GLYCOCONJUGATES INVOLVED IN HOST/PARASITE INTERACTIONS

阴道毛滴虫是人类最常见的性传播疾病之一，滴虫病。滴虫病是一种严重的疾病，会导致公然的阴道炎，并与不孕不育、早产、低出生体重婴儿、宫颈癌和艾滋病毒易感性显著增加有关。细胞表面的糖结合物似乎介导了寄生虫与宿主细胞的结合。建立了一种具有寄生虫-宿主特异性的人阴道上皮细胞体外培养体系。将人血管内皮细胞暴露于寄生虫中可在30小时内破坏血管内皮细胞。经过碘酸盐处理的寄生虫不会损伤血管内皮细胞。阴道毛滴虫具有一种细胞表面糖结合物--脂磷聚糖(LPG)，它可以取代与血管内皮细胞结合的寄生虫，这意味着一种特定的受体-配体相互作用。寄生虫释放的一种相关的水溶性多糖(TV-SG)也得到了纯化。这项建议的长期目标是从分子和细胞水平阐明阴道毛滴虫的病理生物学机制(S)。1)利用人脐静脉内皮细胞培养体系和~(35)S-蛋氨酸标记的寄生虫结合置换实验，研究LPG在宿主-寄生虫黏附中的作用，并鉴定和纯化人脐静脉内皮细胞表面结合寄生虫的“受体”分子。2)完成LPG的结构表征，并研究其与TV-SG的关系，以期将结构分析与生物活性联系起来。结合分析也将被用来分析通过化学和酶处理获得的部分LPG分子。3)寄生虫对人脐静脉内皮细胞的细胞毒作用将通过定量的方法进行研究，并对接触依赖和接触非接触机制进行评价。用酶联免疫吸附试验检测人血管内皮细胞的坏死率和凋亡率。受体的纯化将基于寻找与LPG结合的放射性标记的HVEC表面分子的平移技术，或基于固定化LPG的亲和柱法。液化石油气的结构将使用多种技术进行表征，包括高效液相色谱、糖化表面、MALDI-TOF MS和核磁共振。这些研究将有助于确定阴道毛滴虫致病的机制，最终将导致更好的滴虫病治疗。

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.