This work was supported by grants from the Institute of Science and Technology of the Federal District (ICyTDF, 64/2012 to EO) and the Mexican Council for Science and Technology (Conacyt, 179895 to MS).

1. Creazione e mantenimento di E. Culture histolytica <ol><li> Crescere in condizioni asettiche (completamente libero di tutti gli altri organismi contaminanti) 1 x 10 5 trofozoiti di Entamoeba histolytica ceppo HML: IMSS clonare un 18 a 16 x 125 mm tubi di coltura con teflon tappi a vite marittimo di linea (o 1 x 10 6 trofozoiti in un usa e getta T- 25 beuta) e 15 ml (o 50 ml in T-25 pallone) di TYI-S-33 medio (TYI brodo supplementato con 3% Diamante vitamina miscela, siero inattivato bovino adulto 10% calore, 0,5 UI / ml di penicillina e 35 mg / ml streptomicina) 19 in un incubatore a 37 ° C. </li><li> Trofozoiti raccolto durante la fase di crescita logaritmica solito a intervalli 48-96 hr (Figura 1) raffreddando le provette di coltura per 5-10 minuti in un bagno di ghiaccio-acqua per liberare trofozoiti collegato al tubo di vetro cultura. </li><li> Trasferire la coltura in una provetta conica e capovolgere più volte per disperdere la cells. Determinare il numero di cellule utilizzando un emocitometro (camera di Neubauer), e trasferire un inoculo in una provetta di coltura contenente mezzo fresco TYI-S-33. <ol><li> Utilizzare basso numero di amebe per periodi più lunghi di incubazione (~ 3 x 10 5 cellule per ~ 5 giorni) e un numero più elevato per periodi più brevi (~ 1 x 10 6 cellule per ~ 1 giorno). Mentre inoculi contati sono auspicabili, culture stabiliti diventano prevedibili in modo che i volumi stimati di inoculi sono fattibili. </li><li> Titolare l&#39;importo trofozoiti per ottimizzare il numero di cellule per ogni esperimento. </li><li> Mantenere una cultura duplicato parallelo di avere un back-up in caso di contaminazione accidentale o del tubo rottura. </li></ol></li><li> Tubi tappo e li incubare a 37 ° C in un 5 ° angolo di inclinazione orizzontale. </li><li> Controllare le culture di ispezione visiva regolarmente, dal momento che una crescita eccessiva della cultura può contenere molte cellule lisate. </li></ol> 2. Creazione e mantenimento di MDCK Cultura &lt;/ P&gt; <ol><li> Grow MDCK (Madin Darby Canine Kidney) cellule di tipo I (alta resistenza) in monouso T-75 beute da 10 ml di mezzo DMEM supplementato con penicillina (100 UI / ml), streptomicina (100 mg / ml), insulina (0,08 U / ml) e siero di vitello neonato 10% in atmosfera umidificata al 5% di CO 2 e il 95% aria a 37 ° C. </li><li> Per dividere le celle, li controlla su un microscopio invertito. Dividere le celle quando sono ~ 85-100% confluenti. </li><li> Utilizzare un aspiratore a vuoto in una cappa sterile e sterile Pasteur pipetta di vetro per rimuovere il supporto dalla cultura. Per evitare di raschiare le cellule, girare il pallone di testa in giù e mezzi di aspirare dal basso. </li><li> Pipettare 10 ml di PBS preriscaldata (140 mM NaCl, 2,7 mM KCl, 10 mM Na 2 HPO 4, 1,8 mM KH 2 PO 4, pH 7.4) in un pallone da T-75 (5 ml nel caso di un T-25 pallone ) e rock delicatamente il pallone per lavare le cellule. Rimuovere PBS tramite aspirazione a vuoto. Ampia lavaggio è richiesto dal siero inibisce la tripsina. </li><li> Pipettare 1,5 ml di 0,05% tripsina in un pallone T-75 (per T-25 pallone di usare 0,5 ml) e agitare delicatamente il pallone per coprire lo strato di cellule con tripsina. </li><li> Incubare per 5-10 minuti in incubatrice (il tempo esatto dipende dall&#39;attività tripsina). </li><li> Verificare la presenza di distacco cella tenendo il pallone contro la luce e cercare scorre cellule. Distacco delle cellule (cellule arrotondate) può essere controllato utilizzando un microscopio. Spesso, cellule