Immunology
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21687
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Browsing Immunology by Author "Hodge, Lisa"
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Item Culture with Dextrose Reduced Macrophage Viability in A Dose Dependent Manner: Implications for Prolotherapy(2017-03-14) Castillo, Rudy; Conway, Allison; Rabago, David; Nourani, Booby; Hodge, Lisa; Marvin, HannahPurpose: Prolotherapy, coined from proliferant therapy, is an alternative injection-based therapy that has been used in clinical practice for over 80 years to treat various chronic musculoskeletal conditions. Modern hypotheses suggest prolotherapy promotes growth of normal cells and tissues to improve ligament mechanics, and decrease pain through inflammatory mechanisms. The most common injectant contains dextrose (D-glucose), a natural form of glucose found in the body. Recent literature indicates adults with symptomatic knee pain received the most relief from intra-articular injection of dextrose, compared to saline injections, or exercise. This study aims to begin to explain the mechanism of action of dextrose in the inflammatory response. Specifically, we hypothesized that dextrose would suppress the release of inflammatory mediators from LPS-activated macrophages. Methods: To test this hypothesis, murine RAW 264.7 macrophages were cultured in vitro with phosphate-buffered saline (PBS) or dextrose solution at 2.5%, 5%, and 10% of total volume per well, with or without 500ng lipopolysaccharide (LPS). Twenty-four hours after incubation at 37°C with 5% CO2, culture supernatants were stored and assayed for nitrite (NO2-) using Griess reagent. Macrophage viability was measured using flow cytometry with the markers Annexin V and Propidium Iodide. Results: Dextrose did not significantly alter the production of NO2- in macrophages cultured without LPS. However, in LPS-activated macrophages, dextrose significantly (p2- compared to respective PBS controls. Specifically, 2.5% dextrose suppressed NO2-release by 78.31%, while 5% and 10% dextrose completely inhibited production of NO2- . Cell viability was also significantly (p Conclusions: Dextrose reduced viability and suppressed the production of NO2- by macrophages in vitro. Prolotherapy may protect against inflammation by reducing the inflammatory activities of macrophages. Future studies will examine the effect of dextrose on macrophage function in vivo using animal models.Item Thoracic Duct Lymph Suppresses the Inflammatory Response of Macrophages in vitro(2017-03-14) Hodge, Lisa; Castillo, Rudy A.Purpose: The gastrointestinal lymphatic vessels redistribute a large pool of lymph rich in immune cells, inflammatory mediators, and lipids. Recent literature suggests that during gastrointestinal injury, soluble factors released from the mesentery redistribute duct to the lung via the thoracic duct where they initiate inflammation and contribute to multiple organ dysfunction syndrome (MODS). Alternatively, normal mesenteric lymph has been shown to suppress inflammation in vivo and in vitro. Specifically, under inflammatory conditions, normal mesenteric lymph reduced the expression of cell adhesion molecules and myeloperoxidase in pulmonary tissue and reduced expression of cell adhesion molecules on pulmonary endothelial cells. However, the role of normal lymph on phagocyte function remains unknown. Importantly, macrophages have been to shown to contribute to MODS. The aim of this study was to investigate the effect of normal lymph on macrophage function. Specifically, we hypothesized that normal thoracic duct lymph (TDL) would suppress the release of inflammatory mediators by LPS-activated macrophages. Methods: To test this hypothesis, under anesthesia the thoracic ducts of eight mongrel dogs were cannulated and lymph was collected. The TDL was centrifuged to remove cells and the TDL supernatant was frozen and stored at -80oC. Murine RAW 264.7 macrophages were cultured in vitro with TDL at 0.5, 1, 2, 5 or 10% total volume per well or phosphate-buffered saline at 5 or 10% total volume per well with or without lipopolysaccharide (LPS) for 24 hours at 37°C with 5% CO2. After incubation, cell cultures were centrifuged to remove cells and the supernatants were assayed for nitric oxide (NO) and tumor necrosis factor-alpha (TNF-a) production. Macrophage viability was measured using flow cytometry with the markers Annexin V and Propidium Iodide to distinguish live cells from apoptotic cells. Results: TDL did not augment the production of NO2-, TNF-a or alter cell viability by macrophages cultured in media alone. However, when macrophages were activated with LPS, TDL suppressed the release of NO and TNF-a. Specifically, the addition of TDL at 5% total volume per well suppressed NO2- production (15±0.6 uM) and TNF-a production (5016±425 pg/mL) compared to LPS. Culture with LPS and/or TDL did not alter cell viability. Conclusions: Our data suggests that during stimulation with LPS, a biological factor in lymph suppressed the release of inflammatory mediators by macrophages. Furthermore, cell viability was unaltered, suggesting that that TDL altered macrophage function. Future studies will focus on the ability of lymph to suppress the inflammatory response in disease models.