この仕事は健康（NIH）AI091759とクローン病とアメリカのウィリアム·アンド·シェルビーModellファミリー財団研究賞（MNまで）の大腸炎財団の国民の協会によってサポートされていました。住血吸虫感染マウスは、NIAID住血吸虫リソースセンター（NIAID契約N01 A130026）によって供給された。

1。 マンソン住血吸虫卵を浄化する<ol><li> 住血吸虫のライフサイクル8の感染段階である住血吸虫セルカリア、とスイス-Websterマウスに感染します。また、NIAID住血吸虫症リソースセンター（から住血吸虫感染マウスを得る<a href="http://www.schisto-resource.org/" target="_blank">http://www.schisto-resource.org/~~V</a> ）。その時点で、卵は肝臓内に存在し、後述するように（ 図を参照してください。1）を回復することができ、感染後6から7週間のマウスにしておきます。 </li><li> CO 2でマウスを安楽死させると、70％エタノールでぬれた。背中の上にマウスを置きます。皮膚の基礎となる腹膜を切り取るために鈍い鉗子とハサミを使用しています。消費税​​肝臓、胸郭と横隔膜の下にある。 </li><li>細かく肝臓をミンチし、PBSでコラゲナーゼ/ディスパーゼ（0.5 mg / mL）は、ペニシリン（100 U / mL）およびストレプトマイシン（100μg/ mL）を構成される消化液、肝臓あたり20 mLを追加します。 mixtを置く50mLのファルコンチューブにUREと37°Cシェーカーで一晩インキュベートする。 </li><li> 400 XG / 3分/ 20℃で混合物を遠心分離優しく余分な液体を捨て、PBSでチューブを記入してください。再び遠心分離し、PBS洗浄を繰り返します。最後の遠心分離、25mLのPBSでペレットを再懸濁し以下。 </li><li>細かいふるい（目の細かいふるいを通して混合物をプッシュするために10mLのシリンジを使用して混合物を渡すことによって、続いて標準的なキッチンの金属ストレーナー（ガラスビーカーを介してマッシュ肝臓片に50 mLのシリンジプランジャを使用）を介して再懸濁したペレットを痛める第2のガラスビーカー）。 </li><li> 50mLのファルコンチューブに緊張した混合物を入れてください。 400 XG / 5で遠心分/ 20℃ペレットを失うことなく、上清を注意深く注ぐ。 3 mLのPBSでペレットを再懸濁します。 </li><li>オーバーレイは、20％パーコール、8 mLのパーコールおよび32 mLの0.25Mスクロース（FW ​​= 342.3ので、8.56グラムsucrose/100 mLのH 2 0）を40mLの勾配の上に静かに混ぜる。 </li><li> 10分/ 800 XG / 20℃遠心オフピペットdは、肝細胞のゼラチン最上層を破棄します。ピペットの下部にある卵ペレットを除去し、15mlファルコンチューブに入れられます。 </li><li> 10 mLのPBS / 1mMのEDTA / 1 mMのEGTAを使用して遠心分離設定の洗浄ペレット3X：3分/ 30 XG / 20℃ 500μLのPBSに再懸濁します。 </li><li> 10分/ 800 XG / 20℃：新しい25％パーコール遠心機の設定を使用して10 mLの濃度勾配（2.5 mLをパーコールと7.5mLの0.25Mスクロース）上にオーバーレイミックス</li><li>遠心分離の設定を使用して10mLのPBSで3倍の洗浄：3分/ 30 XG / 20℃ </li><li>最後の10 mLの洗浄では、卵をカウントするために100μLを削除します。卵はすぐに解決する、ファルコンチューブはサンプルを削除する前に十分に混合する必要がありますので。 900μLのPBSに100μLのサンプルを希釈する。ミックスの100μLを解剖顕微鏡（10倍希釈）を使用して、顕微鏡スライド上に液滴としてカウントする必要があります。ワイドボアピペットチップを使用する必要があります。 </li><li> PBSで50,000卵​​/ mLで卵を再懸濁します。卵は-80℃で数ヶ月間保存され、一回またはTWIC解凍することができます使用のためのe。