Le travail dans les laboratoires des auteurs est soutenu par des subventions de recherches en sciences naturelles et en génie du Canada, les Instituts de recherche en santé du Canada d&#39;, et le Programme des chaires de recherche du Canada. JL est soutenu par une bourse de la Groupe d&#39;Etude des Protéines Membranaires (GEPROM) grâce au financement du Fonds de Recherche en santé du Québec.

1. Préliminaire: Les souches bactériennes et les cellules épithéliales. Les manipulations des cellules et des bactéries sont réalisées de façon aseptique, sous une hotte à flux laminaire. <ol><li> Fraîchement isoler l&#39;E. coli 2787, 2787Δ Aida et C600 à partir de stocks de glycérol sur bouillon lysogénie (LB) gélose (1% de tryptone, le chlorure de sodium à 0,5%, extrait de levure 0,5%, 1,5% de gélose) et de croître à 37 ° C. Afin de minimiser la variabilité dans le dosage, il est conseillé de toujours utiliser des souches fraîchement plaquée et de garder les souches à 4 ° C sur des plaques de Pétri scellées seulement pour un maximum de deux semaines. </li><li> Culture cellules HEp-2 (ATCC CCL-23) au moyen de modification de glucose élevé Dulbecco Eagle (DMEM) supplémenté avec 10% inactivé par la chaleur du sérum bovin (inactivation thermique est réalisé à 56 ° C pendant 30 min.) Pendant la culture de routine, nous aussi ajouter 10 U / ml de pénicilline et 10 pg / ml de streptomycine. </li><li> Cultivez cellules Hep-2 à 37 ° C dans une cellule avec une atmosphère contenant 5% de CO 2 incubateur. Utiliser les procédures standard de culture cellulaire pour maintenir les cellules, qui sont cultivés en fioles de 75 cm2 et repiquées chaque fois qu&#39;ils atteignent la confluence. Nous utilisons nos cellules en culture jusqu&#39;à ce qu&#39;ils atteignent 30 à 40 passages et puis les jeter. </li><li> Préparer un essai lorsque les cellules HEp-2 dans un flacon de 75 cm2 sont presque atteindre la confluence et sont donc prêts pour un test d&#39;adhérence. </li></ol> 2. JOUR 1: Préparation de l&#39;inoculum et les cellules épithéliales <ol><li> Laver les-deux cellules Hep dans le ballon une fois avec du phosphate chaude Dulbecco saline tamponnée (DPBS: 8 g / L de NaCl, 0,2 g / L de KCl, 0,2 g / L KH 2 PO 4, 0,21 g / L de Na 2 HPO 4: 7H 2 O) </li><li> Les cellules sont incubées avec 0,05% de trypsine - EDTA pendant 5 min avant d&#39;ajouter fraîcheur milieu chaud complet. Après un milieu frais est ajouté, les cellules sont remises en suspension par aspiration et refoulement. </li><li> Centrifuger la suspension cellulaire à 2000 rpm pendant 5 min et remettre les boulettes dans du DPBS et centrifuger à nouveau. </li><li> Resuspendre les cellules dans un milieu frais supplémenté avec du sérum de 10% mais ne contenant pas d&#39;antibiotiques, à une concentration de 2x10 5 cellules / ml. </li><li> Graines un millilitre de suspension cellulaire dans trois ensembles de deux puits (un pour chaque souche) dans le centre d&#39;une plaque de 24 puits et incuber la plaque pendant la nuit dans un incubateur de culture cellulaire. Remarque: Il est important que le sérum décomplémenté être soigneusement et que les antibiotiques sont omis après cette étape. Ne pas le faire pourrait tuer les bactéries infectant. </li><li> Inoculer une colonie isolée de chaque souche bactérienne (2787, 2787Δ aida et C600) dans 5 ml de bouillon LB (1% de tryptone, le chlorure de sodium à 0,5%, extrait de levure 0,5%) et pousser une nuit à 37 ° C avec une agitation vigoureuse (180 tours par minute ). </li></ol> 3. JOUR 2: L&#39;infection de cellules <ol><li> Inspectez les cellules HEp-2, sous un microscope inversé pour s&#39;assurer qu&#39;ils sont au moins 90% de confluence et