心血管疾患における体液性免疫応答（ - この作品は、ドイツ学術振興協会（DFG）のSonderforschungsbereich Transregio 19（SFB/TR19、C2）は、経済技術プロジェクトZIM-KF 2727801MD0連邦省とイノベーションのコンピテンスセンターでサポートされていたZIK-ハイキング、教育研究、ドイツ連邦省BMBF FKZ 03Z2CN12）。動物とハウジングとの実験は、実験動物学（ゲゼルシャフトエリーゼのためVersuchstierkunde、GV-SOLAS条約）と欧州実験動物学協会（FELASA）連合学会の勧告に従って行われた。

1。患者サンプルからのIgGアイソ<ol><li>空のエコノパックカラムに2ミリリットル患者EDTA血漿当たり3ミリリットル抗IgGセファロースを充填することにより、ミニ免疫吸着カラムを準備します。 IgGの分離は、患者血漿の少なくとも2ミリリットルを必要とします。 </li><li>抗IgGセファロースの上にフィルターを置き、カラムの下部先端を開き、約1滴/秒の流速に到達するためにフィルタを押しカット。欄が不足する保存液してみましょう。 </li><li>プラズマの体積あたりの3倍量の0.9％NaClでカラムを洗浄する。 </li><li>カラム上でプラズマをピペット。プラズマは完全に抗IgGセファロースに浸漬した場合には、同じ体積の0.9％NaClで洗浄します。 </li><li>ピペットカラムに0.2MのグリシンHCl、pHが2.8の1血漿量とセファロースに浸すことができます。カラムにグリシンHClの別のボリュームを追加します。 15mlチューブ内を通る流れを収集し、0.5Mのトリス-HCl、pH 8.0で7.3にpHを調整する。 </li><li>列を再生成するには、3プラズマボリュームグリシンHClで洗浄してください。 2倍量の0.9％NaClで最終洗浄後、カラムは次の血漿試料に使用されるかもしれません。 ℃で4週間までに8 - あるいは、その列は保存液を充填し、4℃で保存することができます。 </li><li>心筋細胞上で使用するために、収集したIgG画分では、実験的なバッファ（EB）に対して透析しなければならない。 EBの調製のために、マグネチックスターラーで1リットルの蒸留水に117 mMのNaCl、2.8のKCl、0.6 mMのMgCl 2、1.2mMのKH 2 PO 4、1.2 mMのCaCl 2、10mMのHEPESおよび20mMグルコースを追加して、pHを調整7.3へ。 </li><li> 100kDaのカットオフ分子量を有するセルロースエステル透析膜チューブにIgG画分を埋める。 1LのEBにチューブを入れて、4℃でマグネチックスターラーでゆっくりかき混ぜる℃、 8時間後、新鮮なEBの3 Lに透析チューブに移し、4℃でさらに20時​​間透析℃、 </li><li>透析後、57℃で30分間inactivatする水浴中でCのために試料を加熱eの補体因子。総IgG濃度を決定し、330μgのIgGのそれぞれを含むアリコートを準備します。測定（ステップ4.3）のための心筋細胞にサンプルを追加するときは、EBで1mlにアリコートを埋める。原液アリコートをさらに使用するまで-20℃で保存することができます。 </li></ol> 2。ラット心筋細胞の単離<ol><li>のCa 2 +遊離、分離バッファ（IB）の調製のために、2.6のKCl、1.2mMのMgSO 4を 、1.2mMのKH 2 PO 4、20mMのHEPESおよび磁気を用いて1Lの蒸留水に11 mMグルコース、110 mM NaClを追加スターラー。滅菌のための0.2μmのボトルトップフィルターを通して室温とフィルターでpHを7.4に調整してください。 </li><li>サーモスタットランゲンドルフ灌流システムの上部貯水池で、IBの80ミリリットルを充填します。 37℃のバッファ温度を維持し、95％O 2/5％30分間、CO 2でバッファを通気するため、サーモスタットの設定値を調整します。 </li><li>約10,000の重量を量る - collag 12,000 Uをenase II型、175 mgの脂肪酸フリーBSAおよび100 mMのCaCl 2ストック溶液22.5μlを含む20ミリリットルIBに溶かす。上部貯水池の分離緩衝液のpHを制御し、必要に応じて調整します。 </li><li>チオペンタール375μg/ kg体重で200グラム - 175のWistarラットをAnaesthetize。チオペンタールソリューションに2500 IUのヘパリンを追加します。無傷の大動脈弓と慎重に開胸、心臓を切除した後、氷冷した0.9％のNaClにそれを転送します。