Detection of M. paratuberulosis in Crohn's Disease

项目摘要 克罗恩病（CD）是胃肠道的慢性炎症性疾病，并且存在强烈的炎症反应。 CD和分枝杆菌感染的易感基因座之间存在相当大的重叠。增加 在全球范围内观察到CD的趋势，据估计， 美方反刍动物中的一种临床和组织病理学相似的疾病，称为约翰病（JD）， 由鸟分枝杆菌副结核亚种（MAP）引起。然而，虽然长期假设， MAP和CD之间的联系尚未得到证实。这在很大程度上是因为缺乏敏感的 和特定的诊断测试，这些测试是稳健的，并且可以容易地应用于筛选足够大量的 组织或临床样品。经过多年的努力，初步研究终于导致了 特异性识别MAP的单克隆抗体（17 A12）。因此，我们在这里提出探索性研究， 为了满足开发高度特异性和灵敏度的检测方法以检测组织中的MAP的公认需求， CD患者提出了两个具体目标。在目标1中，我们将发展和建立绩效 使用免疫组织化学（IHC）测定法检测组织中MAP的特征 MAP特异性单克隆抗体，17 A12。将开发、标准化和验证IHC检测试剂盒 对来自多杆菌或少杆菌感染动物的石蜡包埋组织样品的收集， 和来自实验性接种其它分枝杆菌的小鼠（M. avium和M.肺结核）。 性能特征，如分析灵敏度、特异性、精密度（重复性和 重现性），并将确定新开发的测定法的准确度。在目标2中，我们将应用 新开发的IHC检测试剂盒作为初步筛选，用于检测方便样本中的肠组织 的CD患者和健康对照的MAP的存在，并建立初步估计的分析 以及新开发的检测方法的临床灵敏度和特异性。这些研究将为我们提供 具有长期需要的强大工具包，可以特异性检测CD患者组织中的MAP， 大规模的流行病学和机制研究，可以解决MAP与CD的关联。在 这种高度特异性和敏感性的检测方法的长期可用性可能具有重要的临床意义 通过鉴定可能受益于靶向抗- 分枝杆菌治疗，以及刺激控制策略的发展，以防止污染 牛奶和人类食物供应与MAP。

Project Summary Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract, and there is strong evidence of considerable overlap between susceptibility loci for CD and mycobacterial infections. Increasing trends of CD are observed worldwide, and it is estimated that there are 400,000-600,000 patients with CD in the US. A clinically and histopathologically similar disease in ruminants, called Johne's disease (JD), is caused by Mycobacterium avium subspecies paratuberculosis (MAP). However, although long hypothesized, the association between MAP and CD remains unproven. This is, in large part, because of a lack of sensitive and specific diagnostic tests that are robust and can be easily applied to screen a sufficiently large number of tissue or clinical samples. After many years of effort, preliminary studies have finally led to the development of a monoclonal antibody (17A12) that specifically recognizes MAP. Hence, we here propose exploratory studies to meet the well-recognized need to develop highly specific and sensitive assays to detect MAP in tissues from CD patients. Two specific aims are proposed. In Aim 1, we will develop and establish performance characteristics of an immunohistochemistry (IHC) assay for the detection of MAP in tissues using the MAP-specific monoclonal antibody, 17A12. The IHC assay will be developed, standardized and validated on a collection of paraffin embedded tissue samples from animals with pluribacillary or pauciacillary infections, and from mice experimentally inoculated with other mycobacteria (M. avium and M. tuberculosis). Performance characteristics such as analytical sensitivity, specificity, precision (repeatability and reproducibility), and accuracy of the newly developed assay will be determined. In Aim 2, we will apply the newly developed IHC assay as a preliminary screen to test intestinal tissue from a convenience sample of CD patients and healthy controls for the presence of MAP and establish initial estimates of analytical and clinical sensitivity and specificity of the newly developed assay. Together, these studies will provide us with a long-needed robust toolkit to specifically detect MAP in tissues from patients with CD, and enable future large scale epidemiologic and mechanistic investigations that can address the association of MAP with CD. In the long-term, the availability of this highly specific and sensitive assay may have important clinical implications for the treatment of CD by enabling the identification of individuals that may benefit from targeted anti- mycobacterial therapy, as well as stimulate the development of control strategies to prevent the contamination of milk and the human food supply with MAP.