Early events during infection with anthrax

炭疽感染期间的早期事件

• 批准号: 7028848
• 负责人: Alan S. Cross
• 金额: $ 18.13万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-15 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7028848
• 关键词: anthrax; antibody receptor; antigen antibody reaction; bacteria infection mechanism; biological signal transduction; bioterrorism /chemical warfare; clinical research; host organism interaction; human subject; immunologic memory; laboratory mouse; laboratory rabbit; macrophage; nuclear factor kappa beta; pathologic process; polymerase chain reaction; receptor expression; small interfering RNA; spores; toll like receptor; transfection; western blottings; wild animals

DESCRIPTION (provided by applicant): Following inhalation, anthrax spores are phagocytosed by macrophages from which they germinate into a vegetative form that replicates, disseminates and produces potent virulence factors responsible for the clinical manifestations of the disease. Relatively little is known about an initial step in the innate immune response to anthrax: recognition of spores by host Toll-like receptors (TLR), and their role in the pathogenesis of anthrax infection. We will explore the initial contact of B. anthracis (BA) spores with TLRs expressed on human and murine cells. We hypothesize that TLR and/or FcgammaR activation is required for killing of the ingested spore, but in the absence of immune antibody, the exosporium shields underlying spore structures, particularly the peptidoglycan (PG) from TLR recognition and cell activation. Specific Aim I will add germination-deficient BA spore (to avoid vegetative form contamination) to human embryonal kidney cells transfected with specific human TLR constructs. Induction of IL-8 and NFkappaB activation will be measured following addition of intact BA spores, spores stripped of exosporium, purified PG and purified exosporium. Involvement of specific TLRs will be confirmed in primary non-alveolar and alveolar human and murine macrophages, including those of TLR2-, TLR4- and MyD88 knockout mice. The adaptor protein MyD88 is required for most TLR-mediated signaling. Specific Aim II will assess the potential role of TLRs in FcRgamma-mediated killing of Sterne strain (toxin+, capsule -) BA spores. Antisera raised to spores with and without exosporium will be used to opsonize Sterne BA spores, and the effect of these antisera on BA killing and cytokine induction will be determined in human alveolar and non-alveolar macrophages as well as those from wild type and TLR2-, TLR4- and MyD88 KO mice. These latter studies will address whether killing of ingested BA spores requires cooperativity between TLRs and FcRgammas. These studies using specific human TLR constructs, inhibition of expression/function of endogenous TLR proteins in primary human macrophages with siRNA and cell permeable TLR peptide inhibitors, TLR knockout mice and a unique strain of anthrax spore will define the initial interaction of macrophages with BA spores, and may provide data enabling intellingent manipulation of the cytokine milieu in favor of the host.

描述（由申请人提供）：吸入后，炭疽孢子是由巨噬细胞吞噬的，它们从中发芽成一种营养形式，该形式复制，传播并产生了负责疾病临床表现的有效毒力因子。关于对炭疽的先天免疫反应的第一步，相对较少：宿主收费受体（TLR）对孢子的识别及其在炭疽感染的发病机理中的作用。我们将探索炭疽芽孢杆菌（BA）孢子与在人和鼠细胞上表达的TLR的初始接触。我们假设TLR和/或FCAMMAR激活是杀死摄入的孢子所必需的，但是在没有免疫抗体的情况下，孢子结构上的外孢子盾，尤其是TLR识别和细胞活化的肽聚糖（PG）。具体目的我将在用特定的人类TLR构建体转染的人类胚胎肾细胞中添加缺乏发芽的BA孢子（以避免营养形式污染）。在添加完整的BA孢子，剥离外孢子，纯化的PG和纯化的外孢子后，将测量IL-8和NFKAPPAB激活的诱导。特异性TLR的参与将在原发性非肺泡和肺泡人和鼠巨噬细胞中得到证实，包括TLR2-，TLR4-和MYD88敲除小鼠的参与。大多数TLR介导的信号传导所需的衔接蛋白MYD88。具体的目标II将评估TLR在FCRGAMMA介导的Sterne菌株（毒素+，胶囊 - ）BA孢子中的潜在作用。在有或没有外孢子的孢子上升高的抗血清将用于使Sterne Ba孢子打击，并且这些抗血清对BA杀伤和细胞因子诱导的影响将在人肺泡和非肺泡巨噬细胞中确定，以及来自野生型和TLR2-，TLR2-，TLR4-，TLR4-和MYD88 KO小鼠的抗孢子。这些后者的研究将解决杀死摄入的BA孢子是否需要TLR和FCRGAMMAS之间的合作。这些研究使用特定的人类TLR构建体，抑制内源性TLR蛋白在人类原代巨噬细胞中用siRNA和可渗透的TLR TLR肽抑制剂，TLR敲除小鼠以及炭疽孢子的独特菌株将定义巨噬细胞与BA孢子的初始相互作用，可以在智能中占据良好的智能群体的初始相互作用。