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

通过使用新型子囊后体外模型了解猪带绦虫神经囊尾蚴病的免疫调节

• 批准号: 10396481
• 负责人: Manuela Renee Verastegui
• 金额: $ 13.44万
• 依托单位: UNIVERSIDAD PERUANA CAYETANO HEREDIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10396481
• 关键词: Address; Alkaline Phosphatase; Anions; Anti-Inflammatory Agents; Antigens; Binding; Biological Assay; Brain; CD3 Antigens; Cell Line; Cells; Central Nervous System Infections; Cestoda; Chronic; Clinical; Coupled; Cyst; Data Set; Developing Countries; Development; Disease; Elements; Embryo; FOXP3 gene; Fibrosis; Gel Chromatography; Genes; Genome; Growth; Helminths; Homologous Gene; Immune response; Immunomodulators; Immunotherapy; In Vitro; Infection; Inflammatory; Inflammatory Response; Interleukin-2; Knowledge; Laboratories; Larva; Ligands; Mass Spectrum Analysis; Neurocysticercosis; Neurologic; Parasites; Pathologic; Pathology; Pathway interactions; Patients; Pharmacologic Substance; Play; Proteins; Proteomics; Protozoa; Recombinants; Regulation; Regulatory T-Lymphocyte; Reporter; Reporting; Resources; Role; Severity of illness; Signal Transduction; Symptoms; Taenia solium; Testing; Therapeutic; Tissues; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Tumor stage; acquired epilepsy; angiogenesis; astrogliosis; base; blood-brain barrier permeabilization; candidate identification; cytokine; effective therapy; fast protein liquid chromatography; immunoregulation; improved; in vitro Assay; in vitro Model; member; model development; neuroinflammation; novel; overexpression; prevent; protein expression; secretory protein; transcription factor; transcriptome

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.

神经囊肿（NCC），中心神经系统（CNS）的感染是由 牛ta虫的元素幼虫，猪肉tape虫，在大多数发育中的地方是地方性的 国家被确定为全球癫痫的最常见原因 寄生虫引起慢性神经炎症和病理研究实际反应性 星形胶质病，纤维化，血管生成，脑屏障渗透性的改变和 炎症和抗炎细胞因子的过度表达。 了解NCC患者病理学的基础机制，并且最少 临床手段，以预防这些特定性的神经汇编 NCC仍然是一个挑战，因为人们认为疾病症状的严重程度是 幼虫退化引起的病理炎症反应。 我们开创了t.幼虫发育的体外模型 （肿瘤圈），通过60天的大型圈圈。 寄生虫达到成熟的幼虫或cystemerci，寄生虫的变化是蛋白质表达 个人资料，霍维尔几乎没有关于每个阶段分泌的分子的信息。 TGF-β在原生动物和蠕虫中的大量无菌中起关键作用。 除了宿主TGF-β信号在调节宿主 - 寄生虫相互作用中的重要性外， 大量证据表明，蠕虫寄生虫可能直接影响TGF-β的依赖性。 通过TGF-β受体和配体同源性的表达途径。 我们将进行我们的体外模型的讲话，并检查排泄/分泌（E/S）产品 T. solium开发的不同幼虫阶段以测试免疫调节 功能，从TGF-β开始。 （Oncosphere，15、30和60天的生长和成熟囊肿的共孔术） 通过按摩法分析，表征每个阶段的秘密并比较 我们将在阶段之间进行分泌的分子。 鉴定TGF-β超家族的成员。 幼虫的不同发育阶段将使用凝胶过滤和阴离子分馏 交换快速蛋白质液相色谱仪，以提供随后使用的资源 体外测定，以便在功能上测试和识别新的免疫调节剂。 我们将对TGF-β的测试T. selium e/s在首先使用敏感的活性 TGF-β报告基细胞系的阳性E/s将测试缩小您的分数 鉴定候选分子以在体外筛选，而活性分数将受到对象 质谱法。 使用IL-2和抗CD3来评估转录因子Foxp3的指示， 表明这些细胞已被TGF-β同源物或模拟物诱导。 将鉴定并重组表达具有阳性活性的分子，它们的活性将会。 使用记者MFB-F11生物测定和在体外调节T细胞指示中进行表征 测定。 鉴定TGF-β模拟型幼虫中的模拟蛋白具有改变我们的潜力 了解寄生虫适应宿主并开发可能的免疫疗法 介导的疾病。 成熟可能为感染的药物治疗开辟新的途径。