Vorremmo ringraziare Kerstin Thim (AstraZeneca, Lund, Svezia), Benita Dahlberg e Dr. Anders Eklund (Karolinska Institutet, Stoccolma, Svezia) e Dr. Martin Stampfli (McMaster University, Hamilton, ON, Canada) per l&#39;assistenza abile e la consulenza di esperti.

Gli studi su animali sono state approvate dal comitato etico del benessere degli animali del nord di Stoccolma. Le procedure sperimentali sono state eseguite in conformità con il diritto svedese. 1. Creazione di un LPS Aerosol <ol><li> Sciogliere 0,5 g purificato P. aeruginosa LPS in 50 ml di soluzione fisiologica sterile e scuotendo con cautela e verificare la dissoluzione. Diluire 1 ml LPS disciolti in 9 ml salina sterile ad una concentrazione finale di 1 mg / ml. Proteggere dalla luce con un foglio di alluminio e conservare a -20 ° C. </li><li> Scongelare LPS solubilizzate al buio a temperatura ambiente e mescolare bene immediatamente prima dell&#39;uso. </li><li> In una cappa rischio biologico ventilata livello II, inserire un ingresso rosso in un nebulizzatore, e collegare il nebulizzatore ad aria ambiente pressurizzata attraverso il tubo fornito dal (regime revisione presentando i dispositivi sperimentali in figura 1) produttore. ATTENZIONE: attrezzature protettive adeguate, tra cui un mezzo facciale respirat riutilizzabileo con filtri antiparticolato, occhiali, guanti e indumenti protettivi dovrebbero essere utilizzati nel corso dell&#39;esposizione. </li></ol><img alt="Figura 1" src="/files/ftp_upload/51470/51470fig1highres.jpg" /> Figura 1:. Rappresentazione schematica dei dispositivi sperimentali utilizzati per generare un aerosol L&#39;ingresso del nebulizzatore è collegato ad un&#39;alimentazione di aria. L&#39;uscita del nebulizzatore viene collegato a un flussometro attraverso un tubo di 15,9 millimetri e un filtro dell&#39;aria, e la fornitura di aria viene regolato a 5,0 L / m 2 a pressione kbar. L&#39;uscita è vicino collegato a una scatola in plexiglas dotato di coperchi rimovibili e fori da 5 mm per evitare l&#39;accumulo di pressione. <ol start="4"><li> Collegare l&#39;uscita del nebulizzatore ad un flussimetro di massa tramite un filtro dell&#39;aria. Collegare il flussimetro di massa ad un&#39;alimentazione elettrica. </li><li> Regolare l&#39;alimentazione di aria a 5 l / min, con una pressione rimanendo a 1,0-2,0 bar. </li><li> Rimuovere il misuratore di portata e di massascollegare l&#39;alimentazione dell&#39;aria. </li><li> Collegare l&#39;uscita del nebulizzatore ad un tubo 15,9 millimetri, che si biforca e si collega a due scatole plexiglas con le dimensioni: 150 x 163 x 205 mm, provvista di coperchi amovibili. Ogni scatola dovrebbe avere un foro 5 mm sul lato opposti l&#39;ingresso, per evitare l&#39;accumulo di pressione. </li><li> Luogo fino a 5 topi in ogni scatola di plexiglas e chiudere le palpebre. </li><li> Aprire il nebulizzatore e riempire l&#39;inserto con almeno 4 ml LPS disciolti in soluzione fisiologica sterile o solo veicolo (soluzione fisiologica sterile, il volume non dovrebbe superare 8 ml). Ri-collegare l&#39;ingresso per l&#39;aria di alimentazione. </li><li> Consentire l&#39;aerosol di fluire nelle caselle plexiglas chiuso per 10 min. Monitorare gli animali continua. Assicurarsi che l&#39;alimentazione dell&#39;aria rimane saldamente assicurato all&#39;ingresso del nebulizzatore. </li><li> Staccare l&#39;alimentazione dell&#39;aria. Con i coperchi chiuso, lasciare che gli animali rimangono nelle caselle plexiglas per 2 min. </li><li> Aprire i coperchi e consentire l&#39;aerosol a disperdere, e restituire agli animali di tlui gabbie. Se gli animali appaiono bagnati, posizionare le gabbie su una piastra elettrica impostato su fuoco basso, per evitare l&#39;ipotermia. </li><li> Monitorare gli animali in continuo per i primi 30 minuti, e successivamente ogni 2 ore per le prime 6 ore. Gli animali devono presentare alterazioni della respirazione normale