Biz çocuklar ve ailelerine COPSAC2010 kohort çalışmanın tüm destek ve bağlılık için minnettarlığımızı. Biz kabul ve COPSAC araştırma ekibi ve teknisyen Lisbeth Buus Rosholm, Danimarka Teknik Üniversitesi, teknik yardım sitokinler ve kemokinler ölçüm için benzersiz çaba için teşekkür ederiz.

Helsinki Deklarasyonu yol gösterici ilkeler doğrultusunda çalışmalar yapılmıştır. Etik Komitesi (KF 01-289/96 COPSAC2000 için) ve H-B-2008-093 COPSAC2010için Kopenhag ve Danimarka veri koruma ajansı için onayları elde edilmiştir ve her iki ebeveyn kayıt önce her konunun üzerinden yazılı Onam alındı.
1. deneysel Kur
<ol>
	<li>Adet filtre kağıt kullanın (fibröz hydroxylated-polyester sayfaları, bkz: Malzemeler tablo)6,7.<ol>
		<li>Kesip şeritler 3 x 15 mm 1 aylık çocuk için boyutu ve L şekil büyük çocuk ve yetişkinler (5 x 20 x 10 mm (m kısa kol)).</li>
	</ol>
	</li>
	<li>Bir filtre kağıt parçası forseps kullanarak bir bebek her burun yerleştirin. Filtre kağıdı alt konka (yaklaşık 1 cm içinde Yenidoğan Bebeklerin burun ve diğer tüm yaş için 1,5 cm) ön kısmına yerleştirin.<ol>
		<li>Yenidoğan Bebeklerin bir yatağa yerleştirin ve konfor için şekerli su verin. Not: diğer tüm yaş çocuklar için örnekleme en kolay bir sandalyeye oturmuş konu ile gerçekleştirilir.</li>
		<li>Büyük çocuklar için L şeklindeki filtre kağıdı uzun kolu her burun yerleştirin.</li>
	</ol>
	</li>
	<li>Bir burun kıskacı rahatsızlık en aza indirmek için ve filtre kağıdı yanlışlıkla kaybedilmesini önlemek için burun delikleri çevresinde geçerli.</li>
	<li>Filtre kağıtları emme 2 dk sonra kaldırın.</li>
	<li>Filtre kağıtları etiketli tüplerde yerleştirin ve bunları hemen-80 ° C'de dondurmak</li>
	<li>Herhangi bir çocuk tarafından gösterilen örnekleme gününde hava yolu enfeksiyonu belirtileri not edin.</li>
	<li>Tüm hapşırma, sürekli ağlayan veya örnekleme sırasında 2 dk oluşur epistaksis kaydedin.</li>
</ol>
2. hava yolu bağışıklık-arabulucu miktar
<ol>
	<li>En çok 10 rasgele örnek dondurucudan almam. Kaydın kimlik numarası. Buz üzerinde örnekleri tüm çalışma işlemi sırasında tutmak.</li>
	<li>Buz çözdürme sonra filtre kağıtları (her konu) her iki burun delikleri 300 bırakın µL taze hazırlanmış, tampon (bkz: Malzemeler tablo) içeren bir tam proteaz inhibitörü tablet ( Tablo reçetesigörmek) arabelleği 25 mL başına.<ol>
		<li>Arabellek filtre kağıt boyutuna göre ses düzeyini ayarlayın.<ol>
			<li>300 µL arabellek filtre kağıtları 3 x 15 mm boyutunda için kullanın.</li>
			<li>Sadece bir filtre kağıdı varsa, yarım arabellek birim (150 µL) kullanın.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Örnekleri bir plaka shaker (400 d/d) 5 dk. kullanım için bir zamanlayıcı aktarın.</li>
	<li>Nemli filtre kağıtları ve tahlil arabellek selüloz asetat tüp filtre (0,22 µm gözenek boyutu) bir microcentrifuge içinde yer fincan içine transfer ( Tablo reçetesigörmek) tüp.</li>
	<li>16.000 x g 5 dakika içinde soğutmalı santrifüj (4 ° C'de) için filtre uygulanmış burun hulâsa tüp içinde elde etmek için santrifüj kapasitesi.</li>
	<li>Fincan kaldırın ve buz aliquoting süre tüpler burun özü düşük proteinli bağlama depolama tabak ( Tablo reçetesigörmek) kuyu tutun.</li>
	<li>-80 ° C'de analiz kadar saklayın.</li>
	<li>Sitokinler ve yüksek-duyarlı, electrochemiluminesce tabanlı çoğaltılmış array sistemi kullanılarak nazal özleri kemokinler konsantrasyonları belirleyin (bkz. Tablo malzeme). Not: İnsan 10-plex TH1/TH2 sitokin tahlil, 9-plex Kemokin tahlil ve tek-plex IL-17A, TGF-β1 ve TSLP burada yapıldı.<ol>
		<li>Gecede 4 ° C'de burun özleri deneyleri için kuluçkaya (Bkz. Tablo reçetesi) standart üretici protokol gibi ölçümleri yapmak. Not: Uzun ( Tablo reçetesigörmek) genellikle yüksek dinamik aralık 1 arasında değişen ölçüm için var &#38; 10.000 pg/mL, ama bazı deneyleri için 100.000 pg/mL olabilir. Bu tüm örnekleri, diğer benzer uzun olduğu gibi böylece bireylerde sağlıklı ve hastalıklı birbirine benzemeyen dilutions etkisini sınırlama aynı seyreltme çalıştırılabilir anlamına gelir. Tüm sitokinler için algılama alt sınırı 1 pg/mL ya da daha az ve kemokinler için 1 arasında değişmektedir &#38; 50 pg/mL. TSLP 1 ayda örnekleri % 98'i içinde tespit değildi.</li>
	</ol>
	</li>
</ol>
3. istatistik
<ol>
