Using Host Responses to Neurocysticercosis to Develop Novel, Brain-imaging Free Diagnostics: a US-India Partnership

脑囊虫病(NCC)是一种脑部寄生虫感染，占活动性癫痫病例的34%。 在印度的韦洛尔区。迫切需要诊断NCC的新方法，因为诊断成本 大多数患者无法进行成像，34%的NCC患者被黄金标准遗漏 抗体检测。我们国家神经疾病和中风研究所(R21NS077466)和印度 科技部(BT/MB/BRCP/06/2011)资助的先导研究使用的是血液中的mRNA阵列 单核细胞定量聚合酶链式反应确认NCC患者差异表达基因 相关的癫痫发作/癫痫(称为癫痫)与有癫痫和无癫痫的对照组进行比较。这 方法确定了15个在NCC相关患者中表达水平最高的感兴趣基因 癫痫，其次是缓解的NCC患者，最后是特发性癫痫患者。表达式 不同脑部病变类型NCC患者某些基因表达水平的差异 随着病变的消退而减少。此外，对同一患者的血清进行了电喷雾分析。 电离质谱仪(ESI-MS)识别可区分NCC的质量/电荷峰 病人和对照组。值得注意的是，ESI-MS还将NCC与特发性癫痫/癫痫区分开来，两者都 ESI-MS和基因表达研究证实RAP1A在蛋白质和mRNA水平上都有过表达 在NCC患者中的水平。这些结果对于开发新的诊断方法是非常有希望的 工具。我们的中心假设是NCC相关癫痫可以通过外周生物标志物和 将以两个特定的研究目标进行测试。具体目标1将初步确立 确定了NCC的候选基因。在第一个子目标中，我们将评估暴露于 病患单核细胞和单核细胞中副芽孢子虫抗原和候选基因的表达 就像细胞系一样。第二个子目标将前瞻性地测量NCC患者单核细胞中候选基因的表达。 患者在治疗过程中随着皮损的变化，将与其表达水平进行比较 全血中的基因。特定目标2将识别导致差异表达团块的蛋白质/肽 NCC相关癫痫患者的血清峰值，并证实其对NCC的特异性。串联MS/MS 将用于识别与ESI-MS质谱峰相关的蛋白质，这些蛋白质区分与NCC相关的 癫痫等组患者。能力建设目标1将培训一名初级教员和一名 使用质谱学作为诊断工具的研究员。能力建设目标2将进行培训 所有级别的实验室人员都使用细胞和分子技术来研究寄主对 感染。成功完成这些目标将确定NCC相关癫痫发作的候选生物标记物 用于现场测试，并确定它们预测不同类型NCC相关病变的能力。当地的专业知识将 在印度开发，以进行这些研究并寻求对NCC生物学的新见解。

Neurocysticercosis (NCC) is a parasitic infection of the brain that accounts for 34% of active epilepsy cases in Vellore district, India. New methods for diagnosing NCC are badly needed because the cost of diagnostic imaging is beyond the means of most patients, and 34% of NCC patients are missed by the gold standard for antibody detection. Our National Institute of Neurological Disease and Stroke (R21NS077466) and Indian Ministry of Science and Technology (BT/MB/BRCP/06/2011)-funded pilot study used mRNA arrays of blood monocytes followed by qPCR confirmation to identify differentially expressed genes in patients with NCC- associated seizures / epilepsy (referred to as epilepsy) compared to controls with and without epilepsy. This approach identified 15 genes of interest showing highest expression levels in patients with NCC-associated epilepsy, followed by patients with resolved NCC and finally by those with idiopathic epilepsy. Expression levels of some genes differed among NCC patients with different types of brain lesions with expression decreasing as lesions resolved. In addition, sera from the same patients were analyzed by electrospray ionization mass spectrometry (ESI-MS) to identify mass/charge peaks that could discriminate between NCC patients and controls. Notably, ESI-MS also distinguished NCC from idiopathic seizures / epilepsy, and both ESI-MS and gene expression studies identified overexpression of RAP1A at the protein levels and mRNA levels, respectively, in NCC patients. These results are extremely promising for developing novel diagnostic tools. Our central hypothesis is that NCC-associated epilepsy can be diagnosed by peripheral biomarkers and will be tested with two research specific aims. Specific Aim 1 will establish the relevance of preliminarily identified candidate genes to NCC. In a first sub-aim, we will assess in-vitro relationships between exposure to T. solium metacestodes' antigens and expression of candidate genes in patient monocytes and in monocyte- like cell lines. The second sub-aim will measure candidate gene expression in monocytes prospectively in NCC patients during therapy and as their lesions change, and will be compared with expression levels of the same genes in whole blood. Specific Aim 2 will identify the proteins/peptides causing differentially expressed mass peaks in serum of patients with NCC-associated seizures and confirm their specificity to NCC. Tandem MS/MS will be used to identify proteins linked to ESI-MS mass peaks that discriminate between NCC-associated seizures and other group of patients. Capacity Building Aim 1 will train one junior faculty member and one research fellow in the use of mass spectrometry as a diagnostic tool. Capacity Building Aim 2 will train laboratory personnel at all levels in cellular and molecular techniques used to study host responses to infection. Successful completion of these aims will identify candidate biomarkers of NCC-associated seizures for field testing and determine their ability to predict different types of NCC-related lesions. Local expertise will be developed in India to conduct these studies and pursue novel insights into the biology of NCC.