例示的な例として使用されるすべてのNMRスペクトルは、財政的にネスレでサポートされていた以前に発行された研究7から派生しています。

1。無菌動物とサンプル採取の植民地化<ol><li>無菌アイソレーターと家それらのコントロール（図1）となる従来の動物の前にフィルターを装備したケージの中で、従来の飼育室内から動物を取り外します。 </li><li>無菌動物のくずで制御従来のケージから取り出したゴミ（3日齢）の半分を混ぜる。常に汚れた従来のゴミの1 / 3は、細菌のレベルを（少なくとも3日間それを保つ）を維持するためにそれを更新する必要があるたびにしてください。 </li><li>チューブの上にマウスを処理することにより、1.5 mLのマイクロチューブに尿を収集し、静かに腸をマッサージによって排尿を助ける。スナップインの凍結液体窒素で直ちに。 NMRの分析まで、少なくとも-40℃で保管してください。 20μLの最小体積は5mm NMRプローブで取得するために必要ですが、メタボリックプロファイリングの質を向上させるために30μLを使用することをお勧めします。 </li><li>動物Bすべきeは、麻酔薬の化合物の肝代謝（例えば、放血によって死の確認に続いて頸椎脱臼を使用）によ​​る交絡NMR共鳴を避けるためにどんな麻酔を使用せずに安楽死させ</li></ol> 2。肝生検のコレクションのための勧告<ol><li>汚染を避けるために、アルコールを含む製品を使用しないでください。水のみまたは生理食塩水を使用してツールを洗ってください。 </li><li>胆嚢穿孔しないでください。胆汁の漏れの場合には、水または生理食塩水ですぐに組織を洗う。 </li><li>図2に示すように、左葉から肝生検（15-50約100mg）を収集する。再現可能な生検のために、組織が薄くなって周辺地域を避けて左葉の中心に一貫して収集する。 </li><li>直ちに液体窒素中で凍結生検をパチンと-80に保管しておきます° CのNMR分析まで。 </li></ol>尿微量3。1 H NMRの取得</P&gt; <ol><li> D2O（99.8％）、1mMの3を含む液（pH7.4）中0.2Mリン酸ナトリウム緩衝液を準備- （トリメチルシリル）プロピオン酸- dの4（TSP）。 </li><li>リン酸ナトリウム緩衝液の30μLと尿の30μLを混合。 </li><li>金属針（外径0.5 mm）を装備した50μLガラスシリンジを用いて1.7mmのNMRキャピラリーチューブ（図3（2））に混合液の50μLを移す。気泡を避けるように注意してください。 </li><li>毛細血管を含む尿サンプルの上にキャピラリアダプタ（図3（3））に適合と5mm NMRプローブ（図3（1））のための2.5 mmのNMRのマイクロチューブに入れてください。スペクトル取得のための定期的な5 mmのNMR管にチューブのこの組み合わせを使用してください。 </li><li>それを引き出すために静かにキャピラリアダプタをねじ込んで2.5 mmのNMRチューブからキャピラリーを削除するには、抽出のロッド（図3（4））を使用してください。 </li></ol> 。肝組織生検の4 1 H人事MAS NMR法：試料調製<ol><li>ローターのコンポーネントとツールがあるMAS図4で説明しています。 </li><li>ジルコニウムローターに生検を（15-50約20mg）を挿入します（図4（1））とNMRロック用の純粋なD 2 Oでボリュームの残りを埋める。これはデータ収集のその後のシミングと質の質を変えてしまうので、どんな泡をしないように注意してください。 </li><li>円筒状のネジ（図4（5））を使用して50μLテフロンスペーサー（図4（2））を挿入します。ネジを外し、それと短い側の深さゲージ（図4（8））を使用して、それを校正する。このステップでは、それの部分がスペーサーの穴から漏れる可能性があるため、サンプルに固有の注意を払うことが重要です。このような場合は、その後、生検の一部が破壊され、サンプルの重量は、信頼性はなくなりました。それは、このように最初から再度サンプルの準備を開始する必要があります。 </li><li> THEADピン（図4（3））に置き、ねじ回し（図4（6））で軽く固定します。組織片を持つ任意の残留水を完全に乾かす。 </li><li>でキャップ（図4（4））を配置ローターの上部とローターパッカー（図4（6））に挿入します。キャップが所定の位置になるまでしっかりと押します。ローターとそのキャップの間に残されたスペースは存在しないはずです。 </li><li>光学的スピン速度の検出を可能にするために黒のマーカーペンを使ってローターの底部の半分をマークします。 </li><li> NMR分光計の内部にローターを配置し、5 kHzで回転し始める。メーカーのガイドラインに従ってCPMGパルスシーケンス13を用いて1 H NMRスペクトルを取得する。 </li><li> NMRスペクトルを較正するために5.22 ppmの（二重線）で、αアノマーグルコースの共鳴を使用してください。 </li><li>ローターをアンパックするには、キャップリムーバー（図4（9））を使用してキャップを除去することによって進んでください。ネジを外しTHEADピンと円筒形のネジを使用して削除テフロンスペーサー。徹底的に水と洗剤を使って洗う。 </li></ol> 5。代表的な結果腸内微生物の活動は、尿中の代謝プロファイリングを使用して監視できます。尿中の微生物の数が多い1 H NMRによって同定共同代謝物は、文献7,14-17に記載されている。