Os autores gostariam de agradecer ao Dr. Jeffery Weiser da Universidade da Pensilvânia por seu dom das cepas clínicas de Streptococcus pneumoniae. Este trabalho foi financiado pelos Institutos Canadenses de Pesquisa em Saúde. Cv foi financiado por uma bolsa M. G. DeGroote e uma bolsa da Sociedade Torácica Canadense. Este trabalho foi financiado pela Ontário Lung Association e institutos canadenses de pesquisa em saúde (CIHR). O trabalho no laboratório Bowdish é apoiado em parte por Michael G. DeGroote Centre for Infectious Disease Research e pelo McMaster Imunology Research Centre.

<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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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. -. 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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>

Os autores não têm nada a revelar.

Neste estudo, foram apresentados métodos detalhados para a colonização intranasal de camundongos utilizando uma cepa de streptococcus pneumoniae e o subsequente isolamento e caracterização das células imunes recrutadas para nasofaringe em resposta à bactéria. Demonstramos como um inóculo bacteriano pode ser cultivado em mídia rica em nutrientes e usado para estabelecer um evento de colonização em camundongos, que é inicialmente restrito à nasofaringe. Em seguida, mostramos como os tipos de células...

<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

A colonização nasofaríngea por Streptococcus pneumoniae é um pré-requisito para a invasão aos pulmões ou corrente sanguínea1. Este organismo é capaz de colonizar a superfície mucosa da nasofaringe, onde pode residir, multiplicar e eventualmente superar as defesas hospedeiras para invadir outros tecidos do hospedeiro. O estabelecimento de uma infecção no trato respiratório normalmente inferior resulta em pneumonia. Alternativamente, as bactérias podem se disseminar na corrente sanguínea causando bacteremia, que está associada a altas taxas de mortalidade2, ou então levar diretamente ao desenvolvimento de meningite pneumocócica. Entender a cinética e as respostas imunológicas à colonização nasofaríngea é um aspecto importante dos modelos de infecção por S. pneumoniae.
Nosso modelo de camundongos de colonização intranasal é adaptado dos modelos humanos3 e tem sido usado por vários grupos de pesquisa no estudo de respostas hospedeiras-patógenos na nasofaringe4-7. Na primeira parte do modelo, utilizamos um isolado clínico de S. pneumoniae para estabelecer uma colonização bacteriana auto-limitante que seja semelhante aos eventos de transporte em adultos humanos. O procedimento aqui detalhado envolve a preparação de um inóculo bacteriano, seguido da criação de um evento de colonização através da entrega do inóculo através de uma via intranasal de administração. Macrófagos residentes são o tipo de célula predominante na nasofaringe durante o estado estável. Normalmente, há poucos linfócitos presentes em camundongos não infectados8, no entanto a colonização mucosa levará a inflamações de baixo a alto grau (dependendo da virulência das espécies bacterianas e da cepa) que resultarão em uma resposta imune e o subsequente recrutamento de células imunes hospedeiras. Essas células podem ser isoladas por um lavado do conteúdo traqueal através das nares, e correlacionadas com a densidade das bactérias de colonização para entender melhor a cinética da infecção.

A Figura 1 representa uma visão geral que resume os principais passos do protocolo. As figuras 2-3 fornecem visualização da metodologia microbiológica inerente aos protocolos aqui descritos. A Figura 4 representa o posicionamento adequado de um rato para realizar uma colonização intranasal, enquanto a Figura 5 retrata tipicamente mudanças no peso de camundongos colonizados com cepa de S. pneumoniae P1547. As figuras 6-7 r...

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

A colonização da nasofaringe murina com Streptococcus pneumoniae e a subsequente extração de células aderentes ou recrutadas é descrita. Esta técnica envolve a lavagem da nasofaringe e a coleta do fluido através das nares e é adaptável para várias leituras, incluindo quantificação celular diferencial e análise da expressão mRNA in situ.

Caracterização de Respostas Inflamatórias Durante Colonização Intranasal com Streptococcus pneumoniae

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

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Immunology and Infection

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

12.1K visualizações.  McMaster University. A colonização da nasofaringe murina com Streptococcus pneumoniae e a subsequente extração de células aderentes ou recrutadas é descrita. Esta técnica envolve a lavagem da nasofaringe e a coleta do fluido através das nares e é adaptável para várias leituras, incluindo quantificação celular diferencial e análise da expressão mRNA in situ.

Vídeo: 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

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

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

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Usando imagens Bioluminescent para Investigar sinergismo entre Streptococcus pneumoniae E Vírus da Influenza A em Ratos infantil

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

Imunologia e Infecção

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

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

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

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Imagens de células vivas de Bacillus subtilis E Streptococcus pneumoniae Usando Microscopia lapso de tempo Automated

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

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

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

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

Avaliar as alterações metabólicas hepáticas Durante a colonização progressiva de Germ-free Mouse 1 H NMR Spectroscopy

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

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

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

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Acompanhamento da evolução das Membrane Polaridade, integridade de membrana e intracelulares Ion concentrações no<em&gt; Streptococcus pneumoniae</em&gt; Usando corantes fluorescentes

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

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

A sorotipagem capsular de<em&gt; Streptococcus pneumoniae</em&gt; Usando o Reaction Quellung

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

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsular sorotipagem de<em&gt; Streptococcus pneumoniae</em&gt; Por aglutinação em látex

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

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Um modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonização Vaginal

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

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

Aumento do tempo de recuperação e diminuição da Administração LPS para estudar o nervo vago Estimulação Mecanismos em respostas inflamatórias Limitada

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

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

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

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

Um modelo de mouse transiente Transientized IL-8 para o<em&gt; In Vivo</em&gt; Monitoramento de Longo Prazo de Respostas Inflamatórias

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

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.