我们想感谢帕特里克海登（MatTek），有益的讨论和利比的佐治亚州健康科学大学为他们的EM技能的罗伯特史密斯和佩里。这项研究是由NIDCD授予DC010187到DAD

1。为EpiAirway组织准备生物安全柜<ol><li>穿着专用的白大褂，头发绑背和手套，开始层流。 5分钟后，将在内阁（移液器，小技巧，离心管等），任何一方的工具，喷洒生物安全柜内部和窗框用70％乙醇，并用干净的纸巾擦拭。用70％乙醇再次喷洒清洁的室内，并留下来干。干净的一侧移动工具和重复乙醇的过程​​。 </li><li>在内阁中的气溶胶屏障枪头和专用的移液器。要使用单独包装的无菌血清移液器，通过层流插入年底，破开，滑入机柜的吸管，丢弃的包装外面。 </li><li> EpiAirway插入必须用无菌镊子处理。放置6英寸的细尖弧形分割成自我封闭消毒袋和高压灭菌镊子。准备足够的至少有6个每天准备使用。 </li></ol> 2。开箱EpiAirway组织<ol><li>喷雾用70％乙醇在生物安全柜的工作表面，并用干净的纸巾擦拭。喷一遍，让表面干燥，使用前。打开EpiAirway盒和丢弃的发泡胶及包装材料。 </li><li> EpiAirway不含抗生素的维护媒体，AIR - 100 - MM的亚洲债券基金（MM），是专有的MatTek在插入套件发送。在生物安全柜，媒体类型和日期的标签六个无菌的50 mL离心管，并松开帽。喷雾用70％乙醇的媒体的瓶子，并打开机柜内的。分装成每管每使用一个新的血清无菌吸管管25毫升的MM，收紧的上限，并储存在4 ° C使用新鲜的MM等分的每一天。 </li><li>重复上述新鲜瓶使用1 ×贝科的磷酸盐缓冲液（D - PBS）与钙和镁的2.2。再次重复使用的D - PBS无钙和镁的。所有分装保存在4 ° C。 </li><li> EpiAirway插入空气- 100到达在24孔板中，在4 ° C的固体培养基含有琼脂糖中，必须放置到6孔板使用。标签的日期和使用乙醇耐标记的描述，每块板。将1毫升到每口井的4℃EpiAirway的MM。删除其包装，喷以70％的乙醇和生物安全柜中进行，运输板块。取出磁带和开放的板块。使用高压灭菌镊子，取出EpiAirway插入潮湿的纱布。 </li><li>拿起与使用，扭转运动，以帮助释放琼脂糖蒸压钳插入，并将其放置到6孔板以及。一些琼脂糖可能会坚持到插入的，特别是运输板的温度升高到环境。使用无菌棉签小心地取出插入琼脂糖。避免接触的膜，并检查，无气泡下方插入被困。放入5％的CO 2饱和湿度37℃培养箱中培养板。 </li><li>允许至少24小时的组织，以平衡前开始联合培养的孵化器。 </li></ol> 3。 EpiAirway组织维修<ol><li>使用高压灭菌镊子，拿起一个插入，轻轻吸液管200微升预热的D - PBS到组织中的钙和镁的岩石插入。倾斜角度的插入和对持有膜插入底部的塑料环，无菌P1000的枪头。请勿触摸的组织。吸的D - PBS漂洗，并放入一个标记的无菌离心管。样品可以冷冻在-20 ° C，并在稍后的日期为粘蛋白和/或细胞因子的表达的测定。 </li><li>更改基础MM每天吸和替换用1毫升的新鲜MM。每口井使用一个新的障碍枪头。治疗用10％漂白粉的使用媒体过夜，并丢弃。 </li></ol> 4。接种EpiAirway组织<ol><li> nontypeable 流感嗜血杆菌 （NTHI）从冰冻甘油的新鲜培养了数到巧克力琼脂。成长在加湿5％ 二氧化碳培养箱中培养过夜。 </li><li>第二天，在预热的D - PBS重悬与钙和镁的OD（600 nm）的约0.7 NTHI单菌落。涡大力前的光密度测量。光密度的关系，集落形成单位每毫升（CFU / ml的）的NTHI，外径为0.2（600纳米）约等于1.0 × 10 8 CFU /毫升。因此，外径为0.7（600 nm）的约3.5 × 10 8 CFU / ml的。每个NTHI应变，以接种前，应验证该算法。 </li><li> EpiAirway组织AIR - 100是生长在插入了0.6厘米和0.4微米膜的表面积。组织是由〜8.0 × 10 5〜1.0 × 10 6 &lt;/ SUP&gt;细胞。因此，接种1.0 × 10 7 CFU对应的多重感染了大约10 - 12。接种，冲洗每3.1 EpiAirway插入，然后加入28微升4.2准备在生物安全柜中的每个EpiAirway插入根尖表面的细菌悬液。不要接种基底MM。返回每块板的孵化器。 </li></ol> 5。量化NTHI接种<ol><li> 0.1％的明胶（PBS - G），高压灭菌消毒，准备一个磷酸盐缓冲液（PBS）的解决方案。标签无菌的1.5 ml Eppendorf管所需1:10稀释成每管接种和分装900微升无菌PBS - G OD（600 nm）的基础。 </li><li>确定枚举接种NTHI巧克力琼脂板的数量。