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Regulation of ITK in Lung Allergy

ITK 在肺过敏中的调节

基本信息

批准号：
7843480
负责人：
CONSTANTINE D TSOUKAS
金额：
$ 18.56万
依托单位：
SAN DIEGO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-15 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7843480
关键词：
Adaptor Signaling Protein Affect American Amines Antibodies Area Arginine Asthma Biological Cells Cessation of life Chronic Complex Disease Disease model Drug Design Drug Kinetics Economics Eosinophilia Etiology Event Experimental Models Exploratory/Developmental Grant Extrinsic asthma Human Hyperactive behavior Hypersensitivity ITK gene IgE In Vitro Infiltration Inflammatory Inflammatory Response Interleukin-13 Interleukin-5 Investigation Jurkat Cells LCP2 gene Life Lung Lung Inflammation Lymphocyte Lymphocyte Biology Mediating Modeling Mucous body substance Mus Pathogenesis Patients Peptides Phosphorylation Play Production Public Health Regulation Research Risk Role Running SH3 Domains Signal Pathway Signal Transduction Site Smooth Muscle Social Impacts Symptoms System T-Cell Receptor T-Lymphocyte Testing Th2 Cells United States airway inflammation base cytokine design drug development economic impact emt protein-tyrosine kinase eosinophil in vivo inhibitor/antagonist lipid mediator new technology novel polyproline public health relevance src-Family Kinases synthetic peptide

项目摘要

DESCRIPTION (provided by applicant): Lung allergies, in particular Bronchial Asthma, rank among the most common chronic conditions in the United States, affecting approximately 15 million Americans and causing serious illness and several thousands of deaths. The direct and indirect financial burden created by this disease runs in the billions of dollars. The symptoms underlying this disease involve IgE antibodies, infiltration of lungs by lymphocytes and eosinophils, secretion of mucous, and hyperactivity of the bronchial smooth muscle. Cytokines secreted by Th2 lymphocytes regulate the inflammatory response seen in allergic asthma. In particular, cytokines such as IIL-4, IL-5, and IL-13 are critical for the production of IgE antibodies, recruitment of eosinophils, and the subsequent release of inflammatory vasoactive amines and lipid mediators. The activation of the Inducible T cell Kinase, ITK, has been shown to regulate the production of Th2 cytokines. Interestingly, mice with disrupted ITK genes (ITK-KO mice) display significantly milder symptoms of lung inflammation, as assessed in an experimental model of bronchial asthma. The activation of ITK requires the presence of the adaptor protein SLP 76 and transphosphorylation by the Src kinase LCK. In the absence of SLP 76 ITK does not become transphosphorylated and thus remains enzymatically inactive. In vitro studies have uncovered an interaction between ITK's SH3 domain and the polyproline region (PR) of SLP 76. However, the biological significance of this interaction on ITK activation and subsequent cytokine production in live cells has not been determined. In the present investigation we will use competitive peptide inhibitors in an attempt to disrupt the association between ITK and SLP 76 and inhibit the activation of ITK in live cells. This presumably should create a condition that would mimic the situation with ITK-KO mice. To accomplish this we will assess the feasibility of a novel area of investigation for designing and testing cell-permeable synthetic peptides that represent the specific site in the PR region of SLP 76 with which ITK's SH3 domain interacts. Synthetic peptides representing this site will be rendered cationic by the addition of Arginine residues and tested for their ability to translocate into the cytoplasmic milieu. The effects of such peptides on ITK-SLP 76 association, ITK phosphorylation and enzymatic activity, as well as cytokine production (both Th1 and Th2) will be tested using both the Jurkat cell system and primary murine splenic T cells. In addition, we will inject the polyArg-SLP 76 peptides into mice and test their effects, as above, on splenic T cells ex vivo. Finally, we will test the effects of these peptides on a murine model of Bronchial Asthma. This proposal is novel and in spite of some risk, it has the potential to enhance health-related research and open avenues for the rationale design of drugs for the treatment and management of lung allergies. PUBLIC HEALTH RELEVANCE: Statement Allergic asthma is an immunologically based disease that involves inflammation of the airway. Allergic asthma is considered a serious public health problem worldwide with a significant economic and social impact. Here, we propose studies that may uncover new ways of controlling the mechanisms underlying the pathogenesis of allergic asthma.

描述（由申请人提供）：肺过敏，特别是支气管哮喘，是美国最常见的慢性疾病之一，影响约1500万美国人，造成严重疾病和数千人死亡。这种疾病造成的直接和间接经济负担高达数十亿美元。这种疾病的基本症状包括IgE抗体、淋巴细胞和嗜酸性粒细胞对肺的浸润、粘液分泌和支气管平滑肌的过度活跃。Th 2淋巴细胞分泌的细胞因子调节过敏性哮喘中的炎症反应。特别是，细胞因子如IL-4、IL-5和IL-13对于IgE抗体的产生、嗜酸性粒细胞的募集以及随后的炎性血管活性胺和脂质介质的释放至关重要。诱导性T细胞激酶ITK的活化已显示调节Th 2细胞因子的产生。有趣的是，如在支气管哮喘实验模型中评估的，具有破坏的ITK基因的小鼠（ITK-KO小鼠）显示出显著较轻的肺部炎症症状。ITK的激活需要衔接蛋白SLP 76的存在和Src激酶LCK的转磷酸化。在SLP 76不存在的情况下，ITK不会转磷酸化，因此保持酶活性。体外研究已经揭示了ITK的SH 3结构域和SLP 76的聚脯氨酸区域（PR）之间的相互作用。然而，这种相互作用对ITK活化和随后活细胞中细胞因子产生的生物学意义尚未确定。在本研究中，我们将使用竞争性肽抑制剂，试图破坏ITK和SLP 76之间的关联，并抑制活细胞中ITK的活化。据推测，这应该会创造一种模拟ITK-KO小鼠情况的条件。为了实现这一目标，我们将评估一个新的研究领域的可行性，用于设计和测试细胞可渗透的合成肽，代表与ITK的SH 3结构域相互作用的SLP 76的PR区域中的特定位点。通过添加精氨酸残基使代表该位点的合成肽呈现阳离子，并检测其易位至细胞质环境中的能力。将使用Jurkat细胞系统和原代鼠脾T细胞测试这些肽对ITK-SLP 76结合、ITK磷酸化和酶活性以及细胞因子产生（Th 1和Th 2）的影响。此外，我们将聚Arg-SLP 76肽注射到小鼠中并测试它们对离体脾T细胞的作用，如上所述。最后，我们将测试这些肽对支气管哮喘小鼠模型的作用。这一提议是新颖的，尽管存在一些风险，但它有可能加强与健康相关的研究，并为治疗和管理肺部过敏的药物的合理设计开辟途径。过敏性哮喘是一种以免疫为基础的疾病，涉及气道炎症。过敏性哮喘被认为是一个严重的全球公共卫生问题，具有重大的经济和社会影响。在这里，我们提出的研究，可能会发现新的方法来控制过敏性哮喘的发病机制。