Dominic Mills è stato sostenuto da un Bloomsbury Collegi PhD Studentship (2007-2010). Gli autori desiderano ringraziare sia Ozan Gundogdu e Abdi Elmi per la loro assistenza nello sviluppo del modello VDC.

1. La crescita di monostrati IEC su speciali filtri 0,4 micron <ol><li> Coltura cellule Caco-2 a modificate mezzi essenziali di Dulbecco (DMEM) supplementato con 10% (v / v) di siero fetale bovino, 100 U / ml penicillina, 100 ug / ml streptomicina e 1% (v / v) aminoacidi non essenziali in un standard di coltura tissutale incubatore a 37 ° C in 5% di CO 2 e il 95% di aria. Seme di 4 x 10 5 Caco-2 IECs per 0,4 micron filtro e crescono di polarizzazione di 21 giorni, cambiando i media ogni 2-3 giorni. </li><li> Misurare la TEER per confermare lo stato di polarizzazione del monostrato utilizzando un misuratore di resistenza Volt-Ohm. Nei nostri studi, la TEER di 21 giorni Caco-2 cellule cresciute in questi filtri 0,4 micron è 700-800 Qcm 2. </li></ol> 2. Preparazione di C. jejuni e la Medi batterica per il saggio Coculturing <ol><li> 24 ore prima della partenza desiderata del saggio coculturing VDC, preparare un piatto fresco di agar sangue di C. jejuni e incubare a 37 ° C in condizioni microaerobiche (85% N 2, O 2 5% e 10% CO 2) nella variabile atmosfera incubatrice. </li><li> Allo stesso tempo, Preincubare 30 ml di brodo brucella a 37 ° C in condizioni microaerobiche in un&#39;atmosfera incubatore variabile. </li></ol> 3. Preparazione e sterilizzazione della mezza Chambers VDC prima del test <ol><li> Immergere completamente camere VDC mezzo così come gli O-ring e le spine nella soluzione di sterilizzazione. </li><li> Usando una sterile siringa da 20 ml, lavare la soluzione di sterilizzazione attraverso le prese di gas in entrambe le camere e mezzo. In caso contrario si potrebbe causare la contaminazione dei campioni durante coculturing. </li><li> Lasciare tutte le componenti immersi nella soluzione di sterilizzazione per&gt; 1 ora. </li><li> Risciacquare tutti i componenti con acqua fresca sterile, utilizzando un altro sterile 20 ml siringa per lavare le prese di gas. </li></ol> 4. Preparazionedi inoculo batterico <ol><li> Raccogliere metà un piatto di C. crescita jejuni dalla piastra di agar sangue preparato il giorno precedente in 1 ml di brodo brucella preincubato. Questo dovrebbe produrre ~ 10 10 ufc / ml. </li><li> Misurare la densità ottica della sospensione batterica a 600 nm per semiquantify batteriche unità formanti colonie (CFU). </li><li> Regolare la sospensione batterica al livello desiderato inoculo in 4 ml di brodo brucella preincubato. </li><li> Eseguire una diluizione seriale a questa sospensione batterica finale successiva metallizzazione delle appropriate diluizioni su piastre di agar sangue in triplice copia ed incubare a 37 ° C in atmosfera variabile incubatore per 48 ore per quantificare il numero di CFU batteriche presenti nel inoculo. </li></ol> 5. Impostazione del VDC <ol><li> Posizionare la metà camera inferiore di un VDC piatta sulla panca. Montare un O-ring sulla camera di metà inferiore. </li><li> Staccare un filtro che trasportano i IECs da tegli vettore e lavare tre volte con 400 ml di PBS sterile. Posizionare il filtro sulla camera di metà