La ricerca nel laboratorio di Bragonzi è stato finanziato dalla Fondazione Italiana Fibrosi Cistica (CFaCore) e UE-F7-2009-223.670. Parte di questo lavoro è stato svolto in ALEMBIC, un laboratorio di microscopia avanzata, e il mouse istopatologia è stato eseguito presso l&#39;Unità di Anatomia Patologica (Istituto Scientifico San Raffaele).

1. Batteri Preparazione per l&#39;infezione cronica (tre e due giorni prima del mouse Challenge) <ol><li> Selezionare l&#39;appropriato P. ceppo aeruginosa da testare. </li><li> Seminare un&#39;ansata di P. aeruginosa da -80 ° C delle colture per un piatto Trypticase Soy Agar (TSA) e incubare a 37 ° C durante la notte. </li><li> Scegli una singola colonia e inoculare in 5 ml Trypticase Soy Broth (TSB) in una provetta snap-capped 15 ml ed incubare a 37 ° C per una notte in un incubatore agitazione a 200 rpm. </li></ol> 2. Incorporare batteri in Beads Agar per l&#39;infezione cronica (un giorno prima infezione) <ol><li> Prendere una piccola aliquota della coltura batterica durante la notte, diluire 1:50 in tampone fosfato (PBS) e misurare la densità ottica (OD) a 600 nm. </li><li> Diluire la coltura batterica overnight aggiungendo 2 OD in una nuova provetta con tappo a scatto contenente 20 ml di TSB fresca. </li><li> Incubare a 37 ° C per circa 3 -4 ore di fase di login in un incubatore agitazione a 200 rpm, fino a raggiungere un totale di 10-15 OD. </li><li> Nel frattempo, preparare la TSA, fatta di TSB con 1,5% agar, autoclave ed equilibrare a 50 ° C in un bagno d&#39;acqua. Equilibrare 150 ml di olio minerale pesante preautoclaved in un matraccio di Erlenmeyer a 50 ° C in un bagno d&#39;acqua. </li><li> Una volta P. aeruginosa raggiunge la fase log, raccogliere le cellule batteriche mediante centrifugazione a 2700 xg per 15 min a 4 ° C e scartare il surnatante. </li><li> Risospendere il pellet batterico in 1 ml di PBS sterile e vortice accuratamente per risospendere completamente il pellet. </li><li> Miscelare 1 ml di sospensione batterica con 9 ml di TSA liquido pre-equilibrata a 50 ° C. </li><li> Aggiungere 10 ml TSA-P. aeruginosa miscela di olio minerale pesante (preriscaldata a 50 ° C) e subito mescolare per 6 minuti a temperatura ambiente. L&#39;agitazione deve produrre un vortice visibile nell&#39;olio. </li><li> Raffreddare la miscela a 4 ° C, agitando alla sp minimoeed per 35 min (Figura 1). </li><li> Appoggiare la miscela agar-perle di olio in ghiaccio per altri 20 min. </li><li> Trasferire l&#39;agar-perle in 50 ml provette Falcon e centrifugare a 2.700 xg per 15 min a 4 ° C. </li><li> Accuratamente rimuovere l&#39;olio minerale e lavare con PBS sterile sei volte, come descritto nel passaggio 2.11. Dopo tre lavaggi, le sfere possono essere pellet per gravità invece di utilizzare la centrifuga. Dopo l&#39;ultimo lavaggio, risospendere l&#39;agar-perle in 20-30 ml di PBS. </li><li> Prelevare un &#39;aliquota delle perle (circa 0,5 ml) e asettico omogeneizzare. </li><li> Prendete 100 ml di perline omogeneizzati e diluire in 900 ml di PBS sterile. Serie diluire 01:10 fino al 10 -6. </li><li> Diluizione seriale piastra su piastre di TSA, compreso campione non diluito fino a 10 -6 e incubare le piastre a 37 ° C. </li><li> Misurare il diametro tallone utilizzando un microscopio ottico invertito in vari campi. Il diametro cordone deve essere compreso tra 100-200 micron ( <strong&gt; Figura 1). </li><li> Conservare le perle di una notte a 4 ° C. </li></ol><img alt="Figura 1" fo:content-width="6in" fo:src="/files/ftp_upload/51019/51019fig1highres.jpg" src="/files/ftp_upload/51019/51019fig1.jpg" width="500px" /> Figura 1. Panoramica della preparazione e del mouse perline infezione agar. P. aeruginosa cellule sono risospese in 1 ml di PBS e aggiunti 9 ml di liquido TSA (50 ° C). Questa miscela viene aggiunta a 150 ml di olio minerale pesante a 50 ° C in un pallone e agitata ad alta velocità per 6 minuti a temperatura ambiente. Quando il pallone viene raffreddato a 4 ° C con agitazione lenta per 35 min, l&#39;agar solidifica creando perline, e batteri presenti nella miscela sono incorporati nei branelli agar. Particolare di un cordone agar contenente cellule batteriche è (A) indicato. Dopo aver rimosso l&#39;olio minerale con diversi lavaggi con PBS sterile, la sospensione di agar-perline è pronto fo inoculazione nei polmoni dei topi da un&#39;iniezione endotracheale (B). <a href="https://www.jove.com/files/ftp_upload/51019/51019fig1highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . 