The authors thank Dr. Lynda Partridge for the provision of advice and laboratory facilities.

1) Preparación de la línea celular: <ol><li> El desarrollo de la línea de células RBL-2H3.1 expresar equinos α Fc RI: <ol><li> El uso de las técnicas básicas de cultivo de tejidos para las líneas celulares en monocapa, transfectar células RBL-2H3.1 parentales utilizando el plásmido pEE6, que lleva el gen equino FcεRIα (GenBank: Y18204.1) 28. Añadir 2 g l -1 del ADN del plásmido a 0,8 ml de células a una densidad de 1,2 x10 7 células ml -1. Electroporar las células a 250 V 960 mF usando un electrocuvette 0,4 cm luego incubar inmediatamente en hielo durante 10 min. </li><li> Seleccione las células transformadas utilizando medios que contienen 0,4 g de sulfato de geneticina G418, a continuación, ordenar las células vivas restantes mediante FACS etiquetándolos con un anticuerpo IgE fluorescente. Utilice la RBL-2H3.1 resultante expresar equina línea celular FcεRIα para la investigación 2, 3. </li></ol></li><li> Pre-ensayo de sensibilización de anticuerpos: <ol><li&gt; Cosecha del RBL-2H3.1 células que expresan FcεRIα equino procedente de una placa de Petri confluente. Lavar a continuación, volver a suspender las células en medio de cultivo a una densidad celular de 5x10 ml -1 5 células. </li><li> Añadir la IgE de interés para las células suspendidas a una concentración final de 1 ng ml -1, a continuación, la placa 100 l de células en una placa de 96 pocillos en las columnas 1-6 y se incuba a 37 ° C + 5% de CO 2 + 90% de humedad relativa durante 16 horas. Después del tiempo de incubación, y antes de realizar el ensayo de liberación, compruebe los pozos bajo un microscopio para confluencia bien y la adhesión celular. </li></ol></li></ol> 2) Ensayo de lanzamiento: <ol><li> Lavar las células: <ol><li> Tampón de liberación de calentamiento (tubos de 25 mM, cloruro de sodio 120 mM, cloruro de potasio 5 mM, 0,04 mM de cloruro de magnesio y cloruro de calcio 1 mM) a 37 ° C para permitir el lavado de células suave. </li><li> Lavar las células con un movimiento rápido de la placa para eliminar los medios de comunicación de células y añadiendo 100 l tibia, 37 ° C, tampón de liberación. Repita dos veces. </li></ol></li><li> Desafío Antígeno: <ol><li> Preparar una serie de dilución de antígeno (NIP-HSA o DNP-HSA) de 0 ng ml -1, 0,1 ng ml -1, 1 ng ml -1, 10 ng ml -1, 100 ng ml -1, 1.000 ng ml -1, 10.000 ng ml -1 en tampón de liberación y caliente a 37 ° C. </li><li> Después del segundo lavado celular, descartar los medios de comunicación y reemplazado con 100 l de las soluciones de antígeno. Asegúrese de que los pozos en la misma fila (A1-6 por ejemplo) tienen la misma concentración de antígeno añadido a ellos. </li><li> Establecer un control negativo en la fila A, añadiendo 0 ng antígeno -1 ml. Añadir aumento de la concentración de antígeno por las filas (BG), seguido de triton-x tampón (5% de Triton X-100) en células H fila para lisar las células para ser utilizado como un control positivo. Se incuba a 37 ° C durante 20 minutos para permitir que las células liberen sus mediadores. </li></ol></li><li> Configuración de los controles individuales así: <ol><li> Después de la incubación, transferir 50 l de sobrenadante celular a la otra mitad de la placa (pozos A1-6 a A7-12 pozos, etc.). Desechar los 50 l restantes de sobrenadante y reemplazar con 50 l de tampón de Triton-X para permitir la medición de la cantidad de mediadores liberados en cada pocillo en las columnas 7-12 como un porcentaje del total de los mediadores dentro de las células en la columna de 1-6 . </li></ol></li><li> Sustrato de la enzima: </li><li> Añadir 50 l de sustrato β-hexosaminidasa (50 mM 4-nitrofenil N-acetil-β-D-glucosaminida preparado en DMSO diluido a 2 mM mediante la adición a tampón citrato 0,2 M de ácido cítrico y acetato de sodio 0,2 M, pH 4,5) a todos los pocillos a facilitado la conversión del sustrato a 4-nitrofenol por la enzima β-hexosaminidasa. Las placas se incuban a 37 ° C durante 2 horas. </li></ol><img alt="Figura 2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" width = &quot;400&quot; /&gt; <ol start="5"><li> Terminación de la reacción: <ol><li> Detener la reacción por la adición de 150 l de tampón Tris (1 M Tris-HCl, pH 9) a cada pocillo como el alto pH del tampón se detiene la reacción y se convierte en la 4-nitrofenol en un color amarillo. </li></ol></li><li> La lectura y el análisis de los resultados: <ol><li> Leer la placa usando un espectrofotómetro de placas a 405 nm para medir la absorbancia del color amarillo. Calculó el porcentaje de β-hexosaminidasa liberada usando la siguiente fórmula: </li></ol></li></ol><img alt="Figura 2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" /><ol start="2"><li> Aplicar esta fórmula para cada pozo, después de lo cual se toma un promedio para cada fila. A1 y A7 representan la ubicación de los pozos de la placa de 96 pocillos. Trazar la curva de porcentaje de liberación β-hexosaminidasa (que corresponde a la liberación total de mediador) against antígeno concentración 2, 3. </li></ol>

