ANALYSIS OF T CELL RESPONSES IN HUMAN LEISHMANIASIS

人类利什曼病 T 细胞反应分析

基本信息

批准号：
6098949
负责人：
David Sacks
金额：
--
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

IL-12 is required for immunity to Leishmaniasis. We have previously reported that the infection of mouse bone-marrow- derived or inflammatory macrophages with Leishmania leads to a selective impairment of IL-12-inducing pathways. We have argued that as a consequence, infected macrophages will drive early T cell activation in the absence of the major physiologic inducer of IFN-g, and that this effect explains the delayed onset of cell-mediated control mechanisms that is typical of even self- limiting forms of leishmaniasis. We have extended these studies to include an analysis of cytokine production by infected epidermal Langerhans cells (LC), since it is these cells that have been implicated in the initiation of immune responses in the skin. Using a culture system that allows Langerhans cell-like cells to be routinely expanded and isolated from C57BL/6 fetal skin (fetal skin-derived dendritic cells-FSDDC) we have begun to study dendritic cell-parasite interactions. L. major amastigotes, but not metacyclic promastigotes, were readily internalized by FSDDC. Parasite internalization was associated with activation of FSDDC manifested by upregulation of MHC class I and II surface antigens, increased expression of co-stimulatory molecules (CD40, CD54, and CD86) and IL-12 release all over an 18-hr time period. Although tissue macrophages also readily internalized amastigotes, parasite internalization did not lead to similar changes in surface antigen expression or cytokine production. The data suggest that activation of, and IL-12 induction in, skin dendritic cells by L. major amastigotes is important for development of protective Th1 predominant anti- Leishmania immunity, and that so long as infections are confined to macrophages, the onset of cell-mediated immune responses will be avoided. Studies have been completed that evaluated the toxicity, immunogenicity and efficacy of a clinical grade killed Leishmania vaccine made by Biobras in Brazil plus rhuIL-12 in monkeys (Macaca mulatta). Twelve monkeys receiving two intradermal inoculations of alum absorbed killed vaccine plus IL-12 were completely protected against homologous challenge with L. amazonensis. All but one of 16 animals in the control groups developed typical cutaneous ulcers. The protection was correlated with high levels of IFN-g in serum and in culture supernatants of PBLs stimulated with antigen in vitro. Vaccination with killed antigen using IL-12 and alum as adjuvants was safe and effective in this primate model. Phase I clinical trials are planned. Despite the dramatic increase in the incidence of unresponsiveness to antimony therapy in Indian kala-azar, the possibility that this might be due to the emergence of antimony resistant strains of L. donovani has not been demonstrated. The susceptibility of L. donovani isolates to sodium antimony gluconate (SAG) was compared using infected macrophages in vitro. Isolates were obtained from splenic biopsies of patients in Bihar, India, who either did not respond (18) or did respond (10) to one or more full courses of treatment with SAG. A strong correlation (p < 0.005) with clinical response was observed only when strains were assayed as intracellular amastigotes; responsive isolates ED/50\= 2.5 ?2.8 ?g/ml; unresponsive isolates ED/50\ = 7.2?4 ug/ml. From these data we conclude that treatment failures in India Kala-azar are due to infection with antimony resistant strains of L. donovani. Between July 1, 1997, and June 30, 1998, five patients with suspected leishmaniasis were seen and evaluated at the NIH clinical center. Four of the five were cases of cutaneous disease; one was a Peace Corps volunteer from Mauritania, one was an ornithologist working in the Manu Forest in Peru, one was an elderly woman from Pakistan, and the fourth cutaneous case was a tourist whose lesions healed spontaneously. The most interesting patient was a native Zairean with visceral leishmaniasis, probably acquired in the Aegean Islands of Greece when the patient was attending Athens University. One new feature of the leishmaniasis experience of the past year was the use of liposome-encapsulated amphotericin (Ambisome) for treatment of two of the cutaneous infections as well as the visceral case. The advantage of AmBisome treatment is the shorter duration of treatment (5 or 6 doses over 10 days) as compared to Pentostam (18-20 days).

