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

Die Autoren danken Dr. Jeffery Weiser von der University of Pennsylvania für sein Geschenk der klinischen Stämme von Streptococcus pneumoniae. Diese Arbeit wurde von den Canadian Institutes for Health Research finanziert. CV wurde durch ein M. G. DeGroote Stipendium und ein Stipendium der Canadian Thoracic Society finanziert. Diese Arbeit wurde von der Ontario Lung Association und den Canadian Institutes of Health Research (CIHR) finanziert. Die Arbeit im Bowdish-Labor wird zum Teil von Michael G. DeGroote Centre for Infectious Disease Research und dem McMaster Immunology Research Centre unterstützt.

Bevor Sie beginnen: Alle Schritte werden in einem Biohazard Level 2 (BSL2) Biological Safety Cabinet (BSC) durchgeführt, sofern nicht anders angegeben. Bitte stellen Sie sicher, dass Sie vor Beginn der Experimente die entsprechende Biohazard-Zulassung für die Verwendung infektiöser bakterieller Krankheitserreger gemäß den institutionellen Richtlinien erhalten haben. Bitte stellen Sie außerdem sicher, dass Sie alle Materialien und Reagenzien haben, die für die Durchführung des Verfahrens im Voraus...

<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. U.S.A. 103, 2857-2862 (2006).</li><li>Malley, R., Henneke, P., 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=Recognition+of+pneumolysin+by+Toll-like+receptor+4+confers+resistance+to+pneumococcal+infection.">Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 1966-1971 (2003).</li><li>McCool, T. L., 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=Limited+role+of+antibody+in+clearance+of+Streptococcus+pneumoniae+in+a+murine+model+of+colonization.">Limited role of antibody in clearance of Streptococcus pneumoniae in a murine model of colonization.</a> Infect. Immun. 72, 5807-5813 (2004).</li><li>Gingles, N. 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=Role+of+genetic+resistance+in+invasive+pneumococcal+infection:+identification+and+study+of+susceptibility+and+resistance+in+inbred+mouse+strains.">Role of genetic resistance in invasive pneumococcal infection: identification and study of susceptibility and resistance in inbred mouse strains.</a> Infect. Immun. 69 (1), 426-434 (2001).</li><li>Jeong, D., Jeong, 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=Difference+in+resistance+to+Streptococcus+pneumoniae+infection+in+mice.">Difference in resistance to Streptococcus pneumoniae infection in mice.</a> Lab Anim. Res. 27, 91-98 (2011).</li><li>Wu, H. 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. 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=Visualization+of+Murine+Intranasal+Dosing+Efficiency+Using+Luminescent+Francisella+tularensis:+Effect+of+Instillation+Volume+and+Form+of+Anesthesia.">Visualization of Murine Intranasal Dosing Efficiency Using Luminescent Francisella tularensis: Effect of Instillation Volume and Form of Anesthesia.</a> PLoS ONE. 7 (2), (2012).</li><li>Briles, D. E., Novak, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+Colonization+with+Streptococcus+pneumoniae+includes+subpopulations+of+surface+and+invasive+pneumococci.">Nasal Colonization with Streptococcus pneumoniae includes subpopulations of surface and invasive pneumococci.</a> Infect. Immun. 73 (10), 6945-6951 (2005).</li><li>Wu, H. -. 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>Mo, Y., Wan, 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=Application+of+reverse+transcription-PCR+and+real-time+PCR+in+nanotoxicity+research.">Application of reverse transcription-PCR and real-time PCR in nanotoxicity research.</a> Methods Mol. Biol. 926, 99-112 (2012).</li><li>Kuper, C. F., Koornstra, P. 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=The+role+of+nasopharyngeal+lymphoid+tissue.">The role of nasopharyngeal lymphoid tissue.</a> Trends Immunol. 13, 219-224 (1992).</li><li>Zhang, Q., Leong, S. C., 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=Characterisation+of+regulatory+T+cells+in+nasal+associated+lymphoid+tissue+in+children:+relationships+with+pneumococcal+colonization.">Characterisation of regulatory T cells in nasal associated lymphoid tissue in children: relationships with pneumococcal colonization.</a> PLoS Pathog. 7, (2011).</li><li>Briles, D. E., Novak, L., 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=Nasal+colonization+with+Streptococcus+pneumoniae+includes+subpopulations+of+surface+and+invasive+pneumococci.">Nasal colonization with Streptococcus pneumoniae includes subpopulations of surface and invasive pneumococci.