IMMUNE RESPONSES TO MICROSPORIDIA

对小孢子虫的免疫反应

基本信息

批准号：
7958588
负责人：
Elizabeth Schmidt Didier
金额：
$ 6.05万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7958588
关键词：
Algorithms Animals Cells Complementary DNA Computer Retrieval of Information on Scientific Projects Database Computer software Data Data Set Diarrhea Disease Elements Enterocytozoon bieneusi Exhibits Feces Funding Genes Genetic Transcription Grant Hepatic Histocytochemistry Hour Housing Human Image Immune Immune response Incubated Infection Inflammatory Institution Interferon Type II Interferons Intestines Label Lymphocyte Macaca Macaca mulatta Macaca nemestrina Measurement Microarray Analysis Microsporidia Monkeys Oligonucleotides Papio Pathway Analysis Pathway interactions Primates Printing Publishing RNA Reporting Reproduction spores Research Research Personnel Resources Sampling Signal Transduction Slide Source Technology Tumor Necrosis Factor-alpha United States National Institutes of Health base cytokine human TNF protein nonhuman primate pathogen response wasting

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Enterocytozoon bieneusi is the most prevalent microsporidian infecting humans, yet virtually no studies have been published about the immune responses to this pathogen. Nonhuman primates are natural hosts of E. bieneusi infections, and na¿ve and immune-compromised rhesus macaques exhibit similar signs of disease (eg. diarrhea and wasting) as reported in immune-compromised infected humans. E. bieneusi infections are being reported more frequently in immune-competent humans, and 35-40% of the immune-competent pigtail macaques, rhesus macaques, and baboons housed outdoors at the Tulane National Primate Research Center were detected with E. bieneusi spores in feces by histochemistry and PCR. To begin to study the immune responses to this microsporidian, a preliminary microarray analysis was performed. Duodenal and jejunal intestinal cells from immune-competent E. bieneusi-infected and non-infected rhesus macaques (with and without diarrhea) were incubated with E. bieneusi spores or medium for 24 hours. Total RNA was extracted to generate Cy-labeled cDNA samples using the Low RNA Input Linear Amplification Kit (Agilent Technologies Inc., CA). Labeled cDNA was hybridized overnight to the 44,000 element 60mer oligonucleotide based rhesus macaque microarray printed in a 4x44k format (Agilent Technologies Inc., CA), which can interrogate the transcription of over 18,000 unique macaque genes at once. After hybridization and washing, slides were evaluated in a dual-confocal continuous microarray scanner (GenePix 4000B) using GenePix Pro version 6.1 as the image acquisition and extraction software. Microarray data were based on duplicate measurements. Pathway analyses were performed by uploading significant data sets into Ingenuity Pathways Analysis algorithm. Among the pathways and cytokines related to immune responses that were significantly perturbed (P 0.05) in the E. bieneusi-infected monkey lymphocytes with diarrhea and the uninfected monkeys were those related to inflammatory disease, hepatic disease, and interferon signalling (e.g. TNF-alpha, IFN-gamma). These pathways were not significantly perturbed in intestinal cells of infected animals that were not exhibiting signs of diarrhea, suggesting that healthy immune-competent animals are able to regulate inflammatory pathways in response to E. bieneusi infection.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以出现在其他 CRISP 条目中列出的机构是。对于中心来说，它不一定是研究者的机构。比恩纽氏肠细胞虫是感染人类的最常见的微孢子虫，但实际上尚未发表关于非人灵长类动物是比恩纽氏肠细胞虫感染的天然宿主的免疫反应的研究。免疫功能低下的恒河猴表现出与免疫功能低下的感染人类相似的疾病症状（例如腹泻和消瘦），而在免疫功能正常的人类中，有 35-40% 的人更频繁地报告了 E. bieneusi 感染。 -杜兰国家灵长类研究中心户外饲养的有能力的猪尾猕猴、恒河猴和狒狒均检测到了 E. bieneusi为了开始研究对这种微孢子虫的免疫反应，对免疫活性的 E. bieneusi 感染和未感染的恒河猴（有或没有感染）的十二指肠和空肠肠细胞进行了初步的微阵列分析。腹泻）与 E. bieneusi 孢子或培养基一起孵育 24 小时，提取总 RNA 以生成 Cy 标记的。使用低 RNA 输入线性扩增试剂盒（加利福尼亚州安捷伦科技公司）将 cDNA 样品与以 4x44k 格式打印的基于 44,000 个元件的 60 聚体寡核苷酸的恒河猴微阵列（加利福尼亚州安捷伦科技公司）杂交过夜。一次性询问超过 18,000 个独特的猕猴基因的转录杂交和清洗后，在玻片中进行评估。使用 GenePix Pro 6.1 版作为图像采集和提取软件的双共焦连续微阵列扫描仪（GenePix 4000B）通过将重要数据集上传到 Ingenuity Pathways Analysis 算法中来进行微阵列数据分析。与免疫反应相关的细胞因子在受 E. bieneusi 感染的腹泻猴淋巴细胞和未感染的猴中显着扰动 (P < 0.05)与炎症性疾病、肝脏疾病和干扰素信号传导（例如 TNF-α、IFN-γ）相关的信号通路在未表现出腹泻迹象的受感染动物的肠道细胞中没有受到显着干扰，这表明健康的免疫功能正常。动物能够调节炎症途径以应对 E. bieneusi 感染。