Die Arbeit in den Laboratorien der Autoren wird durch Zuschüsse aus dem Natural Sciences and Engineering Research Council of Canada, der kanadischen Institutes of Health Research, und die Canada Research Chairs-Programm unterstützt. JL wird durch ein Stipendium der Groupe d&#39;Etude des protéines Membranaires (GEPROM) durch Mittel aus dem Fonds de Recherche en Santé du Québec unterstützt.

1. Vorbemerkung: Bakterienstämme und Epithelzellen. Manipulationen von Zellen und Bakterien werden aseptisch unter einer Sterilbank durchgeführt. <ol><li> Frisch isolieren E. coli-Stämme 2787, 2787Δ Aida und C600 aus Glycerin Aktien auf PBS-Broth (LB)-Agar-Platten (1% Trypton, 0,5% Natriumchlorid, 0,5% Hefeextrakt, 1,5% Agar) und wachsen bei 37 ° C. Um die Variabilität zu minimieren in dem Test, ist es ratsam, immer frisch vergoldet Stämme und die Stämme bei 4 ° C zu halten auf versiegelten Petrischalen nur für maximal ein paar Wochen. </li><li> Kultur HEp-2-Zellen (ATCC CCL-23) in Modified Eagle hohen Glucose-Dulbecco Medium (DMEM) mit 10% hitzeinaktiviertem Rinderserumalbumin (Hitzeinaktivierung bei 56 ° C für 30 min durchgeführt) ergänzt. Während der routinemäßigen Kultur, die wir auch hinzufügen, 10 U / ml Penicillin und 10 ug / ml Streptomycin. </li><li> Wachsen Hep-2 Zellen bei 37 ° C in einer Zelle Inkubator mit einer Atmosphäre mit 5% CO 2. Verwenden Sie Standard-Zellkultur Verfahren, um die Zellen, die in 75 cm 2-Kolben-und Subkultur jeder Zeit erreichen sie Konfluenz gezüchtet werden aufrecht zu erhalten. Wir nutzen unsere kultivierten Zellen, bis sie 30 bis 40 Passagen zu erreichen und dann verwerfen. </li><li> Bereiten Sie einen Test, wenn die HEp-2 Zellen in einer 75 cm 2-Kolben fast erreichen Zusammenfluss und sind daher für ein Festhalten Test bereit. </li></ol> 2. TAG 1: Vorbereiten des Inokulums und die Epithelzellen <ol><li> Waschen Sie die HEp-2-Zellen in den Kolben einmal mit warmen Dulbeccos Phosphat-gepufferter Kochsalzlösung (DPBS: 8 g / L NaCl, 0,2 g / L KCl, 0,2 g / L KH 2 PO 4, 0,21 g / L Na 2 HPO 4: 7H 2 O) </li><li> EDTA für 5 min vor der Zugabe von frischem warmem Vollmedium - Die Zellen werden mit 0,05% Trypsin inkubiert. Nach frischem Medium hinzugefügt wird, werden die Zellen durch Auf-und Abpipettieren resuspendiert. </li><li> Centrifuge die Zellsuspension bei 2.000 rpm für 5 min und Resuspendieren der Pellets in DPBS und Zentrifuge wieder. </li><li> Resuspendieren der Zellen in frischem Medium ergänzt mit 10% Serum enthält aber keine Antibiotika, bei einer Konzentration von 2x10 5 Zellen / ml. </li><li> Seed einem Milliliter der Zellsuspension in drei Sätzen von Doppelbestimmungen (eine für jeden Stamm) in der Mitte einer 24-Well-Platte und inkubieren Sie die Platte über Nacht in einem Zellkulturbrutschrank. Hinweis: Es ist wichtig, dass das Serum sorgfältig decomplemented werden und dass Antibiotika sind nach diesem Zeitpunkt verzichtet. Andernfalls könnte töten die Bakterien infizieren. </li><li> Impfen eine isolierte Kolonie von jedem Bakterienstamm (2787, 2787Δ Aida und C600) in 5 ml LB-Medium (1% Trypton, 0,5% Natriumchlorid, 0,5% Hefeextrakt) und wachsen über Nacht bei 37 ° C unter kräftigem Schütteln (180 rpm ). </li></ol> 3. TAG 2: Infektion von Zellen <ol><li> Überprüfen Sie die HEp-2 Zellen unter einem inversen Mikroskop, um festzustellen, dass sie mindestens 90% konfluent und nicht kontaminiert sind. </li><li> Washed die Zellen mit warmem DPBS und in jede Vertiefung, 