Esprimiamo la nostra gratitudine per i bambini e le famiglie dello studio di coorte COPSAC2010 per tutto il loro sostegno e impegno. Riconosciamo e apprezzare gli sforzi unici della squadra di ricerca COPSAC e l'aiuto tecnico da tecnico Lisbeth Buus Rosholm, Technical University of Denmark, per la misurazione di citochine e chemochine.

Gli studi sono stati condotti in conformità con i principi della dichiarazione di Helsinki. Sono state ottenute le approvazioni dal comitato etico per Copenhagen (KF 01-289/96 per COPSAC2000 ) e H-B-2008-093 per COPSAC2010e l'Agenzia danese per la protezione dei dati e consenso informato è stato ottenuto da entrambi i genitori di ogni soggetto prima dell'iscrizione.
1. organizzazione sperimentale
<ol>
	<li>Utilizzare fogli di carta da filtro (fibrosi idrossilati-poliestere fogli, vedere la Tabella materiali)6,7.<ol>
		<li>Tagliare le strisce 3 x 15 mm di dimensione per un 1 mese-vecchio bambino e a forma di L per ragazzi ed adulti (5 x 20 x 10 mm (braccio corto della L)).</li>
	</ol>
	</li>
	<li>Inserire un pezzo di carta da filtro in ciascuna narice di un infante usando il forcipe. Posizionare il filtro di carta sulla parte anteriore del Turbinato inferiore (circa 1 cm dentro la narice per neonati e 1,5 cm per tutte le altre età).<ol>
		<li>Posizionare neonati su un letto e dare loro acqua e zucchero per il comfort. Nota: Per i bambini di tutte le altre età, il campionamento viene eseguito più facilmente con il soggetto seduto su una sedia.</li>
		<li>Per bambini più grandi, è necessario inserire il braccio lungo della carta da filtro a forma di L in ogni narice.</li>
	</ol>
	</li>
	<li>Applicare una clip naso intorno alle narici per ridurre al minimo il disagio e per evitare la perdita accidentale della carta da filtro.</li>
	<li>Rimuovere la carta da filtro dopo 2 min di assorbimento.</li>
	<li>Posizionare la carta da filtro in provette e congelarli immediatamente a-80 ° C.</li>
	<li>Prendere nota di qualsiasi sintomo di infezione delle vie respiratorie indicati dal bambino il giorno del campionamento.</li>
	<li>Registrare qualsiasi starnuto, pianto persistente o epistassi che si verifica durante i 2 min di campionamento.</li>
</ol>
2. quantificazione delle vie respiratorie immunitario-mediatori
<ol>
	<li>Prendere fino a 10 campioni casuali dal freezer. Registrare i numeri di identificazione. Mantenere i campioni sul ghiaccio durante l'intero processo di lavoro.</li>
	<li>Dopo lo scongelamento sul ghiaccio, immergere la carta da filtro (per soggetto) da entrambe le narici a 300 µ l di preparati tampone contenente una compressa da inibitore della proteasi completa (Vedi la Tabella materiali) (Vedi la Tabella materiali) per 25 mL di buffer.<ol>
		<li>Regolare il volume di tampone secondo il formato della carta da filtro.<ol>
			<li>Utilizzare 300 µ l di tampone per filtri di carta 3 x 15 millimetri nel formato.</li>
			<li>Se solo una carta da filtro è disponibile, utilizzare la metà del volume di tampone (150 µ l).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Trasferire i campioni di un agitatore per piastre (400 giri/min) per 5 min, uso un timer.</li>
	<li>Trasferire i filtri di carta umidi e tampone del saggio nella tazza di un filtro di tubo di acetato di cellulosa (dimensione dei pori di 0.22 µm) collocato all'interno di una microcentrifuga tubo (vedere la Tabella materiali).</li>
	<li>Centrifugare a 16.000 x g per 5 min in una centrifuga refrigerata (a 4 ° C) per ottenere Estratto filtrato nasale nel tubo.</li>
	<li>Rimuovere la Coppa e mantenere i tubi sul ghiaccio mentre aliquotare l'Estratto nasale nei pozzetti di piastre di deposito associazione basso contenuto proteico (Vedi la Tabella materiali).</li>
	<li>Conservare a-80 ° C fino all'analisi.</li>
	<li>Determinare le concentrazioni di citochine e chemochine negli estratti nasali utilizzando un sistema ad alta sensibilità, electrochemiluminesce-based array multiplex (Vedi la Tabella materiali). Nota: Un'analisi di umano 10-plex TH1/TH2 cytokine, 9-plex chemokine analisi e IL singolo-plex-17A, TGF-β1 e TSLP sono stati eseguiti qui.<ol>
		<li>Per i dosaggi, Incubare gli estratti nasali durante la notte a 4 ° C. Condurre le misurazioni per il costruttore standard protocollo (Vedi la Tabella materiali). Nota: I test immunologici (Vedi la Tabella materiali) in genere hanno un'alta gamma dinamica per misurazione compreso tra il 1 &#38; 10.000 pg/mL, ma per alcuni test, può essere 100.000 pg/mL. Ciò significa che tutti i campioni possono essere eseguiti con la stessa diluizione, limitando così l'influenza delle diluizioni dissimili in soggetti sani e malati, come è il caso in altri simili test immunologici. Il limite inferiore di rilevamento per tutte le citochine era 1 pg/mL o meno e per chemochine, esso ha variato fra 1 &#38; 50 pg/mL. TSLP non era rilevabile nel 98% dei campioni a 1 mese di età.</li>
	</ol>
	</li>
</ol>
3. statistiche
<ol>
	<li>Registro-trasformare i dati prima dell'analisi per ottenere distribuiti normalmente residui dei livelli di citochine e chemochine.</li>
	<li>Se i campioni sono analizzati in più di un batch, correggere i dati di citochine e chemochine per centraggio per singoli batch sui dati registro-ha trasformato.<ol>
		<li>Incorporare la media complessiva e la trasformazione di anti-registro per ottenere i dati nelle unità di misura originale. Nota: Per ogni serie di analisi fatto, si consiglia di regolare le variabili che influiscono sul livello di citochine e chemochine. Questo potrebbe essere influenze materne (ad esempio, il consumo di alcol e fumo durante la gravidanza), l'allattamento al seno, gli eventi avversi durante il campionamento, fratelli, animali domestici nella casa, ecc.</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>

