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Inmunología y contagio

在 Vivo 感染利什曼病亚马孙病评估小鼠的寄生虫毒性

Published: February 20, 2020
doi:
10.3791/60617
, , , ,
1Department of Physiology, Institute of Bioscience,University of Sao Paulo

Summary

在这里，我们提出了一个编译的协议，以评估小鼠与利什曼病的皮感染。这是研究寄生虫毒性的可靠方法，允许对脊椎动物宿主对感染的反应进行系统观察。

Abstract

利什曼病是原生动物寄生虫，导致利什曼病，疾病呈现从皮肤到内脏病变的各种临床表现。目前，全世界估计有1 200万人感染利什曼病，超过10亿人生活在感染风险之中。利什曼病在中美洲和南美洲流行，通常导致该疾病的皮状形式，可以直接在动物模型中可视化。因此，L. 亚马孙菌株是治疗皮性利什曼病研究的良好模型，因为它们也易于在体外培养。C57BL/6小鼠模仿人类观察到的L.亚马逊-亚马孙病驱动疾病进展，被认为是治疗皮性利什曼病的最佳小鼠菌株模型之一。在脊椎动物宿主中，这些寄生虫栖息于巨噬细胞中，尽管这些细胞具有防御机制。几项研究使用体外巨噬菌体感染测定法来评估不同条件下的寄生虫感染率。然而，体外方法仅限于一个孤立的细胞系统，它无视生物体的反应。在这里，我们编译了一种体内鼠感染方法，该方法提供了宿主-寄生虫相互作用的系统生理概述。C57BL/6小鼠体内感染L.亚马孙病的详细方案包括寄生虫分化成感染性乳腺、小鼠脚垫皮肤接种、病变发展以及寄生虫负荷测定。我们建议这种成熟的方法，作为生理研究宿主免疫和代谢反应对皮性利什曼病的最适当的方法。

Introduction

利什曼病是全世界流行的寄生虫传染病，在发展中国家面临重大挑战，被世界卫生组织1、2号确认为最重要的被忽视的热带病之一。利什曼病的特点是皮面、黏膜和/或内脏表现。皮内利什曼病通常是由L.亚马逊西斯，L.墨西哥，L.巴西兰西斯，L.Guyensis，L.主要，L.热带和L.aethiopica3。这种形式的疾病往往是自我修复在人类由于诱导保护性细胞免疫反应。然而，细胞免疫反应可能失败，并且疾病可以进展到传播皮性利什曼病4，5。由于利什曼病物种和宿主遗传背景的多样性，没有可用的疫苗。治疗方案也有限，因为目前可用的大多数药物要么昂贵，有毒，要么可能需要长期治疗8，9。此外，有报告称，对现有的治疗方法有抗药性10，11。

利什曼病的病原体是原生动物寄生虫利什曼病。寄生虫在其生命周期中呈现两种截然不同的形态形式：在沙蝇中发现的鞭状体;在沙蝇中发现的鞭状体;和乳腺，在哺乳动物宿主巨噬细胞的寄生虫中发现细胞内形式12，13。尽管脊椎动物宿主的巨噬细胞具有防御机制，但阿马斯蒂戈特的入侵、生存和复制能力仍受到许多研究，研究14、15、16、17。因此，一些研究小组一直在描述体外巨噬细胞感染测定，以评估特定环境因素以及寄生虫和宿主基因对寄生虫感染性的影响。这种测定提供了几个优点，如能够适应高通量格式的研究，获得结果的时间相对较短，以及减少牺牲的实验室动物数量。然而，体外测定的发现是有限的，因为它们并不总是在体内研究14，19，20，21复制。体内检测为宿主-寄生虫相互作用提供了系统生理概述，外体测定不能完全模仿。例如，免疫学研究可以通过免疫组织化学测定从收集的足垫组织部分，甚至从波普利特淋巴结进行分析恢复的免疫细胞22。

动物在生物和生物医学研究中经常被用作人类疾病的模型，以更好地了解疾病的基本生理机制23。在利什曼病的情况下，接种的路线、地点或剂量影响疾病结果24，25，26，27。此外，人类和小鼠的易感性和抗药性受到宿主和寄生虫4、5、22、28、29、30、31的遗传背景的高度调节。BALB/c小鼠极易受到L.亚马逊斯皮感染，随着寄生虫传播到淋巴结、脾脏和肝脏32，疾病进展迅速。由于该疾病可能进展到皮质转移，感染可能是致命的。相比之下，C57BL/6小鼠在L.亚马逊感染测定33中经常出现慢性病变，具有持久性寄生虫负荷。因此，L. amazonensis感染这种特殊的小鼠物种一直被认为是研究人类长期形式皮性利什曼病的极佳模型，因为它比BALB/c小鼠感染模型5，34更好地模仿疾病进展。

因此，我们提出，体内的鼠虫感染是利什曼病毒性生理研究适用于人类疾病的有用方法，允许对宿主-寄生虫相互作用的系统看法。重温已建立的测定方法22，我们在此提出一个汇编的C57BL/6小鼠体内感染L.亚马逊酶的分步方案，其中包括寄生虫分化成轴状肌瘤、小鼠脚垫皮肤接种、病变发展以及寄生虫负荷测定。该协议可以适应其他小鼠菌株和利什曼病物种，导致皮性利什曼病。总之，这里介绍的方法对于确定新的抗利什曼病药物靶点和疫苗，以及对宿主免疫和代谢对利什马尼亚感染的生理研究至关重要。

