协助编制本示范的PAL实验室的成员，我们衷心感谢。这项研究是支持小灵通NIH / NIAID的赠款AI076684和AI080615。

1。微量注射若虫肩突硬蜱硬蜱 1。准备针<ol><li>编造通过加热和1毫米的玻璃毛细管（世界精密仪器）拉在一个玻璃微管拉拔设备（Narishige）显微注射针。小心地取出毛细管脆弱。 </li><li>商店在培养皿上胶带拉针（尖朝上）。 </li></ol> 2。准备B。螺旋体 <ol><li>成长B。在BSK培养基7 螺旋体 ，直到浓度每毫升约10 7细胞。通过使用一个佩特罗夫Hausser计数室（Hausser科学）的螺旋体是一个暗视野显微镜下计数。 </li><li>佩莱B。螺旋体 ，在室温为10分钟，在3000 XG离心。 </li><li>去除上清液，并完全重新悬浮颗粒BSK媒体通过200μL无菌微尖 ​​轻轻地传递到一个细胞每毫升10月9日的最后浓度。由于细胞重悬在高密度，可以聚集在一起，细胞悬液应当用于显微注射立即。 </li></ol> 3。准备蜱<ol><li>放置在玻片上明确，双面胶带。 </li><li>在粘尘垫，小心取出若虫蜱，用小刷子的容器。 </li><li>被注入胶带，腹侧，面临着同样的方向，将所需数量的滴答。 </li></ol> 4。注射蜱<ol><li>负载5μL的B.螺旋体进入拉毛细管针使用20μLmicroloader枪头（Eppendorf公司）的文化。检查气泡针​​和针头，如果有必要重新与细菌培养。 </li><li>查看剔双目显微镜下解剖，并专注于打勾的肛门孔径面积。为了打破直径比打勾的肛门光圈稍微小一些，形成的显微​​注射针管，毛细管针的尖端轻轻触摸。这一步是至关重要的，因为任何试图与非适当的针头注入蜱造成伤害和死亡可能注入打勾。 </li><li>双目显微镜下解剖固定蜱，并集中于肛门的光圈，这是由两个可移动的肛门板覆盖。用细镊子，轻轻触摸，非常温和的压力，适用于任何靠近肛门光圈的面积。这将使分离的肛门板和肛孔连接到肠道的开放。小心地插入肛门光圈针尖略，肛门板通过被迫开放。进针应保持在最低限度，玻璃尖可能会损坏半透明肠连接到直肠。使用配备有自动化控制脚（Eppendorf公司）一个微量，注入B。螺旋体的解决方案使用以下参数:1000百帕（HPA）注射压力，注射时间0.2秒和8百帕补偿压力。每个刻度接收单次注射。 </li><li>蜱显微注射后，应做类似的预喷射状态。例如，剔爬在对刺激的反应。 </li><li>我们通常允许蜱在24集的环境室，48小时内恢复16小时/ 8小时，光/暗光照疗法和95％湿度如果不可用，一腔注入蜱可在室温下保存在潮湿条件下，一个密封的容器内，如定期干燥器室，。如果有必要，蜱的恢复时间也可以缩短到几个小时前对小鼠喂养。 </li></ol> 2。共聚焦免疫荧光显微镜的螺旋体本地化 1。解剖蜱<ol><li>液滴放置的磷酸盐缓冲液（PBS）上一个干净的玻璃夹层和另一个幻灯片，在聚- L -赖氨酸涂层滑动显微镜（Sigma公司） 。准备另一载玻片双面胶带。 </li><li>删除从容器中，并用一个小刷子双面胶带打勾。 </li><li>查看和集中在解剖镜下打勾。将锋利的刀片上的第一和第二对腿之间的刻度。按下坚​​决削减两件允许访问的腹部剔。随即，淹没在腹部的PBS液滴。 </li><li>用很细的镊子，切割现场周围抢背，腹的外骨骼。小心地拉出背盾，远离露出了褐色的彩色肠道憩室打勾。要小心，时刻保持下PBS解剖蜱。 </li><li>半透明的唾液腺髻dles位于前的肠道地区的两侧，在这一点上，可以拆卸，如果需要的话。控股钳地方其余的外骨骼，小心地从腹部拉肠道。 </li><li>轻轻地清洁肠道，消除任何被困的组织（例如，气管）。 </li><li>用细镊子的尖端，迅速 ​​转移的PBS液滴在聚- L -赖氨酸的载玻片上剔肠。分成较小的块用细刀片肠道，或用细镊子尖轻轻按下。小心吸肠组织周围多余的PBS。 </li><li>允许肠道组织在室温空气干燥。 </li><li>蜱肠道修复丙酮浸10分钟。允许在室温下干燥空气。幻灯片可以在-20 ° C储存在这一步几个月在一个密封的容器中。 </li></ol> 2。染色<ol><li>使用纸巾，周围的组织中删除多余的水分和子宫颈抹片钢笔或任何疏水屏障衬砌设备画一个圆圈围绕干剔肠。这将有助于保留在随后的孵化步骤染色解决方案。 </li><li>封面与一个或几滴封闭液在室温为30分钟（0.05％Tween - 20的PBS中的5％山羊血清）打勾肠道。取决于阻塞血清抗体的宿主动物源。不要让幻灯片，从这个角度提出干。 </li><li>慢慢吸封闭液。孵育肠道适当的小学和/或继发性的抗体解决方案。我们用异硫氰酸荧光素（FITC） -标记的抗- B。螺旋体抗体（Kirkegaard和佩里实验室）在1:100稀释阻塞在室温下1小时的缓冲。限制暴露在光线下用铝箔盖的容器。 </li><li>卸下温柔的愿望抗体溶液。标签打勾组织，如4，",6 - diamidino - 2 -苯基吲哚（DAPI）或碘化丙啶荧光染料孵育肠道。我们通常使用的PBS 20μg/ mL的碘化丙啶（Sigma公司），5分钟。在室温下。 </li><li> 0.05％吐温20的PBS洗3次。 </li><li>山Slowfade（Invitrogen公司），如抗淬灭剂含有缓冲甘油的幻灯片，并仔细盖上玻璃盖玻片。幻灯片可以存储在一个密封的容器内一步几个月这4 ° C。图像和本地化B。共聚焦显微镜下一个螺旋体 。 </li></ol> 3。代表性的成果若虫蜱为显微注射的位置和图像代表 B。在蜱肠道螺旋体的定位是在图1 。 <img src="/files/ftp_upload/2544/2544fig1.jpg" alt="图1" /> 图1。显微注射法和B 的本地化螺旋体进入蜱肠道。 （一）一个若虫一，腹面观肩突硬蜱的位置进行显微注射。肛门光圈（箭头），用显微注射针插入（箭头）是根据插图的放大。罚款forcep使用，应用温和的压力身体，这使得肛门板块的分离和开放的显微注射肛门孔。针是充满了考马斯亮蓝的解决方案，以提高知名度。（二）代表B的免疫荧光共焦成像结果螺旋体内打勾肠道。肠道憩室前地区。标记与碘化丙啶（红色）或FITC标记的抗B.肠道原子核和螺旋体（箭头） 螺旋体 （绿色）。酒吧= 20微米。