	<li>Günlük-analiz sitokin ve Kemokin düzeyleri normal dağılım kalıntıları elde etmek için önce verileri dönüştürme.</li>
	<li>Eğer birden fazla toplu iş iş işlemde örnekleri analiz edilir, batch-wise günlük dönüştürülmüş veriler üzerinde merkezleme için sitokin ve Kemokin verileri düzeltmek.<ol>
		<li>Genel ortalama ve özgün ölçü birimleriyle verileri elde etmek için anti-günlük dönüştürme dahil. Not: yapılan analizler her dizi için sitokinler ve kemokinler düzeyini etkileyen değişkenleri ayarlama göz önünde bulundurun. Bu anne etkileri (Örneğin, alkol tüketimi ve hamilelik sırasında sigara), emzirme, istenmeyen olaylar olabilir örnekleme, kardeşler, pets içinde ev, vb sırasında</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Bousquet, J., van Cauwenberge, P., Khaltaev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergic+Rhinitis+and+Its+Impact+on+Asthma.">Allergic Rhinitis and Its Impact on Asthma.</a> J Allergy Clin Immunol. 108 (5), 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+monitoring+of+nasal+airway+inflammation+in+rhinitis.">Objective monitoring of nasal airway inflammation in rhinitis.</a> J Allergy Clin Immunol. 115 (3), 414-441 (2005).</li><li>Bensch, G. W., Nelson, H. S., Borish, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cytokines+in+nasal+secretions+after+nasal+antigen+challenge:+lack+of+influence+of+antihistamines.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+and+safety+of+fexofenadine+HCl+and+pseudoephedrine,+alone+and+in+combination,+in+seasonal+allergic+rhinitis.">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., R&#248;meling, F., Mygind, N., Church, M., Holgate, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukotriene+C4+and+histamine+in+early+allergic+reaction+in+the+nose.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+assessing+unchallenged+levels+of+mediators+in+nasal+epithelial+lining+fluid.">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>F&#0248;lsgaard, N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+bacteria+colonizing+the+airways+in+asymptomatic+neonates+stimulates+topical+inflammatory+mediator+release.">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>F&#0248;lsgaard, N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+Cytokine+Profile+in+the+Airway+Mucosal+Lining+Fluid+Is+Skewed+by+Maternal+Atopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Vitamin+D3+Supplementation+During+Pregnancy+on+Risk+of+Persistent+Wheeze+in+the+Offspring:+A+Randomized+Clinical+Trial.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Response+to+A(H1N1)pnd09+Vaccination+in+Pregnant+Women:+A+Single+Blinded+Randomized+Controlled+Trial.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picornavirus-Induced+Airway+Mucosa+Immune+Profile+in+Asymptomatic+Neonates.">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., F&#248;lsgaard, N. V., Rasmussen, M. A., Brix, S., Bisgaard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Siblings+Promote+a+Type+1/Type+17-oriented+immune+response+in+the+airways+of+asymptomatic+neonates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+technique+for+recovery+of+cytokines+from+inflammatory+sites+in+situ.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+bronchoscopic+microsample+probe+to+measure+the+biochemical+constituents+in+epithelial+lining+fluid+of+patients+with+acute+respiratory+distress+syndrome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevation+of+KL-6,+a+lung+epithelial+cell+marker,+in+plasma+and+epithelial+lining+fluid+in+acute+respiratory+distress+syndrome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin-1+level+in+epithelial+lining+fluid+of+patients+with+acute+respiratory+distress+syndrome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine-mediated+xanthine+oxidase+upregulation+in+chronic+obstructive+pulmonary+disease's+airways.">Cytokine-mediated xanthine oxidase upregulation in chronic obstructive pulmonary disease's airways.</a> Pulm Pharmacol Ther. 18 (4), 297-302 (2005).</li><li>Moeller, A., Franklin, P., Hall, G. L., Horak, F., Wildhaber, J. H., Stick, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+exhaled+breath+condensates+in+infants.">Measuring exhaled breath condensates in infants.</a> Pediatr Pulmonol. 41 (2), 184-187 (2006).</li></ol>