non contaminés. </li><li> Lavé les cellules avec du DPBS chaud et, dans chaque puits, ajouter 1 ml de milieu frais supplémenté avec du sérum de 10% mais ne contenant pas d&#39;antibiotiques. Ajoutez également un milieu frais à trois puits sans cellules. Il sera utilisé pour déterminer le nombre total de bactéries présentes dans l&#39;inoculum pour chaque souche. </li><li> Mesurer la densité optique à 600 nm (DO 0,600 nm) des cultures bactériennes. Ajouter une aliquote de chaque culture bactérienne à un ensemble de deux puits contenant les cellules Hep-2 et à un bien ne contenant pas de cellules. Habituellement, nous utilisons un volume de culture de nuit correspondant à 10 6 unités formant colonies (UFC). Cela représente une multiplicité d&#39;infection (MOI) de 5h01 (bactéries: les cellules). Bien que nous utilisons directement nos cultures de la nuit, il est parfois conseillé de centrifuger les bactéries et les remettre en suspension dans du DPBS pour éviter les effets délétères des molécules sécrétées présents dans les cultures de la nuit (comme cytotoxines, par exemple) Remarque: Le MOI peut varier entre 100:1 et 01:10. MOI plus élevé des rendements plus élevés et de la variabilité de fond et les bactéries ont tendance à coller au plastique de la plaque. Basse MOI donne aussi une forte variabilité. Une fois une MOI est choisi, il est impératif d&#39;être cohérent et de garder cette MOI. </li><li> Incuber les cellules infectées dans la culture cellulaire incubateur pendant 3 heures à 37 ° C avec 5% de CO 2. Note: Il est également possible de centrifuger brièvement la plaque à basse vitesse (par exemple 1000 xg pendant 1-2 min), afin d&#39;apporter toutes les bactéries directement en contact avec les cellules. Cela a l&#39;avantage supplémentaire de synchronisation de l&#39;infection, et permettant des temps d&#39;incubation plus courte (aussi peu que 15-30 min). </li><li> Retirez le support à partir des cellules infectées et laver les cellules 3 fois avec du DPBS chaud. À cette étape, les bactéries adhérentes peuvent généralement être vu avec un microscope standard et nous avons l&#39;habitude d&#39;effectuer cette vérification. </li><li> Pour lyser les cellules et détacher le bac respectéestères, ajouter 100 l de 1% de Triton X-100 dans chaque puits contenant les cellules. Autres détergents peuvent être utilisés (tel que la saponine par exemple). </li><li> Incuber les cellules pendant 10 min à température ambiante, puis ajouter 900 ul de milieu LB. </li><li> Doucement homogénéiser les suspensions par répétés de haut en bas de pipetage. Le puits sans cellules contenant l&#39;inoculum sont également homogénéisé doucement. Remarque: Certaines bactéries pourraient être trop sensibles aux détergents, dans ce cas nous détacher les cellules épithéliales par incubation avec la trypsine 0,05% - EDTA pendant 20 min à 37 ° C. Les cellules avec des bactéries peuvent également être respectées raclées des puits avec une pipette. </li><li> Préparer série 10 des dilutions des suspensions de bactéries respectées et l&#39;inoculum à l&#39;aide de bouillon LB et la plaque 100 ul de 3 dilutions (généralement les dilutions 1:1000, 1:10.000 et 1:100.000) sur gélose LB et incuber une nuit à 37 ° C . </li></ol> 4. JOUR 3: comptage ufc et la présentation des données. <ol><li> Le comptage des colonies sur les plaques et de calculer le nombre d&#39;UFC de bactéries adhéré et de l&#39;inoculum en faisant la moyenne de chaque série de dilutions. Seules les plaques avec entre 10 à 300 colonies doivent être comptées. </li></ol> 5. Les résultats représentatifs: Le tableau suivant montre des résultats typiques d&#39;ufc comptage des bactéries respectées