私たちは、ウイルス学研究所の動物施設を使用するための許可をMCMV·リュックとトーマスBaumert（INSERM U748、ストラスブール）を増幅し、滴定のためにリーTuddenham（IBMC、ストラスブール）に感謝。 INSERM、大学·デ·ストラスブールと通信社国立·デ·ラ·ルシェルシュ（ANR-08-MIEN-005-01）からの財政支援が認められている。彼らのマスタープロジェクト間にソニアBeroudとレティシアLelieurの初期の参加も認められている。

1。ウイルス懸濁液の調製<ol><li>ウルリッヒKoszninowskiの研究室からMCMV表現ルシフェラーゼのスミス株（MCMVリュック·15）を入手します。この組換え体では、ルシフェラーゼ遺伝子がゲノムのMCMV IE2遺伝子座に挿入されている。 </li><li> MCMV-リュックを増幅するために、16（MOI、0.001〜1）感染症の異なる多重度MCMVとマウス骨髄ストローマ細胞株（M2-10B4、ATCC＃CRL-1972）を感染させる。このために、37℃で無血清培地中で培養細胞にウイルスを加える℃で1時間後、上清を除去し、DMEMと交換し、10％ウシ胎児血清（FCS）を補充した。 5日後、さらに使用するまで-80℃で培地や店舗アリコート°Cを収穫。 </li><li>プラークアッセイによるウイルス懸濁液の滴定。 <ol><li>ペニシリン（200 IU / ml）とsteptomycin（200 mg / mlのを含む10％FCSを添加したDMEM 1ml中24ウェルプレートに40,000細胞で/ウェルで3T3細胞（ATCC＃CRL-1658）スプリット）と37℃で24時間インキュベートする℃に</li><li> DMEMでMCMV懸濁液のアリコートを連続希釈（10 -1〜10 -8）を準備し、細胞培養培地を除去した後、希釈液200μlで標的細胞（3T3）感染。 37℃で1時間インキュベート℃にトップ培地1 mlを加える（DMEM 3％FCS、2％（v / v）のゲンタマイシン、200 IU / mlのペニシリン、200 mg / mlのストレプトマイシン、8グラム/ Lカルボキシメチルセルロース）および37℃で5日間インキュベート℃で</li><li>各ウェルに200μlの10％ホルムアルデヒド（水で希釈ホルムアルデヒドの毒性吸入を避けるために、化学フード内で行われるべきである）を添加し、室温で6時間インキュベートすることによって、細胞を固定する。アウトピペットで培地+固定具を外し、200μlの細胞染色溶液（20％エタノール中1％のクリスタルバイオレット（重量/容量））を加える。使用前に、9部ミリQ水で1パートを希釈する。室温で2分間インキュベートし、水に浸して3〜4倍皿を洗う、双眼鏡とのプラーク数を乾燥し、カウントすることができます。 </li></ol></li> &lt;李&gt;50μlの50 PFUsを取得し、さらに使用するまで氷上に残すためにDMEM中MCMV-リュックを希釈。新生児は、生後2〜3週間に到達する前に、より高いウイルスの接種を使用すると、致死を誘導する。我々の経験では、500 PFUsは注射後ごと新生児5〜7日で致死性を誘発する。 </li></ol> 2。