Detection of M. paratuberulosis in Crohn's Disease

描述(申请人提供)：克罗恩病(CD)是一种慢性胃肠道炎症性疾病，有强有力的证据表明CD的易感基因与分枝杆菌感染之间有相当大的重叠。CD在世界范围内呈上升趋势，据估计，美国有40万至60万名CD患者。反刍动物的一种临床和组织病理学相似的疾病，称为约翰氏病(JD)，由禽分枝杆菌亚种副结核(MAP)引起。然而，尽管长期以来一直存在假设，MAP和CD之间的联系仍未得到证实。这在很大程度上是因为缺乏敏感和具体的诊断测试，这些测试是可靠的，可以 可方便地应用于筛选足够数量的组织或临床样本。经过多年的努力，初步研究终于开发出了一种能特异性识别MAP的单抗(17A12)。因此，我们建议进行探索性研究，以满足公认的需求，即开发高度特异和敏感的分析方法来检测CD患者组织中的MAP。提出了两个具体目标。在目标1中，我们将发展和建立一种免疫组织化学(IHC)方法，用于使用MAP特异性单抗17A12检测组织中的MAP。IHC检测将在一组石蜡包埋组织样本上进行开发、标准化和验证，石蜡包埋组织样本来自患有多杆菌或少毛杆菌感染的动物，以及实验上接种了其他分枝杆菌(鸟分枝杆菌和结核分枝杆菌)的小鼠。将确定新开发的分析方法的性能特征，如分析灵敏度、特异性、精密度(重复性和再现性)和准确性。在目标2中，我们将应用新开发的IHC检测方法作为初步筛选，以检测CD患者和健康对照的方便样本的肠道组织中MAP的存在，并初步估计新开发的检测方法的分析和临床敏感性和特异性。总之，这些研究将为我们提供一个长期需要的强大工具包，以专门检测CD患者组织中的MAP，并使未来能够进行大规模的流行病学和机械性调查，以解决MAP与CD的关联。从长远来看，这种高度特异和敏感的检测方法的可用性可能对CD的治疗具有重要的临床意义，因为它能够识别可能受益的个体。 从有针对性的抗分枝杆菌治疗，以及刺激制定控制战略，以防止牛奶污染和人类食品供应与MAP。

DESCRIPTION (provided by applicant): 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.