Trabajo en los laboratorios de los autores es apoyado por becas de las Ciencias Naturales e Ingeniería de Investigación de Canadá, el Instituto de Investigación en Salud de Canadá, y el programa de Cátedras de Investigación de Canadá. JL es apoyado por una beca del Groupe d&#39;Étude des Membranaires Protéines (GEPROM) a través de la financiación del Fonds de Recherche en Santé du Québec.

1. Preliminar: Cepas bacterianas y células epiteliales. Manipulaciones de las células y las bacterias se realizado de forma aséptica, bajo una campana de flujo laminar. <ol><li> Recién aislar el E. coli 2787, 2787Δ Aida, y C600 de las existencias de caldo de glicerol Lisogenia (LB) placas de agar (1 triptona%, 0,5% de cloruro sódico, un 0,5% de extracto de levadura, 1,5% de agar) y crecer a 37 ° C. Para minimizar la variabilidad en el ensayo, se aconseja utilizar siempre las cepas recién plateado y para mantener las cepas a 4 ° C en placas de Petri sellada sólo por un máximo de un par de semanas. </li><li> Cultura células HEp-2 (ATCC CCL-23) en el modificado del medio altos de glucosa en Dulbecco Eagle (DMEM) suplementado con 10% inactivado por calor suero bovino (inactivación por calor se realiza a 56 ° C durante 30 min). Durante el cultivo de rutina, también se suman 10 U / ml de penicilina y estreptomicina 10 mg / ml. </li><li> Crecer células Hep-2 a 37 ° C en una celda de la incubadora con una atmósfera que contiene 5% de CO 2. Use los procedimientos estándar de cultivo celular para mantener las células, que se cultivan en 75 cm 2 frascos y se subcultivaron cada vez que llegan confluencia. Usamos nuestras células cultivadas hasta alcanzar 30 a 40 pasajes y luego los desecha. </li><li> Preparar un ensayo cuando las células HEp-2 en un matraz de 75 cm 2 son casi llegar a la confluencia y por lo tanto, están listos para un ensayo de adherencia. </li></ol> 2. DIA 1: Preparación del inóculo y las células epiteliales <ol><li> Lave la células HEp-2 en el matraz una vez con fosfato caliente de Dulbecco buffer salino (DPBS: 8 g / L NaCl, 0,2 g / KCl L, 0,2 g / L KH 2 PO 4, 0,21 g / L de Na 2 HPO 4: 7H 2 O) </li><li> Las células se incubaron con 0,05% de tripsina - EDTA durante 5 min antes de añadir medio fresco completo de calentamiento. Después de un nuevo medio, se añade, las células se resuspenden pipeteando arriba y hacia abajo. </li><li> Se centrifuga la suspensión celular a 2000 rpm durante 5 minutos y volver a suspender las pastillas en DPBS y centrifugar de nuevo. </li><li> Resuspender las células en medio fresco suplementado con 10% de suero, pero que no contengan antibióticos, a una concentración de 2x10 5 células / ml. </li><li> Semilla de un mililitro de suspensión celular en tres series de pocillos (uno para cada cepa) en el centro de una placa de 24 pocillos y se incuba la placa durante la noche en una celda de la cultura incubadora. Nota: Es importante que el suero a fondo decomplemented y que los antibióticos se han omitido después de esta etapa. De lo contrario, podría matar a las bacterias que infectan. </li><li> Inocular una colonia aislada de cada cepa bacteriana (2787, 2787Δ Aida y C600) en 5 ml de medio LB (triptona 1%, 0,5% de cloruro sódico, un 0,5% de extracto de levadura) y crecen durante la noche a 37 ° C con agitación vigorosa (180 rpm ). </li></ol> 3. DIA 2: La infección de las células <ol><li> Inspeccione las células HEp-2 bajo un microscopio invertido para cerciorarse de que son al menos el 90% confluentes y no contaminado. </li><li> Lavar las células con DPBS cálido y, a cada pocillo, añada 1 ml de medio fresco suplementado con 10% de suero, pero que no contengan antibióticos. También añade un nuevo medio de tres pozos sin células. Esto será utilizado para determinar el número total de bacterias en el inóculo de cada cepa. </li><li> Medir la densidad óptica a 600 nm (OD 0.600 nm) de los cultivos bacterianos. Agregar una alícuota de cada cultivo bacteriano a un conjunto de pocillos que contienen células Hep-2 y un bien que no contienen células. Por lo general, se utiliza un volumen de cultivo de una noche que corresponde a 10 6 unidades formadoras de colonias (ufc). Esto representa una multiplicidad de infección (MOI), de 5:1 (bacterias: las células). A pesar de que utilizan directamente los cultivos de una noche, a veces es recomendable centrifugar y resuspender las bacterias en DPBS para evitar los efectos nocivos de las moléculas secretadas presentes en los cultivos durante la noche (como citotoxinas, por ejemplo) Nota: El momento de inercia puede variar entre 100:1 y 01:10. MOI más alto rendimiento y una mayor variabilidad del fondo y las bacterias tienden a pegarse al plástico de la placa. Menor momento de inercia también produce una gran variabilidad. Una vez que una MOI que se elija, es imprescindible para ser coherentes y mantener este momento de inercia. </li><li> Se incuban las células infectadas en el cultivo celular incubadora durante 3 horas a 37 º C con CO2 al 5%. Nota: También es posible centrifugar brevemente la placa a baja velocidad (por ejemplo, 1.000 g durante 1-2 minutos), a fin de que todas las bacterias en contacto directo con las células. Esto tiene el beneficio añadido de la sincronización de la infección, y permitir