血液からの心と周囲の組織と新鮮な0.9％のNaClへの転送を清掃してください。大動脈を露出させ、2-3/secの滴下速度を取得し、カニューレに心臓を添付するランゲンドルフシステムの流れの減速を開きます。 </li><li>最大3分までの残りの血を洗い流すために1滴/秒の流速を持つための心臓を灌流する。ランゲンドルフシステムで、IBを循環させるために起動します。 80ミリリットルに達するために必要に応じて上部貯水池から20mlのバッファーを外し、はるかにバッファとしてコラゲナーゼ溶液とリフィルに記入してください。 cを循環させる<html>別の27分間ollagenaseソリューションを提供します。流量が流れ減速機を使用して、1滴/秒で周期的に制御され、維持されるべきである。 </li><li>心房と大動脈からクリーンカニューレから心臓を、切り離し、小片に切り刻む。コラゲナーゼ溶液を下池に実行できるように、40ミリリットルの最大にボリュームを削減し、さらに10のために刻んだ心室を消化- 95％O 2/5％CO 2で連続曝気下に15分。その後、50 mlチューブに200μmのメッシュサイズのガーゼで細胞懸濁液をろ過する。 </li><li> 200μMのCaCl 2を含有する 10mlのIBの室温再懸濁した細胞で2分間43 xgで細胞懸濁液を遠心：3つのステップで細胞のCa 2 +のコンテンツを復元します。再び遠心分離し、500μMのCaCl 2を10ミリリットルIBに細胞を再懸濁する。最後の遠心分離の後、50,000細胞/ mlの密度に（1.8を参照）を滅菌濾過し、EBで心筋細胞を再懸濁します。 </li></ol>心筋細胞のe_title &quot;&gt; 3。のFura-2染色<ol><li>コー​​トウェルあたり10μgのラミニンとの4ウェルにチェンカバーガラスと、それが完全に乾燥するまで待ちます。 </li><li>よくカットチップ付きマイクロピペットを用いて各々に心筋細胞懸濁液1mlを埋める。細胞は室温で1時間付着することができます。 </li><li> 1 mg / mlののFura-2の10μlを希釈は、5mlのEBにおける原液AM。暗闇の中で染色液を保管してください。 </li><li>カバーガラスと各ウェルに染色液をピペット0.5ミリリットルからバッファと非付着細胞を脱ぐ。適度な揺れで10分間細胞をインキュベートします。続いて、染色液を脱いで1ミリリットルEBで一度細胞を洗浄し、最後に各ウェルに0.5 mlのEBを埋める。染色工程中に直接光を避け、常に暗闇の中で染色した細胞を保つ。 </li></ol> 4。細胞短縮とCa 2 +過渡の記録<ol><li>逆蛍光mにチャンバーカバーガラスを置きicroscopeは筋細胞のカルシウムと収縮記録システムに接続されています。よく最初の流入と流出管が付いているカスタムメイドの電極を配置します。 1ml /分のEBと電気刺激（20V、1Hzで、5ミリ秒の期間）を開始して心筋細胞を表面かん流する。細胞は約2分間刺激に適応することができます。 </li><li>明確なストライエーションと一定の収縮にそのまま棒状の心筋細胞を選択してください。視野の中央にあるセルを置き、カメラのチャンネルを開いてください。セルフレーミングアダプタを回転させ、顕微鏡ステージを移動することによって、ビデオ領域内で水平方向にセルを方向付ける。 </li><li>初期細胞短縮とCa 2 +の一過性を得るために15の収縮-エッジがビデオ領域の制御要素（ 図2）を検出調整、蛍光チャネルおよびレコード10を開きます。 </li><li>蛍光染色の漂白を避けるために顕微鏡光チャネルを閉じます。サンプルにEBから通る流れを切り替える3方弁と患者のIgG 1ミリリットルと表面かん流する心筋細胞でバイパスしてください。空気がよく達する直前にポンプを停止します。 </li><li>光チャネルを開き、別の10記録 - 急性細胞応答を得るために15の収縮を。録音を一時停止し、再び光チャネルを閉じます。 2と5分後に録画を繰り返します。 </li><li>井戸の電極を取り出します。 EBで徹底的に管をきれいにし、チャンバーカバーガラスの次のウェルに進みます。 </li><li>それぞれ細胞短縮とCa 2 + &quot;Edge-Length/Length&quot;を使用してIonWizardソフトウェアと過渡と&quot;フラ2数値は、/比を差し引いた&quot;ウィンドウを、分析します。初期細胞短縮とCa 2 +急性のベースライン（BL％ピーク時間）と呼ばれるピーク高さの過渡、2分、5分間応答からの変化率を計算します。 </li></ol>

