Targeting induction of calcium buffer proteins for treatment of viral encephaliti

项目总结/摘要 治疗病毒性脑炎的一个关键障碍是在患者感染病毒后有效治疗患者的能力。 当病毒可能感染了神经细胞时，他们会向医生报告。不幸的是， 通常不是神经再生的，所以治疗方式应该集中在预防死亡上。 神经元，以及限制病毒复制的化合物，可以有效地渗透血脑 屏障该项目将制定一项战略来实现这一目标。R21阶段的目的 一个项目是确定增加的钙缓冲蛋白是否能明确保护培养中的神经细胞 和动物从西尼罗河病毒（WNV）诱导的死亡，并建立特定的细胞的效用， 代谢物以激活神经元细胞中的钙缓冲蛋白。R33阶段的目的是 基于细胞培养功效和血脑屏障通透性鉴定铅代谢物，以建立 活性的铅化合物在啮齿动物，并探讨这种治疗方法与日本 脑炎病毒（JEV）。具体的目标是基于已发表的结果，即不同地区的西尼罗河病毒感染 细胞系导致Ca++内流，这有利于其复制，但诱导caspase 3切割和增加 细胞死亡的风险。相反，Ca++内流抑制剂使病毒滴度降低>2 log 10， 半胱天冬酶3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 D28 k与神经元存活相关。相反，低基础水平的钙结合蛋白D28 k与 增加的TUNEL染色和细胞损伤或死亡。此外，待研究的特定代谢物 被公认与受体共转运到细胞核以激活钙的产生 缓冲蛋白。具体目标是：R21 SA #1。验证钙缓冲蛋白质可以 保护培养物和动物中的神经元细胞免于WNV诱导的死亡。R21 SA #2。建立一个神经- 评价维生素D3代谢物诱导钙缓冲液的保护细胞培养测定 蛋白质，并用于监测WNV感染的神经元细胞的存活。R33 SA #1。识别电极导线 使用神经保护性细胞培养试验和跨内皮细胞 渗透性测定R33 SA #2。获得临床前数据，支持使用以下药物治疗WNV脑炎： 在动物中具有不同给药途径和治疗方案的主要候选物。R33 SA #3。 确定对JEV的广谱治疗活性。由于在神经病毒学，抗病毒 研究，维生素D代谢和钙信号，我们处于独特的地位，发现治疗 病毒性脑炎本研究为西尼罗河病毒和乙脑病毒的神经基础研究提供了新的途径。 发病机制，并加强临床实践的管理西尼罗河病毒，乙脑病毒，和病毒性脑炎， 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.