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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 * 请注意，下面提到的图表不会以这种格式复制。 请参阅随纸质副本发送的附件。* COBRE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检测方法，以补充即将开展的工作。 功能性免疫学检测方法的建立：使用来自隐孢子虫和减毒活S。伤寒疫苗，我们已经建立了功能性抗体和细胞检测，用于评估人类病原体，并走向标准化，用于未来的人类免疫学核心设施。利用S.伤寒作为病原体模型：调理作用/吞噬作用（图1）;吞噬作用后的细菌杀伤;吞噬指数的测量（PI，图2）;和溶酶体共定位测定。对于细胞免疫学，我们开发了一种七色流式细胞术测定法，以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测定也已正式开发。所有这些检测方法现在都用于研究活减毒登革热疫苗，并将用于未来的隐孢子虫感染的研究。已经开发了专门用于研究病毒病原体的其他测定法。其中包括使用Vero细胞（蚊子细胞系）对登革热血清型1 - 4、西尼罗河、圣路易斯脑炎和黄热病病毒进行标准化空斑减少和中和试验（PRNT）。还开发了病毒扩增测定（登革热血清型1 - 4）。 新的隐孢子虫研究：进一步隐孢子虫研究的机会预计将在COBRE的第5年取得成果。首先，在孟加拉国的达卡，隐孢子虫免疫学的机会是作为一个大的儿童出生队列的一部分。目前，400名儿童已经登记，12%的儿童在6个月内有隐孢子虫感染的证据。临床数据，人类DNA，外周血淋巴细胞和血清是可用的，使这成为一个理想的设置，继续我们的工作到这种感染的人类免疫反应。该队列的工作将集中在三个主要方面：全基因组关联，以扩大我们对隐孢子虫病遗传易感性的观察;对甘露糖结合凝集素（MBL）和其他凝集素/表面活性剂进行了扩展和功能性研究，这些凝集素/表面活性剂在对隐孢子虫感染的免疫中起着先天性成分的作用，并使用7色或8色流式细胞术和ELISPOT测定法评价了对感染的细胞免疫应答第二，我们正在开始招募大量患有终末期艾滋病和隐孢子虫感染的人（乌干达）。我们将在该人群中确认MBL与隐孢子虫的相关性。

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.