inferiore, assicurandosi che l&#39;o-ring rimanga in posizione. </li><li> Abbassare delicatamente la camera di metà superiore in posizione. Una volta che le due mezze camere sono assemblati, fissare insieme con l&#39;anello-morsetti. Collocare il VDC orizzontalmente sul banco, con le due aperture rivolte verso l&#39;alto. </li><li> Aggiungere il 4 ml di inoculo batterico nella camera di metà apicale. </li><li> Aggiungere 4 ml di mezzo di coltura cellulare nella metà camera di basolaterale. </li><li> Mettere i pezzi di fine nelle aperture entrambe le camere e mezzo. </li></ol> 6. Fissaggio del VDC al collettore gas all&#39;interno della variabile Atmosfera Incubator <ol><li> Collocare il VDC nella variabile atmosfera incubatore in prossimità del collettore gas. </li><li> Aprire il regolatore di alimentazione gas collegato al collettore gas. Fissare il tubo dal collettore in ingresso del gas sulla metà camera di basolaterale del VDC. Fissare il gas tubo di uscita che porta fuori dell&#39;atmosfera variabile incubatore a uno dei punti vendita del codolo sulla metà camera di basolaterale. </li><li> Chiudere l&#39;altra presa alla fine-pezzo sulla metà camera di basolaterale. Apertura del flusso del gas sul collettore gas, avendo cura di aprire molto lentamente per evitare la pressione del gas in eccesso, in quanto questo può portare all&#39;espulsione del mezzo basolaterale. </li><li> L&#39;obiettivo è un flusso di gas di 1 bolla ogni 2-5 sec. Controllare periodicamente il flusso di gas durante il corso dell&#39;esperimento e regolare se necessario. </li></ol> 7. Smontaggio del VDC dopo co-coltura <ol><li> Dopo il periodo di co-coltura appropriato, chiudere il regolatore di alimentazione gas collegato al collettore gas. Chiudere il flusso di gas nel Vcc nel collettore. Scollegare l&#39;ingresso del gas, l&#39;uscita del gas ed il fermo dalla VDC. </li><li> Rimuovere il VDC da un&#39;atmosfera incubatore variabile. Rimuovere il surnatante apicale e basolaterale e conservare a -80 ° C per subsequent analisi. </li><li> Rimuovere i morsetti ad anello e delicatamente smontare il VDC. Rimuovere il filtro dalla camera di metà e trasferimento in un piatto di coltura cellulare da 6 pozzetti sterili. Lavare le IXC tre volte con 400 ml di PBS sterile. </li><li> Per il conteggio del numero totale di batteri interagenti, aggiungere 400 ml di PBS sterile contenente 0,1% (v / v) Triton X-100 al IECs e incubare per 20 min a temperatura ambiente per lisare il CEI. Eseguire una diluizione seriale a questa lisato cellulare successiva metallizzazione delle appropriate diluizioni su piastre di agar sangue in triplice copia ed incubare a 37 ° C in atmosfera variabile incubatore per 48 ore per quantificare il numero di interagire CFU batteriche. </li><li> Per il conteggio del numero totale di batteri invasori, aggiungere 400 ml di DMEM cella mezzo di coltura contenente 150 ug / ml di gentamicina ai IECs ed incubare per 2 ore in un incubatore per colture tessuto normale a 37 ° C in 5% di CO 2 e il 95% AIR per uccidere i batteri extracellulari,poi seguire come al punto 7.4 di cui sopra. </li></ol> 8. Pulizia del VDC dopo co-coltura Lavare il VDC con acqua sterile e conservare per la prossima tornata di esperimenti.

