Dominic Mills fue apoyado por una Bloomsbury Colegios Beca de doctorado (2007-2010). Los autores desean dar las gracias a Ozan Gundogdu y Abdi Elmi por su ayuda en el desarrollo del modelo VDC.

1. El crecimiento de monocapas IEC en especial 0.4 Filtros micras <ol><li> Cultura Caco-2 células en medio esencial modificado de Dulbecco (DMEM) suplementado con 10% (v / v) de suero de ternero fetal, 100 U / ml de penicilina, 100 mg / ml de estreptomicina y 1% (v / v) de aminoácidos no esenciales en una tejido estándar de cultivo en estufa a 37 ° C en 5% de CO2 y 95% de aire. Seed 4 x 10 5 células Caco-2 IECs por 0,4 micras filtran y crecen a la polarización durante 21 días, el cambio de los medios de comunicación cada 2-3 días. </li><li> Mida la TEER para confirmar el estado de polarización de la monocapa utilizando un medidor de resistencia de Volt-Ohm. En nuestros estudios, la TEER de 21 días Caco-2 células cultivadas en estos filtros de 0,4 micras es 700-800 Ωcm 2. </li></ol> 2. Preparación de C. jejuni y el Medio bacteriana Para el ensayo de cocultivo <ol><li> 24 horas antes de la punto de inicio deseado del ensayo cocultivo VDC, preparar una placa de agar sangre fresca de C. jejuni y se incuba a 37 ° C en condiciones microaeróbicas (85% N 2, 5% O 2 y CO 2 del 10%) en la atmósfera variable de incubadora. </li><li> Al mismo tiempo, preincubar 30 ml de caldo de Brucella a 37 ° C en condiciones microaerobias en un ambiente incubadora variable. </li></ol> 3. Preparación y esterilización de las medias cámaras VDC antes del ensayo <ol><li> Sumerja completamente las cámaras VDC medio, así como las juntas tóricas y los tapones en la solución de esterilización. </li><li> Mediante una jeringuilla estéril 20, lave la solución de esterilización a través de las entradas de gas en ambas cámaras medio. El no hacerlo puede resultar en la contaminación de las muestras durante el cocultivo. </li><li> Deje todos los componentes inmersos en la solución de esterilización para&gt; 1 hr. </li><li> Enjuague todos los componentes con agua estéril fresco, utilizando otra jeringa estéril de 20 ml para eliminar las entradas de gas. </li></ol> 4. Preparacióndel inóculo bacteriano <ol><li> Cosecha medio de una placa de C. jejuni crecimiento de la placa de agar sangre preparó el día anterior en 1 ml del caldo de Brucella preincubado. Esto debería dar ~ 10 10 ufc / ml. </li><li> Medir la densidad óptica de la suspensión bacteriana a 600 nm para semicuantificar unidades formadoras de colonias bacterianas (CFU). </li><li> Ajustar la suspensión bacteriana al nivel de inóculo deseado en 4 ml de caldo de Brucella preincubado. </li><li> Realizar una dilución en serie a partir de esta suspensión bacteriana final, seguido de chapado de las diluciones apropiadas en placas de agar sangre, por triplicado, a continuación, se incuba a 37 ° C en la atmósfera variable de incubadora durante 48 horas para cuantificar el número de UFC bacterianas presentes en el inóculo. </li></ol> 5. Configuración del VDC <ol><li> Coloque la cámara de mitad inferior de un VDC plana sobre el banco. Coloque una junta tórica en la cámara media más baja. </li><li> Separe un filtro que llevan los IECs de tél portador y lavar tres veces con 400 l de PBS estéril. Coloque el filtro en la cámara media