<ol>
	<li>Steere, A. C., Coburn, J., Glickstein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+Lyme+disease.">The emergence of Lyme disease.</a> J Clin Invest. 113, 1093-1101 (2004).</li><li>Barthold, S. W., Diego, C., Philipp, M. T., Samuels, D. S., Radolf, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Borrelia, Molecular Biology, Host Interaction and Pathogenesis. , 353-405 (2010).</li><li>Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OspC+facilitates+Borrelia+burgdorferi+invasion+of+Ixodes+scapularis+salivary+glands.">OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands.</a> J Clin Invest. 113, 220-230 (2004).</li><li>Yang, X. F., Pal, U., Alani, S. M., Fikrig, E., Norgard, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+OspA/B+in+the+life+cycle+of+the+Lyme+disease+spirochete.">Essential role for OspA/B in the life cycle of the Lyme disease spirochete.</a> J Exp Med. 199, 641-648 (2004).</li><li>Zhang, X., Yang, X., Kumar, M., Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BB0323+function+is+essential+for+Borrelia+burgdorferi+virulence+and+persistence+through+tick-rodent+transmission+cycle.">BB0323 function is essential for Borrelia burgdorferi virulence and persistence through tick-rodent transmission cycle.</a> J Infect Dis. 200, 1318-1330 (2009).</li><li>Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TROSPA,+an+Ixodes+scapularis+receptor+for+Borrelia+burgdorferi.">TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi.</a> Cell. 119, 457-468 (2004).</li><li>Barbour, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+Lyme+disease+spirochetes.">Isolation and cultivation of Lyme disease spirochetes.</a> Yale J Biol Med. 57, 521-525 (1984).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Ixodes+scapularis+anticoagulation+by+using+RNA+interference.">Disruption of Ixodes scapularis anticoagulation by using RNA interference.</a> Proc Natl Acad Sci U S A. 101, 1141-1146 (2004).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tick+antioxidant+facilitates+the+Lyme+disease+agent's+successful+migration+from+the+mammalian+host+to+the+arthropod+vector.">A tick antioxidant facilitates the Lyme disease agent's successful migration from the mammalian host to the arthropod vector.</a> Cell Host Microbe. 2, 7-18 (2007).</li><li>Broadwater, A. H., Sonenshine, D. E., Hynes, W. L., Ceraul, S., DeSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glass+capillary+tube+feeding:+a+method+for+infecting+nymphal+Ixodes+scapularis+(Acari:+Ixodidae)+with+the+Lyme+disease+spirochete+Borrelia+burgdorferi.">Glass capillary tube feeding: a method for infecting nymphal Ixodes scapularis (Acari: Ixodidae) with the Lyme disease spirochete Borrelia burgdorferi.</a> J Med Entomol. 39, 285-292 (2002).</li><li>Policastro, P. F., Schwan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+infection+of+Ixodes+scapularis+larvae+(Acari:+Ixodidae)+by+immersion+in+low+passage+cultures+of+Borrelia+burgdorferi.">Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi.</a> J Med Entomol. 40, 364-370 (2003).</li><li>Fuente, d. e. l. a., Kocan, J., M, K., Almazan, C., Blouin, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+for+the+study+and+genetic+manipulation+of+ticks.">RNA interference for the study and genetic manipulation of ticks.</a> Trends Parasitol. 23, 427-433 (2007).</li></ol>