rilascio veloce con un breve, dolce slap al lato del pallone. </li><li> Aggiungere 15 ml di DMEM supplementato in un pallone T-75 (5 ml per un pallone T-25). Risospendere le cellule pipettando su e giù 4 o 5 volte. </li><li> Per propagare le celle, diluire sospensione cellulare 1:5 con fresco INTEGRAZIONI DMEM. Numero di celle può essere determinato anche, a questo punto con un emocitometro, ma questo è di solito necessario solo se dividere le celle in formati speciali per alcuni saggi. </li></ol> 3. Preparazione di Trofozoite totale lisati <ol><li> Staccare trofozoiti da cutubi lture raffreddando la provetta per 5-10 minuti in un bagno di acqua e ghiaccio. Trasferire asetticamente coltura in una provetta conica e centrifugare a 360 xg per 10 min a 4 ° C. Eliminare attentamente il supernatante per decantazione, aggiungere PBS freddo al pellet, invertire la provetta diverse volte per disperdere le cellule e centrifugare di nuovo a 360 xg per 10 min. Ripetere questa operazione due volte per eliminare ogni traccia di Tyi-S-33 di media. </li><li> Determinare il numero trofozoiti con un emocitometro. </li><li> Lyse trofozoiti dai cicli di gelo-disgelo. Snap-congelare un inoculo misurata di trofozoiti (600.000 trofozoiti / ml) diluito in PBS freddo, in azoto liquido per 1 min. Sbloccare il campione a 4 ° C e agitare vigorosamente. Ripetere gelo e disgelo 3 volte per completare la lisi cellulare. </li><li> Attivare proteasi presenti nei lisati con 0,02% β-mercaptoetanolo. </li></ol> 4. Preparazione di Trofozoite secreto Prodotti <ol><li> Eseguire le operazioni 3,1-3,2. </li><li> Determinarenumero di cellule e vitalità a questo punto con un emocitometro e applicando un test di esclusione del trypan blu 20: <ol><li> Diluire 9 x 10 6 trofozoiti in 3 ml di PBS freddo e trasferire le cellule in una provetta conica da 50 ml. Prendere 10 ml di questa sospensione e aggiungere 1 ml di 0,4% trypan blu stock soluzione a pH 7.2. </li><li> Utilizzare una camera di Neubauer per contare le cellule a basso ingrandimento. </li><li> Contare tutte le cellule e separare il numero di cellule blu, poiché le cellule blu hanno preso il colorante e devono essere considerati morti. </li><li> Calcolare la percentuale di cellule vitali dividendo il numero di cellule vitali per il numero totale di cellule e moltiplicando per 100. </li></ol></li><li> Trasferire il tubo conico contenente la sospensione trofozoita in incubatore a 37 ° C per 2 ore in un 5 ° angolo di inclinazione orizzontale. </li><li> Per raccogliere i prodotti secreti, provette da centrifuga a 360 xg per 10 min. Usare una siringa monouso e sterile e raccogliere la su attenzionepernatant, evitando la contaminazione dei campioni con trofozoiti dal pellet. Eliminare tutte le cellule trasferite passando il surnatante attraverso una membrana di acetato di cellulosa 0,22 micron. Attivare proteasi con il 0,02% β-mercaptoetanolo. </li><li> Per eliminare putativo contaminazione indesiderata con molecole rilasciate dalle cellule morte, riapplicare il test di esclusione del trypan blu per la vitalità cellulare. Numero vitale e totale di celle deve essere lo stesso ottenuto nella fase 4.2. Se questo non è il caso, il supernatante raccolto può non solo contenere proteine ​​secrete e deve essere eliminata. </li></ol> 5. Interazione delle cellule MDCK con Trofozoiti, Trofozoite lisati o secrete Prodotti <ol><li> Incubare confluenti monostrati cellulari MDCK con trofozoiti vivi (un rapporto MDCK-to-ameba 1:1), Trofozoite lisati (un rapporto di MDCK-to-ameba di 1:2) o di prodotti secreti (un rapporto MDCK-to-ameba di 1 : 10) a 37 ° C per 2 min e 30 (Figura 2A). </li><li> Lavare le cellule epiteliali cinque volte con PBS freddo per eliminare le molecole