ご使用になる前に、卵が（ 図1を参照 ）が無傷であることを確認するために顕微鏡下で検査します。 </li></ol> 2。 S.と腹腔内感作マンソンの卵：0日<ol><li> 5mLのsnapcapチューブに5000μlのPBS eggs/100で卵を準備します。注射のために、必要以上に50％以上の卵を推定する。 </li><li>マウス当たりの卵懸濁液100μLを23ゲージ針3/4inch、1 mLシリンジをロードします。卵は、落ち着くので、使用直前に卵で針をロードします。 </li><li>前に、各注射に卵を混ぜて、マウス当たりの懸濁液を腹腔内に100μLを注入する前後にロックシリンジ。 </li></ol> 3。 S.と眼窩静脈チャレンジマンソンの卵：14日<ol><li> 5000 eggs/100μLで上記のように卵を準備します。 </li><li> （4％）イソフルラン、またはキシラジン（10 mg / kg体重）とケタミン（120 mg / kg）を腹腔内で特殊なチャンバー内でマウスを麻酔。 anestの深さhesiaは、足蹠のピンチとは物理的な応答を確保しないで決定されます。 </li><li>卵を1 mLのシリンジと23ゲージ針3/4inchの読み込みに続いて、角度に下向きに面取り90°の角度で針を曲げるために鉗子を使用しています。気泡が存在しないことを確認してください。 </li><li>一方の側にマウスを置きます。目が突出、鼻に45°の角度で眼球の後ろに容器に100μLを注入しますので、皮膚を引き戻します。 </li><li>針を撤回し、出血を制御するために軽い圧力を適用します。 </li></ol>これらの手順のすべてが慎重に行わなければならない、彼らは麻酔から回復するまでマウスを監視する必要があります。これらのプロトコルは、ペンシルベニア州制度動物ケアの大学によって承認され、委員会（IACUC）を使用していた。 4。実験的な収穫：22日目<ol><li> CO 2を持つマウスを安楽死させる。 </li><li>ぬれた70％エタノールでマウス、および背面の場所。 </li><li>鈍ゲストフォースドバイを使用して、EPSは、マウスの腹部を把握し、皮膚に小さな切開を行います。慎重にはさみで皮膚を除去し、胸郭と腹膜を公開します。 </li><li>腹膜を開き、カットします。パスツールピペットを用いて血液を回復するために腹部大動脈を切断します。氷上で保管してください。 </li><li>ダイアフラムを公開するために肝臓を下に移動します。横隔膜をカットし細かいハサミを使用しています。慎重に肺組織を露出させ胸郭を開いてカット。 </li><li>微細な鉗子で肺をドレイン摘除リンパ節を回復します。これらは胸腺9のいずれかの側に、胸郭の下に隠れています。 1mLの滅菌媒体中で氷上店（DMEM、10％熱不活化ウシ胎児血清を補充し、100 U / mlペニシリン、100μg/ mlのストレプトマイシン、2mMのL-グルタミン、50μM2 - メルカプトエタノール、Invitrogen社からのすべての使用可能）。 </li><li> BALを回復し、組織学的分析のために肺を膨らませるために、気管内挿管を実行する必要があります。側に唾液腺を移動することにより気管を露出し、placi気管の下NG細かい鉗子。細かいハサミを使用して、気管の中央に穴をあけて、肺にチューブを挿入します。手術のスレッドと接続することによってチューブを固定します。 </li><li>チューブにPBSで満たされた1 mLのシリンジを接続し、1 mLのPBSで肺を膨らませる。慎重にシリンジで洗浄BALを取得します。氷上で保管してください。 </li><li>チューブにPBSで満たされた1 mLのシリンジに4％パラホルムアルデヒド（PFA）を添付して肺を膨らませる。 </li><li>チューブを外し、PFAの漏洩を防ぐため手術のスレッドを締めます。 </li><li>慎重に5 mLの4％PFAで50mLのファルコンチューブに肺組織と場所を分析する。 </li></ol> 5。回収した組織の準備<ol><li> 血清：血液凝固のために30分〜2時間氷上で以下のストレージ、血清を遠心分離（13000×gで/ 10分/ 4℃）により血液から回収される。