AR、XH、PEはNIH助成金R01HL140398とギルソンロンゲボー財団助成金によって支援されました。DEMMとKKは、NIH助成金R01HL136333とR01HL134880(KYK)、およびヘリス医学研究財団からの助成金によって支援されました。DEMMはまた、ハワードヒューズ医学研究所(HHMI)のギリアム・フェローシップによって支援されています。PEは、精密環境衛生学研修による支援も受けています NIEHS T32 ES027801 フェローシッププログラム。JCとMFは、エピジェネティクス・分子発がん学科のタバコ研究基金とMDアンダーソンのエピジェネティクスセンター(MF賞)によって支援されています。FKおよびYZは、NIH助成金R01 ES029442-01およびR01 AI135803-01ならびにVAメリット助成金CX000104によってサポートされています。このプロジェクトは、ベイラー医科大学のサイトメトリーとセルソーティングコアによって、CPRITコア施設支援賞(CPRIT-RP180672)、NIH(CA125123およびRR024574)、ジョエル・M・セダーストロームの支援を受けて支援されました。

実験とこの技術の開発に関与するすべての動物は、機関動物ケアと使用委員会(IACUC)によって承認された私たちの動物使用プロトコルの下にあり、ベイラー医科大学とMDアンダーソン機関の下で、検査動物ケアの評価と認定協会(AAALAC)によって認定されています。
1. 装置の構築
<ol><li>バルブシステムでエアコンプレッサを組み立てます。
	<ol>
<li>1/8インチのネジ式男性アダプタニップルフィッティングを使用して、流量計(Yバー付き2つの15 L /分と2つのパワーテイクオフ)をミニチュア圧力レギュレータに接続します。スレッド・シール・テープを、すべてのスレッド・エンドで使用してください。</li>
		<li>コンプレッサエアの1/8インチの1/8インチの16進数のニップル、1/8インチのネジ式カップリングフィッティング、圧力レギュレータに接続する1/8インチのスレッド式オスアダプタニップルフィッティングを使用して、組み立てられた圧力レギュレータをフローメーターと医療用エアコンプレッサー機器に接続します。</li>
		<li>酸素スイベル有刺鉄コネクタを各(4)流量計に取り付けます。</li>
		<li>エアコンプレッサの上部エアアウトレット(医療用エアコンプレッサー機器に付属の部品)に男性用アダプターを取り付けます。</li>
	</ol></li>
	<li>露出チャンバの組み立て(4ユニットを作る)
	<ol>
<li>3/4インチ塩素化ポリ塩化ビニル(CPVC)パイプを8つの4インチセグメントに切ります。</li>
		<li>1つの3/4インチ90°エルボCPVC継手に各セグメントを挿入し、3/4インチの直径CPVCオスアダプターに肘のフィッティング側を取り付けます。8 つの CPVC セグメントがあり、それぞれに 1 つの CPVC エルボ フィッティングと 1 つの CPVC 男性アダプターが取り付けられている必要があります。</li>
		<li>8.5 L気密容器(11.25 x 7.75 x 6インチ)の反対側に2つの穴(1 1/4インチの直径)を蓋付きにドリルします( 図1 露光室を参照)。穴の位置は、上から下、左から右に中央に配置されます。</li>
		<li>前に組み立てられたCPVCオスアダプタのネジ面をコンテナの各穴に挿入します。</li>
		<li>容器の内側から、反対側に3/4インチのCPVCキャップ(チェンバースモーク入力)と3/4インチCPVC点滴灌漑女性アダプタを片側に取り付けます(チェンバースモーク出力)。</li>
		<li>チャンバーの煙出力のCPVCキャップの上部に5つの3mmの穴をクインキュックス(シャワーヘッド)パターンでドリルします。これにより、タバコの煙がより速くチャンバーに入り、チャンバー内を全方向に均等に広がるようにします。</li>