menores tiempos de incubación (tan poco como 15-30 min). </li><li> Eliminar el medio de las células infectadas y se lavan las células 3 veces con DPBS caliente. En esta etapa, las bacterias adheridas por lo general se puede observar con un microscopio estándar y de manera rutinaria realizar esta comprobación. </li><li> Para lisar las células y separar la adherido bacrios, añadir 100 ml de 1% de Triton X-100 a cada pocillo que contiene las células. Detergentes se pueden utilizar otros (como la saponina, por ejemplo). </li><li> Se incuban las células durante 10 minutos a temperatura ambiente y luego añadir 900 l de medio LB. </li><li> Suavemente homogeneizar la suspensión por las reiteradas hacia arriba y hacia abajo de pipeteo. Los pozos sin células que contienen el inóculo son igualmente suavemente homogeneizada. Nota: Algunas de las bacterias puede ser muy sensible a los detergentes, en ese caso, separar las células epiteliales de incubación con 0,05% de tripsina - EDTA durante 20 minutos a 37 ° C. Las células con bacterias adheridas también puede ser raspado de los pozos con una punta de la pipeta. </li><li> Prepare serie de 10 diluciones de las suspensiones de bacterias adheridas e inóculo con caldo de LB y placa de 100 l de 3 diluciones (por lo general las diluciones 1:1.000, 1:10.000 y 1:100.000) en agar LB e incubar durante la noche a 37 ° C . </li></ol> 4. DIA 3: contar ufc y presentación de datos. <ol><li> Contar las colonias en las placas y calcular el número de UFC de bacterias adheridas y del inóculo por el promedio de cada serie de diluciones. Placas sólo con entre 10 a 300 colonias deben ser contados. </li></ol> 5. Los resultados representativos: La siguiente tabla muestra los resultados típicos de ufc recuento de bacterias adheridas y el inóculo de 3 experimentos realizados en días diferentes: <img src="/files/ftp_upload/2783/2783table1.jpg" alt="Tabla 1" /> Los resultados pueden ser reportados directamente adherido ufc, como se muestra en la Figura 1A. Dado que el tamaño de inóculo puede variar entre cepas debido a las diferencias en las tasas de crecimiento o los errores de pipeteo, a menudo es recomendable informar de los resultados como porcentaje de bacterias adheridas. El porcentaje de bacterias adheridas se puede calcular dividiendo el número de UFC de bacterias adheridas por el número de ufc del inóculo, como se muestra en la Figura 1B. Al realizar una medida repetidas ANOVA y Bonferroni pruebas post-para comparar todas las columnas de la Figura 1b, se puede observar que existen diferencias significativas (p &lt;0,05) entre 2787 y 2787Δ Aida, así como entre 2787 y el C600, pero no hubo diferencias significativas 2787Δ entre Aida y C600. <img src="/files/ftp_upload/2783/2783fig1.jpg" alt="Figura 1"/> Figura 1. Representación de los resultados en UFC de bacterias adheridas (A) o el porcentaje de bacterias adheridas (B) El porcentaje de cumplimiento observado con frecuencia es muy dependiente del montaje experimental (y, en menor medida, por el experimentador). De particular importancia son las MOI y el número de lavados, por lo que el porcentaje no debe interpretarse literalmente. Las bacterias en el inóculo a veces puede crecer mucho más rápido que las bacterias en la presencia de células, sesgando el tamaño del inóculo en comparación con el número total real de bacterias en los pozos con células. Para solucionar este problema, se recomienda que sea: (i) el uso de pozos con las células para determinar el inóculo: HEp-2 células se siembran en otros así y se infecta. Al final del ensayo, en lugar de desechar el sobrenadante y lavar, 100 l de 10% de Triton X-100 se añade a la fuente. Las células se lisan y el lisado contiene bacterias adheridas y no adheridas-es ligeramente homogeneizada por las repetidas hacia arriba y hacia abajo pipeteo;. O (ii) recoger los sobrenadantes de las células infectadas al final del ensayo, así como los sobrenadantes de los lavados DPBS y determinar el número de ufc en los sobrenadantes combinados. Esto dará lugar a que el número de UFC de bacterias no adheridas. El porcentaje de bacterias adheridas se puede calcular dividiendo el número de UFC de bacterias adheridas por la adición de la cantidad de UFC de bacterias adheridas y no adheridas. Para ilustrar la solidez de la prueba, podemos comparar este protocolo con el ensayo realizado con el S4074, una cepa porcina patógenos adhesiva de Actinobacillus pleuropneumoniae 7, se incubaron con células de una recientemente creada línea de células traqueales lechones, NPTR 8. Aparte de las diferencias en los medios de comunicación necesarios para el crecimiento de las bacterias y las células, la única diferencia es que las bacterias utilizan para la infección son de una cultura de crecimiento exponencial, y no de un cultivo de una noche de la fase estacionaria. Con A. pleuropneumoniae también es muy importante respetar una MOI de 10:1 y de no más de 3 horas de la infección, de lo contrario la secreción de toxinas causa la muerte celular y el sesgo de los resultados. Además, esta cepa de A. pleuropneumoniae puede fácilmente se adhieren al plástico por lo que es importante la utilización de células confluentes y comprobar visualmente que las células no están mudando.