これらの微生物の共同代謝物は、彼らが新たに設立された生態系が安定しているときに推定するために、迅速かつ非侵襲的な方法を提供するように植民地化のプロセスを監視するために特に有用です。図5Aは、明らかに植民地化のプロセスを介し腸の微生物の共同代謝物の外観を示しています。この図は、動物のためのステップ2で説明した手順に従うことによって得られる尿中代謝プロファイルは、ステップ1で説明した手順を使用して20日を植民地化を示しています。この動物は、無菌状態（0日目-青）で任意のインドキシル硫酸とphenylacetylglycine（PAG）及びp -クレゾール硫酸の非常に少ない量を排出していない。植民地化が進むにつれ、腸内細菌によるタンパク質代謝のこの3つのマーカーは、20日目（赤）で平衡に達するために大幅に増加する。 PAGを使用して、図5Bに示すように、これは動物のグループを監視するための特に簡単です。共鳴。この図は7動物のグループのために、PAGの特定の共鳴（三重）に対応する図5Aに灰色で強調表示されて共鳴（δ7.40から7.43）、下の面積を積分して得られた。 1 H高分解能マジック角スピニング（人事MAS）NMR分光法は、生検18の任意の種類の代謝プロファイルを迅速かつ再現性の買収を可能にする非破壊技術です。このプロトコルでは、我々は（青）と後（赤）のコロニー形成（図6）前2マウスの肝代謝のプロファイルを取得するために、この強力な技術を使用していました。この図はよくMAS NMRベースの代謝プロファイルから導出することができる情報を示しています。多数のアミノ酸だけでなく、例えばグルコース、グリコーゲン、乳酸、トリグリセリド、（D）-3 -ヒドロキシ酪酸とnicotinurateなどのエネルギー代謝から得られる代謝産物を可視化することができる。これらのプロファイルはまた、酸化ストレス（すなわちアスコルビンに関連する情報が含まれていますCID、グルタチオン）、ヌクレオチド代謝（すなわちイノシン、ウリジン）およびメチルアミンの代謝（すなわちコリン、トリメチルアミン- N -オキシド）。この例では、それは、無菌マウスはほとんどグリコーゲンやブドウ糖とトリグリセリドの非常に低い量として以前に7を公開したが表示されないことが非常に明確です。 <img src="/files/ftp_upload/3642/3642fig1.jpg" alt="図1。"/> 図1。植民地化のプロトコルの概要。無菌と、従来の動物が側とその仔は、従来の腸内細菌叢（1）からプログレッシブ植民地化を可能にするために交換されることで、フィルタの側面を装備したケージに収容されています。腸内微生物の活動は、1 H NMRベースの代謝プロファイリング（2-3）を使用して監視されます。肝代謝は、1 H人事MAS NMRベースの代謝プロファイリング（4-5）によって評価される。 <img src="/files/ftp_upload/3642/3642fig2.jpg" alt="図2。"/> 図2。マウスのライブRの解剖学。肝臓は、そのような臓器の平らな面として表示されているテーブルに直面している。再現可能な生検の場合は、それは破線の長方形で示されるように常に左葉の中央からサンプルを収集することをお勧めします。 <img src="/files/ftp_upload/3642/3642fig3.jpg" alt="図3。"/> 図3 microvolumesで動作するように1.7ミリメートルNMRキャピラリーキットキー ：。。：1 2.5 mmのNMRマイクロチューブ、2：1.7ミリメートルNMRキャピラリーチューブ、3：キャピラリアダプタ、4：抽出ロッド。 <img src="/files/ftp_upload/3642/3642fig4.jpg" alt="図4。"/> 図4 MASローター装備キー ：。。：1 MASローター、2：50μLテフロンスペーサー、3：THEADピン、4：キャップ、5：円筒形のネジ、6：ドライバー、7：ローターパッカー、8：深さゲージ。 <img src="/files/ftp_upload/3642/3642fig5.jpg" alt="図5。"/> 図5。colonizatio時の尿中代謝プロファイルの進化nの<ol class="ualpha"><li>微生物の共同代謝を可視化できる6.8から7.8 ppmの間のスペクトルの芳香族領域でズーム。1 H NMRスペクトルは0日目（青）で、単一の個体由来した、4（緑）、15（オレンジ）と20 （赤）ポスト植民地化。インドキシル硫酸、彼の：：ヒスチジン、p -クレゾール- S：、：1 - MeHistamine：インドキシル- S 1 - methylhistamineグレーゾーンはBキーで図を作るために統合された領域に対応するp -クレゾール硫酸; PAG ：Phenylacetylglycine。 </li><li>植民地化中の平均PAGの濃度（N = 7）。スチューデントのt検定は、様々なタイムポイントでPAGの濃度差を比較するために使用されていました：P 0日目に比べて&lt;0.05、B：P &lt;0.01は、10日目に比べて。 </li></ol><img src="/files/ftp_upload/3642/3642fig6.jpg" alt="図6。"/> 図6。無菌（青）と元無菌（再由来する肝生検の典型的な600 MHzの1 H人事MAS NMRスペクトルD）マウス。大胆な陽子は、トリグリセリドの共鳴する責任がありますキー ：3 - HB：3 -ヒドロキシ酪酸、GSH：還元型グルタチオン、TGS：トリグリセリド、TMAO：トリメチルアミン- N -オキシド。