Tüm yazarlar hiçbir potansiyel, algılanan ya da gerçek çıkar çatışması bu yazının içeriği ile ilgili olarak ilan ediyorum. Finansmanı kuruluşları tasarım ve çalışma yürütülmesi herhangi bir rolü yoktu; toplanması, yönetim ve verilerin yorumlanması; veya hazırlık, gözden geçirme ve onay şekilde alt alta yazılmalıdır. Hiçbir ilaç firması çalışmaya dahil.

Burada sunulan tekniği ile biz çocuk için 1 ay daha önce bitmiş değil yaş gibi erken vivo içinde üst hava yolu Mukozal bağışıklık profilinde belirlemek başardık. Biz o varlığı belirli hava yolu bakteri ve picornaviruses7,11, yanı sıra diğer öncesi ve perinatal Etkilenmeler, Yenidoğan Bebeklerin hava yolu bağışıklık profilinde yansıtılmış gözlenen. Ayrıca, biz bu hava yolu bağışıklık profil verileri bir randomize mi...

Burun Mukozal astar sıvı üst hava yolu sistem sıvı kısmını yukarı yapar. Epitel ve mikroorganizmaların istila karşı savunma ilk satırı oluşturan bağışıklık hücreleri arasındaki etkileşimi türetilen arabulucu karmaşık matris oluşur. Nazal mukoza kolayca erişilebilir ve burun ve bronşlar1arasında güçlü bir işlevsel ve immünolojik ilişkisi. Bu bölümü çocukluk, astım ve alerjik rinit gibi aynı zamanda diğer solunum bozukluğu daha sonra hayatında daha yaygın bir dizi ortak olan solunum yolu hastalıkları ile ilgili olarak özel ilgi var.
Burada, örnek Mukozal burun boşluğuna sıvı astar bir filtre kağıt tabanlı, noninvaziv teknik yanı sıra bir sonraki çıkarma yordamı kullanarak, protein bazlı analitler filtre kağıtları önce onların miktar üzerinden elute için kullanılan bozulmamış bir yöntemin uygulanması açıklanmaktadır. Bu teknik, vivo sağlıklı bireyler ve bireylerin çeşitli solunum yolu hastalıkları ile bağışıklık imzaları elde etmek için kullanılabilir. Ayrıca, belirli bir bağışıklık imza için önemli Etkilenmeler incelemenize ve predictor veya aracı daha sonra hastalığı gelişme ise değerlendirmek mümkündür.
Mukozal astar sıvı daha önce nazal lavaj2kez bir burun meydan okuma testi ile nerede bir alerjen bir enflamatuar yanıt3,4uyarmak için yüksek seviyelerinde tanıtıldı önce gelmelidir tarafından elde edilmiştir. Ancak, nazal lavaj teknik genç çocuklarda mümkün değildir ve sonuçlar düzeyleri tahlil5algılama sınırın altına düşebilir seyreltilmiş aracı olarak zihnimi karıştıran bir bilinmeyen seyreltme faktörü tanıttı. Ayrıca, bilinmeyen seyreltme faktörü nedeniyle burun