1 ml frisches Medium mit 10% Serum ergänzt, jedoch keine Antibiotika. Fügen Sie auch frisches Medium zu drei Brunnen ohne Zellen. Dies wird verwendet, um die Gesamtzahl der Bakterien im Inokulum für jeden Stamm zu bestimmen. </li><li> Messen der optischen Dichte bei 600 nm (OD 0,600 nm) der Bakterienkulturen. Fügen Sie ein Aliquot jeder Bakterienkultur zu einem Satz von Doppelbestimmungen mit Hep-2 Zellen und einem gut nicht enthaltenden Zellen. Normalerweise verwenden wir ein Volumen von Übernacht-Kultur entsprechend 10 6 Kolonie bildenden Einheiten (KBE). Dies entspricht einer Multiplizität der Infektion (MOI) von 5:1 (Bakterien: Zellen). Obwohl wir direkt an unsere Nacht-Kulturen, ist es manchmal ratsam, die Bakterien zentrifugiert und resuspendiert sie in DPBS zu schädlichen Auswirkungen von sezernierten Moleküle in den Nacht-Kulturen zu vermeiden (wie z. B. Zellgifte, zum Beispiel) Hinweis: Die MOI zwischen 100:1 und 1:10 schwanken. Höhere MOI ergibt höhere Variabilität und Hintergrund und Bakterien dazu neigen, auf den Kunststoff der Platte kleben. Lower MOI ergibt sich auch eine hohe Variabilität. Nach einer MOI gewählt wird, ist es unerlässlich, konsequent zu sein und bewahren Sie diese MOI. </li><li> Inkubieren Sie die infizierten Zellen in der Zellkultur-Inkubator für 3 Stunden bei 37 ° C mit 5% CO 2. Hinweis: Es ist auch möglich, kurz zentrifugieren Sie die Platte bei niedriger Geschwindigkeit (z. B. 1.000 xg für 1-2 min), um alle Bakterien direkt in Kontakt mit den Zellen zu bringen. Dies hat den zusätzlichen Nutzen der Synchronisierung der Infektion und ermöglicht kürzere Inkubationszeiten (so wenig wie 15-30 min). </li><li> Entfernen Sie das Medium aus den infizierten Zellen und die Zellen 3 mal mit warmem DPBS. In diesem Schritt können anhaftende Bakterien in der Regel mit einem Standard-Mikroskop gesehen werden, und wir routinemäßig dieses Kontrollkästchen. </li><li> Um die Zellen zu lysieren und lösen Sie die eingehalten bacterien, 100 ul 1% Triton X-100 in jede Vertiefung, welche die Zellen. Andere Reinigungsmittel können verwendet werden (z. B. Saponin zum Beispiel) werden. </li><li> Inkubieren Sie die Zellen für 10 min bei Raumtemperatur und fügen Sie dann 900 ul LB-Medium. </li><li> Gently homogenisieren den Suspensionen von up-and-down Pipettieren wiederholt. Der Brunnen ohne Zellen, die das Inokulum sind ähnlich vorsichtig homogenisiert. Hinweis: Einige Bakterien könnte zu empfindlich, um das Waschmittel, in diesem Fall werden wir lösen die Epithelzellen durch Inkubation mit 0,05% Trypsin - EDTA für 20 min bei 37 ° C. Cells gemeinsam mit eingehalten Bakterien können auch aus den Vertiefungen mit einer Pipettenspitze abgeschabt werden. </li><li> Bereiten Sie serielle 10-fache Verdünnungen der Suspensionen eingehalten Bakterien und Inokulum mit LB-Medium und die Platte 100 ul von 3 Verdünnungen (in der Regel 1:1.000, 1:10.000 und 1:100.000 Verdünnungen) auf LB-Agar und Inkubation über Nacht bei 37 ° C . </li></ol> 4. TAG 3: cfu Zähl-und Daten-Präsentation. <ol><li> Zählen Sie die Kolonien auf den Platten und berechnen Sie die Anzahl der KBE eingehalten Bakterien und des Inokulums durch Mittelung jeder Serie von Verdünnungen. Nur Platten mit zwischen 10-300 Kolonien gezählt werden sollten. </li></ol> 5. Repräsentative Ergebnisse: Die folgende Tabelle zeigt typische Ergebnisse der Zählung der KBE eingehalten Bakterien und Inokulum aus 3 Versuchen an verschiedenen Tagen