这些板块上下颠倒放置在空气孵化器与上半盘一小时休息的底部。这将使板块既温暖至37 ° C，表面干燥，促进NTHI下降电镀。 </li><li>开始串行1:10稀释的菌悬液加入100微升4.2管头的接种。涡每个稀释大力前5秒，使未来。下降到预热和表面干燥的巧克力琼脂平板10微升等份，每个稀释度板的一半的使用。可容纳五至六个10微升等分之一，每100毫米的钢板的一半，而无需运行起来。当干燥，倒挂在加湿37 ° C的CO 2培养箱中过夜。 </li><li>第二天，确定CFU / ml的计数稀释，每下降15-30独特的殖民地和计算您的实际可行的NTHI接种。 </li></ol> 6。长期合作的文化与NTHI <ol><li>每隔24小时，洗净的插入，然后在3.1池已接种一脉相承的细菌和冻结在-20 ° C在一个标记的离心管，供日后分析每个组织的洗涤。这些样本可检测粘蛋白斑点杂交或用ELISA细胞因子的表达。此外，组织可以为mRNA的收获，并可以就感兴趣的基因进行定量PCR。更改基底MM每天在3.2。继续进行共培养时间。 </li></ol> 7。收获EpiAirway组织<ol><li>准备一个新鲜的1％皂素的解决方案，在没有钙和镁的D - PBS，过滤消毒和温暖至37 ° C。 </li><li>没有钙和镁的温暖的MM和另一个D - PBS管50毫升至37 ° C。 </li><li>准备在5.1稀释管，以及一个空的无菌的1.5 ml Eppendorf管中，每插入，与插入的数目和NTHI应变标记。 </li><li>可收获的EpiAirways任总细胞相关的细菌，其中包括壁和内化的生物体，或只有内在的细菌。 </li><li>巧克力琼脂平板上确定的数量，需要下拉板细胞相关或入侵NTHI，标签和一小时，在空气干燥的孵化器为5.2。 </li><li>收获总细胞相关的细菌，200微升的D - PBS无钙或镁的EpiAirways根尖表面冲洗三次，然后插入倾斜和任何剩余的MM从基底膜冲洗。先洗可以冻结，为以后的分析;丢弃以下的清洗。 </li><li>从7.1到每个组织的根尖表面，放置在一个新的6孔没有MM平板洗插入，和吸液管250微升无菌的1％皂素的解决方案。孵化器10分钟。 </li><li>从孵化器中取出，并使用无菌P1000的枪头，磨砂膜的组织，使用一回的往复运动，圆周运动，删除插入的边缘组织。使用大口径的无菌P1000的枪头，放入7.3编写的空标记管悬挂。插入到根尖表面无钙或镁中加入250微升的D - PBS。 </li><li>检查插入一个倒置相差显微镜，以确定是否有任何剩余的组织收割。如果需要的话，再次与P1000的另一个无菌枪头擦洗，然后添加7.8管悬挂使用大口径的无菌P1000的枪头。管1毫升使用的D - PBS无钙或镁的总量。 </li><li>大力旋涡细胞悬液，在一分钟的最高时速。使用无菌装上了26号针头的1 ml注射器，通过针头注射器吸进暂停。慢慢穿过针3次暂停，注意不要创建气泡。涡大力为一分钟再次。 </li><li>进入第7编写的稀释管大口径枪头，100微升的暂停使用。3。涡大力五秒钟，然后使串行1:10稀释。落板的上表面干燥的巧克力琼脂平板所需的稀释。 </li><li>收获内在的细菌，洗3次，用200微升的D - PBS无钙或镁每个插入。保留和冻结先洗。硫酸庆大霉素的终浓度为100微克/ ml预热的MM。准备1.5毫升收割每次插入媒体。替换与含有庆大霉素的MM基础毫米，300微升含有庆大霉素的MM添加到根尖表面。将板回一小时到的CO 2培养箱。 </li><li>删除从根尖表面含有庆大霉素的MM和洗根尖表面和基底膜广泛预热的D - PBS无钙和镁的插入。继续在7.7-7.11。 </li></ol> 8。代表性的成果： 扫描电子显微镜（A）的未受感染的EpiAirway组织和（二）组织的为期5天的文化合作与NTHI后如图1所示。，不造成重大损害的心尖的长期文化合作与NTHI组织，强调了实用的EpiAirway模型。描绘以上的时间内组织量化的内在细菌的数量是一个图 ，如图2所示，结果是相当重现，EpiAirway 组织一致和生物体外培养的人体上呼吸道模型相关研究NTHI宿主-病原体相互作用。 <img src="/files/ftp_upload/3261/3261fig1.jpg" alt="图1"/> 图1。EpiAirway组织扫描电子显微镜。 A.未感染控制组织。 B.组织后，共培养5天，与NTHI。在受感染的组织观察根尖表面无重大损害。 <img src="/files/ftp_upload/3261/3261fig2.jpg" alt="图2"/> 图2 NTHI内在随着时间的推移，在EpiAirway合作文化。 R2866菌株接种在约1.0 × 10 7 CFU /插入（0天），然后在每一个指定的时间点的内在细菌收获。条代表N = 3至少在重复复制。误差棒的SD。

