子どもと家族の COPSAC2010のコホート研究にすべての彼らのサポートとコミットメントに感謝の意を申し上げます。認識し、COPSAC 研究チームとサイトカインとケモカインの測定技術者 Lisbeth Buus Rosholm、デンマーク工科大学から技術の助けのユニークな努力に感謝します。

ヘルシンキ宣言の基本理念に基づき、研究を行った。(KF 01-289/96 COPSAC2000 ) および H-B-2008-093 COPSAC2010のコペンハーゲン、デンマークのデータ保護局の倫理委員会から承認が得られたし、入学前に各教科の親の両方から書面によるインフォームド コンセントを得た。
1. 実験装置
<ol>
	<li>フィルターの紙のシートを使用して、(線維の水酸化ポリエステル シート材料の表を参照してください)6,7。<ol>
		<li>お子様や大人 (5 × 20 × 10 mm (L の短い腕)) ためストリップ 3 × 15 mm 1 ヶ月歳の子供のための大きさで、L 字型にカットします。</li>
	</ol>
	</li>
	<li>鉗子を用いた幼児各鼻孔にフィルターの紙一枚を挿入します。下鼻甲介 (新生児の鼻と他のすべての年齢のため 1.5 cm 内側約 1 cm) の前方の部分にろ紙を置きなさい。<ol>
		<li>ベッドの上に新生児を置くし、快適さのための砂糖水を与えます。 注: 他のすべての年齢の子供たち、サンプリングが実行されます最も簡単に件名が椅子に座っているの。</li>
		<li>お子様のためには、各鼻孔に L 形のフィルター紙の長い腕を挿入します。</li>
	</ol>
	</li>
	<li>不快感を最小限に抑えるため、フィルター ペーパーの偶然の損失を避けるために鼻孔の周りに鼻クリップを適用します。</li>
	<li>吸収の 2 分後に、フィルター ペーパーを削除します。</li>
	<li>ラベルの付いたチューブでフィルター ペーパーを置き、-80 ° c. ですぐにそれらを凍結</li>
	<li>サンプリングの日に子どもに示す気道感染の症状のメモしておきます。</li>
	<li>どんなくしゃみ、持続的な泣き、またはサンプリングの 2 分の中に発生する鼻出血を記録します。</li>
</ol>
2. 気道免疫メディエーターの定量化
<ol>
	<li>冷凍庫から 10 までのランダムなサンプルを取る。識別番号を記録します。全体の作業工程の中に氷のサンプルを保ちます。</li>
	<li>氷の融解後没頭 (通院) フィルター ペーパー両方の鼻から 300 μ L 作りたてのバッファー (参照材料表) 含まれている 1 つ完全なプロテアーゼ阻害剤タブレット (材料の表を参照してください) 25 mL のバッファーあたり。<ol>
		<li>フィルター ペーパーのサイズに従ってバッファーのボリュームを調整します。<ol>
			<li>フィルター ペーパーのサイズ 3 × 15 mm のバッファーの 300 μ L を使用します。</li>
			<li>フィルター紙の 1 つだけの場合は、半分のバッファー量 (150 μ L) を使用します。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>タイマー 5 分使用プレート シェーカー (400 回転) にサンプルを転送します。</li>
	<li>遠心機内に配置、酢酸セルロース チューブ フィルター (0.22 μ m 孔サイズ) のカップに湿ったフィルター ペーパーおよびアッセイバッファーを転送管 (材料の表を参照してください)。</li>
	<li>(4 ° C) で冷却遠心機で 5 分間 16,000 x g で遠心分離機の管にフィルター処理された鼻エキスを入手します。</li>
	<li>カップを削除し、低蛋白結合記憶板 (材料の表を参照してください) の井戸に早かったしながら氷の上管鼻のエキスを維持します。</li>
	<li>解析まで-80 ° C で保存します。</li>
	<li>高感度、electrochemiluminesce ベースの多重配列を用いた鼻抽出におけるサイトカインとケモカイン濃度を把握 (材料の表を参照してください)。 注: 人間 10 プレックス TH1/TH2 サイトカインのアッセイ、9 プレックス ケモカイン アッセイとシングル プレックス IL 17A、TGF-β 1、TSLP を行ったここ。<ol>
		<li>試金、4 ° C で一晩鼻のエキスを孵化させなさい。標準メーカーごとプロトコル (材料の表を参照してください) として測定を行います。 注: イムノアッセイ測定 1 までの高ダイナミック レンジがある (材料の表を参照してください) 通常 &#38; 10,000 pg/mL である試金 100,000 pg/mL をすることができますが。これは、すべてのサンプルを他の同様のイムノアッセイにおける場合と同様、健康と病気における異種希釈の影響が少なくて、同じ希釈で実行できることを意味します。すべてのサイトカインの検出下限値 1 pg/mL であったまたはそれが 1 間遠隔以下、ケモカイン、&#38; 50 pg/mL。1 カ月の年齢に TSLP がサンプルの 98% で検出可能でした。</li>
	</ol>
	</li>
</ol>
3. 統計
<ol>
	<li>サイトカインおよびケモカイン レベルの正規分布の残差を取得する解析の前にデータを対数変換します。</li>
	<li>サンプルは 1 つ以上のバッチで分析し場合、は、対数変換データを中心に batch-wise のサイトカイン ・ ケモカインのデータを修正します。<ol>
		<li>全体的な平均と元の寸法の単位でデータを取得する逆対数変換を組み込みます。 注: 分析を行うのセットごとに、サイトカインやケモカインのレベルに影響を与える変数を調整することを検討してください。これはサンプリング、兄弟、ホームなどでペットに有害事象、母乳育児における母親の影響 (例えば、アルコール消費量と妊娠中の喫煙) ことがでした。</li>
	</ol>
	</li>
</ol>

