Mitochondrial Kv1.3 potassium channels and lymphocyte apoptosis in sepsis

描述（由申请人提供）：脓毒症是一个主要的卫生保健问题，仅在美国每年的发病率就超过75万例，对人类痛苦和国家卫生保健资源造成巨大损失。导致这种疾病高死亡率的基本机制尚不清楚，但累积数据表明，早期T淋巴细胞凋亡和随后的低免疫阶段的发展在生物体死亡中起着关键作用。如果丢失的T淋巴细胞能得到补充，机体就能抵抗感染，提高生存率。质膜Kv1.3钾通道在T淋巴细胞活化中的关键作用已经被认识了一段时间。我们发现这种离子通道也存在于线粒体中，是T淋巴细胞凋亡的有力调节剂。我们建议通过抑制线粒体Kv1.3钾通道来创造对细胞凋亡具有相对抗性的T淋巴细胞，并探索这些基因工程T淋巴细胞的过继转移作为脓毒症的新治疗方法。工作假设是“抑制淋巴细胞线粒体Kv1.3离子通道可防止细胞凋亡，而抗凋亡工程原代T淋巴细胞的过性转移将提高脓毒症患者的生存率”。提出的三个具体目的是：目的1：研究线粒体Kv1.3在T淋巴细胞凋亡中的作用，目的2：研究线粒体Kv1.3调节细胞凋亡的机制，目的3：在小鼠盲肠结扎-穿刺脓毒症模型中，工程原代T淋巴细胞的体内过养转移和对存活的影响。这一创新的假设将使用广泛的最新技术进行验证，包括基于慢病毒的原代T淋巴细胞体外工程，细胞凋亡的体外评估，各种分子结构的细胞器特异性表达，以及在脓毒症小鼠模型中检测的全动物水平的直接生物学相关性。重点放在对线粒体Kv1.3在细胞凋亡调节中的作用的机制理解上，这一建议与纯粹的临床前现象学研究区别开来。研究团队和顾问组成了执行这一多方面项目所需的独特专业知识集合。

DESCRIPTION (provided by applicant): Sepsis is a major health care problem with incidence exceeding 750,000 cases a year in the United States alone with an enormous cost to human suffering and national healthcare resources. The basic mechanisms responsible for the high mortality rate of this disease remain unknown but cumulative data points to a key role of early T lymphocyte apoptosis and the subsequent development of a hypo-immune phase in the death of the organism. If the lost T lymphocytes can be replenished the organism may be able to fight the infection and increase the survival rate. The key role of plasma membrane Kv1.3 potassium channels in the activation of T lymphocytes has been recognized for some time. We discovered that this ion channel also present in the mitochondria is a powerful regulator of T lymphocyte apoptosis. We propose to create T lymphocytes rendered relative resistance to apoptosis through inhibition of the mitochondrial Kv1.3 potassium channels and explore adoptive transfer of these genetically engineered T lymphocytes as a novel treatment for sepsis. The working hypothesis is "inhibition of lymphocyte mitochondrial Kv1.3 ion channels prevents apoptosis and adoptive transfer of apoptosis-resistant engineered primary T lymphocytes will enhance survival in sepsis". The three specific aims of the proposal are: Aim 1: Examination of the role of mitochondrial Kv1.3 in T lymphocyte apoptosis, Aim 2: Examination of the mechanisms of mitochondrial Kv1.3 regulation of apoptosis, and Aim 3: In vivo adoptive transfer of engineered primary T lymphocytes and examination of the effect on survival in the murine cecal-ligation-puncture model of sepsis. The innovative hypothesis will be tested using a wide range state-of-art techniques including lentivirus-based ex vivo engineering of primary T lymphocytes, ex vivo and in vitro assessment of apoptosis, organelle-specific expression of various molecular constructs, and the direct biological relevance at the whole animal level examined in a mouse model of sepsis. The emphasis placed on gaining a mechanistic understanding of the role of mitochondrial Kv1.3 in the regulation of apoptosis distinguishes this proposal from a purely pre-clinical phenomenological research. The research team, along with the consultants, comprises a unique collection of expertise necessary to execute this multi-faceted project.