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)在中性粒细胞死亡机制中的作用 与牙周病微生物的相互作用及其后续的 宿主对这些微生物的防御。热休克蛋白的一个作用是 被认为是保护中性粒细胞免受自氧化的作用 由于氧的一部分产生的氧自由基- 依赖的杀戮机制。我们的初步研究表明，当 中性粒细胞吞噬牙周病原体，如牙龈假单胞菌， 不表达特定的热休克蛋白(例如，热休克蛋白70)。在本报告中提出的工作 应用程序将解决以下假设：抗氧化 中性粒细胞吞噬HSP后不表达HSP 特定的牙周病原体。HSP表达缺失可能是一种 1)直接抑制HSP合成的结果 转录或翻译水平；或2)未能诱导HSP 吞噬过程中的表达。在其他系统中，未能表达 特定的热休克蛋白与缺乏可检测到的 呼吸爆发与特异性粒细胞过度/不足的产生 促炎症细胞因子(IL-1、IL-6、肿瘤坏死因子α)。 为了验证这一假设，我们提出了以下具体目标：1) 在吞噬过程中表达的热休克蛋白图谱的特征 健康人中性粒细胞对牙周病原体的筛选 供体；2)热休克蛋白基因表达的特征 吞噬选定的牙周病原体；以及，3)测量水平 当时产生的促炎细胞因子消息和产品 吞噬作用。 正常人中性粒细胞表达的热休克蛋白谱特征 在吞噬牙龈假单胞菌等微生物之后， 中性粒细胞的[35S]-蛋氨酸标记 吞噬后合成的蛋白质和鉴定蛋白质 用单抗进行放射自显影和蛋白质印迹分析 人热休克蛋白抗体。筛选出的微生物对生物多样性的影响 用Northern印迹法检测特异性HSP基因的表达 人热休克蛋白特异性cDNAs探针分析抗肿瘤药物 将使用转录和翻译来确定以下级别 哪些特定的微生物诱导或抑制HSP的表达。 促炎症细胞因子的表达将通过检测 吞噬前后部分细胞因子及细胞因子mRNA的表达。

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.