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，如浓度约为10 mM的洗手液，导致消费者在高浓度下广泛暴露。肥大细胞是许多疾病的关键参与者，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，迫切需要有关TCS的哺乳动物毒理学和药理学的信息。Gosse实验室已经表明，TCS抑制脱粒（在大鼠RBL-2 H3细胞和人HMC-1细胞中）和肌动蛋白细胞骨架重排。这两个过程都是通过免疫球蛋白E（IgE）受体Fc？RI和Ca ~（2+）离子载体。本项目将研究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）和微管转运抑制肥大细胞脱粒和F-肌动蛋白皱褶的细胞和分子机制。假设TCS影响PKC和肌动蛋白细胞骨架的活性和细胞定位。将通过ELISA测量PKC活性。初始共聚焦显微镜实验将定义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，超分辨率成像与荧光光活化定位显微镜（FPALM）将被用来研究PKC：肌动蛋白和其他蛋白质结合的相互作用，在传统的显微镜不可见。另一个正在研究的假设是，TCS影响PLD和微管运输的活性和细胞定位。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基因的新方法将用于对TCS对脱粒最后一步的影响进行成像，以直接量化脱颗粒的位置和时间范围。 脱颗粒这项研究将填补缺失的知识TCS对哺乳动物信号的影响，并将允许预测TCS的影响，在不同的细胞类型，共享共同的信号转导元件。这项研究的结果也将通过提供对TCS对人类健康影响的见解来满足迫切需要。

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.