这项工作得到了国家卫生研究院资助 RO1 AI067979 NWA。 YK 是霍华德休斯医学院/马里兰大学帕克分校的本科生奖学金获得者。

所有的实验程序都是按照《国家卫生研究院护理和使用实验动物指南》的建议进行的, 并得到马里兰大学 IACUC 的批准。1至4节所述的所有步骤都应在生物层流柜内灌装进行。应使用个人保护, 在处理所有试验阶段的活的利什曼原虫寄生虫时应谨慎行事。
1. 骨髓源巨噬细胞的分离和分化 (BMMs)8,30,31
<ol><li>
0 天:在 CO2室中牺牲 4-6 周大的雌性C57BL/6或BALB鼠标, 并通过颈椎脱位确认死亡。确保剪刀和镊子的灭菌, 使其浸泡在70% 乙醇。喷雾手套与70% 乙醇, 以允许无菌处理整个骨髓细胞隔离过程。</li>
	<li>通过用70% 乙醇喷洒丰富, 将其前肢固定, 并对其进行消毒。</li>
	<li>在髋关节附近用剪刀做一个小切口 (约1厘米)。小心地在皮肤下插入剪刀以分离底层肌肉, 并在髋关节周围切开皮肤。 注: 解剖板可以旋转, 以允许改善对髋关节的接触。</li>
	<li>小心 deglove 的皮肤从腿部拉向脚踝和删除。在脚踝上切下脚, 小心地在脚踝关节处切开, 使胫骨完全完好无损。 注: 在这个阶段的腓骨是松散的连接, 可以很容易地与镊子分离。</li>
	<li>通过操纵股骨来手动定位髋关节以确定旋转点。小心使用剪刀切断髋关节以上的腿, 使股骨的近端头部完好无损。</li>
	<li>用剪刀从腿部取出尽可能多的肌肉和结缔组织。从脚踝和髋关节上方的任何多余的骨材料中取出任何剩余的皮肤。</li>
	<li>安置被清洗的腿骨头在无菌 RPMI 补充青霉素或链霉素 (1% 最后的集中) 在培养皿在冰。</li>
	<li>重复步骤1.4 至 1.6, 把胫骨和股骨从另一条后腿分离出来。</li>
	<li>用灭菌钳和无菌 #10 刀片从 RPMI 浸泡的骨骼中取出残余的肌肉和结缔组织。</li>
	<li>把骨头放在培养皿盖子上。用刀片在踝关节上方仔细切开胫骨以进入骨髓。将胫骨从腿部的其余部分切除, 然后立即在膝关节下方切开。</li>
	<li>绘制10毫升无菌 RPMI 补充青霉素/链霉素 (1% 最终浓度) 成10毫升注射器和附加25G 针。</li>
	<li>用镊子牢牢地握住胫骨, 在冰上垂直超过50毫升锥形管。用注射器轻轻地注入大约5毫升 RPMI 到骨头的末端, 将骨髓从另一端冲洗成锥形管。用总共10毫升 RPMI 的骨髓冲洗, 直到骨头变成白色。 注: 彻底冲洗后, 骨应变白。如果没有, 继续冲洗, 以去除骨髓。胫骨远端可能需要修剪与刀片, 如果它是太窄, 以插入针。</li>
	<li>立即将股骨置于膝关节上方, 并立即在髋关节下方切开, 露出骨的两端以进入骨髓。用与胫骨相同的方式从股骨中冲洗骨髓, 共10毫升 RPMI。</li>
	<li>重复清洗和冲洗步骤1.6 至1.13 与其余的骨骼和收集骨髓冲洗细胞在一个50毫升锥管 注: 如果骨冲洗前在解剖过程中的任何一点骨折, 不要使用骨髓从这块骨头。</li>
	<li>离心机用于从所有四块骨头中提取骨髓的锥形管, 10 分钟4摄氏度, 300 x g。</li>
	<li>放置60毫升无菌法钢公司培养基 (RPMI 的最终浓度为 20%, 1% 青霉素/链霉素, 1.2 毫米 Na-丙酮酸, 25 µg/毫升人类脑脊液) 成 175厘米2 细胞培养瓶与过滤帽。</li>
	<li>用5毫升的法钢公司培养基从瓶中并用重悬, 在离心后丢弃上清液, 然后用温和向上和向下吹打细胞颗粒。</li>
	<li>将细胞悬浮液转移到培养瓶中, 用法钢公司培养基从培养瓶中冲洗两次锥形管, 以保证细胞的最大回收率。确保细胞悬浮液彻底混合成培养基, 并将30毫升从最初的175厘米2细胞培养瓶中分配到第二175厘米2细胞培养瓶中, 过滤器盖。</li>
	<li>孵化两个含有30毫升细胞悬浮液的烧瓶, 每晚在37摄氏度, 5% CO2 , 以允许分离任何污染的成纤维细胞, 居民巨噬细胞, 和其他黏附体细胞。</li>
	<li>
1 天:将含有未分化骨髓细胞的上清液从烧瓶转移到100毫米 x 15 毫米非 TC 处理的聚苯乙烯培养皿, 约10毫升/碟, 孵育37°c, 5% CO2.扔掉烧瓶。</li>
	<li>
3 或4天:添加一个额外的5毫升法钢公司培养基 (预热到37°c) 到每个培养皿, 并继续孵化37°c, 5%CO2。</li>
</ol>2. 盖玻片感染的电镀 BMMs (7 或8天)
<ol><li>在每个空井的底部放置四 (4) 无菌12毫米玻璃盖玻片 (蒸压并贮存在密闭的玻璃容器中), 准备6井组织培养板。 注: 选择每一个无菌12毫米盖玻片与一个吸气尖端安装在真空线。将盖玻片放入井中, 不加重叠, 通过捏管以中断真空, 释放在所需位置。</li>