<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>

Este protocolo describe un ensayo estándar de la adhesión de bacterias que pueden ser modificados para estudiar la invasión (por ejemplo, usando gentamicina ensayo de protección de 3). El recuento de unidades formadoras de colonias permite cuantificar, en comparación con los enfoques de confiar en las técnicas de visualización estándar bajo un microscopio, como la tinción de Giemsa. Este último sólo da una visión cualitativa de la adherencia, pero es a menudo un complemento útil ya que se pueden ...

<table border="1"><tbody><tr><th> Reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Elevados de glucosa en 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> Suero de crecimiento bovino </td><td> Hyclone </td><td> SH3054 </td></tr><tr><td> Penicilina / estreptomicina </td><td> GIBCO-Invitrogen </td><td> 15140-148 </td></tr><tr><td> Placas de 24 pocillos </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

Para causar infecciones, las bacterias deben colonizar su anfitrión. Los patógenos bacterianos expresan diversas moléculas o estructuras capaces de fomentar el apego a las células huésped 1. Estas adhesinas se basan en la interacción con los receptores de superficie de la célula huésped o proteínas solubles que actúan como un puente entre la bacteria y el huésped. La adhesión es un primer paso crítico antes de la invasión y / o la secreción de toxinas, por lo que es un evento clave para ser estudiado en la patogénesis bacteriana. Además, las bacterias adheridas a menudo inducen a exquisitamente afinado la respuesta celular, los estudios de los cuales han dado a luz al campo de la 2 &#39;microbiología celular. Ensayos robusto para la adhesión bacteriana a las células huésped y su invasión por lo tanto, desempeñan un papel clave en los estudios de la patogénesis bacteriana y han sido utilizados en muchos laboratorios pioneros 3,4. Estos ensayos se practica actualmente por la mayoría de los laboratorios que trabajan en la patogénesis bacteriana. Aquí se describe un ensayo de adherencia tipo que ilustren la contribución de un determinado adhesina. Nosotros usamos la cepa de Escherichia coli 2787 5, una cepa patógena humana que expresa la autotransporter Adhesin que participan en la adhesión difusa (AIDA). Como control, se utiliza una cepa mutante que carecen del gen aida, 2787Δ Aida (F. Berthiaume y Mourez M., inédito), y una cepa de laboratorio comercial de E. coli, C600 (New England Biolabs). Las bacterias son de izquierda a adherirse a las células de los comúnmente utilizados HEp-2 la línea humana de células epiteliales. Este ensayo no ha sido tan ampliamente descritas antes de las 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

Este protocolo es un ensayo simple adhesión bacteriana consiste en contar el número de unidades formadoras de colonias bacterianas que se adhieren a las células cultivadas. El ensayo es sólida, independiente de la adhesina estudiados, y numerosas variantes se utilizan en la mayoría de los laboratorios que trabajan en la patogénesis bacteriana.

Ensayo in vitro de la adhesión bacteriana a las células epiteliales de mamíferos

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 vistas.  Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire. Este protocolo es un ensayo simple adhesión bacteriana consiste en contar el número de unidades formadoras de colonias bacterianas que se adhieren a las células cultivadas. El ensayo es sólida, independiente de la adhesina estudiados, y numerosas variantes se utilizan en la mayoría de los laboratorios que trabajan en la patogénesis bacteriana.

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.

La aplicación de un ratón ligado Ensayo placas de Peyer asa intestinal para evaluar la absorción por las células bacterianas M

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

Investigación

Inmunología e Infección

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.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

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.

Cuantitativo<em&gt; In vitro</em&gt; Ensayo para medir la adhesión de neutrófilos al Activado Primaria células endoteliales microvasculares humanas en condiciones estáticas

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.

Entérica invasión bacteriana de las células epiteliales intestinales<em&gt; In Vitro</em&gt; Se ha mejorado considerablemente gracias a un modelo de cámara de difusión vertical

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 Ensayo de cocultivo para evaluar la migración transepitelial de neutrófilos inducida por patógenos

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.

Ensayo de adhesión de neutrófilos Cámara de flujo humano

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.

Ensayo de adherencia estática para el Estudio de la integrina activación en linfocitos 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.

La electroporación de efectores bacterianos funcionales en células de mamífero

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 modelo para estudiar la adhesión bacteriana a la pared del recipiente en condiciones de flujo

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.

Ensayo de adhesión en condiciones de estrés de cizalla para el Estudio de linfocitos T-molécula de adhesión Interacciones

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