meydan okuma testleri ölçülen analit yanıtlarını bireyler, böylece burun lavaj tekniği ayarlama bir kohort kullanışlılığı sınırlayarak arasında karşılaştırılabilir değildir. Son olarak, alerjen meydan yalnızca duyarlilasmis bireylerde geçerlidir ve histamin sorun gibi başka sorunlar fizyolojik ilgili, potansiyel olarak arabulucu yayın tavan etkisi neden değildir. Bu sorunların sıvı ve analit düzeyleri bireysel salgı arası tek tek farkı etkileyen faktörler nerede Mukozal astar sıvı koleksiyonu için sunulan filtre kağıt tabanlı teknik hile.
Ayıklama işlemi sırasında analitler için tüm örnekleri özdeş birimi arabelleği eklenmesinden sonra filtre gazetelerden eluted. Bu tüm örnekleri benzer ex vivo seyreltme tercih eder. İzotonik tuz solüsyonu albümin tabanlı bir ayıklama arabellek ayıklama adım için kullanılır; Bu protein bazlı arabulucu çıkarma sağlar ve protein miktar önce eluted proteinlerin sonraki donma sırasında denatürasyon sınırlamak için stabilize. Ekstraksiyon aşamasında protein yıkımı önlemek için proteaz inhibitörleri bir kokteyl ayıklama arabelleğe eklenir.
Rahatsız edilmeden vivo içindemiktar için izin teknikleri uygulanması-Mukozal sitelerinde oluşturulan bağışıklık arabulucu olduğunu çok önemli. İlk olarak, vücudun en büyük immünolojik organ Mukozal sitesi oluşturur. İkincisi, burun konum birincil site hava maruz kalma ve1akciğerler solunum immünolojik yuvası için sıkı bir şekilde bağlı. Üçüncü olarak, bu önemli organ invaziv olmayan bir teknik ile ölçme imkanı olasılığı sağlık ve hastalığında solunum yolları ile ilgili olarak önemli mikrop-bağışıklık etkileşim ekseninde bir bolluk bilgi sağlamak için açılır. Dördüncü, bu tekniğin randomize, kontrollü çalışmalarda yerel immünolojik değişiklikler uyuşturucu ve mikro eğitim gibi diğer birçok olası uygulama vardır.
Biz başlangıçta teknik astım tarihinde Kopenhag prospektif çalışmalarda çocukluk2000 (COPSAC2000) kohort Mukozal astar sıvı sağlıklı kontrol13karşı alerjik rinit ile 7 yaşındaki çocuklarda bağışıklık profil belirlendiği yerde, hayata. Daha sonra biz başarılı bir şekilde bu teknik boyuna COPSAC2010 kohort ve biçilen hava yolu bağışıklık profilleri için 1 ay, 2 yıl ve 6 yaşın ve akut solunum yolu semptomları örneklerini uygulanır. Sonuçlar 1 aylık Yenidoğan Bebeklerin bağışıklık imza ve gençliği çevre pozlama7,8,9,10,11,12arasındaki önemli ilişkileri gösterdi.