<ol>
	<li>Mills, D. 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=Increase+in+Campylobacter+jejuni+invasion+of+intestinal+epithelial+cells+under+low-oxygen+coculture+conditions+that+reflect+the+in+vivo+environment.">Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.</a> Infect. Immun. 80, 1690-1698 (2012).</li><li>Marteyn, 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=Modulation+of+Shigella+virulence+in+response+to+available+oxygen+in+vivo.">Modulation of Shigella virulence in response to available oxygen in vivo.</a> Nature. 465, 355-358 (2010).</li><li>Dorrell, N., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+century+of+Campylobacter+research:+recent+advances,+new+opportunities+and+old+problems.">The second century of Campylobacter research: recent advances, new opportunities and old problems.</a> Curr. Opin. Infect. Dis. 20, 514-518 (2007).</li><li>Young, K. T., Davis, L. M., Dirita, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Campylobacter+jejuni:+molecular+biology+and+pathogenesis.">Campylobacter jejuni: molecular biology and pathogenesis.</a> Nat. Rev. Microbiol. 5, 665-679 (2007).</li><li>Hu, L., Kopecko, D. J., Nachamkin, I., Szymanski, C. M., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17.">Chapter 17.</a> Campylobacter. , 297-313 (2008).</li><li>Everest, P. H., 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=Differentiated+Caco-2+cells+as+a+model+for+enteric+invasion+by+Campylobacter+jejuni+and+C.+coli.">Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli.</a> J. Med. Microbiol. 37, 319-325 (1992).</li><li>Konkel, M. E., Hayes, S. F., Joens, L. A., Cieplak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+the+internalization+and+intracellular+survival+of+Campylobacter+jejuni+in+human+epithelial+cell+cultures.">Characteristics of the internalization and intracellular survival of Campylobacter jejuni in human epithelial cell cultures.</a> Microb. Pathog. 13, 357-370 (1992).</li><li>Monteville, M. R., Konkel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-facilitated+invasion+of+T84+eukaryotic+cells+by+Campylobacter+jejuni+occurs+preferentially+at+the+basolateral+cell+surface.">Fibronectin-facilitated invasion of T84 eukaryotic cells by Campylobacter jejuni occurs preferentially at the basolateral cell surface.</a> Infect. Immun. 70, 6665-6671 (2002).</li><li>Friis, L. M., Pin, C., Pearson, B. M., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+culture+methods+for+investigating+Campylobacter+invasion+mechanisms.">In vitro cell culture methods for investigating Campylobacter invasion mechanisms.</a> J. Microbiol. Methods. 61, 145-160 (2005).</li><li>Schuller, S., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerobic+conditions+enhance+type+III+secretion+and+adherence+of+enterohaemorrhagic+Escherichia+coli+to+polarized+human+intestinal+epithelial+cells.">Microaerobic conditions enhance type III secretion and adherence of enterohaemorrhagic Escherichia coli to polarized human intestinal epithelial cells.</a> Environ. Microbiol. 12, 2426-2435 (2010).</li><li>Cottet, S., Corthesy-Theulaz, I., Spertini, F., Corthesy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerophilic+conditions+permit+to+mimic+in+vitro+events+occurring+during+in+vivo+Helicobacter+pylori+infection+and+to+identify+Rho/Ras-associated+proteins+in+cellular+signaling.">Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.</a> J. Biol. Chem. 277, 33978-33986 (2002).</li></ol>

