LSUHSC COBRE:PROJ 3: ALTERNATIONS IN T CELL SIGNAL TRANSDUCTION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 慢性炎症引起的SRNS患者T细胞信号转导的改变： A.具体目标 激素抵抗型特发性肾病综合征(SRINS)是儿童终末期肾病的主要原因。有强有力的证据支持T细胞在SRINS和激素敏感型特发性肾病综合征(SSINS)的发病机制中的作用。申请者的初步研究表明，SRINS患者的T细胞IL-2表达增加，而NF-kB p65表达选择性降低。这是相关的，因为核因子-kB p65的表达减少可以减少T细胞的凋亡，增加IL-2的产生，从而激活STAT5。反过来，STAT5可以与糖皮质激素受体(GCR)结合，阻止其核转位，导致类固醇耐药。我们的初步数据支持这一假说，即SRINS患者存在损害GCR核转位并导致类固醇耐药的信号转导机制的特定改变。患者样本和体外模型将被用来完成拟议的工作。为了检验这一假设，我们提出了以下具体目标 1.确定SRINS与SSINS患者T细胞改变的频率。这一具体目标将扩大最初的观察，并确定与SRINS的联系。 2.验证核因子-kB p65缺失与SRINS T细胞GCR核移植受损有关的假设 A.在患者的T细胞中证明这一现象。 B.建立体外模型以检验分子机制。 3.验证SRINS中IL-2活性增加与STAT-5产生增加和GCR-STAT-5复合体形成增加有关的假设。 B.研究和成果 这些将根据具体目标进行讨论。 1.确定SRINS与SSINS患者T细胞改变的频率。申请人已获得IRB批准，并开始收集患者样本。另外六个中心正在获得IRB的批准。实时聚合酶链式反应的方法已经过验证，可以使用的是所建议的引物和可用于所建议的实验的有限数量的细胞。到目前为止收集的样本已保存在液氮中，以供将来分析。将使用负选择列隔离T细胞。

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. Alterations in T cell signal transduction caused by chronic inflammation in SRNS: A. Specific Aims Steroid-resistant idiopathic nephrotic syndrome (SRINS) is a primary cause for end stage renal disease in children. There is strong evidence supporting the role of T cells in the pathogenesis of SRINS and steroid sensitive idiopathic nephrotic syndrome (SSINS). The applicant preliminary studies have shown that T cells from SRINS patients have an increased expression of IL-2 and a selective decrease in NF-kB p65. This is relevant since decreased expression of NF-kB p65 can decrease T cell apoptosis an increase IL-2 production, which activates STAT5. In turn, STAT 5 can bind the glucocorticoid receptor (GCR) preventing its nuclear translocation and resulting in steroid resistance. Our preliminary data support the hypothesis that patients with SRINS have specific alterations in signal transduction mechanism that impair nuclear tranlocation of GCR and lead to steroid resistance. Patient samples and in vitro models will be used to accomplish the proposed work. To test the hypothesis, we propose the following specific aims 1. Determine the frequency of alterations in T cells from patients with SRINS vs. SSINS. This specific aim will expand initial observations and determine association with SRINS. 2. To test the hypothesis that the absence of NF-kB p65 is associated with impaired nuclear transfer of of the GCR in T cells from SRINS a. Demonstrate this phenomenom in T cells from patients. b. Develop an in vitro model to test molecular mechanism. 3. Test the hypothesis that increased IL-2 activity in SRINS is associated with increased STAT 5 production and increased formation of GCR-STAT 5 complexes. B. Studies and Results These will be discussed based on the specific aims 1. Determine the frequency of alterations in T cells from patients with SRINS vs. SSINS. The applicant has obtained IRB approval and started collecting patient samples. Six additional centers are in the process of obtaining IRB approval. Methodologies for real time PCR have been validated for the proposed primers to be used and the limited amount of cells available for the proposed exeriments. The samples collected so far have been saved on liquid nitrogen for future analysis. T cells will be isolated using negative selection columns.