non legate o trofozoiti. </li></ol> 6. Preparazione dei campioni per immunofluorescenza <ol><li> Cellule Cultura MDCK su vetrini sterili posti all&#39;interno di una piastra di coltura cellulare di 24 multiwell. Ispezionare le celle del microscopio invertito fino a raggiungere il 100% di confluenza. Quindi, sostituire medio di 1 ml di DMEM fresco INTEGRATO medio e trasferire la piastra ad un incubatore a 37 ° C per 24 ore più. </li><li> Rimuovere il mezzo usando un aspiratore a vuoto e una sterile Pasteur pipetta di vetro e aggiungere 1 ml di PBS caldo a ciascun pozzetto. Rimuovere PBS e ripetere il lavaggio con PBS fresco. Fare attenzione a non raschiare cellule dal fondo del pozzetto con la pipetta di vetro. </li><li> Aggiungi diverse condizioni di trofozoiti diluito in 1 ml di PBS caldo: trofozoiti vivi (T), lisati totali trofozoita (TL) e prodotti secreti (SP). <ol><li> Mettere la piastra di coltura in incubatrice per 2 o 30 min. </li><li> Preparare due condizioni di controllo: un tempo denominata 0 min, dove le cellule MDCK non devono essere incubate con E. histolytica ma solo con 1 ml di caldo PBS; e un&#39;altra condizione come controllo anticorpi secondari, in questo caso incubare MDCK con T, TL o SP per 2 o 30 min e omettere incubazione con anticorpi primari. </li></ol></li><li> Lavare le cellule epiteliali cinque volte con PBS freddo per eliminare le molecole non legate o trofozoiti. </li><li> Fissare e permeabilize le cellule con 1 ml di etanolo al 96% per 30 minuti a -20 ° C. </li><li> Per rimuovere l&#39;etanolo, effettuare tre lavaggi rapidi di 1 ml di PBS a temperatura ambiente. </li><li> Effettuare le seguenti operazioni in una camera umida per evitare l&#39;essiccamento dei campioni: <ol><li> Utilizzare qualsiasi scatola piatta disponibile che può essere chiuso e riempito con un tovagliolo di carta bagnata in cui i campioni possono essere inseriti in modo sicuro. Ad esempio, una casella di punta della pipetta vuota serve bene a questo scopo. </li><li> All&#39;interno della camera, in modo uniforme posizionare uno strato Parafilm e pipetta 25 ml di bloccoing soluzione (0,5% BSA) per ogni vetrino. </li><li> Prendere coprioggetto su pozzetti con pinza sottile, rimuovere accuratamente il liquido in eccesso dal bordo con carta da filtro e mettere coprioggetto nella goccia contenente soluzione bloccante (0,5% BSA) con le cellule di fronte la soluzione di saturazione. Incubare i campioni per 1 ora a temperatura ambiente. </li></ol></li><li> Preparare una miscela di anticorpi primari in PBS: IgM anti-topo EhCPADH112 (mα-EhCPADH112; 1:10) e IgG di coniglio anti-ZO-1 (pα-ZO-1; diluizione 1:100). </li><li> Mettere un foglio fresco Parafilm nella casella umida e pipetta 25 ml di soluzione di anticorpi per ogni vetrino. </li><li> Sollevare i coprioggetti dalla soluzione di blocco, asciugare il liquido in eccesso ai bordi con una carta da filtro, e metterli nelle gocce con le cellule di fronte alla soluzione di anticorpi. Incubare i campioni notte a 4 ° C. </li><li> Mettere ogni vetrino in un pozzo di 24 multipozzetto (lato cella in alto) e aggiungere 1 ml di PBS. <ol><li&gt; Lavare cinque volte con 1 ml di PBS per rimuovere le molecole non legate. </li></ol></li><li> Preparare una miscela di anticorpi secondari fluorescenti in PBS: topo di capra anti-IgM accoppiato con FITC (diluizione 1:100) e di capra anti-IgG di coniglio accoppiato TRITC (diluizione 1:50). </li><li> Mettere un foglio fresco Parafilm in area umida e pipetta 25 ml di soluzione di anticorpi secondario per ogni vetrino. </li><li> Trasferire i campioni in 6.10 e incubare per 1 ora a temperatura ambiente al buio per evitare sbianca dei coloranti fluorescenti. Ripetere passo 6.11.1. </li><li> Per la