IL-12 是对利什曼病的免疫力所必需的。此前我们曾报道过小鼠感染骨髓来源的或炎性巨噬细胞利什曼原虫导致 IL-12 诱导的选择性损伤途径。我们认为，结果是，感染在缺乏巨噬细胞的情况下，巨噬细胞将驱动早期 T 细胞激活 IFN-g 的主要生理诱导剂，这种效应解释了细胞介导的控制机制的延迟启动是典型的甚至是利什曼病的自限性形式。我们已经扩展了这些研究包括对感染者细胞因子产生的分析表皮朗格汉斯细胞 (LC)，因为正是这些细胞具有与皮肤免疫反应的启动有关。使用允许朗格汉斯细胞样细胞的培养系统常规扩增并从 C57BL/6 胎儿皮肤中分离（胎儿皮肤源性树突状细胞-FSDDC）我们已经开始研究树突状细胞与寄生虫的相互作用。 L. 大无鞭毛体，但不是后循环前鞭毛体很容易被 FSDDC 内化。寄生虫内化与 FSDDC 激活相关表现为 MHC I 类和 II 类表面抗原的上调，共刺激分子（CD40、CD54、和 CD86) 和 IL-12 在 18 小时的时间内释放。尽管组织巨噬细胞也很容易内化无鞭毛体，寄生虫内化并没有导致表面的类似变化抗原表达或细胞因子产生。数据表明 L. 激活皮肤树突状细胞并诱导 IL-12。主要无鞭毛体对于保护性 Th1 的发育很重要主要的抗利什曼原虫免疫力，只要感染仅限于巨噬细胞，细胞介导的发病将避免免疫反应。研究已完成评估临床药物的毒性、免疫原性和功效巴西 Biobras 生产的级灭活利什曼原虫疫苗 plus 猴子 (Macaca mulatta) 中的 rhuIL-12。十二只猴子接受两次皮内接种明矾吸收灭活疫苗加 IL-12 完全免受同源攻击与 L. amazonensis。对照中 16 只动物中除一只外的所有动物各组出现典型的皮肤溃疡。保护是与血清和培养物中高水平的 IFN-g 相关体外用抗原刺激的 PBL 上清液。疫苗接种使用IL-12和明矾作为佐剂杀死抗原是安全的在此灵长类动物模型中有效。计划进行 I 期临床试验。尽管反应迟钝的发生率急剧增加印度黑热病的锑疗法，这种可能性可能是由于 L. 抗锑菌株的出现。多诺瓦尼尚未得到证实。 L 的敏感性多诺瓦尼分离株转化为葡萄糖酸锑钠 (SAG) 在体外使用受感染的巨噬细胞进行比较。分离株为从印度比哈尔邦患者的脾脏活检中获得，这些患者未对一项或多项完整回复 (18) 或已回复 (10) SAG 的治疗疗程。强相关性 (p < 0.005) 仅在对菌株进行分析时才能观察到临床反应作为细胞内无鞭毛体；响应分离株 ED/50\= 2.5 ?2.8 克/毫升；无反应分离株 ED/50\ = 7.2?4 ug/ml。从这些数据来看我们的结论是，印度黑热病治疗失败的原因是杜氏乳杆菌的锑抗性菌株感染。之间 1997年7月1日和1998年6月30日，5名疑似患者利什曼病在 NIH 临床中心进行观察和评估。五例中有四例是皮肤病；一个是和平来自毛里塔尼亚的军团志愿者，其中一位是正在工作的鸟类学家在秘鲁的马努森林里，有一位来自秘鲁的老妇人。巴基斯坦，第四例皮肤病例是一名游客，其皮损自愈。最有趣的病人是本地人患有内脏利什曼病的扎伊尔人，可能是在爱琴海获得的患者前往雅典就诊时的希腊群岛大学。利什曼病经历的一个新特点去年是使用脂质体封装的两性霉素（Ambisome）用于治疗两种皮肤感染以及内脏的情况。 AmBisome 治疗的优点是治疗持续时间较短（10 天内 5 或 6 剂）与 Pentostam 相比（18-20 天）。