GLYCOCONJUGATES INVOLVED IN HOST/PARASITE INTERACTIONS

阴道毛滴虫引起的滴虫病是人类最常见的性传播疾病之一。滴虫病是一种引起阴道炎的严重疾病，与不孕症、早产、低出生体重婴儿、子宫颈癌和明显增加对艾滋病毒的易感性有关。细胞表面糖缀合物似乎介导寄生虫与宿主细胞的结合。建立了一种具有寄主特异性的人阴道上皮细胞体外培养系统。HVECs暴露于寄生虫会导致HVECs在30小时内被破坏。经高碘酸盐处理的寄生虫不会损害HVECs。阴道绦虫具有细胞表面糖缀合物，脂磷聚糖（LPG），它取代寄生虫与HVECs结合，这意味着特异性受体-配体相互作用。一种由寄生虫释放的相关水溶性聚糖（TV-SG）也得到了纯化。本研究的长期目标是在分子和细胞水平上阐明阴道绦虫的病理机制。具体目的是：1)利用HVEC培养系统和35s -甲硫氨酸标记的寄生虫结合置换实验，研究LPG在宿主-寄生虫粘附中的作用，并鉴定和纯化HVEC表面结合寄生虫的“受体”分子。2)完成LPG的结构表征，研究其与TV-SG的关系；期望结构分析将与生物活性相关联。结合测定也将用于测定由化学和酶处理衍生的LPG分子的部分。3)通过定量分析研究寄生虫对HVECs的细胞毒性，评估接触依赖性和非接触依赖性机制。HVEC破坏将使用ELISA法检测坏死与凋亡。受体纯化将基于筛选技术来寻找与液化石油气结合的放射性标记的HVEC表面分子或固定化液化石油气的亲和柱方法。液化石油气的结构将使用多种技术进行表征，包括HPLC， Glyko-FACE， MALDI-TOF MS和NMR。这些研究将有助于明确阴道滴虫的致病机制，最终导致更好的治疗滴虫病。

Trichomonas vaginalis, causes one of the most common sexually transmitted disease in humans, trichomoniasis. Trichomoniasis is a serious disease that causes flagrant vaginitis and has been linked to infertility, preterm delivery, low birth weight infants, cervical cancer and markedly increased susceptibility to HIV. Cell surface glycoconjugates appear to mediate parasite binding to host cells. An in vitro culture system of human vaginal epithelial cells (HVECs), which exhibits parasite-host specificity, has been developed. Exposure of HVECs to parasites results in destruction of HVECs within 30 h. Periodate treated parasites do not damage HVECs. T. vaginalis possesses a cell surface glycoconjugate, lipophosphoglycan (LPG), which displaces parasites binding to HVECs, implying a specific receptor-ligand interaction. A related water soluble glycan (TV-SG), released by parasites, has also been purified. The long term goal of this proposal is to elucidate the mechanism(s) of T. vaginalis pathobiology on a molecular and cellular level. The specific aims are: 1) to utilize the HVEC culture system and an 35S-methionine-labeled-parasite binding-displacement assay to study the role of LPG in host-parasite adhesion, and to identify and purify "receptor" molecules on the HVEC surface that bind parasites. 2) to complete the structural characterization of LPG and study its relationship to TV-SG; with the expectation that structural analysis will be correlated with bioactivity. The binding assay will also be employed to assay portions of the LPG molecule derived by chemical and enzymatic treatments. 3) Parasite cytotoxicity toward HVECs will be studied by quantitative assays and contact-dependent and contact-independent mechanisms will be evaluated. HVEC destruction will be assayed for necrosis vs. apoptosis using an ELISA assay. Receptor purification will be based on either a panning technique to search for radiolabeled HVEC surface molecules that bind to LPG or an affinity column approach with immobilized LPG. LPG structure will be characterized using a variety of techniques, including HPLC, Glyko-FACE, MALDI-TOF MS and NMR. These studies will help to define the mechanisms of T. vaginalis pathogenicity, which will ultimately lead to better treatments for trichomoniasis.