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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 *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。* 目的4：隐孢子虫感染的遗传易感性：在第三年，我们通过研究人类甘露糖结合凝集素(MBL)扩大了我们对人类遗传在隐孢子虫感染易感性中的作用的观察。MBL是一种进化上保守的蛋白质，它通过结合微生物表面和促进吞噬细胞来发挥人体天然免疫功能。MBL已被证明与隐孢子虫子孢子结合，先前的工作表明MBL的保护作用可能在儿童时期最重要。我们评估了MBL基因(Mbl2)的多态和血清MBL缺陷与儿童隐孢子虫、溶组织内阿米巴和肠贾第鞭毛虫感染的关系。我们使用了一大批孟加拉学龄前儿童的匿名样本，这些儿童被跟踪了三年多。检测临床结果、MBL血清水平、mbl2基因多态性和单倍型。采用聚合酶链式反应(PCR)和直接测序法进行MBL2基因多态性的基因分型。多态分析是从一组14个引物开始的，但由于无法解释不清晰的条带，因此修改为下面的方法。发现了4个单核苷酸多态(SNP)，其中3个位于外显子1的结构区域，1个位于启动子区域，分别位于密码子54(甘氨酸[GGC]到天冬氨酸[GAC]替换)、密码子57(甘氨酸[GGA]到谷氨酸(GAA)替换)和密码子52(精氨酸[CGT]到半胱氨酸[TGT]替换)。野生型等位基因在所有三个位置都称为A，多态分别称为B、C和D。4任何结构多态(B、C、D)统称为“O”多态。研究的启动子多态位于-221位(X/Y变异体)，存在(G？C)核苷酸替换。 在数据分析中，未发现与溶组织性肠杆菌和肠溶血性肠杆菌有显著关联。然而，血清MBL缺乏、-221启动子区域的多态和XA/Yo mbl2单倍型与隐孢子虫感染，特别是反复感染密切相关。反复感染隐孢子虫的儿童更有可能是MBL缺陷(OR 10.45)、携带-221启动子变异(OR 4.02)和Yo/XA单倍型(OR 4.91)。请参见表1和图1。我们已经确定了人类对隐孢子虫感染的先天免疫反应的一个潜在的重要组成部分。MBL对隐孢子虫感染的保护作用机制有待进一步研究。 目的5：未来对隐孢子虫的研究将集中在探索MBL突变在控制人类隐孢子虫病方面的功能意义。利用免疫印迹和化学发光检测，我们将检测具有已知MBL基因变异的儿童血清中MBL的亚型。一旦确认，我们将使用现有的方法将野生型和变异型MBL基因克隆到CHO细胞中。使用基于抗体的技术，我们将评估MBL蛋白变体与隐孢子虫卵囊和子孢子结合的不同能力，并与临床结果相关。与此同时，我们还期待着研究班莱什达卡700名儿童的出生队列中的隐孢子虫，以检查感染前和感染后细胞因子的产生和淋巴细胞表型。与弗吉尼亚大学的合作者合作，将在这700名儿童身上进行全基因组关联，我们希望证实并补充我们的发现，MBL和人类白细胞抗原是改变感染易感性的遗传成分。 *请参阅数字和表格的纸质副本(此处不再转载)* 指导总结： 伊丽莎白·邦尼 我和科布雷初级学院的贝丝·柯克帕特里克都对人类粘膜免疫学和传染病感兴趣，我与她见过面，并就她的项目提供咨询。她大大扩展了她在临床和翻译研究方面的工作，围绕几种重要的感染性病原体开发疫苗。我的计划是每月与她会面，进一步确定职业目标，并鼓励公布结果。她已经获得了大量资金来支持她的工作。 拉尔夫·巴德 在过去的一年里，柯克帕特里克博士在发表论文和疫苗中心进化方面取得了丰硕成果。她现在已经从约翰·霍普金斯大学获得了登革热病毒疫苗试验的分包合同。此外，她还成功地通过海军为弯曲杆菌工作获得了资金，并为伤寒疫苗试验提供了生物技术支持。此外，柯克帕特里克博士还承担了开发人类免疫学核心实验室的任务，这是发展疫苗中心所必需的，但总体上对科布雷团队也是有用的。这将在5月作为T1补充申请提交。 来年的主要问题将是确定哪个项目/有机体可以开发成R21或R01应用程序。柯克帕特里克博士的K08奖将于今年结束，现在有必要将重点放在我们允许她竞争NIH资金的一两个领域。选管会的报告亦有提及这一点。

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. ***Please note the Tables and Figures mentioned below would not reproduce in this format. Please see attachments sent with the paper copy.*** Aim 4: Genetic Susceptibility to Cryptosporidium infection: In year 3, we extended our observations of the role of human genetics in susceptibility to Cryptosporidium infection by studying human mannose binding lectin (MBL). MBL is an evolutionarily conserved protein which functions in human innate immunity by binding microbial surfaces and promoting opsonophagocytosis. MBL has been shown to bind Cryptosporidium sporozoites and prior work has suggested that the protective role of MBL may be most important in childhood. We evaluated the association of polymorphisms in the MBL gene (mbl2) and serum MBL deficiency with childhood infection due to Cryptosporidium, Entamoeba histolytica, and Giardia intestinalis. We used anonymous specimens from a large, prospectively-followed cohort of Bangladeshi preschool children which followed for over three years. Clinical outcomes, MBL serum levels, and mbl2 polymorphisms and haplotypes were determined. Genotype determination of the MBL2 gene polymorphisms were performed by polymerase chain reaction (PCR) and direct sequencing. Analysis of polymorphisms was started with a 14-primer set, but modified to the methods below due to inability to interpret indistinct bands. Four single nucleotide polymorphisms (SNPs) of interest were determined, three located in the structural region on exon 1, and one located in the promoter region, Table 1. The three structural polymorphisms occur at codon 54 (glycine [GGC] to aspartic acid [GAC] substitution), codon 57 (lycine [GGA] to glutamic acid (GAA) substitution) and at codon 52 (arginine [CGT] to cysteine [TGT] substitution). The wild type allele is termed A at all three positions and polymorphisms