3. Topi sfida con Agar-perle Etica Dichiarazione: Questo protocollo e sperimentazione seguono le linee guida della cura degli animali e il comitato etico di Istituto Scientifico San Raffaele. <ol><li> Contare il numero di Unità Formanti Colonie (CFU) sulle piastre di TSA per determinare il numero di CFU / ml nella sospensione agar-perline. Diluire l&#39;agar-perline con PBS sterile per 2-4 x 10 7 UFC / ml per raggiungere un inoculo ottimale di 1-2 x 10 6 in 50 microlitri. </li><li> Anestetizzare C57Bl/6NCr (20-22 g, 6-8 settimane) topi maschi con ketamina (50 mg / ml) e xilazina (5 mg / ml) in 0,9% NaCl somministrata ad un volume di 0,002 ml / g di peso corporeo da iniezione intraperitoneale. NOTA: L&#39;anestesia è considerato adeqlutare quando l&#39;animale rimane ancora in silenzio, non risponde agli stimoli esterni, ed è costante il cuore e la frequenza respiratoria. </li><li> Posizionare il mouse in posizione supina. Disinfettare il cappotto del mouse con il 70% di etanolo. </li><li> Esporre la trachea da un taglio verticale della pelle e intubare la trachea con una sterile, flessibile 22 G 0,9 millimetri x 25 mm catetere IV, mantenendo attenzione per rimuovere il stylette mentre si muove verso il basso nella trachea. Inserire il catetere non troppo in profondità nella trachea. Arrestare prima di raggiungere la carena (biforcazione). </li><li> Immediatamente prendere un volume di 50 ml di sospensione di sferette agar da una siringa da 1 ml e collegarlo al catetere. Premere delicatamente il pistone della siringa, permettendo alle sfere di essere impiantati nel polmone. Chiudere l&#39;incisione utilizzando clip di sutura. </li><li> Posizionare l&#39;animale su una piastra elettrica fino a completamente sveglio. </li></ol> 4. Valutazione Mice <ol><li> Osservare i topi al giorno per segni clinici tra cui la qualità del cappotto, la postura,deambulazione, e lo stato di idratazione. Controllo del peso corporeo al giorno. I topi che perdono ≥ 20% del peso corporeo devono essere eutanasia. </li><li> Seguire il punto 5 per la raccolta del liquido di lavaggio broncoalveolare (BAL) e dei punti 6 e 7 per la raccolta dei polmoni e l&#39;analisi di totale CFU, l&#39;analisi istologica, la risposta infiammatoria in termini di numero totale e differenziale delle cellule nel BAL, analisi di citochine, e mieloperossidasi (MPO) attività. </li></ol> 5. BAL Fluid Raccolta e analisi <ol><li> Euthanize i topi da CO 2 inalazione. </li><li> Posizionare il mouse nella posizione supina. Disinfettare il cappotto del mouse con il 70% di etanolo. </li><li> Esporre la trachea e la gabbia toracica da un taglio verticale della pelle. Esporre i polmoni tagliando il diaframma. </li><li> Inserire un filo di sutura sotto la trachea utilizzando una pinzetta e intubare la trachea con una sterile, flessibile 22 g 0,9 millimetri x 25 mm catetere IV. Tirare le due estremità del filo di sutura di impegnare la catheter alla trachea e annodare il filo attorno alla trachea. </li><li> Prendere un volume di 1 ml di RPMI 1640 utilizzando una siringa da 1 ml e fissarlo al catetere. Spingere lo stantuffo della siringa per lavare i polmoni e immediatamente recuperare il liquido, riporlo in una provetta da 15 ml. NOTA: Se le citochine sono da analizzare, aggiungere inibitori della proteasi per RPMI 1640. </li><li> Ripetere questa operazione tre volte per un totale di 3 ml di RPMI. Da ora in poi, conservare il liquido BAL su ghiaccio. Andare al punto 6 per la raccolta e l&#39;analisi dei polmoni. NOTA: Si prega di notare che non tutto il liquido sarà recuperato (2,8 ml al massimo). </li><li> Per la quantificazione dei batteri presenti nel BAL, campionare una piccola aliquota (300 microlitri), serialmente diluire 1:10 in PBS sterile, piastra su piastre di TSA e incubare a 37 ° C per una notte. </li><li> Contare le cellule totali utilizzando un microscopio ottico invertito luce diluendo un&#39;aliquota del BAL 1:2 con Tuerk in una camera di conta cellulare Burker. </li><li> Centrifugare la remaining BAL a 330 xg per 8 minuti a 4 ° C. Prendere il surnatante per l&#39;analisi di citochine ELISA, riporlo a -80 ° C. Seguire i passaggi 5,10-5,13 per la conta