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The authors declare that they have no competing financial interests in this paper.

En resumen, los resultados de esta investigación mostraron que cuando las células RBL-2H3.1 expresan FcεRIα equinos están sensibilizados con IgE equina y desafiados por un antígeno, que dan una liberación pico mediador de 36.68% ± 4.88% de la cantidad total de mediador dentro de la células, en comparación con el RBL-2H3.1 células parentales no expresan equina FcεRIα. Así, este ensayo proporciona una herramienta útil para la investigación y el estudio de las respuestas alérgi...

Alergia se conoce desde hace milenios. Un tratamiento del asma se describe en el texto médico egipcio antiguo conocido como el Papiro de Ebers (~ 1550 aC) y discutió los remedios herbales para tratarla 7. Hoy en día la alergia se clasifica como una respuesta de hipersensibilidad de tipo I, donde el tipo de células T helper 2 (TH 2) el brazo del sistema inmunitario dirige la producción de inmunoglobulina E (IgE) anticuerpos en respuesta a antígenos ambientales llamadas alérgenos. Estos son diversas sustancias que comúnmente interactúan con las células del sistema inmunológico y estimulan la síntesis y secreción de citoquinas pro-inflamatorias, incluyendo la interleucina-4 e interleucina-13 8, 9 como lo hacen las partículas en partículas de humo del cigarrillo o de escape diesel que potencian la síntesis de IgE 10. El aumento de las manifestaciones alérgicas en los países industrializados en los últimos 50 años se ha atribuido a una combinación del efecto decontaminantes del medio ambiente y una tendencia a un ambiente más saneado, que se combinan para cambiar la respuesta inmune hacia un perfil predominado por Th 2 citoquinas, tal como propone la &quot;hipótesis de la higiene&quot; 11. Como se mencionó anteriormente, los seres humanos no son los únicos mamíferos afectados por la alergia. Cabe destacar que los caballos y los perros también pueden desarrollar respuestas alérgicas clásicas y un estudio de 12 ha demostrado que, al igual que en los seres humanos, la alergia equina se atribuye a factores genéticos y ambientales. Como consecuencia, estos animales presentan buenos modelos para el estudio de la interacción entre las causas genéticas y ambientales de la alergia, su progresión desde la sensibilización a la enfermedad, y las posibles estrategias de intervención una vez que las manifestaciones clínicas se han fijado en En 1887, Stömmer fue la primera persona en describir la similitud entre el ser humano y el asma equina 13, el efecto de la histamina sobre el sistema cardiovascular equina esmuy similar a la de los seres humanos 14. Los caballos son también la piedra angular de la industria de las carreras de caballos, que vale US $ 72 mil millones con un volumen de apuestas de US $ 115 mil millones al año 15. La mayoría de los caballos de carreras contemporáneos son descendientes del pequeño número de caballos árabes criados por Lady Anne Blunt a partir de 1878 en adelante. Caballos de carreras modernos están endogámicas comúnmente para seleccionar para capacidades de rendimiento. Son propensos a trastornos genéticos, uno de los cuales es su susceptibilidad a montar respuestas alérgicas. También tienen 1.000 veces más altos niveles de IgE en suero que incluso los seres humanos más gravemente alérgica 16. Caballos en las respuestas alérgicas se manifiestan generalmente como hipersensibilidad a la picadura de insectos (IBH) 17, 18. Resultados de IBH en la dermatitis debido a las picaduras de insectos forman en el género Culicoides. Otra forma de enfermedad alérgica de las vías respiratorias equina es obstrucciones recurrentes (RAO), esto se manifiesta en los pulmones y las vías respiratorias. Se caracteriza por sibilancias y laboratoriorespiración ORed. RAO ocurre comúnmente en respuesta a las esporas de moho, y los altos niveles de IgE alergeno-específica se han registrado en los caballos que sufren de RAO en un estudio de 19 aunque otra investigación no ha confirmado 20. Estudios sobre la alergia equina giraban alrededor del intento de seguimiento y la neutralización de IgE equina mediante el desarrollo de anticuerpos monoclonales anti-IgE equina (mAbs) 21, 22. Además, el estudio de 23 discute la producción de los dominios extracelulares de α equino de alta afinidad Fc de receptor cadena (FcεRIα) receptor en un intento de detectar y cuantificar suero equino IgE. Un estudio relacionado por Ledin 24 discute un nuevo enfoque destinado a neutralizar la IgE