在这里，我们展示一个基于显微注射过程快速，高效的蜱若虫硬蜱感染的细菌病原体B.螺旋体 。我们还描述了B 的检测共聚焦免疫程序螺旋体在蜱肠道原位 。虽然我们的演示涉及若虫肠道，类似的程序也适用其他蜱的发育阶段，如幼虫或成虫8,9 ，。然而，由于其较小的尺寸，该技术可能会比较具有挑战性的幼虫，但应在成年蜱适用。 与 B蜱人工感染其他方法螺旋体都得到发展，如通过玻璃毛细管10喂养或浸泡在培养基中11。这些方法是高效，简单和相对便宜的的。然而，这些程序依赖于B 的转移相对失控螺旋体成单个蜱，从而有可能被限制在与平等的病原体负担出没蜱的同伙。后者的缺点可能会显着更多的控制下交付程序，如显微注射，克服。在我们的实验室中，我们经常采用这种转移 B.程序成蜱的感染率近100％，而且大多数蜱螺旋体生存的过程。注入蜱可存放在实验室几个星期到几个月，或立即允许大吃对小鼠和传输B。螺旋体感染。 B 的效率和动力学螺旋体从显微注射蜱传播的类似自然感染的蜱，因此，人工蜱感染程序可能在我们努力学习蜱传播的莱姆borreliosis协助。此外，类似的显微注射技术也被应用于相关的实验目的，例如，RNA干扰介导的基因操纵蜱 6,9,12 。 微量注射蜱的不成熟阶段，是一个比较微妙的过程。因此，这是非常重要的验证，蜱与 B注入螺旋体是健康的，然后再继续下一组实验。注射后缩回腿蜱和刺激，如呼出的呼吸或用刷子触摸反应迟钝，不应该用于进一步的实验。这些都是最有可能死亡或注射创伤死亡的边缘。我们已经发现，针头和注射参数的大小的过程中的两个关键因素，较大的针尖和注射量，可能导致高打勾死亡率的肠壁破裂。同样重要的是要注意，这里描述的显微注射设置主要是注射 B.优化螺旋体悬浮在BSK媒体。其他生物材料，如抗体或集中的RNA解决方案，可能与流体的粘度。对于其他材料，最佳显微注射的设置，主要是注射压力和注射时间，需要凭经验确定。

