This work was supported by NIH grants to T.W. (R01AI072060 and R01AI099123). G. Xie was supported by a Sealy Center for Vaccine Development Predoctoral Fellowship. We thank Dr. Richard Flavell (Howard Hughes Medical Institute, Yale University School of Medicine, New Haven) and Dr. Shizuo Akira (Osaka University, Japan) for providing the MyD88&#8211;/&#8211; mice and Dr. Y Cong (UTMB, Galveston) for providing OTII transgenic mice.

Todos los experimentos con animales fueron aprobadas por el Comité de Cuidado y Uso de Animales de la Universidad de Texas Medical Branch. 1. Aislamiento de países en desarrollo de ratones y no infectados infectado con el VNO <ol><li> Partido por edad y sexo 6-10 semanas de edad, de tipo salvaje C57BL / 6 (B6) y MyD88 - / - ratones. Inocular por vía intraperitoneal (ip) con 500 formando la placa unidad (PFU) de WNV NS4B- P38G mutante. En el día 3 después de la infección, la eutanasia B6 y MyD88 - / - ratones con CO 2. Utilice los ratones no infectados de ambos grupos como los controles. Utilice tres ratones de cada grupo. </li><li> Marino mojado en el lado izquierdo del ratón sacrificado utilizando etanol al 70% y cortar la piel a lo largo del lado izquierdo del ratón, a mitad de camino entre la patas delanteras y traseras. Cortar abrir la cavidad del cuerpo. Retirar el bazo utilizando las pinzas. Coloque el bazo en una placa de Petri con medio RPMI. </li><li> Aislar esplénicos DCs mediante el uso de perlas magnéticas anti-CD11c segúnlas instrucciones del fabricante. </li></ol> 2. Purificación y Etiquetado de las células T de ratones transgénicos otii <ol><li> Cosecha un bazo de un ratón otii ingenuo como se describe en el paso 1.2 anterior y homogeneizar el bazo entre los extremos helados de dos diapositivas. Transferir la suspensión celular a un tubo cónico de 15 ml, se añade medio RPMI hasta 14 ml. Deje reposar la suspensión durante 5 min y transferir 13 ml de ella a un nuevo tubo cónico de 15 ml. </li><li> Aislar células T CD4 + esplénicas mediante el uso de un kit de aislamiento de células T CD4 + de acuerdo con las instrucciones del fabricante. </li><li> La transferencia de células en un tubo cónico de 50 ml, se lavan dos veces con PBS. Resuspender las células en PBS con albúmina de suero bovino al 0,5% (BSA) a 1 x 10 6 células por ml, y añadir 0,5 mol / ml de CFSE. Incubar a 37 ° C durante 10 min, protegido de la luz. </li><li> Añadir 5 ml de medio completo frío (RPMI-1640 con 10% de suero bovino fetal inactivado por calor, 1/100 volantibióticos / antimicóticos, 1/100 vol L-glutamina y 1 / 1.000 vol de 1000 x 2-mercaptoetanol). </li><li> Incubar en hielo durante 5 min. Lavar las células una vez con PBS, fijan 0,2 x 10 6 células T CD4 + etiquetados en 2% PFA y adquieren en un citómetro de flujo. Utilice esta muestra para determinar el nivel basal de CFSE. Vuelva a suspender el resto de las células marcadas en medio completo. </li></ol> 3. Co-cultura otii células T con los países en desarrollo de ratones infectados con el WNV <ol><li> Cultura 2 x 10 5 células T CD4 + purificadas de ratones otii solos o con purificados los DC de tipo salvaje o MyD88 - / - ratones (2 x 10 4) en una placa de 24 pocillos con o sin OVA residuos 323-339 (1 g / ml) en 1 ml de medio completo por pocillo. </li><li> Se incuban las células a 37 ° C durante 5 días. Las células de la cosecha, se lavan dos veces en tampón FACS (PBS con 1% de SFB) y fijan en 0,25 ml de 2% PFA. Vortex inmediatamente. </li></ol> 4. Análisis de citometría de flujo in vitro &lt;/ Em&gt; de células T de cebado <ol><li> Para la adquisición, haga doble clic en el icono del software de citometría de flujo. Para un gráfico de densidad lineal de FSC-A vs lineal SSC-A, seleccionar canales de 0 a 200 mil en FSC-A y de 0 a 200 mil en SSC-A. A continuación, crear una puerta en células vivas (P2; véase la Figura 1A). </li><li> Para analizar la intensidad de fluorescencia de CFSE, abra el histograma del canal FL1 y adquirir 20.000 eventos en la población cerrada. Establecer un marcador en muestras de células otii cultivadas solas (Figura 1A). </li><li> Adquirir muestras para células otii co-cultivadas con DCs de tipo salvaje (Figura 1B) o MyD88 - / - DCs (Figura 1C) de una manera similar. </li></ol> 5. Aislamiento y estimulación de esplenocitos de citoquinas Ensayos <ol><li> Infect B6 y MyD88 - / - ratones con WNV NS4B-P38G mutante utilizando el mismo procedimiento como se describe en step 1,1. En el día 30 después de la infección, re-desafío de sobrevivir ratones con 2000 UFP de la de tipo salvaje cepa WNV