We thank Dr. Frank Wolschendorf for access to the Cytation 3 automated imaging plate reader. This work was funded in part by NIH grant R01-AI104499 to OK. Parts of the work were performed in the UAB CFAR facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311.

注：2 6206は、非病原性株17、18である結核菌の MCとしては、このプロトコルですべての作業は、バイオセーフティレベル2の施設（BSL-2）で行うことができます。 結核菌 MC 2を発現する緑色蛍光タンパク質1.培養条件6206（ マウンテンバイク -GFP） 注： ヒト結核菌H37RvはpMN437がこのプロトコル16全体で使用されるプラスミドを発現するGFPで形質転換された栄養要求株MC 2 6206（ΔpanCD、ΔleuCD）に由来しました。研究者のための歪みを簡単にアクセシビリティを有効にするには、野生型株を発現する非GFPでのMtb -GFP株を代用することが可能です。しかし、GFP発現は、食作用とのMtb /マクロファージの凝集体の形成の視覚的確認を可能にすることが望ましいです。経度のためのGの長期保存、Mtbの -GFPを、20％グリセロールを補充し（ステップ1.1参照）完全7H9培地中で-80℃で凍結しました。 <ol><li> 10％ミドルOADC、0.02％チロキサポール、24μgの/ mLのD-パントテン酸、を50μg/ mLのL-ロイシンおよび50μg/ mLのハイグロマイシンBで7H9培地を補充することによって、完全なマウンテンバイク -GFPメディア（7H9-C）を準備</li><li> マウンテンバイク -GFPの1つのバイアルを解凍し、30 mLの正方形の底PETGフラスコ中の7H9-Cの10 mLに追加します。 </li><li>ロータリーシェーカー（50回転）に37℃でインキュベートします。 OD 600が 1.0に達するまで（約5-8日）数日おきに光学密度測定（OD 600）を取ります。 </li><li> 0.2〜1.0の間のOD 600を維持するために必要に応じて希釈して継代培養。 </li><li>感染のために、対数増殖期（OD 0.6から1の600）内のMtb -GFPの確立された文化を使用します。塊状の文化を避けるために、水浴中のMtb培養フラスコを超音波処理（130 W; 3×5秒パルス）ONCeまたは週二回</li></ol> 2.培養条件 THP-1細胞<ol><li> 10％熱不活性化ウシ胎児血清、2mMのL-グルタミン、および10mM HEPESを含むRPMI 1640培地を補充することによって、完全なTHP-1細胞培地（RPMI-C）を調製します。 </li><li> 5％CO 2の加湿雰囲気中37℃でT-75フラスコ中で細胞をインキュベートします。 </li><li>血球計数器を用いて細胞数をカウントしたり、一日おきにフローサイトメトリーおよび0.1 mLの100万〜0.6の間の濃度でTHP-1細胞を維持します。 </li></ol> 3.感染プロトコルのMtb /マクロファージ集計構造を生成するために、 <ol><li> THP-1細胞調製物（96ウェルプレートあたり） <ol><li> 5分間250×gで遠心分離し、7×10 6 THP-1細胞。 7 mLのRPMI-Cでの再懸濁。 </li></ol></li><li> MTB -GFP準備 <ol><li>遠心分離2.8×10 8 のMtb -GFPスイングバケット遠心分離機で3200×gで5分間のuOD 600 1.0 = 3×10 8個の細菌/ mLでの変換を歌います。 </li><li>ステップ3.2.1のようにRPMI-Cと遠心機で一回洗浄します。 </li><li> 10秒のための7 mLのRPMI-Cで再懸濁し、ボルテックス。 </li></ol></li><li> 感染 注：テンプレートについては、 図1を参照してください。 <ol><li>培地の蒸発を防ぐために、水のリムの行AとHと、列1および12に滅菌水200μLを加えることにより、96ウェルプレートを準備します。 </li><li>バックグラウンドコントロール（ブランク）用カラム2（G2へのB2）に200μLのRPMI-Cを追加します。 </li><li>感染させるために、THP-1細胞懸濁液（ステップ3.1）へのMtb -GFP懸濁液（ステップ3.2）を追加し、ピペットでよく混ぜます。最終的なTHP-1細胞密度は、mL当たり5×10 5であり、感染の対応する多重度は40です。 </li><li> 25 mLのリザーバにTHP-1 / マウンテンバイク -GFP懸濁液を注ぎます。 </li><li> usin残りのすべての96ウェル（G11を通じてB3）にTHP-1 / マウンテンバイク -GFP懸濁液200μLを追加GAマルチチャンネルピペット。 注：複数の96ウェルプレートで作業する場合は、定期的にさえ混合物を各ウェルに添加されていることを確認するために、リザーバ内の残りのTHP-1 / MTBサスペンションを再懸濁します。 </li><li> 7〜10日間、5％CO 2で37℃でインキュベートします。 </li><li>ゆっくりと100μLを除去することにより、2日おきにメディアを変更し、マルチチャンネルピペットを用いて100μLに予め温めたRPMI-Cを追加するだけでなく、静かにそれぞれの最上部からメディアを過ごしました。井戸を再懸濁し、ウェルの底にマウンテンバイク /マクロファージの凝集体を邪魔しないでください。 </li><li>視覚のMtb /マクロファージの凝集体の大きさに着目し、毎日の蛍光顕微鏡（4-10X目的）によってウェルを調べます。日7-10では、 マウンテンバイク /マクロファージの凝集体は、薬物の効力試験（第4節）のために進行する（参考のために図2を参照）を十分に大きくなければなりません。 </li><li>所望であれば、自動細胞イメージングシステムを用いて96ウェルのキャプチャー画像が嵌め込ま明視野およびGFPフィルターでマウンテンバイク /マクロファージ凝集体形成を文書化するために設定します。 注：ユーザーが自動化されたイメージングシステムへのアクセスを持っていない場合は、代表ウェルの画像をGFPと明視野能力を有する任意の適切な顕微鏡を用いて撮影することができます。 </li></ol></li></ol> マウンテンバイク /マクロファージ集約由来医薬品に対する効力のMtbを評価する4.成長阻害アッセイ<ol><li> 薬剤の準備（2薬剤のための三重の条件） 注：テンプレートについては、 図1Aを参照してください。 <ol><li>独立した96ウェルプレートでは、G10までB2に7H9-Cメディアの125μLを追加します。 </li><li>ステップ4.2.4での希釈を考慮するために、7H9-Cの1mLに二重最高所望の最終濃度で、二つの薬剤を準備します。 </li><li>それぞれ三連の治療のために、ウェルB11-C11-D11とE11-F11-G11に各薬剤の250μLを追加します。 </li><li>マルチチャンネルピペットを使用して、連続希UTE試験薬物B10-G10にB11-G11から125μLを移動することで2倍。各ステップで5回ピペッティングすることによって混合します。 </li><li>プレートを横切って（右から左）の列に列から125μLを移動させ、カラム4の後に停止し続けます。 </li><li>カラム4を混合した後、廃棄物容器に125μLを捨てます。列2と3は、バックグラウンド（ブランク）とプラス成長コントロールなどの使用を可能にするために、任意の薬を含めることはできません。 注：また、テスト薬剤のライブラリについては、図1（b）のテンプレートに従ってください。各96ウェルプレートは、単一の濃度で58の薬物を収容することができます。また、ステップ4.2.4で半分に希釈されるので、倍の所望の最終濃度を使用して、この薬物プレートを準備します。 </li></ol></li><li> 薬物治療： <ol><li> Mtbを含む96ウェルプレートを取り出し、マクロファージ（ のMtb /マクロファージ凝集体）-infected。 </li><li>慎重にマルチチャンネルピペットを用いて、関連するすべてのウェル（G11を介してB2）をデカント。2段階の方法でこれを実行します。第一プレートを傾けることなく、150μLを削除してから、プレートを傾けるとウェルの底縁にピペットチップを挿入することにより、残りのメディア（〜50μL）を取り外します。 マウンテンバイク /マクロファージの凝集体がウェルの底に付着しているように、何の材料が失われてはなりません。 </li><li>静かマウンテンバイク /マクロファージの凝集体を含むプレートに関連するすべてのウェル（G11を通じてB2）に7H9-Cメディアの100μLを追加します。 </li><li>マルチチャンネルピペットを用いて、感染プレートの対応するウェルに96ウェルプレート（ステップ4.1）を含む薬剤から100μLを移します。 </li><li>密封された袋に入れて、37℃で3日間インキュベートします。 </li></ol></li></ol>レサズリンマイクロタイターアッセイを使用して5薬効の定量<ol><li> H 2 O中の0.8mg / mLの最終濃度でレサズリン原液を準備し滅菌のため0.22μmの細孔径のPVDF膜を通して濾過。</li><li> 1：1の比率で2レサズリンストック溶液、H 2 OおよびTween-80（H 2 O中20％溶液）を混合して溶液を作動レサズリン準備します。最終濃度は0.4 mg / mlでのレサズリン及び5％のTween-80です。 </li><li> 37℃で24時間、530 nm励起および590 nmの発光で30分毎に蛍光を読み取るためのプログラムは、プレートリーダーを使用してセットアップ。 37°Cにプリ暖かいプレートリーダー。 </li><li>マルチチャンネルピペットを用いて薬剤処理プレート（ステップ4.11）に関連するすべてのウェル（G11を通じてB2）にレサズリン作業溶液20μLを追加します。 </li><li>プレートリーダーでプレートを置き、ステップ5.3で設定プログラムを起動します。 注：アッセイプレートの大量の処理を容易にするために、37℃のインキュベーター中でアッセイを開発し、単一の蛍光は24時間毎に読み出し実行するのに十分です。これはまた、運動やインキュベーション機能を備えたプレートリーダーへのアクセス権を持たないユーザーにも適用可能です。 </li></ol>

