这项工作是支持的补助金BAV的克罗恩病和结肠炎基金会和加拿大卫生研究院（CIHR）加拿大（CCFC）。 GB CIHR研究生奖学金的资助。 BAV是的儿童肠和肝脏疾病（儿童）基金会主席儿科IBD的研究和加拿大研究主席在儿科胃肠病学。

1。 枸橼酸rodentium接种物的制备和小鼠灌胃<ol><li>准备和高压灭菌卢里亚Bertani肉汤（LB）。 </li><li>获得可行的C. LB琼脂平板上，用无菌接种环或移液器吸头rodentium从冷冻甘油股票和条纹上。孵育，37℃培养过夜。 ，接种3毫升无菌的LB肉汤猎鹰文化的LB平板上的菌落，用接种环或吸液管头管。应做的接种物的制备，使用无菌技术。 </li><li>孵育C. rodentium文化有氧，在37°C在200转台式培养摇床过夜。接种LB培养基过夜培养后出现混浊。 </li><li>使用一个灯泡放倒连接到1 ml注射器管饲法用100μl的隔夜C.各小鼠灌胃针rodentium文化。鼠标轻轻颈背，紧紧抓住它的脖子和背部松弛的皮肤用拇指和手指。回力动物的头，用食指固定头部和整顿灌胃针插入食道。 </li><li>保持在直立位置的鼠标和指示灌胃针沿它的嘴侧以及全球舌头灯泡小费。轻轻走过针沿着屋顶的嘴和食道推进。如果有阻力，在此过程中感受到，删除灌胃针，并重新将其插入。 </li><li>慢慢注入100μL的细菌接种灌胃针，轻轻取出。监控呼吸速率的鼠标行为后返回到它的笼子。 </li></ol>注： <ul><li>在步骤2和3中，还准备猎鹰培养管中，使用无菌的LB肉汤中培养过夜，用3毫升的无菌技术，以确保有肉汤本身是无细菌污染。 LB培养基过夜培养后出现清晰的。 </li><l&gt;轻轻地搅动文化的Falcon管内装入注射器灌胃同等剂量的细菌传递给每一个鼠标，在步骤4中。 </li><li>应该被丢弃过夜培养，如果它已被超过24小时接种。 </li><li>所有C. rodentium感染和受感染的动物应进行住房在二级生物安全设施。 </li></ul> 2。测量结肠上皮屏障通透性C. rodentium感染的小鼠<ol><li>在期望的时间点感染后，测量屏障的完整性。 </li><li>在测定的当天，准备足够的4 kDa的荧光素异硫氰酸酯（FITC） -葡聚糖溶解在磷酸盐缓冲盐水（PBS）的浓度为80毫克毫升-1灌胃每只小鼠用150μl，并制备标准曲线。 </li><li>浮渣的鼠标和管饲法用150微升的FITC-葡聚糖。在这个时候食物从笼子里取出。 </li><li>麻醉小鼠后4小时gavaging，并收集尽可能多的（450微升）的血液通过心脏穿刺。将血液添加至终浓度为3％酸 - 柠檬酸葡萄糖在微量离心管，以防止凝血。安乐死小鼠，并收集在PBS中的结肠组织细菌计数（步骤3.1 - 3.5）和10％福尔马林固定的组织，并最终免疫荧光染色（步骤4.1 - 4.8）。 </li><li>附带的血液样本在离心机中以1,000 xg离心12分钟，在4℃下收集血清，并稀释至1/10和1/100的PBS。每个样品中加入100μl在这两个稀释到96孔板中复制。 </li><li>为了制备的标准曲线，稀原来的80毫克毫升-1 FITC-葡聚糖用管饲法在PBS的小鼠的浓度如下：800，400，200，100，50，25，12.5，6.25，和0微克/毫升。将100微升各浓度添加到96孔板中，一式三份。 </li><li>使用在激发荧光wav文件，量化各样品的荧光elength表示，485 nm和535 nm的发射波长。 </li><li>分析的原始数据，通过绘制标准曲线。输入的原始数据对每个样品所产生的标准曲线，以确定在每个样品中的浓度的FITC-葡聚糖的线入方程。 </li></ol>注： <ul><li>从第4步向前，保持血液样本，冰，尽量减少暴露在光线下。 </li><li>当测量的时间点之前，或在感染后7天，上皮屏障的完整性是最优的，这一点后，会发生严重的组织损伤。测量前屏障的完整性受到严重损害发生，让您透性C.确定最大的差异小鼠品系之间rodentium感染。 </li><li>确保未感染对照组小鼠上皮屏障的完整性测试。 </li></ul> 3。 C.组织中的细菌负荷测量rodentium感染的小鼠<ol><li>加入1毫升无菌PBS一个第二蒸压一个圆形的金属珠见底2 mL离心管中，用无菌技术。从每个动物收集的单个组织将被放置在单独的PBS管。前称量试管加入组织。 </li><li>删除从鼠标盲肠和结肠。分离盲肠，结肠和推动管腔内容，使用产钳。添加到PBS管的内容。分离后0.5厘米部分，从远端结肠和盲肠的10％福尔马林固定，切开剩余的结肠和盲肠和纵向管组织，用无菌PBS洗涤前。 </li><li>称量PBS管与组织，然后使用珠的搅拌器搅拌6分钟在30 Hz均质化样品。 </li><li>使用无菌技术，180微升无菌PBS中，每孔添加到96孔板中。加入20μl的每个均质样品，每一行的第一口井。搅拌均匀，连续稀释，从每个井获得加入20μl稀释10 -1（第一我们LL）到10 -6。板10微升每种稀释液从每个样品一式三份一个方形的底部与一个网格的LB平板上。孵育过夜，在37°C。 </li><li>计数菌落形成单位（CFU），然后平均3个计数，并记录稀释每个样品计数。乘以平均CFU 50，使用原来的1毫升样品为20微升，和由该样品进行计数得到的以CFU / ml的稀释因子。最后，除以组织CFU / g的重量，以确定。 </li></ol> 4。组织学评估和感染肠组织免疫荧光染色<ol><li>在步骤3.2收集组织。商店在福尔马林隔夜组织，然后用70％的乙醇。嵌入组织中的石蜡和削减5μm切片进行染色。用苏木精 - 伊红染色组织学评估，并进行免疫荧光染色，继续进行下面的步骤。 </li><li>要deparaffinize组织染色前，地点幻灯片一个科普林氏染色缸，放在65°C水浴10分钟。接下来，将幻灯片二甲苯洗涤四次，每2分钟。相片数，然后再水化与两个5分钟的在100％乙醇洗涤，然后由一个5分钟的洗涤在每个下列：95％乙醇，75％乙醇和蒸馏水2 0。这些清洗应在通风橱内进行，以避免暴露于有害气体。 </li><li>将幻灯片中的科普林氏罐预热的柠檬酸钠缓冲液，然后入蒸笼30分钟的地方，然后坐30分钟。此过程会破坏蛋白交联形成的福尔马林固定检索潜在的抗原位点。 </li><li>叠氮化用PBS洗涤5分钟，三次。干片和一个PAP笔创建一个疏水性的障碍，使局部组织染色试剂与周围组织的标志区。 </li><li>组织为1小时，在室温下用封闭缓冲液（ 例如：α-山羊血清）中的免疫染色的湿气室内座。 </li><li>稀释PRIM进制所需浓度的抗体，抗体稀释液。倒掉封闭缓冲液和组织中添加50〜100μl的主要抗体。孵育在4℃孵育过夜或在室温下搅拌2小时。 </li><li>叠氮化用PBS洗涤5分钟，三次。加入50-100微升的抗体稀释缓冲液中稀释到组织中，在室温下孵育1小时，在黑暗中的二次抗体。洗净用dH 2 O 5分钟，三次。 </li><li>脱水幻灯片，的DAPI延长黄金的安装介质和适用的盖玻片。查看幻灯片在荧光显微镜下。 </li></ol>注： <ul><li> ，PBS中与新鲜制备的PBS中，可以被取代的磷酰基叠氮化物，作为一种替代，从步骤4开始，以避免在洗涤介质中的细菌的生长。 </li></ul>