<ol><li>Bousquet, J., van Cauwenberge, P., Khaltaev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allergic+Rhinitis+and+Its+Impact+on+Asthma%5BTitle%5D)+AND+%22J+Allergy+Clin+Immunol%22%5BJournal%5D)">Allergic Rhinitis and Its Impact on Asthma.</a> J Allergy Clin Immunol. 108 (5), Supplement 147-334 (2001).</li>
<li>Howarth, P. H., Persson, C. G. A., Meltzer, E. O., Jacobson, M. R., Durham, S. R., Silkoff, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Objective+monitoring+of+nasal+airway+inflammation+in+rhinitis%5BTitle%5D)+AND+%22J+Allergy+Clin+Immunol%22%5BJournal%5D)">Objective monitoring of nasal airway inflammation in rhinitis.</a> J Allergy Clin Immunol. 115 (3), Supplement 414-441 (2005).</li>
<li>Bensch, G. W., Nelson, H. S., Borish, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+cytokines+in+nasal+secretions+after+nasal+antigen+challenge%3A+lack+of+influence+of+antihistamines%5BTitle%5D)+AND+%22Ann+Allergy+Asthma+Immunol%22%5BJournal%5D)">Evaluation of cytokines in nasal secretions after nasal antigen challenge: lack of influence of antihistamines.</a> Ann Allergy Asthma Immunol. 88 (5), 457-462 (2002).</li>
<li>Sussman, G. L., Mason, J., Compton, D., Stewart, J., Ricard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+efficacy+and+safety+of+fexofenadine+HCl+and+pseudoephedrine%2C+alone+and+in+combination%2C+in+seasonal+allergic+rhinitis%5BTitle%5D)+AND+%22J+Allergy+Clin+Immunol%22%5BJournal%5D)">The efficacy and safety of fexofenadine HCl and pseudoephedrine, alone and in combination, in seasonal allergic rhinitis.</a> J Allergy Clin Immunol. 104 (1), 100-106 (1999).</li>
<li>Bisgaard, H., Robinson, C., Rømeling, F., Mygind, N., Church, M., Holgate, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leukotriene+C4+and+histamine+in+early+allergic+reaction+in+the+nose%5BTitle%5D)+AND+%22Allergy%22%5BJournal%5D)">Leukotriene C4 and histamine in early allergic reaction in the nose.</a> Allergy. 43 (3), 219-227 (1988).</li>
<li>Chawes, B. L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+method+for+assessing+unchallenged+levels+of+mediators+in+nasal+epithelial+lining+fluid%5BTitle%5D)+AND+%22J+Allergy+Clin+Immunol%22%5BJournal%5D)">A novel method for assessing unchallenged levels of mediators in nasal epithelial lining fluid.</a> J Allergy Clin Immunol. 125 (6), 1387-1389 (2010).</li>
<li>Følsgaard, N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenic+bacteria+colonizing+the+airways+in+asymptomatic+neonates+stimulates+topical+inflammatory+mediator+release%5BTitle%5D)+AND+%22Am+J+Respir+Crit+Care+Med%22%5BJournal%5D)">Pathogenic bacteria colonizing the airways in asymptomatic neonates stimulates topical inflammatory mediator release.</a> Am J Respir Crit Care Med. 187 (6), 589-595 (2013).</li>
<li>Følsgaard, N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+Cytokine+Profile+in+the+Airway+Mucosal+Lining+Fluid+Is+Skewed+by+Maternal+Atopy%5BTitle%5D)+AND+%22Am+J+Respir+Crit+Care+Med%22%5BJournal%5D)">Neonatal Cytokine Profile in the Airway Mucosal Lining Fluid Is Skewed by Maternal Atopy.</a> Am J Respir Crit Care Med. 185 (3), 275-280 (2012).</li>
<li>Chawes, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+Vitamin+D3+Supplementation+During+Pregnancy+on+Risk+of+Persistent+Wheeze+in+the+Offspring%3A+A+Randomized+Clinical+Trial%5BTitle%5D)+AND+%22JAMA%22%5BJournal%5D)">Effect of Vitamin D3 Supplementation During Pregnancy on Risk of Persistent Wheeze in the Offspring: A Randomized Clinical Trial.</a> JAMA. 315 (4), 353-361 (2016).</li>