inferior, asegurándose de que la junta tórica permanece en su lugar. </li><li> Baje con cuidado la cámara de medio superior en su lugar. Una vez que las dos medias cámaras están montadas, la abrazadera juntas usando las abrazaderas de anillo. Coloque el VDC horizontalmente en la parte superior del banco, con las dos aberturas hacia arriba. </li><li> Añadir el 4 ml de inóculo bacteriano en la cámara de medio apical. </li><li> Añadir 4 ml de medio de cultivo celular en la cámara de medio basolateral. </li><li> Coloque las piezas de los extremos en las aberturas en ambas cámaras medio. </li></ol> 6. Colocación de la CC al colector de gas dentro de la variable de ambiente Incubadora <ol><li> Coloque el VDC a la atmósfera variable de incubadora en estrecha proximidad con el colector de gas. </li><li> Abra el regulador de suministro de gas conectado al colector de gas. Fije el tubo desde el colector en la entrada de gas en la cámara de medio basolateral de la VCC. Conecte el gas tubo de salida que conduce fuera de la variable ambiente incubadora a uno de los puntos de venta en la pieza final en la cámara de medio basolateral. </li><li> Cierre la otra salida de la pieza final en la cámara de medio basolateral. Abra el flujo de gas en el colector de gas, teniendo cuidado para abrirlo muy lentamente para evitar el exceso de presión de gas, ya que esto puede conducir a la expulsión del medio basolateral. </li><li> El objetivo es un flujo de gas de 1 burbuja cada 2-5 seg. Periódicamente comprobar el flujo de gas durante el transcurso del experimento y ajustar si es necesario. </li></ol> 7. Desmontaje del VDC después Coculture <ol><li> Después del periodo de co-cultivo apropiado, cerrar el regulador de suministro de gas conectado al colector de gas. Cierre el suministro de gas en el VDC en el colector. Desconectar la entrada de gas, la salida de gas y el tapón de la VCC. </li><li> Retire el VDC de un ambiente incubadora variable. Retirar el sobrenadante y almacenar apical y basolateral a -80 ° C para subsequenanálisis de t. </li><li> Retire las abrazaderas del anillo y tome suavemente aparte del VDC. Quite el filtro de la cámara de medio y transferir a un 6-así placa de cultivo celular estéril. Lávese las IECS tres veces con 400 l de PBS estéril. </li><li> Para la enumeración del número total de bacterias que interactúan, añadir 400 l de PBS estéril que contiene 0,1% (v / v) de Triton X-100 a las IECs y se incuba durante 20 min a temperatura ambiente para lisar la IEC. Realizar una dilución en serie de este lisado celular, seguido por chapado de las diluciones apropiadas en placas de agar sangre, por triplicado, a continuación, se incuba a 37 ° C en la atmósfera variable de incubadora durante 48 horas para cuantificar el número de interactuar UFC bacterianas. </li><li> Para la enumeración del número total de bacterias invasoras, añadir 400 l de medio de cultivo celular DMEM que contiene 150 mg / ml de gentamicina a los IECs y se incuba durante 2 horas en un incubador de cultivo de tejido estándar a 37 ° C en 5% de CO 2 y 95% aire para matar las bacterias extracelulares,luego siga como para el paso 7.4 anterior. </li></ol> 8. Limpieza del VDC después Coculture Lave el VDC con agua estéril y tienda para la siguiente ronda de experimentos.