Tutti gli autori non dichiarano alcun conflitto di interessi potenziale, percepita o reale per quanto riguarda il contenuto di questo manoscritto. Le agenzie di finanziamento non hanno avuto alcun ruolo nell'ideazione e la conduzione dello studio; la raccolta, la gestione e l'interpretazione dei dati; o la preparazione, revisione e approvazione del manoscritto. Nessuna azienda farmaceutica è stato coinvolta nello studio.

Con la tecnica presentata qui, siamo stati in grado di determinare l'in vivo della superiore-via aerea mucoso profilo immunitario nei bambini da più presto 1 mese di età, che non è stato fatto in precedenza. Abbiamo osservato che la presenza di batteri specifici delle vie respiratorie e picornavirus7,11, così come altri pre e perinatale esposizioni, sono state rispecchiate nel profilo immunitario delle vie respiratorie dei neonati. Inoltre, abbiamo u...

Il liquido di rivestimento mucoso del naso costituisce la parte liquida del sistema della superiore-via aerea. Si compone di una matrice complessa di mediatori derivati dall'interazione tra l'epitelio e cellule immunitarie che compongono la prima linea di difesa contro l'invasione di microrganismi. La mucosa nasale è facilmente accessibile, e c'è una forte relazione funzionale ed immunologica tra il naso e bronchi1. Questo comparto è di particolare interesse in relazione a malattie delle vie respiratorie che sono comuni nell'infanzia, come l'asma e la rinite allergica, ma anche a una serie di altri disturbi respiratori più prevalente più tardi nella vita.
Qui, descriviamo l'implementazione di un metodo a campione indisturbato mucoso rivestimento liquido dalla cavità nasale usando una tecnica non invasiva basata su carta da filtro, così come una procedura di estrazione successiva, utilizzato per eluire basati su proteine analiti dalla carta da filtro prima della loro quantificazione. Questa tecnica, ad esempio, consente di ottenere in vivo immuni firme di individui sani e di individui con le varie malattie respiratorie. Inoltre, è possibile esaminare le esposizioni di importanza per una firma immune specifica e valutare se è un preannunciatore o mediatore di sviluppo successivo di malattia.
Liquido di rivestimento mucoso è stata ottenuta in precedenza da lavaggio nasale2, che è spesso preceduto da una prova nasale di sfida, dove un allergene è stato introdotto in alti livelli di stimolare una risposta infiammatoria3,4. Tuttavia, la tecnica di lavaggio nasale non è fattibile nei bambini piccoli e introduce un fattore di diluizione sconosciuto, che confonde gli esiti, come mediatore diluito livelli possono scendere sotto il limite di rilevamento del test5. Inoltre, a causa del fattore di diluizione sconosciuto, le risposte di analita misurato dal test di provocazione nasale non sono comparabili tra individui, limitando così l'utilità della tecnica del lavaggio nasale in una coorte di impostazione. Infine, la sfida dell'allergene è applicabile solo in soggetti sensibilizzati, e altre sfide, come la sfida di istamina, non sono fisiologicamente rilevanti, potenzialmente causando un effetto soffitto sul rilascio del Mediatore. Questi problemi vengono eluse la tecnica basata su carta da filtro presentato per accumulazione fluida rivestimento mucoso, dove i singola secrezione di fluidi e i suoi livelli analitici sono gli unici fattori che influenzano la varianza inter-individuale.
