Name: characterization of inflammatory responses during intranasal colonization with streptococcus pneumoniae
Uploaded: 2014-01-17T13:00:00
Description: Watch this Scientific Journal Video about Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae at JoVE.com

作者要感谢宾夕法尼亚大学的杰弗瑞·韦瑟博士赠送肺炎 链球菌临床菌株。这项工作由加拿大卫生研究所资助。简历由M.G.德格罗特奖学金和加拿大胸科学会的奖学金资助。这项工作由安大略肺脏协会和加拿大卫生研究所（CIHR）资助。鲍迪什实验室的工作部分得到了迈克尔·德格罗特传染病研究中心和麦克马斯特免疫学研究中心的支持。

在开始之前：除非另有说明，否则所有步骤均在生物危害 2 级 （BSL2） 生物安全柜 （BSC） 中完成。请确保在开始实验之前，您已获得适当的生物危害批准，以便根据机构指南使用传染性细菌病原体。此外，请确保您拥有进行事先准备的程序所需的所有材料和试剂。这些实验中使用的老鼠包括杰克逊实验室、查尔斯河或塔科尼奇的雌性C57BL/6小鼠，年龄在10-14周（尽管我们尚未发现鼻腔...

<ol>
	<li>Bogaert, D., de Groot, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pneumoniae+colonisation:+the+key+to+pneumococcal+disease.">Streptococcus pneumoniae colonisation: the key to pneumococcal disease.</a> Lancet Infect. Dis. 4, 144-154 (2004).</li><li>Kadioglu, A., Weiser, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Streptococcus+pneumoniae+virulence+factors+in+host+respiratory+colonization+and+disease.">The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease.</a> Nat. Rev. Microbiol. 6 (4), 288-301 (2008).</li><li>McCool, T. L., Cate, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+response+to+pneumococcal+proteins+during+experimental+human+carriage.">The immune response to pneumococcal proteins during experimental human carriage.</a> J. Exp. Med. 195, 359-365 (2002).</li><li>Nelson, A., Roche, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsule+enhances+pneumococcal+colonisation+by+limiting+mucus-mediated+clearance.">Capsule enhances pneumococcal colonisation by limiting mucus-mediated clearance.</a> Infect. Immun. 75, 83-90 (2007).</li><li>van Rossum, A., Lysenko, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+bacterial+factors+contributing+to+the+clearance+of+colonisation+by+Streptococcus+pneumoniae+in+a+murine+model.">Host and bacterial factors contributing to the clearance of colonisation by Streptococcus pneumoniae in a murine model.</a> Infect. Immun. 73, 7718-7726 (2005).</li><li>Barocchi, M. A., Ries, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumococcal+pilus+influences+virulence+and+host+inflammatory+responses.">A pneumococcal pilus influences virulence and host inflammatory responses.</a> Proc. Natl. Acad. Sci. 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Y., Virolainen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+Streptococcus+pneumoniae+nasopharyngeal+colonization+model+in+adult+mice.">Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice.</a> Microb. Pathog. 23, 127-137 (1997).</li><li>Southam, D. S., Dolovich, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+intranasal+instillations+in+mice:+effects+of+volume,+time,+body+position.">Distribution of intranasal instillations in mice: effects of volume, time, body position.</a> Lung Physiol. 282, 833-839 (2002).</li><li>Miller, M. A., Stabenow, J. 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Dis. 5, 83-93 (2005).</li><li>Brueggemann, A., Griffiths, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+Relationships+between+Invasive+and+Carriage+Streptococcus+pneumoniae+and+Serotype+and+Clone+Specific+Differences+in+Invasive+Disease+Potential.">Clonal Relationships between Invasive and Carriage Streptococcus pneumoniae and Serotype and Clone Specific Differences in Invasive Disease Potential.</a> J. Infect. Dis. 187, 1424-1432 (2003).</li><li>Mohler, J., Azoulay-Dupis, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pneumoniae+strain-dependent+lung+inflammatory+responses+in+a+murine+model+of+pneumococcal+pneumonia.">Streptococcus pneumoniae strain-dependent lung inflammatory responses in a murine model of pneumococcal pneumonia.</a> Intensive Care Med. 29, 808-816 (2003).</li><li>Wu, H. Y., Virolainen, A., Mathews, B., King, J., Russell, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+Streptococcus+pneumoniae+nasopharyngeal+colonization+model+in+adult+mice.">Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice.</a> Microb. Pathog. 23, 127-137 (1997).</li><li>Zhang, Z., Clarke, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+effectors+mediating+Th17-dependent+clearance+of+pneumococcal+colonization+in+mice.">Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice.</a> J. Clin. Invest. 119, 1899-1909 (2009).</li><li>Parker, D., Martin, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pneumoniae+DNA+initiates+type+I+interferon+signaling+in+the+respiratory+tract.">Streptococcus pneumoniae DNA initiates type I interferon signaling in the respiratory tract.</a> MBio. 2, (2011).</li><li>Haya, D. L., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+identification+of+serotype+4+Streptococcus+pneumoniae+virulence+factors.">Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors.</a> Mol. Microbiol. 45, 1389-1406 (2002).</li><li>Nakamura, S., Favis, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+stimulation+of+type+I+interferons+during+influenza+virus+coinfection+promotes+Streptococcus+pneumoniae+colonization+in+mice.">Synergistic stimulation of type I interferons during influenza virus coinfection promotes Streptococcus pneumoniae colonization in mice.</a> J. Clin. Invest. 121, 3657-3665 (2011).</li><li>Kim, J. O., Weiser, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+intrastrain+phase+variation+in+quantity+of+capsular+polysaccharide+and+teichoic+acid+with+the+virulence+of+Streptococcus+pneumoniae.">Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae.</a> J. Infect. Dis. 177, 368-377 (1998).</li><li>Roche, A. M., King, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+attenuated+Streptococcus+pneumoniae+strains+induce+serotype-independent+mucosal+and+systemic+protection+in+mice.">Live attenuated Streptococcus pneumoniae strains induce serotype-independent mucosal and systemic protection in mice.</a> Infect. Immun. 75, 2469-2475 (2007).</li><li>Cohen, J. 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在这项研究中，我们提出了使用 肺炎链球菌 临床分离菌株的小鼠的鼻内结肠的详细方法，以及随后针对细菌招募到鼻咽的免疫细胞的隔离和特征。我们演示了如何在营养丰富的介质中培养细菌，并用于在小鼠中建立殖民活动，最初仅限于鼻咽。然后，我们展示了如何通过使用罐装针头，在气管暴露、切口和鼻腔熔洗后分离出招募到鼻咽的免疫细胞类型。鼻腔熔岩样本可在PBS中采集，以分?...

