この研究は、NIH の補助金 HL66299 SL、TS へ HL60024 と mf HL136869、RB、TS によって部分で賄われていた。

動物のすべてのプロシージャを行った機関と動物ケア委員会南アラバマ大学によって承認し、すべての連邦、州、およびローカル規則に従って行われました。初代培養ラット肺微小血管内皮細胞 (PMVECs) の肺生物学南アラバマ大学センターで細胞培養コアから得られました。前に説明した手順16を使用して電池を作製しました。
1. 細胞培養上清の世代
注: ここで、我々 は緑膿菌の 2 つの異なる系統使用: PA103 標的細胞に感染、およびタイプ III の分泌システムに欠けている、∆PcrV の中に ExoU と ExoT exoenzymes を転送することができるそのままタイプ III 分泌システムを持っています。exoenzymes を接種対象セルに転送することができません。
<ol><li>フォーゲル ボナー (VB) 寒天培地プレート17に細菌を連勝し、37 ° C で一晩成長<ol>
<li>プレートを準備するように次の原液 (フォーゲル ボナー塩; 10 x の在庫): 2 g MgSO4、20 g のクエン酸の酸 (遊離酸)、100 g K2PO4、および 35 g NaH2PO4を測定します。1 L と低速排気で 15 分間オートクレーブに ddH2O を追加します。</li>
		<li>450 mL ddH2O で 7.5 g の寒天と上記としてオートクレーブを配置します。</li>
		<li>オートクレーブ、次クールに 50 ° C の水浴に両方のソリューションを配置します。</li>
		<li>寒天に 10 x VB ストック塩溶液 50 mL を追加します。</li>
		<li>(使用されている膿の系統) の 400 μ g/mL に carbenicillin を追加し、フラスコを旋回でよく混ぜます。</li>
		<li>個々 の無菌培養皿に VB の寒天液の場所 5 mL は、固化し、細菌の播種日まで 4 ° C で保存する寒天を許可します。</li>
		<li>細菌の播種日前 37 ° c のプレートを温めるし、プレート滅菌ループを使って細菌を連勝します。37 ° C の定温器のプレートの場所は一晩します。</li>
	</ol>
</li>
	<li>肯定的な染色蛍光グリフォニア simplicifolia PMVEC 純度を確認レクチンおよび否定的な汚損蛍光リンゴマイマイレクチン (血管内皮細胞のマーカー)。細胞分注、液体窒素で凍結。</li>
	<li>実験用セル ダルベッコ変更されたワシの媒体 (DMEM) 10% 牛胎児血清を添加したを維持し、5% CO2を含む 37 の ° C の定温器で細胞を成長します。5-15 の通路でセルを使用します。<ol>
<li>細胞培養上清の世代、個々 の 150 cm 皿に 2 x 10 の6セルをプレート、合流点を達成するまで 3-4 日の拡張します。2 枚実験 (下記参照) を使用します。</li>
		<li>、細胞培養上清の分析プレート 1 × 105セル 6 ウェルの個々 の井戸には皿し、合流が達成されるまで 4 日間の成長します。</li>
	</ol>
</li>
	<li>上清を生成するリン酸緩衝生理食塩水でステップ 1.3.1 から PMVECs の 2 つの 150 cm の料理の一つ、trypsinize、洗ってその後セルをカウント (我々 は、自動化された細胞カウンターを使用して、製造元のプロトコルに従う;材料の表を参照してください)。PMVECs とハンクのバランスの取れた塩ソリューション (HBSS) の他の皿を洗って、20:1 の感染 (MOI) の多様性で膿のいずれかの株に感染してしまいます。これは、次のとおり達成されます。<ol>