durchgeführt: <img src="/files/ftp_upload/2783/2783table1.jpg" alt="Tabelle 1" /> Die Ergebnisse können berichtet direkt als cfu eingehalten werden, wie in Abbildung 1A dargestellt. Da das Inokulum Größen zwischen Stämmen wegen der Unterschiede in den Wachstumsraten oder Pipettierfehler variieren kann, ist es oft ratsam, die Ergebnisse als Prozentsatz eingehalten Bakterien Bericht. Der Anteil der Einhaltung Bakterien können, indem die Anzahl der KBE eingehalten Bakterien durch die Anzahl der KBE des Inokulums berechnet werden, wie in Abbildung 1B gezeigt. Durch die Durchführung einer wiederholten Messung ANOVA und Bonferroni post-Prüfungen, um alle Spalten der Abbildung 1B vergleichen, können wir sehen, dass es signifikante Unterschiede (p &lt;0,05) zwischen 2787 und 2787Δ aida, sowie zwischen 2787 und C600, aber keinen signifikanten Unterschied zwischen 2787Δ Aida und C600. <img src="/files/ftp_upload/2783/2783fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Darstellung der Ergebnisse als KBE eingehalten Bakterien (A) oder in Prozent vom eingehalten Bakterien (B) Der Anteil der Einhaltung beobachtet ist oft sehr abhängig von der Versuchsanordnung (und in geringerem Umfang, auf dem Experimentator). Besonders wichtig sind die MOI und die Anzahl der Wäschen, so der prozentuale Anteil sollte nicht wörtlich verstanden werden. Die Bakterien im Inokulum kann manchmal viel schneller wachsen als die Bakterien in Gegenwart von Zellen, Neigen der Größe des Inokulums durch Vergleich mit der realen Anzahl der Bakterien in den Vertiefungen mit Zellen. Zur Linderung dieses Problems ist es, entweder empfohlen: (i) Vertiefungen mit Zellen des Inokulums bestimmen: HEp-2-Zellen werden in einem weiteren gut ausgesät und infiziert. Am Ende des Tests, statt der Überstand verworfen und das Waschen, 100 ul 10% Triton X-100 ist es, die gut aufgenommen. Die Zellen werden lysiert und das Lysat mit eingehalten und nicht eingehalten Bakterien wird sanft durch wiederholtes Auf-und Ab-Pipettieren homogenisiert;. Oder (ii) sammeln die Überstände von infizierten Zellen am Ende des Tests, sowie die Überstände aus die DPBS wäscht und bestimmt die Anzahl der KBE in den vereinigten Überständen. Daraus ergibt sich eine Anzahl der KBE nicht eingehalten Bakterien. Der Anteil der Einhaltung Bakterien können dann durch Division der Anzahl der KBE eingehalten Bakterien durch die Zugabe von der Anzahl der KBE eingehalten und nicht eingehalten Bakterien berechnet werden. Zur Veranschaulichung der Robustheit des Tests können wir dieses Protokoll mit dem Assay mit S4074 durchgeführt, ein Klebstoff Schweine pathogenen Stamm von Actinobacillus pleuropneumoniae 7, mit Zellen aus einer kürzlich gegründeten tracheale Ferkel-Zelllinie, nptr 8 inkubiert vergleichen. Abgesehen von Unterschieden in den Medien für das Wachstum der Bakterien und den Zellen erforderlich, ist der einzige Unterschied, dass die Bakterien zur Infektion verwendet aus einer exponentiell wachsenden Kultur sind, und nicht von einer stationären Phase Übernacht-Kultur. Mit A. pleuropneumoniae es ist auch sehr wichtig, eine MOI von 10:1 zu respektieren und nicht mehr als 3 Stunden nach der Infektion, da sonst die Sekretion von Toxinen wird Zelltod und Bias der Ergebnisse führen. Darüber hinaus dieser Sorte von A. pleuropneumoniae können ohne weiteres auf Kunststoff haften so ist es wichtig, konfluenten Zellen verwenden, und überprüfen Sie visuell, dass die Zellen nicht Abstossung.