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特別な利害関係は宣言されません。

提示された方法は不明起源のDCM患者において、機能的に有効な心臓の自己抗体を検出するのに適した方法を提供しています。他の方法と比較して、cAMPレベル6への影響により、β1-アドレナリン受容体に対して機能アクティブ抗体の検出を、例えば 、我々の手法は、特定のエピトープとは無関係です。もちろん、文献に記載されている他のエピトープの独立したアッセイは、パ?...

<table border="1"><tbody><tr><td> 試薬/機器の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> Immunosorba保存液</td><td>フレゼニウスメディカルケア</td><td> 903609211 </td><td></td></tr><tr><td>コラゲナーゼタイプ2 </td><td>細胞系</td><td> LS004176 </td><td></td></tr><tr><td> BSA、脂肪酸フリー</td><td>シグマアルドリッチ</td><td> A6003 </td><td></td></tr><tr><td>ラミニン</td><td>シグマアルドリッチ</td><td> L2020 </td><td></td></tr><tr><td>のFura-2 AM </td><td>シグマアルドリッチ</td><td> F0888 </td><td> DMSO中で1 mg / mlの</td></tr><tr><td>エコノパッククロマトグラフィーカラム</td><td> BioRad社</td><td> 732-1010 </td><td></td></tr><tr><td>スペクトラ/ポアバイオセルロースエステル透析MEMBRAN電子</td><td> Spectrumlabs </td><td> 131417 </td><td></td></tr><tr><td> 4ウェルチェンカバーガラス</td><td>ヌンク</td><td> 155383 </td><td></td></tr><tr><td>筋細胞のカルシウムと収縮レコーディングシステム</td><td> IonOptix </td><td></td><td></td></tr><tr><td> IonWizard 6.0解析ソフトウェア</td><td> IonOptix </td><td></td><td></td></tr></tbody></table>