<ol>
	<li>Mills, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+Campylobacter+jejuni+invasion+of+intestinal+epithelial+cells+under+low-oxygen+coculture+conditions+that+reflect+the+in+vivo+environment.">Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.</a> Infect. Immun. 80, 1690-1698 (2012).</li><li>Marteyn, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Shigella+virulence+in+response+to+available+oxygen+in+vivo.">Modulation of Shigella virulence in response to available oxygen in vivo.</a> Nature. 465, 355-358 (2010).</li><li>Dorrell, N., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+century+of+Campylobacter+research:+recent+advances,+new+opportunities+and+old+problems.">The second century of Campylobacter research: recent advances, new opportunities and old problems.</a> Curr. Opin. Infect. Dis. 20, 514-518 (2007).</li><li>Young, K. T., Davis, L. M., Dirita, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Campylobacter+jejuni:+molecular+biology+and+pathogenesis.">Campylobacter jejuni: molecular biology and pathogenesis.</a> Nat. Rev. Microbiol. 5, 665-679 (2007).</li><li>Hu, L., Kopecko, D. J., Nachamkin, I., Szymanski, C. M., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17.">Chapter 17.</a> Campylobacter. , 297-313 (2008).</li><li>Everest, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+Caco-2+cells+as+a+model+for+enteric+invasion+by+Campylobacter+jejuni+and+C.+coli.">Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli.</a> J. Med. Microbiol. 37, 319-325 (1992).</li><li>Konkel, M. E., Hayes, S. F., Joens, L. A., Cieplak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+the+internalization+and+intracellular+survival+of+Campylobacter+jejuni+in+human+epithelial+cell+cultures.">Characteristics of the internalization and intracellular survival of Campylobacter jejuni in human epithelial cell cultures.</a> Microb. Pathog. 13, 357-370 (1992).</li><li>Monteville, M. R., Konkel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-facilitated+invasion+of+T84+eukaryotic+cells+by+Campylobacter+jejuni+occurs+preferentially+at+the+basolateral+cell+surface.">Fibronectin-facilitated invasion of T84 eukaryotic cells by Campylobacter jejuni occurs preferentially at the basolateral cell surface.</a> Infect. Immun. 70, 6665-6671 (2002).</li><li>Friis, L. M., Pin, C., Pearson, B. M., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+culture+methods+for+investigating+Campylobacter+invasion+mechanisms.">In vitro cell culture methods for investigating Campylobacter invasion mechanisms.</a> J. Microbiol. Methods. 61, 145-160 (2005).</li><li>Schuller, S., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerobic+conditions+enhance+type+III+secretion+and+adherence+of+enterohaemorrhagic+Escherichia+coli+to+polarized+human+intestinal+epithelial+cells.">Microaerobic conditions enhance type III secretion and adherence of enterohaemorrhagic Escherichia coli to polarized human intestinal epithelial cells.</a> Environ. Microbiol. 12, 2426-2435 (2010).</li><li>Cottet, S., Corthesy-Theulaz, I., Spertini, F., Corthesy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerophilic+conditions+permit+to+mimic+in+vitro+events+occurring+during+in+vivo+Helicobacter+pylori+infection+and+to+identify+Rho/Ras-associated+proteins+in+cellular+signaling.">Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.</a> J. Biol. Chem. 277, 33978-33986 (2002).</li></ol>