<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>

Dieses Protokoll beschreibt eine Standard-bakteriellen Adhärenz-Assay, modifiziert Invasion Studie (zB mit Gentamicin Protection Assay 3) werden kann. Die Zählung der koloniebildenden Einheiten ermöglicht die Quantifizierung durch Vergleich mit Ansätzen unter Berufung auf Standard-Visualisierungs-Techniken unter einem Mikroskop, wie Giemsa. Letztere gibt nur einen qualitativen Blick Haftung wird aber oft eine sinnvolle Ergänzung, da es verschiedene Muster der Haftung kann differenzieren und geben mechani...

<table border="1"><tbody><tr><th> Reagens </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> Hohe Glukose 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> Bovine Growth Serum </td><td> Hyclone </td><td> SH3054 </td></tr><tr><td> Penicillin / Streptomycin </td><td> GIBCO-Invitrogen </td><td> 15140-148 </td></tr><tr><td> 24 Well-Platten </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

Um Infektionen verursachen, müssen Bakterien besiedeln die Gastgeber. Bakterielle Krankheitserreger Ausdruck verschiedener Moleküle oder Strukturen in der Lage, um die Befestigung zu fördern, um Zellen 1 Host. Diese Adhäsine setzen auf Interaktion mit dem Host Rezeptoren auf der Zelloberfläche oder lösliche Proteine, die als eine Brücke zwischen Bakterien und Wirt. Die Haftung ist ein entscheidender erster Schritt vor der Invasion und / oder Sekretion von Toxinen, daher ist es eine der wichtigsten Veranstaltungen in bakterielle Pathogenese untersucht werden. Darüber hinaus haften Bakterien oft exquisit induzieren Feinabstimmung zellulären Reaktionen, die Studien, die entbunden haben auf dem Gebiet der &quot;zellulären Mikrobiologie &#39;2. Robust-Assays für die bakterielle Adhäsion an Wirtszellen und ihre Invasion spielen daher eine Schlüsselrolle in bakterielle Pathogenese Studien und seit langem in vielen Labors Pionier 3,4 verwendet worden. Diese Assays sind mittlerweile von den meisten Labors arbeiten bakterielle Pathogenese praktiziert. Hier beschreiben wir eine Standard-Einhaltung Assay illustrieren den Beitrag eines spezifischen Adhäsin. Wir nutzen die Escherichia coli-Stamm 2787 5, eine humanpathogene Stamm, der Autotransporter Adhesin in Diffuse Adherence (AIDA) beteiligt. Zur Kontrolle verwenden wir eine Mutante fehlt die Aida-Gen, 2787Δ Aida (F. Berthiaume und M. Mourez, unveröffentlicht), und ein kommerzielles Labor-Stamm von E. coli, C600 (New England Biolabs). Die Bakterien werden nach links, um die Zellen aus dem häufig verwendeten HEp-2 menschlichen epithelialen Zell-Linie zu halten. Dieser Assay wurde weniger intensiv vor 6 beschrieben.

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

Dieses Protokoll ist eine einfache bakterielle Adhäsion Assay aus der Zählung die Zahl der bakteriellen Kolonie bildende Einheiten, die auf kultivierten Zellen eingehalten werden. Der Test ist robust, studierte unabhängig von der Adhäsin und zahlreiche Variationen sind in den meisten Labors arbeiten bakterielle Pathogenese.

In-vitro-Assay der bakteriellen Adhäsion auf Mammalian Epithelzellen

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 Aufrufe.  Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire. Dieses Protokoll ist eine einfache bakterielle Adhäsion Assay aus der Zählung die Zahl der bakteriellen Kolonie bildende Einheiten, die auf kultivierten Zellen eingehalten werden. Der Test ist robust, studierte unabhängig von der Adhäsin und zahlreiche Variationen sind in den meisten Labors arbeiten bakterielle Pathogenese.

Serie: 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.

Die Anwendung eines Maus Ligierte Peyer-Patch-Darmschlinge Assay zu bakterielle Aufnahme von M-Zellen auswerten

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

Forschung

Immunologie und Infektion

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.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

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 auf Neutrophile Haftung auf Aktiviert primären humanen mikrovaskulären Endothelzellen unter statischen Bedingungen messen

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.

Enteric bakterielle Invasion von intestinalen Epithelzellen<em&gt; In Vitro</em&gt; Ist erheblich verstärkt unter Verwendung eines vertikalen Diffusion Chamber Modell

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 Coculture Assay zur Bewertung pathogeninduzierter Neutrophiler Transepithelmigration

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.

Humanen neutrophilen Flusskammer 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 ...

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.

Statische Haftung Assay für das Studium der Integrin-Aktivierung in T-Lymphozyten

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.

Elektroporation von Functional bakterielle Effektoren in Säugerzellen

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- und In-vivo-Modell, um die bakterielle Adhäsion an der Gefäßwand unter Strömungsbedingungen Studieren

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.

Test der Haftung unter Scherspannung für das Studium der T-Lymphozyten-Adhäsions-Molekül-Wechselwirkungen

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