作者感谢兽药提供该项目的核心和技术支持的VA-MD地区学院。这项工作是由美国国立卫生研究院的职业发展奖（K01DK092355）的支持。

所有的研究都根据机构护理和使用委员会（IACUC）的弗吉尼亚理工大学的批准，并按照健康指南的国家研究院实验动物的护理和使用进行。 1。 LPS使用口咽管理气管内（它）接种<ol><li>确保每个动物的特征通过使用一个耳朵打孔，耳标或其他制度上认可的方法进行唯一标识。 </li><li>记录基线体重和体温的每个动物。 </li><li>准备LPS的工作股票。每只小鼠将在1x磷酸盐缓冲盐水（PBS）中得到1毫克/公斤的LPS的50微升的剂量。模拟处理的动物将接受1×PBS中的50微升的剂量。 </li><li>准备一个合适的腔异氟醚的使用下面滴法的管理。机构准则变化有关使用异氟醚和其他麻醉剂和前向initiatING任何研究，应联络动物保健/福利的机构的部门，以确保所有指导方针得到充分满足。如果降法异氟醚是不是一种选择，其他麻醉剂是可以接受的替代品。 <ol><li>在一个适当的安全柜，申请约3毫升异氟醚的一种折叠的吸收纸毛巾，并装在500毫升的烧杯的底部。 </li><li>放置一小片铝箔的纸巾上的顶部，并盖上一个透明盖的烧杯中。 </li></ol></li><li>准备将它在接种过程中使用的工具和试剂。定位橡皮筋，将确保动物头插管立场。这个过程需要一对直的镊子，一对成角度的或弯曲的钳子，和一个P200移液管与提示。 </li><li>负载50微升的LPS到枪头和发生在气管插管支架旁边一个容易到达的位置。 </li><li>麻醉鼠标通过将动物放入异氟烷室与覆盖所述腔室的透明盖子。观察动物的呼吸方式，这将是快速而浅时，首先放置在室内。动物将被充分麻醉时的呼吸率接近1的呼吸/ 2秒，这通常是在30秒内将在腔室的到达。如使用滴法异氟醚在批准的动物协议规定的鼠标应该被麻醉。 </li><li>从试验箱中取出麻醉小鼠，并暂停在插管动物站在它前面的门牙。确保该动物被牢固地约束和口腔和舌头的访问。 </li><li>轻轻的安全与直钳舌。抓住舌倾斜的镊子，轻轻拉出口中，直到稍微感觉到阻力。这个动作是足以阻挡会厌并获得气管。 </li><li>继续按住舌头在伸出位置时，管理LPS的50微升剂量到喉咙的后部，并立即覆盖动物的鼻孔用戴手套的手指。该LPS是可见的喉咙后部，直到鼠标吸入。 </li><li>继续覆盖鼻孔按住延长5-10额外呼吸的舌头。 </li><li>从插管支架移开鼠标，然后将动物放回一个新的笼子，直到它从麻醉中恢复。 </li><li>监测与疾病进展相关的替代指标。体重和体温，应监测，每4-8小时的学习时间。同样，评价行为特征和发病率升高相关的临床症状。 </li><li>以评估疾病进展，收获小鼠在以下脂多糖暴露特定的时间点。以下LPS曝光典型时间点收获包括0，6，12，18，24，和48小时。为了评估恢复和生存，评估老鼠7哒YS后接种。 </li></ol> 2。血清和支气管肺泡灌洗液（BALF）收集<ol><li>使用制度上认可的方法牺牲鼠标。 </li><li>将动物在它的后面，并将其固定到一个尸检板，（1.27公分）0.5理想的高度。 </li><li>湿整个鼠标用70％乙醇。 </li><li>用1ml注射器，用27号针头，进行心脏穿刺进行任何切口之前。它应该是可能的退出500-800微升的全血从单个小鼠。 </li><li>从注射器取下针头和全血转移到一个预先标记的1.5 ml离心管。对于血清的收集，允许全血在室温下凝结至少30分钟。除了血清集合，这个过程也可以通过加入抗凝血剂的管和注射器，采集全血，来研究循环免疫细胞之前修改。 </li><li>做一个横向切口一穿过腹膜腔的长度和从腹膜腔的纵切口，鼠标的下颚。 </li><li>使用镊子，把握切口的两侧，轻轻拉扯皮肤从底层的腹腔和胸腔。 </li><li>使一个大切口沿腹膜腔从阴部到胸骨的长度，同时注意避免切割膜片。轻轻移肠子在腹腔内，以允许访问肾脏中的一个。 </li><li>切肾静脉通往肾脏作为一个排水点灌注。 </li><li>尼克隔膜用剪刀，小心避免肺部和心脏，在心脏的左侧露出。 </li><li>手动灌注使用10毫升注射器用27号针头和1x PBS溶液的心脏。避免灌注过程中用力过猛确保食盐不被强迫进入肺部。剩下的血要排出来自门静脉切开。</li><li>仔细沿切使用钝/钝剪刀胸骨肋骨，同时注意避免切割的心脏和肺。在此过程中不小心切割肺部将显著降低BALF中所收集的量，并会导致无法正确地膨胀与固定液肺部。 </li><li>轻轻分离心脏和肺部远离使用镊子肋骨。小心切开胸腔的使用钝/钝剪刀尽量靠近脊椎尽可能每个部分和完全删除这两个部分。 </li><li>单独使用镊子唾液腺或用剪刀将其删除，以露出气管。 </li><li>小心切开用剪刀的覆盖气管肌肉。至关重要的是，气管不能在此过程中切断。 </li><li>用剪刀，分离锁骨上覆气管。 </li><li>使用镊子轻轻抓住胸腺和解除组织离心脏。用剪刀取出胸腺，同时注意避免切割心脏或肺部。 </li><li>做一个小切口水平在1-2环下面使用有角度的剪刀（45-90度角，锋利/锋利）喉气管。切口应足够大，以牢固地固定在套管。 </li><li>插入套管，使锥形端延伸下方切口2-3气管环，并用丝线缝合固定到位的套管。确保缝合气管和食道之间穿过并拉紧的套管。 </li><li>填写1毫升注射器1毫升汉克斯缓冲盐溶液（HBSS），并轻轻插入其鲁尔进入套管。 </li><li>使用缓慢，但持续的运动，注入〜900μL进气管，充气肺，并立即撤回HBSS。将回收的BALF中15毫升锥形管中。 </li><li>重复此灌洗2次额外收取约3毫升支气管肺泡灌洗液中，供日后分析。存储支气管肺泡灌洗液在冰上或4℃，直到准备使用。 </li></ol><ol><li>将10毫升注射器进入肺部通胀的立场。装配油管路厄氏，鲁尔到活门，并在活栓到注射器。确保活塞处于关闭位置。 10 ml注射器将作为一个重力流水库。贮存应附着在充气站4.75在上述动物和储层的顶部应在动物上延伸10.5。 </li><li>用中性缓冲福尔马林溶液填充注射器。确保该注射器是完全充满的固定液。 </li><li>放置吸水巾管子的开口端之下并打开活塞，使固定剂来填充管道。一旦固定液开始从管道流出，立即关闭活塞，重新填充注射器与固定液的顶部。重要的是，在注射器被完全填充，以提供压力的合适量为肺膨胀。 </li><li&gt;轻轻通过第二片缝合线的气管下，约1-2个气管环下方的套管的端部。 </li><li>绑在缝合一个松散的结;但是，不要拉结紧。 </li><li>从插入鼠标通胀管子站进入套管。 </li><li>打开活塞，使肺部受重力通胀来填补。 </li><li>一旦肺部达到其最大的通胀水平，拉动缝线紧，配合第二结。 </li><li>关闭活塞和从所述套管移除导管。 </li><li>轻轻抓住缝线有一对镊子和持套管用手指从气管取出套管。小心地将缝线下来，对胸腔和插管背对着鼠标的鼻子。 </li><li>轻轻抓住气管或缝线用钳子和解除气管远离颈腔。 </li><li>割断气管尾用剪刀钝了并列缝合。 </li><li>一旦trache一个是免费的从下层组织，开始拉气管远离鼠标。轻轻提起肺部出了胸腔。 </li><li>小心切开结缔组织保持肺在胸腔。继续拉动气管远离鼠标的身体和应用稳步向上的力，同时降低相关的连接。