<li>膿の540 0.25 = 2 × 108 CFUs/mL を OD します。この希釈を準備するには、1 mL の使い捨てのキュベットに 1 mL HBSS を追加します。分光光度計を使用して測定したとき、0.25 の外径540が得られるまで手順 1.1 で準備されたプレートから十分な細菌をこすり。次のステップは、細菌が必要とどのくらいの計算になります。</li>
		<li>培養皿の PMVECs の数が決定される (1.4 上記の手順)、適切な PMVECs を含む 2 番目、非トリプシン培養皿に追加する細菌数を決定します。たとえば、PMVECs の培養皿は 10 の7セル x 1.3 が含まれているし、1.3 × 107セル x 20 細菌/携帯 x 1 mL/108 CFUs の準備と判断された場合は、1.3 mL の細菌を = します。</li>
		<li>20 mL の最終巻には HBSS に細菌の 1.3 mL を希釈する、150 センチメートルの皿の表面全体液で覆われることができますし、PMVECs のプレートに希釈した細菌を追加します。</li>
		<li>プレートは、顕微鏡で観察するときセル単分子膜の形成、ギャップを見ることができるまで、4-5 時間 5% CO2インキュベーターで 37 ° C で細菌を接種した PMVECs (ギャップ形成の適切なレベルを参照してください図 1 ) 孵化させなさい。</li>
		<li>、上清を収集し、細胞の残骸を削除する卓上遠心分離機で 2,000 x g で 10 分間遠心します。 注: 十分な g を生成することができる任意のテーブル トップ遠心強制ペレット unlysed 細胞及び大細胞断片はこの手順で使用できるには。</li>
		<li>上清を上澄みの細菌を除去するフィルターを通過し、その末尾に添付 0.2 μ m フィルターを持つ注射器に注ぐ。</li>
		<li>無菌培養上清の因数を使用して細胞毒性をテスト (2.1 を参照) し、将来使用するため-80 ° C で培養上清の残りの部分を凍結します。</li>
	</ol>
</li>
</ol>2. 細胞培養上清の分析
<ol><li>細胞毒性の試金<ol>
<li>合流点まで 6 よく料理の PMVECs を成長します。洗浄井戸一度 HBSS で追加 1.5 mL (どちらか PA103 または ∆PcrV 接種の細胞採取) フィルター滅菌培養上清の個々 の井戸。また一つに陰性対照として滅菌 HBSS を追加します。</li>
		<li>21-24 h の CO2インキュベーターにプレートを配置し、セル殺害/細胞毒性のための観察 (次の手順を参照してください)。さらに実験のため細胞毒性を示す培養上清を使用し、細胞の殺害を表示しないものを破棄します。</li>
		<li>セル殺害の定量<ol>
<li>市販の LDH アッセイ キットと製造元を使用して死んだ細胞からメジャー乳酸脱水素酵素 (LDH) のリリースでは、手順をお勧めします。</li>
			<li>また、顕微鏡的 ImageJ ソフトウェアを使用してセル殺害を定量化します。このため、顕微鏡画像とそのままセルを含む培養皿のエリアを記録、セル (単層の細胞死を示す) に欠けている地域を定量化するカスタム ImageJ (健康の国民の協会) マクロ (を参照してくださいのコーディングの補足ファイルを使用).必要な場合は、手動で次のようにマクロのステップを実行します。<ol>
<li>マクロに画像 (RGB または Tiff 形式) を入力し、15% 飽和ピクセルのコントラストを調整します。コントラスト調整後の画像を複製し、ビューのフィールド内のセルとギャップ領域のコントラストの高い画像を得る「背景を引く」コマンドを実行します。</li>