et l&#39;inoculum à partir de 3 expériences réalisées à des jours différents: <img src="/files/ftp_upload/2783/2783table1.jpg" alt="Tableau 1" /> Les résultats peuvent être rapportés directement comme respectées ufc, comme le montre la figure 1A. Depuis les tailles d&#39;inoculum peut varier entre les souches en raison de différences dans les taux de croissance ou des erreurs de pipetage, il est souvent conseillé de rapporter les résultats en pourcentage des bactéries collées. Le pourcentage de bactéries respectées peut être calculé en divisant le nombre d&#39;UFC de bactéries respectées par le nombre de cfu de l&#39;inoculum, comme montré dans la figure 1B. En effectuant une mesure répétée ANOVA et post-tests de Bonferroni pour comparer toutes les colonnes de la figure 1B, on peut voir qu&#39;il ya des différences significatives (p &lt;0,05) entre 2787 et 2787Δ aida, ainsi qu&#39;entre 2787 et C600, mais aucune différence significative entre 2787Δ aida et C600. <img src="/files/ftp_upload/2783/2783fig1.jpg" alt="Figure 1"/> Figure 1. Représentation des résultats en UFC de bactéries respectées (A) ou le pourcentage de bactéries respectées (B) Le pourcentage d&#39;adhésion observée est souvent très dépendante de l&#39;expérimental (et, dans une moindre mesure, sur l&#39;expérimentateur). Particulièrement importantes sont la MOI et le nombre de lavages, de sorte que le pourcentage ne doit pas être interprétée littéralement. Les bactéries présentes dans l&#39;inoculum peut parfois se développent beaucoup plus vite que les bactéries dans la présence de cellules, biaisant la taille de l&#39;inoculum par comparaison avec le nombre réel total de bactéries dans les puits avec des cellules. Pour remédier à ce problème, il est recommandé de: (i) l&#39;utilisation des puits avec des cellules afin de déterminer l&#39;inoculum: cellules HEp-2 sont ensemencées dans un puits supplémentaire et infectées. A la fin de l&#39;essai, au lieu de jeter le surnageant et laver, 100 ul de 10% de Triton X-100 est ajoutée à la bien. Les cellules seront Lyse et le lysat contenant des bactéries respectées et non respecté est doucement homogénéisé par répétés de haut en bas de pipetage;. Ou (ii) de recueillir les surnageants de cellules infectées à la fin de l&#39;essai, ainsi que les surnageants de les lavages DPBS et déterminer le nombre d&#39;UFC dans les surnageants mis en commun. Cela donnera le nombre de cfu de bactéries non respectées. Le pourcentage de bactéries respectées peut alors être calculé en divisant le nombre d&#39;UFC de bactéries respectées par l&#39;ajout du nombre d&#39;UFC de bactéries respectées et non-respectées. Pour illustrer la robustesse de l&#39;analyse, nous pouvons comparer ce protocole avec le test effectué avec S4074, une souche porcine adhésive pathogènes d&#39;Actinobacillus pleuropneumoniae 7, incubées avec des cellules d&#39;une lignée récemment créé porcelets trachéale cellulaire, nptr 8. Mis à part les différences dans les médias nécessaires à la croissance des bactéries et des cellules, la seule différence est que les bactéries utilisées pour l&#39;infection d&#39;une culture sont en croissance exponentielle, et non d&#39;une culture en phase stationnaire pendant la nuit. Avec A. pleuropneumoniae, il est également très important de respecter une MOI de 10h01 et à ne pas dépasser 3 heures de l&#39;infection, sinon la sécrétion de toxines peut entraîner la mort cellulaire et biaiser les résultats. De plus, cette souche de A. pleuropneumoniae peut facilement adhérer au plastique de sorte qu&#39;il est important d&#39;utiliser des cellules confluentes et vérifier visuellement que les cellules ne sont pas desquamation.

