Role of B. anthracis Toxins in Human Inhalation Anthrax

炭疽杆菌毒素在人类吸入炭疽中的作用

• 批准号: 7695607
• 负责人: JORDAN PATRICK METCALF
• 金额: $ 37.57万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7695607
• 关键词: Adverse effects; Agreement; Alveolar; Alveolar Macrophages; Anthrax disease; Bacillus anthracis; Bacillus anthracis spore; Bacteria; Binding; Biological Models; Bioterrorism; Breathing; Cell Line; Cell Separation; Cells; Complement Factor B; Dendritic Cells; Diagnosis; Disease; Dose; Down-Regulation; Duct (organ) structure; Edema; Epithelial Cells; Equus caballus; Event; Flow Cytometry; Follow-Up Studies; Functional disorder; Funding; General Population; Goals; Grant; Health; Human; Immune response; Immune system; Immunofluorescence Microscopy; Immunologics; Immunosuppression; Infection; Inhalation Therapy; Institutes; Lead; Learning; Life; Lung; Lung diseases; Lymph; Methods; Modeling; Modification; Molecular; Mouse Cell Line; Mus; Occupational Exposure; Organ Culture Techniques; Papio; Pathogenesis; Patients; Phase; Play; Process; Recruitment Activity; Reproduction spores; Resistance; Resources; Role; Site; Staging; Structure of lymph node of thorax; Structure of parenchyma of lung; Techniques; Testing; Time; Tissue Model; Toxin; Vaccines; Virulence Factors; Work; anthrax lethal factor; anthrax toxin; anthrax toxin receptors; base; cell type; chemokine; cytokine; killings; lymph nodes; macrophage; monocyte; mouse model; neutrophil; novel; pathogen; prevent; programs; research study; response; tissue culture

The lung is the critical site of entry for the most deadly form of anthrax infection, inhalation anthrax. Inhalation anthrax s relatively unique in that germinated vegetative bacteria of the etiologic agent of this disease, B.anthracis (Ba), do not cause disease at the site of entry. Instead Ba spores are taken up by resident lung cells and carried through lung ymphatic ducts to the thoracic lymph nodes (TLN), from which site Ba disseminates to cause the highly lethal, terminal phase of the disease. There remain many unanswered questions about this deadly disease. It is not known why vegetative Ba do not cause disease at the site of entry. It is not known what cells take the pathogen out of the lung to the TLN. Most importantly, it s not known what role Ba toxins (lethal toxin, LT; edema toxin, ET) play in this process. The overall goal of this proposal is to answer these questions. The current paradigm, based on mouse models and mouse cell lines, is that LT and ET do not play a significant role in the pathogenesis of inhalation anthrax. This may be correct for mice, but may not apply to humans. Mouse macrophage cell lines are very sensitive to the immunosuppressive effects of LT and express anthrax toxin receptors (ATR). In the last granting period we have determined that human alveolar macrophages (HAM) efficiently kill Ba vegetative bacteria, do not express ATR and are resistant to immunosuppressive effects of LT. We will test a new paradigm that holds that Ba toxins are very important in the early stages of inhalation anthrax and that the role of key lung cells is due to variable expression of the Ba toxins. We will test our new paradigm and answer the unanswered questions regarding inhalation anthrax in three Aims using a novel human lung organ culture model and a baboon inhalation model that is being developed by our colleague, Dr. Kurosawa. In the first Aim we will determine the human lung cells that internalize Ba and the state of the pathogen in these cells by exploiting our human lung organ culture model and by using flow cytometry, cell sorting, and quantitative confocal immunofluorescence microscopy. In the second Aim we will use a modification of the lung organ culture model, and tissue from Dr. Kurosawa's model to determine and confirm the cells that facilitate escape of Ba from the lung using techniques developed in the first Aim. In the final Aim, we will determine the role that Ba toxins play in inhalation anthrax, and whether downregulation of ATR in the lung organ culture model decreases internalization, survival and escape of the pathogen in human lung. This last set of experiments should provide a proof of concept to determine whether or not modulation of anthrax toxin receptors in human lung may be useful as preventative therapy for inhalation anthrax.

肺部是最致命的炭疽感染（吸入性炭疽）进入的关键部位。吸入性炭疽 其相对独特之处在于，该疾病的病原体炭疽芽孢杆菌 (Ba) 的发芽营养细菌不 在进入部位引起疾病。相反，Ba 孢子被常驻肺细胞吸收并通过肺 淋巴管通向胸淋巴结 (TLN)，Ba 从该部位传播，导致高度致命的终末感染 疾病的阶段。 关于这种致命疾病仍有许多未解答的问题。目前尚不清楚为什么植物性Ba不会引起 进入部位的疾病。目前尚不清楚是什么细胞将病原体从肺部带到 TLN。最重要的是，它 目前尚不清楚Ba毒素（致死毒素，LT；水肿毒素，ET）在此过程中发挥什么作用。本次活动的总体目标 建议就是回答这些问题。当前基于小鼠模型和小鼠细胞系的范例是 LT ET在吸入性炭疽的发病机制中不起重要作用。这对于小鼠来说可能是正确的，但也可能 不适用于人类。小鼠巨噬细胞系对 LT 和 LT 的免疫抑制作用非常敏感 表达炭疽毒素受体（ATR）。在上一个授权期间，我们确定人类肺泡 巨噬细胞 (HAM) 能有效杀死 Ba 营养菌，不表达 ATR，并且对免疫抑制具有抵抗力 LT 的影响。 我们将测试一种新的范式，该范式认为 Ba 毒素在吸入性炭疽的早期阶段非常重要 关键肺细胞的作用是由于 Ba 毒素的可变表达所致。我们将测试我们的新范例并 使用新型人体肺器官培养物回答三个目标中有关吸入性炭疽的未解答问题 模型和我们的同事黑泽博士正在开发的狒狒吸入模型。在第一个目标中，我们将 通过利用我们的人类肺细胞来确定内化 Ba 的人类肺细胞以及这些细胞中病原体的状态 肺器官培养模型并使用流式细胞术、细胞分选和定量共聚焦免疫荧光 显微镜。在第二个目标中，我们将使用肺器官培养模型的修改，以及来自 Dr. 的组织。 黑泽明的模型利用技术确定和确认促进 Ba 从肺部逃逸的细胞 在第一个目标中开发。在最终目标中，我们将确定 Ba 毒素在吸入性炭疽中所起的作用，以及 肺器官培养模型中 ATR 的下调是否会降低肺器官培养模型中 ATR 的内化、存活和逃逸 人类肺部的病原体。最后一组实验应该提供一个概念证明，以确定是否 调节人肺中的炭疽毒素受体可能有助于预防吸入性炭疽。