<ol>
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没有利益冲突的声明。

这种方法允许的长期宿主 - 病原体相互作用的首要人权组织在气 - 液界面的呼吸生物学相关背景调查。在这里，我们使用了感染的有机体NTHI，但用这种方法可以量化的任何细菌的相互作用，不引入不可接受的细胞毒作用随着时间的推移。 EpiAirway模式也可以被用于研究病毒，药物或化学物质，影响人类的上呼吸道5 6 7 8。我们一直保持着超过40天，与NTHI感染的组织至少10天未感染的组织。我...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录＃ </th><th>评论</th></tr><tr><td>皂素</td><td> Calbiochem </td><td> 558255 - 25GM </td><td>在D - PBS无钙或镁的1％，过滤消毒</td></tr><tr><td> 1 + X贝科的磷酸盐缓冲生理盐水与钙和镁</td><td>龙沙</td><td> 17 - 513Q </td><td></td></tr><tr><td> 1 + X贝科的磷酸盐缓冲生理盐水无钙或镁</td><td>龙沙</td><td> 17 - 515Q </td><td></td></tr><tr><td> EpiAirway抗生素自由组织</td><td> MatTek </td><td> AIR - 100 - ABF </td><td></td></tr><tr><td> EpiAirway抗生素免费维修媒体</td><td> MatTek </td><td> AIR - 100 - MM - ABF </td><td>提供试剂盒</td></tr><tr><td> 10X磷酸盐缓冲盐溶液</td><td>电解二氧化锰</td><td> 6506 </td><td>使用前稀释成1X </td></tr><tr><td>明胶</td><td> JT贝克</td><td> 2124-01 </td><td>在1 × PBS和高压灭菌，加入终浓度为0.1％ </td></tr><tr><td> DIFCO GC中等基地（巧克力琼脂） </td><td> VWR </td><td> 90002-016 </td><td>高压灭菌36克在500毫升DDH 2 O和冷却到60 ° C </td></tr><tr><td> BBL血红蛋白（巧克力琼脂） </td><td> VWR </td><td> 90000-662 </td><td>高压灭菌10克，500毫升DDH 2 O，冷却至60 ° C和混合与GC以上中型基地</td></tr><tr><td> BD BBL IsoVitaleX浓缩（巧克力琼脂） </td><td> VWR </td><td> 90000-414 </td><td> GC中型基地和血红蛋白的混合物冷却至55℃，添加10毫升水合IsoVitaleX，倒入巧克力琼脂平板</td></tr><tr><td>解剖钳，尖端细，弯曲</td><td> VWR </td><td> 82027-406 </td><td></td></tr><tr><td>自密封的消毒袋</td><td> VWR </td><td> 89140-802 </td><td></td></tr><tr><td>硫酸庆大霉素，10毫克/毫升</td><td>龙沙</td><td> 17 - 519Z </td><td>加入10微升/毫升EpiAirway的MM庆大霉素杀死</td></tr></tbody></table>