<li>Bischoff, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+Response+to+A%28H1N1%29pnd09+Vaccination+in+Pregnant+Women%3A+A+Single+Blinded+Randomized+Controlled+Trial%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Altered Response to A(H1N1)pnd09 Vaccination in Pregnant Women: A Single Blinded Randomized Controlled Trial.</a> PloS One. 8 (4), 56700(2013).</li>
<li>Wolsk, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Picornavirus-Induced+Airway+Mucosa+Immune+Profile+in+Asymptomatic+Neonates%5BTitle%5D)+AND+%22J+Infect+Dis%22%5BJournal%5D)">Picornavirus-Induced Airway Mucosa Immune Profile in Asymptomatic Neonates.</a> J Infect Dis. 213 (8), 1262-1270 (2016).</li>
<li>Wolsk, H. M., Chawes, B. L., Følsgaard, N. V., Rasmussen, M. A., Brix, S., Bisgaard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Siblings+Promote+a+Type+1%2FType+17-oriented+immune+response+in+the+airways+of+asymptomatic+neonates%5BTitle%5D)+AND+%22Allergy%22%5BJournal%5D)">Siblings Promote a Type 1/Type 17-oriented immune response in the airways of asymptomatic neonates.</a> Allergy. 71 (6), 820-828 (2016).</li>
<li>Alam, R., Sim, T. C., Hilsmeier, K., Grant, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+new+technique+for+recovery+of+cytokines+from+inflammatory+sites+in+situ%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Development of a new technique for recovery of cytokines from inflammatory sites in situ.</a> J Immunol Methods. 155 (1), 25-29 (1992).</li>
<li>Ishizaka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+bronchoscopic+microsample+probe+to+measure+the+biochemical+constituents+in+epithelial+lining+fluid+of+patients+with+acute+respiratory+distress+syndrome%5BTitle%5D)+AND+%22Crit+Care+Med%22%5BJournal%5D)">New bronchoscopic microsample probe to measure the biochemical constituents in epithelial lining fluid of patients with acute respiratory distress syndrome.</a> Crit Care Med. 29 (4), 896-898 (2001).</li>
<li>Ishizaka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elevation+of+KL-6%2C+a+lung+epithelial+cell+marker%2C+in+plasma+and+epithelial+lining+fluid+in+acute+respiratory+distress+syndrome%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Mol+Physiol%22%5BJournal%5D)">Elevation of KL-6, a lung epithelial cell marker, in plasma and epithelial lining fluid in acute respiratory distress syndrome.</a> Am J Physiol Lung Cell Mol Physiol. 286 (6), 1088-1094 (2004).</li>
<li>Nakano, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelin-1+level+in+epithelial+lining+fluid+of+patients+with+acute+respiratory+distress+syndrome%5BTitle%5D)+AND+%22Respirology%22%5BJournal%5D)">Endothelin-1 level in epithelial lining fluid of patients with acute respiratory distress syndrome.</a> Respirology. 12 (5), 740-743 (2007).</li>
<li>Komaki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytokine-mediated+xanthine+oxidase+upregulation+in+chronic+obstructive+pulmonary+disease%27s+airways%5BTitle%5D)+AND+%22Pulm+Pharmacol+Ther%22%5BJournal%5D)">Cytokine-mediated xanthine oxidase upregulation in chronic obstructive pulmonary disease's airways.</a> Pulm Pharmacol Ther. 18 (4), 297-302 (2005).</li>
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すべての著者には、この原稿の内容に関する潜在的な知覚、または実質利益相反の宣言しません。資金の機関設計と試験の実施で、役割はありませんでした。コレクション、管理、およびデータの解釈または、準備、レビュー、および原稿の承認。研究に関与する製薬会社がありませんでした。