No tenemos nada que revelar.

El uso de métodos de cultivo de células in vitro para estudiar las interacciones de patógenos bacterianos con células huésped es una técnica ampliamente utilizada en muchos laboratorios de investigación. Sin embargo, como estos ensayos de cultivo celular se realizan bajo condiciones aeróbicas, estos modelos in vitro pueden no representar con precisión el entorno in vivo en el que las interacciones huésped-patógeno se llevan a cabo. El ampliamente utilizado en model...

Las interacciones de patógenos bacterianos con células huésped se han investigado extensamente en el uso de métodos de cultivo celular in vitro. El uso de estos ensayos de cultivo celular, la adhesión bacteriana a las células huésped, la identificación de los receptores de la célula huésped, célula huésped vías de señalización y la invasión bacteriana de células huésped todas han sido estudiados en detalle, lo que resulta en muchas observaciones importantes. Sin embargo, tales ensayos de cultivo celular se realizan bajo condiciones aeróbicas que pueden no ser representativos de la ambiente in vivo. Una limitación importante de los modelos in vitro utilizados para estudiar infecciones gastrointestinales es que las condiciones de cultivo, incluyendo altos niveles de oxígeno generalmente favorecen la supervivencia celular eucariota. Sin embargo las condiciones en el lumen intestinal serán casi anaeróbica. Patógenos entéricos en un entorno muy poco oxígeno expresan genes de virulencia cuya expresión cambia en condiciones aeróbicas 2. Como tal, los datos obtenidos utilizando el estándar de cultivo celular modelos may dar una indicación inexacta de las interacciones con las células huésped bacterianas. Campylobacter jejuni es el agente causal principal de gastroenteritis aguda bacteriana en todo el mundo, con síntomas que van desde diarrea leve hasta enteritis inflamatoria severa. La mayoría de C. jejuni infecciones producen gastroenteritis sin complicaciones, sin embargo C. jejuni es también el agente infeccioso identificado con mayor frecuencia en las neuropatías periféricas, incluyendo el síndrome de Guillain-Barré (GBS). En el Reino Unido, se estima que hay más de 500.000 casos de enteritis causadas por C. jejuni infección cada año con un coste previsto de la economía del Reino Unido de £ 580 millones. En el mundo en desarrollo, C. jejuni es la causa principal de mortalidad entre los niños. A pesar de la indudable importancia de la infección por Campylobacter y décadas de investigación, incluido el análisis basada en la genómica completa, C. jejuni patogenia es aún mal comprendood, en contraste con otros patógenos entéricos, tales como Salmonella, Escherichia coli, Shigella y Vibrio cholerae. La falta de un modelo animal pequeño conveniente es una razón principal para este 3. También el ampliamente utilizado en modelos de infección in vitro son más apropiado para el estudio de microaerophilic C. jejuni que para otros patógenos entéricos que son anaerobios facultativos. Mientras que C. jejuni es reconocido como un patógeno invasor, los mecanismos de C. jejuni invasión de las células epiteliales intestinales (IECS) aún no están claros 4,5. C. invasión jejuni ha demostrado ser dependiente de cualquiera de microfilamentos, microtúbulos, una combinación de ambos o ninguno 5. La confusión en esta zona es muy probablemente debido a la utilización de no apropiado en condiciones de ensayo in vitro. Un número de diferentes ensayos de cultivo celular se han utilizado para investigar las interacciones de C. jejunicon las células huésped. Caco-2 6, INT 407 7, y 8 líneas de células T84 han todos han utilizado para estudiar las capacidades de adherencia y la invasión de diferentes C. jejuni cepas. Sin embargo, los niveles de la adhesión bacteriana y la invasión de C. jejuni con IECs son considerablemente inferiores a los de otros patógenos entéricos 9. El cocultivo de C. jejuni con IECs se realiza normalmente en un incubador de CO2 en condiciones próximas a los niveles de