The authors would like to acknowledge Dr. Ellen M. Fox for her work regarding this model of local anaphylaxis. This work was supported by the Uniformed Services University of the Health Sciences grant number R073UE.

1. Ratones Sensibilizar <ol><li> Preparar una solución madre de OVA mediante la dilución de OVA en PBS 1X a una concentración final de 20 mg / ml. Congele por alícuotas en crioviales y almacenar a -80 ° C para su uso futuro. </li><li> Traiga una o más alícuotas de 20 mg / ml OVA de stock a temperatura ambiente. Diluir OVA en PBS 1X a una concentración final de 1 mg / ml en un tubo de poliestireno de 5 ml de fondo redondo con una tapa. Vortex brevemente para mezclar. </li><li> Mientras sostiene el tubo de poliestireno que contiene el 1 mg / ml de OVA en un vórtice a velocidad media, añadir un volumen igual de bien homogeneizada Imject alumbre (40 mg / ml) gota a gota mientras se continúa vórtice del tubo para producir una proporción de 1: 1 de alumbre para inmunógeno. </li><li> Coloque el tapón de tubo de tubo y cinta para vórtice. Gire vórtice en posición más baja y deje OVA para adsorber al alumbre durante 30 min. <ol><li> No permita que una gran cantidad de tiempo que pase entre la preparación de OVA / Alum y sensibilizar a los ratones. Si sensibilización no se puede completar directamente después de OVA / Alum preparation, almacenar OVA / Alum a temperatura ambiente. Vortex durante 1 min antes de usar. </li></ol></li><li> Cargue una jeringa de insulina de 1 ml con mezcla de OVA / Alum. Tape la jeringa con aguja de calibre 27. </li><li> Inyectar 100 l de OVA / Alum en el peritoneo de ratones BALB / c para una dosis por ratón de 50 g de OVA y 2 mg de Alum. </li><li> Para un control negativo, sensibilizar a otro grupo de ratones a PBS. Preparar una mezcla 1: 1 de alumbre y PBS de la misma manera como se indica anteriormente, la omisión de OVA. </li><li> Sensibilizar a todos los ratones de control (OVA y PBS) una vez por semana durante 3 semanas para un total de 3 inyecciones. Después de la inyección final, espere por lo menos 2 semanas antes de realizar el ensayo de anafilaxis local. </li></ol> 2. Ensayo anafilaxia local <ol><li> Traiga OVA solución madre a temperatura ambiente. En un tubo de microcentrífuga de 1,5 ml, diluir 20 mg / ml de OVA de stock a una concentración final de 5 mg / ml con PBS (dilución 1: 4 de OVA en PBS 1X). Vortex brevemente para mezclar. </li><li> Anestesiar a los ratones utilizando RC 2 &lt;/ Sup&gt; Roedores Sistema de Anestesia (o sistema de entrega vaporizado comparable). Llene el depósito con isoflurano, y encienda el O 2 del flujo de aire. Coloque los ratones en cámara de inducción y ajustar dial de control de isoflurano al 5% para iniciar la anestesia. Una vez que los ratones se anestesiaron completamente, se indica por la falta de movilidad, ajuste dial de control de isoflurano al 3% para mantener la anestesia profunda. El propósito de la anestesia en este procedimiento es para inmovilizar el animal. Las inyecciones de la oreja y la cola de la vena no causan dolor significativo o trastornos a los animales. NOTA: El isoflurano puede tener efectos negativos sobre el sistema reproductivo humano. Asegúrese de utilizar en una habitación bien ventilada y adjuntar un bote de barrido a la cámara de inducción para neutralizar los gases residuales. Si usted está embarazada mientras está usando isoflurano, use una máscara de filtro de carbono para minimizar la exposición. </li><li> Cargar 10 l de 5 mg / ml de OVA en una jeringa de insulina 3/10 cc tapado con una aguja 31 G. Cargar otra jeringa de insulina 3/10 cc con 10 lPBS 1x. </li><li> Usando un microscopio de disección, inyectar 10 l de OVA en el oído derecho de un ratón anestesiado para una dosis de prueba de 50 g de OVA. Otros dosis, como 20 mg, también son aceptables. <ol><li> Para estabilizar el oído para