Durante la procedura di estrazione, analiti vengono eluiti dai giornali filtro dopo l'aggiunta di volumi identici del buffer per tutti i campioni. Questo favorisce la diluizione simile ex vivo di tutti i campioni. Un tampone di estrazione di soluzione salina isotonica basata su albumina viene utilizzata per la fase di estrazione; consente l'estrazione dei mediatori a base di proteine e stabilizza le proteine per limitare la denaturazione durante il successivo congelamento delle proteine eluite prima della quantificazione. Per evitare la degradazione della proteina durante la fase di estrazione, viene aggiunto un cocktail di inibitori della proteasi per il tampone di estrazione.
L'attuazione di tecniche che consentono per la quantificazione del indisturbati, in vivo-generati mediatori immuni alle mucose siti è della massima importanza. In primo luogo, il sito della mucosa costituisce il più grande organo immunologico nel corpo. In secondo luogo, la posizione nasale è il sito primario di esposizione dispersa nell'aria ed è strettamente collegata al vano respiratorie immunologico del polmoni1. In terzo luogo, la possibilità di rilievo di questo importante organo con una tecnica non invasiva apre la possibilità di fornire una pletora di informazioni sull'asse di interazione di microbo-immunitario importante in materia di salute e malattia delle vie aeree. In quarto luogo, ci sono molte altre possibili applicazioni di questa tecnica, come studiare le alterazioni immunologiche locali in randomizzato, studi controllati di farmaci e micronutrienti.
Inizialmente abbiamo implementato la tecnica in studi prospettivi Copenaghen sull'asma nella coorte di infanzia2000 (COPSAC2000), dove abbiamo determinato il profilo immunitario del rivestimento mucoso fluido nel 7-anno-vecchi bambini con rinite allergica rispetto a controlli sani13. Successivamente, abbiamo applicato con successo questa tecnica al gruppo di2010 COPSAC longitudinale e profili immuni valutati delle vie respiratorie a 1 mese, 2 anni e 6 anni di età e alle istanze di sintomi respiratori acuti. Risultati dai neonati 1-mese-vecchio hanno dimostrato importanti associazioni tra la firma immune e l'esposizione ambientale di presto-vita7,8,9,10,11,12.

<table border="0" cellpadding="0" cellspacing="0" width="1688">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Fibrous hydroxylatedpolyester sheets</td>
			<td style="width:103px;">Accuwik Ultra&#160;</td>
			<td style="width:119px;">SPR0730</td>
			<td style="width:1084px;">Filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milliplex Assay Buffer&#160;</td>
			<td>Millipore</td>
			<td>L-AB</td>
			<td>Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">low-protein binding storage plates&#160;</td>
			<td>Thermo Scientific</td>
			<td>CLS8161</td>
			<td>Plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor</td>
			<td style="width:103px;">Roche</td>
			<td align="right" style="width:119px;">11873580001</td>
			<td>complete EDTA-free Protease Inhibitor Cocktail</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Reader of multi-spot plates</td>
			<td style="width:103px;">Mesoscale</td>
			<td style="width:119px;">NA</td>
			<td>Sector imager 6000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Assays&#160;</td>
			<td style="width:103px;">Mesoscale</td>
			<td style="width:119px;"></td>
			<td>Human 10-plex TH1/TH2 cytokine assay and 9-plex chemokine assay, and singleplex IL-17A, TGF-&#946;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