				<li>元のイメージからこのイメージを減算し、イメージ電卓"AND"関数を使用して元のイメージと結果のイメージを結合します。ブラック (ギャップ) と白 (セル) マスク (最低 0、最大 5) しきい値関数を得られる画像に変換し、画像からノイズを除去する「バイナリをむしばむ」関数を使用します。</li>
				<li>「面積率」測定結果のイメージ内の白いピクセルを黒の比率を測定します。プロットし、最大ギャップ領域の % として各治療時点の小数のエリアを表現します。</li>
				<li>手段を計算し、テューキー事後テストで重要と見なされる 0.05 未満のp値と、一方向の分散分析を使用して比較します。</li>
			</ol>
</li>
		</ol>
</li>
		<li>培養上清中の a β オリゴマー タウ、アミロイドのプレゼンスを確立するのにイムノブロット解析を使用します。標準的な手順10、11,12、電気泳動前に、少なくとも 10 倍培養上清を集中以外を用いたイムノブロット解析を実行します。<ol>
<li>上清を集中、その膜が 10 kDa の分子量カットオフ遠心フィルター ユニットに 1-2 mL の培養上清を配置します。その後、フィルター ユニットを配置テーブル トップ遠心分離機、遠心分離 2,000 x に g まで濃度の必要度は達成 (通常 45-60 分)。</li>
			<li>11濃縮培養上清中のタンパク質の量を決定し、ゲルの個々 の井戸にサンプルの同量を読み込みます。</li>
			<li>ゲルを実行し、硝酸セルロースに蛋白質を転送、A11 抗アミロイド抗体、T22 反オリゴマー タウ抗体または適切な二次抗体に続いて MOAB2 抗 a β 抗体でしみをプローブします。化学発光の標準的なプロシージャを使用してしみを開発します。</li>
		</ol>
</li>
		<li>Thioflavin T アッセイを行う、上清中にアミロイドを定量化するのに蛍光 thioflavin T (ThT) を使用します。これらの測定はフィルター滅菌培養上清を使用します。<ol>
<li>10 mL のリン酸緩衝生理食塩水 (PBS) に 8 mg thioflavin T (ThT) を停止することによって thioflavin T の原液 (50 倍) を準備します。混合した後、粉じんを除去する 0.22 μ m のフィルターを通してソリューションをフィルターします。</li>
			<li>測定は、1 mL の PBS で 1 mL 分光光度計のキュベットに ThT 株式の 20 μ L を追加します。希釈したサンプルを蛍光測定装置に配置します。</li>
			<li>425 nm 励起 2 nm 刻みで 450-575 nm の蛍光性の放出をスキャンしてベースライン蛍光発光を測定します。</li>
			<li>425 nm 励起を用いたコマ撮りのスキャンを実行し、482 nm 発光、データ取得ごとの 0.2 s 60 s。</li>
			<li>初期の 20 コマのスキャンの s 対策空白キュベットで蛍光します。20 s、スキャンを一時停止し、フィルター滅菌清 10 μ L をキュベットに追加。逆解析によるキュベットをミックスし、蛍光測定装置に置きます。</li>
			<li>再開最終 40 を取得、時間ベースのスキャン データの s。</li>
			<li>コマのスキャンが完了したら、2.1.5.3 の詳細として同一の設定を使用して最終的な蛍光発光スペクトル スキャンを実行します。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