小心避免切肺。注意，心脏应由附加到使用这种方法肺部。 </li><li>一旦肺部已被删除，将它们放置在10毫升福尔马林缓冲液中。 </li><li>拔下鼠标尾部的基因分型一节。 </li><li>以下适当的制度规定进行处置鼠标胴体。 </li></ol> 4。细胞因子的评价<ol><li>离心分离全血，在步骤2.5收集，以12,000×g离心5分钟以分离血清。血清转移至预先标记的1.5 ml离心管中并储存于-80℃。 </li><li>评估塞鲁米的细胞因子水平通过ELISA或其他类似的试验。稀释血清1点05 - 1:20适当的稀释缓冲液，这取决于测定中，下列的制造的说明。这些稀释液应在运行批量样品之前，根据经验来确定。 </li><li>由于血清收集量低，减少了一半装载到ELISA板的样品体积。例如，大多数商业ELISA试剂盒利用的标准和样品100微升体积。为了节省标本，负荷标准50微升稀释的血清，每孔。 </li><li>离心机在冷冻台式离心机在200 XG 5分钟的支气管肺泡灌洗液。转移的无细胞上清液分成两个预先标记的1.5 ml离心管中并储存于-80℃。 </li><li>不稀释用ELISA或其他类似的分析评估BALF中细胞因子。 </li><li>将未使用的血清和支气管肺泡灌洗液在-80°C。 </li></ol> 5。鉴别染色法和BAL细胞性评价<ol><li>以下BALF离心和完整的HBSS中去除，溶解用低渗盐水沉淀的红细胞。重新悬浮于900微升蒸馏水中的细胞。立即加入100微升10倍的PBS。 </li><li>单独裂解每个样品。如果样品含有红细胞过多，细胞可在400×g离心5分钟，将红细胞溶解协议可以反复进行沉淀在台式离心机。 </li><li>确定在1毫升悬浮液的总BALF细胞含量下使用10X或20倍放大用台盼蓝染色法血细胞计数器。评估和展示这些数据作为细胞/ ml。 </li><li>准备材料的细胞离心涂片和鉴别染色。用铅笔或耐溶剂笔标注标准显微镜载玻片。确保滑入一个细胞离心涂片支架和漏斗。固定滑动组件中的细胞离心涂片转子。 </li><li>细胞离心涂片器150微升BALF中，在100×g离心5分钟。如果细胞密度过大，有效地评估细胞形态，减少降速到幻灯片的体积。允许的幻灯片空气干燥过夜。 </li><li>鉴别染色的幻灯片下面的生产协议。允许的幻灯片空气干燥过夜。用Permount盖玻片的载玻片并用配有20倍和40倍物镜的显微镜评价的幻灯片。 </li><li>收获供后续分析，其余的细胞，如流式细胞仪，电子显微镜，共聚焦显微镜，RNA提取用于基因表达的评估，和/或蛋白质提取用于蛋白印迹分析。 </li></ol> 6。组织病理学评估<ol><li>准备肺部组织病理学评估。后24-48小时，福尔马林固定的腹侧定向整个充气肺和石蜡包埋。切产生的块暴露的主要传导气道。 </li><li>以提高评分的精度，定位在相同位置的肺和修剪每个块以得到吨的最大纵向的可视化他肺内主要轴向气道。从这个角度，切5微米连续切片和染色用苏木精和伊红（H＆E）。额外的部分可以被切割，并使用标准方案制备用于原位杂交。 </li><li>使用基于以下的炎性参数，这些参数计分0（不存在）和3（重度）之间的半定量评分系统评价组织病理学：单核和多形核细胞浸润;气道上皮细胞增生和损伤;外渗;血管周围和peribroncheolar袖套;和涉及炎症的肺的百分比。肺组织病理学应该由有经验的病理医师进行评估。 </li><li>平均的所有参数的分数，以产生一个总的组织病理学得分或使用个人的分数量化疾病进展的特定方面。进行所有得分在双盲的方式，与审稿蒙蔽两个基因型和治疗。此评分系统已经前情Ÿ描述6,7,10。 </li></ol>

