Lavoro nei laboratori degli autori è sostenuto da finanziamenti della scienze naturali e ingegneria Research Council del Canada, gli Istituti di Canada Health Research, e il Canada Research Chairs programma. JL è sostenuto da una borsa di studio dal Groupe d&#39;Étude des Membranaires Protéines (GEPROM) attraverso i finanziamenti del Fonds de Recherche en Santè du Québec.

1. Preliminare: ceppi batterici e le cellule epiteliali. Manipolazioni di cellule e batteri vengono eseguite in modo asettico, sotto una cappa a flusso laminare. <ol><li> Appena isolare la E. coli 2787, 2787Δ Aida, e C600 dalle scorte glicerolo in brodo lisogenia (LB) piastre di agar (1 triptone%, cloruro di sodio allo 0,5%, estratto di lievito 0,5%, 1,5% agar) e crescono a 37 ° C. Per minimizzare la variabilità nel test, si consiglia di utilizzare sempre ceppi appena placcato e per mantenere i ceppi a 4 ° C in piastre di Petri chiusa solo per un massimo di un paio di settimane. </li><li> Cultura cellule HEp-2 (ATCC CCL-23) in alto glucosio Dulbecco modificato (DMEM) supplementato con 10% siero bovino inattivato con il calore (inattivazione termica viene eseguita a 56 ° C per 30 min). Durante la cultura di routine aggiungiamo anche 10 U / ml di penicillina e 10 mg / ml di streptomicina. </li><li> Crescere le cellule Hep-2 a 37 ° C in una cella incubatore con un&#39;atmosfera contenente il 5% di CO 2. Utilizzare le procedure standard di coltura cellulare per mantenere le cellule, che vengono coltivate in 75 cm 2 palloni e subcoltura ogni volta che raggiungere la confluenza. Noi usiamo le nostre cellule in coltura fino a raggiungere 30-40 passaggi e poi disfarsene. </li><li> Preparare un test quando la cellule HEp-2 in un centimetro 75 2 fiasco sono quasi raggiungendo confluenza e quindi sono pronti per un test di aderenza. </li></ol> 2. GIORNO 1: Preparare l&#39;inoculo e le cellule epiteliali <ol><li> Lavare i-2 HEp cellule nel pallone una volta con fosfato caldo Dulbecco salina tamponata (DPBS: 8 g / L di NaCl, 0,2 g / L di KCl, 0,2 g / l KH 2 PO 4, 0,21 g / L di Na 2 HPO 4: 7H 2 O) </li><li> Le cellule vengono incubate con il 0,05% tripsina - EDTA per 5 minuti prima di aggiungere nuovi media caldo completo. Dopo mezzo fresco viene aggiunto, le cellule sono risospese pipettando su e giù. </li><li> Centrifugare la sospensione cellulare a 2.000 giri per 5 minuti e risospendere il pellet in DPBS e centrifugare di nuovo. </li><li> Risospendere le cellule in terreno fresco supplementato con 10% di siero, ma che non contiene antibiotici, ad una concentrazione di 2x10 5 cellule / ml. </li><li> Semi di un millilitro di sospensione cellulare in tre serie di pozzetti in doppio (uno per ogni ceppo) al centro di un 24-pozzetti ed incubare la piastra di notte in una coltura cellulare incubatore. Nota: è importante che il siero venga accuratamente decomplemented e che gli antibiotici sono stati omessi, dopo questa fase. In caso contrario potrebbe uccidere i batteri infettante. </li><li> Inoculare una colonia isolata di ogni ceppo batterico (2787, 2787Δ aida e C600) in 5 ml di brodo LB (1 triptone%, cloruro di sodio allo 0,5%, estratto di lievito 0,5%) e crescono durante la notte a 37 ° C con agitazione vigorosa (180 giri al minuto ). </li></ol> 3. 2 ° GIORNO: infezione delle cellule <ol><li> Ispezionare la cellule HEp-2 sotto un microscopio invertito per accertare che sono almeno il 90% confluenti e non contaminati. </li><li> Lavato le cellule con DPBS caldo e, in ogni pozzetto, aggiungere 1 ml di terreno fresco supplementato con 10% di siero, ma che non contiene antibiotici. Aggiungete anche mezzo fresco di tre pozzi senza cellule. Questo sarà utilizzato per determinare il numero totale di batteri nel inoculo per ogni ceppo. </li><li> Misurare la densità ottica a 600 nm (OD 0,600 nm) di colture batteriche. Aggiungere un&#39;aliquota di ogni coltura batterica in una serie di pozzetti in doppio che contiene le cellule Hep-2 e ad un bene che non contengono cellule. Di solito, si usa un volume di cultura durante la notte che corrisponde a 10 6 unità formanti colonia (ufc). Questo rappresenta una molteplicità di infezione (MOI) di 5:1 (batteri: le cellule). Anche se siamo utilizzare direttamente le nostre culture durante la notte, a volte è consigliabile centrifugare i batteri e risospendere in DPBS per evitare gli effetti deleteri di molecole secrete presenti nelle culture durante la notte (come citotossine, per esempio) Nota: il MOI può variare tra 100:1 e 1:10. MOI più alto rendimento maggiore variabilità e lo sfondo e batteri tenderà ad aderire alla plastica della piastra. MOI rendimenti inferiori anche elevata variabilità. Una volta che un MOI è scelto, è indispensabile essere coerenti e mantenere questa MOI. </li><li> Incubare le cellule infette nella cella cultura incubatore per 3 ore a 37 ° C con 5% di CO 2. Nota: E &#39;anche possibile di centrifugare brevemente la piastra a bassa velocità (ad esempio 1.000 xg per 1-2 min), al fine di portare tutti i batteri direttamente a contatto con le cellule. Questo ha l&#39;ulteriore vantaggio di sincronizzare