私たちは、カースティン·ティム（アストラゼネカ、ルンド、スウェーデン）、ベニータダールバーグ博士アンダースエクランド（カロリンスカ研究所、ストックホルム、スウェーデン）だけでなく、熟練した援助のためのドクターマーチンシュテンプフリ（マクマスター大学、ハミルトン、ON、Canada）を感謝したいと思いますと専門家の助言。

動物研究は、北ストックホルム動物福祉倫理委員会によって承認された。実験手順は、スウェーデンの法律に準拠して行った。 1. LPSエアロゾルの生成<ol><li> P.精製を0.5g溶解穏やかに撹拌しながら50ミリリットル滅菌生理食塩水中の緑膿菌 LPS及び溶解を確認します。 1mg / mlの最終濃度に9 mlの滅菌生理食塩水にLPSを溶解した1 mlで希釈する。 -20℃でアルミ箔とストアとの光から保護します。 </li><li>室温で暗所に可溶化されたLPSを解凍し、使用直前によく混ぜる。 </li><li>換気されたレベルIIバイオハザードフード中で、噴霧器に赤の入口を挿入し、メーカーが提供するチューブ（ 図1の実験装置を提示するレビュースキーム）を介して加圧された室内空気に噴霧器を接続してください。 注意：適切な個人保護具を、半分の顔ピース再利用可能なrespirat含むまたは粒子フィルタ、ゴーグル、手袋、防護服との暴露の過程で使用されるべきである。 </li></ol><img alt="図1" src="/files/ftp_upload/51470/51470fig1highres.jpg" /> 図1：エアロゾルを生成するために用いた実験装置の概略プレゼンテーション噴霧器の入口は、空気供給源に接続されている。噴霧器の出口は、第15.9ミリメートル管及びエアフィルタ、及び空気供給を介して流量計に接続された2キロバールの圧力で5.0 L /メートルに調整する。出口は、圧力上昇を防止するために取り外し可能な蓋と、5mmの穴を取り付けプレキシガラス箱に接続次ある。 <ol start="4"><li>エアフィルタを介して質量流量計に噴霧器の出口に接続します。電源に質量流量計を接続します。 </li><li> 1.0-2.0バーで残りの圧力で、5 L / minに空気の供給を調整します。 </li><li>質量流量計を取り外し、空気供給を切断します。 </li><li> 150×163 X 205ミリメートル、取り外し可能な蓋を装備：分岐すると寸法を持つ2つのプレキシガラスのボックスに接続する15.9ミリメートルのチューブに噴霧器の出口に接続します。各ボックスは、圧力上昇を防止するために、入口に対向する側に5mmの穴を有するべきである。 </li><li>各プレキシグラスボックス内の5匹のマウスにまで入れて、蓋を閉じます。 </li><li>噴霧器を開き、滅菌生理食塩水または単独の車両に溶解し、少なくとも4ミリリットルのLPSでの挿入を埋める（滅菌生理食塩水;ボリュームは、8ミリリットルを超えないようにしてください）​​。空気供給への入口を再接続します。 </li><li>エアゾールを10分間閉鎖さプレキシグラスの箱に流入することを許可する。継続的に動物を監視します。空気供給が噴霧器の入口にしっかりと保護されたままにしてください。 </li><li>空気供給を外します。蓋を閉じた状態で、動物は2分間プレキシグラスの箱に残ってみましょう。 </li><li>蓋を開き、エアロゾルが分散することを可能にし、tに動物を返す彼のケージ。動物は湿っ表示された場合は、低体温症を防ぐために、弱火に設定された加熱パッドの上にケージを置く。 </li><li>最初の30分間連続して動物を監視し、その後、最初の6時間ごとに2時間。動物は、噴霧手順の間および後に正常呼吸パターンおよび活性を示すべきである。 </li></ol> 2.気管支肺胞洗浄（BAL） <ol><li>実験の終了時点で、深くあなたの機関の獣医スタッフによって推奨されているように達成するためにイソフルランで動物を麻酔。大手術を行うために、麻酔の十分な深さを確保するために引っ込め反射を確認するために後肢をピンチ。 70％エタノールを用いた動物の毛皮ダウンスプレー。 </li><li>はさみを使用して腹部を開き、動物を放血する大動脈を切断。血液を吸収するために腹部の上に組織片を置きます。 </li><li>安楽死をFollwingにハサミのシングル前後カットを使用胸部を公開。胸骨の前方先端で胸郭を持ち上げ、すべての肺葉に切断することなく、ほとんどの腹側時点でダイヤフラムを穿刺するはさみを使用しています。前後方向（顎下の会合）で2カットを行うことで胸郭を開きます。 </li><li>静かに鉗子を用いて胸郭を引き離す、および喉頭下に気管をカット。 </li><li>鉗子で気管を持ち上げ、胸腔へのローブを接続する靭帯を切断し、そして優しく脂肪と心臓組織によって引っ張って肺を取り除く。 </li><li>気管に及び絹糸の文字列を使用してチューブを固定し;：ポリエチレンチューブ（0.965ミリメートル、外径0.58ミリメートル内径）を挿入します。 </li><li>絹糸で（右肺の4のローブ）マルチローブをオフに接続します。 </li><li>ポリエチレンチューブに23ゲージの針を挿入し、ゆっくりと1ミリリットルの注射器を持つ単一の葉に氷冷滅菌PBS250μlのを注入する。 </li><li>慎重に肺をタップする30回と注射器を介して液体を集める。 200μlのPBS（約300μlのPBSを450μlの総注入量から単一の葉から回収する必要があります）を使用して手順を繰り返します。 </li><li>氷上で気管支肺胞洗浄液（BALF）を保持、またはすぐにBAL細胞を列挙。細胞11を染色するトルコ溶液を使用して、heamocytometerで細胞を数える。染色溶液の希釈係数とheamocytometerカウントフィールド内の体積で細胞数を乗じることによって総細胞数を計算する。 </li><li>他の場所で11説明したように細胞遠心細胞から差動細胞数を準備します。 </li></ol>組織学的評価のための肺組織の3ホルマリン固定<ol><li>マルチローブを削除し、スナップ凍結をドライアイス上。 -80℃で保管してください。 </li><li>金属支持スタンドに取り除かジャーで60ミリリットル注射器を取り付けます。 10％ホルマリンで注射器を充填一定圧力の20センチメートルを表す実験台上20cmの高さは、。 </li><li>ホルマリンの流れを制御するバルブでプラスチックチューブを経由して60ミリリットル注射器に気管に固定さ23ゲージの針を接続します。 </li><li> 5分間ホルマリンで肺葉を吹き込み。針を外し、気管を閉じて、肺の中の圧力を保持するために絹糸に引きながら気管からポリエチレンチューブと一緒にそれを削除します。 </li><li>ホルマリンで肺葉水没し、4℃で24時間固定します。 </li><li> 70％エタノールで少なくとも20分間固定した組織を3回洗浄し、そして以下のレジメン（1時間毎）を介してキシレンに脱水する。 <ul><li> 3倍の70％エタノール</li><li> 3倍の95％エタノール</li><li> 3倍の100％エタノール</li><li> 3Xキシレン</li><li> 1×（液体）パラフィン</li></ul></li><li>埋め込み脱水4-5ミクロン切片に切断し、パラフィン組織、および組織学的評価を可能にするためにヘマトキシリン​​およびエオシンで染色する。</li>
</ol>