cellulare differenziale cytospin. </li><li> Se il pellet è rosso, lisare gli eritrociti risospendere il pellet in 250-300 ml di tampone di lisi diluito 1:10 in acqua distillata ultrapura per 3 min. Neutralizzare con 2 ml di PBS e centrifugare a 330 xg per 8 minuti a 4 ° C. </li><li> Eliminare il surnatante e risospendere il pellet in RPMI 10% di siero fetale bovino (FBS). Utilizzare un volume che fornirà 1 x 10 6 cellule / ml, in base al numero di celle totale. </li><li> Posizionare vetrini da microscopio e dei filtri in apposite cave nel cytospin con i filtri di cartone di fronte al centro del cytospin. Pipettare 150 ml di ciascun campione nei pozzetti appropriati del cytospin e centrifugare in una citocentrifuga a 300 xg per 5 min. </li><li> Colorare i vetrini da Romanowsky colorazione utilizzando un kit commerciale, secondo la maistruzioni di nufacturer e come descritto in precedenza 12. Seguire i passaggi 5,14-5,17 per l&#39;analisi delle attività MPO. </li><li> Centrifugare la rimanente quantità di BAL a 380 xg per 5 minuti a 4 ° C. Eliminare il supernatante e risospendere il pellet in 250 ml di cloruro di esadeciltrimetilammonio 0,5% in acqua distillata ultrapura per lisare le cellule. La sospensione può essere congelato a -20 ° C per diversi giorni prima dell&#39;esecuzione del test. </li><li> Centrifugare a 16.000 xg per 30 min a 4 ° C e utilizzare il surnatante effettuare test MPO in piastre a 96 pozzetti, aggiungere il campione in duplicato a ciascun pozzetto e, se necessario, anche vere diluizioni del campione. </li><li> Aggiungere a ciascun campione nei pozzetti un volume uguale di 3,3 &#39;, 5,5&#39;-tetrametilbenzidina (TMB) come substrato per la perossidasi. Lasciare che la reazione abbia luogo al buio per almeno 5 minuti e fino a quando non vi è un ulteriore sviluppo nel colore. </li><li> Arrestare la reazione aggiungendo 2 MH 2 SO 4 unand misurare la densità ottica a 450 nm. Il valore di assorbanza sarà direttamente proporzionale all&#39;attività perossidasi. </li></ol> 6. Misurazione della carica batterica nei polmoni e citochine analisi <ol><li> Subito dopo la raccolta BAL, polmoni accise del topo, li lavare in PBS sterile, lobi distinti, metterli in un tubo a fondo rotondo con 2 ml di PBS sterile e memorizzare sul ghiaccio. NOTA: Se citochine sono da analizzare, aggiungi inibitori della proteasi di PBS ai tubi. </li><li> Asetticamente omogeneizzare i polmoni, prendere una piccola aliquota (300 microlitri) dall&#39;omogenato, serie diluire 1:10 in PBS, la piastra su piastre di TSA e incubare a 37 ° C durante la notte. Si prega di notare che il carico totale delle vie aeree batterica sarà la somma dei CFU trovati in BAL e del polmone. </li><li> Centrifugare l&#39;omogeneizzato rimanendo a 16.000 xg per 30 min a 4 ° C. Prendete il surnatante per l&#39;analisi di citochine ELISA, e conservare a -80 ° C. </li></ol> 7. Esame istologico &lt;ol&gt; <li> Effettuare analisi istologica solo sui polmoni in cui BAL non sono stati raccolti, per preservare aspetto e le proprietà del polmone. </li><li> Euthanize il mouse CO 2 inalazione. </li><li> Esporre la gabbia toracica da un taglio verticale della pelle, esporre i polmoni tagliando il diaframma. </li><li> Accise polmoni, li lavare in PBS, separano i lobi, metterli in una provetta contenente formalina 5-10 ml 10% neutra tamponata (4% formaldeide) e conservare a 4 ° C al riparo dalla luce. </li><li> Polmoni Incorpora in paraffina, utilizzando le procedure standard. </li><li> Tagliare 5 sezioni micron di spessore utilizzando un microtomo. </li><li> Diapositive macchia con ematossilina e eosina e coprire le diapositive con un coprioggetto, secondo le procedure standard. </li><li> Esaminare vetrini utilizzando un microscopio invertito campo chiaro e acquisire le immagini collegando al microscopio una telecamera. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Le fasi critiche del P. aeruginosa-perline preparazione e la sfida del mouse sono riportati di seguito. Il ceppo P. aeruginosa utilizzato per i topi sfida è critica. Mortalità, infezione cronica o di liquidazione potrebbero differire significativamente a seconda del ceppo batterico utilizzato per la sfida. Il P. aeruginosa RP73 ceppo clinico è stato preferito rispetto al ceppo di laboratorio di riferimento PAO1 quanto ha determinato una mortalità relat...