新生児の注射<ol><li>新生児（4〜20時間齢のマウス）は母親にストレスと仔の拒絶反応を引き起こすであろう、 &quot;外国の&quot;匂いによる汚染を避けるために慎重に子犬を取り扱う前に手袋とゴミとケージの糞を操作し、利用可能な場合。 </li><li> 図1Aに示すように、インスリンシリンジ（29 G）を用いて腹腔内注射を行う。背部皮膚で新生児を保持します。腹側を上にして、前肢の下に針を導入し、優しく腹腔を（臍下少し）達成するために皮下に押し込みます。我々は現在DPBSで希釈し、1％メチレンブルーを注入し、pに生存を監視ウイルス感染を行う前にractice手法。メチレンブルーは、射出プロセス中にはっきりと見えているので、我々は腹腔内ウイルス懸濁液の適切な配信を保証するために、最初にこの &quot;訓練&quot;注射を行うことをお勧めします。 </li><li>ケージに新生児バックを発生し、配置することがあり小さな血滴をなくす。 </li></ol> （3） 生体内イメージングで <ol><li> 7日目に、同じように、前述の注意と新生児を扱う。各動物に50μlの腹腔内麻酔薬の混合物（4 mg / mlのケタミン、0.8 mg / mlのキシラジン）とルシフェリン（0.15ミリグラム/ gの体重）を注入。動物の平均体重は1〜2 gおよび麻酔あたり動物の用量です：ルシフェリンは排出の最大に達するまでケタミン40μgの、キシラジン8μgのは12-15分待ちます。これは、事前に決定されなければならないとルシフェリンプロバイダおよび使用される撮像システムに依存する。 </li><li>子犬はanesthetiですがゼット、将来の識別のためにそれらをタグ付けし、変異動物が使用される場合、将来の遺伝子型決定のために使用することができる組織の小片を収穫。 </li><li>イメージングシステムで子犬を置き、全身から放射される光の買収を行う。七日間感染後、ウイル​​スは、肝臓、腎臓、肺や唾液腺などの標的臓器に多数のコピーに複製されているので、露出の期間は短くする必要があります。我々のケースでは、我々は10〜20秒（ 図1B）の取得を行う。撮像装置の取得室内のプラットフォームを麻酔した動物の体温の過剰な冷却を回避するために加熱する必要がある。また、買収の1ラウンド内のいくつかの動物の低解像度の画像を得るために有用である、その後、特定の動物や孤立臓器次郭清の一部に集中するために、プラットフォーム近いカメラレンズまで移動。ソフトウェアには、急速な変化を有効にする必要がありますパラメーター（露光時間、距離サンプル/レンズ、高感度）、関心とスナップショットイメージの将来の輸出の定義された領域の定量。 </li><li>頭だけから光信号を取得するために、高いウイルス複製が発生した臓器のレベル（肝臓、肺、唾液腺）で放出された光子をマスクし、実行します厚い、濃い紙で横方向に横たわっている新生児の体をカバー5〜10分（ 図1C）の取得。同じ条件での動物の対向側の買収を実行します。 </li><li>ケージに子犬を再導入し、麻酔新生児の過剰な冷却を避けるために、上部の加熱ランプを配置。定期的にポスト麻酔回復を監視します。指示された用量で、麻酔は、現在30分続きます。 </li><li>日9、11と14で同じ手順を繰り返します。日11と14日、麻酔薬の用量（6 mg / mlのケタミン、1.2 mg / mlのキシラジン）を変更。 </li></ol>