血清は-20℃で保存されており、サイトカインのELISA または Sに使用することができますマンソン卵抗原特異的IgGアイソタイプELISA法<sup&gt; 6、10。 </li><li> リンパ節：単一細胞懸濁液を調製し、細胞数は免疫応答の大きさを評価するために使用されています。 Th2細胞の偏光を調べるために、細胞がSで再刺激されている72時間マンソン卵抗原。 Intracellarサイトカイン染色、フローサイトメトリーにより検討することができ、上清を回収し、-20℃で保存できますTh2サイトカインのELISA（IL-4、IL-5、IL-13）6 C。 </li><li> BAL：細胞は（400 XG / 5分/ 4℃）でペレット化しています。 BAL液はELISA法によるTh2サイ​​トカインの分析のために回収し、-20℃で保存されています。 BAL細胞数は気道炎症の読み出しとして使用されています。 Diffquik染色に続いて10,000-100,000細胞（500回転/ 5分/ 4°C）のCytocentrifuge製剤は、炎症性浸潤物4の列挙、6が可能になります。また、BAL細胞はフローサイトメトリー6で調べることができます。 </li><li> 肺組織：肺組織はSTORです。一晩4℃のED固定組織はパラフィン包埋し、切片と組織学的および免疫蛍光分析のためのスライドにマウントされています。 </li></ol> 6。代表的な結果このプロトコルの詳細は、（i）in vivoでの使用のためのマンソン住血吸虫卵 （ 図1）、（II）増感し、これらの卵を持つマウスに挑戦し、精製、および（iii）肺の炎症反応の検査のために臓器を回復する準備をするために必要なすべての手順を実行します。 in vivoモデルこれは、再現性、強力なTh2の肺の炎症反応を駆動します。これは、BAL細胞数とcytocentrifuge製剤（ 図2）によって可視化した気道炎症と好酸球増加によって示されている。 H＆E染色肺セクションは、卵誘発性肉芽腫（ 図3a）を調べ、肺切片の免疫蛍光染色は、代わりに活性化マクロファージや抵抗などのTh2サイトカイン誘導性遺伝子の可視化を可能にすることができます錫のような分子（RELM）α（ 図3b）。抗原特異的CD4 Th2サイトカイン応答は、IL-5、IL-4および抗原刺激摘除リンパ節細胞（ 図4）のIL-13 ELISAによって検査されます。 <img alt="図1" src="/files/ftp_upload/3905/3905fig1.jpg" /> 図1はマンソン住血吸虫卵の注入モデル代表高S.mansoni卵負担の特性肉芽腫肝臓の画像、およびS.mansoniの卵が示されている- 。平均サイズ（L）（W）は50μmから120μmの。 <img alt="図2" src="/files/ftp_upload/3905/3905fig2.jpg" /> 図2。S.マンソンの卵注入、気道の炎症を駆動します。A.ナイーブ（N）またはS.mansoni（SM）卵注入したマウスからのBAL細胞数（×5）。 * P &lt;0.05。 BAL細胞のBのCytocentrifugeの準備。マック、マクロファージ、EOの好酸球、リンパ液、リンパ球。バーは10μm。 <img alt="図3" src="/files/ftp_upload/3905/3905fig3.jpg" /> 図3肺肉芽腫の可視化とS.mansoni卵を囲む代わりに活性化マクロファージ。A. Smの卵（黒い矢印）を取り囲む肺肉芽腫は、H＆E染色肺切片で可視化した。 RELMαのためにB.免疫染色（緑）、マンノース受容体（赤）およびDAPI（青）の自家蛍光のSm卵（黒い矢印）周囲の肉芽腫で交互に活性化マクロファージ（白矢印）を明らかにする。バーは50μm。 <img alt="図4" src="/files/ftp_upload/3905/3905fig4.jpg" /> 図4。S.mansoni卵抗原特異的Th2サイトカイン応答。卵注入ナイーブ（N） や Smマウスから摘除リンパ節細胞の排水が SMで刺激した&lt;/ IL-4の上清のELISA、IL-5およびIL-13に続く72時間のem&gt;卵抗原、。スケール、ng / mLで。* P &lt;0.05。