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なしにレポートします。

ここでは、簡単な培養方法の生物を引き起こす肺炎感染中の細胞毒性アミロイドの生成のデモンストレーションをするとおりです。これらのメソッドは、細胞培養の細胞毒性の試金、免疫ブロット、顕微鏡法と ThT バインドを使って細胞の定量。細胞毒性薬の分析を示した (図 2 と図 4) は本質的にアミロイドが、プリオンの<sup c...

肺炎を生き残るための患者は次の病院退院1,ヶ月で死亡率を上昇している2,3,4,5,6。ほとんどの場合、死は、ストローク5,6と同様、腎、肺、心臓を含むエンド臓器不全や肝のイベントのいくつかのタイプで発生します。この患者集団で高い死亡率の原因は確立されていません。
肺炎がコミュニティ買収されるどちらかとして分類されるまたは院内 (院内)、および肺炎を引き起こすことができるエージェントは、細菌、ウイルス、菌類および化学薬品。院内肺炎の主要な原因の 1 つは緑膿菌です。膿はタイプ III の分泌システムを使用して、ターゲット セル7,8の細胞質に直接 exoenzymes と呼ばれる、様々 なエフェクター分子を転送するグラム陰性生物です。肺血管内皮細胞の感染、時に、exoenzymes は微小管関連蛋白タウ9,10、11,12 の内皮のフォームを含む、各種の細胞内タンパク質をターゲットします。、肺機能と、多くの場合、死に低下した重度の肺浮腫の結果血管内皮バリア破壊に至る、です。
前述したように、初期の肺炎を生き残るための患者は退院後最初の 12 ヶ月で死亡率を上昇しています。この現象を説明するための潜在的なメカニズムは、長期的な予後につながる初期の感染時に長命毒素のいくつかの種類が生成されることです。2 つの観測は、この可能性をサポートします。膿と最初に扱われる最初、培養肺血管内皮細胞は、細菌が抗生物質13によって殺された後の週までの増殖に失敗します。第二に、長命のプリオンとプリオンの特性を持つエージェントは、様々 な人間や動物疾患、特に病気に関連付けられている神経系14,15で実証されています。
肺感染症の中に長寿命の細胞毒性薬の潜在的な生産の調査の方法を記載されていること。ここで簡単な生体外の試金のシリーズのとおりグリコプロテイン生産と一般的なエージェントの肺炎原因、膿を使用して感染活動を調査するために使用できます。これらの試金は調査可能グリコプロテイン誘導感染、肺炎を引き起こす他のエージェントを使用して、次に容易に適応する必要があり、生成される清面も全部 cytotoxins の効果を調査するため役に立つはず臓器や動物。最後に、ここで最も可能性の高い説明は、アッセイは肺炎の中に cytotoxins の生産のための動物と人間の体液をテストに適応可能であります。

<table><tbody><tr><td>Rabbit anti beta amyloid</td><td>Thermo</td><td>71-5800</td><td></td></tr><tr><td>A11 amyloid oligomer antibody</td><td>StressMarq</td><td>SPC-506D</td><td></td></tr><tr><td>T22 anti-tau oligomer antibody</td><td>EMD Millipore</td><td>ABN454</td><td></td></tr><tr><td>Thioflavin T</td><td>Sigma Aldrich</td><td>T3516</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025-092</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>0.22 micron syringe filters</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>HRP Goat antirabbit IgG</td><td>Abcam</td><td>ab6721-1</td><td></td></tr><tr><td>Strains PA103 and &amp;Delta;PcrV</td><td></td><td></td><td>These strains of P. aeruginosa were obtained from Dr. Dara Frank, University of Wisconsin Medical College, Milwaukee, WI</td></tr><tr><td>FITC Griffonia lectin</td><td>Sigma Aldrich</td><td>L9381</td><td></td></tr><tr><td>TRITC Helix pomatia lectin</td><td>Sigma Aldrich</td><td>L1261</td><td></td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td></td></tr><tr><td>0.22 micron nitrocellulose</td><td>BioRad</td><td>162-0112</td><td></td></tr><tr><td>Type 2 collagenase</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone</td><td>SH30898.03IH</td><td></td></tr><tr><td>PenStrep</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>Carbenicillin Disodium salt</td><td>Sigma&amp;nbsp;</td><td>C1389</td><td></td></tr><tr><td>Microcetrifugation concentrators- 10,000 MW cut-off</td><td>Millipore</td><td>UFC801008</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma&amp;nbsp;</td><td>795496-500G</td><td></td></tr><tr><td>Magnesium phosphate heptahydrate</td><td>MP Biomedicals</td><td>MP021914221</td><td></td></tr><tr><td>Citric Acid</td><td>G Biosciences</td><td>50-103-5801</td><td></td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Sigma</td><td>S9506-500G</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10277</td><td></td></tr></tbody></table>

methods for detecting cytotoxic amyloids following infection of pulmonary endothelial cells by pseudomonas aeruginosa

