VERMONT COBRE: PROJECT 2: INNATE IMMUNE RESPONSES TO CRYPTOSPORIDIUM PARVUM

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 *请注意，下面提到的表格和数字不会以这种格式复制。请参阅随纸质副本一起发送的附件。* 科布雷第4年的工作重点是探索人类隐孢子虫病研究的新途径，以及开发新的免疫学分析来补充这项即将进行的工作。 建立功能免疫学分析方法：利用隐孢子虫和伤寒沙门氏菌减毒活疫苗临床研究的标本，我们已经建立了用于评估人类病原体的功能性抗体和细胞分析方法，并朝着标准化方向发展，以便在未来的人类免疫学核心设施中使用。以伤寒沙门氏菌为模型病原体，发展了四种新的功能抗体检测方法：调理/吞噬作用(图1)；吞噬后杀菌；吞噬指数测定(PI，图2)；以及溶酶体共定位试验。对于细胞免疫学，我们开发了一种七色流式细胞术来评估感染或疫苗接种前后的细胞内细胞因子(图3)。B细胞记忆ELISPOT分析也已正式开发出来。所有这些检测方法现在都被用于研究登革热减毒活疫苗，并将用于未来对隐孢子虫感染的研究。还开发了专门用于研究病毒病原体的其他分析方法。这些措施包括使用Vero细胞(蚊子细胞系)对登革热1-4型、西尼罗河病毒、圣路易斯脑炎和黄热病病毒进行标准化的空斑减少和中和分析(PRNT)。病毒扩增试验(登革热血清型1-4型)也已开发出来。 新的隐孢子虫研究：进一步研究隐孢子虫的机会预计将在科布雷的第5年取得成果。首先，孟加拉国达卡有机会获得隐孢子虫免疫学，这是一大批出生儿童的一部分。目前，已有400名儿童登记在册，其中12%的儿童在出生6个月时有证据表明隐孢子虫感染。临床数据、人类DNA、外周淋巴细胞和血清都是可用的，这使得这是我们继续研究人类对这种感染的免疫反应的理想环境。这个队列的工作将集中在三个主要途径上：扩大我们对隐孢子虫病遗传易感性的观察的全基因组关联；对甘露糖结合凝集素(MBL)和作为隐孢子虫免疫的固有成分的其他集合素/表面活性物质的扩大和功能性工作，以及使用7或8色流式细胞术和ELISPOT分析评估感染的细胞免疫反应(见上文)。第二，我们正在开始招募一大批终末期艾滋病和隐孢子虫感染者(乌干达)。我们将在该人群中确认MBL与隐孢子虫的关联。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ***Please note the Tables and Figures mentioned below would not reproduce in this format. Please see attachments sent with the paper copy.*** Work in year 4 of the COBRE was focused on exploring new avenues for human cryptosporidiosis research as well as development of new immunology assays to compliment this upcoming work. Establishment of functional immunology assays: Using specimens from clinical research studies on Cryptosporidium and live attenuated S. Typhi vaccines, we have established functional antibody and cellular assays for evaluation of human pathogens and toward standardization for use in a future human immunology core facility. Four new functional antibody assays have been developed using S. typhi as a model pathogen: opsonization/ phagocytosis (Figure 1); bacterial killing post-phagocytosis; measures of phagocytic index (PI, Figure 2); and a lysosome-colocalization assay. For cellular immunology, we have developed a seven-color flow cytometry assay to evaluate intracellular cytokines pre-and post-infection or vaccination (Figure 3). A B-cell memory ELISPOT assay has also been formally developed. All these assays are now in use for studying live attenuated dengue vaccines and will be used for future study of Cryptosporidium infection. Other assays, specifically for the study of viral pathogens, have been developed. These include standardized plaque reduction and neutralization assays (PRNT) using Vero cells (mosquito cell line) for Dengue serotype 1-4, West Nile, St. Louis Encephalitis and Yellow Fever viruses. Viral amplication assays (Dengue serotypes 1-4) have also been developed. New Cryptosporidium studies: opportunities for further Cryptosporidium work are anticipated to come to fruition in year 5 of the COBRE. First, opportunities for Cryptosporidium immunology are available in Dhaka, Bangladesh as part of a large birth cohort of children. Presently, 400 children have been enrolled and 12% have evidence of Cryptosporidium infection by 6 months of life. Clinical data, human DNA, peripheral lymphocytes and sera are available, making this an ideal setting for continuing our work into human immune responses to this infection. The work from this cohort will focus on three major avenues: genome-wide associations to expand our observations of genetic susceptibility to cryptosporidiosis; expanded and functional work on mannose-binding lectin (MBL) and other collectins/surfactants which function as an innate components in immunity to Cryptosporidium infection and evaluation of cellular immune responses to infection using 7 or 8-color flow cytometry and ELISPOT assays (see above).Secondly, we are initiating enrollment of a large cohort of persons with end-stage AIDS and Cryptosporidium infection (Uganda). We will be confirming the MBL association with Cryptosporidium in this population.