Protocol

圣保罗大学生物科学研究所动物护理和使用委员会（CEUA 342/2019）核准了所有实验程序，并根据圣保罗州保护和使用实验室动物的建议和政策（Lei Estadual 11.977， de 25/08/2005）和巴西政府（莱联邦11.794，de 08/10/2008）。第 1-5 节中描述的所有步骤都应无菌地在层流柜内执行。在处理活的利什曼病寄生虫时，应使用个人防护设备。 1. 在体外分化L. 亚马孙普罗马斯蒂戈特到…

Representative Results

利什曼原生动物寄生虫在无脊椎动物和脊椎动物宿主的生命周期中以两种发育形式存在：原生动物，在雌性沙蝇的流明中发现的增殖形式;和乳腺，在哺乳动物宿主细胞的寄生虫中发现的增殖形式。前列腺有一个长长的体，宽约1.5微米，长20微米，通常从前肢出现一个旗杆。Amastigots 的体形为圆形或卵形体，长度为 2-6 μm，宽度为 1.5-3 μm，并具有明显的旗杆<sup class="xr…

Discussion

本议定书中描述的体内感染测定允许任何研究人员评估体内皮性利什曼病，考虑在系统场景中的宿主-寄生虫相互作用。这些测定方法已被许多组使用22，24，27，29，31，32，34，49，在这里我们编译了一个分步协议，以标准化这种方法，同时考虑一些组可能有的基础设施限制。…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

我们要感谢圣保罗大学生物医学研究所动物中心的尼尔斯·奥尔森·萨拉伊瓦·塞马拉教授的支持和西尔维亚·雷尼·乌利亚纳教授提供玻璃组织研磨机。这项工作得到了圣保罗研究基金会（FAPESP – MFLS赠款2017/23933-3）的支持。

Materials

96-well plate Greiner bio-ne 655180 A flat-bottom plate for limiting dilution assay
adenine Sigma A8626 Supplement added to M199 cell culture media
caliper Mitutoyo 700-118-20 A caliper to measure the thickness of footpad
cell culture flask Corning 353014 A 25 cm2 volume cell culture flask to cultivate Leishmania parasite
centrifuge Eppendorf 5804R An equipament used for separating samples based on its density
CO2 incubator 34 °C Thermo Scientific 3110 An incubator for amastigotes differentiation
ethanol Merck K50237083820 A disinfectant for general items
fetal bovine serum Gibco 12657-029 Supplement added to M199 cell culture media
glass tissue grinder tube Thomas Scientific 3431 E04 A tube to collect and disrupt infected footpad tissue
glucose Synth G1008.01.AH Supplement added to M199 cell culture media
GraphPad Prism Software GraphPad A software used to plot the data and calculate statistical significance
hemin Sigma H-2250 Supplement added to M199 cell culture media
HEPES Promega H5303 Supplement added to M199 cell culture media
incubator 25 °C Fanem 347CD An incubator for promastigotes cultivation
inverted microscope Nikon TMS An equipament used to visual analyze the promastigote and amastigote cultures
isoflurane An inhalant anesthetics for mice (3-5%)
laminar flow cabinet Veco VLFS-09 A biosafety cabinet used for aseptical work area
M199 cell culture media Gibco 31100-035 A cell culture media for Leishmania cultivation
microcentrifuge tube Axygen MCT150C A microtube used for sample collection, processing and storage
multichanel pipette Labsystems F61978 A multichannel pipette used for limiting dilution assay
NaHCO3 Merck 6329 Supplement added to M199 cell culture media
NaOH Sigma S8045 Supplement added to M199 cell culture media
Neubauer chamber HBG 2266 A hemocytometer to count the parasite suspension
optical microscope Nikon E200 An optical equipament used to count parasite
parafilm Bemis 349 A flexible and resistant plastic to seal the plate
penicillin/streptomycin Gibco 15140122 Supplement added to M199 cell culture media
Petri dishes TPP 93100 A sterile dish to dissect the footpad tissue
pipetman kit Gilson F167360 A micropipette kit containing four pipettors (P2 P20 P200 P1000)
scale Quimis BG2000 An equipament used to weigh collected footpad lesions
scalpel Solidor 10237580026 A scalpel to cut and collect footpad tissue
serological pipette 10 mL Nest 327001 A sterile pipette used for transfering mililiter volumes
tips Axygen A pipette tip used for transfering microliter volumes
Trypan blue Gibco 15250-061 A dye used to count viable parasites
trypticase peptone Merck Supplement added to M199 cell culture media
tuberculin syringe BD 305945 A syringe with 27G needle to inoculate the parasite suspension
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Leishmania AmazonensisIn Vivo InfectionVirulenceCutaneous LeishmaniasisMouse ModelPromastigotesAmastigotesSubplantar InjectionLesion ProgressionParasite Quantification

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Aoki, J. I., Hong, A., Zampieri, R. A., Floeter-Winter, L. M., Laranjeira-Silva, M. F. In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice. J. Vis. Exp. (156), e60617, doi:10.3791/60617 (2020).
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