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L&#39;uso di metodi di coltura cellulare in vitro per studiare le interazioni di patogeni batterici con cellule ospiti è una tecnica largamente impiegata in molti laboratori di ricerca. Tuttavia, come tali analisi di colture cellulari vengono eseguite in condizioni aerobiche, questi modelli in vitro possono non rappresentare accuratamente l&#39;ambiente in vivo in cui le interazioni ospite-patogeno si svolgono. Il ampiamente usato in modelli di infezione in vitro sono partic...

Le interazioni di patogeni batterici con cellule ospiti sono stati studiati estesamente utilizzando metodi di coltura cellulare in vitro. L&#39;uso di tali saggi di coltura cellulare, adesione batterica alle cellule ospiti, l&#39;identificazione dei recettori della cellula ospite, cellula ospite vie di segnalazione e l&#39;invasione batterica delle cellule ospiti sono stati tutti studiati in dettaglio, con conseguente molte importanti osservazioni. Tuttavia tali analisi di colture cellulari vengono eseguite in condizioni aerobiche che possono non essere rappresentativi della ambiente in vivo. Una limitazione importante di modelli in vitro utilizzati per studiare le infezioni gastrointestinali è che le condizioni di coltura tra cui alti livelli di ossigeno in generale favoriscono la sopravvivenza delle cellule eucariotiche. Tuttavia le condizioni nel lume intestinale saranno quasi anaerobica. Patogeni enterici in un ambiente povero di ossigeno esprimono geni di virulenza cui espressione cambia in condizioni aerobiche 2. Come tale, i dati ottenuti utilizzando standard di coltura cellulare modelli may dare un&#39;indicazione imprecisa delle interazioni batteriche con le cellule ospiti. Campylobacter jejuni è il principale agente eziologico della gastroenterite acuta batterica in tutto il mondo, con sintomi che vanno dalla diarrea lieve a grave enterite infiammatoria. La maggioranza di C. jejuni infezioni provocano gastroenterite non complicata, tuttavia C. jejuni è anche l&#39;agente infettivo più comunemente identificato in neuropatie periferiche, incluse la sindrome di Guillain-Barré (GBS). Nel Regno Unito, si stima che ci siano oltre 500.000 casi di enteriti causate da C. jejuni infezione ogni anno, con un costo previsto per l&#39;economia del Regno Unito di £ 580.000.000. Nel mondo in via di sviluppo, C. jejuni è una delle principali cause di mortalità tra i bambini. Nonostante l&#39;indubbia importanza di infezione da Campylobacter e di decenni di ricerca, tra cui l&#39;analisi genomica basata approfondita, C. jejuni patogenesi è ancora poco understood, a differenza di altri patogeni enterici come Salmonella, Escherichia coli, Shigella e Vibrio cholerae. La mancanza di un comodo piccolo modello animale è una delle ragioni principali per questo 3. Anche il ampiamente utilizzato in modelli di infezione in vitro sono più appropriati per lo studio microaerofili C. jejuni che per altri patogeni enterici che sono anaerobi facoltativi. Mentre C. jejuni è riconosciuto come un agente patogeno invasivo, i meccanismi di C. jejuni invasione delle cellule epiteliali intestinali (IECs) sono ancora poco chiari 4,5. C. jejuni invasione ha dimostrato di essere dipendente sia microfilamenti, microtubuli, una combinazione di entrambi o nessuno 5. La confusione in questa zona è molto probabilmente dovuta all&#39;uso di appropriati nelle condizioni di saggio in vitro. Un certo numero di differenti saggi di coltura cellulare sono stati utilizzati per studiare le interazioni di C. jejunicon le cellule ospiti. Caco-2 6, 7 INT 407 e T84 8 linee cellulari sono stati tutti utilizzati per studiare le capacità di adesione e invasione di diversa C. ceppi jejuni. Tuttavia, i livelli di adesione batterica e invasione per C. jejuni con IECs sono notevolmente inferiori rispetto ad altri patogeni enterici 9. La coculturing di C. jejuni con IECs viene