Questo protocollo descrive campionamento non invadente di liquido di rivestimento mucoso delle vie respiratorie superiore indisturbato. E ' anche in dettaglio la procedura di estrazione utilizzata prima dell'analisi di mediatori immuni in eluati fluidi per lo studio della firma immuni topica delle vie aeree, senza la necessità di procedure di stimolazione (spesso utilizzato da altre tecniche). Il liquido di rivestimento mucoso viene campionato su una striscia di carta da filtro posizionato sulla parte anteriore del inferior turbinate e sinistro per 2 min di assorbimento. Analiti vengono eluiti dalla carta da filtro, e gli eluati di base di proteine estratti vengono analizzati da un immunoassay elettrochemiluminescenza-based, che consente per la quantificazione di alto-sensibilità di analiti a basso e alto livello nello stesso campione. Abbiamo misurato i livelli in vivo di 20 mediatori immuni preselezionati legate alla specifica immunitario vie nella mucosa delle vie respiratorie superiore di segnalazione, ma la tecnica non è limitata a quel pannello specifico o il sito di campionamento. La tecnica innanzitutto è stata implementata nei bambini di 7-year-old da studi prospettivi Copenaghen sull'asma nella coorte di2000 (COPSAC2000) di infanzia con rinite allergica. Da allora in poi è stato usato nella coorte di nascita2010 COPSAC longitudinale, provata a 1 mese, 2 anni e 6 anni di età e alle istanze di sintomi respiratori acuti. Abbiamo correttamente acquisiti e analizzati campioni da 620 (89%) di 700 1-mese-vecchi bambini; alcuni campioni erano sotto il limite di rilevamento del test (segnalato come la mediana (Inter-Quartile Range (IQR)). Il numero di campioni di sotto del limite di rilevazione (cioè da 0 al set point per il limite inferiore di rilevamento) per ogni Mediatore era 29 (7,25-119.5). Questa tecnica consente la quantificazione del profilo di in vivo delle vie respiratorie mucose immuni dalla nascita, può essere applicata longitudinalmente e può essere applicata agli studi sull'effetto di genetica e le esposizioni ambientali di presto-vita, patofisiologia, endotyping e il monitoraggio di malattie respiratorie, sviluppo e valutazione di nuove terapie.

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

Questo protocollo descrive una tecnica non invasiva per il campionamento del liquido di rivestimento mucoso indisturbati dalle vie respiratorie superiori. Può essere utilizzato per eseguire la quantificazione dei livelli in vivo dei mediatori di proteina, quali citochine e chemochine, in soggetti di tutte le età.

Campionamento non invasivo del rivestimento mucoso fluido per la quantificazione dei livelli di Immune-mediatore In Vivo delle vie respiratorie superiore

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

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 Views.  University of Copenhagen. Questo protocollo descrive una tecnica non invasiva per il campionamento del liquido di rivestimento mucoso indisturbati dalle vie respiratorie superiori. Può essere utilizzato per eseguire la quantificazione dei livelli in vivo dei mediatori di proteina, quali citochine e chemochine, in soggetti di tutte le età.

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

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Test di cocultura per valutare la migrazione transeteliale neutrofila indotta da agenti patogeni

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Ricerca

Immunologia e infezioni

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Dissezione Innate Immune Signaling in Evasion virale di produzione di citochine

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Modellazione mucosa Candidosi in Zebrafish larvale da vescica natatoria Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Intranasale somministrazione di ricombinante Influenza vaccini nei modelli chimerico topo per studiare immunità delle mucose

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Intra-tracheale Amministrazione<em&gt; Haemophilus influenzae</em&gt; In modelli murini per studiare infiammazione delle vie aeree

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Tampone Metodo di campionamento per la rilevazione di Norovirus umana sulle superfici

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Formazione di Biofilm indotta da sali biliari in patogeni enterici: tecniche di identificazione e quantificazione

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantificazione del fagosoma da microscopia di fluorescenza di cella singola

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Quantificazione dell'attività chemiotatica monocite in vivo e caratterizzazione dei macrofagi derivati monociti del sangue

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.