colorazione nucleare, incubare per 3 minuti con 200 ml di soluzione DAPI 0,05 mm a temperatura ambiente e proteggere dalla luce. Ripetere passo 6.11.1. </li><li> Per conservare i coloranti fluorescenti, posizionare i coprioggetti (cellule rivolti verso il basso) in 5 ml Vecta Shield antifade soluzione di montaggio su vetrini per microscopio. Sigillare le coprioggetto utilizzando smalto per evitare l&#39;essiccazione del campione. </li><li> Per la conservazione a lungo termine, mantenere le diapositive di unabox vetrino da microscopio a -20 ° C. </li><li> Analizzare preparati mediante microscopia confocale attraverso tratti Z-stack e XZ-piani (Figura 2A). </li></ol> 7. Incubazione di Trofozoiti con inibitori della proteasi o anticorpo specifico <ol><li> Ripetere i passaggi 3,1-3,2. Per ciascuna condizione sperimentale, incubare 1 x 10 5 trofozoiti (risospese in 50 microlitri di PBS) con inibitori della proteasi (1 mM completi e 40 mcg / ml E-64) o 30 mg di anticorpo monoclonale contro EhCPADH112 (mαEhCPADH112) 21 per 20 min a 4 ° C (Figura 2B). Utilizzare questi preparativi per saggi di co-incubazione come descritto al punto 8.5. </li></ol> 8. Misura della resistenza elettrica transepiteliale (TER) <ol><li> Cultura cellule MDCK su sterili transwell supporti permeabili (0,4 micron di dimensione dei pori). Nel vano superiore, posizionare 100 microlitri di sospensione cellulare e nel vano inferioreVersare 600 ml di mezzo DMEM integrato. <ol><li> Controllare il livello medio periodicamente. Mezzo fresco può essere aggiunto come richiesto. </li><li> Ispezionare le cellule su un microscopio invertito fino a raggiungere il 100% di confluenza. Cambiare medio ogni 2-3 giorni. </li><li> Per migliorare l&#39;adesione delle cellule (se necessario), equilibrare i filtri transwell notte a 37 ° C in DMEM prima dell&#39;aggiunta della sospensione cellulare. </li></ol></li><li> Sterilizzare elettrodi stx2 nella cappa per immersione in etanolo e poi in PBS sterile per 15 min ciascuno, sotto luce UV. Collegare gli elettrodi ad un epiteliale voltohmmeter Evom e selezionare la modalità di resistenza. </li><li> Raccogliere mezzo dal vano superiore di ogni transwell e piscina. Introdurre 50 ml di questa miscela Mezzi e combinarlo con 50 ml di sospensione trofozoiti (1 x 10 5 trofozoiti in PBS) o con solo PBS (controllo). Utilizzare una miscela simile per ogni transwell. </li><li> Aggiungere miscele verso la camera superiore di ogni contenent transwellg cellule MDCK. Condizioni sperimentali includono cellule MDCK senza trofozoiti (controllo), una co-coltura con trofozoiti o con trofozoiti pre-incubate con inibitori della proteasi o mαEhCPADH112. Per eliminare la resistenza fornita dai filtri, utilizzare transwells incubate con solo mezzo. Per ciascuna condizione sperimentale usare almeno tre transwells. </li><li> Immediatamente, misurare TER immergendo gli elettrodi stx2 in ogni transwell. <ol><li> Assicurarsi che l&#39;elettrodo non tocchi il fondo della camera inferiore e che l&#39;elettrodo inferiore è coperta dal fluido nel vano superiore (vedere la Figura 2B). </li><li> Assicurati di tenere gli elettrodi perpendicolarmente ogni volta. Ciò permetterà di migliorare la riproducibilità, in modo significativo. Evitare di spostare o inclinare l&#39;elettrodo durante le misurazioni in quanto questo porterà alla variazione dei dati. </li></ol></li><li> Questa misura dovrebbe essere considerato il valore iniziale TER. Poi, monitorare il TER durante la seguente30 min. </li><li> Per calcolare il valore TER finale, sottrarre il valore TER di soli filtri. Per confrontare i risultati di diversi esperimenti di normalizzare ogni punto dati ai valori iniziali, prendendo questi come 100% (Figura 2B). </li></ol>