e l&#39;attività durante e dopo la procedura di nebulizzazione. </li></ol> 2. lavaggio broncoalveolare (BAL) <ol><li> Al punto finale sperimentale, profondamente anestetizzare gli animali con isoflurano ad effetto come raccomandato dal personale veterinario del proprio istituto. Pizzica la zampa posteriore per controllare un riflesso di ritiro per garantire una sufficiente profondità dell&#39;anestesia per condurre interventi di chirurgia maggiore. Spray giù la pelliccia degli animali con il 70% di etanolo. </li><li> Aprire l&#39;addome con le forbici, e tagliare l&#39;aorta per Exsanguinate l&#39;animale. Posizionare un pezzo di tessuto sopra l&#39;addome per assorbire il sangue. </li><li> Follwing eutanasia, utilizzare un unico taglio antero-posteriore delle forbici peresporre il torace. Sollevare la gabbia toracica dalla punta anteriore dello sterno e usare le forbici per forare il diaframma nel punto più ventrale, senza intaccare alcun lobo polmonare. Aprire la gabbia toracica, facendo due tagli in direzione antero-posteriore (riunioni sotto la mascella). </li><li> Tirare delicatamente a parte la gabbia toracica con pinze, e tagliare la trachea sotto la laringe. </li><li> Sollevare la trachea con le pinze e rimuovere i polmoni tagliando i legamenti che collegano i lobi alla cavità toracica, e tirando delicatamente dal adiposo e tessuto cardiaco. </li><li> Inserire un tubo di polietilene (diametro interno: 0,58 millimetri; diametro esterno: 0,965 millimetri) nella trachea e fissare il tubo con una stringa di filo di seta. </li><li> Legare il multilobe (i quattro lobi del polmone destro) con filo di seta. </li><li> Inserire un ago calibro 23 nel tubo di polietilene e iniettare lentamente 250 ml di ghiaccio freddo PBS sterile nel singolo lobo con una siringa da 1 ml. </li><li> Toccare con attenzione i polmoni30 volte e raccogliere il liquido attraverso la siringa. Ripetere la procedura con 200 microlitri di PBS (circa 300 ml PBS deve essere recuperato dal singolo lobo dal volume totale iniettato 450 ml). </li><li> Tenere il liquido di lavaggio broncoalveolare (BAL) su ghiaccio, o elencare immediatamente le cellule BAL. Contare le cellule con un heamocytometer, usando la soluzione Turk per colorare le cellule 11. Calcolare il numero totale di cellule moltiplicando il numero di cellule con il fattore di diluizione della soluzione colorante e il volume nel campo conteggiato del heamocytometer. </li><li> Preparare la conta cellulare differenziata dalle cellule cytocentrifuged come descritto altrove 11. </li></ol> 3. formalina Fissazione di tessuto polmonare per la valutazione istologica <ol><li> Rimuovere il multilobe e snap-freeze in ghiaccio secco. Conservare a -80 ° C. </li><li> Montare una siringa da 60 ml con lo stantuffo rimosso su un cavalletto di sostegno metallico. Riempire la siringa con il 10% di formalina perl&#39;altezza di 20 cm sopra il banco di laboratorio, in rappresentanza di 20 cm di pressione costante. </li><li> Collegare l&#39;ago calibro 23 fissata alla trachea alla siringa 60 ml con un tubo di plastica con una valvola per controllare il flusso di formalina. </li><li> Insufflare il lobo polmonare con formalina per 5 min. Scollegare l&#39;ago e rimuoverlo insieme al tubo di polietilene dalla trachea tirando sul filo di seta per chiudere la trachea e mantenere la pressione nel polmone. </li><li> Immergere il lobo polmonare in formalina e fissare per 24 ore a 4 ° C. </li><li> Lavare il tessuto fissato tre volte per almeno 20 min con 70% di etanolo, e disidratare a xilene attraverso il seguente schema (1 ora ciascuno): <ul><li> 3x 70% di etanolo </li><li> 3x 95% di etanolo </li><li> 3x 100% di etanolo </li><li> 3x xilene </li><li> 1x (liquido) paraffina </li></ul></li><li> Tessuto Incorpora disidratati in paraffina, tagliato a 4-5 micron sezioni, e macchia con ematossilina e eosina per consentire la valutazione istologica.</li>
</ol>

