Mechanism of Triclosan Disruption of Mast Cell Function

描述（由申请人提供）：三氯生（TCS）是一种抗菌剂，广泛用于医院、消费品和个人护理产品，如洗手皂，浓度约为10毫米，导致消费者广泛接触高浓度。肥大细胞是许多疾病的关键因素，包括过敏、哮喘、自身免疫、传染病、癌症、炎症性肠病，甚至许多中枢神经系统疾病，如自闭症、焦虑症和多发性硬化症。由于肥大细胞在无数生理过程和疾病中的中心地位，以及美国人口普遍暴露于这种化学物质，迫切需要关于TCS的哺乳动物毒理学和药理学的信息。Gosse实验室已经证明TCS抑制脱颗粒（在大鼠RBL-2H3细胞和人hcc -1细胞中）和肌动蛋白细胞骨架重排。两种抗原通过免疫球蛋白E （IgE）受体Fc？RI和Ca2+离子载体。该项目将研究TCS通过蛋白激酶C （PKC）和肌动蛋白细胞骨架以及磷脂酶D （PLD）和微管运输抑制肥大细胞脱颗粒和f -肌动蛋白褶皱的细胞和分子机制。假设TCS影响PKC和肌动蛋白细胞骨架的活性和细胞定位。通过ELISA检测PKC活性。最初的共聚焦显微镜实验将确定TCS对活肥大细胞中PKC、肌动蛋白和相关蛋白的一般亚细胞定位和动力学的影响。接下来，将使用荧光光激活定位显微镜（FPALM）的超分辨率成像来研究PKC：肌动蛋白和其他在传统显微镜下不可见的蛋白质结合相互作用。另一个正在研究的假设是，TCS影响PLD的活性和细胞定位以及微管运输。使用共聚焦和FPALM， TCS对微管蛋白动力学和PLD定位的影响将被研究。一种利用荧光葡聚糖作为报告者的新方法将用于成像TCS在脱粒的最后一步的影响，以直接量化的位置和时间框架

DESCRIPTION (provided by applicant): Triclosan (TCS) is an antimicrobial that is used widely in hospitals, consumer goods, and personal care products such as hand soaps at concentrations ~10 mM leading to wide exposure at high concentrations to consumers. Mast cells are critical players in numerous diseases, including allergy, asthma, autoimmunity, infectious disease, cancer, inflammatory bowel disease, and even many central nervous system disorders such as autism, anxiety, and multiple sclerosis. Due to the centrality of mast cells in myriad physiological processes and diseases and the ubiquitous exposure of the U.S. population to this chemical, there is an urgent need for information on the mammalian toxicology and pharmacology of TCS. The Gosse lab has shown that TCS inhibits both degranulation (in rat RBL-2H3 cells and human HMC-1 cells) and actin cytoskeletal rearrangement. Both of these processes are stimulated by both antigen via the Immunoglobulin E (IgE) receptor Fc?RI, and by Ca2+ ionophore. This project will investigate the cellular and molecular mechanisms by which TCS suppresses mast cell degranulation and F-actin ruffling via Protein Kinase C (PKC) and the actin cytoskeleton and also through Phospholipase D (PLD) and microtubule transport. The hypothesis is that TCS affects the activity and cellular localization of PKC and actin cytoskeleton. PKC activity will be measured through an ELISA. Initial confocal microscopy experiments will define TCS effects on general sub-cellular localization and dynamics of PKC, actin, and related proteins in live mast cells. Next, super-resolution imaging with fluorescence photoactivation localization microscopy (FPALM) will be used to investigate PKC:actin and other protein binding interactions not visible in traditional microscopy. Another hypothesis being studied is that TCS affects the activity and cellular localization of PLD and microtubule transport Using both confocal and FPALM, TCS effects on tubulin dynamics and PLD localization will be investigated. A new method that utilizes fluorescent dextran as a reporter will be used to image TCS effects on the final step of degranulation to directly quantify the locations and timeframes of degranulation. This research will fill in missing knowledge on the effects of TCS on mammalian signaling, and will allow prediction of TCS effects in disparate cell types that share common signal transduction elements. The results of this study will also fulfill an urgent need by providig insights into the impact of TCS on human health.