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作者宣称没有竞争的财务权益。

成功地评估在小鼠肺部宿主的免疫反应中最关键的步骤如下：1）选择合适的小鼠品系，性别为被评估的模式; 2）优化PAMP输送到肺部; 3）正确收集和处理的支气管肺泡灌洗液; 4）固定好，并准备对肺组织病理学评估。 小鼠品系的选择是在评价宿主免疫反应的重要因素。 C57BL / 6小鼠通常被认为是最佳的鼠标背景用于研究天然免疫由于大多数的PAMP其Th1型偏斜和强有力的反应。同?...

与致病菌种引起的肺部感染是全球发病率和死亡率的常见原因。确定开车到这些病原体的宿主免疫反应的机制将促进新的预防策略和治疗药物，这将削弱这些感染影响的发展。这里所描述的协议的总体目标是为用户提供一种灵活的方法，使用一种病原体相关分子模式（PAMP）来替代活细菌来评价对病原体感染宿主先天免疫反应。大多数以往的研究评估，以细菌宿主先天免疫反应都集中在腹膜车型由于相对容易执行。虽然这些模型是非常有用的，并导致宿主 - 病原体相互作用和全身性炎症领域的显著进步，从这些模型产生的数据并不总是适合于研究involvi纳克呼吸系统。在这里，急性肺部炎症肺模型，提出为经典的腹腔内（IP）注射模型的实用性和临床相关的扩展。所提出的技术允许在一个特定器官模型系统的先天免疫应答的局部评估。 这里描述的方法的目的是提供一个简单而强大的技术，以允许用户评估为脂多糖，这是一种常见的PAMP宿主的免疫反应。该方法是基于气管内毒素，其诱导小鼠和模拟物中的许多人患呼吸道感染和急性肺损伤患1中观察到的病理生理特征的肺部一个健壮的先天免疫反应的（它）滴入。该技术的主要优点是，它允许用户评估宿主的免疫应答，而不与导电体内研究相关 ​​的混杂因素和安全问题用活细菌。同样，在本协议中所述曝光的口咽它的给药途径比其他常用的技术，包括鼻内（中）管理和手术IT管理显著优点。例如，口咽施用它允许相对准确dosaging和肺沉积相比，在施用，其通常从肺沉积的变异性增加患有由于代理在鼻腔的损失和鼻窦2-4。在它的给药途径规避这些空腔，并允许直接进入气管和呼吸道。同样，它的手术方法是一个显著更加病态的给药方法，需要掌握大量的培训。这里所描述的协议还包括一个描述的常用技术，并用于评估炎症的进展，并与描述正确的技术准备禄协议最终替代指标NGS的组织病理学评估。这些协议都集中在最小化通过最大限度地从每个单独的动物所产生的数据需要为每个研究的小鼠的数目。 所描述的协议是非常灵活的，可以很容易地修改，以评估各种不同的PAMP及相关损害的分子模式（D-AMPS）。此外，有一些额外的修改，这些协议也可以被应用到研究评估过敏性气道疾病的进展或与活的细菌，病毒或真菌5-10宿主-病原体相互作用。

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the utilization of oropharyngeal intratracheal pamp administration and bronchoalveolar lavage to evaluate the host immune response in mice

对病原体的宿主免疫应答是一个复杂的生物过程。大多数体内研究的经典来表征宿主-病原体相互作用取小鼠选择细菌或病原体相关的分子模式（PAMP）的腹膜内注射的优点。虽然这些技术已经取得了与感染性疾病相关的病理生物学巨大的数据，腹腔注射模型并不总是适合于肺宿主 - 病原体相互作用的研究。利用小鼠的急性肺部炎症模型，可以进行先天免疫反应，利用脂多糖（LPS）的主机的高分辨率分析。在这里，我们描述的方法使用非手术口咽气管内给药管理脂多糖，监测与疾病发病机理相关的临床参数，并利用支气管肺泡灌洗液评估宿主的免疫反应。所描述的技术广泛适用于学习到的PAMP和致病菌的多元化宿主先天免疫反应。同样，用小的修改，这些技术也可以用在研究评估过敏性气道炎症和药理用途应用。

革兰氏阴性菌的细胞壁是由LPS，这是环境中的高丰度的。 LPS的敏感人群吸入加剧气道反应性，并能引发强烈的免疫response11的。 LPS也是在小鼠模型中用于引发一个健壮的先天免疫反应的共同PAMP。在这里描述的协议中，小鼠接受LPS的剂量是孤立的从大肠杆菌埃希氏菌 （血清型0111：B4）使用口咽IT管理。在LPS暴露的车型，无论是体温和体重是动物发病和病情恶化的典型的替代指标。在LPS刺激会...

Watch this Scientific Journal Video about The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice at JoVE.com

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

病原菌感染宿主的免疫反应是一种严格的监管过程。利用小鼠脂多糖肺曝光模式，也能够进行与疾病的发病机制相关联的复杂机制的分辨率高的评价。

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

the-utilization-oropharyngeal-intratracheal-pamp-administration

Research

JoVE Journal

Immunology and Infection

16.5K 次观看.  Virginia Polytechnic Institute and State University. 病原菌感染宿主的免疫反应是一种严格的监管过程。利用小鼠脂多糖肺曝光模式，也能够进行与疾病的发病机制相关联的复杂机制的分辨率高的评价。 

视频: The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

科研

免疫与感染

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

接种<em&gt;冈比亚按蚊</em&gt;珠蚊子诱发和测量黑化免疫反应

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.