<ol>
	<li>King, J. D., Kocincova, D., Westman, E. L., Lam, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Lipopolysaccharide+biosynthesis+in+Pseudomonas+aeruginosa.">Review: Lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.</a> Innate Immun. 15, 261-312 (2009).</li><li>Grommes, J., Soehnlein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+neutrophils+to+acute+lung+injury.">Contribution of neutrophils to acute lung injury.</a> Mol Med. 17, 293-307 (2011).</li><li>Pesci, 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=Inflammatory+cells+and+mediators+in+bronchial+lavage+of+patients+with+chronic+obstructive+pulmonary+disease.">Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease.</a> Eur Respir J. 12, 380-386 (1998).</li><li>Hoogerwerf, J. J., 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=Lung+Inflammation+Induced+by+Lipoteichoic+Acid+or+Lipopolysaccharide+in+Humans.">Lung Inflammation Induced by Lipoteichoic Acid or Lipopolysaccharide in Humans.</a> Am. J. Respir. Crit. Care Med. 178, 34-41 (2008).</li><li>Scheuchenzuber, W. J., Eskew, M. L., Zarkower, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+humoral+responses+to+Escherichia+coli+and+sheep+red+blood+cell+antigens+introduced+via+the+respiratory+tract.">Comparative humoral responses to Escherichia coli and sheep red blood cell antigens introduced via the respiratory tract.</a> Exp Lung Res. 13, 97-112 (1987).</li><li>Asti, C., 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=Lipopolysaccharide-induced+lung+injury+in+mice.+I.+Concomitant+evaluation+of+inflammatory+cells+and+haemorrhagic+lung+damage.">Lipopolysaccharide-induced lung injury in mice. I. Concomitant evaluation of inflammatory cells and haemorrhagic lung damage.</a> Pulm Pharmacol Ther. 13, 61-69 (2000).</li><li>Brand, P., 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=Total+deposition+of+therapeutic+particles+during+spontaneous+and+controlled+inhalations.">Total deposition of therapeutic particles during spontaneous and controlled inhalations.</a> Journal of pharmaceutical sciences. 89, 724-731 (2000).</li><li>Liu, F., Li, W., Pauluhn, J., Trubel, H., Wang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+acute+lung+injury+in+rats:+comparative+assessment+of+intratracheal+instillation+and+aerosol+inhalation.">Lipopolysaccharide-induced acute lung injury in rats: comparative assessment of intratracheal instillation and aerosol inhalation.</a> Toxicology. 304, 158-166 (2013).</li><li>Skerrett, S. J., 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=Role+of+the+type+1+TNF+receptor+in+lung+inflammation+after+inhalation+of+endotoxin+or+Pseudomonas+aeruginosa.">Role of the type 1 TNF receptor in lung inflammation after inhalation of endotoxin or Pseudomonas aeruginosa.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 276, L715-L727 (1999).</li><li>Roos, A. B., 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=Lung+epithelial-C/EBPbeta+contributes+to+LPS-induced+inflammation+and+its+suppression+by+formoterol.">Lung epithelial-C/EBPbeta contributes to LPS-induced inflammation and its suppression by formoterol.</a> Biochem Biophys Res Commun. 423, 134-139 (2012).</li><li>Didon, 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=Lung+epithelial+CCAAT/enhancer-binding+protein-beta+is+necessary+for+the+integrity+of+inflammatory+responses+to+cigarette+smoke.">Lung epithelial CCAAT/enhancer-binding protein-beta is necessary for the integrity of inflammatory responses to cigarette smoke.</a> Am J Respir Crit Care Med. 184, 233-242 (2011).</li><li>Silverpil, E., 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=Negative+feedback+on+IL-23+exerted+by+IL-17A+during+pulmonary+inflammation.">Negative feedback on IL-23 exerted by IL-17A during pulmonary inflammation.</a> Innate Immunity. 19, 479-492 (2013).</li><li>Mercer, P. F., 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=Proteinase-Activated+Receptor-1,+CCL2+and+CCL7+Regulate+Acute+Neutrophilic+Lung+Inflammation.">Proteinase-Activated Receptor-1, CCL2 and CCL7 Regulate Acute Neutrophilic Lung Inflammation.</a> American Journal of Respiratory Cell and Molecular Biology. , (2013).</li><li>Korkmaz, B., Horwitz, M. S., Jenne, D. E., Gauthier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases. Pharmacological Reviews. 62, 726-759 (2010).</li><li>Bafadhel, 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=Acute+Exacerbations+of+COPD:+Identification+of+Biological+Clusters+and+Their+Biomarkers.">Acute Exacerbations of COPD: Identification of Biological Clusters and Their Biomarkers.</a> Am. J. Respir. Crit. Care Med. , 201104-200597 (2011).</li><li>Hurst, J. R., Perera, W. R., Wilkinson, T. M. A., Donaldson, G. C., Donaldson, G. C., Wedzicha, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+and+Upper+and+Lower+Airway+Inflammation+at+Exacerbation+of+Chronic+Obstructive+Pulmonary.">Systemic and Upper and Lower Airway Inflammation at Exacerbation of Chronic Obstructive Pulmonary.</a> Am. J. Respir. Crit. Care Med. 173, 71-78 (2006).</li><li>Rabe, K. F., 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=Global+Strategy+for+the+Diagnosis,+Management,+and+Prevention+of+Chronic+Obstructive+Pulmonary+Disease:+GOLD+Executive+Summary.">Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary.</a> Am. J. Respir. Crit. Care Med. 176, 532-555 (2007).</li></ol>

