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.

神经囊尾蚴病 (NCC)，由囊尾蚴引起的中枢神经系统 (CNS) 感染 猪绦虫（猪肉绦虫）的后绦虫幼虫在大多数发展中国家流行 国家并确定为全世界获得性癫痫的最常见原因。这 寄生虫会引起慢性神经炎症，病理学研究表明反应性 星形胶质细胞增生、纤维化、血管生成、脑血屏障通透性改变和 炎症和抗炎细胞因子的过度表达。但为此我们却做得很差 了解 NCC 患者病理学的潜在机制，并尽可能减少 预防这些患者神经系统并发症的临床手段。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 退化幼虫引起的病理炎症反应。 我们开创了从感染阶段开始的猪幼虫发育的体外模型 （oncosphere），经历了 60 天的后 oncosphere。在这些阶段期间直至 寄生虫到达成熟的幼虫或囊尾蚴，寄生虫本身会改变其蛋白质表达 然而，我们对每个阶段分泌的分子几乎没有信息。 TGF-β 在多种原生动物和蠕虫感染中发挥着关键作用。 除了宿主 TGF-β 信号传导在调节宿主-寄生虫相互作用中的重要性之外， 大量证据表明，蠕虫寄生虫可能直接影响 TGF-β 依赖性 通过TGF-β受体和配体同系物的表达途径。基于这些研究， 我们将利用我们的体外模型并检查排泄/分泌 (E/S) 产品 T. solium 不同幼虫发育阶段的免疫调节测试 功能，从 TGF-β 开始。猪幼虫发育的五个不同阶段的 E/S （生长 15 天、30 天和 60 天的癌球、癌后球和成熟包囊），到蛋白质组学 通过质谱分析，表征每个阶段的分泌蛋白组并比较 各阶段之间分泌分子的光谱。我们将询问这个新资源 鉴定 TGF-β 超家族成员的同源物。此外，E/S 蛋白来自 幼虫的不同发育阶段将使用凝胶过滤和阴离子进行分级 交换快速蛋白液相色谱，为后续研究提供资源 体外测定，以功能测试和鉴定新的免疫调节剂。 然后，我们将首先利用敏感的酶来测试 T. solium E/S 的 TGF-β 样活性。 TGF-β报告细胞系。然后，将测试阳性 E/S 的分数分布，以缩小范围 鉴定候选分子进行体外筛选，并以活性组分为对象 到质谱分析。随后，E/S 将在幼稚 CD4+ T 细胞的体外培养物中进行测试 用 IL-2 和抗 CD3 来评估转录因子 Foxp3 的诱导， 表明这些细胞已被 TGF-β 同源物或模拟物诱导产生 Tregs。 具有阳性活性的分子将被鉴定并重组表达，其活性将 使用报告分子 MFB-F11 生物测定和体外调节性 T 细胞诱导进行表征 化验。 鉴定 T. solium 幼虫中的 TGF-β 模拟蛋白有可能改变我们的 了解寄生虫对宿主的适应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 成熟可能为感染的药物治疗开辟新途径。