	</ol></li>
	<li>タバコ室の組み立て(最大4つのマウス暴露ユニットを構成)
	<ol>
<li>1ホールのゴム栓(メーカーサイズ8.5)を取り、広い側に1/4インチの有刺鉄線Yコネクタを挿入し、狭い側にストレート有刺鉄線のフィッティング(8mm開口部)を挿入します。喫煙手続き(タバコ台座)の間に、タバコはここに置かれます。</li>
		<li>長さ12インチの医療グレードのビニールパイプの一方の端を、ゴム栓に取り付けられたYコネクタの有刺鉄線コネクタの1つに接続し、もう一方の端を1/4インチのフィッティングに接続し、このフィッティングの反対側を1穴ゴムストッパー(メーカーサイズ1)に挿入します。</li>
		<li>別のゴム製ストッパー(メーカーサイズ8.5)に、ストッパーの広い側に1/4インチのストレートチューブコネクタを1つ挿入し、フィッティングの外側の端を7フィートの医療グレードのビニールパイプに接続します。</li>
		<li>前に組み立てた2つのゴム製ストッパー構造を、実験室用ガラス製のドレインチューブから8インチx 1.75インチのガラスシリンダーに接続します。</li>
	</ol></li>
	<li>バルブ制御システム
	<ol>
<li>システムはタバコの煙およびきれいな空気の吸入(膨らむ)を模倣するソレノイド弁のリズミカルな開閉によって制御される。ソレノイドバルブを制御するシステムは、商業的に設計されました ( 材料表を参照)。</li>
	</ol></li>
	<li>すべてのコンポーネントをまとめて組み立てる( 図1を参照)
	<ol>
<li>1インチファスナーを使用して、バルブ制御システムの側面に4つのソレノイドバルブを取り付けます。</li>
		<li>ソレノイドバルブを、メーカーの指示に従ってバルブ制御システムに接続します。</li>
		<li>ソレノイドバルブの排気(EXH)接続に10-32(M)ネジストレートコネクタを接続し、同じソレノイドバルブの「IN」接続と「OUT」接続にネジポートアダプタを接続します。</li>
		<li>7フィートの医療グレードのビニールチューブを使用して、「OUT」接続を介してソレノイドバルブにコンプレッサーに取り付けられた流量計を接続します。</li>
		<li>ステップ1.3.3でゴム栓で組み立てられた7フィートのビニール管を接続します。ソレノイドバルブの「IN」コネクタに接続します。</li>
		<li>チャンバーの煙の入力にタバコの部屋の小さいゴム栓を挿入します。</li>
		<li>手順1.3.1で組み立てられたタバコ台の有刺鉄線Yコネクタの2番目の接続にソレノイドバルブを接続します。</li>
	</ol></li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61793/61793fig01.jpg"> 図1:CSへの暴露のためのWBISの接続の概略図。この図は、すべてのコンポーネントがどのように組み立てられ、作業装置を形成するかを示しています。図は機械が作動することができる4つの1つの組み立てられた喫煙室だけを示している。 <a href="https://www.jove.com/files/ftp_upload/61793/61793fig01large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>
2. タバコの煙の暴露
注意:タバコの煙への第二および第三の手の暴露を避けてください。タバコおよび露出室はクラスIIタイプB2の層流生物安全キャビネットの中で使用されるべきである。適切なPPEは、煙露実験(マスク、手袋、ヘアネット、ガウン)を実施しながら着用する必要があります。
<ol><li>
圧力と気流の設定
	<ol>
<li>すべてのコンポーネントが 図 1のように組み立てられると、空気圧縮機の電源を入れ、安全アラームが単独でオフになるまで待ちます。</li>