Abraham Roos ha ricevuto i diritti docente da Boehringer Ingelheim, borse viaggio da AstraZeneca R &amp; S e di un assegno di ricerca da AstraZeneca R &amp; S. Magnus Nord è un dipendente a tempo pieno di AstraZeneca R &amp; S e ha ricevuto assegni di ricerca da AstraZeneca R &amp; S. Johan Grunewald e Tove Berg sono co-ricercatori su un progetto di ricerca finanziato dalla correlato AstraZeneca R &amp; S.

Inalato LPS genera una risposta infiammatoria nelle vie aeree, caratterizzata dai neutrofili nella sottomucosa epiteliale, spazi circostanti vie aeree di conduzione, nonché gli spazi alveolari. Questo è, unitamente alla maggiore contenuto proteico totale BALF, indicative di perdite plasma, rappresentativo della patologia del danno polmonare acuto. Come LPS induce una infiammazione sterili, la reazione è indipendente della risposta immunitaria adattativa, e ci sono limitazioni alla rilevanza alle infezioni batteriche....

Lipopolisaccaride (LPS) è un componente della parete cellulare di batteri gram negativi 1. Sfida a LPS è un modello ben documentato di danno polmonare acuto, una sindrome caratterizzata da infiammazione neutrofila acuta e l&#39;edema 2. Inoltre, neutrofilia polmonare è anche un segno distintivo della broncopneumopatia cronica ostruttiva (BPCO) 3, e LPS sfida negli esseri umani è stato utilizzato per modellare riacutizzazioni della BPCO 4. Così, modelli sperimentali di esposizione LPS sono clinicamente rilevanti e preziosi strumenti per comprendere la patologia umana. L&#39;obiettivo della consegna polmonare di LPS aerosol qui descritte è quello di generare una risposta infiammatoria neutrofili nella conduzione e vie respiratorie, senza coinvolgimento sistemico. Diverse tecniche di LPS sfida sono stati descritti in precedenza. Iniezione intra-venosa di LPS è il percorso più comunemente usato di somministrazione. Sebbene questa tecnica è facilmente accessibile, tche danno primario è all&#39;endotelio, distruzione secondaria dell&#39;epitelio polmonare conseguente migrazione dei neutrofili al polmone. Somministrazione intra-venosa induce anche l&#39;infiammazione sistemica 2, che può complicare il quadro clinico in modelli animali. Infiammazione sistemica è in contrasto non osservati con la somministrazione intra-tracheale. Questa tecnica, tuttavia, è laborioso e richiede anestetici e notevole formazione 5, 6. Inoltre, la deposizione polmonare da questa via di somministrazione dipende respirazione 7. Così, la deposizione polmonare è influenzato dalla profondità dell&#39;anestesia necessari per la somministrazione intra tracheale e la deposizione nelle vie aeree variabili possono essere osservate. Al contrario, la consegna polmonare con LPS aerosol richiede un addestramento minimo, e può essere facilmente realizzata su un gran numero di animali con poca o nessuna variazione tra individui 5, 8.Un recente studio conferma che la consegna aerosol è superiore al percorso intra-tracheale in materia di deposito, e che dosi più rilevanti di LPS induce infiammazione neutrofila con questo modello 8. Studi precedenti hanno dimostrato che sfida per aereosol Psuedomonas aeruginosa LPS genera una risposta infiammatoria marcato nel lume delle vie aeree e del parenchima polmonare, inclusi gli spazi alveolari 9, 10. L&#39;infiammazione è caratterizzata da una predominanza di neutrofili e presenza di edema polmonare, e può quindi essere utilizzato per affrontare patogenesi del danno polmonare acuto e acquisire ulteriori conoscenze dei meccanismi che contribuiscono alla patologia malattia.

<table border="0" cellpadding="0" cellspacing="0" style="width:931px;" width="929">
