TEMPORAL ANALYSIS OF MRNA AND PROTEIN EXPRESSION IN CELLS OF INNATE IMMUNITY AN

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。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证明RNA和蛋白质表达的变化与脓毒症引起的ALI的进展显着相关。假设1：脓毒症诱导的ALI的进展与Th 1和Th 2淋巴细胞和血小板之间的关键相互作用显著相关。具体目标1。我们将从20名机械通气的脓毒症早期ALI儿童患者的外周血中性粒细胞、单核细胞、TH 1和TH 2以及CD 8 + T淋巴细胞和血小板中收集总RNA。将在沿着72小时内的6个单独时间点采集血样，并进行疾病严重程度评分，以确定疾病进展程度。将对3名回顾性鉴定的ALI进展的示例性患者的所有6种细胞类型的时间序列进行表达谱分析，并与3名非进展性ALI患者和3名正常对照的谱进行比较，以生成潜在细胞-细胞相互作用的图谱。假设二：对脓毒症引起的ALI的进展具有重要功能的基因和蛋白质的外周血细胞表达模式将反映肺水肿细胞和液体中的基因和蛋白质表达模式。具体目标2。使用目标1中的优先级模型，我们将选择2种显示有希望的细胞类型作为“指示”细胞类型。从目标1中的前瞻性细胞收集和该目标中更集中的收集中，我们将随机选择15例进行性和15例非进行性ALI受试者，根据本提案文本中概述的标准确定。我们将在所有30名受试者中通过QMF RT-PCR验证在目标1中发现的选择指示基因表达变化（例如，在具有最显著倍数变化或最功能相关性的20个基因中）。将通过流式细胞术和/或外周血和肺水肿液中蛋白质的ELISA在蛋白质水平上在整个队列中确认基因表达的功能性重要变化。最近发表的数据支持从ALI到AHRF（急性低氧性呼吸衰竭）的定义变化。虽然这不会以任何方式显著改变研究，但它确实更容易纳入适当的患者。ALI定义一直是入组的障碍。自这一定义变化以来，入学率有了显著提高。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 ALI 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. Recently published data supports a definitional change from ALI to AHRF (Acute Hypoxemic Respiratory Failure). Although this does not significantly alter the study in any way, it does make it easier to include appropriate patients. The ALI definition had been an impediment to enrollment. Since this definitional change, enrollment has improved significantly.