		<li>圧力調整器のノブを回して、空気圧縮機の圧力を40~50 psiに調整します。</li>
		<li>空気圧縮機からの気流を、流量計を使用して5 Lに調整します。</li>
		<li>バルブコントローラの電源を入れます。</li>
		<li>タイマーの最初の桁で UP キーを押しながら SET/LOCK キーを押して、バルブ コントローラのデジタル タイマーを PULSE-C (画面に「Pu-c」と表示)動作モードに調整します。次に、PU-c モードに達するまで UP キーを押します。RESET キーを押して、表示される動作モード(つまり Pu-C)を作業モードとして設定します。</li>
		<li>SET/LOCK を押してタイマー 1 を変更します(画面に「T1」と表示されます)。</li>
		<li>上または下のキーを押して T1 を 20 s に設定します。</li>
		<li>SET/LOCK を押してタイマー 2 を変更します(画面に「T2」と表示されます)。</li>
		<li>上または下のキーを押して T2 を 3 s に設定します。 注: ステップ 2.1.5 ~ 2.1.9 は、特定のタイマーで使用するように調整されています ( 資料一覧を参照)。この製品の他の用途の詳細については、対応するユーザーマニュアルを参照してください。</li>
	</ol>
</li>
	<li>
タバコの煙の治療 注:このシステムは1-4マウスの露出の部屋の同時使用を可能にする。<ol>
<li>エアコンプレッサの電源を入れ、安全アラームが自らオフになるまで待ちます。</li>
		<li>バルブコントローラの電源を入れます。</li>
		<li>8.5 Lの容積の気密な取り外し可能なふたを備えた4つの露出室のそれぞれに5匹のマウスを移す。</li>
		<li>ラミナーフロー生物学的安全キャビネットの中で、タバコを点灯し、タバコ室の中にタバコを挿入します。ケンタッキー州3RF4研究用タバコ(9.5 mg/cigタールおよび0.73 mg/cigニコチン)9と比較して、15mg/cigタールおよび1.1 mg/cigニコチン8を含む市販のタバコを使用してください。</li>
		<li>現在使用中のチャンバーに対応するバルブコントローラのバルブをオンにします。露出は2段階に分けられる:(T1)きれいな空気が20 sのために暴露室にポンプで送られ、(T2)空気の流れはタバコの部屋からの燃焼および煙を3sの露出室にポンプで送り出す。タバコがフィルターに達するまで完全に燃え尽きるようにします。<ol>
<li>タイマー設定を調整して、約 4 分の期間で平均 10 個のパフ/タバコを実行します。タイマーとシステムは、研究者の研究ニーズに応じてCS投与レジメンを強化または下げるために簡単にカスタマイズ可能であることに注意してください。</li>
		</ol>
</li>
		<li>タバコフィルターを取り外し、火炎を消火し、臭いを弱めるために水でガラスビーカーにタバコの吸殻を入れることによってそれを処分します。</li>
		<li>タバコ室がタバコなしで再び閉じられていることを確認してください。機械は10分間きれいな空気をポンプでくむ。CSに曝露される脊椎動物の常時モニタリングを維持することが最も重要である。この暴露レジメンは、曝露チャンバあたり9週間以上生後5匹の雌マウスに最適化されている。</li>
		<li>1室あたり1日に合計4本のタバコについて、ステップ2.3.4~2.3.7を3回繰り返します。この手順は、研究者が実験に必要な限り、週5日繰り返されます。</li>
		<li>マウスを露出チャンバーから取り外し、対応するケージに戻します。</li>
		<li>バルブコントローラとエアコンプレッサーの電源を切ります。</li>
		<li>露出とタバコの部屋を取り除き、タールの残留物を取り除くために水と石鹸で洗います。</li>
		<li>チャンバーを完全に乾燥させてから再び使用してください。</li>
	</ol>
</li>
</ol>