</a> Infect. Immun. 73, 6945-6951 (2005).</li><li>Weinberger, D. M., Trzcinski, K., 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=Pneumococcal+capsular+polysaccharide+structure+predicts+serotype+prevalence.">Pneumococcal capsular polysaccharide structure predicts serotype prevalence.</a> PLoS Pathog. 5, (2009).</li><li>Bryant, W. P., 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=Which+Pneumococcal+Serogroups+Cause+the+Most+Invasive+Disease:+Implications+for+Conjugate+Vaccine+Formulation+and+Use,+Part+I..">Which Pneumococcal Serogroups Cause the Most Invasive Disease: Implications for Conjugate Vaccine Formulation and Use, Part I..</a> Clin. Infect. Dis. 30, 100-121 (2000).</li><li>Hausdorff, W. P., Feikin, D. 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=Epidemiological+differences+among+pneumococcal+serotypes.">Epidemiological differences among pneumococcal serotypes.</a> Lancet Infect. 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. M., Khandavalli, S., Camberlein, E., Hyams, C., Baxendale, H. E., Brown, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+contributions+against+invasive+Streptococcus+pneumoniae+pneumonia+of+antibody+and+Th17-Cell+responses+to+nasopharyngeal+colonisation.">Protective contributions against invasive Streptococcus pneumoniae pneumonia of antibody and Th17-Cell responses to nasopharyngeal colonisation.</a> PLoS One. 6 (10), (2011).</li><li>Cohen, J. M., Khandavalli, S., Camberlein, E., Hyams, C., Baxendale, H. E., Brown, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+contributions+against+invasive+Streptococcus+pneumoniae+pneumonia+of+antibody+and+Th17-Cell+responses+to+nasopharyngeal+colonisation.">Protective contributions against invasive Streptococcus pneumoniae pneumonia of antibody and Th17-Cell responses to nasopharyngeal colonisation.</a> PLoS One. 6 (10), (2011).</li><li>Richards, L., Ferreira, D. M., Miyaji, E. N., Andrew, P. W., Kadioglu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunising+effect+of+pneumococcal+nasopharyngeal+colonisation;+protection+against+future+colonisation+and+fatal+invasive+disease.">The immunising effect of pneumococcal nasopharyngeal colonisation; protection against future colonisation and fatal invasive disease.</a> Immunobiology. , 215-251 (2010).</li><li>Lanie, J. A., Ng, W. L., 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=Genome+sequence+of+Avery's+virulent+serotype+2+strain+D39+of+Streptococcus+pneumoniae+and+comparison+with+that+of+unencapsulated+laboratory+strain+R6.">Genome sequence of Avery's virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6.</a> J. Bacteriol. 189, 38-51 (2007).</li><li>Robertson, G. T., Ng, W. L., Foley, J., Gilmour, R., Winkler, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+transcriptional+analysis+of+clpP+mutations+of+type+2+Streptococcus+pneumoniae+and+their+effects+on+physiology+and.">Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and.</a> 184, 3508-3520 (2002).</li><li>Orihuela, C. J., Gao, G., 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=Tissue-specific+contributions+of+pneumococcal+virulence+factors+to+pathogenesis.">Tissue-specific contributions of pneumococcal virulence factors to pathogenesis.</a> J. Infect. Dis. 190, 1661-1669 (2004).</li><li>Orihuela, C. J., Gao, G., 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=Organ-specific+models+of+Streptococcus+pneumoniae+disease.">Organ-specific models of Streptococcus pneumoniae disease.</a> Scand. J. Infect. D. 35, 647-652 (2003).</li><li>Swirski, F. K., Nahrendorf, 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=Identification+of+splenic+reservoir+monocytes+and+their+deployment+to+inflammatory+sites.">Identification of splenic reservoir monocytes and their deployment to inflammatory sites.</a> Science. 325, 612-616 (2009).</li></ol>

In dieser Studie präsentierten wir detaillierte Methoden für die intranasale Besiedlung von Mäusen unter Verwendung eines klinischen Isolatestamms von Streptococcus pneumoniae und die anschließende Isolierung und Charakterisierung der Immunzellen, die als Reaktion auf die Bakterien an Nasopharynx rekrutiert wurden. Wir demonstrierten, wie ein bakterielles Inokulum in nährstoffreichen Medien kultiviert werden kann und verwendet werden, um ein Kolonisationsereignis bei Mäusen zu etablieren, das zunächst auf...