	<li>从培养皿上涂上粘附骨髓巨噬细胞的培养基 (以及任何非粘附单元), 轻轻冲洗黏附细胞2x 无菌 Dulbecco 磷酸盐缓冲盐水无钙和氯化镁 (PBS-/) 预预热到37°c。</li>
	<li>添加滴状, 5 毫升无菌 1x PBS-/与最终浓度为1毫米 EDTA 每培养皿和孵化5分钟在37°c, 以分离黏附 BMMs. 验证单元格是否使用显微镜分离。</li>
	<li>收集所有分离的细胞悬浮在 1x PBS-/补充与1毫米 EDTA 在50毫升圆锥管。用5毫升 PBS 冲洗每道菜 2x, 并加入细胞悬浮池。 注: 细胞悬浮可能被稀释与额外的 PBS--以保护巨噬细胞从 EDTA 毒性在收集过程中。</li>
	<li>离心机在 300 x g 10 分钟在4摄氏度。用吹打将上清和并用重悬巨噬细胞在 5-10 毫升法钢公司培养基中丢弃。在冰上放置悬浮细胞, 以防止附着在管子和结块。</li>
	<li>在 hemocytometer 中使用台盼蓝排除计数可行细胞。 注: 要计数法钢公司细胞悬浮在5毫升培养基中, 稀释混合25µL 细胞悬浮在445µL PBS-/和30µL 0.4% 台盼蓝解决方案通常允许简单的量化在 hemocytometer (考虑到20x 稀释因子的最终计算).仅计数没有被台盼蓝染色的细胞 (可行细胞)。如果这种混合物过于浓缩或稀释, 细胞悬浮量可能会改变。一般情况下, 每个准备法钢公司的收率在 2 x10 7 和 5 x 10 7 单元格之间从单个鼠标中变化.</li>
	<li>在所需的电镀浓度 (5 x 105/毫升) 和分散悬浮到6井板中, 在2毫升介质中准备法钢公司介质中的悬浮液, 以便每个井接收 1 x10 6 巨噬细胞。 注意: 这是一个关键步骤, 确保每一个井得到足够数量的巨噬细胞均匀地覆盖盖玻片。检查井中的盖玻片不重叠或漂浮在介质中。如果是这样, 用不育的吸管尖轻轻调整。</li>
	<li>孵化过夜在37°c, 5% CO2。</li>
</ol>3. 对感染形式的amazonensis的纯化
注意:使用标准 L 前鞭毛体从固定 promastigote区域性8、13或将区域性中的 amastigote 区分为 amazonensis 形式, 为感染-净化 metacyclic 前鞭毛体准备利什曼原虫。无菌差异协议 6, 8.
<ol><li>
使用密度梯度介质 (材料) 对利什曼原虫metacyclic 前鞭毛体进行纯化33<ol>
<li>在无菌无内毒素水中制备40% 种密度梯度介质的溶液。</li>
		<li>稀释密度梯度溶液在 10x M199 培养基 (不含血清) 中制备10% 浓度的 M199 培养基。</li>
		<li>通过0.22 µm 过滤器筛选所有解决方案。 注意: 库存解决方案可以在黑暗中存储 4 oC, 不超过1月。</li>
		<li>在15毫升锥形离心管的底部添加2毫升40% 密度梯度介质溶液。</li>
		<li>2毫升的10% 密度梯度介质溶液在 M199 上的40% 密度梯度介质层仔细, 使用巴斯德吸管, 以避免任何混合在两层之间。</li>
		<li>从固定相培养中收集 1 x 109寄生虫, 离心在 1900 x g 处以10分钟。</li>
		<li>并用重悬细胞在6毫升 Dulbecco 的修饰必需培养基 (DMEM)。</li>
		<li>2毫升的细胞悬浮在10% 密度梯度介质层的顶端, 轻轻地用巴斯德吸管避免在层间混合。</li>
		<li>在室温下, 离心机在 1300 x g 处的梯度为10分钟。 注意: 由于利什曼原虫种之间的物理特性不同, 每个可能需要稍微调整离心条件以确保最大产量。</li>
		<li>收集寄生虫 (丰富的 metacyclic promastigote 形式) 从形成在上层10% 密度梯度介质边界 (介面之间的0% 和10% 密度梯度介质层)。</li>
		<li>稀释寄生虫与一容量 DMEM 和收集通过离心 (1900 x g 10 分钟在室温)。</li>
		<li>并用重悬在500µL DMEM 培养基中, 计数 hemocytometer 以量化产量。</li>
	</ol>
</li>
	<li>
在无菌区域性条件下生成 amazonensis amastigotes (低 pH 值/高温 ) 6, 8, 13<ol>
<li>混合5毫升的对数相 promastigote 培养 (ph 7.4 在 26 oC) 与等量的 amastigote 培养基 (ph 4.5), 使用25厘米2烧瓶 (总10毫升培养基) 和孵化在26°c 过夜。</li>
		<li>将烧瓶从26°c 移至32摄氏度。</li>
		<li>在3或4天以后, 分裂文化1:5 在 amastigote 媒介在32°c。</li>