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The authors have nothing to disclose.

ここでは、詳細に薬効試験に適した代替のMtb感染モデルを説明してきました。このモデルは、アカウント初期の結核治療薬の開発プロセスの間に、より配慮を与えられるべき二つの重要な要因を考慮に入れる：薬剤浸透と感染時のMtbの代謝変化に生理学的に関連の障壁の存在。我々は以前に私たちの感染モデルの利点を示し、スループットの互換性16の?...

<html>結核（TB）は、40年以上にわたって1用の抗結核化学療法レジメンの利用可能性にもかかわらず、世界的に深刻な健康への脅威のまま。これは、非準拠2を患者につながる複数の薬剤の組み合わせを使用して6ヶ月以上の長い治療期間の要件に一部起因します。近年の薬剤耐性結核の出現は、さらに臨床的に承認された医薬品の開発に成功が事実上存在しない3分野で問題を配合しました。確かに、徹底的な抗結核薬の開発にもかかわらず、唯一の単一の薬物は、FDAは、過去40年4臨床使用が承認されました。したがって、抗結核薬の新しい世代が緊急にこの問題に対処するために必要とされます。 結核治療薬の発見において重要な問題は、臨床設定における有効性へのin vitroでの活性を有する化合物からの転送成功の欠如であります= &quot;外部参照&quot;&gt; 5、6、7。最初に、ターゲットベースのアプローチは、細菌細胞全体に変換することができなかった抗Mtbの薬剤 5をスクリーニングするために使用されました。 Mtbの細胞を用いた場合でも、それは多くの場合正確にインビボ 8,9 において薬効を予測していないブロス増殖させた培養物を用いて行われます。これらの問題は認識されているとのMtbを含むマクロファージまたは潜在性のMtbに対する薬物スクリーニングアッセイは、正常8に確立された、10、11、12。しかし、これらのより高度なアッセイは、薬は、非血管新生した肺病変における、および感染部位に壊死巣で遭遇する浸透障壁に十分な配慮を与えることはありません。確かにでも最初の行の結核治療薬のリファンピシンのため、次善の投与は、 生体組織や脳脊髄液（CSF） で不十分なに疑問視されている13、14、15だけでなく、細胞内のMtb 8、9に対して有効性を減少を浸透。このように、初期のリード開発プロセスの間に考慮に入れ、これらのパラメータを取る新モデルおよびアッセイは間違いなく結核治療薬の発見の努力を改善するであろう。 このニーズに応えるために、我々は最近、安価で迅速、かつMtbの薬効試験の16のためのBSL-2互換の代替感染モデルを確立しました。生理学的に関連する細胞透過障壁を再現し大マクロファージの結合構造体、および生成されたマクロファージ継代以内に密集したMtbの生産この感染モデル<e潜在マウンテンバイクに似ている、変化した生理状態を持つメートル&gt;マウンテンバイク。この感染モデルから派生したMtbは、他の細胞内感染モデルと一貫性のある結果を生成した薬効を評価するためにレサズリンマイクロタイターアッセイ（REMA）と合わせ、血清濃度に比べて高いCSF濃度を達成するための一般的な結核治療薬の報告能力とよく相関していました16。 ここでは、 マウンテンバイク /マクロファージの結合構造体の生成は、REMAを使用して、薬剤感受性試験に適したマクロファージ継代のMtbを生成するために、詳細に説明します。特に、我々は、この感染系を候補の抗結核薬のスループットスクリーニングとの互換性のために96ウェル形式に適合させることができる方法を示します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7H9</td>
			<td>BD Difco</td>
			<td>271310</td>
			<td>Follow manufacturer&#39;s recommendations</td>
		</tr>
		<tr>
			<td>Middlebrook OADC</td>
			<td>BD Biosciences</td>
			<td>212351</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyloxapol</td>
			<td>Sigma</td>
			<td>T8761</td>
			<td>Prepare 20% stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>D-Pantothenic acid hemicalcium salt</td>
			<td>Sigma</td>
			<td>P5710</td>
			<td>Prepare 24 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>MP Biomedicals</td>
			<td>194694</td>
			<td>Prepare 50 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>EMD Millipore</td>
			<td>400051</td>
			<td>Prepare 200 mg/ml&#160; stock solution in H2O</td>
		</tr>
		<tr>
			<td>Nalgene Square PETG media bottle</td>
			<td>Thermo Fisher</td>
			<td>2019-0030</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Hyclone</td>
			<td>SH30027.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning</td>
			<td>MT25005CI</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Hyclone</td>
			<td>SH30237.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 3 plate reader</td>
			<td>Biotek</td>
			<td></td>
			<td>Interchangable with any fluorescent plate reader and microscope</td>
		</tr>
		<tr>
			<td>Gen5 Software</td>
			<td>Biotek</td>
			<td></td>
			<td>Recording and analysis of rezasurin coversion</td>
		</tr>
		<tr>
			<td>Rifampicin&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP2679250</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Moxifloxacin Hydrochloride</td>
			<td>Acros Organics</td>
			<td>457960010</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Resazurin Sodium Salt</td>
			<td>Sigma</td>
			<td>R7017</td>
			<td>Prepare 800 &#956;g/mL stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher Scientific</td>
			<td>T164500</td>
			<td>Prepare 20% stock solution in H2O; filter sterilize</td>
		</tr>
	</tbody>
</table>