<ol>
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Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 879, 1467-1475 (2011).</li><li>Sanchez-Patan, F., Monagas, M., Moreno-Arribas, M. V., Bartolome, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+microbial+phenolic+acids+in+human+faeces+by+UPLC-ESI-TQ+MS.">Determination of microbial phenolic acids in human faeces by UPLC-ESI-TQ MS.</a> J. Agric. Food. Chem. 59, 2241-2247 (2011).</li><li>Roux, A., Lison, D., Junot, C., Heilier, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+liquid+chromatography+coupled+to+mass+spectrometry-based+metabolomics+in+clinical+chemistry+and+toxicology:+A+review.">Applications of liquid chromatography coupled to mass spectrometry-based metabolomics in clinical chemistry and toxicology: A review.</a> Clin. Biochem. 44, 119-135 (2011).</li><li>Ryan, D., Robards, K., Prenzler, P. D., Kendall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+and+potential+developments+in+the+analysis+of+urine:+a+review.">Recent and potential developments in the analysis of urine: a review.</a> Anal. Chim. Acta. 684, 8-20 (2011).</li><li>Nagayama, K., Wuthrich, K., Bachmann, P., Ernst, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+J-resolved+1H+n.m.r.+spectroscopy+for+studies+of+biological+macromolecules.">Two-dimensional J-resolved 1H n.m.r. spectroscopy for studies of biological macromolecules.</a> Biochem. Biophys. Res. Commun. 78, 99-105 (1977).</li><li>Aue, W. P., Bartholdi, E., Ernst, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+spectroscopy.+Application+to+nuclear+magnetic+resonance.">Two-dimensional spectroscopy. Application to nuclear magnetic resonance.</a> J. Chem. Phys. 64, 2229-2246 (1975).</li><li>Bodenhausen, G., Ruben, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+abundance+15N+NMR+by+enhanced+heteronuclear+spectroscopy.">Natural abundance 15N NMR by enhanced heteronuclear spectroscopy.</a> Chemical. Physics. Letters. 69, 185-189 (1980).</li><li>Fan, T. W. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolite+profiling+by+one-+and+two-dimensional+NMR+analysis+of+complex+mixtures.">Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures.</a> Progress in nuclear magnetic resonance spectroscopy. 28, 161-219 (1996).</li><li>Fan, T., Lane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+profiling+of+metabolites+and+isotopomers+by+NMR.">Structure-based profiling of metabolites and isotopomers by NMR.</a> Progress in Nuclear Magnetic Resonance Spectroscopy. 52, 48-48 (2008).</li><li>Fonville, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+partial+least+squares+models+and+related+chemometric+approaches+in+metabonomics+and+metabolic+phenotyping.">The evolution of partial least squares models and related chemometric approaches in metabonomics and metabolic phenotyping.</a> Journal of Chemometrics. 24, 636-649 (2010).</li><li>Merrifield, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metabolic+system-wide+characterisation+of+the+pig:+a+model+for+human+physiology.">A metabolic system-wide characterisation of the pig: a model for human physiology.</a> Mol. Biosyst. , (2011).</li><li>Tugnoli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+human+gastric+mucosa+by+HR-MAS+magnetic+resonance+spectroscopy.">Molecular characterization of human gastric mucosa by HR-MAS magnetic resonance spectroscopy.</a> International Journal of Molecular Medicine. 14, 1065-1071 (2004).</li><li>Sitter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+HR+MAS+MR+spectroscopic+profiles+of+breast+cancer+tissue+with+clinical+parameters.">Comparison of HR MAS MR spectroscopic profiles of breast cancer tissue with clinical parameters.</a> NMR Biomed. 19, 30-40 (2006).</li><li>Beckonert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiling,+metabolomic+and+metabonomic+procedures+for+NMR+spectroscopy+of+urine,+plasma,+serum+and+tissue+extracts.">Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.</a> Nat. Protoc. 2, 2692-2703 (2007).</li></ol>