		<li>检查寄生虫在未来 3-4 天 (最多7天), 看看他们是否可以使用的感染。 注意: 健康的无菌 amastigotes 应该有一个椭圆形的形状, 没有可见的鞭毛。部份地被区分的 amastigotes 有一个大卵形与短鞭毛。这种文化不应该有大量的团簇--许多团簇是不生长的、垂死的寄生虫的征兆。amastigote 文化可以被分裂1:5 和保持至多3星期。</li>
	</ol>
</li>
</ol>4. 感染L. amazonensis
<ol><li>根据所需的语言 (通常为每巨噬细胞 3-5 metacyclic 前鞭毛体 (1:3 或 1:5) 和 1 amastigote 每巨噬细胞 [语言 1:1]) 稀释寄生虫悬浮液。在 PBS 中添加利什曼原虫--在体积为 50-100 µL 的每一个已包含 2 mL 介质的井中。</li>
	<li>用 metacyclic 前鞭毛体34o C BMMs 1 小时的 amastigotes 和3小时孵育, 以进行感染。 注: 最佳感染温度可能因种类而异。</li>
	<li>跟随孵化, 彻底地洗涤在每个井的自由寄生虫3x 与2毫升 PBS-预热到37°c。 注: 轻轻分散 PBS, 确保盖玻片不漂浮在一起。轻轻地旋转盘子, 然后吸出液体。如果使用多种寄生虫, 避免交叉污染, 使用单独的吸气尖端。</li>
	<li>固定1小时或3小时的时间点样品通过孵化每个井与 1.5-2 毫升2% 粉煤灰在 pbs-/-为10分钟洗3x 与 pbs-/。不要在 PBS 中吸入最后的洗涤和冷藏的盘子, 直到染色盖玻片。</li>
	<li>增加2毫升新鲜法钢公司培养基到每个井在板材为进一步时间点和孵化在34°c。</li>
	<li>按照步骤4.3 中的描述, 修正剩余的时间点, 并冷藏盘子直到染色。</li>
</ol>5. DAPI 染色和盖玻片安装
<ol><li>从含有盖玻片的水井中吸出 pbs, 并添加1.5 毫升 pbs--与0.1% 非离子洗涤剂。在室温下孵育10分钟, 然后用2毫升 PBS-/-进行3x 洗涤。</li>
	<li>添加1毫升 PBS-/-与2µg/毫升 DAPI (稀释从5毫克/毫升溶液在水中) 到含盖玻片的水井, 在室温下孵育另1小时。 注意: DAPI 的潜伏期对细胞内利什曼原虫寄生虫的正确可视化至关重要。巨噬细胞细胞核染色迅速, 由于其大小和 DNA 含量较大, 但细胞内寄生虫的细胞核染色较慢。因此, 延长孵化时间超出所需的可视化法钢公司核是必要的, 以允许 DAPI 渗透和寄生虫的等离子膜, 和寄生虫核膜。通过适当的 DAPI 染色, 除了核 DNA 外, 寄生在鞭毛口袋附近的线粒体 kinetoplast dna 也可以被可视化。</li>
	<li>用2毫升 PBS 冲洗每一个含盖玻片3x 的井, 然后用镊子抬起并翻转盖玻片, 将细胞侧向下并安装在玻璃显微镜上, 并提供商用 antifade 安装试剂。 注意: 盖玻片非常脆弱, 需要谨慎处理。避免给他们施加压力, 尤其是在提升时对井侧壁施加压力。添加几滴 PBS--减少了盖玻片和井之间的表面张力, 促进了提升过程。细量规针可用于帮助从井底窥探盖玻片。在幻灯片上放置一个盖玻片后, 轻轻地按下钳子, 以推出安装介质中的气泡。如果盖玻片在起重过程中损坏或有可能翻转, 请勿重新装载;只需使用备用的第四盖玻片。</li>
	<li>将幻灯片冷藏至量化。</li>
</ol>6. 感染量化
<ol><li>在荧光显微镜下检查 DAPI 染色的幻灯片, 方法是使用100X 物镜与浸没油 (358 nm 的激发) 来聚焦巨噬细胞; 最佳排放量为 461 nm。 确保焦点在玻璃滑块和盖玻片之间的巨噬细胞层上。</li>
	<li>量化的巨噬细胞数量 (与 DAPI 染色的大核) 和小的 amastigote 核聚集在每个巨噬细胞核周围 (参见图 2A, DAPI) 为每个视野, 使用手动计数器。 注: Amastigote 核由于体积小, 在一平面上可能不会全部可见。计算那些在巨噬细胞核上可见的, 然后用细焦点检查在巨噬细胞核上方和下方的飞机上的寄生虫核。</li>
	<li>移动到另一个视野, 重复量化。使用单独的计数器键跟踪计数的字段数。在每个盖玻片中通过并行行中的可视字段移动, 以防止计数重叠。</li>
	<li>每盖玻片的最小200只巨噬细胞数量。量化每一次感染的时间点 (3 盖玻片)。如果前一个不适合计数由于盖玻片重叠, 安装不良, 玻璃破损, 等, 请计算第四盖玻片。 注: 如果200只巨噬细胞不能在任何一个盖玻片上计数, 请计算备用的第四。</li>
	<li>计算感染率为 amastigotes/巨噬细胞或 amastigotes/100 巨噬细胞, 并确定从原始量化数据中感染的巨噬细胞百分比。</li>
</ol>