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Appendix 3, (2001).</li><li>Arroyo, R., Orozco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+identification+of+an+Entamoeba+histolytica+adhesin.">Localization and identification of an Entamoeba histolytica adhesin.</a> Mol Biochem Parasitol. 23, 151-158 (1987).</li><li>Diamond, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axenic+cultivation+of+Entamoeba+hitolytica.">Axenic cultivation of Entamoeba hitolytica.</a> Science. 134, 336-337 (1961).</li><li>Diamond, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+of+axenic+cultivation+of+Entamoeba+histolytica+Schaudinn,+1903+and+E.+histolytica-like+amebae.">Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae.</a> J Parasitol. 54, 1047-1056 (1968).</li><li>Dukes, J. D., Whitley, P., Chalmers, 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=The+MDCK+variety+pack:+choosing+the+right+strain.">The MDCK variety pack: choosing the right strain.</a> BMC Cell Biol. 12, (2011).</li><li>Espinosa-Cantellano, M., Martinez-Palomo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+intestinal+amebiasis:+from+molecules+to+disease.">Pathogenesis of intestinal amebiasis: from molecules to disease.</a> Clin Microbiol Rev. 13, 318-331 (2000).</li><li>Martinez-Palomo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+bases+of+the+cytolytic+mechanisms+of+Entamoeba+histolytica.">Structural bases of the cytolytic mechanisms of Entamoeba histolytica.</a> J Protozool. 32, 166-175 (1985).</li><li>Martinez-Lopez, 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=The+EhADH112+recombinant+polypeptide+inhibits+cell+destruction+and+liver+abscess+formation+by+Entamoeba+histolytica+trophozoites.">The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites.</a> Cell Microbiol. 6, 367-376 (2004).</li><li>Ocadiz-Ruiz, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+silencing+of+the+Ehcp112+gene+on+the+in+vitro+virulence+of+Entamoeba+histolytica.">Effect of the silencing of the Ehcp112 gene on the in vitro virulence of Entamoeba histolytica.</a> Parasit Vectors. 6, 248 (2013).</li><li>Johnson, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+imaging+techniques+to+studies+of+epithelial+tight+junctions.">Applications of imaging techniques to studies of epithelial tight junctions.</a> Adv Drug Deliv Rev. 57, 111-121 (2005).</li></ol>

