Understanding Immune Modulation in Taenia solium neurocysticercosis by using a novel postoncosphere in vitro model

脑囊虫病（NCC），由脑囊虫引起的中枢神经系统（CNS）感染， 猪带绦虫的后绦虫幼虫，即猪肉绦虫，是大多数发展中国家的地方病， 癫痫病是世界上最常见的癫痫病之一。的 寄生虫引起慢性神经炎症，病理研究表明， 星形胶质细胞增生、纤维化、血管生成、脑血屏障通透性的改变和 炎症和抗炎细胞因子的过度表达。然而，为了达到这个目的， 了解NCC患者病理学的潜在机制， 预防这些患者神经系统并发症的临床方法。有效治疗 NCC仍然是一个挑战，因为疾病症状的严重程度被认为是由于 由退化幼虫引起的病理性炎症反应。 我们开创了T.幼虫发育，从感染阶段 (the六钩蚴），通过大的60天后六钩蚴。在这些阶段，直到 当寄生虫到达成熟幼虫或囊尾蚴时，寄生虫本身改变其蛋白质表达， 然而，我们几乎没有关于每个阶段分泌的分子的信息。 TGF-β在原生动物和蠕虫的大范围感染中起关键作用。 除了宿主TGF-β信号在宿主-寄生虫相互作用调节中的重要性外， 许多证据表明，蠕虫寄生虫可能直接影响TGF-β依赖性 通过表达TGF-β受体和配体同源物来调节TGF-β途径。根据这些研究， 我们将利用我们的体外模型，并检查排泄/分泌（E/S）产品 不同发育阶段的T.免疫调节试验 从TGF-β开始。从T.幼虫发育 （六钩蚴、生长15、30和60天的六钩蚴后和成熟包囊）， 通过质谱分析，表征每个阶段的分泌组，并比较 阶段之间的分泌分子谱。我们将审问这个新资源， 鉴定TGF-β超家族成员的同源物。此外，E/S蛋白从 将使用凝胶过滤和阴离子分离不同发育阶段的幼虫。 交换快速蛋白质液相色谱法，为后续研究提供资源。 体外试验，以便功能性地测试和鉴定新的免疫调节剂。 然后我们将测试T。solium E/S用于TGF-β样活性，在第一种情况下利用敏感的 TGF-β报告细胞系。然后，阳性E/S将对其分数谱进行测试，以缩小 鉴定候选分子以进行体外筛选，并且将对活性级分进行体外筛选。 到质谱分析。随后，将在初始CD 4 + T细胞的体外培养物中检测E/S 用IL-2和抗CD 3来评估转录因子Foxp 3的诱导， 表明这些细胞已被TGF-β同源物或模拟物诱导为TGF-β。 具有阳性活性的分子将被鉴定并重组表达，它们的活性将 使用报告基因MFB-F11生物测定和体外调节性T细胞诱导进行表征 测定。 为了鉴定T. solium幼虫有可能改变我们的 了解寄生虫对宿主的适应性，并开发可能的免疫疗法， 介导的疾病。此外，了解寄生虫所需的发育信号， 成熟可以为感染的药物治疗开辟新的途径。

Neurocysticercosis (NCC), the infection of the central nervous system (CNS) caused by the metacestode larva of Taenia solium, the pork tapeworm, is endemic in most developing countries and identified as the most common cause of acquired epilepsy worldwide. The parasites cause a chronic neuroinflammation and pathological studies reveal reactive astrogliosis, fibrosis, angiogenesis, alteration of the brain blood barrier permeability and overexpression of both inflammatory and anti-inflammatory cytokines. Yet to this end we poorly understand the mechanisms underlying the pathology in NCC patients, and have minimal clinical means to prevent neurological complications in these patients. Effective treatment for NCC remains a challenge, as the severity of disease symptoms is thought to be a result of pathologic inflammatory response induced by the degenerating larvae. We have pioneered an in vitro model of T. solium larval development, from the infectious stage (the oncosphere), through large 60-day post-oncospheres. During these stages and until the parasite reaches a mature larva or cysticerci, the parasite itself changes its protein expression profile, however we have little to no information on the molecules secreted by each stage. TGF-β plays a pivotal role in a large spectrum of infections with protozoa and helminths. Besides the importance of host TGF-β signaling in the regulation of host-parasite interactions, much evidence has shown that helminth parasites might directly influence the TGF-β dependent pathway via the expression of TGF-β receptor and ligand homologues. Based on these studies, we will take advantage of our in vitro model and examine the excretory/secretory (E/S) products of the different larval stages of development of T. solium to test for immunomodulatory functions, starting with TGF-β. E/S from the five different stages of T. solium larval development (oncosphere, postoncospheres at 15, 30 and 60 days of growth and mature cysts), to proteomic analysis by mass spectrometry, characterize each stage’s secretome and compare the spectrum of secreted molecules between the stages. We will interrogate this new resource to identify homologues of members of the TGF-β superfamily. Additionally, E/S proteins from the different development stages of the larvae will be fractionated using both gel filtration and anion exchange Fast Protein Liquid Chromatography to provide a resource for use in subsequent in vitro assays in order to functionally test for and identify new immunomodulators. We will then test T. solium E/S for TGF-β like activity utilizing in the first instance a sensitive TGF-β reporter cell line. Positive E/S will then have its fraction profile tested to narrow down the identification of candidate molecules to screen in vitro, and the active fraction(s) will be subject to mass spectrometry. Subsequently, E/S will be tested in in vitro cultures of naïve CD4+ Tcells with IL-2 and anti-CD3 in order to assess the induction of the transcription factor Foxp3, that indicates that these cells have been induced to Tregs by a TGF-β homologue or mimic. Molecules with positive activity will be identified and recombinantly expressed, their activity will be characterized using the reporter MFB-F11 bioassay and in in vitro regulatory T cell induction assays. To identify TGF-β mimic proteins in the T. solium larvae has the potential both to change our understanding of parasite adaptation to the host and to develop possible therapies for immune mediated disease. In addition, understanding developmental signals required for parasite maturation may open new avenues for pharmaceutical treatment of infection.