作者声明他们没有竞争的金融利益,

上述法钢公司感染试验所产生的定量数据, 使调查人员能够在相对较短的时间内获得感染率和可靠地测定毒力特性的变化 (最多2周, 比2体内实验所需的月份)。这种方法依赖于 DNA 特异染料 DAPI, 专门染色巨噬细胞和寄生虫细胞核, 并允许快速识别和量化感染细胞。相比之下, 其他污渍, 如姬姆萨绑定到大量不同的细胞分量, 具有不同的强度, 复杂的视觉分析37。使用 DAPI 允许识别胞内寄生虫及其与细胞结构的明显区别 (只有宿主细胞的细胞核也被染色, 它们的大小比寄生细胞核大数级), 允许更容易和更快地感染的量化。
与任何实验过程一样, 此方法有一些限制, 需要仔细执行的几个关键步骤。体内利什曼原虫感染在吞噬功能之前涉及复杂的先天免疫反应, 这决定了感染的终极过程38。因此, 模型小鼠应变的选择在研究设计中具有重要的意义。此协议中使用的C57BL/6和BALB/c小鼠菌株都承担了基因编码中的突变, 即天然抵抗相关巨噬细胞蛋白 (Nramp1), 一个质子外流泵, translocates Fe2 +和锰2 +从巨噬细胞溶酶/phagolysosomes 到细胞质的离子。这导致在巨噬细胞的吞室内复制的病原体的易感性增加8,30,31。成功地在巨噬细胞中的殖民化还取决于感染的利什曼原虫寄生虫的独特能力, 以操纵免疫反应, 以抑制/生存巨噬细胞激活39,40。用于利什曼原虫法钢公司感染的分化巨噬细胞有点模仿巨噬细胞的非活化状态体内, 但该化验并不解释更复杂的宿主成分, 影响毒力在动物模型。
隔离健康感染的利什曼原虫表单是准确的毒力测定的另一个绝对关键的要求。在低 pH 值/高温条件下41, 并非所有利什曼原虫菌株的所有前鞭毛体都能有效地区分为 amastigotes。因此, 感染是经常进行的 metacyclic 前鞭毛体纯化的固定 promastigote 文化。为了有效隔离 metacyclic 前鞭毛体, 一些纯化策略利用了不同生命期内寄生虫表面糖萼的变化, 包括 lipophosphoglycan (LPG) 的广泛修改。结构42,43,44。例如, 花生凝集素 (肽), 一个凝集素, 有选择地绑定到 procyclic 但不 metacyclic 液化石油气已有效地用于否定选择协议, 以净化L. 少校metacyclic 前鞭毛体45。Metacyclic promastigote 纯化策略L. amazonensis通常涉及单克隆抗体 mAb3A. 1 可以凝集 procyclic 前鞭毛体通过靶向特定的表面蛋白表位, 在 Metacyclic 无法访问表单由于表面糖萼修改 8, 13, 32.利用密度梯度介质将 metacyclic 前鞭毛体从前鞭毛体中分离出来, 这是一种很有吸引力的方法, 因为它不依赖于寄生虫表面配体的物种特异性变化。此方法最初为L. 主要metacyclic promastigote 纯化33, 已成功地通过了其他几个利什曼原虫物种, 主要是通过对离心条件的修改在密度-梯度沉淀46,47,48,49。
同样重要的是, 要了解可能会使分析的实施复杂化的问题, 这可能发生在过程中的几个步骤中。这些问题可能包括法钢公司提取过程中的污染、BMMs 的不成功分化、不一致的巨噬细胞电镀、感染性寄生虫形态的不完全纯化、不良或不一致的感染以及在显微镜幻灯片上安装玻璃盖玻片困难。这些可能性已在议定书中得到解决, 并可通过仔细评估感染程序的每一步骤来确定问题来解决。例如, 在分化过程中法钢公司文化的污染可能表明在提取过程中需要改进无菌技术。仔细注意纯化试剂的纯度和新鲜度, 并精确实施纯化协议, 可纠正不成功或不完全纯化所需的寄生虫形态。不良或不一致的感染可能是由于不准确的定量的寄生虫或教学语言太高或过低的特定实验条件。在提取, 操作和安装玻璃盖玻片可以说是最具技术挑战性的过程中, 这一技术;个人可能会发现他们更喜欢使用不同的工具来方便盖玻片的处理。DAPI 染色核的清晰可视化是显微镜下准确计数寄生虫的关键。微弱的染色和不适当的聚焦是两个主要的罪魁祸首在不一致的定量。这可以通过质量控制的 DAPI 染色步骤, 限制光暴露时间, 以减少照片漂白, 并迅速改变目标焦点, 以占所有寄生虫核的领域。DAPI 荧光亮度也可以通过保证样品的适当通透和洗涤剂的浓度来提高, 并且在染色过程中增加 DAPI 的含量。一种可以促进量化过程的替代方法是在显微检查过程中获得样品的数字图像。这些图像可用于以后的量化, 并可由独立调查员验证/量化。然而, 与这一进程相关的技术困难需要仔细考虑。由于 PV 的球形性质, 寄生虫并不总是在同一焦点平面上。这就需要在每个显微领域使用不同的焦平面获取多个图像, 然后组装起来, 以解释所有的寄生虫。否则, 从照片中量化是很有误导性的。在感染过程中, 巨噬细胞/场和 amastigotes/场的计算对于确保一致的巨噬细胞电镀和寄生虫的传播是至关重要的。由于 BMMs 是完全分化的细胞, 它们的数量不应该随着时间的推移而增加。如果宿主巨噬细胞的数量随着时间的推移而增加, 那就意味着巨噬细胞仍然不成熟。在这种情况下, 应检查脑脊液的来源和浓度。测定受感染的巨噬细胞的比例也有助于提供对宿主细胞-病原体相互作用的全面看法, 特别是在感染率和寄生虫负荷不遵循类似趋势的情况下20,21.
此处详述的体外法钢公司感染方法可根据具体的实验需要进行修改。可以对过程的任何基本步骤进行修改-法钢公司提取和分化, 纯化感染性寄生虫形态, 并量化体外巨噬细胞感染, 以及实施不同的感染mois 》杂志和调整感染期和时间点进行分析。培养基的成分也可以通过补充或消耗特定的营养素而容易地被修改, 并且可以同时评估多种不同的菌株或形式的寄生虫。这些修改可能需要进一步调整技术程序;例如, 在不同的利什曼原虫种中, amastigote 准备和 metacyclic promastigote 纯化的程序将有所不同。除了 DAPI 或荧光标记的蜂窝组件之外, 还可以使用其他荧光染料来可视化一系列宿主寄生虫交互过程6,24。此外, 这种分析可以适应高通量系统 (高温超导) 的格式, 这将允许快速筛选复合库, 以确定新的和有效的药物线索治疗利什曼病24。