sérica cebando el sistema inmune utilizando un inmunógeno sí / no-yo. Todos estos estudios, sin embargo, carecían de un ensayo eficaz para probar la seguridad y eficacia de sus protocolos. En este artículo, presentamos ahora una sys tales ensayotem aplicable al estudio de las estrategias de diagnóstico y terapéuticas relevantes para el sistema equino, donde la liberación β-hexosaminidasa, como un indicador de la desgranulación de mediador celular, se evaluó en células RBL-2H3.1 que expresan equina FcεRIα. Este protocolo se basa en publicaciones anteriores 25, 4, 5, 2, 3 que describen la ingeniería de células RBL transfectadas con el gen que codifica el dominio de unión a IgE del receptor de alta afinidad para IgE de diferentes especies. El protocolo explica cómo realizar un ensayo de liberación de β-hexosaminidasa, cuyos resultados se presentan como la media ± desviación estándar de tres experimentos. El ensayo de liberación se desarrolló por primera vez por Siraganian y el gancho 25 para estudiar la alergia humana. El grupo de laboratorio dirigido por el Dr. Rubén Siraganian también desarrolló la línea celular RBL. Estas células RBL se desarrollaron para expresar la FcεRIα humana y el protocolo se publicó por 4. La pieza finalel ensayo de vino con el desarrollo del plásmido pSV en el documento de Neuberger 26 que describe la producción de un anticuerpos IgE mediante la clonación de su gen de la cadena pesada aguas abajo de un gen de ratón para una región variable de IgE que se dirige el hapteno 4-hidroxi-3 nitro-fenacetilo (NP), el anticuerpo quimérico resultante era completamente funcional. La capacidad para desarrollar cualquier IgE focalización el mismo hapteno, mientras que también la clonación de su receptor en la superficie de las células RBL dado lugar a la normalización del ensayo de lo que es un protocolo útil para medir la desgranulación de basófilos. El ensayo tiene pros y contras. Los pros del ensayo es su adaptabilidad para ser utilizado en cualquier sistema de mamífero, nuestro laboratorio así lo ha utilizado para probar la degranulación en los sistemas humanos, caninos y equinos, y esto se puede lograr simplemente mediante la síntesis de IgE del organismo y la clonación de su receptor sobre la superficie de las células RBL. Por otra parte,los contras del ensayo es que las células RBL son muy sensibles a los cambios térmicos, mecánicos y de pH, lo que les dan una variación de los niveles de desgranulación dentro del mismo ensayo. Por tanto, se recomienda encarecidamente que los ensayos siempre se repiten por triplicado y luego se toma una media de ellos. Además, las células RBL tienden a cambiar hacia un fenotipo no liberar si se dejan en cultivo de tejidos durante tiempos prolongados (&gt; 10 semanas) 27, por lo que su mantenimiento engorroso. También son propensos a las infecciones por bacterias de micoplasma, que no son visibles desde un microscopio de luz y no cambian la morfología de la célula, sino que cambiar drásticamente sus niveles de desgranulación. Por lo tanto se necesitan pruebas de micoplasma regulares.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RBL-2H3.1 Expressing Equine Fc&#949;RI&#945;</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>Equine IgE anti NIP-HSA</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Sigma</td>
			<td>CLS3595</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Multi Channel Pipette</td>
			<td>Anachem</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Galaxy R</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4Hydroxy-5-iodo-3-nitrophenylacetic acid</td>
			<td>Cambridge Research Biochemicals</td>
			<td>N-1070-1</td>
			<td>NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab</td>
		</tr>
		<tr>
			<td>Dinitrophenyl Conjugated to Human Serum Albumin</td>
			<td>Sigma</td>
			<td>A6661</td>
			<td>Abbreviated DNP-HSA</td>
		</tr>
		<tr>
			<td>Plate Spectrophotometer</td>
			<td>Anthos Labtec HT2</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipes</td>
			<td>Sigma</td>
			<td>P1851</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Triton x100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4-nitrophenyl N-acetyl-&#946;-D-glucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>Stock solution called &#946;-hexosaminidase substrate was 50mM prepared in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma</td>
			<td>251275</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma</td>
			<td>S7670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fc&#949;ri

