我々は彼らの技術支援のためにブランドンハセット、カルロス·オルティスに感謝します。この作品は、NSFの助成金IOB-0544428、IOS-0951272、博士マイケルKolomietsにし、食と農の米農務省国立研究所（NIFA）、AFRIの育種と教育グラント＃2010から85117でIOS-0925561によってサポートされていました-20539博士へ。セスマレー、トーマスIsakeit、とマイケルKolomiets。

1。トウモロコシカーネルのバイオアッセイ<ol><li>二週間前までに、文化28でポテトデキストロース寒天（PDA）上での真菌病原体℃、 </li><li>彼らはバイオアッセイバイアルの底レベル、50mlファルコンチューブ内の場所を築くため、好ましくは、平坦化され、同様の大きさと形でカーネルを選択します。選択したカーネルは、類似した種子の年齢及び代謝物の組成を保証するために、同じ環境で同時に製造されている必要があります。 </li><li>表面は70％エタノールで5分間、滅菌水で1分間、6％次亜塩素酸ナトリウムで10分間室温でチューブを振とうしてカーネルを滅菌する。継続的な振とうしながら滅菌水で、毎回5分間、3回すすいでください。オートクレーブタオルで乾かしカーネル。 </li><li> アスペルギルスフラバスの感染を容易にするために、0.5cmの深さに18 G針で胚側の ​​小さな傷を作成します。しかし、我々の経験では大きな傷がフザリウムverticillioides感染に必要とされる。番目のerefore、0.5ミリメートルの深さで胚側にカットするためにかみそりの刃を使用しています。 </li><li>胞子を解放し、オートクレーブチーズの少なくとも4つの層を介してフィルタリング重力によって菌糸を除去するオートクレーブ0.01パーセントのTween-20溶液の約5 mlの培養プレートを削って接種懸濁液を準備します。血球と胞子濃度を計算し、Aの10 6胞子/ mlに調整フラバスまたは F verticillioides。病原体またはホストの他の種を使用している場合は、適切な感染接種の濃度を評価することは重要である。 </li><li>紙タオルや滅菌水100 mlの5枚が並んでプラスチック容器（29.2センチ×18.7センチメートルX 8.3センチメートル）内の湿度チャンバーを作成します。チャンバーは、水または気密と追加の水ではありませんペーパータオルの潤いを保つために、実験を通して追加する必要があります。 </li><li>オートクレーブ20mlのガラス製シンチレーションバイアルに4カーネルを配置し、その質量を記録します。 、200μlの胞子懸濁液を接種するキャップ、懸濁液と均一にコートカーネルの渦。自由に湿度チャンバーへの空気の交換と場所を可能にするためにキャップを緩めます。 </li></ol> 注：これらのメソッドに記載されている以外の種子や真菌の場合、必要な接種量が異なる場合があり、実験的に導出する必要があります。 <ol start="8"><li> °Cで7日間で28または所望されるまで12-h-light/12-h-dark日長下のカーネルをインキュベートします。 </li></ol> 2。分生子の列挙<ol><li>感染期間の後、1分間徹底的に感染したカーネルと渦を含むシンチレーションバイアルにオートクレーブ0.01パーセントのTween-20の5ミリリットルを追加します。 </li><li>ワイドボアピペットチップを使用して、すぐに0.01パーセントのTween-20（または感染症に応じて、必要に応じて希釈する）の1.8 mlを含む2.0ミリリットルエッペンドルフチュー​​ブに胞子懸濁液の2つの独立した200μlのアリコートを転送することにより希釈する。 </li><li>血球4に二回、各200μlのアリコートを列挙すると治療の間の分生子のレベルを比較します。 </li></ol> 3。アフラトキシンの定量<ol><li> 80％メタノールと0.05グラムのNaCl、1分間高速でカバーとブレンド20mlで1サンプルから50ミリリットルブレンダーカップにカーネルを追加します。 </li><li>きれいな回収容器を介して溝濾紙を配置し、フィルタに抽出液を注ぐ。 </li><li>きれいな容器にろ液10 mlを20 mlの蒸留H 2 Oを追加して、よく混ぜる。 </li><li>クリーンカップに1.5μmのガラスマイクロファイバーフィルタを通してフィルタサンプル。 </li><li>アフラトキシン検査の列にフィルタ抽出液を1 mlを適用すると、圧縮空気を使用して、空になるまで1〜2滴/秒の速度でカラムを通して濾過し抽出物を強制します。 </li><li>蒸留H 2 O 1 mlで2回カラムを洗浄し、ドロップ/秒の空気が伝わってくるまでは1-2にカラムを通過します。 </li><li>ドロップ/ガラスキュベットに採取二流1-2に1ミリリットル100％HPLCグレードのメタノールで溶出するカラム。 </li><li>加える1溶出液にアフラトキシン試験Developerのmlとよく混ぜる。場所は、キャリブレーション蛍光光度計キュベットに、60秒で読む。 </li></ol> 注：Aflatest FGISプロトコルを使用する場合、蛍光光度計からの測定は100ミリリットルで抽出したサンプルの最初の50グラムに基づいて計算されます。あなたは水試料の希釈を考慮した場合は、1ミリリットルは、サンプルの0.166グラムを表す列に適用されます。したがって、プロトコルを変更するとき、あなたのアカウントにサンプルサイズの違いや初期抽出液を取る必要があります。たとえば、カーネルの2グラムを20mlの80％メタノールで抽出されています。これは0.1グラム/ mlである。このサンプルの1 mlを2 mlの水と混合されたときに、液体のサンプルの割合は、現在0.033グラム/ mlである。このサンプルでは100 ppbの蛍光光度計の読みを与える場合は、実際の濃度は、ppbの=（0.166グラムXは100 ppb）/ 0.1グラム）、または166 ppbである0.166のサンプルの割合に基づいています。別のアフラトキシン、定量のためにカチオン方法は、参考文献7を参照してください。 4。フモニシンB1（FB1）の分析<ol><li>菌はトウモロコシの穀粒の上に成長されている場合、撹拌せずに室温でアセトニトリル/水（50/50、v / v）を10 mlの一晩サンプルを抽出します。その後、脱イオン水（6 ml）で抽出（2 ml）を混合し、C-18固相抽出に直接（8 ml）にこのミックスを適用します。 </li><li>水2ml、続いてアセトニトリル2mlで洗浄し、サンプル、前提条件C-18固相抽出カラムをロードする前に。 </li><li>サンプルをロードした後、カラムをアセトニトリル/水2ミリリットル（15/85、v / v）を、続いて水2mlで洗浄する。 HPLC分析（FB1を含む）のサンプルは、アセトニトリル/水2ml（70/30、v / v）で溶出させる。 </li><li> 0.1ミリリットルホウ酸緩衝液（0.05Mホウacid/0.05 Mホウ酸ナトリウム[50/50、V / V]は、pH 8.5）および0.1を含むバイアルにカラム溶出液の0.1 mlを転送することにより、O-フタルアルデヒド（OPA）でFB1を誘導体化ミリリットルOPA（アセトンで0.1 mg / mlの0.5％itrile - メルカプトエタノール）。 </li><li> acetonitrile/0.01 Mホウ酸（40/60、v / v）を0.5 mlを加えることによって10分後に反応を停止します。 </li><li>分析ZORBAX ODSカラム（4.6 150ミリメートル）と、可変波長Shimatzu RF-10Axl蛍光検出器（励起335 nmで/発光440 nm）を装備島津HPLC LC-20ATシステム上でFB1を分析します。 </li><li>線形グラデーションを使用し（溶媒：アセトニトリルMリン酸ナトリウム（40/60）は、pH 3.3;溶媒B：アセトニトリル/ 0.1 Mリン酸ナトリウム（60/40）は、pH 3.3）されており、勾配プログラムは次のとおりです。 100パーセントで5分間、10分間、100％Bの100％Bへ。 </li><li> HPLCでFB1の基準を分析し、ピーク面積の測定は、標準曲線を生成するために使用されています。その後、FB1標準曲線とピーク面積を比較することによって、サンプル中のFB1のレベルを定量化する。 </li></ol> 5。エルゴステロールの分析<ol><li>文化7〜14日のためにトウモロコシの穀粒に菌。 </li><li>メタ：インキュベーション期間の後、クロロホルム10mlを加え各バイアルにanol（2:1、v / v）で。一度追加された、よくサンプルを振ると、その後24時間室温で暗所でバイアルをインキュベートします。 </li><li> 24時間後、サンプルを遠心し、上清を収集し、0.45 umのナイロン膜を通してそれをフィルタリングします。 </li><li> 4.6 U ODS-C18カラム（200Å、250±4.6 mm）と282 nmでモニターするように設定島津SPD-20A UV / VIS検出器を備えた島津HPLC LC-20ATシステムに直接サンプルを注入します。 </li><li>移動相として1.5 mL / minの流量でメタノール（100％）を使用します。 HPLCグレードのエルゴステロールから生成された標準曲線と試料のピーク面積を比較することにより、サンプル中のエルゴステロールの定量化を決定します。 </li></ol> 6。代表的な結果二から三日間の湿度チャンバー内接種およびインキュベーションの後、真菌の増殖は、カーネルに表示を開始する必要があります。七日間の後処理、処理された植物で植物の成長がはっきりと見えるはずです、薬用電子モックコントロール（ 図1B、上）感染していないことがあります。長い潜伏期間は、より豊富な栄養成長（ 図1B、下）を助長しています。条件の下で、本明細書に記載します（ 図2G）、A. フラバス 、野生型NRRLそれぞれ8、6、8日、植民地化で、アフラトキシンの蓄積、及び分生子の生産の3357表示された最大値（ 図2、AC）。しかし、マイコトキシン汚染と分生子はエルゴステロールの単位当たりで比較した場合には、最大レベルは（ 図2、DE）はそれぞれ、4および6日目に観察された。 図2Fは、これらの菌のバイオマスに依存した最大値の観測をまとめたものです。興味深いことに、4-6日後接種の間に、カーネルは時間経過（ 図2H、下）上のサンプルからのトータルRNAで見られるように、核酸の分解を受けています。 我々自身を含むいくつかの研究は、examinを持っているED F. verticilliodiesは、トウモロコシのカーネル2,8,9,10,11,12に感染して正常時ポイント観測用として7月13日日使用されます。メソッドを使用して、ここに3,500-8,000 ng / gでカーネル4,13と5,000-10,000 ng / gのカーネル14,13からエルゴステロールレベルの範囲からフモニシンレベルの範囲を説明しました。 図3は、フモニシン（上）とエルゴステロール（下の代表的なピークを示しています。 ）HPLCクロマトグラムから。エルゴステロールの測定は、吸光度スペクトル15を介して行うことができます。 <img alt="図1" src="/files/ftp_upload/3727/3727fig1.jpg" /> 図1：真菌バイオマス、減数し、マイコトキシンの生産とカーネルのバイオアッセイから、栄養成長のための代表的な結果の定量化のためのフローチャート。トウモロコシのカーネルで菌類の病原性を評価するために使用する基本的な生物学的パラメータの定量化のために説明した方法を概説A）フローチャート。 B）代表者カーネルのバイオアッセイの結果。トップ、A.フラバス （NRRL3357）B73遺伝的背景のトウモロコシのカーネルでは栄養成長。種子は7日後に接種を受けた10 6胞子/ mLと写真の200μlで接種した。底部、F. B73背景13日感染後に接種したカーネルをverticillioides。カーネルは10 6胞子/ mlを200μlを接種した。 <img alt="図2" src="/files/ftp_upload/3727/3727fig2.jpg" /> 図2：アスペルギルスフラバスカーネルバイオアッセイ時間コース。 B73カーネルは10 6胞子/ ml アスペルギルスフラバス分生子懸濁液200μlを接種し、2〜8日（G）インキュベートした。 （;平均値±SE、N = 3-4）のすべての値は、4カーネルの乾燥重量の平均値から決定した。 A）植民地（エルゴステロールに基づいて）、（B）アフラトキシン、および（C）分生子は、上記の方法を用いて定量した。 E）アフラトキシン及び（F）分生子が表示されます。hのエルゴステロールを介して測定された菌類のバイオマスの関数として。 F）エルゴステロール、アフラトキシンの蓄積、および真菌バイオマスの関数としての分生子の最大値 - それぞれの量の最大値は100％に設定されて観察した。 （H）時間コースからの全RNAのレーン当たり1μgの。 <img alt="図3" src="/files/ftp_upload/3727/3727fig3.jpg" /> 図3。フモニシンB1（上）とフザリウムverticillioides（M3125）に感染したカーネルから分離されたエルゴステロール（下）の代表的な高性能液体クロマトグラフィ（HPLC）のクロマトグラム。