利什曼病是指由利什曼原虫属的原虫寄生种类引起的广泛的人类疾病。目前约有1200万人感染了全球范围内的利什曼原虫, 3.5亿多名患者面临风险。疾病病理学依赖于利什曼原虫物种和寄主因素, 症状不同于无害的自我愈合皮肤病变和致命的 visceralizing 形式。如果未经治疗, 内脏利什曼病是致命的, 只有在疟疾后才会成为感染了原生动物寄生虫的最致命的人类疾病1。尽管疾病病理和症状存在广泛的差异, 但所有的利什曼原虫物种分别在昆虫和脊椎动物寄主的 promastigote 和 amastigote 阶段之间有 digenic 的生命周期交替。在脊椎动物体内,利什曼原虫目标宿主巨噬细胞侵入, 并诱导 parasitophorous 液泡 (PVs) 的形成, 酸性隔层具有高度剧毒 amastigote 形态复制的 phagolysosomes 特性。Amastigotes 持续在宿主组织中的慢性感染, 可以传递到未感染的白蛉, 完成传输周期。因此, 在人类疾病发展的背景下, amastigotes 是最重要的利什曼原虫生命周期表单2。调查 amastigotes 如何在巨噬细胞 PVs 中复制对于了解利什曼原虫毒力3,4,5,6,7和新的有效疗法的发展。
我们在这里描述了我们的实验室经常使用的方法来研究在骨髓源巨噬细胞 (BMMs) 中的利什曼原虫感染和复制, 这涉及到定量评估细胞内的利什曼原虫的数量随着时间的推移。该过程包括从小鼠骨髓中获取单核细胞和分化为培养的巨噬细胞,体外感染与感染型 (metacyclic 前鞭毛体或 amastigotes)的利什曼原虫和量化的每隔24小时胞内寄生虫的数量在感染后 72-96 小时内。本试验已在实验室中使用, 以确定几种环境因素和寄生虫基因的影响, 包括鉴定铁在促进L. amazonensis毒力方面的关键作用, 进一步验证了都安小鼠病变发育研究6,8,9,10,11,12,13,14,15.由于所有致病性的利什曼原虫物种都在宿主巨噬细胞内建立了它们的复制利基, 这种检测方法可普遍用于所有利什曼原虫物种的毒力测定。
执行法钢公司感染可以分析单个细胞层面上的寄主寄生虫相互作用, 从而更广泛地了解利什曼原虫寄生虫如何与他们的首选宿主微环境、巨噬细胞的 PVs 相互作用。巨噬细胞感染化验已成功使用多个组16,17,18,19,20,21,22 , 探索宿主巨噬细胞和利什曼原虫特定基因的功能, 以及它们在细胞内感染的复杂相互作用中的潜在参与。法钢公司感染允许将寄生虫生长量量化为对影响细胞内生存的宿主因素的影响的读出, 如杀菌剂一氧化氮生产、产生活性氧种类和其他不利条件在溶酶体样 PVs23中遇到。巨噬细胞感染的检测也被用来识别潜在的抗 leishmanial 药物治疗发展的线索13,24。
法钢公司感染的体外性质比其他评估利什曼原虫毒力的方法具有多种优势。然而, 以前的几项研究表明, 随着时间的推移, 细胞内寄生虫存活的机制并没有量化感染率为20,21,24。此外, 许多研究侧重于以下的体内感染随着时间的推移, 通过测量皮肤病变大小和其他生理症状只是间接相关的寄生虫复制25,26,27.体内感染是一种严格的评估寄生虫毒力的方法, 但仅基于都安肿胀的病变大小测量往往是不够的, 因为它们反映了感染组织的炎症反应, 而不是寄生虫的绝对数量。因此, 都安病变的发展化验必须遵循量化的寄生虫负荷的感染组织, 这一程序需要冗长的限制稀释化验28。此外,体内研究通常涉及在不同时间点牺牲多个动物以提取感兴趣的组织6,8,9,10,11,13. 相比之下, 大量的 BMMs 可以从一只动物身上获得, 这些细胞可以在允许在不同时间点对感染进行评估的条件下进行镀膜。此外, 与体内研究相比, 执行体外法钢公司感染可以更好地控制实验条件。量化的巨噬细胞感染与寄生虫本身允许精确控制感染的多样性 (语言) 和文化条件。对这些因素的精细控制是识别离散细胞通路特征和了解它们对感染过程的影响的关键。
考虑到这些优势, 对于研究利什曼原虫毒力的少数群体迄今充分利用细胞内复制的定量评估, 这一点令人吃惊。在本文中, 我们讨论了可能妨碍更广泛地利用此检测的常见缺陷, 并提供了一步一步的协议, 以促进其适当的实施。考虑到它的精确性和通用性, 我们在这里描述的法钢公司感染检测方法不仅可以用来探索影响利什曼原虫毒力的寄主病原体相互作用, 而且还能研究在体内复制的其他微生物。巨噬细胞29。重要的是, 这种检测也可以作为一种快速、经济的临床前筛选方法, 用于抗 leishmanial 药物的开发。