l&#39;infezione, e consentendo tempi di incubazione più breve (non più di 15-30 min). </li><li> Rimuovere il supporto da parte delle cellule infettate e lavare le cellule 3 volte con DPBS caldo. In questa fase, i batteri aderenti di solito può essere visto con un microscopio standard e di routine che esegue questo controllo. </li><li> Per lisare le cellule e staccare il aderito bacteri, aggiungere 100 ml di 1% Triton X-100 in ciascun pozzetto contenente le cellule. Altri detergenti possono essere utilizzati (come ad esempio saponina, per esempio). </li><li> Incubare le cellule per 10 minuti a temperatura ambiente e poi aggiungere 900 ml di LB medium. </li><li> Delicatamente omogeneizzare le sospensioni da ripetute su e giù pipettaggio. I pozzi senza cellule contenenti l&#39;inoculo sono altrettanto delicatamente omogeneizzato. Nota: alcuni batteri potrebbe essere troppo sensibile al detergente, in questo caso si staccano le cellule epiteliali di incubazione con tripsina 0,05% - EDTA per 20 minuti a 37 ° C. Insieme le cellule con i batteri aderito possono anche essere raschiato dai pozzi con la punta della pipetta. </li><li> Preparare seriale 10-diluizioni delle sospensioni di batteri aderito e l&#39;inoculo utilizzando brodo LB e la piastra di 100 ul da 3 diluizioni (di solito il diluizioni 1:1.000, 1:10.000 e 1:100.000) su agar LB ed incubare una notte a 37 ° C . </li></ol> 4. GIORNO 3: conteggio cfu e presentazione dei dati. <ol><li> Contare le colonie sulle piastre e calcolare il numero di cfu di batteri aderito e dell&#39;inoculo la media ciascuna serie di diluizioni. Piatti unici con tra 10-300 colonie dovrebbero essere contate. </li></ol> 5. Rappresentante dei risultati: La tabella seguente mostra i risultati tipici di ufc conteggio dei batteri aderito e l&#39;inoculo da 3 esperimenti eseguiti in giorni diversi: <img src="/files/ftp_upload/2783/2783table1.jpg" alt="Tabella 1" /> I risultati possono essere riportati direttamente come aderito cfu, come mostrato nella Figura 1A. Dal momento che le dimensioni inoculo può variare tra i ceppi a causa delle differenze nei tassi di crescita o errori di pipettamento, è spesso consiglia di segnalare i risultati come percentuale di batteri aderito. La percentuale di batteri aderito può essere calcolato dividendo il numero di cfu di batteri aderito per il numero di cfu di inoculo, come mostrato nella Figura 1B. Eseguendo una misura ripetuta ANOVA e Bonferroni post-test per confrontare tutte le colonne di Figura 1B, possiamo vedere che ci sono differenze significative (p &lt;0,05) tra il 2787 e 2787Δ aida, così come tra 2787 e C600, ma nessuna differenza significativa tra 2787Δ aida e C600. <img src="/files/ftp_upload/2783/2783fig1.jpg" alt="Figura 1"/> Figura 1. Rappresentazione dei risultati come UFC di batteri aderito (A) o la percentuale di batteri aderito (B) La percentuale di adesione osservati è spesso molto dipende dal set-up sperimentale (e, in misura minore, sul sperimentatore). Particolarmente importanti sono il MOI e il numero di lavaggi, per cui la percentuale non deve essere interpretata letteralmente. I batteri nel inoculo a volte può crescere molto più velocemente rispetto ai batteri in presenza di cellule, alterando le dimensioni dell&#39;inoculo rispetto al reale numero totale di batteri in pozzi con le cellule. Per ovviare a questo problema, si consiglia di: (i) utilizzare i pozzi con le cellule per determinare l&#39;inoculo: cellule HEp-2 sono seminati in un pozzo supplementare e infetti. Alla fine del test, invece di scartare il surnatante e lavare, 100 ml di 10% di Triton X-100 si aggiunge al pozzo. Lisare le cellule e il lisato contenente batteri aderito e non è gentilmente aderito omogeneizzato da ripetuti su e giù pipettaggio;. O (ii) raccogliere i surnatanti delle cellule infettate, alla fine del test, così come i surnatanti di la lava DPBS e determinare il numero di cfu in quelle surnatanti pool. Questo produrrà il numero di cfu di batteri non aderito. La percentuale di batteri aderito può essere calcolato dividendo il numero di cfu di batteri aderito con l&#39;aggiunta del numero di cfu di batteri aderito e non aderito. Per illustrare la robustezza del test, possiamo confrontare questo protocollo con il test effettuato con S4074, un adesivo suina di ceppo patogeno Actinobacillus pleuropneumoniae 7, incubate con cellule provenienti da una linea di recente costituzione tracheale cellula maialino, NPTr 8. A parte le differenze nel supporto richiesto per la crescita dei batteri e le cellule, l&#39;unica differenza è che i batteri sono usati per l&#39;infezione da una cultura in crescita esponenziale, e non da una fase stazionaria cultura durante la notte. Con A. pleuropneumoniae è anche molto importante rispettare un MOI di 10:1 e di non superare 3 ore di infezione, altrimenti la secrezione di tossine che causano la morte cellulare e pregiudizi dei risultati. Inoltre, questo ceppo di A. pleuropneumoniae possono facilmente aderire alla plastica quindi è importante utilizzare le cellule confluenti e verificare visivamente che le cellule non sono sloughing off.