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作者宣称，他们没有竞争的金融利益。

枸橼酸rodentium感染传染病的小鼠结肠炎的研究提供了一个有价值的模型。这种独特的模式允许两个宿主反应，以及细菌的致病特性的表征。这种模式的成功使用该协议的细节中列出的步骤。 有几个关键的步骤，在此协议时，请记住诱导结肠炎和分析反应。首先，准备一个新鲜的过夜C. rodentium在LB肉汤接种感染小鼠的成功。由于需要的剂量为10 8 -10 9</s...

肠道细菌病原体感染引发胃肠道（GI）的炎症，以及肠道病理学与病理生理学，包括腹泻和肠上皮屏障功能障碍。细菌病原体的殖民他们的主机，以及具体的抵御这种感染的宿主反应，胃肠道的致病机制了解甚少，但最近的进步已经开始在肠道细菌感染的造型，以帮助我们了解这些过程。肠道细菌也被连接到的炎症性肠病（IBDs）。 IBDs克罗恩病（CD）和UC复杂疾病的病因不明的慢性肠道炎症和组织损伤，其特征。许多模型小鼠肠道炎症的存在，从在遗传修饰的菌株，如IL-10 - / - 小鼠的自发炎症的化合物，如葡聚糖硫酸钠（DSS）和d，化学挑战initrobenzene磺酸（DNBS）1。据推测，适应不良的慢性炎症反应在IBD患者在遗传易感个体发展后肠黏膜免疫系统的异常暴露的肠道细菌，因此感染性结肠炎模型的研究也提供了显着的潜力，确定潜在的致病主机肠道细菌的反应。 枸橼酸杆菌rodentium诱导的结肠炎是罕见的模式已感染性结肠炎，以及其特征在于1,3之一，允许对分析的宿主反应，以肠溶性的细菌和潜在的IBD发病机制的进一步理解，一个重要的步骤在开发新的预防和治疗方法。 C. rodentium是革兰氏阴性安装和谦虚（A / E），小鼠的特异性细菌性病原体是密切相关的人体重要病原体肠致病性大肠杆菌大肠杆菌 （EPEC），肠出血性大肠杆菌大肠杆菌 （EHEC）3-8。的家庭的A / E病原体密切附加到根尖盲肠和结肠上皮细胞的宿主细胞的膜，形成一种非侵入性的台座状结构的宿主细胞。口腔挑战C. rodentium 10 8 -10 9生物体产生一个强大的感染性结肠炎模型，其特征在于由结肠的隐窝，单核的免疫细胞浸润和杯状细胞耗竭3,4增生或伸长。初始站点的殖民统治，几个小时后，面临的挑战，是在盲肠修补程序，然后发展到远端结肠感染后2〜3天，3。在免疫功能正常的小鼠品系中，间隙实现的病原体感染后3〜4周1,3,4。然而，许多转基因的菌株， 即基因缺失或基因敲除小鼠（ - / - ），已被发现显示增加sed的感染导致夸张的损坏和/或慢性感染和炎症9-14的易感性。这在这些基因敲除株的感染性结肠炎模型，许多人缺乏与生俱来的信号蛋白，一直是不可或缺的分辨率的肠道感染和炎症的组成部分露出几个宿主蛋白。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>卢里亚肉汤</td><td> ABM </td><td> G247 </td><td>添加LB粉25克至1L水。高压灭菌后方可使用。 </td></tr><tr><td>广场底板与电网</td><td>费舍尔</td><td> 08-757-11A </td><td></td></tr><tr><td>猎鹰文化管</td><td>萨尔斯塔特</td><td> 62.515.006 </td><td></td></tr><tr><td>灯泡放倒灌胃针</td><td>精细科学工具</td><td> 18060-20 </td><td></td></tr><tr><td> 1ml注射器</td><td> BD Biosciences公司</td><td> 309659 </td><td></td></tr><tr><td> 4 kDa的FITC-葡聚糖</td><td>西格玛</td><td> FD-4 </td><td></td></tr><tr><td>柠檬酸</td><td>西格玛</td><td> C7129 &lt;/ TD&gt; <td></td></tr><tr><td>柠檬酸钠</td><td>费舍尔</td><td> S279-500 </td><td></td></tr><tr><td>葡萄糖</td><td>费舍尔</td><td> D16.1 </td><td></td></tr><tr><td>柠檬酸盐葡萄糖</td><td></td><td></td><td>的20mM ctiric酸，110 mM柠檬酸钠，5mM的葡萄糖</td></tr><tr><td>黑色96 - 孔板</td><td>费舍尔</td><td> 07-200-762 </td><td></td></tr><tr><td>金属珠（5毫米） </td><td> Qiagen公司</td><td> 69989 </td><td></td></tr><tr><td> 10％的福尔马林</td><td>费舍尔</td><td> 5F93-4 </td><td></td></tr><tr><td> 5毫升小瓶</td><td> DiaMed </td><td> STK3205 </td><td></td></tr><tr><td>苏木</td><td>费舍尔</td><td> H345-23 </td><td></td></tr><tr><td>曙红</td><td>费舍尔</td><td> E511-100 </td><td></td></tr><tr><td>二甲苯</td><td>费舍尔</td><tD&gt; HC700-1GAL </td><td></td></tr><tr><td>吐温20 </td><td>西格玛</td><td> P5927 </td><td></td></tr><tr><td>科普林氏染色缸</td><td> VWR </td><td> 47751-792 </td><td></td></tr><tr><td>柠檬酸钠缓冲液</td><td></td><td></td><td> 10mM柠檬酸钠，0.05％吐温20，pH 6.0的</td></tr><tr><td>巴氏笔</td><td> CEDARLANE </td><td> Mu22 </td><td></td></tr><tr><td>山羊血清</td><td>西格玛</td><td> G902-3 </td><td></td></tr><tr><td>牛血清白蛋白（BSA） </td><td>费舍尔</td><td> BP1600-100 </td><td></td></tr><tr><td>的Triton X-100的</td><td>西格玛</td><td> T8532 </td><td></td></tr><tr><td>叠氮化钠</td><td>西格玛</td><td> SZ002 </td><td></td></tr><tr><td>封闭缓冲液</td><td></td><td></td><td> 2％山羊血清，1％BSA，0.1％Triton X-100，0.05％吐温20，0.05％的氢氧化钠叠氮钠，0.01 M PBS，pH值7.2，混合储存于4°C。 </td></tr><tr><td>抗体稀释液</td><td></td><td></td><td> 0.1％Triton X-100，0.1％BSA，0.05％叠氮钠，0.04％EDTA </td></tr><tr><td>阻断缓冲液和抗体稀释液TIR </td><td></td><td></td><td>与上述相同的配方，但不添加的洗涤剂（TRITON X-100和吐温20） </td></tr><tr><td>延长Gold抗淬灭试剂与DAPI </td><td> Invitrogen公司</td><td> P-36931 </td><td></td></tr><tr><td>盖玻片</td><td>费舍尔</td><td> 12.54SE </td><td></td></tr><tr><td>台式孵化振动筛</td><td> Barnstead实验室线</td><td>最大Q4000 </td><td></td></tr><tr><td>荧光计</td><td> Perkin Elmer公司</td><td> Victor2D </td><td></td></tr><tr><td>冷冻离心机</td><td> Beckman Coulter公司</td><td>微型离心22R</td><td></td></tr><tr><td>汽船</td><td> Black＆Decker公司</td><td></td><td></td></tr><tr><td>荧光显微镜</td><td>蔡司</td><td> Axio上Image.Z1 </td><td></td></tr></tbody></table>

