作者要感谢仪酯蚊子滋生。这项工作是由内部资金来自研究中心博斯特尔支持和莱布尼兹中心感染加盟资金。

安全措施及操守准则本文介绍的研究工作涉及与人类病原体结核分枝杆菌和啮齿动物疟疾寄生虫恶性疟原虫（伯氏疟原虫）。实验与结核分枝杆菌 （菌株H37Rv）和P.疟原虫 ，必须在适当的生物安全的条件下进行。生物安全水平（BSL）2实验室所需要的灭鼠疟疾寄生虫如P。疟原虫和BSL 3对Mtb的 ，因此，对于所有共感染的研究本文所述。注意：感染了结核杆菌的小鼠没有在附近传播感染到其他小鼠（我们自己的观察）。蔓延到实验者因此可以排除，但是相应的防护服具有内BSL 3实验室可以穿在任何时候。按照我们的规定，实验者戴上手术袍，发网，一次性口罩，两副手套，其中外层被丢弃随时一有效值是从机柜排出。新的问题也提上再次进入内阁之前。两个容器，一个为2％Buraton（注：核准以失活Mtb的其它消毒剂可用于在核准的浓度）为液体和传染性废物材料和一种用于表面去污，实验前准备和放置在机柜内。此外，塑料袋将用于固体废物的非感染性。据德国规定，涉及到从结核杆菌衍生结核杆菌培养或组织的处理和加工的所有工作感染小鼠必须进行的2级生物安全柜中的BSL3实验室。虽然处理分枝杆菌，措施要注意避免在任何时候都气溶胶的产生。 雌性C57BL / 6小鼠（Charles River）6〜8周龄之间用于所有合并感染实验和具体的屏障条件BSL 3设备下维持。动物护理和实验是PErformed按照该部的动物实验为农业，环境和石勒苏益格 - 荷尔斯泰因州的农村经伦理委员会的协议为了获得抗疟疾蚊子阶段，NMRI小鼠从Charles River实验室，Sulzfeld，德国购买和海德堡大学动物设施（IBF）内无特定病原体条件下保存。所有动物实验均按照欧洲法规的规定执行，并经巴登 - 符腾堡（Regierungspräsidium卡尔斯鲁厄）的国家机关。 1。使用GLAS-上校气雾室中与小鼠结核杆菌气溶胶感染标准化实验动物与结核杆菌的气溶胶感染的最佳方法是使用由结核分枝杆菌冷冻的库存与已知的CFU滴度。为了准备股票文化，文化结核杆菌在米德尔7H9肉汤补充OADC（油酸酸，白蛋白，葡萄糖，过氧化氢酶）富集培养基和0.05％吐温80​​中，碳源为分枝杆菌和测量，以避免细菌聚集在同一时间。文化应该有一个外径≤1在收获的时候。在更高的外径，细菌丛生的增加和生存能力下降。店铺1ml等份在-80℃下确定可行的菌落形成单位（CFU）的冷冻的库存通过电镀一系列的上补充有0.5％甘油，1 g / L的天冬酰胺7H11琼脂平板上三个独立的小瓶中10倍稀释液的数量，并OADC和4之后列举的菌落周的潜伏期在37°C。执行如下所述的气溶胶感染。 <ol><li>已知的CFU滴度解冻结核杆菌股票和混合仔细暂停五次用1毫升注射器配有27号针头驱散细菌团块。避免产生气溶胶。 </li><li>取决于所需的剂量感染，转移分枝杆菌库存的需要的体积成在50ml管中的无菌PBS中。最终体积为6毫升注意：通过改变微生物的数量在此悬浮液中，细菌轴承气溶胶液滴的比例是变化的。我们通常的目标是每肺癌（低剂量感染）100存活菌的摄取。在我们的经验，这需要大约1-2×10 6的Mtb /毫升在6ml其中5.5毫升被雾化的总体积（见下文）。建议在做了一系列的不同浓度的细菌实验气溶胶挑战，找到适合您感染了理想的条件。而高剂量的感染具有高达5000的分枝杆菌可以做，以加快感染过程中，少至5个细菌可成功地用于感染小鼠的气雾剂。感染率一般为100％。 </li><li>电镀确定接种滴度删除足量（超过所需的最终体积的准备），我们通常除去500微升直接制版技术一式三份。 </li><li>将动物一个隔间网状筐（每筐一个鼠标）之通函气雾室中，并关闭气雾室的盖子。注：其他车型都配备了一个饼形篮子五个单独隔间，每个可容纳20只小鼠组成。 </li><li>附上文丘里雾化器单元的三个不锈钢套筒接头。 </li><li>从50毫升管一个10毫升注射器装有一个18G的钝针取出分枝杆菌悬浮和携带注射器气溶胶室在一个封闭的运输箱。 </li><li>取出喷雾器单元的螺丝帽，并仔细的分枝杆菌悬浮液注入到雾化器。避免气溶胶的产生。丢弃注射器插入含有2％Buraton一个尖锐物品容器。密封该雾化器与螺旋盖。 </li><li>关闭主电源开关，紫外线灯上。在控制面板上的显示屏显示“GLAS-上校器械有限公司”。 </li><li>打开程序开关。显示的意志显示“是对雾化器准备好了？”“是篮装？”按回车键时准备好“。按Enter键。 </li><li>显示屏显示“请输入预热时间900”，这意味着预热时间为焚烧炉（其中decontaminates排风）为900秒。按Enter键。 </li><li>显示屏将显示“输入雾化1,800时间”，这意味着雾化时间设置为1,800秒为默认值。为了延长雾化时间，输入“2400”，然后按回车。注：在雾化周期，在压力下雾化空气悬挂，从而产生含有细小的分枝杆菌气溶胶液滴。与主气流，这些液滴（大小约为2-5微米）被带入气雾室中。 </li><li>显示屏将显示“输入的CD时间1,800”，这意味着衰变周期为1800秒。设置为“2400”，然后按回车。注意：在这个周期中，云已建成ü在雾化循环中的p气溶胶室可衰减。小液滴由实验动物吸入。 </li><li>显示屏将显示“输入减速时间900”，这意味着紫外线消毒周期将采取900秒。按Enter键。机器将开始通过预热，雾化，云衰减和紫外线消毒循环。 注意：真空流量计应注明60立方英尺/小时（检查时预热周期的开始，如果需要调整真空控制阀）和压缩空气流量计10立方英尺/小时（检查雾化周期开始时，相应地调整空气控制阀）。 </li><li>当循环完成时，键盘会显示“过程完成 - 取出标本”。关闭程序，UV和主电源开关。 </li><li>检查分枝杆菌悬浮液已被完全雾化，如果没有，通过仔细除去悬浮装有一个适当的注射器记录的剩余量18G的钝针。剩余的体积应不超过1毫升</li><li>从接头取下​​雾化器，并将其放置在含2％Buraton最少2小时的锅，消毒，通常是做的O / N。此后，雾化器转移到一个新的锅，用清水彻底冲洗。离开雾化器风干。 </li><li>打开气雾室中，并返回到动物的笼子。袋子篮子在釜袋和高压灭菌。擦拭干净气雾室用2％Buraton 注意的内表面：出于安全考虑，电动送风过滤式呼吸器应在此过程中被磨损。 </li><li>使用剩余的500微升的分枝杆菌悬浮液（参见步骤1.3）在技术一式三份（3×100微升）在7H11琼脂平板上的板10倍稀释。 </li></ol> 2。验证所需的CFU吸收的肺实验动物的气溶胶感染后一天确定细菌LO广告中指定的对照动物的肺中，以便验证所要的CFU的吸收。注：我们通常指定3-5小鼠的额外组第1天CFU的决心。 以监测结核分枝杆菌感染随时间的过程中，分枝杆菌组织负担可以通过电镀的全器官匀浆物为菌落形成单位测定系列稀释液进行检查。肺是肺结核原发部位疾病的表现却脾，肝，淋巴结，通常相应的分析。待镀稀释依赖于预期的分枝杆菌负载的器官，这依赖于初始接种物（低剂量与高剂量），这就会引起对不同细菌负荷在组织中，以及在器官和时间点到进行分析。经低剂量感染， 结核分枝杆菌 H37Rv的在肺中的细菌负荷，通常达到25-30天之间的平台期。 <ol><li>将实施安乐死的动物（安乐死： 二氧化碳窒息或终端麻醉）对解剖板在II类生物安全柜吸收纸和消毒小鼠用70％乙醇。注：70％的酒精仅供表面去污，并不会杀死结核杆菌。 </li><li>做一个小的切口在腹部正中和缩回小鼠皮肤的头部上方。 </li><li>打开腹部和胸廓用手术剪和去除胸壁，使肺的访问。 </li><li>取出肺并转移到15ml试管中，在均化缓冲液（无菌水/ 1％体积/体积的吐温80/1％w / v的白蛋白）。 </li><li>通过100微米的筛子用5毫升注射器株肺部的柱塞成一个小培养皿。 </li><li> （;确保他们有一个良好的干燥表面约 250微升/板），使用几次1毫升吸管配备了屏障尖和分发8琼脂平板之间的肺组织匀浆冲洗筛子。 </li><li>小心使用被丢弃到2％的可支配吊具板样品Buraton。 </li><li>离开琼脂平板上以干燥柜内。每封单盘用封口膜，包装在铝箔直立孵育在37℃至少4周。 </li></ol> 3。的逃生防蚊子笼施工啮齿类疟原虫菌株对人体无害。然而，由于结核杆菌感染小鼠的寄生虫和工作的受助人是在BSL 3条件下进行，预防措施是必要的，以防止蚊虫逃逸他们的笼子。取决于计划感染疗法，高压灭菌并重复使用的金属框架的笼子或自制的一次性笼都可以使用。