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	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Name of the material/equipment&#160;</td>
			<td style="width:244px;">Company</td>
			<td style="width:145px;">Catalog number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:307px;">Purified Pseudomonas aeruginosa LPS&#160;</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Harmful. Recomended purification. LPS purified from other bactria may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pari LC sprint star nebulizer</td>
			<td style="width:244px;">PARI Respiratory Equipment Inc.&#160;</td>
			<td style="width:145px;">023G1250</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">TSI mass flowmeter 4040</td>
			<td style="width:244px;">TSI</td>
			<td style="width:145px;">4040</td>
			<td style="width:235px;">Alternative product from supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Saint-Gobain 15.9 mm Tygon tube</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">Z685704</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Plexiglas boxes with removable lids</td>
			<td style="width:244px;">Custom built</td>
			<td style="width:145px;">N/A</td>
			<td style="width:235px;">150 x 163 x 205 mm (a 2 mm hole on the side).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Half Facepiece Reusable Respirator</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">7503</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Advanced Particulate Filters (P100)&#160;</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">2291</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sissors</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">233-1104</td>
			<td style="width:235px;">Preferred scissors may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Forceps&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">232-1313</td>
			<td style="width:235px;">Preferred forceps may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Intramedic PE50 polyethylene tube</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">427411</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ethicon 2-0 Perma-hand silk tread&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">95056-992</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">26 &#189;&#160; gage needle&#160;</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">1 mL BD slip-tip syringe, non-sterile</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=301025">301025</a></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">60 mL BD Luer-Lok syringe, non-sterile, polypropolene&#160;</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">301035</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fluka Hematoxylin-Eosin</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">3972</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T&#252;rk&#39;s solution</td>
			<td style="width:244px;">Merck Millipore</td>
			<td style="width:145px;">109277</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Table top centrifuge</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative manufacturers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytospin 4 cytocentrifuge</td>
			<td style="width:244px;">Thermo Scientific</td>
			<td style="width:145px;">A78300003</td>
			<td style="width:235px;">Alternative centrifuge can be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEMA-3 stat pack</td>
			<td style="width:244px;">Fisher Scientific</td>
			<td style="width:145px;">23-123-869</td>
			<td style="width:235px;">Alternative staining kits exists.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">HT501128&#160;</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
	</tbody>
</table>

