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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 SRNS中慢性炎症引起的T细胞信号转导的改变： a.具体目标 激素抵抗性特发性肾病综合征（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和类固醇敏感性特发性肾病综合征（SSINS）发病机制中的作用。申请人的初步研究表明，来自SRINS患者的T细胞具有增加的IL-2表达和选择性降低的NF-κ B p65。这是相关的，因为NF-kB p65的表达降低可以减少T细胞凋亡，增加IL-2的产生，从而激活STAT5。反过来，STAT 5可以结合糖皮质激素受体（GCR），防止其核转位，并导致类固醇耐药性。我们的初步数据支持这一假设，SRINS患者有特定的信号转导机制的改变，损害核转位的GCR和导致类固醇抵抗。患者样本和体外模型将用于完成拟定工作。为了验证这一假设，我们提出了以下具体目标 1. 确定SRINS与SSINS患者的T细胞变化频率。这一具体目标将扩大初步观测范围，并确定与SRINS的联系。 2. 为了检验以下假设，即NF-kB p65的缺失与SRINS的T细胞中GCR的核转移受损有关 a. 在患者的T细胞中证明这种现象。 B. 建立体外模型以测试分子机制。 3. 检验SRINS中IL-2活性增加与STAT 5产生增加和GCR-STAT 5复合物形成增加相关的假设。 B。研究和结果 这些将根据具体目标进行讨论 1. 确定SRINS与SSINS患者的T细胞变化频率。申请人已获得IRB批准，并开始采集患者样本。另有6家临床试验机构正在获得IRB批准。真实的时间PCR的方法学已针对拟使用的拟定引物和可用于拟定实验的有限细胞量进行了验证。迄今收集的样本已保存在液氮中，以备将来分析。将使用阴性选择柱分离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.