<ol>
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著者らは開示するものは何もない。

ここでは、マウスのWBIS用装置の構築に必要な情報をCSに提供する。システムの設置後、研究者が動物のニコチンまたはコチニンの送達用量に基づいてシステムを較正することが非常に重要である。装置は、タバコのパフのボリューム、パフ周波数、組み合わせた煙の暴露期間、および動物が各タバコの間に受け取る休息間隔を調整するために使用できるタイマーと圧力計が含まれています。?...

タバコの喫煙は、過去50〜60年の間にタバコを吸う成人の数が着実に減少しているにもかかわらず、米国の予防可能な疾患の主な原因の1つです。喫煙は、慢性閉塞性肺疾患(COPD)を含む肺および血液の複数の疾患に関連していることが広く知られている、肺気腫および慢性気管支炎2、3、4を含む疾患のグループ。米国疾病管理センター(CDC)によると、2014年、COPDは米国で3番目に主要な死因であり、1,500万人以上のアメリカ人がこの病気に苦しんでいました5。
CSはまた最近、クローン造血(CH)6、7、単一の造血幹細胞が不釣り合いに人の末梢血の大部分を産生する状態を発症するリスクが高いと関連している。この知見は、喫煙と骨髄機能との間の潜在的な関係を示す。CSの広範かつ非常に重要な健康への影響を考えると、病気のマウスモデルが生物医学研究の進歩の基礎であることを考えると、マウスのCSをモデル化するための効率的で手頃な価格のシステムを開発することは有用である。
ここでは、肺気腫や骨髄恒常症に対するCSの生体内効果を治療し、研究するための手頃なシステムを構築するためのステップバイステップガイドを提供します。この装置のアセンブリは、ユーザーが専門的な知識を持っている必要はありませんので、DIYアセンブリを可能にします。

<table>
	<tbody>
		<tr>
			<td>1 in fastener</td>
			<td>Lowes</td>
			<td>756990</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 in Barbed Y connector</td>
			<td>VWR</td>
			<td>89093-282</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 in straight tubing connector</td>
			<td>VWR</td>
			<td>62866-378</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 hex nipple</td>
			<td>Lowes</td>
			<td>877221</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 in threaded coupling fitting</td>
			<td>Lowes</td>
			<td>877208</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 in threaded male adapter nipple fitting</td>
			<td>Lowes</td>
			<td>877243</td>
			<td></td>
		</tr>
		<tr>
			<td>10/32 (M) threaded straight connector</td>
			<td>Bimba</td>
			<td>EB60</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in 90-degree elbow CPVC fitting</td>
			<td>Lowes</td>
			<td>22643</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in chlorinated polyvinyl chloride (CPVC) pipe</td>
			<td>Lowes</td>
			<td>23814</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in CPVC cap</td>
			<td>Lowes</td>
			<td>23773</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in CPVC Drip irrigation female adapter</td>
			<td>Lowes</td>
			<td>194629</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in diameter CPVC male adapter</td>
			<td>Lowes</td>
			<td>23766</td>
			<td></td>
		</tr>
		<tr>
			<td>8.5 L airtight container with lid (11.25in x 7.75in x 6 in)</td>
			<td>Komax</td>
			<td>N/A</td>
			<td>Listed as &#34;Komax Biokips Large Bread Box | (280-oz) Large Storage Container&#34;</td>
		</tr>
		<tr>
			<td>Glass drain tube (1.75 in diameter x 8 in length)</td>
			<td>KIMAX</td>
			<td>6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isonic Solenoid Valves</td>
			<td>Bimba</td>
			<td>V2A02-AW1</td>
			<td></td>
		</tr>
		<tr>
			<td>Marlboro Red 100&#39;s</td>
			<td>Marlboro</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen swivel barbed connector</td>
			<td>Global Medical Solutions</td>
			<td>RES002</td>
			<td></td>
		</tr>
		<tr>
			<td>Panasonic Timer LT4H-W</td>
			<td>Panasonic</td>
			<td>LT4HW</td>
			<td>Item was built-in the valve controller by Shepherd Controls &#38; Associates</td>
		</tr>
		<tr>
			<td>Pressure regulator</td>
			<td>Allied Electronics and Automation</td>
			<td>70600552</td>
			<td>Also listed as &#34;Norgren R07-100-RGKA&#34;</td>
		</tr>
		<tr>
			<td>Rubber stopper # 1 (one hole)</td>
			<td>VWR</td>
			<td>59581-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber stopper # 8.5 (one hole)</td>
			<td>VWR</td>
			<td>59581-389</td>
			<td></td>
		</tr>
		<tr>
			<td>Scireq inExpose system</td>
			<td>Scireq and Emka Technologies</td>
			<td>N/A</td>
			<td>Commercial system used for comparison with our DIY WBIS</td>
		</tr>
		<tr>
			<td>Straight barbed fitting (8mm opening)</td>
			<td>VWR</td>
			<td>10028-872</td>
			<td></td>
		</tr>
		<tr>
			<td>Thread Sealant tape</td>
			<td>Lowes</td>
			<td>1184243</td>
			<td></td>
		</tr>
		<tr>
			<td>Threaded port adaptor</td>
			<td>Bimba</td>
			<td>P1SA1</td>
			<td></td>
		</tr>
		<tr>
			<td>Timeter Aridyne 2000 Medical Air Compressor</td>
			<td>MFI Medical</td>
			<td>AHC-TE20</td>
			<td></td>
		</tr>
		<tr>
			<td>Timeter flowmeter</td>
			<td>Allied Healthcare Products</td>
			<td>15006-03YP2</td>
			<td>Also listed as &#34;Puritan Air Meter&#34;</td>
		</tr>
		<tr>
			<td>Valve Control system</td>
			<td>Shepherd Controls and Associates</td>
			<td>N/A</td>
			<td>Company custom designed the valve control system for this model.</td>
		</tr>
		<tr>
			<td>Vinyl pipes</td>
			<td>Vitality Medical</td>
			<td>RES3007</td>
			<td></td>
		</tr>
	</tbody>
</table>