较后的会建议，如果通过实验动物的叮咬感染是必需的，以进行由所定义的每只小鼠的蚊子数。如果自制的笼子是必需的，准备笼子确切规格的蚊子​​和实验室的安全的健康取决于他们的源程序ND建设（ 图2）。 <ol><li>拿一个纸箱，如半品脱冰淇淋纸盒或纸咖啡杯（ 图2）。 </li><li>切一小口进入纸盒的侧面和盖在胶乳的双层带切成每片的狭缝，以创建用于蚊子的安全入口。注意：该开口具有2厘米×2厘米的整体尺寸，管（吸气装置），我们使用带内和/或取出的蚊子是连接到真空泵的改性15毫升Falcon管中，因此可作为吸气器，其直径为1.5厘米。 </li><li>磁带上的​​一片滤纸上的纸箱来吸收水滴的内底。 </li><li>关闭关闭冰淇淋杯网（尼龙网眼双层，尺寸1毫米×1毫米），并用胶带和松紧带固定在纸箱。 </li><li>感染实验中，需要每鼠蚊子定义号码，准备笼中10-15感染的蚊子，其中每一个理想包含一个AV10000野生型唾液腺P. erage 疟原虫子孢子。 </li><li>理想情况下，饿死在执行血粉之前蚊子24小时。 </li></ol> 4。小鼠通过蚊虫叮咬感染疟疾本文中使用的啮齿动物疟疾寄生虫是P.然而疟原虫任何感兴趣的其他啮齿类动物疟原虫菌株都可以使用。为了获得感染的蚊子传播孢子寄生虫的整个生命周期保持在这两个脊椎动物宿主（鼠）和蚊子。寄生虫的维护是在一个养虫。对于天然传输实验，每唾液腺子孢子的最低人数应不低于10000。如需详细的协议，对寄生虫维护我们提到的方法在疟疾研究由莫尔等人 ，2013年。 <ol><li>麻醉天真的小鼠或动物preinfected 40天用Mtb的氯胺酮（100毫克/千克）通过腹膜内注射（200微升/小鼠）和甲苯噻嗪（7毫克/千克）的溶液。注：结核杆菌和疟原虫感染之间的时间可以根据潜在的研究问题进行调整。同样，病原体挑战的顺序可以颠倒，即小鼠可以接触到感染蚊子叮咬之前，结核杆菌感染。 </li><li>将麻醉小鼠到蚊子笼（每笼蚊界定蚊子鼠标比一个鼠标）的网，让蚊子通过膜料10-15分钟。注：血液中的胆蚊子的存在意味着馈送，因此，子孢子的传输。 </li><li>转移小鼠放回笼子和监督不断，直到他们从麻醉中醒来。注：将小鼠在纸巾上，而不是在床上用品（窒息的危险），并保持温暖（地方紧密地结合在一起，并覆盖机构用纸巾）。 </li><li>通过在净价用70％乙醇或其它消毒剂喷洒他们杀蚊子ing的笼子。如果使用一次性的人反应釜笼并丢弃。 </li></ol> 5。原虫监测<ol><li>穿刺尾静脉处用针快结束，并收集一滴血在幻灯片的每一端。 </li><li>使用另一张幻灯片拉一滴血超过一半的幻灯片的长度。翻转吊具使用其他边缘第二涂片。离开幻灯片风干。 </li><li>姬姆萨染色<ol><li>将幻灯片在幻灯片持有人，并在无水甲醇短暂浸泡固定血涂片。注：由于使用玻璃洁具应保持在最低限度在BSL 3，我们使用的聚丙烯试管的所有解决方案。 </li><li>沉浸（去离子水1:10）在姬姆萨涂片。 10分钟后，浸在染色试管装满去离子水几次冲洗掉多余的污渍进出的幻灯片。 </li><li>最后，空气干燥载玻片在垂直位置。 </li></ol></li><li>替代染色：赖特＆＃180;氏染色<ol><li>溶解瑞氏染色以1毫克/毫升的甲醇溶液的浓度。 </li><li>通过在使用前折叠滤芯过滤器。 </li><li>浸血涂片染色溶液，孵育4分钟（注：涂片甲醇固定是没有必要，因为染色的解决方案是基于甲醇）。 </li><li>通过漂洗幻灯片如上​​所述去离子水清洗并留下幻灯片风干。 </li><li>一旦姬姆萨/赖特的染色完成后，轻轻地转用成含Buraton处置锅的所有试剂。 </li></ol></li><li>血涂片分析<ol><li>通过光学显微镜在100倍放大倍率的油浸分析染色血涂片。 </li><li>计数未感染和感染的红细胞在红细胞被安排在一个单分子层的血涂片的区域。 </li><li>为了实现一个良好定义的寄生虫血症，计数的视图显示红细胞单层至少10种不同的字段。 </li><li>演算除以寄生红细胞的数量由红细胞的数目再乘以100吃的相对原虫（以％表示）。 </li></ol></li></ol>

