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    The Effect of Lymph Isolated during Osteopathic Lymphatic Pump Treatment on the Immune Response against Acute Pneumonia
    (2019-03-05) House, Sara; Hodge, Lisa M.; Rudolph, Erika
    Purpose: Community acquired pneumonia (CAP) accounts for over 1,000,000 hospital admissions yearly. Osteopathic physicians use lymphatic pump techniques (LPT) as a tool to mobilize lymph and treat infectious disease, such as CAP. Recent research supports LPT as an adjunctive therapy in the treatment of CAP, such as streptococcal pneumonia; however, the exact mechanism by which LPT is protective in this setting is unknown. As the first line of defense, resident alveolar macrophages respond to pathogens by engulfing bacteria and secreting antimicrobials such as nitric oxide and inflammatory mediators such as TNF-α. Thus, alveolar macrophages are key regulators in the immune response to airway pathogens. The overall objective of this study is to identify the biological effect of the thoracic duct lymph (TDL) mobilized during LPT on the immune response against streptococcal pneumonia. In this study, we hypothesized that lymph mobilized during LPT would suppress the inflammatory effect of alveolar macrophages against lipoteichoic acid (LTA), a component of the cell wall of S. pneumoniae. Methods: To test our hypothesis, TDL was collected from 6 mongrel dogs during 4 minutes of baseline (baseline TDL), during 4 minutes of LPT (LPT TDL), and during 10 minutes following LPT (post-LPT TDL). The murine alveolar macrophage cell line, MH-S, was cultured in media or media plus 5% phosphate buffer saline (PBS), 5% baseline TDL, 5% LPT TDL, or 5% post-LPT TDL and co-cultured with or without LTA. After 24 hours of culture, the supernatant was collected to measure the production of nitrite and TNF-α. Alveolar macrophage viability was measured by flow cytometry using the markers annexin V and propidium iodide. Results: Alveolar macrophages did not produce nitrite or TNF-a in the absence of stimulation with LTA. During culture with LTA, the addition of baseline, LPT, or post-LPT TDL significantly (P0.05). Furthermore, there were no differences in TNF-α and IL-10 production by MH-S macrophages cultured with baseline, LPT, or post-LPT TDL with or without LTA. Conclusions: In vitro, TDL reduced some of the inflammatory activity of macrophages that are associated with the immune pathology caused by S. pneumoniae. By mobilizing lymph into circulation, LPT may mobilize protective factors to the lung to reduce inflammation, thereby protecting from pulmonary disease. A better understanding of the physiological effects of LPT will allow us to expand translational and clinical research and guide osteopathic practitioners in their clinical practice.
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    MOG-specific Treg generation is potentially affected by thymic atrophy
    (2019-03-05) Thomas, Rachel; Oh, Jiyoung; Su, DongMing; Wang, Weikan
    Purpose: Increased chronic inflammation in the elderly is partially attributed to the disruption of central immune tolerance, comprising thymocyte negative selection and regulatory T cell (Treg) generation due to age-related thymic atrophy. Mechanistically, decreased self-antigen expression by thymic epithelial cells (TECs) in the atrophied thymus is unable to induce strong T cell receptor (TCR) signaling so that thymocyte negative selection is defective and polyclonal Treg generation is relatively enhanced. However, it was reported that certain specific clonal Treg generation is defective. Given the fact that aging aggravates relapsing-remitting multiple sclerosis (mouse model experimental autoimmune encephalomyelitis, EAE) and myelin oligodendrocyte glycoprotein (MOG) specific Treg is a key to resolve EAE, we ask whether the generation of monoclonal MOG-specific Treg cells are defective during thymic atrophy. Methods: We used inducible thymic atrophy mice reconstituted with MOG-specific TCR transgenic bone marrow to investigate the alteration of generation and function of MOG-specific Treg cells. Results: We found that the mog gene expression in TECs is decreased in the atrophied thymus, implying a potential contributor to the alteration of MOG-specific T cell development. At the peak stage of EAE, the ratio of MOG-specific Treg cells to MOG-specific conventional T cells in the central nervous system of thymic atrophy mice is decreased, with a decreased trend of Foxp3 expression in these Treg cells. Although we did not find obvious clinical differences in EAE between the thymic atrophy and normal thymus groups at the small sample numbers, we will continue to investigate the clinical and pathological differences via increasing animal numbers. Conclusion: This study suggests that even though in the aged thymus the generation of polyclonal Treg cells is enhanced, certain tissue-specific Treg cell generation could be reduced, leaving holes in the Treg-TCR repertoire.