normalmente eseguita in un incubatore CO 2 in condizioni vicine a livelli di ossigeno atmosferico, necessaria per la sopravvivenza di IECs. C. espressione genica jejuni sarà molto differente nell&#39;ambiente ossigeno basso del lume intestinale rispetto a condizioni di ossigeno atmosferico. L&#39;uso di un sistema di camera di diffusione verticale (VDC) è stato sviluppato che permette la co-coltura di batteri e cellule ospiti sotto diverse condizioni medie e gas 1,10,11. Questo sistema simula le condizioni nell&#39;intestino umano, dove i batteri saranno ONUder condizioni di tessuto mentre molto basso ossigeno saranno forniti con l&#39;ossigeno dal flusso sanguigno. Monostrati polarizzati IEC coltivate in speciali 0,4 micron filtri sono stati collocati in un VCC creando un compartimento apicale e basolaterale, che sono stati riempiti singolarmente con brodo batterica e coltura cellulare rispettivamente (Figura 1). Il VDC è stata posta in atmosfera variabile incubatore contenente 85% N 2, O 2 5%, e 10% di CO 2 a 37 ° C, con condizioni ottimali per C. jejuni. Il compartimento apicale stata lasciata aperta ed esposta all&#39;atmosfera microaerobica all&#39;interno della variabile atmosfera incubatrice, mentre il vano basolaterale sigillata è stata fornita con l&#39;ossigeno da una costante somministrazione di 5% 95% O 2 miscela di gas con un tubo di uscita evitando accumuli di pressione CO 2 / . Caco-2 sopravvivenza cellulare e integrità monostrato in queste condizioni sono state confermate dal monitoraggio ele transepitelialeResistenza ctrical (TEER) attraverso il monostrato oltre 24 ore per dimostrare sopravvivenza della separazione Caco-2 monostrato cellulare e fisica dei compartimenti apicale e basolaterale in condizioni di bassa ossigeno nel compartimento apicale. La TEER di monostrati in VDCs mantenuto in atmosfera incubatrice variabile (condizioni microaerobiche) ed in una coltura cellulare di CO 2 incubatore standard (condizioni aerobiche) non ha evidenziato differenze significative, indicando integrità del monostrato cellulare in diverse condizioni meteorologiche 1. In condizioni microaerobiche, giunzioni strette sono rimasti presenti e uniformemente distribuito tra i bordi delle celle con un pattern di colorazione occludina simili alle cellule mantenute in condizioni aerobiche 1. Le interazioni di C. jejuni con Caco-2 e T84 cellule del VDC sono stati studiati per valutare l&#39;interazione batterica (adesione e invasione) e l&#39;invasione. Due diversi C. jejuni wild-type straiNS sono stati utilizzati 1. C. jejuni 11168H è un derivato hypermotile dell&#39;originale ceppo sequenza NCTC11168. Il ceppo 11168H mostra molto più alti livelli di colonizzazione in un modello di colonizzazione pulcino ed è quindi considerato un ceppo adatto da utilizzare per studi di interazione ospite-patogeno. 81-176 è un umano isolare ed è uno dei più invasive ceppi di laboratorio ampiamente studiate. C. jejuni ceppi sono stati aggiunti al compartimento apicale di un VDC in condizioni sia microaerobiche o aerobico. Abbiamo osservato tassi più elevati sia per l&#39;interazione e l&#39;invasione sono stati registrati per C. jejuni in condizioni microaerobiche 1. L&#39;aumento C. interazioni jejuni non erano dovuti ad un aumento del numero di batteri in condizioni microaerobiche 1. Questi dati supportano la nostra ipotesi che l&#39;ambiente a basso ossigeno nel compartimento apicale del VDC migliora interazioni batteri-ospite e indica che le proprietà invasive di C. jejuni sono incrfacilitato in queste condizioni. Questo era il primo rapporto di utilizzo del modello VCC per studiare un patogeno batterico invasiva e mette in evidenza l&#39;importanza di eseguire saggi di infezione in vitro in condizioni che imitare maggiormente la situazione in vivo. Il modello VDC potrebbe essere usato per studiare le interazioni ospite-patogeno per molte differenti specie batteriche.