<table>
	<tbody>
		<tr>
			<td>6 well cell culture plate</td>
			<td>Cellstar</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Acros Organics</td>
			<td>AC147440250</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipet Tips (100-1000 &#956;L)</td>
			<td>Fisherbrand</td>
			<td>02-707-404</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipet Tips (20-200 &#956;L)</td>
			<td>Fisherbrand</td>
			<td>02-707-430</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipet Tips (2-20 &#956;L)</td>
			<td>Fisherbrand</td>
			<td>02-707-432</td>
			<td></td>
		</tr>
		<tr>
			<td>Bard-Parker Rib-Back Carbon Steel Surgical Blade #10</td>
			<td>Aspen Surgical</td>
			<td>371110</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Luer-Lok Tip 10 mL Syringe</td>
			<td>Becton Dickinson (BD)</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Precisionglide Needle, 25G&#160;</td>
			<td>Becton Dickinson (BD)</td>
			<td>305124</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish 35 mm x 10 mm</td>
			<td>Cellstar</td>
			<td>627 160</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Flask</td>
			<td>Cellstar</td>
			<td>660175</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glasses: 12 mm circles</td>
			<td>Fisherbrand</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Biotin</td>
			<td>J.T. Baker</td>
			<td>C272-00</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol 200 proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 100 mm x 15 mm non-TC-treated polystyrene Petri dish</td>
			<td>Corning</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Seradigm</td>
			<td>1500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll400</td>
			<td>Sigma Aldrich</td>
			<td>F8016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Nikon</td>
			<td>E200</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG Texas red</td>
			<td>Invitrogen</td>
			<td>T-862</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG AlexaFluor488</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Tokyo Chemical Industry Co. LTD</td>
			<td>H0008</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoton II Diluent</td>
			<td>Beckman Coulter</td>
			<td>8546719</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gemini</td>
			<td>400-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199 (10X)</td>
			<td>Gibco</td>
			<td>11825-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Na pyruvate (100 mM)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Alfa Aesar</td>
			<td>43368</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gemini</td>
			<td>400-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (-/-)</td>
			<td>ThermoFisher</td>
			<td>14200166</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropyline conical Centrifuge Tubes 15 mL</td>
			<td>Cellstar</td>
			<td>188 271</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropyline conical Centrifuge Tubes 50 mL</td>
			<td>Cellstar</td>
			<td>227 261</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Lamp-1 antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>1D4B</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>PeproTech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Reichert Bright-Line&#160; Hemocytometer&#160;</td>
			<td>Hausser Scientific</td>
			<td>1492</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640 (1X)</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 Surfactant</td>
			<td>Millipore Sigma</td>
			<td>TX1568-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Scissors, 4 1/2&#34;</td>
			<td>VWR</td>
			<td>82027-582</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Forceps, Fine Tip</td>
			<td>VWR</td>
			<td>82027-386</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>VWR</td>
			<td>16004-368</td>
			<td></td>
		</tr>
		<tr>
			<td>Z1 Coulter Particle Counter, Dual Threshold</td>
			<td>Beckman Coulter</td>
			<td>6605699</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of intracellular growth inside macrophages is a fast and reliable method for assessing the virulence of leishmania parasites

利什曼原虫的生命周期, 利什曼病的致病剂, 分别在昆虫和脊椎动物寄主 promastigote 和 amastigote 阶段交替。虽然利什曼病的致病症状可能有很大差异, 从良性皮肤病变到高度致命的内脏疾病的形式取决于感染的物种, 所有的利什曼原虫物种驻留在宿主巨噬细胞在脊椎动物阶段他们的生命周期。因此,利什曼原虫传染性直接关系到它在巨噬细胞内 parasitophorous 泡 (PVs) 中侵入、生存和复制的能力。因此, 评估寄生虫复制 intracellularly 的能力是确定毒力的可靠方法。研究利什曼病的发展使用动物模型是费时, 繁琐, 往往是困难的, 特别是与 pathogenically 重要的内脏形式。我们这里描述了一个方法来遵循的细胞内发育的利什曼原虫骨髓源性巨噬细胞 (BMMs)。细胞内寄生虫的数量是确定的24小时间隔为 72-96 h 继感染后。这种方法可以可靠地确定各种遗传因素对利什曼原虫毒力的影响。举例来说, 我们展示了单个等位基因删除利什曼原虫线粒体铁转运基因 (LMIT1) 如何削弱利什曼原虫 amazonensis突变株LMIT1/ΔLmit1在 BMMs 内生长的能力,反映了与野生型相比, 毒力的急剧减少。这种检测还允许精确控制实验条件, 可以单独操作, 分析各种因素 (营养素、活性氧种类、等等) 对寄主-病原体相互作用的影响。因此, 适当的执行和量化的法钢公司感染研究提供了一个非侵入性, 快速, 经济, 安全和可靠的替代传统动物模型研究。