The authors have nothing to disclose.

Per studiare in vitro le interazioni ospite-patogeno in corso di infezione da E. epiteliale histolytica, è fondamentale lavorare con le culture consolidate di entrambe le cellule epiteliali e trofozoiti. Ad esempio, in passato, E. culture histolytica erano stati di solito stabiliti in associazione con alcune specie di batteri o trypanosomatids 22,23. Tuttavia, co-coltivazione di E. culture histolytica è controproducente per lo studio de...

Entamoeba histolytica è un singolo protozoo responsabile della amebiasi umana, un&#39;infezione intestinale delle cellule causando infiammazione e diarrea. E. histolytica infetta fino a 50 milioni di persone ogni anno, ma solo circa il 10% delle persone infette sviluppano i sintomi associati a amebiasi 1. L&#39;infezione avviene per ingestione di alimenti contaminati o acqua contenente E. cisti histolytica. Nell&#39;intestino, cisti producono trofozoiti vivi che aderiscono al colon mucina e proliferano 2. Trofozoiti di solito si formano le cisti che vengono escreti con feci. In altri casi e per ragioni ancora sconosciute, trofozoiti rompere lo strato epiteliale intestinale e invadono i tessuti sottostanti. Nel peggiore dei casi, entrano nel flusso sanguigno e colpiscono altri organi come il fegato 3. Rompere la barriera epiteliale richiede interruzione di strutture transmembranal epiteliali che mantengono le cellule unite. Cellule epitelialicontatti sono formati dal complesso giunzionale apicale costituito stretto (TJ) e adherens giunzioni (AJ), e desmosomi 4. Le giunzioni più apicali sono TJ, e quindi, sono la prima barriera offeso da E. histolytica e di alcuni altri agenti patogeni durante l&#39;invasione host. TJ sono costituiti da recettori di adesione transmembranal come claudine, occludina e molecole di adesione giunzionale (JAM), che si impegnano in interazioni eterofili omo-o con i recettori della cellula vicina. Sono intracellulare vincolati da molecole impalcatura delle occludere zonula (SO) della famiglia che collegano recettori di adesione al citoscheletro di actina per fornire ulteriore resistenza meccanica all&#39;epitelio. TJ sono responsabili per sigillare tessuto intestinale dal lume intestinale, impedendo all&#39;acqua eccessiva e perdita soluto. Così, dopo TJ sono interrotti dal parassita, i tessuti sono invase. E. histolytica secerne diverse molecole quali: (i) quelli coinvolti nella adesione di amebe a targcellule et 5; (Ii) fattori membrana attiva partecipano uccisione delle cellule ospiti per esocitosi, per esempio, i peptidi che formano canali ionici chiamati amoebapores 6,7; e (iii) proteinasi che degradano le proteine ​​della matrice extracellulare e mediano tessuto disintegrazione 5,8,9. La cisteina proteasi EhCP112 e la molecola di adesione EhADH112 che insieme formano il complesso EhCPADH112 sono due E. histolytica virulenza proteine ​​che svolgono un ruolo importante nello smontaggio di TJ 10. Trofozoiti vivi, loro lisati totali e prodotti secreti inducono cambiamenti molecolari nel TJ complesso e funzionale disturbo della barriera epiteliale. In questo studio, è dimostrato che EhCP112 e EhADH112 interagiscono con occludina e claudin-1 proteine ​​che portano alla internalizzazione e la degradazione delle proteine ​​cellulari, facilitando così E. ingresso histolytica attraverso la via paracellulare. I nostri dati e quelli of altri gruppi di 11-17 suggeriscono con forza la necessità di interazioni ospite-patogeno specifiche che consentono parassita invasione. Svelare le basi molecolari di queste interazioni è della massima importanza per una migliore comprensione della patogenesi amebiasi. Disturbo selettivo di TJ da trofozoiti, caratterizzate da un aumento della permeabilità paracellulare, può essere misurata da una diminuzione della resistenza elettrica transepiteliale (TER). Il trasferimento delle proteine ​​parassite verso epiteli ospitanti può essere determinato da immunofluorescenza e microscopia confocale, un metodo che può anche rivelare co-localizzazione di fattori di virulenza amebe con marcatori epiteliali giunzionale indicano possibili interazioni dirette. In questo articolo, descriviamo in dettaglio come le cellule epiteliali e trofozoiti sono coltivati, raccolti e manipolati per esaminare le interazioni ospite-patogeno e le loro conseguenze.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Entamoeba histolytica HM1:IMSS, Clone A&#160;</td>
			<td>IMSS Hospital, Mexico</td>
			<td>Without/number</td>
			<td>Virulent trophozoites18&#160;</td>
		</tr>
		<tr>
			<td rowspan="8">TYI broth</td>
			<td rowspan="8">Becton, Dickinson and Company 
			Merck 
			Merck 
			Merck 
			J.T. Baker 
			Reproquifin 
			SIGMA-Aldrich 
			SIGMA-Aldrich</td>
			<td rowspan="8">211862 
			K35625437 626 
			21578 
			4873 
			3252-01 
			CAS 50-81-7 
			C7880 
			F-5879</td>
			<td rowspan="8">3.45% BBL Biosate peptone 
			58 mM glucose 
			39 mM NaCl 
			5 mM KH2PO4 
			6.5 mM K2HPO4 
			16.3 mM ascorbic acid 
			8.1 mM L-cysteine 
			&#160;0.1 mM ferric ammonium citrate, adjust pH 6.819</td>
		</tr>
		<tr>
			<td>Bovine serum adult</td>
			<td>Microlab , Labs., Mex.</td>