the citrobacter rodentium mouse model: studying pathogen and host contributions to infectious colitis

该协议列出的步骤需要产生一个强大的感染性疾病和肠炎的模型，以及用来描述枸橼酸rodentium感染的小鼠的方法。C. rodentium是一种革兰氏染色阴性，小鼠特异性细菌性病原体，临床上重要的人类病原体肠致病性大肠杆菌是密切相关的大肠杆菌和肠出血性大肠杆菌大肠杆菌 。一旦感染C. rodentium，免疫小鼠患上温和和短暂的体重减轻和腹泻。组织学观察，肠隐窝伸长率，免疫细胞浸润，杯状细胞耗竭。实现间隙感染后3〜4周。肠上皮屏障的完整性，细菌量，以及组织损伤，感染后的不同时间点的测量，让易受感染的小鼠品系的特征。 致病机制s的细菌病原体定植于肠道他们的主机，以及具体的抵御这种感染的宿主反应，却知之甚少。因此，C. rodentium作为一个有价值的工具，以帮助我们了解这些过程模型肠道细菌感染。肠道细菌也被连接到的炎症性肠病（IBDs）。据推测，在肠黏膜免疫系统的异常暴露的肠道细菌基因易感个体适应不良的慢性炎症反应的IBD患者中发展。因此，感染性结肠炎模型的研究提供了重要C.确定潜在的致病性肠道细菌的宿主反应的潜力。 rodentium诱导的结肠炎就是这样一个难得的模式，让肠道细菌的宿主反应的分析，我们进一步了解鸡传染性法氏囊病发病的潜在机制;新的预防和治疗方法的发展至关重要。

在标准的感染实验，小鼠灌胃100μL过夜C.感染者约2.5×10 8 CFU通过rodentium文化。 与 C的C57BL / 6小鼠的感染rodentium结果，在温和和短暂的体重减轻和腹泻。虽然很少发生C57BL / 6小鼠，动物可能生病和需要euthanization。因此，鼠标应该减肥的程度和症状的困扰，如piloerect毛皮和驼背姿势进行监控，以确定不同菌株在何种程度上受到感染。 结果呈现?...

Watch this Scientific Journal Video about The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis at JoVE.com

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

枸橼酸rodentium感染提供了一个有价值的模型来研究肠道细菌感染以及宿主的免疫反应和结肠炎的小鼠。该协议概述了屏障的完整性测量，病原体负荷和病理损害，从而为深入鉴定病原体和宿主小鼠的感染性肠炎的贡献。

“<em&gt;枸橼酸rodentium</em&gt;鼠标产品型号：研究感染性结肠炎的病原体和宿主的贡献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

the-citrobacter-rodentium-mouse-model-studying-pathogen-host

Research

JoVE Journal

Immunology and Infection

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

26.3K 次观看.  BC Children's Hospital. 枸橼酸rodentium感染提供了一个有价值的模型来研究肠道细菌感染以及宿主的免疫反应和结肠炎的小鼠。该协议概述了屏障的完整性测量，病原体负荷和病理损害，从而为深入鉴定病原体和宿主小鼠的感染性肠炎的贡献。

视频: The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

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

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

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

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 ...

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 ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

鼠标型号病原体引起的慢性炎症局部及全身站点

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

实验性感染<em&gt;李斯特菌</em&gt;作为研究主机干扰素γ响应模型

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

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 ...