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Immunol. 40, 396-405 (2010).</li><li>Craig, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+animal+models+for+research+on+severe+malaria.">The role of animal models for research on severe malaria.</a> PLoS Pathog. 8, e1002401 (2012).</li><li>de Souza, J. B., Hafalla, J. C., Riley, E. M., Couper, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+malaria:+why+experimental+murine+models+are+required+to+understand+the+pathogenesis+of+disease.">Cerebral malaria: why experimental murine models are required to understand the pathogenesis of disease.</a> Parasitology. 137, 755-772 (2010).</li><li>Van den Steen, P. 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作者什么都没有透露。

我们描述了如何老鼠可以高效感染结核杆菌和P.通过受感染的自然路线疟原虫 。我们应用建立感染协议研究实验肺结核13或12疟疾和最近通过他们学习结核杆菌和恶性之间的合并感染的小鼠模型1。 对于成功的感染中最关键的步骤是两种病原体在所需的号码的传输。分枝杆菌的股票不应该是2岁以上的，因为他们将失去生存能力随着时间的推移，和股票的最初确定的滴度将极有可能不再准确。其结果是，感染的剂量将远低于预期。因此，股市文化的CFU滴度应确定每隔几个月。同等重要的是规范化栽培和代结核杆菌股票摆在首位。培养条件如培养基，营养补充剂，时间和体积应该标准化，以便能够使用不同的股票的Mtb的实验数据进行比较。分枝杆菌倾向于培养过程中结块，因此，保持培养条件标准化，收获细菌在相同的生长阶段和分装过程中频繁地重悬是很重要的。否则，就不可能在CFU滴度和感染的结果差异很大。 的均匀受感染的蚊子批次的产生是另一个关键的一步。因为不同的蚊子将饲料上每个单独的鼠标，也都用于一个实验感染蚊子的来自同一批次，只有那些其中超过90％的蚊子感染批次使用是很重要的。 目前的模型可以用来通过比较单一感染的宿主免疫反应来剖析免疫反应和免疫调制在合并感染的主机。我们可以应用VArious完善的方法，如组织学，免疫组织化学，流式细胞术的免疫细胞群的分析或PCR和ELISA对细胞因子和趋化因子的检测到所关注的组织或体液中。应当指出，这种小鼠模型中具有一定的局限性。而大多数人感染了结核杆菌培养潜伏感染的临床症状体征，小鼠发展为慢性疾病，进行性肺部疾病。尽管如此，C57BL / 6小鼠是结核杆菌感染相对性和不开发经典肉芽肿是人类结核病的患者中观察到。其结果是，在小鼠的免疫病理反应也适当地反映潜既不积极也不人类肺结核病。尽管在人类和小鼠潜伏和慢性疾病之间的差异，小鼠已被广泛用于鉴定并研究控制结核杆菌感染，是宝贵的，不可或缺的工具，调查宿主因素在感染性疾病的免疫应答。重要的是，易患结核分枝杆菌感染的常用近交系小鼠品系，更敏感菌如DBA或C3H之间有很大的差别可以被包括在共同和单一感染的研究。此外，除了本文所述的病原体种类，可以使用任何其他的分枝杆菌属物种的兴趣（ 例如临床分离株）。同样，虽然没有单一的疟疾模型复制人类疾病不同的小鼠模型的各个方面都酷似自然获得的人类疟疾感染的不同方面。例如， 第疟原虫安卡和P.约氏疟原虫 YM/17XL被广泛研究人类（ 恶性疟原虫引起的）重症疟疾的车型，与体育伯氏疟原虫安卡被视为人类大脑malari s 14,15一个很好的模型。伯氏疟原虫 NK65是hyperparasitaemia和急性疟疾贫血一个很好的模型，并在最近被描述作为实验模型疟疾相关性急性呼吸窘迫综合征（MA-ARDS 16）。使用所描述的共感染模型，我们可以最近表明，并行P.伯氏疟原虫 NK65感染加重慢性肺结核1。 除了使用不同的啮齿动物疟疾寄生虫的感染事件的顺序和时间可根据基础研究问题改编。在现场，伴随疟原虫感染可能存在于结核杆菌感染时或其后收购。在这两种方案中的免疫应答的Mtb和疟原虫可能受到不同的影响。因此，老鼠可以之前或结核杆菌感染后，被感染疟原虫子孢子。此外，小鼠可以在不同的时间被挑战后的Mtb感染，评估疟原虫引起的免疫应答的急性与慢性的影响肺结核。重要的是，在选择疟原虫的研究疟疾肺结核合并感染，人们应该知道，一些啮齿类疟原虫株引起的急性疾病，某些小鼠品系，并屈从于感染仅在几天或几周，而结核杆菌引起的慢性和持久的感染免疫小鼠。因此，共感染的时序是至关重要的，以避免实验动物的过早死亡。 宿主免疫受感染的同时与多个病原体种类调制的了解甚少。我们的实验合并感染模型提供了一个功能强大的工具来调查分枝杆菌 - 恶性互动与共同感染宿主的免疫系统，将有助于我们了解如何合并感染可影响发病机制，疾病转归，疫苗接种，免疫诊断和治疗。

