POLYCLONAL B CELL ACTIVATION BY F NUCLEATUM CELL WALLS

核梭杆菌(FN)细胞壁是一种强有力的刺激物 人多克隆B淋巴细胞活化(PBA)。的确有 越来越多的证据表明，FN和其他革兰氏剂诱导的PBA 阴性斑块细菌使慢性炎症持续存在，并 导致体内异常大量的B细胞被激活 牙周炎损害。作为我们持续研究的一部分 非特异性B细胞活化在牙周病中的作用 检查FN细胞壁中分离的大分子和 表征这些成分对B细胞的激活作用。 这项提议有两个具体目标。第一个目标是 用标准生化技术分离FN细胞壁 分成以下六个组分：细胞质膜， 肽聚糖，蛋白质/脂蛋白，脂多糖，类脂A， 和多糖类。这些分数是其他革兰氏阴性的 细菌是强有力的PBA刺激物，因此很可能 FN诱导的PBA的候选者。每一部分都将被测试 在人淋巴细胞培养中诱导PBA的能力。 活性馏分将被表征，并在随后的 学习。 第二个目标是在细胞水平上进行研究 纯化的FN细胞壁诱导多克隆B细胞活化 组件。这些研究的淋巴细胞将从以下来源获得 健康献血者的外周血。使用单抗， 荧光激活细胞分选(FACS)和标准玫瑰花环 程序中，B和T淋巴细胞将被彻底分离 耗尽了污染细胞类型。初步研究将确定 B细胞的哪个亚群(S)被激活，这些细胞的百分比 外周血中的细胞。接下来，对B细胞的生长进行多种检测 并将利用差异化来描述事件的特征 这发生在B细胞刺激期间或之后。最后， 将研究静息或激活的T细胞对PBA的调节 用分级剂量的未分离T细胞培养受刺激的B细胞 细胞、T细胞亚群或T细胞来源的淋巴因子。

Fusobacterium nucleatum (FN) cells walls are potent stimulants of human polyclonal B lymphocyte activation (PBA). There is increasing evidence that PBA induced by FN and other gram negative plaque bacteria perpetuates chronic inflammation and is responsible for the unusually large number of activated B cells in periodontitis lesions. As part of our continuing study of nonspecific B cell activation in periodontal diseases, we intend to examine isolated macromolecules from FN cell walls and characterize B cell activation by these components. This proposal has two specific objectives. The first objective is to fractionate cell walls of FN by standard biochemical techniques into the following six fractions: cytoplasmic membranes, peptidoglycan, proteins/lipoproteins, lipopolysaccharide, lipid A, and polysaccharide. These fractions in other gram negative bacteria are potent PBA stimulants and, therefore, are likely candidates of FN-induced PBA. Each fraction will be tested for the capacity to induce PBA in human lymphocyte cultures. Active fractions will characterized and used in subsequent studies. The second objective is to investigate at the cellular level polyclonal B cell activation induced by the purified FN cell wall components. Lymphocytes for these studies will be obtained from peripheral blood of healthy donors. Using monoclonal antibodies, fluorescence activated cell sorting (FACS), and standard rosetting procedures, B and T lymphocytes will be separated and thorougly depleted of contaminating cell types. Initial studies will identify which subset(s) of B cells is activated and the percentage of these cells in peripheral blood. Next, multiple assays of B cell growth and differentiation will be utilized to characterize the events which occur during or following stimulation of B cells. Finally, regulation of PBA by resting or activated T cells will be studied by culturing stimulated B cells with graded doses of unseparated T cells, T cell subsets, or T cell-derived lymphokines.