著者らは、ペンシルベニア大学のジェフリー・ワイザー博士が 肺炎球菌の臨床株の贈り物に感謝したいと思います。この研究は、カナダ保健研究所によって資金提供されました。CVは、M.G.デグルートフェローシップとカナダ胸部協会からのフェローシップによって資金提供されました。この研究は、オンタリオ肺協会とカナダ保健研究所(CIHR)によって資金提供されました。ボウディッシュ研究所での研究は、マイケル・G・デグルート感染症研究センターとマクマスター免疫学研究センターによって部分的にサポートされています。

<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. 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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. 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Dis. 187, 1424-1432 (2003).</li><li>Mohler, J., Azoulay-Dupis, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pneumoniae+strain-dependent+lung+inflammatory+responses+in+a+murine+model+of+pneumococcal+pneumonia.">Streptococcus pneumoniae strain-dependent lung inflammatory responses in a murine model of pneumococcal pneumonia.</a> Intensive Care Med. 29, 808-816 (2003).</li><li>Wu, H. Y., Virolainen, A., Mathews, B., King, J., Russell, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+Streptococcus+pneumoniae+nasopharyngeal+colonization+model+in+adult+mice.">Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice.</a> Microb. Pathog. 23, 127-137 (1997).</li><li>Zhang, Z., Clarke, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+effectors+mediating+Th17-dependent+clearance+of+pneumococcal+colonization+in+mice.">Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice.</a> J. Clin. Invest. 119, 1899-1909 (2009).</li><li>Parker, D., Martin, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pneumoniae+DNA+initiates+type+I+interferon+signaling+in+the+respiratory+tract.">Streptococcus pneumoniae DNA initiates type I interferon signaling in the respiratory tract.</a> MBio. 2, (2011).</li><li>Haya, D. L., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+identification+of+serotype+4+Streptococcus+pneumoniae+virulence+factors.">Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors.</a> Mol. Microbiol. 45, 1389-1406 (2002).</li><li>Nakamura, S., Favis, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+stimulation+of+type+I+interferons+during+influenza+virus+coinfection+promotes+Streptococcus+pneumoniae+colonization+in+mice.">Synergistic stimulation of type I interferons during influenza virus coinfection promotes Streptococcus pneumoniae colonization in mice.</a> J. Clin. Invest. 121, 3657-3665 (2011).</li><li>Kim, J. O., Weiser, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+intrastrain+phase+variation+in+quantity+of+capsular+polysaccharide+and+teichoic+acid+with+the+virulence+of+Streptococcus+pneumoniae.">Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae.</a> J. Infect. Dis. 177, 368-377 (1998).</li><li>Roche, A. M., King, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+attenuated+Streptococcus+pneumoniae+strains+induce+serotype-independent+mucosal+and+systemic+protection+in+mice.">Live attenuated Streptococcus pneumoniae strains induce serotype-independent mucosal and systemic protection in mice.</a> Infect. Immun. 75, 2469-2475 (2007).</li><li>Cohen, J. 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著者らは開示するものは何もない。

本研究では、 肺炎球菌 の臨床単離株を用いたマウスの鼻腔内浸着の詳細な方法と、その後の細菌に応答して鼻咽頭に採用された免疫細胞の単離および特徴付けについて明記した。我々は、細菌接種物を栄養豊富な培地で培養し、マウスの植民地化イベントを確立するために使用する方法を実証した。次に、鼻咽頭に採用された免疫細胞型の応答が、口蓋暴露、切開および鼻洗浄に続?...

<table><tbody><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>Anti-Mouse CD4 eFluor 605NC</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 1 ml Syringe</td><td>Becton Dickinson</td><td>309659</td><td></td></tr><tr><td>BD 26 G 3/8 Intradermal Bevel</td><td>Becton Dickinson</td><td>305110</td><td></td></tr><tr><td>Buffer RLT Lysis Buffer</td><td>Qiagen</td><td>79216</td><td></td></tr><tr><td>Difco Tryptic Soy Agar</td><td>Becton Dickinson</td><td>236950</td><td></td></tr><tr><td>Defibrinated Sheep Blood</td><td>PML Microbiologicals</td><td>A0404</td><td></td></tr><tr><td>RNAqueous-Micro Kit</td><td>Ambion</td><td>AM1931</td><td></td></tr><tr><td>M-MuLV Reverse Transcriptase</td><td>New England Biolabs</td><td>M0253L</td><td></td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6001</td><td></td></tr></tbody></table>

characterization of inflammatory responses during intranasal colonization with streptococcus pneumoniae

肺炎球菌による鼻咽頭コロニー形成は、肺または血流1に侵入する前提条件である。この生物は、鼻咽頭の粘膜表面を植民地化することができ、宿主の他の組織に侵入するために宿主防御を克服し、増殖し、最終的に克服することができる。通常下気道に感染を起こすと肺炎が生じる。あるいは、細菌は、高い死亡率2に関連する細菌血症を引き起こす血流に広がり得るか、またはそれ以外の場合は肺炎球菌性髄膜炎の発症に直接導く。鼻咽頭コロニー形成の運動学および免疫応答を理解することは、S.肺炎感染モデルの重要な側面である。
我々の鼻腔内コロニー形成のマウスモデルは、ヒトモデル3 から適応され、鼻咽頭4-7における宿主病原体応答の研究において複数の研究グループによって使用されている。モデルの最初の部分では 、S.肺炎 の臨床的分離株を使用して、ヒト成人のキャリッジイベントに似た自己制限細菌コロニー形成を確立する。本明細書で詳述される手順は、細菌接種の調製を含み、続いて鼻腔内投与経路を介して接種を送達することによってコロニー形成事象を確立する。居住マクロファージは、定常状態の間に鼻咽頭の主要な細胞型である。典型的には、未感染マウス8に存在するリンパ球はほとんど存在しないが、粘膜コロニー形成は、免疫応答とその後の宿主免疫細胞の動員をもたらす低位から高等級の炎症(細菌種および株の病原性に応じて)をもたらす。これらの細胞は、ナレスを通して気管内容物の洗浄によって単離され、感染の動態をよりよく理解するためにコロニー形成細菌の密度と相関することができる。