			<td>SU146</td>
			<td>Use at 10% and inactivated to 56&#176; C for 30 min</td>
		</tr>
		<tr>
			<td>Diamond&#160; vitamin&#160; mixture- Tween 80</td>
			<td>In vitro</td>
			<td>SR-07</td>
			<td>Use at 3%</td>
		</tr>
		<tr>
			<td>Penicillin&#160;</td>
			<td>Lakeside,&#160; M&#233;x.</td>
			<td>34564SSA IV</td>
			<td>0.5 IU/mL</td>
		</tr>
		<tr>
			<td>Streptomycin&#160;</td>
			<td>Lakeside,&#160; M&#233;x.</td>
			<td>75757SSA IV</td>
			<td>35 &#181;g/ mL</td>
		</tr>
		<tr>
			<td>Pyrex 15 mL screw cap culture tubes with PTFE lined phenolic caps</td>
			<td>Corning-Pyrex</td>
			<td>9826-16X</td>
			<td>16x125 mm, capacity 15 mL and caps fabricated from special formula resistant to effects of temperature</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6 Well</td>
			<td>Corning-Costar</td>
			<td>3516</td>
			<td>Sterile plates, well diameter 34.8 mm and growth area 9.5 cm2.&#160; Rings on lid prevent cross-contamination</td>
		</tr>
		<tr>
			<td>25cm2 cell culture flask</td>
			<td>Corning-Costar</td>
			<td>430168</td>
			<td>Canted neck flasks</td>
		</tr>
		<tr>
			<td>MDCK (Madin Darby canine kidney) type I</td>
			<td>American Type Culture Collection</td>
			<td>CCL34</td>
			<td>Kidney epithelial cells grown between the 60th and 90th passage</td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Gibco&#160;</td>
			<td>12800-017</td>
			<td>Dulbecco&#39;s Modified Eagle Medium with high glucose.</td>
		</tr>
		<tr>
			<td>Neonate Calf Serum</td>
			<td>In vitro</td>
			<td>S-02</td>
			<td>Use at 10%.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin mixture&#160;</td>
			<td>In vitro&#160;</td>
			<td>A-01</td>
			<td>Stock solution 10,000 U/&#181;g/mL</td>
		</tr>
		<tr>
			<td>Insulin&#160;&#160;</td>
			<td>AMSA</td>
			<td>398MJ94SSA IV</td>
			<td>Stock solution 100 IU/mL</td>
		</tr>
		<tr>
			<td>Trypsin solution&#160;</td>
			<td>In vitro</td>
			<td>EN-005</td>
			<td>0.05% enzyme solution without calcium and magnesium</td>
		</tr>
		<tr>
			<td>75cm2 cell culture flask</td>
			<td>Corning-Costar</td>
			<td>430720</td>
			<td>Canted neck flasks for trophozoite culture in TYI-S-33 medium</td>
		</tr>
		<tr>
			<td>Transwell permeable supports</td>
			<td>Corning-Costar</td>
			<td>3470</td>
			<td>0.4. &#181;m polyster membrane, 6.5 mm insert in 24 well plate, growth area 0.3 cm2</td>
		</tr>
		<tr>
			<td>24 well cell culture dish&#160;&#160;</td>
			<td>Corning-Costar</td>
			<td>3524</td>
			<td>Clear polystyrene, treated for optimal cell attachment, sterilized by gamma radiation and certified non-pyrogenic</td>
		</tr>
		<tr>
			<td>Complete Mini</td>
			<td>Roche</td>
			<td>11836 153 001</td>
			<td>Protease inhibitor cocktail inhibits a broad spectrum of serine, cysteine and metallo-proteases. Final concentration 1 mM&#160;</td>
		</tr>
		<tr>
			<td>Trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64)</td>
			<td>SIGMA-Aldrich</td>
			<td>E3132</td>
			<td>Cystein protease inhibitor, final concentration 40 &#181;g/mL</td>
		</tr>
		<tr>
			<td>p&#945;ZO-1&#160;</td>
			<td>Invitrogen</td>
			<td>402200</td>
			<td>IgG rabbit policlonal&#160; antibody&#160; against&#160; a synthetic peptide in the middle region of the ZO-1 human protein</td>
		</tr>
		<tr>
			<td>m&#945;EhCPADH112</td>
			<td>Homemade antibody</td>
			<td>Without/ Number</td>
			<td>IgM mouse monoclonal antibody&#160; against&#160; 444-601 epitope located at C-terminal of EhCPADH11221,27</td>
		</tr>
		<tr>
			<td>FITC-goat anti-mouse IgM</td>
			<td>Zymed</td>
			<td>62-6811</td>
			<td>Fluorescein isotiocyanate (FITC)-labelled goat anti-mouse secondary&#160; antibody</td>
		</tr>
		<tr>
			<td>TRITC- goat anti-rabbit IgG (H+L)</td>
			<td>Zymed</td>
			<td>816114</td>
			<td>Tetramethyl-rhodamine isothiocyanate (TRITC)-labelled&#160; goat anti-rabbit IgG&#160; secondary antibody.</td>
		</tr>
		<tr>
			<td>STX2 Electrode</td>
			<td>World Precision Instrument&#160;</td>
			<td>102711</td>
			<td>Consists of a fixed pair of double electrodes, 4 mm wide and 1 mm thick. Each stick of the electrode pair contains a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current. For use with EVOM</td>
		</tr>
		<tr>
			<td>EVOM epithelial voltohmmeter</td>
			<td>World Precision Instrument&#160;</td>
			<td>12111</td>
			<td>Use in resistance mode and maintain unplugged during TER measurements</td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>MEARIENFELD</td>
			<td>610610</td>
			<td>Hemocytometer&#160;</td>
		</tr>
		<tr>
			<td>Leica TCS_SP5_MO</td>
			<td>Leica</td>
			<td>Without/number</td>
			<td>Laser confocal microscopy with Leica microsystems CMS Gmbh/leica Las af Lite/BIN software</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories, Inc.</td>
			<td>H-1000</td>
			<td>Mounting medium for fluorescence</td>
		</tr>
		<tr>
			<td>4&#180;, 6-diamino-2-phenylindole (Dapi)</td>
			<td>SIGMA</td>
			<td>D-9542</td>
			<td>0.05 mM final concentration</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological</td>
			<td>A-1310</td>
			<td>0.5%&#160; final concentration for blocking solution</td>
		</tr>
	</tbody>
</table>