La fibrosi cistica (CF) è una malattia genetica causata da mutazioni nel transmembrana della fibrosi cistica regolatore della conduttanza (CFTR). Questo gene codifica per un canale del cloro espressi sulla membrana della maggior parte delle cellule epiteliali. Bronchiectasia, muco tamponamento e parenchimale distruzione causata principalmente da infezioni da Pseudomonas aeruginosa progressivamente portare a grave malattia polmonare e la mortalità nella maggior parte dei pazienti CF 1. Capire patogenesi CF e l&#39;ulteriore sviluppo di nuove terapie si basano su modello animale con le caratteristiche di CF. Diversi topi, geneticamente modificati per il gene CFTR, sono stati generati, ma limitazioni nella capacità di queste specie di ricapitolare malattia polmonare CF-simili e diverse altre anomalie di organi visto in pazienti CF sono stati ampiamente documentati 2. Sviluppo di infezione è una delle principali sfide CF modello animale. I cl letteraturasuggerisce presto che una infezione cronica che dura più di un mese può essere raggiunto solo se i topi vengono inoculati con i batteri incorporati in un agente di immobilizzazione come agar, agarosio, o alghe alginato 3-5. Questi agenti immobilizzanti forniscono le condizioni microaerobica / anaerobiche che permettono ai batteri di crescere in forma di microcolonie, analogamente alla crescita nel muco dei pazienti FC 6. Questo modello di infezione cronica porta alla persistenza dei batteri nei polmoni causando infiammazione delle vie respiratorie e danni 7. Tuttavia, a seconda del metodo utilizzato, il ceppo batterico e la dose inoculato nei polmoni, la percentuale di topi infettati croniche e la carica batterica recuperato nei polmoni a differenti tempi possono differire considerevolmente. In particolare, la sfida principale per l&#39;infezione cronica a lungo termine rimane la bassa carica batterica da P. aeruginosa e la bassa percentuale di infetti settimane topi dopo sfida, indicando that cellule batteriche vengono progressivamente cancellati dall&#39;host. Selezionando il P. aeruginosa RP73 ceppo clinico da una raccolta di CF isolati 8 abbiamo ottenuto con successo una bassa mortalità, le lesioni più gravi, e l&#39;alta percentuale di infezione cronica con una carica batterica stabile fino a un mese nei topi C57Bl/6NCrl. Dettagli Questo documento metodologia per l&#39;incorporamento P. aeruginosa nelle perle di agar, abbiamo infettato topi mediante instillazione, misurata la carica batterica e citochine nei polmoni, raccolta BAL ed eseguito un esame istologico. Nel complesso, questo protocollo sarà di aiuto ai ricercatori per affrontare questioni di fondamentale importanza sulla patogenesi 8,9 e testare nuove terapie contro il P. aeruginosa infezione cronica 10,11.

