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所必需的。我们以前已经报道了小鼠的感染骨髓派生或炎症性巨噬细胞利什曼尼亚导致IL-12诱导的选择性损害途径。我们认为，因此感染了巨噬细胞将在没有的情况下驱动早期T细胞激活 IFN-G的主要生理诱导剂，这种影响解释了典型的细胞介导的控制机制的发作延迟甚至是利什曼病的自我限制形式。我们已经扩展了这些研究包括感染的细胞因子生产分析表皮Langerhans细胞（LC），因为是这些细胞的与皮肤中免疫反应的开始有关。使用允许Langerhans细胞样细胞为的培养系统经常从C57BL/6胎儿皮肤（胎儿）扩展和孤立皮肤衍生的树突状细胞fsddc）我们已经开始研究树突状细胞寄生虫相互作用。 L.主要的amastigotes，但不是 FSDDC很容易将Metacyclic前寄生虫化。寄生虫内在化与FSDDC的激活有关由MHC I类和II表面抗原的上调表现出来，共刺激分子的表达增加（CD40，CD54，以及CD86）和IL-12在整个18小时的时间内释放。尽管组织巨噬细胞也很容易内在化的杂物，但寄生虫的内在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。主要的amastigotes对于开发保护性TH1很重要主要的抗利什曼原虫免疫，只要感染仅限于巨噬细胞，细胞介导的发作将避免免疫反应。研究已经完成评估了临床的毒性，免疫原性和功效等级杀死了Biobras在巴西生产的利什曼原虫疫苗猴子中的rhuil-12（Macaca Mulatta）。十二只猴子接收校友吸收的两种皮内接种被杀的疫苗加上 IL-12受到完全保护，免受同源挑战与L. Amazonens。除了控制中的16只动物中的一只组患有典型的皮肤溃疡。保护是与血清和培养中的高水平IFN-G相关用抗原在体外刺激的PBL的上清液。疫苗接种使用IL-12和明矾作为佐剂杀死的抗原是安全的，并且在这个灵长类动物模型中有效。计划进行I期临床试验。尽管无响应率急剧增加在印度卡拉 - 阿萨尔（Kala-Azar）进行锑疗法，这是这种可能性可能是由于L. Donovani尚未得到证明。 L的敏感性。 Donovani分离株与钠葡萄糖酸钠（SAG）为在体外使用感染的巨噬细胞进行比较。分离株是从印度比哈尔邦的患者的脾脏活检获得要么没有响应（18），要么对一个或多个响应（10） SAG治疗课程。强相关性（p <0.005）仅在分析菌株时观察到临床反应作为细胞内膜片；响应式隔离材料/50 \ = 2.5？2.8 ？g/ml;无反应的隔离株ED/50 \ = 7.2？4 ug/ml。从这些数据中我们得出的结论是，印度的治疗失败是由于多诺瓦氏乳杆菌的抗抗毒性菌株感染。之间 1997年7月1日和1998年6月30日，五名疑似患者在NIH临床中心看到并评估了利什曼病。五个中有四例是皮肤病的病例。一个是和平来自毛里塔尼亚的军团志愿者是一名鸟类学家在秘鲁的马努森林中，一个是一个老妇巴基斯坦和第四个皮肤案是一名游客自发治愈。最有趣的病人是本地人 Zairean具有内脏利什曼病，可能是在爱琴海获得的当患者参加雅典时，希腊岛大学。利什曼病经历的一项新功能过去的一年是使用脂质体封装的两性霉素（Ambisome）治疗两种皮肤感染的治疗以及内脏的情况。 Ambisome治疗的优势是治疗的持续时间较短（10天内5或6剂）与Pentostam相比（18-20天）。