<ol>
	<li>Kline, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesins+in+host-microbe+interactions.">Bacterial adhesins in host-microbe interactions.</a> Cell Host Microbe. 5, 580-580 (2009).</li><li>Cossart, P., Boquet, P., Normark, S., Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+microbiology+emerging.">Cellular microbiology emerging.</a> Science. 271, 315-315 (1996).</li><li>Elsinghorst, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+invasion+by+gentamicin+resistance.">Measurement of invasion by gentamicin resistance.</a> Methods Enzymol. 236, 405-405 (1994).</li><li>Pizarro-Cerda, J., Lecuit, M., Cossart, P., Sansonetti, P., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cellular Microbiology. , 161-161 (2002).</li><li>Srivastava, A., Isberg, R. R., Sansonetti, P., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cellular Microbiology. , 179-179 (2002).</li><li>Benz, I., Schmidt, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+an+adhesin+(AIDA-I)+involved+in+diffuse+adherence+of+enteropathogenic+Escherichia+coli.">Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.</a> Infect Immun. 57, 1506-1506 (1989).</li><li>Charbonneau, M. E., Berthiaume, F., Mourez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+processing+is+not+essential+for+multiple+functions+of+the+Escherichia+coli+autotransporter+adhesin+involved+in+diffuse+adherence+(AIDA-I).">Proteolytic processing is not essential for multiple functions of the Escherichia coli autotransporter adhesin involved in diffuse adherence (AIDA-I).</a> J Bacteriol. 188, 8504-8504 (2006).</li><li>Jacques, M., Paradis, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesin-receptor+interactions+in+Pasteurellaceae.">Adhesin-receptor interactions in Pasteurellaceae.</a> FEMS Microbiol Rev. 22, 45-45 (1998).</li><li>Auger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+of+Actinobacillus+pleuropneumoniae+with+porcine+lung+and+tracheal+epithelial+cells.">Host-pathogen interactions of Actinobacillus pleuropneumoniae with porcine lung and tracheal epithelial cells.</a> Infect Immun. 77, 1426-1426 (2009).</li><li>Jacques, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lipo-oligosaccharides+and+lipopolysaccharides+in+bacterial+adherence.">Role of lipo-oligosaccharides and lipopolysaccharides in bacterial adherence.</a> Trends Microbiol. 4, 408-408 (1996).</li><li>Nataro, J. P., Kaper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diarrheagenic+Escherichia+coli.">Diarrheagenic Escherichia coli.</a> Clin Microbiol Rev. 11, 142-142 (1998).</li><li>Jallat, C., Darfeuille-Michaud, A., Rich, C., Joly, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+clinical+isolates+of+diarrhoeogenic+Escherichia+coli:+diffusely+adhering+E.+coli+strains+with+multiple+adhesive+factors.">Survey of clinical isolates of diarrhoeogenic Escherichia coli: diffusely adhering E. coli strains with multiple adhesive factors.</a> Res Microbiol. 145, 621-621 (1994).</li></ol>

Nessun conflitto di interessi dichiarati.

