MENTORED PATIENT-ORIENTED RESEARCH CAREEER DEVELOPMENT AWARD

描述(由申请人提供)： 基于对ALI分子过程的新见解，我们假设，对先天免疫和血栓形成细胞的纵向分析将显示与脓毒症ALL进展显着相关的RNA和蛋白质表达的变化。假设1：脓毒症诱导的ALI的进展与Th1和Th2淋巴细胞和血小板之间的关键相互作用显著相关。具体目的1.收集20例机械通气早期败血症ALI患儿外周血中性粒细胞、单核细胞、TH1、TH2、CD8+T淋巴细胞和血小板的总RNA。血液样本将在超过72小时的6个个体时间点进行，并对病情严重程度进行评分，以确定疾病的进展程度。对3例ALI进展的典型患者的所有6种细胞类型的时间序列进行表达谱分析，并与3例非进展性ALI患者和3例正常对照进行比较，以生成潜在的细胞-细胞相互作用图。假设2：外周血细胞对脓毒症ALI进展具有重要作用的基因和蛋白的表达模式将反映肺水肿细胞和肺液中基因和蛋白的表达模式。具体目标2.使用目标1中的优先排序模型，我们将选择显示Promise的2个单元格类型作为“指示器”单元格类型。从目标1的预期细胞集合和本目标的更有重点的集合中，我们将根据本提案案文中概述的标准，随机选择15名进展性ALI受试者和15名非进展性ALI受试者。我们将通过QMF RT-PCR在所有30名受试者中验证在AIM 1中发现的精选指示基因的表达变化(例如，在20个具有最显著折叠变化或最大功能相关性的基因中)。通过流式细胞术和/或外周血和肺水肿液中蛋白质的酶联免疫吸附试验，将在整个队列的蛋白质水平上证实基因表达的重要功能变化。

DESCRIPTION (provided by applicant): Based on new insights into the molecular processes of ALI we hypothesize that a longitudinal analysis of cells of innate immunity and thrombosis will demonstrate changes in RNA and protein expression that are significantly associated with progression of All from sepsis. Hypothesis 1: The progression of sepsis-induced ALI is significantly associated with critical interactions between Th1 and Th2 lymphocytes and platelets. Specific Aim 1. We will collect total RNA from peripheral blood neutrophils, monocytes, TH1 and TH2 and CD8+ T lymphocytes and platelets from 20 mechanically ventilated pediatric patients with early ALI from sepsis. Blood sampling will take place at 6 individual time points over 72 hours along with severity of illness scoring to determine degree of disease progression. The temporal series of all 6 cell types banked for 3 retrospectively identified patients exemplary of ALI progression will be expression profiled and compared to profiles in 3 non-progressive ALI patients and 3 normal controls in order to generate a map of potential cell-cell interactions. Hypothesis 2: The pattern of peripheral blood cell expression of genes and proteins functionally important to the progression of ALI from sepsis will reflect the pattern of gene and protein expression in pulmonary edema cells and fluid. Specific Aim 2. Using the prioritization model from Aim 1, we will select the 2 cell types that show promise as "indicator" cell types. From the prospective cell collections in Aim 1 and more focused collections in this aim, we will choose, at random, 15 progressive and 15 non-progressive ALI subjects as determined by criteria outlined in the text of this proposal. We will verify select indicator gene expression changes found in Aim 1 by QMF RT-PCR in all 30 subjects (e.g. in the 20 genes with the most significant fold changes or most functional relevance.) Functionally important changes in gene expression will be confirmed in the entire cohort on the protein level by flow cytometry, and/or ELISA of proteins in peripheral blood and pulmonary edema fluid.