人类群体中很少暴露于仅一种病原体。特别是在地区，感染，如撒哈拉以南非洲地区的发病率很高，占混合感染的黄金，但非常被低估的公共卫生问题。结核病和疟疾是人类，分别为最普遍的细菌和寄生虫感染，并继续成为发病率和死亡率在热带地区贫困人口的主要原因。尽管在合并感染的风险肺结核和疟疾以及大量的个人之间的广泛的地理重叠，很少有人知道有关各种与常抵消免疫调节和效应同时引发针对疟疾寄生虫和结核菌双重感染中个体之间的相互作用。 混合感染的啮齿动物模型允许在一台主机对特征明显的不同病原体的免疫反应，从而脱落光的相互十字形ENCES调节病理和个人疾病的临床结果，这也将有助于确定治疗和预防的新建议。我们已经建立了一个实验性小鼠模型，使我们在同一个主机1中，调查引起并发感染结核分枝杆菌 （ 结核杆菌 ）的后果和啮齿动物种疟原虫 。重要的是，以模拟自然获得的人类感染尽可能地，我们的模型实现了感染都使用病原体的天然路由。结核病是一种空气传播，因此这主要表现在肺部。病原体， 结核杆菌是由人与疾病活动通过气溶胶途径，当他们咳嗽或打喷嚏时排出的传染性气溶胶飞沫传播。因此，气溶胶感染是选择的方法，以模仿自然感染。含有结核杆菌的气载粒子的生成可以通过使用可以实现的吸入暴露系统。这全身暴露室允许暴露实验动物的Mtb含有传染性气溶胶液滴（ 图1），其被吸入其中感染是引发2肺部的肺泡。 疟疾，另一方面是由原生动物，顶复门寄生虫恶性那自然是由按蚊雌性蚊子叮咬传播媒介传播的疾病。在血粉，传染性的子孢子被沉积在宿主的皮肤下，并随后经由血液流到达肝脏。肝细胞内他们开发并迅速繁衍成肝阶段裂殖。最终，成熟的裂殖体破裂并释放数以千计的致病第一代裂殖子进入血流，他们发起红细胞浸润，复制，红细胞破裂，并再侵入3的累进周期。 疟原虫生命周期康廷UE的一些裂殖子发育成性的寄生虫阶段，男性和女性配子体，可在血液饭菜就由蚊子。在蚊子体内疟原虫居住在蚊子的中肠卵囊内发展，并最终迁移到唾液腺，以便转交到另一台主机3,4。 而在实验动物模型的结核杆菌通过气溶胶，因此最相关的路由传递是很普遍的，许多实验啮齿类疟原虫感染的研究，包括疟疾，结核病合并感染5-7的研究已经进行了小鼠感染与寄生虫感染的红细胞，从而引发要同时排除了临床上沉默的肝段相血液阶段的疟疾感染。然而，肝期是感染和相关的期间强制性步骤，用于抗疟原虫免疫8-11。我们认为，因此，重要的是包括肝pH值疟疾传播，以及在这两个疟疾和疟疾肺结核合并感染，分别研究维修的酶。另外，它已经表明，自然发射的子孢子比那些通过针头感染12，这促使我们通过蚊子建立，而不是注入隔离唾液腺派生的子孢子的感染疟疾咬发送的传染性。获得蚊子传染疟疾寄生虫的自然传播的惟一途径是保持寄生虫的整个生命周期中都脊椎动物宿主（此处鼠标）和蚊子。因此，获得的养虫的寄生虫维修是不可避免的，以便通过叮咬进行自然传播。 我们这里所描述的协议的开发是为了研究如何合并感染疟原虫孢子对慢性肺结核1的影响。这样做，将小鼠气溶胶感染了M。肺结核和40天后，当M。结核感染已达到慢性期，将小鼠暴露于疟疾感染的蚊子。无论疟疾和结核病的双重感染动物的结果可以跟随，监测血原虫和细菌负荷在组织分别。在我们的协议中，我们将通过他们的自然感染途径，以及如何确认成功病原体传播详细描述如何感染小鼠两种病原体。在我们的手中，一般达致的感染率为实验感染为100％。这些协议可以应用到研究均单独感染或，和我们一样，两个最具挑战性的人类传染病的建模和研究合并感染。这种模式也适用于其他分枝杆菌和啮齿类疟原虫种类超过本协议所描述的。

