Gli autori ringraziano il Dr. Jeffery Weiser dell'Università della Pennsylvania per il suo dono dei ceppi clinici di Streptococcus pneumoniae. Questo lavoro è stato finanziato dal Canadian Institutes for Health Research. CV è stato finanziato da una borsa di studio M. G. DeGroote e da una borsa di studio della Canadian Thoracic Society. Questo lavoro è stato finanziato dalla Ontario Lung Association e dal Canadian Institutes of Health Research (CIHR). Il lavoro nel laboratorio bowdish è supportato in parte dal Michael G. DeGroote Centre for Infectious Disease Research e dal McMaster Immunology 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. 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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>

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In questo studio abbiamo presentato metodi dettagliati per la colonizzazione intranasale dei topi utilizzando un ceppo isolato clinico di Streptococcus pneumoniae e il successivo isolamento e caratterizzazione delle cellule immunitarie reclutate in rinofaringe in risposta ai batteri. Abbiamo dimostrato come un inoculo batterico possa essere coltivato in mezzi ricchi di nutrienti e utilizzato per stabilire un evento di colonizzazione nei topi, che è inizialmente limitato al rinofaringe. Abbiamo quindi mostrato c...

<table border="1">
<tbody>
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 <td>Name of Reagent/Material</td>
 <td>Company</td>
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 <td>Anti-Mouse Ly6C FITC</td>
 <td>BD Pharmingen</td>
 <td>553104</td>
 <td></td>
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 <td>Anti-Mouse Ly6G PE</td>
 <td>BD Pharmingen</td>
 <td></td>
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 <td>Anti-Mouse CD45.1 eFluor 450</td>
 <td>eBioscience</td>
 <td>48-0453-82</td>
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 <td>Anti-Mouse F4/80 Antigen APC</td>
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 <td>Anti-Mouse CD11c PerCP-Cy5.5</td>
 <td>eBioscience</td>
 <td>45-0114-82</td>
 <td></td>
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 <td>Anti-Mouse CD11b PE-Cy7</td>
 <td>eBioscience</td>
 <td>25-0112-82</td>
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 <td>Anti-Mouse CD3 Alexa Fluor 700</td>
 <td>eBioscience</td>
 <td>56-0032-82</td>
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 <td><a href="http://us.ebioscience.com/mouse-cd4-antibody-efluor-605nc-gk15.htm" target="_blank">Anti-Mouse CD4 eFluor 605NC</a></td>
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 <td>Intramedic Polyethylene Tubing - PE20</td>
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 <td>BD 26G3/8 Intradermal Bevel</td>
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 <td>Buffer RLT Lysis Buffer</td>
 <td>Qiagen</td>
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 <td>M-MuLV Reverse Transcriptase</td>
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 <td>GoTaq qPCR Master Mix</td>
 <td>Promega</td>
 <td>A6001</td>
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 </tbody>
</table>

characterization of inflammatory responses during intranasal colonization with streptococcus pneumoniae

La colonizzazione nasofaringea da parte di Streptococcus pneumoniae è un prerequisito per l'invasione ai polmoni o al flusso sanguigno1. Questo organismo è in grado di colonizzare la superficie mucosa del rinofaringe, dove può risiedere, moltiplicarsi e infine superare le difese ospiti per invadere altri tessuti dell'ospite. L'accertamento di un'infezione nelle vie respiratorie normalmente inferiori provoca polmonite. In alternativa, i batteri possono diffondersi nel flusso sanguigno causando la batteriemia, che è associata ad alti tassi dimortalità 2, oppure portare direttamente allo sviluppo della meningite pneumococcica. Comprendere la cinetica e le risposte immunitarie alla colonizzazione nasofaringea è un aspetto importante dei modelli di infezione da S. pneumoniae.
Il nostro modello di topo di colonizzazione intranasale è adattato daimodelli umani 3 ed è stato utilizzato da più gruppi di ricerca nello studio delle risposte ospite-patogeno nel nasofaringe4-7. Nella prima parte del modello, usiamo un isolato clinico di S. pneumoniae per stabilire una colonizzazione batterica autolimitante che è simile agli eventi di trasporto negli adulti umani. La procedura qui dettagliata prevede la preparazione di un inoculo batterico, seguito dall'istituzione di un evento di colonizzazione attraverso la consegna dell'inoculo attraverso una via intranasale di somministrazione. I macrofagi residenti sono il tipo di cellula predominante nel nasofaringe durante lo stato stazionario. In genere, ci sono pochi linfociti presenti nei topi non infetti8, tuttavia la colonizzazione della mucosa porterà a infiammazioni di basso-alto grado (a seconda della virulenza delle specie batteriche e del ceppo) che si tradurrà in una risposta immunitaria e nel successivo reclutamento delle cellule immunitarie ospiti. Queste cellule possono essere isolate da un gabinetto del contenuto tracheale attraverso i narici e correlate alla densità dei batteri della colonizzazione per comprendere meglio la cinetica dell'infezione.

La figura 1 rappresenta uno schema di panoramica che riassume i passaggi principali del protocollo. Le figure 2-3 forniscono la visualizzazione della metodologia microbiologica inerente ai protocolli descritti nel presente documento. La figura 4 rappresenta il corretto posizionamento di un topo per eseguire una colonizzazione intranasale, mentre la figura 5 descrive tipicamente i cambiamenti di peso dei topi colonizzati con ceppo S. pneumoniae P...

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

Viene descritta la colonizzazione del nasofaringe murino con streptococco pneumoniae e la successiva estrazione di cellule aderenti o reclutate. Questa tecnica prevede il lavaggio del nasofaringe e la raccolta del fluido attraverso i nares ed è adattabile per varie letture, tra cui la quantificazione differenziale delle cellule e l'analisi dell'espressione dell'mRNA in situ.

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco 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 Views.  McMaster University. Viene descritta la colonizzazione del nasofaringe murino con streptococco pneumoniae e la successiva estrazione di cellule aderenti o reclutate. Questa tecnica prevede il lavaggio del nasofaringe e la raccolta del fluido attraverso i nares ed è adattabile per varie letture, tra cui la quantificazione differenziale delle cellule e l'analisi dell'espressione dell'mRNA in situ.