oxígeno atmosférico, necesario para la supervivencia de IECs. C. la expresión de genes jejuni será muy diferente en el entorno de bajo oxígeno de la luz intestinal en comparación con las condiciones de oxígeno atmosférico. El uso de un sistema de cámara de difusión vertical (VCC) ha sido desarrollado que permite el co-cultivo de las bacterias y las células huésped bajo diferentes condiciones de medio y de gas 1,10,11. Este sistema imita las condiciones en el intestino humano, donde las bacterias no sufriráder condiciones de muy bajo, mientras que los tejidos de oxígeno se suministran con el oxígeno de la corriente de la sangre. Monocapas de IEC polarizadas cultivadas en filtros de 0,4 micras especiales se colocaron en una VCC la creación de un compartimento apical y basolateral, que se llenaron individualmente con caldo bacteriano y medio de cultivo celular, respectivamente (Figura 1). El VDC se colocó en la atmósfera variable de incubadora que contiene 85% de N 2, 5% de O 2, y CO 2 al 10% a 37 ° C, que representa las condiciones óptimas para C. jejuni. El compartimento apical se dejó abierta y expuesta a la atmósfera microaerobia dentro de la atmósfera variable de incubadora, mientras que el compartimento basolateral sellada fue suministrado con oxígeno mediante la administración constante de 5% de CO 2/95% de O 2 mezcla de gas con un tubo de salida prevención de la acumulación de la presión . Caco-2 y la supervivencia celular integridad de la monocapa en estas condiciones se confirmaron mediante la supervisión del elemento transepitelialresistencia ctrical (TEER) a través de la monocapa de más de 24 horas para demostrar la supervivencia de células Caco-2 monocapa celular y separación física de los compartimentos apical y basolateral en condiciones bajas de oxígeno en el compartimiento apical. La TEER de monocapas en los CDA mantenidos en la atmósfera incubadora variables (condiciones microaeróbicas) y en un cultivo de células de CO 2 incubadora estándar (condiciones aeróbicas) no mostró diferencias significativas, indicando la integridad de la monocapa de células en diferentes condiciones atmosféricas 1. En condiciones microaeróbicas, uniones estrechas se mantuvo presente y distribuida de manera uniforme entre los bordes de las celdas con un patrón de tinción similar a la ocludina células mantenidas en condiciones aeróbicas 1. Las interacciones de C. jejuni con células Caco-2 y células T84 en el VDC fueron investigados mediante la evaluación de la interacción bacteriana (adherencia y la invasión) y la invasión. Dos diferentes C. jejuni wild-type strains se utilizaron 1. C. jejuni 11168H es un derivado de la cepa hypermotile secuencia original NCTC11168. La cepa 11168H muestra los niveles de colonización mucho más altos en un modelo de colonización de pollo y por lo tanto se considera una cepa adecuada a utilizar para estudios de interacción huésped-patógeno. 81-176 es un aislado de humanos y es una de las cepas de laboratorio ampliamente estudiados más invasivos. C. jejuni cepas fueron añadidos al compartimiento apical de una VCC ya sea bajo condiciones microaeróbicas o aeróbico. Hemos observado tasas más altas tanto para la interacción y la invasión se registraron para C. jejuni en condiciones microaeróbicas 1. El aumento de la C. interacciones jejuni no se debieron a un aumento en el número de bacterias en condiciones microaerobias 1. Estos datos apoyan nuestra hipótesis de que el entorno de bajo nivel de oxígeno en el compartimento apical de la VCC mejora las interacciones bacteria-huésped e indica que las propiedades invasivas de C. jejuni son incraliviado en estas condiciones. Este fue el primer informe del uso del modelo de VCC para estudiar un patógeno bacteriana invasiva y pone de relieve la importancia de llevar a cabo en ensayos de infección in vitro en condiciones que más estrechamente imita la situación in vivo. El modelo VCC podría ser utilizado para estudiar las interacciones huésped-patógeno para muchas especies bacterianas diferentes.