図 1 は、プロトコルの主要な手順を要約した概略図を示しています。 図2〜3は 、本明細書に記載されたプロトコルに固有の微生物学的方法論の可視化を提供する。 図4 は、鼻腔内コロニー形成を行うマウスの適切な位置を示し、図 5 は典型的に S.肺炎株 P1547でコロニー形成されたマウスの体重の変化を示している。 図6〜7は</str...

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

肺炎球菌によるマウス鼻咽頭のコロニー形成および後の付着またはリクルートされた細胞の抽出について説明する。この技術は、ナアレスを通して鼻咽頭および流体の収集を洗い流し、その場所でのmRNA発現の差動細胞定量および分析を含む様々な読み出しに適応可能である。

肺炎球菌による鼻腔内コロニー形成時の炎症反応の特徴

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

characterization-inflammatory-responses-during-intranasal

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

12.3K ビュー.  McMaster University. 肺炎球菌によるマウス鼻咽頭のコロニー形成および後の付着またはリクルートされた細胞の抽出について説明する。この技術は、ナアレスを通して鼻咽頭および流体の収集を洗い流し、その場所でのmRNA発現の差動細胞定量および分析を含む様々な読み出しに適応可能である。

ビデオ: Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

実験的な人間の肺炎球菌キャリッジ

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

医学

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用肺炎球菌植民に対するプロバイオティクスの影響を調査<em&gt;体外</em&gt;接着アッセイ

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

免疫・感染学

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

生物発光細菌を使用してリアルタイムに急性肺炎のマウスモデルにおける肺炎球菌の毒性因子の影響を以下の

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

マウスにおける宿主の免疫応答を評価する口腔咽頭気管内PAMP管理および気管支肺胞の活用

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

微生物群集を学びます<em&gt;インビボ</em&gt;：ホスト間の媒介の相互作用のモデル<em&gt;カンジダ</em&gt;と<em&gt;緑膿菌</em&gt;航空で

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

マウスにおける実験的細菌性肺炎の誘導と表現型のための非侵襲的および技術的に非集中的な方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin &#38; eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

の可視化<em&gt;肺炎球菌</em&gt;心臓Microlesionsとその後の心臓リモデリングの中

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

敗血症誘発免疫抑制のセカルライゲーションと穿刺および鼻腔感染の二重モデルの設計

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

インフルエンザ菌に対する呼吸器免疫応答の評価

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

肺炎 連鎖 球菌のウイルス重感染によるコロニー形成者から病原体への移行に関するマウスモデルが加齢増悪疾患を再現

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

肺炎球菌による鼻腔内コロニー形成時の炎症反応の特徴

要約

さらに動画を探す

この動画の章

実験的な人間の肺炎球菌キャリッジ

使用肺炎球菌植民に対するプロバイオティクスの影響を調査

生物発光細菌を使用してリアルタイムに急性肺炎のマウスモデルにおける肺炎球菌の毒性因子の影響を以下の

マウスにおける宿主の免疫応答を評価する口腔咽頭気管内PAMP管理および気管支肺胞の活用

微生物群集を学びます

マウスにおける実験的細菌性肺炎の誘導と表現型のための非侵襲的および技術的に非集中的な方法

の可視化

敗血症誘発免疫抑制のセカルライゲーションと穿刺および鼻腔感染の二重モデルの設計

インフルエンザ菌に対する呼吸器免疫応答の評価

肺炎連鎖球菌のウイルス重感染によるコロニー形成者から病原体への移行に関するマウスモデルが加齢増悪疾患を再現

肺炎球菌による鼻腔内コロニー形成時の炎症反応の特徴

要約

さらに動画を探す

この動画の章

実験的な人間の肺炎球菌キャリッジ

使用肺炎球菌植民に対するプロバイオティクスの影響を調査

生物発光細菌を使用してリアルタイムに急性肺炎のマウスモデルにおける肺炎球菌の毒性因子の影響を以下の

マウスにおける宿主の免疫応答を評価する口腔咽頭気管内PAMP管理および気管支肺胞の活用

微生物群集を学びます

マウスにおける実験的細菌性肺炎の誘導と表現型のための非侵襲的および技術的に非集中的な方法

の可視化

敗血症誘発免疫抑制のセカルライゲーションと穿刺および鼻腔感染の二重モデルの設計

インフルエンザ菌に対する呼吸器免疫応答の評価

肺炎 連鎖 球菌のウイルス重感染によるコロニー形成者から病原体への移行に関するマウスモデルが加齢増悪疾患を再現

肺炎連鎖球菌のウイルス重感染によるコロニー形成者から病原体への移行に関するマウスモデルが加齢増悪疾患を再現