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse Ly6C FITC</td>
 <td>BD Pharmingen</td>
 <td>553104</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse Ly6G PE</td>
 <td>BD Pharmingen</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse CD45.1 eFluor 450</td>
 <td>eBioscience</td>
 <td>48-0453-82</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse F4/80 Antigen APC</td>
 <td>eBioscience</td>
 <td>17-4801-82</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse CD11c PerCP-Cy5.5</td>
 <td>eBioscience</td>
 <td>45-0114-82</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse CD11b PE-Cy7</td>
 <td>eBioscience</td>
 <td>25-0112-82</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Mouse CD3 Alexa Fluor 700</td>
 <td>eBioscience</td>
 <td>56-0032-82</td>
 <td></td>
 </tr>
 <tr>
 <td><a href="http://us.ebioscience.com/mouse-cd4-antibody-efluor-605nc-gk15.htm" target="_blank">Anti-Mouse CD4 eFluor 605NC</a></td>
 <td>eBioscience</td>
 <td>93-0041-42</td>
 <td></td>
 </tr>
 <tr>
 <td>Intramedic Polyethylene Tubing - PE20</td>
 <td>Becton Dickinson</td>
 <td>427406</td>
 <td></td>
 </tr>
 <tr>
 <td>BD 1ml Syringe</td>
 <td>Becton Dickinson</td>
 <td>309659</td>
 <td></td>
 </tr>
 <tr>
 <td>BD 26G3/8 Intradermal Bevel</td>
 <td>Becton Dickinson</td>
 <td>305110</td>
 <td></td>
 </tr>
 <tr>
 <td>Buffer RLT Lysis Buffer</td>
 <td>Qiagen</td>
 <td>79216</td>
 <td></td>
 </tr>
 <tr>
 <td>Difco Tryptic Soy Agar</td>
 <td>Becton Dickinson</td>
 <td>236950</td>
 <td></td>
 </tr>
 <tr>
 <td>Defibrinated Sheep Blood</td>
 <td>PML Microbiologicals</td>
 <td>A0404</td>
 <td></td>
 </tr>
 <tr>
 <td>RNAqueous-Micro Kit</td>
 <td>Ambion</td>
 <td>AM1931</td>
 <td></td>
 </tr>
 <tr>
 <td>M-MuLV Reverse Transcriptase</td>
 <td>New England Biolabs</td>
 <td>M0253L</td>
 <td></td>
 </tr>
 <tr>
 <td>GoTaq qPCR Master Mix</td>
 <td>Promega</td>
 <td>A6001</td>
 <td></td>
 </tr>
 </tbody>
</table>