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			<td>Speciality vertical diffusion system for use with Snapwell inserts</td>
			<td>Harvard Apparatus</td>
			<td>66-0001</td>
			<td>Manifold &#38; six Snapwell chambers</td>
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			<td>Snapwell filters (pore size 0.4 &#956;m)</td>
			<td>Corning Costar</td>
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			<td>Millicell ERS-2 Volt-Ohm resistance meter</td>
			<td>Millipore</td>
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			<td>WPA Lightwave II spectrophotometer</td>
			<td>Biochrom</td>
			<td>80-3003-72</td>
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enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model

Le interazioni di patogeni batterici con cellule ospiti sono stati studiati estesamente utilizzando metodi di coltura cellulare in vitro. Tuttavia, come tali analisi di colture cellulari vengono eseguite in condizioni aerobiche, questi modelli in vitro possono non rappresentare accuratamente l&#39;ambiente in vivo in cui le interazioni ospite-patogeno si svolgono. Abbiamo sviluppato un modello in vitro di infezione che permette la co-coltura di batteri e cellule ospiti sotto diverse condizioni medie e gas. La camera di diffusione verticale (VDC) modello simula le condizioni nell&#39;intestino umano, dove i batteri saranno in condizioni di tessuto, mentre molto basso di ossigeno sarà fornita con l&#39;ossigeno dal flusso sanguigno. Posizionamento polarizzati cellulari (IEC) monostrati epiteliali intestinali cresciute in Snapwell inserisce in un VDC crea compartimenti apicale e basolaterale separati. Il vano basolaterale è riempito con mezzo di coltura cellulare, sigillato e perfusi con l&#39;ossigeno wHilst compartimento apicale è pieno di brodo, tenute aperte e incubate in condizioni microaerobiche. Sia Caco-2 e T84 IECs possono essere mantenuti nel VDC in queste condizioni, senza apparenti effetti negativi sulla sopravvivenza delle cellule o l&#39;integrità monostrato. Esperimenti Coculturing eseguiti con differenti C. jejuni ceppi wild-type e diverse linee IEC nel modello VDC con condizioni microaerobiche nel compartimento apicale riproducibile risultano in un incremento del numero di interagire (quasi 10 volte) e intracellulari (quasi 100 volte) batteri rispetto a condizioni di coltura aerobiche 1. L&#39;ambiente creato nel modello VDC imita più da vicino l&#39;ambiente incontrate da C. jejuni nell&#39;intestino umano e mette in evidenza l&#39;importanza di esibirsi in saggi di infezione in vitro in condizioni che mimano più da vicino la realtà in vivo. Noi proponiamo che l&#39;uso del modello VDC consentirà nuove interpretazioni delle interazioni scommettonoween batteri patogeni e cellule ospiti.

Esperimenti Coculturing eseguite con il C. jejuni ceppo selvatico e IECs nel modello VDC con condizioni microaerobiche nel compartimento apicale hanno dimostrato un aumento del numero di interagire (quasi 10 volte) e intracellulari (quasi 100 volte) batteri rispetto a condizioni di coltura aerobiche in un momento maniera dipendente dalla 1. Questa osservazione era riproducibile utilizzando due diversi C. jejuni ceppi wild-type (11168H e 81-176) e due linee IEC diversi (Caco-2 e T84), mettend...

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Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Esecuzione di test di coltura cellulare per indagare adesione batterica e l&#39;invasione in condizioni aerobiche è di solito non rappresentativo della<em&gt; In vivo</em&gt; Ambiente. Un modello di camera di diffusione verticale permette lo studio delle interazioni del patogeno umano<em&gt; Campylobacter jejuni</em&gt; Con le cellule epiteliali intestinali in più<em&gt; In vivo</em&gt; Condizioni simili, con conseguente maggiore invasione batterica.

Enterico invasione batterica della cellule epiteliali intestinali<em&gt; In Vitro</em&gt; È drammaticamente migliorata utilizzando una camera di diffusione verticale Modello

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

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Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

13.7K Views.  London School of Hygiene & Tropical Medicine. Esecuzione di test di coltura cellulare per indagare adesione batterica e l'invasione in condizioni aerobiche è di solito non rappresentativo della In vivo Ambiente. Un modello di camera di diffusione verticale permette lo studio delle interazioni del patogeno umano Campylobacter jejuni Con le cellule epiteliali intestinali in più In vivo Condizioni simili, con conseguente maggiore invasione batterica.

Video: Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

Nella formazione di biofilm in vitro in un 8-bene scorrere Camera

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

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Immunologia e infezioni

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Utilizzando luciferasi di infezioni batteriche immagine in topi

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Invasione di cellule umane da un agente patogeno batterico

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

In vitro di Adesione batterica su mammiferi cellule epiteliali

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Applicazione di un mouse legatura Patch Peyer Assay intestinale Loop per valutare assorbimento da parte delle cellule batteriche M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. 
We found the optimum fatty acid concentrations to be between 1-5 ng/&mu;l, and that for cholera toxin to be &lt; 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

Determinazione degli acidi grassi tollerabile e Concentrazioni tossina colerica Uso Umano cellule epiteliali intestinali e BALB / c macrofagi del mouse

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

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

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

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

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

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

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

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Valutazione dell'invasione batterica delle cellule cardiache in coltura e colonizzazione cardiaca nei topi infetti utilizzando listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Analisi dell&#39;immunofluorescenza della formazione di granuli di stress dopo la sfida batterica delle cellule dei mammiferi

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un High-throughput Shigella-specifica analisi battericida

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...