Molecular characterization of a hookworm Macrophage Migration Inhibitory Factor

描述(申请人提供)：钩虫感染是发展中国家营养不良和贫血的主要原因。全世界有近10亿人感染了这些吸血线虫寄生虫，这些寄生虫附着在肠道粘膜上，吞噬血液和组织。慢性钩虫病与严重贫血、营养不良和生长/认知迟缓有关。钩虫附着在肠粘膜上存活多年，因此长期以来一直认为，这种寄生虫必须有效地抑制宿主的免疫反应，才能延长生存时间。在人类和动物模型中都表明，钩虫感染与不同程度的免疫抑制有关，表现为淋巴细胞对钩虫和异种抗原的反应减弱。然而，到目前为止，钩虫在宿主组织迁移和随后的发育过程中利用哪些特定的进化策略来逃避宿主的免疫反应，以建立或维持感染，目前还知之甚少。我们已经从人钩虫中鉴定出炎性哺乳动物细胞因子巨噬细胞移动抑制因子(MIF)的同源基因。这种新的蛋白AceMIF存在于成虫的可溶性提取物和排泄/分泌(ES)蛋白中，但其在疾病发病机制中的潜在作用尚未确定。初步研究表明，AceMIF与人MIF受体(CD74)结合，具有与人MIF相似的巨噬细胞趋化活性和互变构象酶催化活性。与人类蛋白质不同，AceMIF对哺乳动物MIF的小分子抑制剂ISO-1具有抵抗力，为病原体特异性抑制剂的设计提供了概念证明。我们认为，AceMIF通过有效地调节宿主小肠内附着部位的宿主免疫反应来促进寄生虫的存活。AceMIF的免疫调节作用将通过基于细胞的体外实验来研究，以阐明CD74受体参与的远端信号转导事件。宿主MIF在体内的表达动力学将通过组织特异性ELISA来推断，免疫组织化学研究将确定宿主和寄生虫MIF表达的空间关系。宿主MIF在钩虫感染中的作用将通过中和抗体和ISO-1的抑制来研究，使用完全允许的头孢曲霉动物模型。钩虫MIF在贫血和生长延迟的发病机制中的作用将通过动物疫苗试验来表征，该试验旨在中和AceMIF在体内的活性，并损害成虫在肠道内的生存能力。这些研究将使开发旨在控制这种重要人类病原体的新方法成为可能。

DESCRIPTION (provided by applicant): Hookworm infection is the leading cause of malnutrition and anemia in the developing world. Nearly one billion individuals worldwide are infected with these bloodfeeding nematode parasites, which attach to the intestinal mucosa and ingest blood and tissue. Chronic hookworm disease is associated with severe anemia, malnutrition, and growth/cognitive delay. Hookworms survive for years attached to the intestinal mucosa, therefore it has long been theorized that the parasites must effectively dampen the host immune response in order to prolong survival. It has been shown both in humans and animal models that hookworm infection is associated with varying degrees of immunosuppression, manifested by impaired lymphocyte responses to both hookworm and heterologous antigens. However, to date, little is known about the specific evolutionary strategies utilized by hookworms to evade the host immune response during host tissue migration and subsequent development in order to establish or maintain an infection. We have identified an orthologue of the inflammatory mammalian cytokine macrophage migration inhibitory factor (MIF) from the human hookworm Ancylostoma ceylanicum. This novel protein, AceMIF, is present in soluble extracts and excretory/secretory (ES) proteins from adult hookworms, but its potential role in disease pathogenesis has not been defined. Preliminary data reveal that AceMIF binds the human MIF receptor (CD74), and possesses macrophage chemotaxis and tautomerase catalytic activity similar to that of human MIF. Unlike the human protein, AceMIF is resistant to the small molecule inhibitor of mammalian MIFs, ISO-1, establishing proof of concept for the design of pathogen specific inhibitors. We propose that AceMIF promotes parasite survival by effectively modulating the host immune response at the site of attachment within the host small intestine. The immunomodulatory role of AceMIF will be investigated using cell based in vitro assays to elucidate signal transduction events distal to CD74 receptor engagement. The kinetics of host MIF expression in vivo will be deduced through tissue-specific ELISA and immunohistochemical studies will define the spatial relationship of host and parasite MIF expression. The role of host MIF during hookworm infection will be investigated by inhibition with neutralizing antibodies and ISO-1 using a fully permissive animal model of A. ceylanicum. The role of hookworm MIF in the pathogenesis of anemia and growth delay will be characterized using animal vaccine trials aimed at neutralizing the activity of AceMIF in vivo and impairing the ability of adult worms to survive within the intestine. These studies will allow for the development of new methods aimed at controlling this important human pathogen.