<ol>
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利害の衝突が宣言されません。

ここでは、2型肺の炎症を誘導するマンソン住血吸虫卵を採用したモデルが記載されています。このモデルの特徴は、強力なTh2免疫応答は、気道の炎症や肺の肉芽腫形成が含まれています。これらのパラメータはCD4 Th2細胞応答を11に依存しているので、これはTh2細胞応答に関するタンパク質固有または系統特異的欠失の影響を調べるために有用なモデルである。ここでは読み出?...

<table border="1"><tbody><tr><td> 試薬 </td><td> サイズ/説明 </td><td> 会社 </td><td> カタログ＃ </td></tr><tr><td>コラゲナーゼ/ディスパーゼ</td><td> 500 mgの</td><td>ロシュ·ダイアグノスティックス</td><td> 11 097 113 001 </td></tr><tr><td>差分Quikのヘマ3染色</td><td>パック</td><td>フィッシャー·サイエンティフィック（株）LLC </td><td> 122から911 </td></tr><tr><td> DMEM </td><td>液体</td><td>ギブコ</td><td> 11965 </td></tr><tr><td>ファルコン50mLを</td><td>パック</td><td>ファルコン</td><td> 352070 </td></tr><tr><td> Intramedic PEチューブ</td><td> 0.58ミリメートル</td><td>ベクトン·ディッキンソン·アンド·カンパニー</td><td> 427410 </td></tr><tr><td>イソフルレン</td><td> 250 mLの</td><td>アボットアニマルヘルス</td><td> 52600405 </td></tr><tr><td>ケタミン</td><td> 100 mg / mLの</td><td>フォートドッジ、アニマルヘルス</TD&gt; <td> N / A </td></tr><tr><td>マンノース受容体抗体</td><td>ビオチン</td><td> ABD Serotec </td><td> MCA2235B </td></tr><tr><td>金属製のストレーナー</td><td>標準のキッチンストレーナー</td><td>ターゲット</td><td>ラウレンティス</td></tr><tr><td>パラホルムアルデヒド</td><td> 16パーセント</td><td>電子Microsc。科学</td><td> 15710 </td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td> 100倍</td><td>ギブコ</td><td> 15070 </td></tr><tr><td>パーコール</td><td> 1リットル</td><td> GEヘルスケアバイオサイエンス社</td><td> 17 0891 01 </td></tr><tr><td> RELMα抗体</td><td> 50μgの</td><td> Peprotech社製</td><td> 500-P214 </td></tr><tr><td>手術のスレッド</td><td> 2.0 USP 100ヤード</td><td>手術の専門株式会社</td><td> SP118 </td></tr><tr><td>注射針</td><td> 23グラム、3/4インチ</td><td> BDバキュテイナラボ。 MED。 </td><td>305143 </td></tr><tr><td>米国標準試験ふるい</td><td> 150μm以下、0.0059 &quot; </td><td> WSタイラー社</td><td> ASTME-11仕様。 100号</td></tr><tr><td>キシラジン</td><td> 20 mg / mLの</td><td>バトラー</td><td> N / A </td></tr></tbody></table>

using eggs from schistosoma mansoni as an in vivo model of helminth-induced lung inflammation

