Targeting induction of calcium buffer proteins for treatment of viral encephaliti

项目摘要/摘要 治疗病毒性脑炎的一个关键障碍是有效治疗患者的能力 当病毒可能感染了神经细胞时，向他们的医生展示。不幸的是，神经元 通常不是神经再生的，所以治疗方法应该侧重于预防 神经元，以及限制病毒复制的化合物，可以有效地穿透血脑 障碍。该项目将制定一项实现这一目标的战略。R21阶段的目的是 项目是确定增加的钙缓冲蛋白是否可以在培养中明确地保护神经细胞。 和动物从西尼罗河病毒(WNV)诱导的死亡，并建立特异性细胞的效用 代谢物激活神经细胞中的钙缓冲蛋白。R33阶段的目的是 根据细胞培养效果和血脑屏障通透性鉴定铅代谢产物，以建立 先导化合物在啮齿动物体内的活性，并对日本人的这种治疗方法进行了研究 脑炎病毒(JEV)具体目标是基于已发表的结果，即西尼罗河病毒在不同地区的感染 细胞系导致钙离子内流，有利于其复制，但诱导caspase3裂解和增加 细胞死亡的风险。相反，钙离子内流的抑制剂使病毒滴度降低2log10，并降低 Caspase3裂解。在WN神经系统疾病的创新仓鼠模型中，钙结合蛋白的诱导 D28k与神经元存活相关。相反，基础水平较低的Calbindin D28k与 TUNEL染色增加，细胞损伤或死亡。此外，待研究的特定代谢物 已被公认为与受体共转位到细胞核以激活钙的产生 缓冲蛋白。具体目标是：R21 SA#1。检验钙缓冲蛋白可以 保护培养中的神经细胞和动物免受西尼罗河病毒诱导的死亡。R21 SA#2.建立神经- 保护细胞培养法评价维生素D3代谢产物对钙缓冲的诱导作用 用于监测西尼罗河病毒感染的神经细胞的存活情况。R33 SA#1.确定销售线索 应用神经保护细胞培养试验和经内皮细胞培养的维生素D3代谢物的候选 通透性测定。R33 SA#2.获得临床前数据，支持使用 在不同给药途径和治疗计划的动物中处于领先地位。R33 SA#3。 测定乙脑病毒的广谱治疗活性。因为在神经病毒学方面的专业知识，抗病毒 研究、维生素D代谢和钙信号，我们在发现治疗方法方面具有独特的优势 病毒性脑炎。本项目可为WNV和JEV神经细胞的基础研究提供新的途径。 西尼罗河病毒、乙脑和病毒性脑炎的发病机制和加强临床实践 将军。

PROJECT SUMMARY/ABSTRACT A critical barrier for the treatment of viral encephalitis is the ability to effectively treat patients after they present to their physicians when the virus has likely infected neuronal cells. Unfortunately, neurons are typically not neuro-regenerative, so therapeutic modalities should focus on preventing the death of neurons, as well as to limit virus replication with compounds that can effectively penetrate the blood brain barrier. This project will develop a strategy to accomplish this. The purpose of the R21 phase of the project is to determine if increased calcium buffer proteins can definitively protect neuronal cells in culture and animals from West Nile virus (WNV)-induced death, and to establish the utility of specific cellular metabolites to activate calcium buffer proteins in neuronal cells. The purpose of the R33 phase is to identify the lead metabolite based on cell culture-efficacy and blood brain barrier permeability, to establish activity of the lead compound in rodents, and to investigate this therapeutic approach with Japanese encephalitis virus (JEV). The specific aims are based on published results that WNV infection in different cell lines leads to Ca++ influx, which benefits its replication, but induces caspase 3 cleavage and increase the risk of cell death. Conversely, inhibitors of Ca++ influx decreases viral titer by >2 log10, and decreased caspase 3 cleavage. In an innovative hamster model of WN neurological disease, induction of calbindin D28k correlates with neuronal survival. Conversely, low basal levels of calbindin D28k correlates with increased TUNEL staining, and cell injury or death. Moreover, the specific metabolites to be investigated are well recognized to co-translocate to the nucleus with the receptor to activate production of calcium buffer proteins. The Specific Aims are: R21 SA #1. Test the hypothesis that calcium buffer proteins can protect neuronal cells in culture and animals from WNV-induced death. R21 SA #2. Establish a neuro- protection cell culture assay for evaluation of vitamin D3 metabolites for induction of calcium buffer proteins, and for monitoring the survival of WNV-infected neuronal cells. R33 SA #1. Identify lead candidate of vitamin D3 metabolites using the neuro-protective cell culture assay and a transendothelial permeability assay. R33 SA #2. Obtain preclinical data supporting the treatment of WNV encephalitis with lead candidate in animals with different routes of administration and treatment schedules. R33 SA #3. Determine the broad-spectrum therapeutic activity in JEV. Because of expertise in neurovirology, antiviral research, vitamin D metabolism, and calcium signaling, we are uniquely positioned to discover therapy for viral encephalitis. This project could provide new avenues of basic research for WNV and JEV neuro- pathogenesis, and enhance clinical practice for the management of WNV, JEV, and viral encephalitis in general.