characterization of inflammatory responses during intranasal colonization with streptococcus pneumoniae

肺炎链球菌的鼻咽结肠是侵入肺部或血液的先决条件。这种生物体能够殖民鼻咽的粘膜表面，在那里它可以居住，繁殖，并最终克服宿主防御入侵到宿主的其他组织。在通常的下呼吸道中发现感染会导致肺炎。或者，细菌可以传播到血液中，导致细菌血症，这与高死亡率2有关，否则直接导致肺炎球菌脑膜炎的发展。了解鼻咽结肠的动能和免疫反应是S.肺炎感染模型的一个重要方面。
我们的小鼠内殖民化模型改编自人类模型3， 并被多个研究小组用于鼻咽4-7宿主病原体反应的研究。在模型的第一部分，我们使用 肺炎 的临床分离建立一个自我限制的细菌殖民化，类似于人类成人的运输事件。此处详细说明的程序包括准备细菌接种，然后通过通过鼻内管理途径传递接种库建立殖民化事件。在稳定状态下，居民巨噬细胞是鼻咽的主要细胞类型。通常，在未受感染的小鼠8中，淋巴细胞很少，但粘膜结肠会导致低至高档炎症（取决于细菌物种和菌株的毒性），从而产生免疫反应，并随后招募宿主免疫细胞。这些细胞可以通过气管内含物的厕所进行分离，并与殖民化细菌的密度相关，以更好地了解感染的动能。

图 1 表示概述示意图，总结了协议的主要步骤。 图2-3 提供了本文所述协议所固有的微生物方法的可视化。 图4 表示小鼠进行鼻内殖民化的正确定位，而 图5 则描绘了与 S.肺炎 菌株P1547结肠的小鼠体重的典型变化。 图6-7 表示过程鼻腔熔洗部分的特定阶段，用于辅助可视化这两种技术。 图8-11 包括对从鼻腔清洗后从...

Watch this Scientific Journal Video about Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae at JoVE.com

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

描述了 肺炎链球菌 的鼻咽结肠化以及随后对附体或招募细胞的提取。该技术涉及冲洗鼻咽和收集流体通过鼻孔，并适应各种读数，包括微分细胞定量和分析的mRNA表达 原位。

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Research

JoVE Journal

Immunology and Infection

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

在无菌无鼠标进定植评估肝代谢的变化 1 H核磁共振光谱

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

荚膜血清型<em&gt;肺炎链球菌</em&gt;使用荚膜肿胀反应

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

荚膜血清型<em&gt;肺炎链球菌</em&gt;乳胶凝集

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

A Murine Model of Group B Streptococcus Vaginal Colonization

A Murine Model of Group B Streptococcus Vaginal Colonization

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

增加了恢复时间，并减少LPS管理研究迷走神经刺激机制的有限炎症反应

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

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

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

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 ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

成像 Ca2 +在志贺菌感染上皮细胞期间的响应

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...