<ol>
	<li>Loewendorf, A., Benedict, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+host+innate+and+adaptive+immune+defenses+by+cytomegalovirus:+timing+is+everything.">Modulation of host innate and adaptive immune defenses by cytomegalovirus: timing is everything.</a> J. Intern. Med. 267, 483-501 (2010).</li><li>Lischka, P., Zimmermann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiviral+strategies+to+combat+cytomegalovirus+infections+in+transplant+recipients.">Antiviral strategies to combat cytomegalovirus infections in transplant recipients.</a> Curr. Opin. Pharmacol. 8, 541-548 (2008).</li><li>Johnsen, J. I., Baryawno, N., Soderberg-Naucler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+human+cytomegalovirus+a+target+in+cancer+therapy?.">Is human cytomegalovirus a target in cancer therapy?.</a> Oncotarget. 2, 1329-1338 (2011).</li><li>Morein, B., Abusugra, I., Blomqvist, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+in+neonates.">Immunity in neonates.</a> Vet. Immunol. Immunopathol. 87, 207-213 (2002).</li><li>Zaghouani, H., Hoeman, C. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathogenesis+of+congenital+cytomegalovirus+infection:+disease+mechanisms+and+prospects+for+intervention.">Neuropathogenesis of congenital cytomegalovirus infection: disease mechanisms and prospects for intervention.</a> Clin. Microbiol. Rev. 22, 99-126 (2009).</li><li>Tsutsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cytomegalovirus+infection+on+embryogenesis+and+brain+development.">Effects of cytomegalovirus infection on embryogenesis and brain development.</a> Congenit. Anom. 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著者らは競合する経済的利害関係を宣言しません。 倫理声明動物は、学院のd&#39;ImmunologieエトHématologieの動物のケア施設で無菌条件下で維持された。マウスと実験手順の処理は実験動物の保護のためのフランスの法律に基づいて実施された。手順は、承認番号、67から345の下にサービスvéterinaireデラ県デュバラン（フランス）により承認された。