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			<td>Speciality vertical diffusion system for use with Snapwell inserts</td>
			<td>Harvard Apparatus</td>
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			<td>Manifold &#38; six Snapwell chambers</td>
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			<td>Snapwell filters (pore size 0.4 &#956;m)</td>
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			<td>Millicell ERS-2 Volt-Ohm resistance meter</td>
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			<td>WPA Lightwave II spectrophotometer</td>
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enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model

Las interacciones de patógenos bacterianos con células huésped se han investigado extensamente en el uso de métodos de cultivo celular in vitro. Sin embargo, como estos ensayos de cultivo celular se realizan bajo condiciones aeróbicas, estos modelos in vitro pueden no representar con precisión el entorno in vivo en el que las interacciones huésped-patógeno se llevan a cabo. Hemos desarrollado un modelo in vitro de la infección en que permite que el co-cultivo de las bacterias y las células huésped bajo diferentes condiciones de medio y de gas. La cámara de difusión vertical (VCC) modelo imita las condiciones en el intestino humano, donde las bacterias estarán en condiciones de muy baja, mientras que los tejidos de oxígeno serán suministrados con el oxígeno del torrente sanguíneo. La colocación de monocapas de células epiteliales intestinales polarizadas (IEC) obtenidas en los prospectos Snapwell en una VDC crea compartimentos apical y basolateral separados. El compartimento basolateral se llena con medio de cultivo celular, sellado y perfundidos con oxígeno wHilst el compartimiento apical está lleno de caldo, mantienen abiertas y se incubaron bajo condiciones microaeróbicas. Tanto Caco-2 y T84 IECs se pueden mantener en el VDC bajo estas condiciones, sin efectos perjudiciales aparentes sobre la supervivencia celular o integridad de la monocapa. Experimentos de cocultivo a cabo con diferentes C. jejuni cepas de tipo salvaje y diferentes líneas de IEC en el modelo VCC con condiciones microaeróbicas en el compartimiento apical reproducible resultan en un aumento en el número de interactuar (casi 10 veces) y las bacterias intracelulares (casi 100 veces) en comparación a las condiciones de cultivo aerobio 1. El ambiente creado en el modelo VDC imita más de cerca el ambiente encontrado por C. jejuni en el intestino humano y pone de relieve la importancia de llevar a cabo en ensayos de infección in vitro en condiciones que imitan más estrechamente la realidad in vivo en el. Proponemos que el uso del modelo VDC permitirá nuevas interpretaciones de las interacciones apuestaween patógenos bacterianos y células huésped.

Experimentos de cocultivo realizaron con un C. jejuni cepa de tipo salvaje y los IEC en el modelo VCC con condiciones microaeróbicas en el compartimiento apical han demostrado un aumento en el número de interactuar (casi 10 veces) y las bacterias intracelulares (casi 100 veces) en comparación con las condiciones de cultivo aerobias en un momento que dependen de forma 1. Esta observación fue reproducible usando dos diferentes C. jejuni cepas de tipo salvaje (11168H y 81-176) y dos líneas...

Watch this Scientific Journal Video about Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model at JoVE.com

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

Realización de ensayos de cultivo celular para investigar la adhesión bacteriana y la invasión en condiciones aerobias es generalmente representativo de la<em&gt; In vivo</em&gt; Medio ambiente. Un modelo de cámara de difusión vertical permite el estudio de las interacciones del patógeno humano<em&gt; Campylobacter jejuni</em&gt; Con las células epiteliales del intestino más bajo<em&gt; In vivo</em&gt; Condiciones similares, lo que resulta en una mayor invasión bacteriana.

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 ...

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

13.7K vistas.  London School of Hygiene & Tropical Medicine. Realización de ensayos de cultivo celular para investigar la adhesión bacteriana y la invasión en condiciones aerobias es generalmente representativo de la In vivo Medio ambiente. Un modelo de cámara de difusión vertical permite el estudio de las interacciones del patógeno humano Campylobacter jejuni Con las células epiteliales del intestino más bajo In vivo Condiciones similares, lo que resulta en una mayor invasión b...

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

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

En la formación de biofilm in vitro en una diapositiva Cámara de 8 pocillos

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Investigación

Inmunología e Infección

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Luciferasa utilizando a las infecciones bacterianas de la imagen en ratones

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

La invasión de las células humanas por un patógeno bacteriano

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

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 ...

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 ...

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. 
We found the optimum fatty acid concentrations to be between 1-5 ng/&mu;l, and that for cholera toxin to be &lt; 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

Determinación de los ácidos grasos tolerables y Concentraciones toxina del cólera El uso de células epiteliales intestinales humanas y de ratón BALB / c Macrófagos

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

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 ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Evaluación De La Invasión Bacteriana De Las Células Cardíacas En Cultivo Y Colonización Del Corazón En Ratones Infectados Usando Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Análisis de inmunofluorescencia de la formación de gránulos de estrés tras el desafío bacteriano de las células de mamíferos

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un alto rendimiento Shigella-ensayo bactericida específico

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...