ここで示した手法、体内上部気道粘膜免疫プロファイル以前行われていない時代の 1 ヶ月という早い時期から子供を決定することができました。気道細菌がその存在や周産期曝露と同様、ピコルナ ウイルス7,11, pre が新生児の気道の免疫プロファイルにミラー化されたを見ました。さらに、我々 は高用量ビタミン d の微量栄養素の無作為化?...

鼻の粘膜内層液上部気道系の液体の一部を占めています。調停に由来する上皮および侵入の微生物に対する防御の最初の行を構成する免疫細胞間の相互作用の複雑なマトリックスで構成されています。鼻の粘膜は簡単にアクセスできる、鼻と気管支1の免疫学的機能の強い関係があります。この区画は幼年期、生命に後でより普及しているその他の呼吸器系疾患の範囲にも喘息やアレルギー性鼻炎などでは一般的な気道疾患に関連して特別な関心。
ここでは、サンプル邪魔されず鼻腔から流体をライニング粘膜フィルター紙ベース、非侵襲的手法としてそれに続く抽出プロシージャを使用して、その定量化する前にフィルター ペーパーからの検体の蛋白質ベースの溶出に使用するメソッドの実装について述べる.この手法は、生体内で健康な個人と様々 な呼吸器疾患を持つ個人の免疫の署名を取得するたとえば、使用できます。さらに、特定免疫署名の重要性の露出を確認し、予測または発病後の調停の場合を評価することが可能です。
粘膜内層液は、鼻洗浄2がよく付いている鼻チャレンジ テスト、炎症性応答3,4を刺激するために高いレベルでのアレルゲンが導入される以前受けた。ただし、鼻洗浄技術は、幼児に適さないことがし、5アッセイの検出限界値以下のレベルが落ちることができる希薄化後の調停者として、結果を混同する不明な希釈率を紹介します。また、不明な希釈倍率により鼻チャレンジ テストから測定試料応答が設定コホートで鼻洗浄法の有用性を制限すること、個人間に匹敵します。最後に、アレルゲンの挑戦は、鋭敏化の分野で適用されるだけ、ヒスタミンの挑戦など、その他の課題は生理学的関連性の高い、メディエーター遊離の天井の効果を引き起こす可能性がありますありません。これらの問題は、流体と検体レベルの個々 の分泌が個体間の差異に影響を与える唯一の要因を粘膜内層流体コレクションの紹介したフィルター紙ベース テクニックで回避します。
抽出中に、検体は、すべてのサンプルに、バッファーの同じボリュームの付加の後でフィルター ペーパーから溶出されます。これは、すべてのサンプルの希釈前のヴィヴォ同様優先されます。抽出手順にアルブミン ベース等張食塩水の抽出バッファーを使用します。蛋白質ベースのメディエーターの抽出と定量化の前に溶出蛋白質の以降凍結変性を抑えるタンパク質を安定できます。抽出段階でのタンパク質分解を避けるためには、プロテアーゼ阻害剤のカクテルは抽出バッファーに追加されます。
邪魔されない生体内での定量化を可能にする技術の実装-粘膜のサイトで生成された免疫メディエーターが非常に重要です。最初に、粘膜のサイトは体内最大の免疫臓器をなります。第二に、鼻の位置の気中暴露のプライマリ サイトで、肺1呼吸器免疫学的区画にしっかりと接続されています。第三に、非侵襲的手法でこの重要な器官の調査の可能性は健康と気道の病気に関連して重要な微生物免疫の相互作用軸情報の茄多を提供する可能性を開きます。第四に、この技術は、医薬品と栄養素のランダム化された、制御された試験のローカル免疫学的変化などの他の多くの可能なアプリケーションがあります。
喘息のコペンハーゲンの前向き研究の手法を実装されて最初幼年期2000 (COPSAC2000) コホート研究において、7 歳児健常者13対アレルギー性鼻炎の粘膜内層液の免疫のプロフィールを調べた。その後、我々 は正常に縦 COPSAC2010コホートと評価気道免疫プロファイルにこの手法を適用 1 ヶ月、2 歳、6 歳の時、急性呼吸器症状のインスタンスで。1 歳 1 か月の新生児からの結果は、免疫の署名と初期の生活環境暴露7,8,9,10、11,12間の重要な関連を示しています。