マウス新生児にMCMV-リュックの普及を監視する生体イメージング技術で使用して、我々は、野生型とは対照的に、変異動物の脳にウイルスの拡散を観察することができました。脳の動物とex vivoイメージングのさらなる切開は、中枢神経系における発光ウイルスの存在を確認した。さらに、我々はまた、MCMV E1初期タンパク質に特異的な抗体と脳薄切片上（図示せず）免疫組...

ヒトサイトメガロウイルス（HCMV/HHV-5）は、β-ヘルペスウイルスファミリーのメンバーである。 HCMVは、通常、無症候性感染1は早く生命の間に取得された高度に普及している、日和見病原体である。すべてのヘルペスウイルスと同様に、HCMVは、免疫システムしっかりウイルス複製を制御するホストの全生涯にわたって持続します。ウイルスの再活性化のエピソードはほとんど移植患者は、移植片拒絶反応2を防ぐために薬を受け取るなど、免疫不全の人に起こる。成人では、HCMVも神経膠芽腫3にリンクされています。さらに、HCMVは未熟免疫4-6と新生児のための著名な病原体である。発達中の胎児や新生児に初感染は深刻な結果を持つことができます。 HCMV感染症は先進国では先天性先天性欠損症と小児疾患の最も一般的な感染原因となっています。なお、新生児HCMV感染症の発生率は0.5から1パーセントプレーヤーに影響を及ぼしていると推定されるfは5〜10％がこのような小頭症や小脳の形成不全などの重篤な症状に悩まされますそのうち全て出生。また、無症候性ウイルス感染と感染児の10％が後損失、視覚的な欠陥や発作、てんかん7,8を聞いて 、精神遅滞につながる後遺症を開発する。 として注射9の異なる経路を経由してマウスに接種することができるような単純ヘルペス1（HSV-1/HHV-1）などの他のヒトヘルペスウイルスとは対照的に、サイトメガロウイルスの複製は種特異的である。この機能は、厳しくHCMVの異なる動物モデルで行われ病因（マウス、ラット、モルモット、アカゲザル）とそれぞれの本物のホスト固有CMVsの調査を妨げている。すべてCMVsは、ゲノムサイズと組織、組織親和性および遺伝子発現の調節の有意な類似性を示す。彼らはまた、それぞれのホストで同じような病態を引き起こす。ゲノムの多様性にもかかわらずであるトゥイーンHCMVおよびマウスサイトメガロウイルス（MCMV）（ORFの50％マウスCMVにおいて識別されるヒトウイルス中に存在する）は、マウスモデルは、最近ほとんど変異株は、ウイルスを制御する能力について試験することができるので、有利であることが証明されているin vivoでの複製。これは、このウイルスは、10〜 &quot;resistome&quot;を構成する成虫期で発現するマウス遺伝子の数の推定を可能遺伝学的スクリーニングにつながっている。要するに、これは、MCMV-感染マウスは、成人におけるホストウイルスの相互作用の研究のための魅力的なモデルを表して示している。人間とマウスの間に胎盤層組織の違いは、マウスにおけるウイルス感染の母に胎児伝送を損なうため、先天性CMV感染症の調査はより複雑です。最近では、妊娠12.5日に胎盤におけるMCMVの直接注入は、減損11聴力につながったマウス新生児の脳の感染を可能にしました。しかし、ほとんどの一nvestigationsは現在、潜在的に血行性脳の感染症につながる全身ウイルス普及、頭蓋内注射に比べてより関連性のあるモデルを提供するために4-20 HR-古い新生児の腹腔内注射を使用しています。このプロトコルは、CMVの病因に重要な洞察を提供し、より具体的には、マクロファージ12のような単核細胞で浸潤される炎症病巣部に位置ニューロンおよびグリア細胞におけるウイルス複製における新生児の結果とMCMV感染を実証した。このレポートには、減少粒状ニューロンの増殖と遊走と複数インターフェロン刺激遺伝子の誘導を伴う小脳の変化した形態形成を説明しました。中枢神経系におけるMCMVを制御するためのCD8 + T細胞の本質的な役割についても同じグループ13によって報告された。微生物の病理学的効果を分析する際に考慮すべき重要な側面は、感染のダイナミクスですイオン。 MCMVの場合には、将来の神経生物学的傷害の大きさを理解し、予測するために、発達中の脳へのウイルス伝播の進行を探索し、定量化することが特に重要である。伝統的に、感染の進行の定量化は、感染した動物の定期的な犠牲を、そうでなければアクセスできない脳などの組織中の病原体を、滴定する必要があります。プロトコルのこのタイプは、今必要な動物福祉の向上と3Rの（交換して、絞込み、リデュース）原則14によって挑戦されています。 生体内画像化技術において使用したインビボの感染実験において必要とされる動物の数の大幅な削減を可能にすることができる。ここでは、報告し、マウス新生児への腹腔内MCMVリュック·注入時に脳内にウイルスの普及の経時的分析について説明します。同じ動物を使用して、我々は強烈なVIのサイトを追跡し、 生体内で監視2週間の期間中にRAL複製。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Fisher Scientific</td>
			<td>W3523A</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Methylene blue</td>
			<td>Sigma Aldrich</td>
			<td>319112</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Insulin needles</td>
			<td>VWR</td>
			<td>613-4897</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ketamine</td>
			<td>CentraVet</td>
			<td>Ket 201</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Xylazine/Vetranal</td>
			<td>Sigma Aldrich</td>
			<td>46995</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DPBS</td>
			<td>DUTSCHER</td>
			<td>P0436500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Luciferin</td>
			<td>Caliper</td>
			<td>760504</td>
			<td></td>
		 </tr>
		 <tr>
			<td>gentamycin</td>
			<td>Sigma Aldrich</td>
			<td>G1272</td>
			<td></td>
		 </tr>
		 <tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>carboxymethylcellulose</td>
			<td>Sigma Aldrich</td>
			<td>C4888</td>
			<td></td>
		 </tr>
		 <tr>
			<td>formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		 </tr>
		 <tr>
			<td>crystal violet</td>
			<td>Sigma Aldrich</td>
			<td>C3886</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>IVIS 50</td>
			<td>Caliper/Perkin Elmer</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

use of in vivo imaging to monitor the progression of experimental mouse cytomegalovirus infection in neonates

