BACTEROIDES LPS/CYTOKINE REGULATION IN INFLAMED GINGIVAL TISSUE

发炎牙龈组织中拟杆菌脂多糖/细胞因子的调节

• 批准号: 6270281
• 负责人: Suzanne M. Michalek
• 金额: $ 20.82万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6270281
• 关键词: Bacteroides; Bacteroides gingivalis; bactericidal immunity; biopsy; cytokine; enzyme linked immunosorbent assay; fibroblasts; gene expression; gingiva; human tissue; immunocytochemistry; immunopathology; in situ hybridization; inflammation; interleukin 1; interleukin 6; interleukin 8; laboratory mouse; leukocyte activation /transformation; lipopolysaccharides; macrophage; messenger RNA; monocyte; neutralizing antibody; oral bacteria; periodontitis; polymerase chain reaction; tissue /cell culture; tumor necrosis factor alpha

Periodontitis is a chronic inflammatory disease of periodontal tissues in which the black-pigmented Bacteroides species, particularly Porphyromonas (Bacteroides) gingivalis, have been implicated as etiologic agents. Microbial components of the Bacteroides, including their lipopolysaccharide (LPS), have been implicated in the initial infiltrate of lymphocytes, monocytes/macrophages and neutrophils which is characteristic of this disease. However, the microbial and host related mechanisms involved are not fully understood. Bacterial LPS has the ability to activate host cells for the production of inflammatory factors such as interleukin-1 (IL-1), tumor-necrosis factor-alpha (TNF-alpha), IL-6, IL-8 and IL-1 inhibitor. IL-8 is a chemoattractant for PMNs and T lymphocytes and its production is induced by TNF-alpha, IL-1 and LPS. Therefore, IL-8 along with other cytokines and microbial LPS may be involved in self-amplifying loops in promoting cellular events associated with inflammatory disease. It is likely that modulation of cellular function is mediated by changes in gene expression which can result from direct stimulation by LPS and/or by cellular products such as IL-1, TNF- alpha or IFN-gamma. Classical LPS (e.g., LPS derived from Escherichia coli) is a potent inducer of cytokines, especially IL-1, but only recently have we begun to understand the molecular mechanisms involved in LPS activation of cells and in the induction and regulation of cytokine production. The LPS of the Bacteroides is structurally different from and is less potent than classical LPS. Therefore, the manner in which these different LPS molecules interact with cells for activation is likely to be different. the overall objectives of the studies proposed in this application are to demonstrate that Bacteroides LPS stimulates host cells to produce inflammatory cytokines; that the mechanisms of stimulation differ from those involved with classical LPS; and that eh developmental stage and the source of cells influence the profile of cytokines which can be produced. Specifically, we will (1) determine the differences in the ability of Bacteroides LPS compared to classical LPS to stimulate human monocytes/macrophages and gingival and dermal fibroblasts to produce the inflammatory cytokines IL-1, TNF-alpha, IL-6 and IL-8, as well as IL-1 inhibitor. Dose response and time course studies will be performed to determine the induction sequence of message and protein and the amount of each factor produced following in vitro incubation of cells with LPS or LPS and cytokines. Cytokine-specific mRNA will be identified by the reverse PCR method and the levels of cytokines produced will be assessed in functional assays and by ELISA. We will also (2) determine whether differences exist in the profile of cytokines produced by gingival fibroblasts derived from healthy and granulomatous tissue and from the periodontal ligament following in vitro stimulation with Bacteroides LPS. Finally, we will (3) determine the cellular production of IL-1, IL-1 inhibitor, TNF-alpha, IL-6 and IL-8 by in situ hybridization for cytokine-specific mRNA in gingival biopsies obtained from patients with periodontitis and determine if detection of cytokine mRNA correlates with the level of each cytokine detected in crevicular fluid samples obtained from these subjects. Immunohistologic techniques will be used to define the cellular composition of tissue biopsies. These studies will provide evidence for the mechanism(s) by which Bacteroides LPS stimulates host cells and will define the contribution of this microbial LPS, cytokines and their inhibitors in mediating events occurring in inflamed periodontal tissues. Taken together, these studies should help us understand the cellular events that take place in inflamed tissue which will help in the development of better methods for treatment and prevention of periodontitis as well as other inflammatory diseases.

牙周炎是牙周组织的慢性炎症性疾病 其中黑色色素的杀菌物种，特别是 卟啉症（杀菌剂）牙龈牙龈已被认为是病因 代理商。 细菌的微生物成分，包括它们 脂多糖（LPS）已与初始浸润有关 淋巴细胞，单核细胞/巨噬细胞和中性粒细胞 这种疾病的特征。 但是，微生物和宿主与 涉及的机制尚未完全理解。 细菌LP具有 激活宿主细胞以产生炎症因子的能力 例如白介素-1（IL-1），肿瘤 - 丝不症因子-Alpha（TNF-Alpha）， IL-6，IL-8和IL-1抑制剂。 IL-8是PMN和 TNF-Alpha，IL-1和LPS诱导T淋巴细胞及其产生。 因此，IL-8以及其他细胞因子和微生物LP可能是 参与自我扩增的回路，以促进相关的细胞事件 患有炎症性疾病。 细胞的调节可能很可能 功能是由基因表达的变化介导的，这可能是由 LP和/或细胞产物（例如IL-1，TNF-）直接刺激 alpha或ifn-gamma。 古典有限元（例如，来自Escherichia的LPS 大肠杆菌是细胞因子的有效诱导剂，尤其是IL-1，但仅 最近，我们是否开始了解涉及的分子机制 在LPS激活细胞以及诱导和调节中 细胞因子产生。 细菌的LPS在结构上是 与经典唱片不同，并且不如经典唱片。 因此， 这些不同的LPS分子与细胞相互作用的方式 激活可能会有所不同。 总体目标 在本应用中提出的研究是为了证明杀菌剂 LPS刺激宿主细胞产生炎性细胞因子；那是 刺激机制与经典LPS涉及的机制不同。 EH发育阶段和细胞来源影响 可以产生的细胞因子的谱。 具体来说，我们将（1） 确定菌洛伊德斯LP的能力的差异与 经典的LP刺激人类单核细胞/巨噬细胞和牙龈和 皮肤成纤维细胞产生炎性细胞因子IL-1，TNF-alpha， IL-6和IL-8，以及IL-1抑制剂。 剂量响应和时间课程 将进行研究以确定消息的诱导顺序 和蛋白质以及在体外产生的每个因子的量 用LPS或LPS和细胞因子孵育细胞。 细胞因子特异性 mRNA将通过反向PCR方法和水平来识别 产生的细胞因子将在功能测定和ELISA中评估。 我们还将（2）确定在轮廓中是否存在差异 由健康和 体外后的肉芽肿组织和牙周韧带 用细菌型LP刺激。 最后，我们将（3）确定 IL-1，IL-1抑制剂，TNF-Alpha，IL-6和IL-8的细胞产生 牙龈活检中细胞因子特异性mRNA的原位杂交 从牙周炎患者中获得，并确定是否检测 细胞因子mRNA与检测到的每个细胞因子的水平相关 从这些受试者获得的缝隙流体样品。 免疫组织学 技术将用于定义组织的细胞组成 活检。 这些研究将通过 哪些杀菌型LP刺激宿主细胞，并将定义 该微生物LP，细胞因子及其抑制剂的贡献 介导在发炎的牙周组织中发生的事件。 拍摄 这些研究共同帮助我们了解细胞事件 那是在发炎的组织中发生的，这将有助于发展 更好的治疗和预防牙周炎以及 其他炎症性疾病。