Questo protocollo descrive un dosaggio standard di aderenza dei batteri che possono essere modificati per studiare l&#39;invasione (ad esempio usando gentamicina saggio di protezione 3). Il conteggio delle unità formanti colonie consente la quantificazione, a confronto con approcci affidamento su tecniche di visualizzazione standard di sotto un microscopio, come la colorazione Giemsa. Quest&#39;ultimo fornisce una visione qualitativa di adesione, ma spesso è un complemento utile dal momento che può differe...

<table border="1"><tbody><tr><th> Reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Alta glucosio DMEM </td><td> GIBCO-Invitrogen </td><td> 12430-054 </td></tr><tr><td> DDPBS </td><td> GIBCO-Invitrogen </td><td> 14190-144 </td></tr><tr><td> Crescita siero bovino </td><td> Hyclone </td><td> SH3054 </td></tr><tr><td> Penicillina / streptomicina </td><td> GIBCO-Invitrogen </td><td> 15140-148 </td></tr><tr><td> 24 tavole ben </td><td> Corning </td><td> 3337 </td></tr><tr><td> Triton X-100 </td><td> Fisher Scientific </td><td> BP151-100 </td></tr></tbody></table>

in vitro assay of bacterial adhesion onto mammalian epithelial cells

A causare infezioni, i batteri devono colonizzare il loro ospite. Batteri patogeni esprimere varie molecole o strutture in grado di promuovere l&#39;attaccamento alle cellule ospite 1. Queste adesine si basano su interazioni con i recettori della cellula ospite superficie o proteine ​​solubili che funge da ponte tra batteri e ospite. L&#39;adesione è un primo passo fondamentale prima di invasione e / o secrezione di tossine, perciò è un evento chiave per essere studiato nella patogenesi batterica. Inoltre, i batteri aderito spesso squisitamente indurre risposte cellulari messo a punto, gli studi che hanno dato vita al campo di 2 &#39;microbiologia cellulare&#39;. Test robusti per l&#39;adesione batterica su cellule ospiti e la loro invasione quindi giocare un ruolo chiave negli studi di patogenesi dei batteri e sono da tempo utilizzati in molti laboratori pioniere 3,4. Questi test sono ora praticata dalla maggior parte dei laboratori che lavorano su patogenesi batterica. Qui, descriviamo un test standard di aderenza che illustrano il contributo di uno specifico adesina. Utilizziamo il ceppo di Escherichia coli 2787 5, un ceppo patogeno umano che esprime la autotransporter Adhesin Coinvolto in aderenza diffusa (AIDA). Come controllo, si utilizza un ceppo mutante privi del gene aida, 2787Δ Aida (F. e M. Berthiaume Mourez, inedito), e un ceppo di laboratorio commerciale di E. coli, C600 (New England Biolabs). I batteri sono lasciati di aderire alle cellule dal comunemente usati HEp-2 linea di cellule epiteliali umane. Questo test è stato meno ampiamente descritto in precedenza 6.

Watch this Scientific Journal Video about In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells at JoVE.com

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

Questo protocollo è un test semplice adesione batterica che consiste nel contare il numero di unità formanti colonie batteriche che vengano rispettate su cellule in coltura. Il test è robusto, indipendente dal adesina studiato, e numerose varianti vengono utilizzate nella maggior parte dei laboratori che lavorano su patogenesi batterica.

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 ...

in-vitro-assay-of-bacterial-adhesion-onto-mammalian-epithelial-cells

Research

JoVE Journal

Immunology and Infection

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

25.2K Views.  Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire. Questo protocollo è un test semplice adesione batterica che consiste nel contare il numero di unità formanti colonie batteriche che vengano rispettate su cellule in coltura. Il test è robusto, indipendente dal adesina studiato, e numerose varianti vengono utilizzate nella maggior parte dei laboratori che lavorano su patogenesi batterica.

Video: In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

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 ...

Ricerca

Immunologia e infezioni

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitative<em&gt; In vitro</em&gt; Assay misurare Adesione neutrofili attivati ​​per primarie umane endoteliali microvascolari in condizioni statiche

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

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

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

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

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

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 ...

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 ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Umano camera di flusso neutrofili saggio

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Static Adesione saggio per lo Studio della integrina attivazione nei linfociti T

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Elettroporazione di effettori batterici funzionali in cellule di mammifero

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In vitro e in vivo modello per studiare l&#39;adesione batterica alla parete del serbatoio in condizioni di flusso

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Saggio di adesione Sotto Shear stress per lo studio di linfociti T-Adhesion Molecule Interazioni

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...