ip En los días 8 y 21 de infección por el VNO post-primaria o en el día 4 después de la infección secundaria, eutanasia ratones con CO 2 para la recolección de bazos . NOTA: Se utilizó 2-3 ratones por grupo para cada punto de tiempo. </li><li> Hacer una suspensión de células de esplenocitos como se describe anteriormente en el paso 1.2 y paso 2.2. Contar las células usando un hemocitómetro y re-suspender en 10 ml de medio completo. </li><li> Diluir las células con medio completo a 2,5 x 10 6 células / ml. Añadir 1 ml de esplenocitos en un tubo de microcentrífuga de 1,5 ml. </li><li> Para simular las células T CD8 +, diluir NS4B y E-WNV péptidos específicos (SSVWNATTA y IALTFLAV) a 1 mg / ml en DMSO. Para la estimulación de las células T CD4 +, diluir NS3 y péptidos E-WNV específica (RRWCFDGPRTNTILE y PVGRLVTVNPFVSVA) a 1 mg / ml en DMSO. Añadir 10 l de péptidos a las células followed por 1 l de solución de Brefeldina A. Utilice células sin péptidos tratamiento como controles. Mezclar las células. </li><li> Haga dos agujeros con un 18 G aguja en el tapón del tubo. Se incuban las células a 37 ° C durante 5 horas. </li></ol> 6. intracelular de citoquinas tinción <ol><li> La transferencia de células a un nuevo tubo de microcentrífuga. Haga girar las células durante 5 minutos a 300-400 xg y verter el líquido sobrenadante. Añadir 1 ml de tampón FACS, vuelta 5 min a 300-400 xg y volver a suspender las células mediante pipeteo con 120 l de tampón FACS. </li><li> Añadir 2 l Fc bloqueador de células y se incuba a temperatura ambiente durante 10 min. Añadir 1 ml de tampón FACS a cada tubo. Girar 5 min a 300-400 x g. </li><li> Vuelva a suspender las células en 300 l de tampón FACS y dividirlos en tres tubos (alrededor de 0,8 x 10 6 células por tubo). Como se muestra en la Tabla 1, reservar el primer tubo de cada condición de cultivo para la rata tinción de IgG-PE. Para el segundo tubo, añadir 3 l de anti-CD4 APC a las células CD4-péptidos tratados, 3 μl de anti-CD8 FITC a las células tratadas con péptidos CD8 o ambos anticuerpos a las células sin tratamiento péptidos. Utilice el tercer tubo para la tinción de compensación. Para cada condición de cultivo, utilizar tres tubos de células teñidas con APC-conjugado CD4, CD8 conjugado con FITC o PE anticuerpos marcados CD3 solos (3 l por tubo) como controles de compensación para FL1, FL2 y canales FL4. </li><li> Células Vortex brevemente. Deja en hielo durante 20 min y protegerlo de la luz. Añadir 1,4 ml de tampón FACS. Haga girar 5 min a 300-400 xg y verter el líquido sobrenadante. </li><li> Agitar para interrumpir sedimento celular (o vórtice brevemente). Vuelva a suspender sedimento de células en 250 l de fijación / permeabilización solutionby pipeta. Incubar a temperatura ambiente durante 20 min y proteger de la luz. </li><li> Añadir 1,2 ml de tampón FACS. Girar 5 min a 300-400 xg y volver a suspender las células en tampón FACS 300 l. NOTA: En este punto, las células se pueden transferir a un laboratorio BL2 para su posterior procesamiento o almacenado en un refrigerador y protegido de la luz para up a tres días. </li><li> Añadir 500 l de tampón FACS. Girar 5 min a 300-400 xg y volver a suspender las células en 500 l de tampón de permeabilización / lavado 1x. Girar 5 min a 300-400 x g. </li><li> Células en 100 l 1x Perm / Wash Vuelva a suspender, añadir 3,5 l anti-IFN-PE al tubo previamente adicionado con anticuerpos para CD4 y / o marcadores de células T CD8 en el paso 6.3 y 3.5 l rata IgG-PE en el tubo reservados para el control de IgG durante 25 minutos en hielo y protegerlo de la luz. Lavar las células mediante la adición de 1,4 ml de 1x tampón de permeabilización / lavado. Girar 5 min a 300-400 x g. Repita el paso de lavado añadiendo 1.4 ml 1x tampón de permeabilización / lavado. </li><li> Haga girar 5 min a 300-400 xg y decantar el sobrenadante. Lavar las células mediante la adición de 1,4 ml de tampón FACS y re-suspender en 400 l de tampón FACS para la adquisición final. </li></ol> 7. Análisis de citometría de flujo intracelular de citoquinas tinción <ol><li> Utilice la misma configuración de adquisición como en el paso 4.1. Create una puerta en células vivas (P2; ver Figura 2A). Ajuste los voltajes que utilizan los tubos de compensación para cada grupo. </li><li> Abra dos nuevos gráficos de puntos, uno para mostrar los datos recogidos para FL1 frente. FL2 (CD8 vs. IFN-γ), y el otro es para mostrar los datos recopilados para FL4 vs. FL2 (CD4 vs. IFN-γ). Adquirir 50.000 eventos para la población privada de cada muestra. </li></ol>