<table border="0" cellpadding="0" cellspacing="0" width="605">
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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Bacto Tryptic Soy Broth</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:143px;">211823</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Difco Agar, granulated</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:143px;">214510</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Heavy mineral oil</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">330760-1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">S-(+)-Ketamine hydrochloride</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">K1884</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine hydrochloride</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">X1251</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:272px;">1 ml syringe 25 G 5/8&#39;&#39;&#160;0.5 x 16 mm</td>
			<td style="width:191px;">PIC</td>
			<td style="width:143px;">3071250300350</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Catheter 22GA 0.9 x 25 mm</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:143px;">381223</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Graefe Forceps - 0.5 mm Tips Curved</td>
			<td style="width:191px;">Fine Science Tools</td>
			<td style="width:143px;">11152-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Scissors, Iris, 11 cm, straight</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:143px;">501758</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Suture clips</td>
			<td style="width:191px;">Fine Science Tools</td>
			<td style="width:143px;">12040-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Suture thread</td>
			<td style="width:191px;">Fine Science Tools</td>
			<td style="width:143px;">18020-40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">RPMI 1640</td>
			<td style="width:191px;">Lonza</td>
			<td style="width:143px;">BE12-167F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Complete protease inhibitor cocktail</td>
			<td style="width:191px;">Roche</td>
			<td style="width:143px;">11836145001</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:272px;">Fast-Read 102 Burker disposable chamber</td>
			<td style="width:191px;">Biosigma</td>
			<td style="width:143px;">390497</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Tuerk solution</td>
			<td style="width:191px;">Fluka</td>
			<td style="width:143px;">93770</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">RBC lysis buffer</td>
			<td style="width:191px;">Biolegend</td>
			<td style="width:143px;">420301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fetal bovine serum</td>
			<td style="width:191px;">Lonza</td>
			<td style="width:143px;">DE14-801F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">EZ cytofunnel</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:143px;">A78710021</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Superfrost ultra plus microscope slides</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:143px;">J3800AMNZ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Diff-Quik Romanowsky staining set</td>
			<td style="width:191px;">Medion Diagnostics</td>
			<td style="width:143px;">130832</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:272px;">Hexadecyltrimethylammonium chloride</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">52366-10G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">96-well EIA/RIA plate</td>
			<td style="width:191px;">Costar</td>
			<td style="width:143px;">3590</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">3,3&#8217;,5,5&#8217;- tetramethylbenzidine</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">T8665-1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sulfuric acid</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">320501-1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">10% neutral buffered formalin</td>
			<td style="width:191px;">Bio-optica</td>
			<td style="width:143px;">05-01005Q</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Harris haematoxylin non Papanicolau</td>
			<td style="width:191px;">Bio-optica</td>
			<td style="width:143px;">05-M06004</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Eosin plus alcoholic solution</td>
			<td style="width:191px;">Bio-optica</td>
			<td style="width:143px;">05-M11007</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
<td>[header]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;"></td>
			<td style="width:191px;"></td>
			<td style="width:143px;"></td>
<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Shaking incubator</td>
			<td style="width:191px;">Amerex Instruments</td>
			<td style="width:143px;">Steady Shake 757</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Water bath</td>
			<td style="width:191px;">Grant</td>
			<td style="width:143px;">SUB14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Homogenizer</td>
			<td style="width:191px;">Ystral</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Precision balance</td>
			<td style="width:191px;">KERN</td>
			<td style="width:143px;">440-47N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Cytocentrifuge</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:143px;">A78300003</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Low Cost Heating Pad</td>
			<td style="width:191px;">2biol</td>
			<td style="width:143px;">LCHP</td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:272px;">Homogenization probe</td>
			<td rowspan="2" style="width:191px;">Ystral</td>
			<td style="width:143px;">2366931(great)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:143px;">2366925(small)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Inverted optical microscope</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:143px;">Axioplan2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Camera (microscope)</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:143px;">Axiocam MRc5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Rotary microtome</td>
			<td style="width:191px;">Leica</td>
			<td style="width:143px;">RM2255</td>
		</tr>
	</tbody>
</table>

