exosomes released were analyzed by Nanotrack analysis (NTA). Mutant cells treated with Rapamycin, mTORC1(Mammalian Target of Rapamycin Complex 1) inhibitor, were analyzed for the cargo and exosomal secretion. Mutant cells were injected in nude mice to generate tumors. Serum exosomes were isolated and analyzed for cargo and number of exosome release by NTA. Results: In this study, we found that phosphorylated Annexin A2 at tyrosine 23 increases exosome secretion. It loads proteins like AnxA2, CD9 (Cluster of Differentiation 9), LC3B, and Tsg101(Tumor susceptibility gene 101), and AnxA2 and mTOR mRNA into the exosomes. Moreover, secretion and loading of cargo into the exosomes is regulated by increased phosphorylation of AnxA2 and reduced downstream mTORC1 activity. Conclusions: Phosphorylation of AnxA2 at tyrosine 23 regulates exosome secretion and cargo loading into the exosomes in TNBC.Item Pursuit of an Optimal Murine Model for Early Life Stress: Does Diet Matter?(2022) Choe, Jamie Y.; Jones, HarlanEarly life stress (ELS) is known to have negative effects on long-term human health. Neglect is a significant source of ELS during childhood and accounts for 80% of reported abuse and over 30% of maltreatment-related deaths in the United States. A major challenge of studying the impact of stress on immune competency has been difficulty developing a reliable mouse model with reproducible stress effects. Animal models of ELS emulate the nature of childhood neglect through scheduled separation. Although variations of maternal separation in rodents have been published, the reported results are inconsistent. This may in part be attributed to variations in animal housing conditions between research institutions, such as diet. In the present study, we describe a modified version of the maternal separation with early weaning (MSEW) paradigm using C57BL/6 mice and compare the effects of two commercially available diets (ClearH2O® DietGel® and PicoLab® 5058) on peripheral stress hormones and cytokine profiles of select primary and secondary lymphoid tissues. Pups were produced via in-house breeding procedures and subjected to our modified MSEW protocol. Euthanasia occurred at postnatal day (PD) 21 for tissue harvest and blood collection via cardiac puncture. Cytokines and serum catecholamine and corticosterone levels were detected using commercially available ELISA. This pilot study sheds light on the impact dietary variations have on immune outcomes in the context of stress. We describe an updated MSEW protocol in C57BL/6 mice and demonstrate diet is a critical component of our stress model. Preliminary data suggests diet affects cytokine production within select lymphoid tissues at PD21. This work provides insight into the need for MSEW diet standardization to improve the reliability and reproducibility of murine models designed to study ELS.Item Radiation-mediated effect on exosomal and non-exosomal-derived microRNA-21 (miR21) gene expression by Triple Negative Breast Cancer cell line MDA-MB-231(2019-03-05) Chaudhary, Pankaj; Jones, Harlan; Choe, Jamie Y.Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype which lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor-2 receptor (HER2). TNBC is therefore not responsive to hormonal treatment and currently lacks targeted therapies. Overexpression of miR21 is routinely observed with TNBC and correlates with advanced tumor stage and lymph node metastasis. MDA-MB-231 is a metastatic human TNBC cell line with high recurrence rate via metastasis to secondary sites. Specialized extracellular vesicles called exosomes are involved in intercellular communication and have been postulated to have roles in tumor metastasis. Objective: In this study, we evaluate the effect of high-dose radiation on viability of MDA-MB-231 and identify changes in miR21 expression in cells and exosomes released in response to ionizing radiation. Methods: MDA-MB-231 cells (obtained from American Type Culture Collection) were cultured and irradiated with single dose exposure to 8.6 Gy (low dose) and 17.2 Gy (high dose). At 24h post-irradiation, cells were assessed for viability, proliferation, and wound healing. Exosomes were isolated from culture medium at 48h post-irradiation using differential ultracentrifugation method and evaluated for size and purity. Western blot confirmed isolation of exosomes by determining expression of established exosome membrane protein markers CD81 and TSG101. RT-qPCR evaluated expression of miR21 in cells and exosomes. Results: High-dose (17.2 Gy) radiation suppressed MDA-MB-231 proliferation based on MTT and wound healing assays. MDA-MB-231 cells exposed to 8.6 Gy showed marked upregulation of cellular miR21 and relative downregulation of exosomal miR21; exposure to 17.2 Gy resulted in downregulation of both cellular and exosomal miR21 relative to the control. Conclusion: This pilot study demonstrated that tumor cells may display compartmental differential expression of miRNA in response to radiation and suggests that miRNA expression in cells may not be predictive of exosomal cargo.