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<td>Glass capillary tubes</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Vertical glass puller</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>PC-10</td>
<td>&nbsp;</td>
<tr>
<td>Petroff-Hausser counting chamber</td>
<td>&nbsp;</td>
<td>Hausser scientific</td>
<td>3900</td>
<td>&nbsp;</td>
<tr>
<td>Microloader pipette tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>930001007</td>
<td>&nbsp;</td>
<tr>
<td>Femtojet microinjector</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>920010504</td>
<td>&nbsp;</td>
<tr>
<td>Foot control FemtJet</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>920005098</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP665-1</td>
<td>Filter-sterilized</td>
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methods for rapid transfer and localization of lyme disease pathogens within the tick gut

莱姆病是由螺旋体的病原体伯氏疏螺旋体 ，保持它在本质上是由蜱灭鼠感染周期1感染引起的。蜱传播的小鼠模型已发展到在实验室中研究莱姆病。虽然天真蜱可感染与 B 螺旋体感染小鼠喂养，蜕皮过程需要几个星期到几个月才能完成。因此，发展更迅速，更高效的蜱感染技术，如显微注射为基础的程序，是为莱姆病3,4研究的重要工具。过程需要几个小时，以产生感染的蜱，并允许在同等数量的螺旋体的蜱队列交付的控制。这是B 的一代尤为重要螺旋体感染的蜱自然喂养利用老鼠进行的过程中失败，以确保100％的感染率和病原体的负担变化的潜在的结果除美联储蜱。此外，显微注射，可用于感染蜱与 B 螺旋体分离的情况下，弱毒株不能建立在小鼠体内的感染，并因此不能自然由蜱5收购。这种技术也可用于成蜱提供多种其他生物材料，例如，特异性抗体或双链RNA 6。在这篇文章中，我们将证明在体外生长B.若虫蜱显微注射螺旋体 。我们还将描述为莱姆病的病原体在蜱肠道使用共聚焦免疫荧光显微镜的本地化方法。

Watch this Scientific Journal Video about Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut at JoVE.com

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

莱姆病的研究往往需要一代感染的蜱与莱姆病螺旋体的病原体，这一过程通常需要几个星期的。在这里，我们展示了一个基于显微注射蜱感染过程，可在数小时内完成。我们也证明内蜱免疫荧光法在B的螺旋体原位本地化。

莱姆病的病原体的蜱肠道内的快速传递和本地化的方法

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

methods-for-rapid-transfer-localization-lyme-disease-pathogens-within

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.5K 次观看.  University of Maryland. 莱姆病的研究往往需要一代感染的蜱与莱姆病螺旋体的病原体，这一过程通常需要几个星期的。在这里，我们展示了一个基于显微注射蜱感染过程，可在数小时内完成。我们也证明内蜱免疫荧光法在B的螺旋体原位本地化。

视频: Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

"发光法"的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

科研

免疫与感染

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

从救援的cDNA重组新城疫病毒

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

方法基因水平转移的研究<em&gt;金黄色葡萄球菌</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

从正式固定组织检测利沙病毒抗原的免疫化学测试

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

在 Vivo 增强的古特-霍姆调节 T 细胞诱导

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...