NEUTROPHIL HSP MODULATION BY PERIODONTAL PATHOGENS

这个实验室的长期目标是确定热的作用 休克蛋白（HSP）参与中性粒细胞的机制 与牙周病微生物的相互作用及其随后的 宿主对这些微生物的防御。 HSP的一个作用是 建议保护中性粒细胞免受自氧化 由于作为其氧的一部分产生的氧自由基， 依赖性杀伤机制 我们的初步研究表明，当 嗜中性粒细胞吞噬牙周病原体，例如牙龈卟啉单胞菌， 特异性HSP（例如，HSP 70）不表达。 在此提出的工作 本申请将解决以下假设： 嗜中性粒细胞在吞噬 特定的牙周病原体。 缺乏热休克蛋白的表达可能是一个 结果是：1）直接抑制热休克蛋白合成， 转录或翻译水平;或2）不能诱导HSP 在吞噬过程中表达。 在其他系统中，未能表达 特异性HSP与缺乏可检测的 呼吸爆发和特定的过度/不足的生产 促炎细胞因子（IL-1、IL-6、TNF α）。 为了验证这一假设，我们提出了以下具体目标：1） 表征在吞噬过程中表达的hsp谱， 选择牙周病原体的中性粒细胞获得健康 2）检测热休克蛋白mRNA的表达， 选择的牙周病原体的吞噬作用;和，3）测量水平 促炎细胞因子的信息和产品产生的时间 吞噬作用。 正常人中性粒细胞hsp表达谱的特征 在微生物如牙龈卟啉单胞菌的吞噬作用之后， 通过对中性粒细胞进行[35 S]-甲硫氨酸标记来完成 吞噬作用后合成的蛋白质和识别蛋白质 使用单克隆抗体通过放射自显影和Western印迹分析合成 人HSP的抗体。 选择的微生物对 通过北方印迹进行特异性hsp mRNA的表达 使用特异性人HSP的cDNA探针进行分析。 的抑制剂 转录和翻译将被用来确定水平， 所述特定微生物诱导或抑制HSP表达。 将通过检测促炎细胞因子的表达来检查促炎细胞因子的表达。 选择细胞因子和细胞因子mRNA之前和之后的吞噬。

The long-term goals of this laboratory are to identify the role of heat shock proteins (hsp) in the mechanisms involved in the neutrophil's interaction with periodontal disease microorganisms and in its subsequent defense of the host against these microbes. One role of hsp has been proposed to be the protection of the neutrophil from the autooxidation due to the oxygen free radicals that are generated as part of its oxygen- dependent killing mechanism. Our preliminary studies indicate that when neutrophils phagocytize periodontal pathogens, such as P. gingivalis, specific hsp (e.g., hsp 70) are not expressed. The work proposed in this application will address the following hypothesis: Oxidation-protective hsp are not expressed by neutrophils following the phagocytosis of specific periodontal pathogens. Absence of hsp expression may be a result of either 1) direct inhibition of hsp synthesis at the transcriptional or translational levels; or 2) failure to induce hsp expression during phagocytosis. In other systems, the failure to express specific hsp has been correlated with the absence of a detectable respiratory burst and the over/under production of specific proinflammatory cytokines (IL-1, IL-6, TNFalpha). To test this hypothesis we propose the following specific aims: 1) to characterize the hsp profiles expressed during the phagocytosis of selected periodontal pathogens by neutrophils obtained from healthy donors; 2) to characterize the expression of hsp mRNA following phagocytosis of selected periodontal pathogens; and, 3) to measure levels of proinflammatory cytokine messages and products produced at the time of phagocytosis. Characterization of hsp profiles expressed by normal human neutrophils following the phagocytosis of microbes such as P. gingivalis will be accomplished by performing [35S]-methionine labeling of neutrophil proteins synthesized following phagocytosis and identifying proteins synthesized by autoradiography and Western blot analysis using monoclonal antibodies to human hsp. The effect of selected microorganisms on the expression of specific hsp mRNA will be performed by Northern blot analysis using cDNA probes for specific human hsp. Inhibitors of transcription and translation will be employed to identify the levels at which specific microorganisms induce or inhibit hsp expression. Expression of proinflammatory cytokines will be examined by detecting selected cytokines and cytokine mRNA before and after phagocytosis.