<table border="0" cellpadding="0" cellspacing="0" width="650">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Buraton</td>
			<td style="width:124px;">Sch&#252;lke</td>
			<td style="width:119px;"></td>
			<td style="width:191px;">active ingredients: aldehyds (formaldehyde, glutaraldehyde, oxalaldehyde, ethyl hexanal)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Middlebrook 7H9&#160;</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:119px;">M0178</td>
			<td style="width:191px;">For Mtb broth cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Middlebrook 7H11&#160;</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:119px;">283810</td>
			<td style="width:191px;">Agar medium for Mtb culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Middlebrook OADC enrichment medium</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:119px;">212240</td>
			<td style="width:191px;">Add to 7H9 and 7H11 for Mtb culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Staining Dish</td>
			<td style="width:124px;">Science Services</td>
			<td style="width:119px;">E62542-12</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24-Slide Holder w/Handle</td>
			<td style="width:124px;">Science Services</td>
			<td style="width:119px;">E62543-06</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Giemsas Azur-Eosin-Methylene blue solution</td>
			<td style="width:124px;">Merck Millipore</td>
			<td style="width:119px;">109204</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Wright&#180;s stain</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:119px;">W0625</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inhalation Exposure System</td>
			<td style="width:124px;">Glas-Col</td>
			<td style="width:119px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nebulizer-Venturi</td>
			<td style="width:124px;">Glas-Col</td>
			<td style="width:119px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ice cream cups</td>
			<td style="width:124px;">H&#228;agen-Dazs</td>
			<td style="width:119px;"></td>
			<td style="width:191px;">Used as mosquito cages</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal-frame mosquito cages</td>
			<td style="width:124px;">BioQuip Products</td>
			<td style="width:119px;">1450A</td>
			<td></td>
		</tr>
	</tbody>
</table>