a high-throughput compatible assay to evaluate drug efficacy against macrophage passaged mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

96ウェルプレートフォーマットにこの感染モデルを適合させるの頑健性を確認するために、ここでは、図に示したテンプレートに従ってリファンピシン（RIF）及びモキシフロキサシン（MOXI）に当社の96ウェル適応感染モデル由来のMtbの薬剤感受性を調べました1A。我々は、このアッセイの鍵のMtb /マクロファージ集合構造の発生?...

Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

マクロファージ継代に対する薬効を評価するために、ハイスループットアッセイの互換性<em&gt;結核菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

a-high-throughput-compatible-assay-to-evaluate-drug-efficacy-against

Research

JoVE Journal

Immunology and Infection

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

7.7K ビュー.  University of Alabama at Birmingham. New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening. 

ビデオ: A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

の成長結核バイオフィルム

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

免疫・感染学

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

自然送信時に結核マラリア重複感染を研究する実験モデル結核菌とマラリアベルゲイ</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

多剤耐性を実証<em&gt;結核菌</em&gt;増幅マイクロアレイ

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

設立し、研究するために、高スループットの設定の最適化<em&gt;表皮ブドウ球菌</em&gt;と<em&gt;マイコバクテリウムmarinumの</em創薬のためのモデルとして&gt;感染症

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

ハイスループット定量的リアルタイムRT-PCRアッセイのタイプIおよびIIIインターフェロンサブタイプの発現プロファイルを決定するための

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

免疫担当げっ歯類で堅牢な肺炎モデルはに対して抗菌有効性を評価するために<i&gt; S。ニューモニエ</i&gt;、<i&gt; H。インフルエンザ</i&gt;、<i&gt; K。ニューモニエ</i&gt;、<em&gt; P。緑膿菌</em&gt;または<em&gt; A。バウマニ</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム<em&gt;結核菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

アン<em&gt;インビトロ</em&gt;結核病変における薬物侵入を予測するカゼウム結合アッセイ

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

レポーター酵素蛍光を用いたマウスにおける結核菌をイメージング

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

結核抗酸菌 marinum感染成人ゼブラフィッシュをモデル化

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

マクロファージ継代に対する薬効を評価するために、ハイスループットアッセイの互換性

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の成長結核バイオフィルム

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レポーター酵素蛍光を用いたマウスにおける結核菌をイメージング

結核抗酸菌 marinum感染成人ゼブラフィッシュをモデル化