<table><tbody><tr><td>Fibrous hydroxylatedpolyester sheets</td><td>Accuwik Ultra&amp;nbsp;</td><td>SPR0730</td><td>Filter paper</td></tr><tr><td>Milliplex Assay Buffer&amp;nbsp;</td><td>Millipore</td><td>L-AB</td><td>Buffer</td></tr><tr><td>Low-protein binding storage plates&amp;nbsp;</td><td>Thermo Scientific</td><td>CLS8161</td><td>Plates</td></tr><tr><td>Protease Inhibitor</td><td>Roche</td><td>11873580001</td><td>complete EDTA-free Protease Inhibitor Cocktail</td></tr><tr><td>Reader of multi-spot plates</td><td>Mesoscale</td><td>NA</td><td>Sector imager 6000</td></tr><tr><td>Assays&amp;nbsp;</td><td>Mesoscale</td><td></td><td>Human 10-plex TH1/TH2 cytokine assay and 9-plex chemokine assay, and singleplex IL-17A, TGF-&amp;beta;1and TSLP. A description can be found online on www.mesoscale.com</td></tr></tbody></table>

noninvasive sampling of mucosal lining fluid for the quantification of in vivo upper airway immune-mediator levels

このプロトコルでは、妨げられていない上部気道粘膜内層流体の非侵襲的サンプリングについて説明します。それはまた刺激プロシージャ (よく、他の技術によって使用される) を必要とせず、気道局所の免疫署名に関する流体波における免疫メディエーターの分析の前に使用される抽出手順の詳細します。フィルター ペーパーのストリップの粘膜内層液をサンプリング下鼻甲介と吸収の 2 分間左の前部で置かれました。検体用濾紙から溶出され、同じサンプルの低レベルと高レベル分析の高感度定量を可能にする、電気化学発光を用いたイムノアッセイ法によって抽出された蛋白質基づいた溶出を分析します。上部気道粘膜における情報伝達システム特定の免疫に関連する 20 あらかじめ免疫メディエーターの体内レベルを測定したが、技術はその特定のパネルまたはサンプリング サイトに制限されていません。技術は、アレルギー性鼻炎小児2000 (COPSAC2000) コホート研究における喘息のコペンハーゲンの前向き研究から 7 歳児に最初実装されました。それはその後 1 ヶ月、2 歳、6 歳、急性呼吸器症状のインスタンスをサンプリング、縦の COPSAC2010出生コホートで使用されました。いくつかのサンプル (中央 (四分範囲 (IQR)) として報告測定検出限界以下であった我々 は正常に取得し 700 1 か月児の 620 (89%) からサンプルを分析した; します。各調停の (すなわち0 から検出下限値の設定ポイント) の検出限界以下のサンプル数は、29 (7.25 119.5) だった。この技術は、誕生からin vivo気道粘膜免疫プロファイルの数量化できる、縦、適用することができます、遺伝学と初期の生活環境暴露、病態、endotyping、および呼吸器疾患と開発と新規治療薬の評価の監視の影響に関する研究に適用することができます。

Baseline Characteristics of the Airway Immune Profiles: 
Complete data on upper airway mucosal immune mediator levels at 1 month of age was obtained in 620 (89%) of the 700 children enrolled in the COPSAC2010 cohort. Ten neonates were enrolled before the technique was established, and 19 did not attend the 1-month visit. Additionally, 47 samples were excluded because they were extracted and measured in another laboratory used in a pilot study, and 4 sample...