analysis of the epithelial damage produced by entamoeba histolytica infection

Entamoeba histolytica è l&#39;agente eziologico di amebiasi umana, una delle principali cause di diarrea e di ascesso epatico nei paesi tropicali. L&#39;infezione è avviata da interazione del patogeno con le cellule epiteliali intestinali. Questa interazione porta alla rottura delle strutture intercellulari come giunzioni strette (TJ). TJ assicurare la chiusura dello strato epiteliale per separare tessuto ospite dal lume intestinale. Recenti studi forniscono prove che la rottura del TJ dalla proteina EhCPADH112 parassita è un prerequisito per E. invasione histolytica che è accompagnato da disfunzione della barriera epiteliale. Pertanto, l&#39;analisi dei meccanismi molecolari coinvolti nella TJ smontaggio durante E. histolytica invasione è di fondamentale importanza per migliorare la nostra comprensione della patogenesi amebiasi. Questo articolo presenta un modello semplice che consente la valutazione delle interazioni iniziali ospite-patogeno e il potenziale parassita invasione. I parametri da analizzare sono transepithelial resistenza elettrica, l&#39;interazione di EhCPADH112 con i recettori di superficie epiteliali, cambiamenti di espressione e la localizzazione di marcatori giunzionale epiteliali e la localizzazione delle molecole del parassita all&#39;interno delle cellule epiteliali.

Per un E. successo cultura histolytica, due importanti condizioni devono essere soddisfatte: la crescita in condizioni axeniche e raccolto in fase logaritmica. In precedenza, colture di E. histolytica erano facilmente stabilita in associazione con alcune specie di batteri o trypanosomatids 22. Tuttavia, al giorno d&#39;oggi è comune avere la coltivazione axenic di questo parassita che significa un subcoltivazione indefinito di amebe in un ambiente privo di batteri che metabolizzano...

Watch this Scientific Journal Video about Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection at JoVE.com

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

L&#39;infezione umana da Entamoeba histolytica porta ad amebiasi, una delle principali cause di diarrea nei paesi tropicali. L&#39;infezione è avviata da patogeno con cellule epiteliali intestinali, provocando l&#39;apertura dei contatti cellula-cellula e di conseguenza diarrea, a volte seguita da infezione fegato. Questo articolo fornisce un modello per valutare le interazioni primi ospite-patogeno per migliorare la nostra comprensione della patogenesi amebiasi.

Analisi del danno epiteliale Prodotto da<em&gt; Entamoeba histolytica</em&gt; Infezione

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

analysis-epithelial-damage-produced-entamoeba-histolytica

Research

JoVE Journal

Immunology and Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

18.6K Views.  Center for Research and Advanced Studies of the National Polytechnic Institute.  L'infezione umana da Entamoeba histolytica porta ad amebiasi, una delle principali cause di diarrea nei paesi tropicali. L'infezione è avviata da patogeno con cellule epiteliali intestinali, provocando l'apertura dei contatti cellula-cellula e di conseguenza diarrea, a volte seguita da infezione fegato. Questo articolo fornisce un modello per valutare le interazioni primi ospite-patogeno per migliorare la nostra comprensione della patogenesi amebias...

Video: Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Test in vitro Metilazione tRNA con l&#39;Entamoeba histolytica del DNA e Dnmt2 tRNA metiltransferasi (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

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Immunologia e infezioni

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Sviluppo di un marcatore negativo selezionabile per Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

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 ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Flusso Isolamento citometrica di primari murini di tipo II cellule alveolari epiteliali per gli studi funzionali e molecolari

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Analisi della accessibilità al solvente dei residui di cisteina in<em&gt; Mais Rayado Fino virus</em&gt; Particelle virus-simili Prodotto in<em&gt; Nicotiana benthamiana</em&gt; Piante e Cross-linking di peptidi a VLP

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsulare Sierotipizzazione di<em&gt; Streptococcus pneumoniae</em&gt; Da agglutinazione al lattice

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

L&#39;utilizzo della tecnica di sovrapposizione Soft-agar per lo screening composti inibitori bacterially Prodotto

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2 + risposte di imaging durante l'infezione delle cellule epiteliali Shigella

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caratterizzazione dei sottoinsiemi di monociti umani da sangue intero flusso Cytometry analisi

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...