<ol>
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<p class="jove_content"&gt;私は開示することは何もありません。</p

<p class="jove_content"&gt;ここで説明する方法は広範囲にテストされ、真菌のコロニー形成、減数し、マイコトキシンの生産のための定量的な結果の生成に堅牢であることが証明されています。また、これらのメソッドはmycotoxigenic菌類（例えば、ピーナッツ、小麦、綿花、ピスタチオなど）による汚染の影響を受けやすい他の植物種の種子に適用する必要があります。有能な植物 - 病原体相互作用分析のために、そ...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ＃ </td></tr><tr><td>ポテトデキストロース寒天</td><td>フィッシャーScientifc </td><td> S71659A </td></tr><tr><td> Tween-20を</td><td>フィッシャーScientifc </td><td> BP337-100 </td></tr><tr><td>プラスチック製の培養容器</td><td> Sterilite </td><td> 1713LAB06 </td></tr><tr><td>ブレンダー</td><td> Vicam </td><td> 20200 </td></tr><tr><td> 24センチメートル溝付き濾紙</td><td> Vicam </td><td> 31240 </td></tr><tr><td> 1.5μmのガラスマイクロファイバー</td><td> Vicam </td><td> 31955 </td></tr><tr><td>アフラトキシン試験カラム</td><td> Vicam </td><td> G1024 </td></tr><tr><td> Afrlaテスト開発者</td><td> Vicam </td><td> 32010 </td></tr><tr><td>メタノール</td><td> Vicam </td><td> 35016 </td></tr><tr><td>アセトニトリル</td><td>フィッシャーScientifc </td><td> AC14952-0025 </td></tr><tr><td>エタノール</td><td>フィッシャー科学者TIFC </td><td> AC39769-0025 </td></tr><tr><td> C-18固相抽出カラム（準備9月​​SPE C18カラム） </td><td>フィッシャーScientifc </td><td> 60108-304 </td></tr><tr><td> O-フタルアルデヒド（OPA） </td><td>シグマケミカル社</td><td> 79760〜5グラム</td></tr><tr><td>ホウ酸</td><td>フィッシャーScientifc </td><td> BP168-500 </td></tr><tr><td>ホウ酸ナトリウム</td><td>フィッシャーScientifc </td><td> RDCS0330500 </td></tr><tr><td>メルカプトエタノール</td><td>フィッシャーScientifc </td><td> 45-000-231 </td></tr><tr><td>島津HPLC LC-20AT（ポンプ） </td><td>島津科学機器株式会社</td><td> LC-20AT </td></tr><tr><td> ZORBAX ODSカラム（4.6x150mm） </td><td>アジレント·テクノロジー</td><td> 443905-902 </td></tr><tr><td> Shimatzu RF-10Axl蛍光検出器</td><td>島津科学機器株式会社</td><td> RF-10AXL </td></tr><tr><td>リン酸ナトリウム</td><td>フィッシャーScientifc </td><td> AC38987-0010 </td></tr><tr><td> FB1の基準</td><td>シグマケミカル社</td><td> F1147-1mgの</td></tr><tr><td>クロロホルム</td><td> VWR </td><td> MK444410 </td></tr><tr><td> 0.45 umのナイロンメンブレン（HPLC）で13​​ mmのシリンジフィルター</td><td>ポールライフサイエンス</td><td> 4426 </td></tr><tr><td>エルゴステロール</td><td> Sigma-Aldrich社</td><td> 45480-50G-F </td></tr><tr><td>シンチレーションバイアル</td><td> VWR </td><td> 66021-602 </td></tr><tr><td>塩化ナトリウム</td><td> Vicam </td><td> G1124 </td></tr></tbody></table>