Watch this Scientific Journal Video about Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels at JoVE.com

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

このプロトコルでは、上気道から邪魔されず粘膜被覆液のサンプリングのための非侵襲的技術について説明します。それは、すべての年齢層の被験者でタンパク質メディエーター、サイトカイン、ケモカインなどの生体内レベルの定量化を実行する使用できます。

体内上部気道免疫メディエーター レベルの定量化のための粘膜内層流体の非侵襲的サンプリング

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

noninvasive-sampling-mucosal-lining-fluid-for-quantification-vivo

Research

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Immunology and Infection

Unit 1

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Upper Respiratory Tract Infections

SCIENTISTS IN ACTION

10.3K ビュー.  University of Copenhagen. このプロトコルでは、上気道から邪魔されず粘膜被覆液のサンプリングのための非侵襲的技術について説明します。それは、すべての年齢層の被験者でタンパク質メディエーター、サイトカイン、ケモカインなどの生体内レベルの定量化を実行する使用できます。

ビデオ: Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

アレルゲン誘発性喘息のマウスモデル

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

免疫・感染学

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

初代ヒト気道上皮細胞の気道表面液 pH をリアルタイムで半自動で蛍光測定

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

生化学

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

気道抵抗測定とマウスにおける気管支肺胞洗浄液のサンプリングのためのリバーシブル、非侵襲的方法

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

生物学

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

免疫組織化学およびフローサイトメトリーを用いた気管支生検における空間分布と表現型の同定：ヒト肺樹状細胞

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

炎症性細胞の浸潤を分析するための肺胞の気管支肺胞洗浄

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

閉ループ慣性マイクロ流路を用いた患者由来気道分泌からラベル無料好中球の濃縮

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

鼻粘膜および気管支の流体の吸収: 人間の呼吸器粘膜とサンプル室加工高精度サンプリング

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

医学

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

喀痰誘発および実験室の処理のための方法論

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

インフルエンザ菌に対する呼吸器免疫応答の評価

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

体内上部気道免疫メディエーターレベルの定量化のための粘膜内層流体の非侵襲的サンプリング

要約

さらに動画を探す

アレルゲン誘発性喘息のマウスモデル

初代ヒト気道上皮細胞の気道表面液 pH をリアルタイムで半自動で蛍光測定

気道抵抗測定とマウスにおける気管支肺胞洗浄液のサンプリングのためのリバーシブル、非侵襲的方法

免疫組織化学およびフローサイトメトリーを用いた気管支生検における空間分布と表現型の同定：ヒト肺樹状細胞

炎症性細胞の浸潤を分析するための肺胞の気管支肺胞洗浄

閉ループ慣性マイクロ流路を用いた患者由来気道分泌からラベル無料好中球の濃縮

鼻粘膜および気管支の流体の吸収: 人間の呼吸器粘膜とサンプル室加工高精度サンプリング

喀痰誘発および実験室の処理のための方法論

インフルエンザ菌に対する呼吸器免疫応答の評価

In Vivo 光シート顕微鏡観察に基づくアレルギー性炎症の実験的肺ホーミングヒトリンパ球のイメージング高度な

体内上部気道免疫メディエーター レベルの定量化のための粘膜内層流体の非侵襲的サンプリング

要約

さらに動画を探す

アレルゲン誘発性喘息のマウスモデル

初代ヒト気道上皮細胞の気道表面液 pH をリアルタイムで半自動で蛍光測定

気道抵抗測定とマウスにおける気管支肺胞洗浄液のサンプリングのためのリバーシブル、非侵襲的方法

免疫組織化学およびフローサイトメトリーを用いた気管支生検における空間分布と表現型の同定：ヒト肺樹状細胞

炎症性細胞の浸潤を分析するための肺胞の気管支肺胞洗浄

閉ループ慣性マイクロ流路を用いた患者由来気道分泌からラベル無料好中球の濃縮

鼻粘膜および気管支の流体の吸収: 人間の呼吸器粘膜とサンプル室加工高精度サンプリング

喀痰誘発および実験室の処理のための方法論

インフルエンザ菌に対する呼吸器免疫応答の評価

In Vivo 光シート顕微鏡観察に基づくアレルギー性炎症の実験的肺ホーミング ヒトリンパ球のイメージング高度な

体内上部気道免疫メディエーターレベルの定量化のための粘膜内層流体の非侵襲的サンプリング

In Vivo 光シート顕微鏡観察に基づくアレルギー性炎症の実験的肺ホーミングヒトリンパ球のイメージング高度な