BACTEROIDES LPS/CYTOKINE REGULATION IN INFLAMED GINGIVAL TISSUE
BACTEROIDES LPS/CYTOKINE REGULATION IN INFLAMED GINGIVAL TISSUE
批准号:
6238401
负责人:
Suzanne M. Michalek
金额:
$20.69万
依托单位国家:
美国
项目类别:
财政年份:
1996
资助国家:
美国
项目状态:
已结题
起止时间:
1996-12-01 至 2000-04-30
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
中文摘要
牙周炎是一种慢性牙周组织炎症性疾病。
其中黑色素类杆菌物种,特别是
牙龈卟啉单胞菌(类杆菌)已被认为是病因
探员们。类杆菌的微生物成分,包括它们的
脂多糖(LPS)参与了最初的浸润性病变
淋巴细胞、单核/巨噬细胞和中性粒细胞
这种疾病的特点。然而,与微生物和宿主相关的
其中涉及的机制还不完全清楚。细菌内毒素具有
激活宿主细胞产生炎症因子的能力
如白介素1(IL-1)、肿瘤坏死因子-α(TNF-α)
IL-6、IL-8和IL-1抑制物。IL-8是一种趋化中性粒细胞和
T淋巴细胞及其产生是由肿瘤坏死因子-α、白介素1和内毒素诱导的。
因此,IL-8与其他细胞因子和微生物内毒素一起可能是
参与促进相关细胞事件的自我放大环路
患有炎症性疾病。很可能是细胞的调制
功能是通过基因表达的变化来调节的,这种变化可能导致
由内毒素和/或细胞产物如IL-1、TNF-1等直接刺激
阿尔法或干扰素-伽马。经典脂多糖(例如,源自大肠杆菌的脂多糖
是一种有效的细胞因子诱导剂,尤其是IL-1,但仅限于
最近我们开始了解其中的分子机制了吗?
在细胞的内毒素激活以及在诱导和调节
细胞因子的产生。类杆菌的内毒素在结构上是
与经典的内毒素不同,效力也不如经典的内毒素。因此,
这些不同的内毒素分子与细胞相互作用的方式
激活可能会有所不同。该计划的总体目标
本申请中提出的研究旨在证明类杆菌
内毒素刺激宿主细胞产生炎性细胞因子;
刺激机制不同于经典的内毒素;
而EH发育阶段和细胞来源影响着
可产生的细胞因子的概况。具体地说,我们将(1)
确定类杆菌脂多糖的能力与
经典内毒素刺激人单核/巨噬细胞和牙龈
真皮成纤维细胞产生炎性细胞因子IL-1,TNF-α,
IL-6和IL-8,以及IL-1抑制剂。剂量反应和时间进程
将进行研究以确定消息的诱导序列
和蛋白质以及在体外产生的每种因子的量
细胞与脂多糖或脂多糖和细胞因子孵育。细胞因子特异性
采用逆转录-聚合酶链式反应(RT-PCR)方法鉴定基因的表达水平。
产生的细胞因子将通过功能分析和酶联免疫吸附试验进行评估。
我们还将(2)确定在配置文件中是否存在差异
健康人牙龈成纤维细胞产生的细胞因子
肉芽肿性组织和牙周膜的体外培养
类杆菌脂多糖刺激。最后,我们将(3)确定
细胞产生IL-1、IL-1抑制物、肿瘤坏死因子-α、IL-6和IL-8
牙龈活检组织中细胞因子特异性mRNA的原位杂交检测
从牙周炎患者那里获得,并确定是否检测到
细胞因子mRNA与检测到的每种细胞因子的水平相关
从这些受试者身上采集的绒毛膜液样本。免疫组织学
技术将被用来定义组织的细胞组成
活组织检查。这些研究将通过以下方式为该机制(S)提供证据
哪种类杆菌脂多糖刺激宿主细胞并将定义
细菌内毒素、细胞因子及其抑制物在细菌感染中的作用
在发炎的牙周组织中发生的中介事件。已被占用
总之,这些研究应该有助于我们理解细胞事件
发生在发炎的组织中,这将有助于
更好的治疗和预防牙周炎的方法以及
其他炎症性疾病。
英文摘要
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.
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海外基金