are referred to as B, C, and D respectively.4 Any structural polymorphism (B, C, D) are collectively referred to as an "O" polymorphism. The promoter polymorphism studied is located at position -221 (X/Y variant) where there is a (G¿C) nucleotide substitution. In analysis of data, significant associations with E. histolytica and G. intestinalis were not found. However, serum MBL deficiency, polymorphisms in the -221 promoter region and the XA/YO mbl2 haplotype were strongly associated with Cryptosporidium infections, particularly recurrent infection. Children with multiple infections with Cryptosporidium were more likely to be MBL deficient (OR 10.45), carry the -221 promoter variant (OR 4.02), and the YO/XA haplotype (OR 4.91). Please see Table 1 and Figure 1. We have identified a potentially important component of the human innate immune response to Cryptosporidum infection. Further work is needed to evaluate the mechanism of protection of MBL in Cryptosporidium infections. Aim 5: Future work on Cryptosporidium will be focused on exploring the functional significant of the MBL mutations in control of human cryptosporidiosis. Using western blotting and chemoluminesent detection, we will examine the isoforms of MBL in the sera of children with known MBL genetic variants. Once confirmed, we will then clone the wild-type and variant MBL genes into CHO cells using established methods. Using antibody-based techniques, we will then evaluate the differing abilities of the MBL protein variants to bind both Cryptosporidium oocysts and sporozoites and correlate binding with clinical outcomes. Concurrently, we are also looking forward to studying Cryptosporidium in a birth-cohort of 700 children in Dhaka, Banglaesh in order to examine pre-and post-infection cytokine production and lymphocyte phenotypes. Working with collaborators at the University of Virginia, where genome-wide associations will be performed on these 700 children, we hope to confirm and add to our findings of MBL and HLA as genetic components changing susceptibility to infection. ***Please see paper copy for figures and tables (would not reproduce here)*** Mentoring Summaries: Elizabeth Bonney Beth Kirkpatrick, a COBRE Junior Faculty and I share an interest in human mucosal immunology and infectious disease, and I have met with her and provided consultation on her projects. She has greatly expanded her work in clinical and translation research around the development of vaccines to several important infectious agents. My plan is to meet with her monthly to further define career goals and encourage the publication of results. She has already obtained significant funding to support her work. Ralph Budd Dr. Kirkpatrick has been productive during the past year in terms of publications and success with the vaccine center evolution. She now has received a subcontract from Johns Hopkins for a dengue virus vaccine trial. In addition, she has been successful in obtaining funding through the Navy for the Campylobacter work, and biotech support for a typhoid vaccine trial. In addition, Dr. Kirkpatrick has taken on the task of developing a human immunology core laboratory, which is needed to grow the vaccine center, but will also be useful for the COBRE group in general. This will be submitted in May as a T1 supplement request. The main issue for the coming year will be defining which project/organism can be developed into an R21 or R01 application. Dr. Kirkpatrick's K08 award ends this year and there is a need to now focus on the one or two areas that we allow her to compete for NIH funding. This was also mentioned in the EAC report.