<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

Bu iletişim kuralı rahatsız edilmeden üst solunum yolu Mukozal astar sıvı noninvaziv örnekleme açıklar. Ayrıca sıvı eluates bağışıklık arabulucu analizini önce (genellikle diğer teknikleri tarafından kullanılan) stimülasyon yordamlar için gerek kalmadan havayolu topikal bağışıklık imza çalışma için kullanılan ayıklama yordam ayrıntıları. Mukozal astar sıvı bir şerit filtre kağıdı üzerindeki örneklenir alt konka ve emme 2 min için sol ön kısmına yerleştirilmiş. Analitlerin filtre gazetelerden eluted ve ayıklanan protein bazlı eluates düşük ve yüksek seviye analitler aynı örnek içinde yüksek-duyarlı miktar için izin veren bir electrochemiluminescence tabanlı immunoassay tarafından incelenir. Biz 20 önceden seçilmiş bağışıklık arabulucu yolları üst solunum yolu mukozasında sinyal belirli bağışıklık ile ilgili vivo içinde düzeyleri ölçüldü, ama tekniği o belirli paneli veya örnekleme sitesine sınırlı değildir. Bu teknik ilk 7 yaşındaki çocuklarda astım üzerine Kopenhag prospektif çalışmalar çocukluk2000 (COPSAC2000) kohort alerjik rinit ile uygulanmıştır. Bundan sonra 1 ay, 2 yıl ve 6 yaşın ve akut solunum yolu semptomları örneklerini örneklenmiş boyuna COPSAC2010 doğum kohort, kullanıldı. Biz başarıyla elde edilen ve 620 (% 89) 700 1 aylık çocukların örnekleri analiz; (Medyan (arası DÖRTTEBİRLİK aralığı (IQR)) bildirdi. tahlil algılama sınırın altındaki birkaç edildi Her aracı için (Yani 0 algılama alt sınırı için set noktasına) algılama sınırın altındaki örnekleri sayısı 29 (7.25-119.5) oldu. Bu teknik doğum içinde vivo hava yolu Mukozal bağışıklık profilden miktar sağlar, boyuna uygulanabilir ve çalışmalar genetik ve gençliği çevre pozlama, patofizyolojisi, endotyping ve solunum yolu hastalıkları ve geliştirme ve roman therapeutics değerlendirilmesi izleme etkisi üzerinde uygulanabilir.

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

Bu iletişim kuralı için üst solunum yolları rahatsız edilmeden Mukozal astar sıvı örnekleme invaziv olmayan bir teknik anlatılmaktadır. Vivo düzeyleri sitokinler ve kemokinler, gibi protein arabulucu miktar her yaştan bireylerde gerçekleştirmek için kullanılabilir.

Mukozal astar sıvı noninvaziv örnekleme için In Vivo üst solunum yolu bağışıklık arabulucu düzeyleri miktar

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

Universidad de Cartagena UDEC

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

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

10.2K Görüntüleme.  University of Copenhagen. Bu iletişim kuralı için üst solunum yolları rahatsız edilmeden Mukozal astar sıvı örnekleme invaziv olmayan bir teknik anlatılmaktadır. Vivo düzeyleri sitokinler ve kemokinler, gibi protein arabulucu miktar her yaştan bireylerde gerçekleştirmek için kullanılabilir.

Video: 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.

Alerjen Bağlı Astım murin Modeli

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, ...

İmmünoloji ve Enfeksiyon

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.

Primer ınsan hava yolu epitel hücrelerinin hava yolu yüzeyi sıvı pH 'Sı gerçek zamanlı, yarı otomatik floresan ölçümü

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

Biyokimya

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.

Havayolu Direnci Ölçümleri ve Fare Bronkoalveolar Lavaj Sıvısı Örnekleme için, geri dönüşümlü, Non-invaziv Yöntemi

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

Biyoloji

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Alternatifi olarak dilaltı immünoterapi Akut Solunum Enfeksiyonları Karşı Koruma tetikleyebilecek

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Tıp

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.

İmünohistokimya ve Akım Sitometri Kullanımı Endobronşiyal Biyopsilerinde Mekansal Dağılımı ve Fenotipik Kimlik: İnsan Akciğer dendritik hücrelerin

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.

İnflamatuvar Hücre İnfiltrasyonunu İncelemek İçin Murin Akciğerlerin Bronkoalveoler Lavajı

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.

Kapalı çevrim atalet Havacilik kullanarak hasta kaynaklı hava yolu salgı dan etiket içermeyen nötrofil zenginleştirme

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.

Burun ve bronş sıvıları emilimini: hassas örnekleme insan solunum mukoza ve laboratuvar işleme örnekleri

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

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.

Metodoloji balgam indüksiyon ve laboratuvar için

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

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.