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アブラハムルースはベーリンガーインゲルハイム、アストラゼネカのR＆DとアストラゼネカのR＆Dからの研究助成金からの移動補助金から講師料を受け取った。マグナスノールは、アストラゼネカのR＆Dのフルタイムの従業員で、アストラゼネカ、R＆Dからの研究助成金を受けている。ヨハン·グリューネとトーベベルクは、アストラゼネカのR＆Dが出資関係のない研究プロジェクトの共同研究者である。

エアロゾル化LPSは、上皮粘膜下組織中の好中球、誘導気道のほか、肺胞腔を囲むスペースで特徴付けられる気道における炎症反応を、生成されます。これは、一緒に血漿漏出、急性肺障害の病理学の代表を示すBAL​​Fにおける増加し、総タンパク質含有量、である。 LPSは、無菌性炎症を誘導するように、反応は、適応免疫応答とは無関係であり、細菌感染との関連性には限界がある。技術?...

リポ多糖（LPS）は、グラム陰性菌1の細胞壁成分である。 LPSへの挑戦は、急性肺障害、急性好中球性炎症と浮腫2を特徴とする症候群のよく文書モデルです。また、肺好中球増加はまた、COPD増悪4をモデル化するために使用されている慢性閉塞性肺疾患（COPD）3、およびヒトにおけるLPSチャレンジの特徴である。このように、LPS曝露の実験モデルは、臨床的にヒト病理を理解するための関連、貴重なツールです。 ここで説明するエアロゾル化LPSの肺送達の目的は、全身性の関与なしに、導電性、呼吸気道における好中球の炎症性応答を生成することである。 LPSチャレンジのいくつかの技術が、以前に記載されている。 LPSの静脈内注射は、投与の最も一般的に使用される経路である。この技術は、tは容易にアクセス可能であるが彼の主な損傷は、肺への好中球の移行以下の肺上皮の二次破壊に、内皮にある。静脈内投与は、動物モデルにおいて臨床像を複雑にする全身性炎症2を誘導する。全身性炎症は、気管​​内投与では観察されないとは対照的である。しかしながら、この技術は、労働集約的であり、麻酔剤、ならびにかなりの訓練5,6を必要とする。さらに、この投与経路による肺沈着は、呼吸7に依存している。したがって、肺沈着は、気道内、気管内投与、変数析出が観察され得るために必要な麻酔の深さに影響される。対照的に、エアロゾル化したLPSによる肺送達は、最小限の訓練を必要とし、容易に個人5,8の間にほとんど又は全く変動が多数の動物で行うことができる。最近の研究では、エアロゾル送達が堆積に関して気管内経路よりも優れていること、及びLPSのより適切な用量は、このモデル8と好中球性炎症を誘導することを確認する。 これまでの研究では、エアロゾル化した緑膿菌 LPSへの挑戦は、肺胞腔9、10を含む気道内腔および肺実質の著しい炎症反応を生成し、実証した。炎症は、好中球および肺水腫の存在が優位を特徴とし、従って、急性肺損傷の病因に対処し、疾患の病理学に寄与するメカニズムのさらなる知識を得るために使用することができる。

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Name of the material/equipment&#160;</td>
			<td style="width:244px;">Company</td>
			<td style="width:145px;">Catalog number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:307px;">Purified Pseudomonas aeruginosa LPS&#160;</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Harmful. Recomended purification. LPS purified from other bactria may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pari LC sprint star nebulizer</td>
			<td style="width:244px;">PARI Respiratory Equipment Inc.&#160;</td>
			<td style="width:145px;">023G1250</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">TSI mass flowmeter 4040</td>
			<td style="width:244px;">TSI</td>
			<td style="width:145px;">4040</td>
			<td style="width:235px;">Alternative product from supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Saint-Gobain 15.9 mm Tygon tube</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">Z685704</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Plexiglas boxes with removable lids</td>
			<td style="width:244px;">Custom built</td>
			<td style="width:145px;">N/A</td>
			<td style="width:235px;">150 x 163 x 205 mm (a 2 mm hole on the side).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Half Facepiece Reusable Respirator</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">7503</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Advanced Particulate Filters (P100)&#160;</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">2291</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sissors</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">233-1104</td>
			<td style="width:235px;">Preferred scissors may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Forceps&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">232-1313</td>
			<td style="width:235px;">Preferred forceps may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Intramedic PE50 polyethylene tube</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">427411</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ethicon 2-0 Perma-hand silk tread&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">95056-992</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">26 &#189;&#160; gage needle&#160;</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">1 mL BD slip-tip syringe, non-sterile</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=301025">301025</a></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">60 mL BD Luer-Lok syringe, non-sterile, polypropolene&#160;</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">301035</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fluka Hematoxylin-Eosin</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">3972</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T&#252;rk&#39;s solution</td>
			<td style="width:244px;">Merck Millipore</td>
			<td style="width:145px;">109277</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Table top centrifuge</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative manufacturers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytospin 4 cytocentrifuge</td>
			<td style="width:244px;">Thermo Scientific</td>
			<td style="width:145px;">A78300003</td>
			<td style="width:235px;">Alternative centrifuge can be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEMA-3 stat pack</td>
			<td style="width:244px;">Fisher Scientific</td>
			<td style="width:145px;">23-123-869</td>
			<td style="width:235px;">Alternative staining kits exists.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">HT501128&#160;</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
	</tbody>
</table>