use of the epiairway model for characterizing long-term host-pathogen interactions

Nontypeable 流感嗜血杆菌 （NTHI）是人类适应革兰氏阴性菌，可引起复发和慢性感染的呼吸道粘膜1 2。要研究的机制，这些生物生存和呼吸组织内，一个是必需的，可以进行长期成功的细菌和人类细胞的共培养模式。我们使用初级人类呼吸道上皮组织，提高气 - 液界面，EpiAirway模型（MatTek，阿什兰，马萨诸塞州）。这些都是非永生化，分化良好，3维的组织，包含紧密连接，纤毛和nonciliated细胞，杯状细胞产生的粘蛋白，并保留产生细胞因子对感染的反应能力。 有关这种生物在人类上呼吸道的体外模型可以用在许多方面;这种方法的总体目标是，随着时间的推移执行长期NTHI和定量EpiAirway组织共培养细胞相关的和内在的细菌。同时，受感染的共同文化的生产和粘蛋白，细胞因子的个人资料才能确定。这种方法提高了，许多当前的协议后，现有的方法，利用人体细胞淹没单层或Transwell小文化，这是不能够支持长时间的细菌感染超过3。例如，如果一个有机体，可以在覆媒体复制，这可能导致在宿主细胞的细胞毒性和损失不可接受的水平，逮捕了实验。 EpiAirway模型允许长期宿主 - 病原体相互作用的特性。此外，由于EpiAirway源是人体正常细胞，而不是一个永生行气管支气管，每一个实际人类上呼吸道组织的优秀代表，在结构和功能4 ，。 对于这种方法，EpiAirway组织断奶2天交货前的抗微生物和抗真菌化合物，和所有的程序都无抗生素的条件下进行。这就需要特殊的考虑，因为这两种细菌和小学的人体组织在同一生物安全柜使用，并长时间共同培养。

Watch this Scientific Journal Video about Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions at JoVE.com

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

此方法允许EpiAirways，初级人类呼吸道上皮组织生长在气 - 液界面，生物有关延长细菌共同培养的表征<em&gt;在体外</em&gt;车型。该方法可用于任何微生物，是经得起长期的合作文化。

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

use-epiairway-model-for-characterizing-long-term-host-pathogen

Research

JoVE Journal

Immunology and Infection

11.6K 次观看.  Mercer University School of Medicine. 此方法允许EpiAirways，初级人类呼吸道上皮组织生长在气 - 液界面，生物有关延长细菌共同培养的表征在体外车型。该方法可用于任何微生物，是经得起长期的合作文化。

视频: Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

科研

免疫与感染

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

果蝇蛹中炎症细胞动力学的长期体内追踪

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...