quantification of fungal colonization, sporogenesis, and production of mycotoxins using kernel bioassays

種子感染菌による穀物の腐敗は、人間と動物の健康に深刻なリスクに言及しないように、穀物生産の世界的な最大の経済課題の一つを引き起こします。穀物生産のうち、トウモロコシは穀物の整合性とマイコトキシン汚染種子における病原体によって誘発される損失のため、おそらく最も影響を受けた作物である。トウモロコシ生産者と食品および飼料のプロセッサに2つの最も普及していると問題のマイコトキシンは、それぞれアスペルギルスフラバスとフザリウムverticillioidesによって生成されたアフラトキシンやフモニシン、である。 分子植物-病原体相互作用における最近の研究では、真菌感染症とマイコトキシン汚染1,2,3,4,5,6に対する植物の応答に関連付けられた特定のメカニズムを理解する上で約束を示している。多くのラボでは、植物 - 病原体相互作用を研究するためにカーネルのアッセイを使用しているので、異なる生物学的パラメータを定量化するための標準的な方法の必要性がそう、そこです。別の研究室からの結果はクロスと解釈することができます。種子の定量分析するための堅牢で再現性のある手段のために、私たちは真菌の増殖、バイオマス、マイコトキシン汚染を定量化するためにラボでのカーネル·アッセイおよび後続のメソッドを開発しました。四滅菌トウモロコシのカーネルは、菌の懸濁液（10 6）をガラスバイアルに接種し、所定の期間インキュベートする。サンプルバイアルは、その後、HPLCにより血球、エルゴステロールベースの​​高性能液体クロマトグラフィー（HPLC）によるバイオマスの分析、AflaTest蛍光法を用いてアフラトキシンの定量化、およびフモニシンの定量化による分生子の列挙のために選択されています。

Watch this Scientific Journal Video about Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays at JoVE.com

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

種子感染菌による穀物の荒廃は、より良い植物 - 病原体相互作用を理解するための多数の研究努力を求めている。実験室の設定で種子菌の相互作用を研究するために、我々は真菌の繁殖、バイオマス、カーネルのバイオアッセイを用いたマイコトキシン汚染の定量化のための堅牢な方法を開発した。

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 ...

quantification-fungal-colonization-sporogenesis-production-mycotoxins

Research

JoVE Journal

Immunology and Infection

18.0K ビュー.  Texas A&M University. 種子感染菌による穀物の荒廃は、より良い植物 - 病原体相互作用を理解するための多数の研究努力を求めている。実験室の設定で種子菌の相互作用を研究するために、我々は真菌の繁殖、バイオマス、カーネルのバイオアッセイを用いたマイコトキシン汚染の定量化のための堅牢な方法を開発した。

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

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 ...

免疫・感染学

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

真菌病原体の食作用の生細胞ビデオ顕微鏡

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

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 ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

機能研究のためのマウス骨髄好中球と養子移入実験の分離、精製とラベリング

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

設立<em&gt;インビトロ</em&gt;システムの細胞内挙動を研究するために、<em&gt;カンジダ·グラブラタ</em&gt;人間のTHP-1マクロファージにおける

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

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 ...

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 ...

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 ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

アスペルギルス症では実験的肺真菌負担を決定する組織の定量化

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

リアルタイム イメージングと二相循環フロー システムを用いた菌バイオ フィルム開発の定量

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...