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    Chemotherapeutic drugs increase CS1 (CD319) expression on multiple myeloma cells enhancing NK cell mediated killing
    (2019-03-05) Malaer, Joseph; Mathew, Porunelloor A.; Sun, Yuanhong
    Background: Multiple myeloma (MM) is characterized by malignant plasma cells. The clinical diagnosis includes end-organ damage, such as hypercalcemia and renal insufficiency. CS1, a member of the Signaling Lymphocyte Activation Molecule (SLAM) family of receptors, is expressed on natural killer (NK) cells as an activation receptor. CS1-CS1 binding could activate natural killer cell cytotoxicity. In multiple myeloma cells, CS1 is highly expressed and therefore offers a remarkable target for antibody dependent cell-mediate cytotoxicity (ADCC) by NK cells. Elotuzumab (Empliciti), is a humanized monoclonal antibody against CS1. Clinical trials suggest Empliciti combined with chemotherapeutic drugs has increased efficacy than using Empliciti only. In this study we investigated the mechanism of enhanced killing of MM by NK cells. Hypothesis: Chemotherapeutic drugs up-regulate the CS1 expression on multiple myeloma cell surface and enhance NK cell mediated ADCC. Material and Methods: Multiple myeloma cells, NCI-H929 were treated with vehicle control, lenalidomide and anti-CS1 mAb, doxorubicin and dexamethasone, or dexamethasone and anti-CS1 mAb for 24h and 48h. NCI-H929 cells were labeled with antibody to detect CS1 expression on cell surface by flow cytometry. CS1 mRNA expression in multiple myeloma cells were investigated by RT-qPCR. NK cell mediated killing was determined by chromium release assay. Results: Compare with vertical group, CS1 expression levels are increased in all experiment groups, but only doxorubicin and dexamethasone treatment 24h group show significantly increase, same results in anti-CS1 mAb and lenalidomide treatment 48h group. After treating NCI-H929 cell with dexamethasone and anti-CS1 mAb for 24h and 48h, mRNA expression levels are significantly decreased. No statistically variation is observed among the other groups. Natural killer cell killing abilities are significantly improved in chemotherapeutic drugs combination with anti-CS1 mAb treatment groups, no significant increase in doxorubicin and dexamethasone group. Conclusion: In conclusion, our results suggest chemotherapeutic drugs could increase the CS1 expression on NCI-H929 cell. Combining clinical trial observations and our data, it appears chemotherapeutic drugs may increase the anti-mAb treatment efficacy by up-regulating the CS1 expression level in multiple myeloma cells resulting in enhanced NK cell mediated cytotoxicity.
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    Conceptualizing Stress as a Mediator of Lung Microbiota’s Influence on Respiratory Health
    (2019-03-05) Jones, Harlan P.; Ndjom, Colette; Tirloni, Adrianna
    Purpose: Stress responses have been shown to alter the microbial ecosystem along the respiratory tract affecting the elimination and migration of microbes. However, to date little is known about the mechanisms through which bacteria function as a resident flora within the respiratory system, especially when confronted to stress. Using an experimental murine model, the purpose of the current study was to assess whether bacteria could be recovered from the lower respiratory tract (e.g. lung tissue), as well as assess the quality and quantity of bacterial species given stress exposure. Methods: C57 B6 mice strain were assigned to either a control group or a stress group and were subjected to a restraint stress paradigm reported to elicit a neuroendocrine stress response. Mice weight was recorded daily as an indicator of stress sensitivity over the course of the study. Following the stress paradigm, mice lungs were harvested, homogenized, and plated onto prepared Brain Heart Infusion agar plates. Eighteen (18) hours later, bacterial numbers were quantified by colony forming unit (CFU) techniques. Gram staining methods were also performed on lung bacterial isolates to access Gram (+) and Gram (-) species. In addition, 16S DNA amplification of lung bacterial isolates was performed for future bacterial sequence analysis. Results: The weights recorded throughout the stress paradigm showed stressed mice had greater variations in weight fluctuations than non-stressed mice. Quantification of bacteria isolated from the lungs demonstrated that while bacteria were recovered from both stressed and non-stressed mice; 8 of 9 stressed mice demonstrated quantifiable colonies where only 3 of 9 non-stressed mice had bacterial counts above the limit of detection. Qualitatively differences in the proportion of Gram (+) and Gram (-) bacteria were observed in the lungs of stressed mice compared to non-stressed mice. Amplification of 16S Illumina V4 primer and gel electrophoresis confirmed the presence of bacterial DNA necessary for downstream sequence analysis to identify species-specific differences present in the lungs of stressed and non-stressed mice. Conclusions: Initial data indicates that stress could be a factor that regulate lung microbiota. Future research will investigate microbiota diversity in the lung and how changes due to stress impact asthma disease pathogenesis.