<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>

Ce protocole décrit un dosage standard de l&#39;adhérence des bactéries qui peuvent être modifiés pour étudier l&#39;invasion (par exemple en utilisant la gentamicine protection de test 3). Le comptage des unités formant colonie permet la quantification, par comparaison avec les approches s&#39;appuyant sur des techniques de visualisation standard sous un microscope, comme la coloration de Giemsa. Ce dernier ne donne qu&#39;une vue qualitative de l&#39;adhérence, mais est souvent un complément utile ...

<table border="1"><tbody><tr><th> Réactifs </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> De glucose à haute 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> Sérum de croissance bovine </td><td> Hyclone </td><td> SH3054 </td></tr><tr><td> Pénicilline / streptomycine </td><td> GIBCO-Invitrogen </td><td> 15140-148 </td></tr><tr><td> Plaques de 24 puits </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

Pour provoquer des infections, les bactéries colonisent doit leur hôte. Les bactéries pathogènes d&#39;exprimer différentes molécules ou de structures capables de favoriser l&#39;attachement aux cellules hôtes 1. Ces adhésines s&#39;appuyer sur les interactions avec les récepteurs de l&#39;hôte à la surface cellulaire ou des protéines solubles agissant comme un pont entre les bactéries et l&#39;hôte. L&#39;adhésion est une première étape essentielle avant l&#39;invasion et / ou la sécrétion de toxines, il est donc un événement clé pour être étudiés dans la pathogénie bactérienne. Par ailleurs, les bactéries induisent souvent respectées délicieusement affiné les réponses cellulaires, les études qui ont donné naissance au domaine de la 2 &#39;microbiologie cellulaire. Dosages robuste pour l&#39;adhérence des bactéries sur les cellules hôtes et leur invasion par conséquent jouer un rôle clé dans la pathogenèse bactérienne et les études ont longtemps été utilisés dans de nombreux laboratoires pionniers 3,4. Ces tests sont maintenant pratiquée par la plupart des laboratoires travaillant sur la pathogenèse bactérienne. Ici, nous décrivons un test standard de l&#39;adhésion illustrant la contribution d&#39;une adhésine spécifique. Nous utilisons la souche d&#39;Escherichia coli 2787 5, une souche pathogène humaine exprimant la Autotransporter impliquées dans l&#39;adhérence diffuse (ACRA) Adhesin. Comme contrôle, on utilise une souche mutante dépourvue du gène aida, 2787Δ aida (F. Berthiaume et M. Mourez, non publié), et une souche de laboratoire commercial de E. coli, C600 (New England Biolabs). Les bactéries sont de gauche à adhérer aux cellules de l&#39;couramment utilisés HEp-2 lignées cellulaires épithéliales humaines. Ce test a été moins largement décrite avant 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

Ce protocole est un test simple adhésion bactérienne consistant à compter le nombre de colonies bactériennes formant des unités qui sont collées sur des cellules cultivées. Le test est robuste, indépendante de l&#39;adhésine étudié, et de nombreuses variations sont utilisés dans la plupart des laboratoires travaillant sur la pathogenèse bactérienne.

Test in vitro de l&#39;adhésion bactérienne sur des mammifères cellules épithéliales

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

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Research

JoVE Journal

Immunology and Infection

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

25.2K vues.  Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire. Ce protocole est un test simple adhésion bactérienne consistant à compter le nombre de colonies bactériennes formant des unités qui sont collées sur des cellules cultivées. Le test est robuste, indépendante de l'adhésine étudié, et de nombreuses variations sont utilisés dans la plupart des laboratoires travaillant sur la pathogenèse bactérienne.

Vidéo: 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.

Application d&#39;une souris ligaturés Assay Peyer Patch anse intestinale pour évaluer absorption par les cellules bactériennes M

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

Recherche

Immunologie et infection

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.

Présentation de contrainte de cisaillement dans l&#39;étude de l&#39;adhésion bactérienne

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; Test pour mesurer l&#39;adhésion des neutrophiles à Activé cellules endothéliales microvasculaires humaines primaires dans des conditions statiques

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.

Entérique invasion bactérienne des cellules épithéliales intestinales<em&gt; In Vitro</em&gt; Est considérablement améliorée en utilisant une chambre diffusion verticale du modèle

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 Essai de coculture pour évaluer la migration transépithéliale des neutrophiles induite par l’agent pathogène

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.

Human neutrophiles Chambre flux Essai d&#39;adhésion

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.

Statique Essai d&#39;adhésion pour l&#39;étude de l&#39;intégrine activation dans les lymphocytes 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.

Électroporation d&#39;effecteurs bactériens fonctionnels dans des cellules mammifères

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 et in vivo modèle pour étudier l&#39;adhésion bactérienne au navire muraux dans des conditions de débit

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.

Dosage de Adhérence Sous contrainte de cisaillement pour l&#39;étude des lymphocytes T-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 ...