cigarette smoke exposure in mice using a whole-body inhalation system

米国の成人の14%近くが2018年にタバコを吸ったと報告されています。タバコの煙(CS)が肺や心血管疾患に及ぼす影響は広く研究されているが、血液や骨髄などの他の組織や器官におけるCSの影響は未だ完全に定義されていない。げっ歯類のCSの効果を研究するための適切なシステムを見つけることは非常に高価であり、市販のシステムの購入を必要とします。このように、我々は、マウスにおけるCSの病理学的影響を研究するための手頃な価格で信頼性の高い、汎用性の高いシステムを構築することに着手しました。この全身吸入暴露システム(WBIS)のセットアップは、CSときれいな空気への露出を交互にすることによってタバコの呼吸とふくらみを模倣する。ここでは、この日曜大工(DIY)システムが4ヶ月間のタバコの煙暴露の後にマウスの気道炎症および肺気腫を誘発することを示す。この装置を用いた骨髄における造血幹細胞および前駆細胞(HSPCs)に対するCSの全身吸入(WBI)の影響も示されている。

CS曝露の主な特徴の1つは肺の空気嚢(肺胞)の損傷および破壊によって特徴付けられる肺気腫である。したがって、最初の実験は、CSへの全身暴露を繰り返した際に、雌マウスの肺の肺の肺の変化を引き起こすDIYシステムの能力に焦点を当てた。CS投与レジメンは、CSを用いたマウスを治療し、肺気腫10、11、12、13、14、15、16の分子病態生理を研究するために...

Watch this Scientific Journal Video about Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System at JoVE.com

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

このプロトコルは、体内に構築された全身吸入(WBI)暴露システム(WBI)を用いたタバコの煙(CS)の病態生理学的影響の研究を示す。このシステムは、肺気腫および造血液に対するCS媒介効果の研究のために、制御された反復可能な条件下で動物をCSにさらす可能性がある。

全身吸入システムを用いたマウスのタバコの煙暴露

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

cigarette-smoke-exposure-in-mice-using-a-whole-body-inhalation-system

Research

JoVE Journal

Immunology and Infection

6.1K ビュー.  Baylor College of Medicine. このプロトコルは、体内に構築された全身吸入(WBI)暴露システム(WBI)を用いたタバコの煙(CS)の病態生理学的影響の研究を示す。このシステムは、肺気腫および造血液に対するCS媒介効果の研究のために、制御された反復可能な条件下で動物をCSにさらす可能性がある。

ビデオ: Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

の間に相乗効果を調査するために生物発光イメージングを使用して肺炎球菌幼児マウスで>とインフルエンザウイルス

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

免疫・感染学

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

のための負の選択マーカーの開発赤痢アメーバ</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3の人口ベースのシーケンシングを用いたHIV - 1トロピズムの遺伝子型推論

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

マウスでイメージ細菌感染にルシフェラーゼを用いた

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

新生児と大人の中枢神経系から培養ミクログリア

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

レポーターマウスを用いたIL-22を発現するリンパ球の可視化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

麻痺、視神経脊髄炎自己抗原アクアポリン 4 の T 細胞特定のマウスの視覚系障害の誘導

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

膣内HIV曝露とHIV感染治療に対するヒト化NOGマウス

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

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 ...

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 ...