measurement of antibody effects on cellular function of isolated cardiomyocytes

拡張型心筋症（DCM）は、若年成人1で心不全のための主要な原因の一つです。有毒物質に対する遺伝的気質と博覧会は、患者の約3分の1で、この病気の原因を知られているが、DCMの起源は、主に不明なままである。これらの患者の相当数は、心臓エピトープに対する自己抗体が検出されており、2,3病の発症と進行に重要な役割を果たすことが疑われている。心筋自己抗体の重要性は、免疫吸着3月5日で自己抗体を除去した後、DCM患者において観察された血行動態の改善によって強調される。特定の種々の抗原は、すでに2,3同定されており、これらのターゲットに対する抗体は免疫アッセイによって検出することができる。しかし、これらのアッセイは、刺激（したがって、機能的に有効な）と自己抗体をブロッキングを区別することはできません。 evidenが高まっているこの区別は6,7重要であることをCE。またcardiotropic抗体の数のターゲットはまだ正体不明であるため、免疫アッセイでは検出できないことが想定される。したがって、我々はそれぞれの抗原とは独立し、機能的に活性なcardiotropic抗体の検出のための方法を確立した。法の背景には、通常哺乳類8,9間に心臓のタンパク質の機能領域について観測と高い相同性があります。これは、ヒト抗原に対する抗体が心臓になる成体ラットの心筋細胞のDCM患者からのIgGのテストを許可する非ヒト標的細胞と交差反応することを示唆している。本手法は3つのステップで構成されています：最初に、IgGは免疫吸着カラム（PlasmaSelect、テーテロー、ドイツ）から入手セファロース結合抗IgG抗体を用いて患者血漿から分離されています。第二に、成体心筋細胞は、APを使用してランゲンドルフ灌流装置にコラゲナーゼ灌流によって分離されているrotocolは、以前の作品から、10,11を変更しました 。得られた心筋細胞は容易に細胞内カルシウム（Ca 2 +）の内容12を観察するための細胞内に持ち込むことのできるカルシウム選択的蛍光色素、ラミニンコートチャンバーcoverglassesに取り付け、のFura-2で染色されています。最後のステップでは、細胞短縮とCa 2上の患者IgGの効果フィールドの+過渡刺激心筋細胞は、商業筋細胞のカルシウムと標準蛍光逆に接続されている収縮性監視システム（IonOptix、ミルトン、MA、USA）を使用してオンラインで監視されている顕微鏡。

フィールドでの携帯変力作用を測定するための二つの例は以下のとおりである筋細胞のCa 2 +および収縮記録システムを用いて成体心筋細胞を（ 図2）を刺激した。 図3は、制御測定の印象を与え、 図4の間cardiodepressive抗体の効果DCMを使用した患者の図が示されている。 どちらの例でも、初期の細胞短縮とCa 2 + EBで灌流...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

提示された方法は、細胞の短縮と分離したラットの心筋細胞の細胞内カルシウムトランジェントの隔離患者の免疫の影響を分析することで、にかかわらず、特定の抗原の、拡張型心筋症患者の血漿中で機能的な効果cardiotropic自己抗体を検出する方法を提供します。

隔離された心筋細胞の細胞機能に対する抗体の効果測定

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

measurement-antibody-effects-on-cellular-function-isolated

Research

JoVE Journal

Immunology and Infection

11.8K ビュー.  University Medicine Greifswald. 提示された方法は、細胞の短縮と分離したラットの心筋細胞の細胞内カルシウムトランジェントの隔離患者の免疫の影響を分析することで、にかかわらず、特定の抗原の、拡張型心筋症患者の血漿中で機能的な効果cardiotropic自己抗体を検出する方法を提供します。

ビデオ: Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

細胞質のCaの測定 2 +絶縁収縮リンパ管における

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

免疫・感染学

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

RBL-2H3マスト細胞脱顆粒に対する化学的影響を評価するために、マイクロプレートアッセイ：有機溶剤を使用せずに、トリクロサンの影響

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

赤血球上の抗体誘導性補体活性化の定量的検出のための方法

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

フローから白血球の補充に間質細胞の影響を分析する

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10における粒子結合のBet V 1アレルゲンのフローサイトメトリー分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

モノクローナル抗体認識リニアB細胞エピトープのペプチドスキャンアシスト識別

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

腹腔内のブドウ糖負荷試験、肥満や呼吸器アウトカムに対する代謝が異常の影響を研究する肺機能の測定や肺の固定

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

マウス骨髄と腹腔マクロファージ活性化抗体を用いた薬剤効果の評価

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

マウスの唾液腺機能の測定法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

合成のバクテリオファージの建設は合わせた多様性と fab 抗体ライブラリーを表示

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...