an experimental model to study tuberculosis-malaria coinfection upon natural transmission of mycobacterium tuberculosis and plasmodium berghei

自然混合感染，由于不同类型的病原微生物的地理重叠发生。并发感染最容易调节到每个单一病原体各自的免疫反应，从而可能影响发病机理和疾病的结果。合并感染的患者也可能差异响应抗感染干预措施。造成的分枝杆菌和疟原虫 ，这两者都是coendemic在撒哈拉以南非洲的许多地方，结核病合并感染之间尚未详细研究。为了达到尽可能的具有挑战性的，但科学和如何疟疾肺结核合并感染调节宿主免疫和每一种疾病的过程中，我们建立了一个实验性小鼠模型，使我们能够剖析诱发免疫反应两种病原体的混合感染宿主临床高度相关的问题。值得注意的是，为了最准确地模仿自然获得人类感染，进行实验小鼠的两种病原体的感染者感染的自然路线， 即气溶胶和蚊虫叮咬，分别。

小鼠结核分枝杆菌和P的感染通过自然的途径， 即气溶胶和蚊虫叮咬，分别，结果所有动物（ 表1）的一致性和可重复性感染疟原虫 1。我们可以监视成功的感染，都疟疾和肺结核通过确定初次发生（prepatency）和寄生虫的数量在血液（ 伯氏疟原虫的寄生虫血症）和结核杆菌在不同器官的负担，分别的过程。由感染性蚊子叮咬成功疟原虫传输的一个例子示于表1和图3。对每只小鼠10子孢子感染的蚊子摄食导致100％的感染发生率证实了的血液阶段寄生虫的存在下4-5天之后（ 表1和图3A）。重要的是，我们发现类似的寄生虫数在相同的实验克的个别小鼠的血牛津大学出版社，指示由蚊子叮咬的结果一致和可重复感染，子孢子传播。在天真的对照组小鼠的prepatency与结核分枝杆菌中双重感染的动物进行比较，我们可以看到，prepatency稍微延迟已被preinfected与结核分枝杆菌 （ 表1，图3B）小鼠1。正如我们以前1日报道，我们可以判断， 如图3B 结核分枝杆菌混合感染的疟疾由日常监测原虫在姬姆萨染色血涂片过程的影响。 感染小鼠结核分枝杆菌通过与个别小鼠（ 图4A）之间的相对低的变异性肺部使用吸入暴露系统导致细菌的成功的沉积。我们可以按照负载分枝杆菌肺部以及由CFU测定随着时间的推移感兴趣的其他器官。在肺分枝杆菌负载为例结核分枝杆菌感染的C57BL / 6小鼠在P的存在或不存在疟原虫 如图4B所示 。 表1中。 Prepatency通过蚊子叮咬传播孢子后，所有小鼠发生血液阶段的感染P的自然传播后伯氏疟原虫子孢子蚊虫叮咬。从1 PLoS ONE的 ，7（10），e48110经许可后改编。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> 实验组小鼠（C57BL / 6） </td><td> 10感染蚊子叮咬挑战 </td><td> 每组动物的血液阶段阳性动物数目/ </td><td> 平均prepatency并[d] </td></tr><tr><td>天真</td><td> 伯氏疟原虫 </td><td> 7/7 </td><td> 4,4 </td></tr><tr><td> 结核分枝杆菌感染</td><td> 伯氏疟原虫 </td><td> 7/7 </td><td> 5,5 </t<html>开发&gt; </tr></tbody></table><img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50829/50829fig1highres.jpg" src="/files/ftp_upload/50829/50829fig1.jpg" /> 图1。实验过程的总体方案。C57BL / 6小鼠暴露在Glas-上校气雾室中含有结核杆菌的气溶胶液滴。一天后，喷雾感染， 结核分枝杆菌的摄取是由肺CFU分析验证。 40天之后的Mtb挑战（当结核已成为慢性感染小鼠），将动物暴露于P。伯氏疟原虫感染的蚊子。确认成功的子孢子传播，并遵循疟原虫感染的过程中，原虫在姬姆萨染色血涂片蚊虫叮咬后以3天的日监测。 / files/ftp_upload/50829/50829fig2highres.jpg“SRC =”/ files/ftp_upload/50829/50829fig2.jpg“/&gt; 图2。一蚊笼的草图。感染蚊子都包含在一个纸箱内，如一个半品脱冰淇淋纸盒。一个小开口被切成纸箱的侧面和覆盖有胶乳的双层带切成每片的狭缝以创建蚊子的安全入口。一块滤纸录音的纸箱来吸收水滴的内底部。冰淇淋杯都关闭了与净额结算（尼龙网），这是由带和松紧带固定纸箱。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/50829/50829fig3highres.jpg" src="/files/ftp_upload/50829/50829fig3.jpg" /> 图3。一赖特单曲染色血涂片显示寄生虫感染的红细胞（比例尺，20＆＃ 血原虫A）示例181;米）B）原虫在小鼠血液通过分析日常姬姆萨染色血涂片监测。合并感染小鼠具有降低原虫相比， 疟原虫感染的单一小鼠（n = 10），结果显示为平均值±标准差; *** P &lt;0,001。从1 PLoS ONE的 ，7（10），e48110经许可后转载。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/50829/50829fig4highres.jpg" src="/files/ftp_upload/50829/50829fig4.jpg" /> 图4。在肺结核杆菌检测的。C57BL / 6小鼠气溶胶挑战与结核杆菌的100 CFU和40天后，合并感染P.疟原虫通过蚊子叮咬A）一天后的气溶胶感染结核杆菌的所需数量的摄取电镀全肺匀浆的CFU测定进行了验证。 二）细菌升负荷在合并感染的时间是在3小鼠肺裂解液通过CFU分析（ 结核杆菌 d40/d0）确定。共感染后12天，肺裂解物镀CFU分析，以确定疟原虫共感染对Mtb的控制的影响。请注意，在肺结核杆菌合并感染人数在增加，P。疟原虫 NK65。 X轴标签：合并感染结核分枝杆菌后后-infection/time时间。从1 PLoS ONE的 ，7（10），e48110经许可后改编。

Watch this Scientific Journal Video about An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei at JoVE.com

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

结核病和疟疾是两个人类和发病率和死亡率在热带地区贫困人口的主要原因最普遍的感染。我们建立的实验模型系统，通过受感染的自然路线的挑战后，研究疟疾结核病合并感染的结果小鼠两种病原体。

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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JoVE Journal

Immunology and Infection

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

19.7K 次观看.  University Hospital Heidelberg. 结核病和疟疾是两个人类和发病率和死亡率在热带地区贫困人口的主要原因最普遍的感染。我们建立的实验模型系统，通过受感染的自然路线的挑战后，研究疟疾结核病合并感染的结果小鼠两种病原体。 

视频: An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

科研

免疫与感染

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

一种新型显微切割方法恢复<em&gt;结核分枝杆菌</em&gt;从福尔马林固定，石蜡包埋肺肉芽肿特异性转录

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...