Multiplex in situ gene expression and phenotypical profiling of inflamed tissues

 描述(申请人提供)：目前肾移植排斥反应的金标准组织诊断是基于活检的光镜评估，结合有限的免疫表型和电子显微镜分析的半定量评分系统。该系统的主要缺点包括观察者间重复性差，致病过程过于简单化，以及应用任意的组织学标准来定义疾病类别。此外，尽管移植肾中相当大比例的病理改变与细胞和/或抗体介导的抗移植物免疫活性有关，但目前还没有全面的基于组织的分析方法来评估这些过程的细胞和分子基础。新兴的分子诊断方法，如基因表达谱，有其自身的缺点，特别是缺乏细胞和结构背景，对疾病过程的机制认识有限。因此，移植肾活检的潜在诊断能力目前尚未得到充分利用。在这项建议中，我们的目标是1)开发多重免疫荧光和原位杂交(MiFish)分析方法来表征移植肾活检组织中的炎症和结构变化，以及2)对照当前的Banff评分和分子图谱的金标准来验证miFish分析方法，以更好地确定疾病类别。总体而言，这项提案中将致力于开发的miFish分析将有助于确定肾移植病理的预后标志物和治疗靶点。T还可用于重新定义Banff中一些有问题的类别的当前诊断标准，例如边界变化。虽然我们建议开发的miFish分析将针对肾脏的移植环境进行优化，但同样的分析也应该适用于(经过一些修改)在其他环境中评估炎症，例如多个器官的各种炎症状况，包括自身免疫性疾病，以及肿瘤病变。

 DESCRIPTION (provided by applicant): Current gold standard tissue diagnosis for renal allograft rejection is based on light microscopic evaluation of biopsies aided by a semi-quantitative scoring system combined with limited immunophenotypical and electron microscopic analysis. Major weaknesses of this system include poor inter-observer reproducibility, over- simplification of the pathogenetic processes, and applying arbitrary histologic criteria to define disease categories. Furthermore, in spite of the fact that a significnt proportion of the pathologic changes in the transplanted kidneys are linked to cellular and/or antibody-mediated anti-graft immunologic activity, no comprehensive tissue based assays are available to assess the cellular and molecular underpinnings of these processes. The emerging molecular diagnostic approaches such as gene expression profiling have their own shortcomings particularly in their lack of cellular and structural context and limited mechanistic insights of the disease process can be gleaned. Therefore, the potential diagnostic power of allograft kidney biopsies is currently under-utilized. In this proposal, we aim to 1) develop multiplex immunofluorescence and in situ hybridization (miFish) assays to characterize inflammation and structural alterations in the kidney allograft biopsies and 2) and validate the miFish assays against the current gold standard of Banff scoring and molecular profiling to better define disease categories. Overall, the miFish assays will aim to develop in this proposal will help to identify prognostic markers and therapeutic targets for kidney allograft pathologies. t may also be used to re-define the current diagnostic criteria for some of the problematic categories in Banff, such as borderline changes. Although the miFish assays we propose to develop will be optimized to a transplant environment in the kidneys, the very same assays should also be applicable (with some modifications) to assess inflammation in other settings, such as various inflammatory conditions in multiple organs, including autoimmune diseases, and also in neoplastic lesions.