<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

Nasopharyngeale Kolonisation durch Streptococcus pneumoniae ist eine Voraussetzung für die Invasion in die Lunge oder Blutkreislauf1. Dieser Organismus ist in der Lage, die Schleimhautoberfläche des Nasopharynx zu besiedeln, wo er sich aufhalten, sich vermehren und schließlich wirtsabwehren überwinden kann, um in andere Gewebe des Wirts einzudringen. Die Einrichtung einer Infektion in den normalerweise unteren Atemwegen führt zu einer Lungenentzündung. Alternativ können sich die Bakterien in den Blutkreislauf verbreiten und Bakteriämie verursachen, die mit hohen Sterblichkeitsraten verbunden ist2, oder direkt zur Entwicklung von Pneumokokken-Meningitis führen. Das Verständnis der Kinetik und immunioniert auf die nasopharyngeale Kolonisation ist ein wichtiger Aspekt der S. pneumoniae-Infektionsmodelle.
Unser Mausmodell der intranasalen Kolonisation ist an die menschlichen Modelle3 angepasst und wurde von mehreren Forschungsgruppen bei der Untersuchung von Wirtspathogen-Antworten im Nasopharynx4-7verwendet. Im ersten Teil des Modells verwenden wir ein klinisches Isolat von S. pneumoniae, um eine selbstbegrenzende bakterielle Kolonisation zu etablieren, die den Beförderungsereignissen bei menschlichen Erwachsenen ähnelt. Das hier beschriebene Verfahren beinhaltet die Vorbereitung eines bakteriellen Inokulums, gefolgt von der Etablierung eines Kolonisationsereignisses durch Lieferung des Inokulums über einen intranasalen Verabreichungsweg. Residente Makrophagen sind der vorherrschende Zelltyp im Nasopharynx während des stationären Zustands. Typischerweise gibt es nur wenige Lymphozyten in nicht infizierten Mäusen8, aber Schleimhautbesieonisation führt zu niedrigen bis hohen Grad Entzündungen (abhängig von der Virulenz der bakteriellen Arten und Stamm), die zu einer Immunantwort und der anschließenden Rekrutierung von Wirtsimmunzellen führen. Diese Zellen können durch eine Lavage des Trachealinhalts durch die Nares isoliert werden und korrelieren mit der Dichte von Besiedlungsbakterien, um die Kinetik der Infektion besser zu verstehen.

Abbildung 1 stellt eine Übersichtschaltplan mit den wichtigsten Schritten des Protokolls dar. Die Abbildungen 2-3 enthalten eine Visualisierung der mikrobiologischen Methodik, die den hier beschriebenen Protokollen innewohnt. Abbildung 4 stellt die richtige Positionierung einer Maus dar, um eine intranasale Kolonisation durchzuführen, während Abbildung 5 typischerweise Gewichtsänderungen von Mäusen zeigt, die mit dem S. pneumoniae-Stamm P15...

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

Die Kolonisation des murinen Nasopharynx mit Streptococcus pneumoniae und die anschließende Extraktion von anhaftenden oder rekrutierten Zellen wird beschrieben. Diese Technik beinhaltet das Spülen des Nasopharynx und die Sammlung der Flüssigkeit durch die Nares und ist anpassungsfähig für verschiedene Auslesungen, einschließlich Differentialzellquantifizierung und Analyse der mRNA-Expression in situ.

Charakterisierung von Entzündungsreaktionen während der intranasalen Kolonisation mit Streptococcus pneumoniae

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

Mit Bioluminescent Imaging, um Synergie von Untersuchen Streptococcus pneumoniae Und Influenza A Virus in Infant Mäuse

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

Forschung

Immunologie und Infektion

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

Live Cell Imaging von Bacillus subtilis Und Streptococcus pneumoniae Verwendung von Automated Zeitraffer-Mikroskopie

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

Die Beurteilung der Leber metabolischen Veränderungen während Progressive Colonization von Keimfrei Maus durch 1 H-NMR-Spektroskopie

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

Überwachen von Änderungen in Membran-Polarity, Membrane Integrity und intrazelluläre Ionenkonzentrationen in<em&gt; Streptococcus pneumoniae</em&gt; Verwenden von Fluoreszenzfarbstoffen

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

Kapsel Serotypisierung von<em&gt; Streptococcus pneumoniae</em&gt; Verwenden der Quellung Reaktion

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

Kapsel Serotypisierung<em&gt; Streptococcus pneumoniae</em&gt; Durch Latexagglutination

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

Ein Mausmodell der Gruppe B<em&gt; Streptococcus</em&gt; Vaginal Colonization

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

Erhöhte Erholzeit und abnehmend LPS-Verabreichung die Vagusnervstimulation Mechanisms in beschränkten Entzündungsreaktionen zu studieren

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

Ein IL-8 transientes transgenisiertes Mausmodell für die<em&gt; In vivo</em&gt; Langzeitüberwachung von entzündlichen Reaktionen

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

Ca2 + Antworten Imaging während der Shigella Infektion von Epithelzellen

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