a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide

Danno polmonare acuto (ALI) è una grave malattia caratterizzata da neutrofilia alveolare, con opzioni di trattamento limitate e alta mortalità. Modelli sperimentali di ALI sono fondamentali nel migliorare la nostra comprensione della patogenesi della malattia. Lipopolisaccaride (LPS) derivato da batteri Gram-positivi induce infiammazione neutrofila delle vie aeree e del parenchima polmonare dei topi. Consegna polmonare efficiente di composti quali LPS è tuttavia difficile da raggiungere. Nell&#39;approccio qui descritto, consegna polmonare nei topi si ottiene sfida di aerosol di Pseudomonas aeruginosa LPS. Disciolto LPS era aerosol da un nebulizzatore collegato ad aria compressa. I topi sono stati esposti a un flusso continuo di LPS aerosol in una scatola plexiglas per 10 minuti, seguito da 2 minuti condizionata, dopo l&#39;aerosol è stato interrotto. L&#39;intubazione tracheale e successivo lavaggio broncoalveolare, seguita da perfusione formalina è stata eseguita successiva, che consente caratterizzazione del p steriliinfiammazione ulmonary. Inalato LPS genera una infiammazione polmonare caratterizzata da neutrofilia alveolare, rilevata in lavaggio broncoalveolare e da una valutazione istologica. Questa tecnica può essere impostato ad un piccolo costo con pochi elettrodomestici, e richiede un minimo di formazione e competenza. Il sistema di esposizione può quindi essere eseguita di routine in qualsiasi laboratorio, con la possibilità di migliorare la nostra comprensione della patologia polmonare.

Sfida di aerosol P. aeruginosa LPS solito produce una risposta infiammatoria marcata nel lume delle vie aeree e lo spazio alveolare, caratterizzato da una predominanza di neutrofili in entrambi i punti temporali precoce e tardiva. Inalato LPS induce neutrofilia polmonare C57BL / 6BY e BALB / c topi sono stati esposti ad aerosol P. aeruginosa LPS o solo veicolo e neutrofili sono stati enumerati in BALF. Il numero totale di cellule in BALF di C57BL /...

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

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Descriviamo un metodo per indurre l&#39;infiammazione polmonare neutrofila dalla sfida di lipopolysaccharide aerosol per nebulizzazione, per modellare danno polmonare acuto. Inoltre, sono anche descritti tecniche chirurgiche di base per l&#39;isolamento del polmone, l&#39;intubazione tracheale e lavaggio broncoalveolare.

Un metodo per generare polmonare Neutrofilia Uso inalato Lipopolisaccaride

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

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

Research

JoVE Journal

Immunology and Infection

11.6K Views.  Karolinska Institutet.  Descriviamo un metodo per indurre l'infiammazione polmonare neutrofila dalla sfida di lipopolysaccharide aerosol per nebulizzazione, per modellare danno polmonare acuto. Inoltre, sono anche descritti tecniche chirurgiche di base per l'isolamento del polmone, l'intubazione tracheale e lavaggio broncoalveolare. 

Video: A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

A Method for Labeling Vasculature in Embryonic Mice

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

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

Un metodo per l&#39;etichettatura vasi embrionali nei topi

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

Ricerca

Immunologia e infezioni

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

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

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

Diagnosticare la tubercolosi polmonare con la MTB / RIF test Xpert

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

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

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

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

Due-fotone videomicroscopia Imaging dei leucociti durante la risposta immunitaria nel fegato Lipopolysaccharide-trattati del topo

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

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Generazione di vettori ricombinanti Herpesvirus aviaria con CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

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

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

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

Analisi di co-immunoprecipitazione per lo studio delle interazioni funzionali tra recettori e gli enzimi

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

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

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

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

Nuovo protocollo per la generazione di cellule dendritiche fisiologiche immunogene

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

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

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

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

Visualizzazione della resistenza batterica utilizzando sonde antibiotici fluorescenti

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

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

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

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

Esposizione al fumo di sigaretta nei topi con un sistema di inalazione per tutto il corpo

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

Generazione di transgenici e knockout in specie di Strongyloides mediante microiniezione

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

Microscopia a fluorescenza intravitale a campo largo della microcircolazione polmonare polmonare polmonare acuta sperimentale utilizzando un sistema di imaging stabilizzato sotto vuoto

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...