TOXOPLASMA GONDII--DIAGNOSIS AND PREVENTION IN AIDS

这一竞争性更新的总体具体目标是进一步改进 弓形虫感染者的免疫治疗 艾滋病。在我们目前的资金期内，我们开发了大量的 关于免疫学、分子生物学和 几种重要寄生虫抗原的血清学，特别是P30， 主要膜和排泄/分泌抗原(ESA) 细胞内病原体。在本提案中，我们计划使用这些 观察以阐明两个不同但互补的特定 目标：寄生虫附着在宿主细胞上，并被人类内化 吞噬细胞。第一个具体目标是评估P30在 寄生虫附着。我们的初步观察表明，P30是 弓形虫的一个重要的依附因素。我们计划将其描述为 用变异体在分子水平上研究这种寄生虫配体的性质 P30的分子结构。我们还计划识别和表征 这种寄生虫分子的宿主细胞受体。在我们的研究中 实验室证明某些新糖蛋白能够阻断 人成纤维细胞的寄生虫感染。这一机制通过它 这些分子会阻止附着到细胞外基质上 调查过了。此外，糖结合分子在糖链中的作用 宿主：寄生虫附着的特征将使用宿主细胞处理 用各种糖基化抑制剂和突变的宿主细胞 糖基化产物中已知的变化。第二个具体问题 目的是确定双特异性抗体(BsAb)是否可以针对T。 弓形虫对人吞噬细胞的杀伤作用，并通过 这些细胞。我们已经证明，细胞外弓形虫的杀灭 当寄生虫被感染时，人的吞噬细胞会显著增加 用bsAb调理。这些双重识别抗体包括 一种与多种吞噬细胞受体交叉连接的寄生虫特异性臂 包括FcGammaR。我们计划使用bsAb将P30与 不同的吞噬细胞受体来确定哪些受体最好 调节寄生虫的内化。我们还证明了 寄生性空泡不能与其他内胞融合 主动渗透后的细胞器可以通过靶向 寄生于宿主细胞FcGammaRII。一旦重要的吞噬细胞 鉴定了速殖子附着和内化的受体， 我们将研究内体和溶酶体与寄生虫的融合- 被这些受体摄取后含有空泡的，以及 以激活杀菌活性的机制。 这将包括对各种O2依赖和O2非依赖的分析 被认为参与弓形虫杀灭活性的机制 吞噬细胞。所获得的信息将与以下方面直接相关 改善弓形虫病患者的免疫治疗 艾滋病。

The overall specific aim of this competing renewal is to further improve the immunotherapy of Toxoplasmosis in those individuals afflicted with AIDS. During our current funding period we have developed a substantial body of information regarding the immunology, molecular biology and serology of several important parasite antigens, in particular P30, the major membrane and excreted/secreted antigen (ESA) of this obligated intracellular pathogen. In this proposal we plan to use these observations to elucidate two different, but complementary specific aims: parasite attachment to host cells and internalization by human phagocytes. The first specific aim is to evaluate the role of P30 in parasite attachment. Our preliminary observations suggest that P30 is an important attachment factor for T. gondii. We plan to characterize the nature of this parasite ligand at the molecular level using variant molecular constructs of P30. We also plan to identify and characterize the host cell receptor for this parasite molecule. Studies in our laboratory demonstrate that certain neoglycoproteins are able to block parasite infection of human fibroblast cells. The mechanism by which these molecules block attachment to extracellular matrix will be investigated. Moreover, the role of sugar binding molecules in host:parasite attachment will be characterized using host cells treated with various inhibitors of glycosylation and mutant host cells with known alterations in their glycosylation products. The second specific aim will be to determine if bispecific antibodies (bsAb) can target T. gondii to human phagocytes and enhance the killing of the parasite by these cells. We have shown that killing of extracellular T. gondii by human phagocytes can be significantly increased when parasites are opsonized with bsAb. These dual recognition antibodies are comprised of a parasite specific arm cross linked with various phagocytic receptors including FcgammaR. We plan to use bsAb that cross link P30 with various phagocyte receptors to determine which receptors can best mediate parasite internalization. We have also demonstrated that the parasitophorous vacuole inability to fuse with other endocytic organelles following active penetration can be overcome by targeting the parasite to a host cell FcgammaRII. Once the important phagocyte receptors for tachyzoite attachment and internalization are identified, we will investigate both endosome and lysosome fusion with the parasite- containing vacuole following uptake by these receptors, and the ability of these receptors to activate the mechanisms for microbicidal activity. This will include analysis of various O2-dependent and O2-independent mechanisms believed to be involved in toxoplasmacidal activity by phagocytes. The information obtained will be directly related toward improving the immunotherapy of toxoplasmosis in those individuals with AIDS.