我々は、開示することは何もない。

このプロトコルでは、我々はさらに無傷の生検の1 H人事MAS NMRのプロファイリングにより評価された肝代謝で腸内細菌の影響を調査するためにオープンな環境で進歩的な植民地化の手順を説明した。植民地化の様々な方法が文献に記載されている。定義された細菌を持つ動物を植民地化する最も一般的な方法は強制経口投与または汚染された飲料水19,20です。糞便の接種は、前...

特定の試薬と機器の表： <table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td> 2.5mmのマイクロチューブ</td><td>新時代</td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1.7ミリメートル毛細管</td><td>シグマアルドリッチ</td><td> NORS175001 </td><td></td></tr><tr><td>キャピラリアダプタ</td><td>新時代</td><td> NE-325-5/1.7 </td><td></td></tr><tr><td>抽出ロッド</td><td>新時代</td><td> NE - 341から5 </td><td></td></tr><tr><td> 50μLとHR - MASローターBL4 球状​​のテフロンスペーサキット</td><td>ブルカー</td><td> HZ07213 </td><td></td></tr><tr><td> 50μLを挿入するためのツールキット</td><td>ブルカー</td><td> B2950 </td><td></td></tr><tr><td>アドバンスIII 600MHzのNMR </td><td>ブルカー</td><td></td><td></td></tr><tr><td> 1 H人事MAS NMR固体プローブ</td><td>ブルカー</td><td></td><td></td></tr><tr><td>重水99.9パーセント</td><td>シグマアルドリッチ</td><td> 530867 - 1L </td><td></td></tr><tr><td> 3 - （トリメチルシリル）プロピオン 酸の- d 4（TSP） </td><td>シグマアルドリッチ</td><td> 269​​913 </td><td></td></tr></tbody></table>

assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1h nmr spectroscopy

それは、腸内細菌がこのような免疫保護とビタミンの合成など、さまざまな利点を提供し、ホストの恒常性に大きく貢献することはよく知られています。彼らはまた、このエコシステムに不可欠な代謝の臓器作り、栄養素のかなりの量のホストを指定します。ホストとその腸内細菌叢との間の代謝相互作用を理解する腸内細菌叢とメタボリックシンドロームとの間のリンク、の増加する証拠の文脈では、現代生物学の重要な課題となってきています。1-4 植民地化は（また、正規化のプロセスとも呼ばれる）の元無菌動物における微生物の設立を指定します。それは、出生時に発生する自然なプロセスですが、それはまた腸花生態系を制御し、さらにホストの代謝への影響を判断するために成人無菌動物で使用されています。植民地化のプロセスを制御するための一般的な手順は、singlで強制経口投与の方法を使用することです。eまたは微生物の混合物。このメソッドは、非常に迅速に植民地化の結果、5非常にストレスであることの不利を提示。したがって、ストレスを最小限に抑え、徐々にホストの代謝上の細菌の設立の影響を観察するために低速の植民地化のプロセスを取得すると便利です。 この原稿では、我々は非破壊メタボリックプロファイリング技術を使用して徐々に植民地化の過程で肝代謝の変更を評価する手順を説明します。我々は、1 H NMRベースの代謝プロファイリングによる共同代謝微生物の尿中排泄により反射された腸内微生物の代謝活性を評価することによって、腸内微生物のコロニー形成を監視するために提案する。これは通常、DGGE（勾配ゲル電気泳動を変性）による糞便細菌を監視することにより評価した腸内微生物生態系の安定した設置を超える腸内微生物活性の安定性の理解が可能になります。6植民地化は、従来のオープンな環境で行われ、汚れたゴミ対照となる従来の動物、で汚れたによって開始されます。糞食性の動物がいるげっ歯類は、これは前述のように均質な植民地化を保証します 肝代謝プロファイリングは、NMR分光法をスピニング1 H高分解能マジック角を使用して無傷の肝生検から直接測定されます。この半定量的な手法は、さらに植民地化のプロセスと肝代謝70から10の間の複雑な相互作用を推定するために、セル構造に損傷を与えることなく、そのようなトリグリセリド、グルコースとグリコーゲンなどの主要な代謝物を評価するための素早い方法を提供しています。このメソッドは任意の組織生検11,12に適用することもできます。

Watch this Scientific Journal Video about Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy at JoVE.com

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

プログレッシブ植民地化の手順は、さらにホストの肝代謝への影響を評価するために記述されています。肝代謝が高分解能マジック角スピニング（人事MAS）無傷の生検のNMRプロファイリングによって評価されている間植民地化は、NMRベースの代謝プロファイリングによる共同代謝微生物の尿中排泄を評価することにより、侵襲的、非監視されます。

無菌マウスでのプログレッシブ植民地時代に肝代謝変化を評価する 1 H NMR分光法

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

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Immunology and Infection

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

13.0K ビュー.  The University of Reading. プログレッシブ植民地化の手順は、さらにホストの肝代謝への影響を評価するために記述されています。肝代謝が高分解能マジック角スピニング（人事MAS）無傷の生検のNMRプロファイリングによって評価されている間植民地化は、NMRベースの代謝プロファイリングによる共同代謝微生物の尿中排泄を評価することにより、侵襲的、非監視されます。

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

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

真菌の植民地、減数、およびカーネルのバイオアッセイを用いたマイコトキシンの生産の定量化

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

免疫・感染学

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

の植民地化 Euprymna scolopesイカでビブリオフィシャリ</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

ノーマルCNSのリアルタイムPCRによると神経炎症時のミクログリアにおけるマイクロRNAの検出

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

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 ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

共焦点顕微鏡による絶縁型肺胞マクロファージの抗真菌活性を評価する

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

リステリア単球遺伝子を用いた感染マウスにおける培養および心臓コロニー形成における心臓細胞の細菌浸潤の評価

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-肝白血球の選択的ハーベスティング

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

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.

グループBのマウスモデル<em&gt;連鎖球菌</em&gt;膣植民地化

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

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

蛍光相関分光法により測定初代リンパ球のプラズマ膜における分子拡散

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵襲体内生物発光システムによってマウスのホストである腸管出血性大腸菌の植民地化の検出

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...