利什曼原虫有两种受感染的窗体-metacyclic 前鞭毛体, 它们区别于区域性的固定阶段的 procyclic 前鞭毛体, amastigotes 是胞内阶段(图 1)。在某些利什曼原虫种 (如L. amazonensis) 中, amastigotes 也可以通过将 promastigote 细胞转移到较低的 pH 值 (4.5) 和高温 (32 °c), 与法钢公司中发现的条件类似, 在无菌文化中进行区分 PVs8,34. 只有 metacyclic promastigote 或 amastigote 形式的利什曼原虫才能诱导形成 PVs 和复制 intracellularly 后被宿主巨噬细胞35吞噬。未分化的测井阶段前鞭毛体是无毒性的, 无法促进光伏形成 (图 2)。
在感染 metacyclic 前鞭毛体的情况下, 通常有24小时的延迟, 然后才增加胞内寄生虫数量。发生这种情况的原因是 metacyclic 前鞭毛体首先在开始复制之前将其区分为 amastigotes。因此, metacyclic 前鞭毛体引发的感染需要更长的时间来显示胞内寄生虫总数的增加, 因此, 可能需要孵化较长的点。
图 2A表示成功的感染, 使用 axenically 分化 amastigotes, 显示在初始感染 (1 小时) 后, 在 PVs 形成之前, 细胞内定位的利什曼原虫(以红色), 融合 phagosomes 与溶.在48小时, 可以观察到有多种寄生虫的不同的大 PVs。PVs 内寄生虫数量的稳步增加是致命的利什曼原虫感染的特征。相反, 非致命的对数相位前鞭毛体(图 2B) 无法启动 PV 开发 (48 小时), 无法复制, 最终在宿主巨噬细胞内被杀死, 即使在可比较数量的宿主巨噬细胞占去时metacyclic 前鞭毛体或 amastigotes。
为了演示此方法的有效性, 我们将野生类型的amazonensis与LMIT1/ΔLmit1寄生虫进行比较, 其中包含LMIT1 (Leishmania Mitochondrial我ron T 的单个副本)。ransporter 1) 基因, 导致线粒体铁进口受损13。当与野生型前鞭毛体13 相比, 丢失一个LMIT1 等位基因不会导致LMIT1/ΔLmit1 前鞭毛体的线粒体活动发生重大改变或降低纯化 metacyclic 的产量。从野生类型 (metacyclic) 和 LMIT1/ΔLmit1固定区域性纯化的前鞭毛体在入侵 BMMs 中同样有效, 其基础是在感染后量化1小时的胞内寄生虫数量 (图 3A, 1 h)。在初始24小时滞后后, metacyclic 前鞭毛体的适应和分化为 amastigote 形式, 细胞内野生型寄生虫数量的稳步增加 (约3倍于24小时和72小时之间) 是观察.相比之下, 观察到LMIT1/ΔLmit1寄生虫 (比较1小时和 72 h 时间点) 的胞内生长很少或没有, 这表明单个LMIT1等位基因的丢失会显著影响这种菌株在体内生长的能力。噬.LMIT1 蛋白在补充菌株中的 Episomal 表达 (LMIT1/ΔLmit1+LMIT1) 将胞内寄生虫的生长从野生型水平中解救出来, 确认LMIT1对 amastigote 细胞内复制和毒力13。
巨噬细胞感染进行与无菌 amastigotes, 由转移 promastigote 文化 (在 ph 值 7.4; 26 o c) amastigotes 生长条件 (ph 4.5; 32 o c) 34, 36, 显示细胞内生长 模式 (图 3B) 类似于用 metacyclic 前鞭毛体观察到的(图 3A)。在初始吸收的可比较级别 (图 3B、1 h) 野生类型 (小波) 和 LMIT1 补充 (LMIT1/ΔLmit1+LMIT1) amastigotes 稳步增长, 而LMIT1/ΔLmit1 amastigotes 再次未能显示细胞内生长。无菌 amastigotes 更具感染性 (语言1:1 比1:5 为 metacyclic 前鞭毛体), 并在 PVs 更有效地复制。
我们的数据显示了 metacyclic promastigote 感染的初始24小时延迟, 在胞内寄生虫数量增加之前 (在图 3A和3B中的 24 h 时间点之间进行比较)。滞后表示内部化 metacyclic 前鞭毛体在开始复制之前先区分为 amastigotes 所需的时间。人们在使用 metacyclic 前鞭毛体感染时犯的一个常见的错误是, 等待的时间不够长。在毒力变化不剧烈的情况下, 进行了96小时以上而不是72小时的实验, 产生了更可靠的测定。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57486/57486fig1.jpg"> 图 1: 扫描电子显微图像的各种L. amazonensis生命期.从固定相和无菌 amastigote metacyclic promastigote 的对数相 promastigote。酒吧 = 10 µm.<a href="//cloudflare.jove.com/files/ftp_upload/57486/57486fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57486/57486fig2.jpg"> 图 2: 无菌 amastigotes (a) 和非致命的对数相前鞭毛体 (B) 的法钢公司感染的代表性例证 L. amazonensis。从BALB小鼠中分离出的巨噬细胞免疫荧光图像, 感染后1小时和48小时。被感染的巨噬细胞被处理为免疫荧光, 如议定书所述。利用大鼠抗鼠 Lamp1 单克隆抗体 (1:1, 000 稀释) 对其 1 h 进行染色, 然后用抗兔荧光 IgG (1:500 稀释) 进行1小时孵化。寄生虫染色是通过孵化盖玻片与无菌L. amazonensis amastigotes 产生的小鼠多克隆抗体, 其次是抗鼠 IgG 红色染料 (1:500稀释) 6。用 DAPI 对细胞核进行染色, 进一步治疗盖玻片。(A) 在 48 h 时间点形成具有多个 amastigotes 的不同 PVs, 这是一个成功的利什曼原虫感染无菌 amastigotes 的特点。(B) 没有明显的光伏形成和复制 amastigotes 在48小时点的代表性是缺乏毒力在前鞭毛体从日志阶段文化。红色表示反利什曼原虫染色, 绿色表示 anti-Lamp1, 蓝色表示 DAPI 染色的 DNA 和黄色表示 anti-Lamp1 和 DAPI 污渍的合并。酒吧, 5µm。此数字已从 Mittra (B. et) (2013) 中修改。最初在j. Exp中发布。<a href="//cloudflare.jove.com/files/ftp_upload/57486/57486fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57486/57486fig3.jpg"> 图 3: 删除一个LMIT1等位基因严重损害突变体的胞内生长LMIT1/Δlmit1寄生虫.法钢公司被感染的指示时间与 L . amazonensis, 或立即固定或进一步孵化 24, 48 或72小时之前, 他们是固定的, 染色的 DAPI, 和细胞内寄生虫的数量在显微镜下确定。(A) BMMs 感染了纯化野生型 ()、单击除 (LMIT1/Δlmit1) 和补充单挖空 (LMIT1/Δlmit1+LMIT1) metacyclic 前鞭毛体 (语言 1:5) 为3小时, 并立即修复或孵化进一步为被表明的时间点, 并且细胞内寄生虫的数量在显微镜下被确定了。这些数据代表了三种测定的平均值, 并代表了三独立实验的结果。星号表示小波和LMIT1/Δlmit1寄生虫 (学生的双尾t测试 48 h, p = 0.017; 72 h, p = 0.008) 在传染性方面存在显著差异。(B)无菌 amastigotes 从野生类型 ()、单挖空 (LMIT1/Δlmit1) 和互补的单挖空 (LMIT1/Δlmit1+LMIT1) 区域性进行测试, 以检测其感染 BMMs 的能力. BMMs 感染1h (语言 1:1), 或立即固定 (1 小时) 或在进一步孵化 24, 48 或 72 h, 并在显微镜下确定胞内寄生虫的数量。这些数据代表了三种测定的平均值, 并代表了三多个独立实验。P 值 (学生的二尾 t 测试) 在各自小组之间被表明。此数字已从 Mittra (B. et) (2016) 中修改。最初在公共图书馆 Pathog中发布。<a href="//cloudflare.jove.com/files/ftp_upload/57486/57486fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites at JoVE.com