a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide

急性肺損傷（ALI）限られた治療選択肢と高い死亡率と、肺胞好中球増加によって特徴付け重篤な疾患である。 ALIの実験モデルは、疾患の病因の我々の理解を高める上で鍵となります。グラム陽性細菌由来のリポ多糖（LPS）は、マウスの気道および肺実質における好中球性炎症を誘導する。 LPSのような化合物の効率的な肺送達は、しかし、達成することは困難である。ここに記載されたアプローチでは、マウスでの肺送達は、エアロゾル化された緑膿菌 LPSをチャレンジすることによって達成される。溶存LPSは、圧縮空気に接続ネブライザーによりエアロゾル化した。マウスは、エアロゾルを中止した後、2分間コンディショニングした後、10分間のプレキシグラスボックス内LPSエアロゾルの連続流に暴露した。気管挿管し、滅菌Pの特性評価を可能にし、ホルマリン灌流に続いて、その後の気管支肺胞、次の行われた、ulmonary炎症。エアロゾル化LPSは、気管支肺胞洗浄および組織学的評価によって検出された歯槽好中球増加によって特徴づけられる肺の炎症を、生成されます。この手法は、少数の家電の小さなコストで設置し、最小限のトレーニングと専門知識を必要とすることができます。露光システムは、このように日常的に肺の病理学の理解を向上させる可能性のある、任意の実験室で行うことができる。

エアロゾル化P.への挑戦緑膿菌 LPSは、通常、両方の初期および後期の時点で好中球の優位性によって特徴づけられる気道内腔および肺胞腔における顕著な炎症反応を、得られます。 エアロゾル化LPSは、肺好中球増加を誘発する C57BL / 6byとBALB / cマウスを、エアロゾル化P.に曝露した緑膿菌 LPS単独車両と好中球がBALF中で数えた?...

Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

我々は、急性肺損傷をモデル化するために、噴霧によりエアロゾル化したリポ多糖にチャレンジすることにより好中球の肺の炎症を誘導するための方法を記載している。また、肺の分離、気管挿管および気管支肺胞洗浄のための基本的な外科技術も記載されている。

エアロゾル化リポ多糖を使用して生成肺好中球増加のための方法

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

a-method-for-generating-pulmonary-neutrophilia-using-aerosolized

Research

JoVE Journal

Immunology and Infection

11.6K ビュー.  Karolinska Institutet. 我々は、急性肺損傷をモデル化するために、噴霧によりエアロゾル化したリポ多糖にチャレンジすることにより好中球の肺の炎症を誘導するための方法を記載している。また、肺の分離、気管挿管および気管支肺胞洗浄のための基本的な外科技術も記載されている。 

ビデオ: A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

マウス胎児の血管系を標識する方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

免疫・感染学

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

XpertのMTB / RIFのテストで肺結核の診断

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

ペプチドエピトープ特異的T細胞のMHCテトラマーベースの濃縮

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

マウスにおけるオートファジーを研究するためのモデルとして、盲腸結紮穿刺誘発性敗血症

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

リポ多糖投与マウス肝臓における免疫応答時に白血球の二光子励起生体イメージング

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

受容体と酵素間の機能的相互作用を研究するため Co 免疫沈降分析

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

生理学的免疫原性樹状細胞を生成するための新規プロトコル

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

蛍光系抗生物質プローブを用いた細菌耐性の可視化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

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

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 diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

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 ...