LIPOPOLYSACCHARIDE ENDOTOXIN RESPONSE GENE IN CELLS

描述(改编自申请人的摘要)： 细菌的外膜成分，宿主细胞以这样或那样的形式存在 在过去的几十年里得到了广泛的调查。这个 革兰氏阴性菌脂多糖内毒素就是一个例子 这些生物体外膜的一种基本元素， 两亲性分子具有结合和刺激多种分子的性质 哺乳动物细胞的类型。这种互动的结果是多方面的 寄主的病理生理、药理和免疫反应。 在过去的几年里，我们的长期目标是获得更好的 了解内毒素如何影响宿主细胞。《知识》 寄主反应受基因控制是理解这一点的关键。 因此，我们一直追求通过使用该基因来分离内毒素基因的目标 C3H/HeJ小鼠品系，其细胞对内毒素具有特异性缺陷。 通过使用差异功能筛选方法，我们分离出 C3H/OuJ应答小鼠脾B细胞基因的表达 C3H/OuJ应答小鼠脾B细胞及其在脾B细胞中的表达 C3H/HeJ无应答小鼠细胞对多克隆B细胞的激活作用 以响应内毒素刺激。在这项建议中，我们的具体目标是 是这一发现的合乎逻辑的结果，如下：1)完成 验证性分析表明，我们分离到的cDNAs的表达 C3H/HEH来源的巨噬细胞系的功能重建 小鼠，2)细化图谱分析，表明该基因已定位 在小鼠的第四染色体上，3)为了定义LPSD基因的性质，我们 从C3H/HeH小鼠中分离，4)研究其基因组结构和 验证在C3H/hej cDNA中发现的两个单碱基替换，5)至 将Lpsn基因导入C3H/HeJ小鼠多种原代细胞 无反应小鼠，并测量它们在暴露于脂多糖后的反应性， 6)将Lpsn基因导入C3H/HeJ无应答小鼠 逆转录病毒载体及内毒素反应性关键参数的测定 包括B细胞有丝分裂、巨噬细胞活化和 细胞因子的合成和对内毒素血症或致死性休克的抵抗力，以及7) LPsn在革兰氏阴性杆菌耐药中的作用 感染。

DESCRIPTION (Adapted from applicant's abstract): The interaction of the outer membrane components of bacteria with host cells in one form or another has been investigated extensively over the past decades. The lipopolysaccharide endotoxin (LPS) of Gram-negative bacteria is an example of an essential element of the outer membrane of these organisms that as amphipathic molecule has the property of binding to and stimulating various types of mammalian cells. The results of this interaction are multiple pathophysiological, pharmacological and immunological responses by the host. Over the preceding years, our long term objective has been to gain a better understanding of how LPS endotoxin affects cells of the host. The knowledge that host responses are under genetic control is key to this understanding. Consequently, we have pursued the goal of isolating the LPS gene by the use of the C3H/HeJ mouse strain whose cells possess a specific defect for LPS. By employing a differential functional screening approach, we have isolated a cDNA from splenic B cells of C3H/OuJ responder mice, and its expression in splenic B cells of C3H/OuJ responder mice, and its expression in splenic B cells of C3H/HeJ non-responder mice resulted in polyclonal B cell activation in response to LPS stimulation. In this proposal, our specific aims, which are a logical consequence of this finding, are as follows: 1) to complete the confirmatory analysis that expression of the cDNA we isolated can functionally reconstitute a macrophage cell line derived from a C3H/HeH mouse, 2) to refine the mapping analysis indicating that the gene is located on mouse chromosome four, 3) to define the nature of the Lpsd gene we isolated from C3H/HeH mice, 4) to investigate its genomic organization and to verify two single-base substitutions found in C3H/HeJ cDNA, 5) to introduce the Lpsn gene into various primary cells of the C3H/HeJ non-responder mouse and measure their responsiveness after exposure to LPS, 6) to introduce the Lpsn gene into C3H/HeJ non-responder mice using a retrovirus vector and measure the key parameters of endotoxin responsiveness in these mice including B cell mitogenesis, macrophage activation and cytokine elaboration, and resistance to endotoxemia or lethal shock, and 7) to assess the role of Lpsn in resistance to Gram negative bacterial infection.