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Los autores no tienen nada que revelar.

WNV es un patógeno BL3. Debido a las regulaciones de seguridad, ensayos inmunológicos con muestras infectadas con el VNO son a menudo limitadas por la disponibilidad de equipos en BL3 instalaciones o más largo y tedioso de realizar. En un estudio reciente, se utilizaron dos métodos basados ​​en la citometría de flujo para estudiar respuestas inmunitarias celulares durante la infección por VNO 5. En ambos ensayos, las células infectadas con el VNO fueron tratados con 2.1% PFA directa o con una soluc...

Virus del Nilo Occidental (VNO), una neurotrópico, plus-detectados flavivirus, es una amenaza emergente para la salud pública. Actualmente, no hay vacunas han sido aprobados para uso humano 1. Una cepa atenuada de virus del Nilo Occidental, que tiene una sustitución en el P38G no estructural (NS) proteína 4B, se sabe que inducen no letalidad en ratones, pero citoquinas innatas superior y respuestas de células T en ratones de tipo salvaje que WNV NY99 cepa 2. Los ratones inmunizados con el mutante NS4B-P38G fueron protegidos de un desafío secundario con letal de tipo salvaje WNV. Esto sugiere que el mutante NS4B-P38G tiene características adecuadas para un candidato a vacuna ideal. Los mecanismos por los que el mutante NS4B-P38G induce una alta inmunidad adaptativa protectora aún no se conocen del todo. Los receptores tipo Toll (TLRs), que reconocen patrones moleculares asociados a patógenos, juegan un papel esencial en la iniciación de la inmunidad innata a la infección viral. La vía de señalización TLR núcleo utiliza respuesta primaria de diferenciación mieloide gene 88 (MyD88) como adaptador principal 3,4. En un estudio reciente, la señalización de MyD88 ha demostrado desempeñar un papel importante en el desarrollo de la inmunidad mediada por células durante la infección por WNV NS4B- P38G mutante en ratones 5. Células dendríticas (DC) son una de las células presentadoras de antígenos más importantes que presentan la capacidad única de iniciar respuestas de células T durante la infección viral primaria 6,7. Las células T CD4 + y CD8 + tanto contribuyen a la inmunidad protectora de larga duración y son importantes para la supervivencia de host seguido de tipo salvaje infección por el VNO 8,9. Dos ensayos inmunológicos se utilizaron en este estudio para evaluar las funciones de estas células en el mutante ratones infectados NS4B-P38G. En primer lugar, un ensayo de cebado de células T in vitro se utilizó para comparar la capacidad presentadora de antígeno de las DC de WNV-infectados de tipo salvaje y MyD88 - / - ratones. Para aumentar la sensibilidad del ensayo, ingenuo CD4 &lt;sup&gt; + células T se aislaron de otii ratones transgénicos, que expresan una específica Vα2 / Vβ5 TCR para el péptido ovoalbúmina de pollo (OVA) 323 - 339. DCs de los ratones infectados de WNV se purificaron, y co-cultivadas con carboxifluoresceína succinimidil Pascua (CFSE ) las células T CD4 + -etiquetados en presencia de péptido OVA. Después de 5 días de co-cultivo, las células se recogieron y se fijaron con paraformaldehído (PFA) y se analizaron por citometría de flujo. Los ensayos de proliferación se han realizado tradicionalmente a través de la incorporación de 5-bromo-2&#39;-desoxiuridina (BrdU) o desoxirribósido timina tritiada (3HTdR) 10. Sin embargo, estos ensayos son o radiactivo y / o en la necesidad de equipos especiales en el nivel de bioseguridad (BL) 3 instalaciones, donde se llevan a cabo estudios de WNV. El análisis de citometría de flujo de la proliferación de linfocitos por reducción a la mitad de serie de la intensidad de fluorescencia del colorante vital CFSE se ha vuelto más comúnmente utilizado en ensayos inmunológicos como el colorante es más establey uniformemente incorporados en las células, fácilmente detectado por citometría de flujo, y es no radiactivo 11. El ensayo también tiene la capacidad de evaluar el número de divisiones celulares. Una ventaja importante de usar este