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

基本信息

批准号：
10642693
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
项目类别：
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/10642693
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 beta Transforming Growth Factor beta Receptors acquired epilepsy angiogenesis astrogliosis 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

项目摘要

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。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然而，我们对此却做得很差。了解 NCC 患者病理学的潜在机制，并尽可能减少临床上采取有效的治疗措施来预防这些患者的神经并发症。 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），通过 60 天的 post-oncosphere 期间，直到寄生虫到达成熟幼虫或囊尾蚴时，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 依赖性基于这些研究，通过TGF-β受体和配体同系物的表达途径。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 T. solium 幼虫发育的五个不同阶段的 TGF-β 开始。（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我们将询问这个新资源以了解各个阶段之间的分泌分子谱。此外，还鉴定了 TGF-β 超家族成员的同源物。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。然后对阳性 E/S 报告细胞系进行分数谱测试以缩小范围。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，其活性将使用报告基因 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此外，寄生虫所需的发育理解信号。成熟可能为感染的药物治疗开辟新途径。