long term chronic pseudomonas aeruginosa airway infection in mice

Un modello murino di infezione cronica delle vie aeree è un asset fondamentale nella fibrosi cistica (CF) di ricerca, anche se ci sono una serie di preoccupazioni per quanto riguarda il modello stesso. Prime fasi di infiammazione e infezione sono stati ampiamente studiati utilizzando il modello di agar-perle del mouse Pseudomonas aeruginosa, mentre solo pochi studi si sono concentrati sulla infezione cronica a lungo termine in vivo. La sfida principale per l&#39;infezione cronica a lungo termine rimane la bassa carica batterica da P. aeruginosa e la bassa percentuale di infetti settimane topi dopo sfida, indicando che le cellule batteriche vengono progressivamente cancellati dall&#39;host. Questo articolo presenta un metodo per ottenere efficiente infezione cronica a lungo termine in topi. Questo metodo si basa sul radicamento del P. aeruginosa ceppi clinici nei agar-perle in vitro, seguita da instillazione nei topi C57Bl/6NCrl. Infezione polmonare bilaterale è associato SEVrali misurabili read-out, tra cui la perdita di peso, la mortalità, infezione cronica, e la risposta infiammatoria. Il P. aeruginosa RP73 ceppo clinico è stato preferito rispetto al ceppo di laboratorio di riferimento PAO1 quanto ha determinato una mortalità relativamente bassa, le lesioni più gravi, e l&#39;infezione cronica più elevata. P. colonizzazione aeruginosa può persistere nel polmone per oltre tre mesi. Murini patologia polmonare assomiglia a quella di pazienti CF con malattia avanzata cronica polmonare. Questo modello murino imita più da vicino il corso della malattia umana e può essere utilizzato sia per studi sulla patogenesi e per la valutazione di nuove terapie.

Quando il protocollo è fatto correttamente, il P. aeruginosa agar-perle misureranno tra 100-200 micron e può essere osservato con un microscopio ottico invertito pipettando un piccolo volume della sospensione di agar-perline su un vetrino. Singole cellule batteriche sono visibili nelle perline agar, come mostrato in dettaglio in figura 1. La scelta di P. ceppo aeruginosa usati nella preparazione agar-perline è critica. Figura 2 ...

Watch this Scientific Journal Video about Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice at JoVE.com

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Descriviamo il metodo di agar-perle di stabilire persistente a lungo termine infezione delle vie respiratorie Pseudomonas aeruginosa cronica nel modello di topo.

Long Term cronica<em&gt; Pseudomonas aeruginosa</em&gt; Infezione delle vie aeree nei topi

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

long-term-chronic-pseudomonas-aeruginosa-airway-infection-in-mice

Research

JoVE Journal

Immunology and Infection

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

22.2K Views.  San Raffaele Scientific Institute.  Descriviamo il metodo di agar-perle di stabilire persistente a lungo termine infezione delle vie respiratorie Pseudomonas aeruginosa cronica nel modello di topo. 

Video: Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-coltura Modelli di Pseudomonas aeruginosa Biofilm Cresciuto in diretta cellule delle vie aeree umane

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Ricerca

Immunologia e infezioni

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa e biofilm Saccharomyces cerevisiae in celle di flusso

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Isolamento del RNA Pseudomonas aeruginosa Colonizzare la Murine tratto gastrointestinale

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso di un terreno dell&#39;espettorato artificiale per testare l&#39;efficacia degli antibiotici contro<em&gt; Pseudomonas aeruginosa</em&gt; In condizioni più rilevanti al polmone Fibrosi Cistica

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Pseudomonas aeruginosa Induced Lung Injury Modello

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

Vieni nel lato di luce: In Vivo monitoraggio di Pseudomonas aeruginosa Biofilm infezioni nelle ferite croniche in un modello murino Hairless diabetico

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Metodi per rilevare amiloidi seguente infezione di polmonare endoteliali cellule citotossiche da Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

Generazione di mutanti di eliminazione genica in-frame in Pseudomonas aeruginosa e test per l'attenuazione della virulenza in un semplice modello di infezione del mouse

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Cultura della piccola colonia Variante di Pseudomonas aeruginosa e Quantitazione del suo Alginato

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Un modello di inoculazione ritardata dell'infezione cronica di Pseudomonas aeruginosa Wound

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...