住血吸虫の寄生虫は1世界中で推定200万人に感染する血液吸虫です。 住血吸虫 、肝線維症と脾腫を含む重篤な病態、慢性感染症ではなく、寄生虫自体は2よりも寄生虫の卵に対する免疫応答によって引き起こされます。寄生虫の卵IL-4の産生によって特徴づけTh2応答を誘導し、IL-5およびIL-13、マクロファージおよび好酸球の動員の代替活性化。ここでは、寄生虫に固有の肺におけるTh2サイトカイン応答および所属リンパ節、卵を周囲の肺肉芽腫の形成、気道の炎症を調べるためのモデルとしてマンソン住血吸虫卵の注入を説明します。 腹腔内感作および静脈内チャレンジ、S.以下マンソンの卵は、それらが肺実質内に閉じ込められている肺動脈を経由して肺に運ばれる肉芽腫でリンパ球、好酸球と交互に活性化マクロファージ3-6の構成。気管支肺胞スペースで肉芽腫形成、炎症に関連付けられている、所属リンパ節およびCD4の拡大は、T細胞の活性化を観察することができる。ここでは詳細は、感染した肝臓（7から変更された）から、 マンソン住血吸虫卵を分離したマウスを感作し、挑戦し、分析のための臓器（気管支肺胞洗浄（BAL）、肺と所属リンパ節）を回復するためのプロトコルです。我々はまた、追加の免疫学的分析のための代表的な組織学的および免疫学的データとの提案が含まれています。 全体的に、このメソッドは、in vivoモデル寄生虫感染症、線維症、アレルギー性炎症や喘息を含むTh2の炎症性疾患の研究に広く適用可能である肺に寄生虫によって誘発される免疫応答を調査するために用意されています。標準の研究のためのこのモデルの利点肺のE 2炎症が肺の中の強力なTh2の炎症反応の再現性と所属リンパ節、卵を周囲の肉芽腫の組織学的検査によって炎症の評価の容易さ、および寄生虫の長期貯蔵の可能性を含む卵。

Watch this Scientific Journal Video about Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation at JoVE.com

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

マンソン住血吸虫の卵は、Tヘルパー2型（Th2型）免疫応答、寄生虫感染症、喘息とアレルギー性炎症の特性の強力な刺激剤である。このプロトコルはS.を利用卵の周りの肺肉芽腫形成、好酸球とマクロファージの代替活性化により特徴付けられる肺のCD4 Th2サイトカインによって誘導される炎症反応を生成するために、 マンソンの卵注入。

から卵を使用して、<em&gt;マンソン住血吸虫</em&gt;として<emインビボ&gt;</em蠕虫によって誘発される肺の炎症の&gt;モデル

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

using-eggs-from-schistosoma-mansoni-as-an-vivo-model-helminth-induced

Research

JoVE Journal

Immunology and Infection

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

13.7K ビュー.  University of Pennsylvania. マンソン住血吸虫の卵は、Tヘルパー2型（Th2型）免疫応答、寄生虫感染症、喘息とアレルギー性炎症の特性の強力な刺激剤である。このプロトコルはS.を利用卵の周りの肺肉芽腫形成、好酸球とマクロファージの代替活性化により特徴付けられる肺のCD4 Th2サイトカインによって誘導される炎症反応を生成するために、 マンソンの卵注?...

ビデオ: Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

セルカリア変換と in vitroで</emの>栽培マンソン住血吸虫 Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

免疫・感染学

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植片対宿主病の誘導および<emインビボで&gt;</emMHCマッチしたマウスモデルを使用する&gt; T細胞のモニタリング

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

アン<em&gt;エクスビボ</em急性脳スライスにおけるオリゴデンドロサイト指向T細胞攻撃の&gt;モデル

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

鶏卵卵におけるインフルエンザウイルスの伝播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

マウスの脾臓から不変ナチュラルキラーT細胞を単離および拡張するための最適化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

共焦点レーザー Endomicroscopy Ex Vivoひと肺組織の新規細菌固有の光学検査プローブします。

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

細胞毒性の評価と肺癌精密カット スライスで物質の免疫調節効果

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

分離と大人住血吸虫から細胞外小胞のキャラクタリゼーション

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...