ensayo en los estudios de virus del Nilo Occidental es que la fijación de las células infectadas con 1-2% PFA podría inactivar WNV 12, lo que permitirá la adquisición de muestras con un citómetro de flujo en un laboratorio BL2. A continuación, se utilizó un procedimiento de tinción intracelular de citoquinas modificado (ICS) para estudiar el papel de MyD88 de señalización en la regulación de las respuestas de células T específicas de VNO en ratones mutantes infectados NS4B-P38G. En este ensayo, los esplenocitos aislados de ratones infectados fueron tratados in vitro con péptidos específicos del WNV. Brefeldina A se añadió a retener las citoquinas dentro de la célula. Después de 5 horas de incubación, las células se recogieron, se lavaron y se tiñeron para subconjuntos de células T. Las células fueron fijadas en PFA, permeabilizaron y se tiñeron para el interferón (IFN) -γ y se analizaron por citometría de flujo.Como con otro ensayo basado en citometría de flujo, una vez que las células se tratan con la fijación y permeabilización de amortiguación que contiene PFA, muestras infectadas pueden ser transferidos a un laboratorio BL2 para su posterior procesamiento y análisis. En varios estudios publicados, hemos utilizado ICS para medir las funciones efectoras de células T en ratones infectados con el VNO 13,14. Aunque está bien establecido, una desventaja importante de este ensayo es que el procedimiento es muy largo y podría ser más tiempo cuando se realiza dentro de las instalaciones BL3. Aquí, un método basado en ICS tubo de microcentrífuga se demostró que era más factible, más fácil de proceder y consume menos tiempo cuando se realiza dentro de un laboratorio BL3.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:153px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">RPMI 1640</td>
			<td style="width:153px;">Invitrogen</td>
			<td align="right" style="width:119px;">11875</td>
			<td>warm up at 37C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD11c magnetic beads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-001</td>
			<td>follow the manufacturer&#8217;s instructions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD4 magnetic beads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-248</td>
			<td>follow the manufacturer&#8217;s instructions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFSE</td>
			<td style="width:153px;">Invitrogen</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OVA residue 323-339</td>
			<td>Genscript Corporation</td>
			<td>RP10610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Peptides</td>
			<td style="width:153px;">Proimmune</td>
			<td>PC0AD-D</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Brefeldin A solution</td>
			<td style="width:153px;">BD Bioscience</td>
			<td align="right">555029</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:216px;">Mouse Fc Blocker</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>14-0161-85</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">APC-conjugated CD4&#160;</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>17-0041-81</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">FITC-conjugated CD8&#160;</td>
			<td style="width:153px;">e- bioscience</td>
			<td>11-0081-82</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Fixation/Permeabilization Solution</td>
			<td style="width:153px;">BD- Bioscience</td>
			<td align="right" style="width:119px;">554722</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Permeabilization/wash buffer</td>
			<td style="width:153px;">BD- Bioscience</td>
			<td align="right">554723</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">anti-IFNg-PE&#160;</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>12-7311-82</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:216px;">Accuri flow cytometer</td>
			<td style="width:153px;">BD Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
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in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection

Un virus atenuado del Nilo Occidental (VNO), una no estructural (NS) 4B-P38G mutante, inducida por citoquinas superior innata y respuestas de células T que la de tipo salvaje WNV en ratones. Recientemente, factor de diferenciación mieloide 88 (MyD88) de señalización ha demostrado ser importante para el cebado de células T inicial y el desarrollo de células T de memoria durante WNV NS4B-P38G infección mutante. En este estudio, dos métodos basados ​​en citometría de flujo-- un ensayo in vitro de células T en el cebado y una tinción intracelular de citoquinas (ICS) - se utilizaron para evaluar las células dendríticas (DCs) y funciones de células T. En el ensayo de cebado de células T, la proliferación celular se analizó mediante citometría de flujo después de co-cultivo de DCs de ambos grupos de ratones con carboxifluoresceína succinimidil éster (CFSE) - marcado células T CD4 + de ratones transgénicos otii. Este enfoque proporciona una determinación exacta del porcentaje de proliferación de células T CD4 + con mejorado significativamente la sensibilidad general que la tradiciónal ensayos con reactivos radiactivos. Un sistema de tubo de microcentrífuga se utilizó en ambos procedimientos de cultivo celular y tinción de citocinas del protocolo ICS. En comparación con el sistema tradicional basado en la placa de cultivo tisular, este procedimiento modificado fue más fácil de realizar en el nivel de bioseguridad (BL) 3 instalaciones. Por otra parte, las células infectadas WNV- fueron tratados con paraformaldehido en ambos ensayos, lo que permitió su posterior análisis fuera de las instalaciones BL3. En general, estos ensayos inmunológicos in vitro se pueden utilizar para evaluar de manera eficiente las respuestas inmunes mediadas por células durante la infección WNV.

En el ensayo de células T de cebado, las células T CD4 + CFSE etiquetados fueron cultivadas con DCs purificados a partir del mutante infectados de tipo salvaje NS4B-P38G y MyD88 - / - ratones en la presencia o ausencia de péptidos OVA. Células T marcadas cultivadas solas con o sin OVA se utilizaron para 5 días como controles negativos. Como se muestra en la Figura 1A, las células T totales fueron cerrada para el análisis de la intensidad de fluorescencia en el canal FL1. El ...

Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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Research

JoVE Journal

Immunology and Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

8.1K vistas.  The University of Texas Medical Branch. Two flow cytometry-based methods – an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Video: In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

Multicolor análisis de citometría de flujo de la respuesta inmune celular en macacos rhesus

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Investigación

Inmunología e Infección

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Siete pasos para células estrelladas

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Una introducción a las avispas parasitarias de<em&gt; Drosophila</em&gt; Y la respuesta inmune antiparasitario

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

PRP como un nuevo enfoque para prevenir la infección: Preparación y<em&gt; In vitro</em&gt; Propiedades antimicrobianas de PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

El uso de ARN de interferencia para Investigar la respuesta inmune innata en macrófagos de ratón

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Un<em&gt; In Vitro</em&gt; Modelo para medir la respuesta inmune a la malaria en el contexto del VIH Co-infección

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Aislamiento de neutrófilos y análisis para determinar su papel en el linfoma de células sensibles a los agentes terapéuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un enfoque simple y eficiente de construcción de mutantes de virus vaccinia Vectores

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Un modelo de ratón para evaluar respuesta inmune innata a la infección por Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...