ヒトサイトメガロウイルス（HCMVまたはHHV-5）は、免疫低下個体における生命を脅かす病原体である。先天性または新生児感染すると、ウイルスが難聴と精神遅滞を含む重篤な神経学的損傷を誘発する可能性がある、発達中の脳に感染し、複製することができます。症状の潜在的な重症度にもかかわらず、治療の選択肢は、ワクチンと特定抗ウイルス療法の有無を利用できないことによって制限されます。観測は主に感染した子供の剖検に由来するため、また、中枢神経系（CNS）の感染の間に発生する分子事象の正確な記述がまだ欠けている。このようなアカゲザルCMVなどのいくつかの動物モデルは、CNSにCMVの病因に重要な洞察力を開発し、提供されている。しかし、人間とその進化の接近にもかかわらず、このモデルは動物や短所を感染させるために使用頭蓋内接種法によって制限されていたistently CNSの感染症を誘発する。さらに、倫理的配慮は、マウスサイトメガロウイルス（MCMV）と生まれたばかりのマウスの新生児の感染は、最近大きな進歩につながっている、そのうち、別のモデルの開発を推進してきました。例えば、それをBalb / cの新生児にMCMVを腹腔内注射は、脳の特定の領域におけるニューロンおよびグリア細胞の感染をもたらすことが報告された。これらの知見は、マウスの実験的な接種が子供でHCMV感染によって誘導される赤字を繰り返す可能性が示唆された。それにもかかわらず、新生児のMCMV感染の動的解析は、古典的な手法は、ウイルス負荷および/または免疫関連のパラメータを分析するために異なる時点で動物はかなりの数の犠牲を必要とするため行うことは困難である。このボトルネックを回避するために、希少変異動物の将来の調査を可能にするために、我々はビラの経時的解析を実行する生体内画像化技術に適用C57BL / 6新生児にルシフェラーゼを発現する組換えMCMVの末梢注射時の脳内リットル普及。

代表的な実験は、 図1に示されている。 MCMV-リュックの50 PFUs（パネル針の皮下経路を可視化するためにメチレンブルーで行われた同様の注射を示しています）の腹腔内注射すると、新生児は麻酔し、同時にルシフェラーゼ基質（ルシフェリン、キャリパー）0.3mgのを受けた。 15分後、動物をIVIS 50の取得室（生体イメージングシステムでは、キャリパー）の腹側を上にして配置さ?...

Watch this Scientific Journal Video about Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates at JoVE.com

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

新生児のヒトサイトメガロウイルス（HCMV）感染症は、精神遅滞の重要な原因を表し、まだウイルス誘発病因につながる分子事象はまだよく理解されていない。脳の感染症のダイナミクスを調べるために、我々は全体の動物の適応<em生体内で&gt;</em&gt;イメージングルシフェラーゼ組換えウイルスに感染した新生児の経時的分析を実行する。

活用<em生体内で&gt;</em新生児の実験マウスサイトメガロウイルス感染の進行を監視する&gt;イメージング

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

use-vivo-imaging-to-monitor-progression-experimental-mouse

Research

JoVE Journal

Immunology and Infection

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

14.8K ビュー.  Université de Strasbourg. 新生児のヒトサイトメガロウイルス（HCMV）感染症は、精神遅滞の重要な原因を表し、まだウイルス誘発病因につながる分子事象はまだよく理解されていない。脳の感染症のダイナミクスを調べるために、我々は全体の動物の適応イメージングルシフェラーゼ組換えウイルスに感染した新生児の経時的分析を実行する。

ビデオ: Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

ライブホストのトランスジェニックリーシュマニア原虫のin vivoイメージング

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

免疫・感染学

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白血球ホーミングと移行の非侵襲的イメージング in vivoで</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

リンパ球増殖を監視するためのカルボキシフルオレセインジアセテートスクシンイミジルエステル（CFSE）の使用

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

に対する抗生物質の有効性をテストするための人工痰培地の使用<em&gt;緑膿菌（Pseudomonas aeruginosa）</em&gt;肺嚢胞性線維症へのより適切な条件で

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

神経侵襲性脳炎ウイルスのin vivoイメージングシステム（IVIS）検出で

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

急性及び慢性炎症を区別するために生体内イメージング法における

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

T細胞応答の評価のための蛍光ターゲット配列の使用<em&gt;インビボ</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

リステリア単球遺伝子感染症後のマウス脾臓におけるインシチュインターフェロンガンマ産生のイメージング

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

胃腸管における カンジダ・アルビカンス ・ヒphphal形態形成を研究するエクスビボアッセイ

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

マウスにおけるガードネレラ膣膀胱曝露によって引き起こされる再発性大腸菌尿路感染症

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...