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

Los parentales células RBL-2H3.1 y las transfectadas con el gen del receptor FcεRIα equina fueron sensibilizados primero con IgE de ratón anti- DNP-HSA y desafiados con el antígeno DNP-HSA. IgE de ratón se une al receptor endógeno de rata en ambas líneas celulares y por lo tanto actúa como un control para comprobar la viabilidad liberación de ambas líneas celulares para liberar mediadores (Figura 1 A). Este es un control importante y debe llevarse a cabo desde rutinariamente sobre extendido (...

Watch this Scientific Journal Video about Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÃŽÂµRI at JoVE.com

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Desarrollo de una<em&gt; In vitro</em&gt; Sistema modelo para estudiar la interacción de<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE con su receptor de alta afinidad Fc RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

development-an-vitro-model-system-for-studying-interaction-equus

Research

JoVE Journal

Immunology and Infection

12.4K vistas.  King Abdulaziz University. The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Video: Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Desarrollo de un marcador negativo seleccionable para Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Investigación

Inmunología e Infección

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Establecimiento de un<em&gt; In vitro</em&gt; Sistema para estudiar el comportamiento intracelular de<em&gt; Candida glabrata</em&gt; En humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

Una Novela<em&gt; In vitro</em&gt; Modelo para el estudio de las interacciones entre Whole Sangre humana y del endotelio

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

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

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 mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

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 ...

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. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

El estudio de las comunidades microbianas<em&gt; En Vivo</em&gt;: Un Modelo de anfitrión mediada por la interacción entre<em&gt; Candida Albicans</em&gt; Y<em&gt; Pseudomonas Aeruginosa</em&gt; En las vías respiratorias

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Un<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro mediante Espectroscopia de Impedancia: Un enfoque en la interacción de células T de la célula endotelial

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

La visualización de los Efectos de esputo en el desarrollo del biofilm mediante un modelo de Septadas cubreobjetos

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Desarrollo de un sistema de entrega de ADN económico por &quot;Acufection&quot; y su aplicación a la investigación de la piel

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.