生き残る肺炎患者は、退院後数ヶ月で死亡率を上昇しています。肺炎肺組織の感染結果後続エンド臓器不全につながることができる長寿命の cytotoxins の生産をえられています。肺感染症の中に cytotoxins が生産されている仮説をテストするための in vitroアッセイを開発しました。ラット肺血管内皮細胞と細菌緑膿菌をモデル システムとして使用され、続いて細胞培養を用いた細菌による内皮細胞の伝染に続く cytoxins の生産を示す乳酸脱水素酵素アッセイと ImageJ 技術を活用した新規顕微鏡法を用いた直接定量から。これらの cytotoxins のアミロイドの性質を示した thioflavin T 結合の試金、イムノブロットおよび A11 抗アミロイド抗体を用いた immunodepletion。イムノブロット分析は、オリゴマー タウと a β が制作され膿によって感染内皮細胞が発表したことを示した。これらのメソッドは、ひと臨床サンプルの解析に容易に適応する必要があります。

簡単な生体外の試金は、緑膿菌に感染細胞の上清中に cytotoxins の存在を試金するためにしました。基本的には、感染細胞から培養液、細菌の添加後 4 h を収集、細菌、培養上清、フィルター殺菌によって削除されます、無菌培養上清がセルの新しい人口に追加されます。細胞上清添加後に 21-24 時間観察し、細胞の殺害を定量化します。
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Watch this Scientific Journal Video about Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa at JoVE.com

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

検出細胞毒性アミロイド次感染症の肺による内皮細胞緑膿菌法

簡単な方法は、緑膿菌による肺血管内皮細胞の感染細胞傷害性のアミロイドの生産の「論証の説明します。

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

methods-for-detecting-cytotoxic-amyloids-following-infection

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

5.5K ビュー.  University of South Alabama. 簡単な方法は、緑膿菌による肺血管内皮細胞の感染細胞傷害性のアミロイドの生産の「論証の説明します。

ビデオ: Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

調製と<em&gt;インビボ</em&gt;アクティビティベースのプローブの使用のために<em&gt; N</em&gt; -acylethanolamine酸アミダーゼ

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

生化学

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

細胞内病原体によって引き起こされる液胞破裂のシングルセル測定

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

免疫・感染学

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

緑膿菌誘発肺傷害モデル

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

一定の流れの原発性ヒト内皮細胞の肺炎球菌感染

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

緑膿菌の小コロニー変異体の培養とアルギン酸の定量

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. アフルギノーサ 抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

肺上皮細胞における シュードモナス 感染に及ぼすタバコの煙の影響の研究

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

嚢胞性線維症患者の喀痰中の 緑膿菌 の可視化

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

嚢胞性線維症肺における 緑膿菌 および 黄色ブドウ球 菌バイオフィルムのEx生体モデルにおける抗生物質有効性試験

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

ホスト細胞への細菌の付着を自動的に、ハイスループットで検出

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...

検出細胞毒性アミロイド次感染症の肺による内皮細胞緑膿菌法

要約

さらに動画を探す

この動画の章

調製と

細胞内病原体によって引き起こされる液胞破裂のシングルセル測定

緑膿菌誘発肺傷害モデル

一定の流れの原発性ヒト内皮細胞の肺炎球菌感染

緑膿菌の小コロニー変異体の培養とアルギン酸の定量

P. アフルギノーサ抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー

肺上皮細胞におけるシュードモナス感染に及ぼすタバコの煙の影響の研究

嚢胞性線維症患者の喀痰中の緑膿菌の可視化

嚢胞性線維症肺における緑膿菌および黄色ブドウ球菌バイオフィルムのEx生体モデルにおける抗生物質有効性試験

ホスト細胞への細菌の付着を自動的に、ハイスループットで検出

検出細胞毒性アミロイド次感染症の肺による内皮細胞緑膿菌法

要約

さらに動画を探す

この動画の章

調製と

細胞内病原体によって引き起こされる液胞破裂のシングルセル測定

緑膿菌誘発肺傷害モデル

一定の流れの原発性ヒト内皮細胞の肺炎球菌感染

緑膿菌の小コロニー変異体の培養とアルギン酸の定量

P. アフルギノーサ 抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー

肺上皮細胞における シュードモナス 感染に及ぼすタバコの煙の影響の研究

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