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

所有致病性的利什曼原虫物种在其脊椎动物宿主的巨噬细胞内栖息和复制。在这里, 我们提出了一个协议, 以感染小鼠骨髓源性巨噬细胞的文化与利什曼原虫, 然后精确定量的细胞内生长动力学。该方法对于研究影响寄主病原体相互作用和利什曼原虫毒力的个体因素非常有用。

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

quantification-intracellular-growth-inside-macrophages-is-fast

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

10.4K 次观看.  University of Maryland. 所有致病性的利什曼原虫物种在其脊椎动物宿主的巨噬细胞内栖息和复制。在这里, 我们提出了一个协议, 以感染小鼠骨髓源性巨噬细胞的文化与利什曼原虫, 然后精确定量的细胞内生长动力学。该方法对于研究影响寄主病原体相互作用和利什曼原虫毒力的个体因素非常有用。

视频: Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书"

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are affected both by the doubling time of the microorganism and by any delay in growth upon transfer from one condition to another, the lag. The ScanLag method enables the characterization of these two independent properties at the level of colonies originating each from a single cell, generating a two-dimensional distribution of the lag time and of the growth time. In ScanLag, measurement of the time it takes for colonies on conventional nutrient agar plates to be detected is automated on an array of commercial scanners controlled by an in house application. Petri dishes are placed on the scanners, and the application acquires images periodically. Automated analysis of colony growth is then done by an application that returns the appearance time and growth rate of each colony. Other parameters, such as the shape, texture and color of the colony, can be extracted for multidimensional mapping of sub-populations of cells. Finally, the method enables the retrieval of rare variants with specific growth phenotypes for further characterization. The technique could be applied in bacteriology for the identification of long lag that can cause persistence to antibiotics, as well as a general low cost technique for phenotypic screens.

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

ScanLag:高通量的菌落生长定量和滞后时间问题

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...