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    Surface PCNA enables pancreatic and colon cancer stem cells to inhibit NK cell effector function
    (2019-03-05) Mathew, Porunelloor A.; Malaer, Joseph
    Purpose: Cancer stem cells (CSC) are a subset of tumor cells that have a stem-cell-like phenotype and are thought to facilitate metastasis by evading peripheral NK cell effector function. NK cell function is regulated by the balance of activating and inhibitory receptors binding to ligands on the surface of target cells. Cancer cells may escape NK cell killing by expressing or secreting ligands for NK cell inhibitory receptors. NKp44, a member of the natural cytotoxicity receptor family, can function as either an activating or an inhibitory receptor depending on ligand interaction. Proliferating cell nuclear antigen (PCNA) associates with MHC class I and forms an inhibitory ligand for NKp44, resulting in the inhibition of NK cytotoxicity. We hypothesize cell surface PCNA can be used as a marker for CSC and as a potential immunotherapeutic target for pancreatic and colon cancer. Methods: Pancreatic (Panc-1) and colon (HCT 116) cancer cell lines were labeled with antibodies against PCNA, CD44, and CD133 and flow cytometry was performed to determine surface expression. CSC were identified as being CD44+CD133+. Cells were labeled and sorted via FACS; CSC transcription factors NANOG, SOX2, and Oct-4 were analyzed by qRT-PCR from sorted populations. NK receptor-ligand interactions were blocked by incubating cells with anti-PCNA, anti-NKp44, or control antibodies; interferon gamma and chromium release assays were performed. Results: In both Panc-1 and HCT 116 cells, a PCNA+CD44+CD133+ population was detected and enriched in naturally detached cells. Furthermore, cell sorting and qRT-PCR determined cells with cell surface PCNA have increased expression of CSC transcription factors compared to cells lacking surface PCNA. Blocking the PCNA-NKp44 interaction enhanced the specific lysis of target cells by NK cells and altered the release of interferon gamma. Conclusions: Cell surface PCNA is associated with co-expression of CD44 and CD133 as well as increased CSC transcription factor expression. Additionally, cell surface PCNA alters interferon gamma secretion and facilitates escape from NK cell killing. Our data suggest that blocking NKp44-PCNA interactions may provide a novel immunotherapeutic target for pancreatic and colon cancer stem cells and prevent metastasis.
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    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.
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    Neutrophils are more effective than monocytes at containment and clearance of Listeria monocytogenes
    (2019-03-05) Berg, Rance E.; Okunnu, Busola
    Neutrophils and monocytes are phagocytic cells that have previously been shown to be important for host protection during infection with the intracellular bacteria, Listeria monocytogenes. Previous studies have shown that simultaneous depletion of neutrophils and monocytes with the Gr-1 antibody leads to susceptibility to Listeria infection. However, the literature is divided on the necessity of neutrophils for host protection during infection. The purpose of these studies is to delineate differences in function between neutrophils and monocytes during intracellular bacterial infection. The mean fluorescence intensity (MFI), obtained with a flow cytometer, of the antibody against Listeria was measured as a determinant of the total bacteria phagocytosed by the cells. It was observed that neutrophils obtained from the bone marrow, liver and spleen of C57Bl/6 mice were more effective at phagocytosis of Listeria in comparison to monocytes as they had a higher total bacteria MFI than the monocytes. To determine differences in the ability of the cells to keep bacteria contained in the phagosome, the cells were infected with a strain of Listeria that only expresses GFP when the bacteria escapes out of the phagosome into the cytosol. Comparison of the MFI of total bacteria present vs escaped bacteria showed that monocytes from the bone marrow, liver and spleen of mice phagocytosed less bacteria and allowed for more bacteria to escape in comparison to neutrophils. Therefore, monocytes are less effective at bacterial containment in comparison to neutrophils. To ascertain differences in killing ability, bone marrow neutrophils and monocytes were sorted for a killing assay. Neutrophils were also observed to be more effective than monocytes at bacterial killing. In conclusion, although both cell types are important for protection during Listeria infection, neutrophils appear to be essential for protection as they are more effective at phagocytosis, phagosomal containment and bacteria killing. Future studies, including measurement of ROS/RNS and cytokine production, will aid in further defining specific functional differences between neutrophils and monocytes during intracellular bacterial infection.
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    Atrophied thymus creates tTreg repertoire holes diminishing an antigen-specific population in the periphery
    (2019-03-05) Oh, Jiyoung; Wang, Weikan; Su, Dong-Ming; Thomas, Rachel
    Purpose: We have shown that there is an increased ratio of total thymic T regulatory (tTreg) cells to thymic T conventional (tTcon) cells generated by the atrophied thymus related to aging. This observation, coupled with the accumulation of peripheral Treg (pTreg) cells with age, poses the difficult question of why Tregs in the periphery of the elderly are unable to suppress amplified self-reactivity-induced inflammaging. Methods: We utilized a chimeric mouse model with immune system reconstitution in which lethally irradiated rat insulin promotor-driven (RIP) mOVA host mice received mixed OT-II TCR transgenic and wild-type bone marrow, each expressing distinct congenic identifiers (CD45.1 vs CD45.2). In this system, OVA serves as a mock self-antigen, which is expressed mainly in the pancreas of the host mice. Further, our mOVA host mice carried a FoxN1-floxed gene, for induction of conditional FoxN1 knock-out, resulting in thymic atrophy analogous to age-related thymic atrophy. Results: We observed that chimeric mice with induced thymic atrophy exhibited significantly decreased OVA-specific (OT-II) Tregs, but not total (pan) Tregs, in the spleen and pancreas. The specific Treg cells of mice with thymic atrophy were also more instable, showing relatively decreased FoxP3 expression, and had greater plasticity, showing an increased Th1-like (IFNγ+FoxP3+) Treg phenotype. Conclusions: Overall, our preliminary results suggest that thymic atrophy creates changes in the antigen-specific repertoire during tTreg generation resulting in potential “holes” that may contribute to inflammaging in the elderly and negatively impact Treg cell-mediated regulation in the aged immune system.