inyección, envuélvalo con cinta scotch (parte adhesiva hacia arriba) en torno a un tubo cónico de 15 ml. Asegure el lado dorsal de la oreja derecha a la cinta. Inserte el lado bisel de la aguja entre las dos hojas de la oreja. Inyecte lentamente OVA en el centro del tejido de la oreja, con cuidado de no golpear a cualquier vaso sanguíneo. </li></ol></li><li> Usando el mismo método descrito anteriormente, se inyectan 10 l de PBS en la oreja izquierda como control negativo. </li><li> Espere 3 min. <ol><li> Durante este tiempo, coloque el ratón en un retenedor y sostenga la cola bajo una lámpara de calor o en un baño de agua tibia durante 1 min para dilatar los vasos sanguíneos. </li></ol></li><li> Usando una jeringa de 1 ml de insulina tapado con una aguja de 27 G, inyectar lentamente 200 l de 0,5% de colorante azul de Evans en la cola vein del ratón. Se administra por inyección rápida puede destruir la estructura de los vasos antes de la totalidad de los 200 l. Si esto ocurre, intente inyectar el volumen restante del medio de contraste en la vena en el lado opuesto de la cola. </li><li> Espere 10 min. </li><li> La eutanasia el ratón y quite los dos oídos utilizando pinzas y tijeras quirúrgicas. <ol><li> Agarrar los bordes de la oreja con fórceps, asegurándose de que no se aplica presión al sitio de inyección en el centro de la oreja. Quite la mayor cantidad de tejido del oído como sea posible mediante la reducción de cerca, pero no a través de la cordillera de cartílago presentes en la base de la oreja. Pequeñas cantidades de la piel pueden estar presentes en el oído extirpado; esto no afectará el ensayo. </li></ol></li><li> Alícuota de 700 l de formamida en tubos de 1,5 ml de microcentrífuga. Preparar un tubo por el oído (dos tubos por ratón). NOTA: La formamida es teratogénico, mutagénico, y un irritante. Usar guantes, protección para los ojos y la cara. Las mujeres embarazadas deben tener cuidado al manipular unas formamida puede ser fetotóxica. </li><li> Añadir oídos para formamida, tapar los tubos, y se incuba durante la noche en un baño de calor seco ajustado a 63 ° C. Orejas todavía pueden aparecer ligeramente azul después de la incubación; sin embargo la mayoría del colorante se extrajo en la formamida. </li><li> Añadir 300 l de cada muestra, por duplicado, a una placa de 96 pocillos. Evitar la transferencia de la piel, si está presente en la muestra. Asegúrese de que no hay burbujas presentes en los pozos, ya que afectarán a la lectura de absorbencia. </li><li> Añadir 300 l de formamida, por duplicado, a la placa de 96 pocillos. Estos pozos se utilizarán como espacios en blanco. </li><li> Utilice un espectrofotómetro para medir la absorbancia a 620 nm. Pozos de formamida Restar de cada lectura de la muestra. NOTA: Todos los experimentos se realizaron bajo los protocolos aprobados por el Comité Institucional Cuidado de Animales y el empleo de los Servicios Uniformados Universidad. </li></ol>

<ol>
	<li>Kojima, T., 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=Mast+cells+and+Bbasophils+are+selectively+activated+in+vitro+and+in+vivo+through+CD200R3+in+an+IgE-independent+manner.">Mast cells and Bbasophils are selectively activated in vitro and in vivo through CD200R3 in an IgE-independent manner.</a> The Journal of Immunology. 179 (10), 7093-7100 (2007).</li><li>Parikh, S. A., Cho, S. H., Oh, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preformed+enzymes+in+mast+cell+granules+and+their+potential+role+in+allergic+rhinitis.">Preformed enzymes in mast cell granules and their potential role in allergic rhinitis.</a> Current Allergy and Asthma Reports. 3 (3), 266-272 (2003).</li><li>Amin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mast+cells+in+allergic+inflammation.">The role of mast cells in allergic inflammation.</a> Respiratory Medicine. 106 (1), 9-14 (2012).</li><li>Nakaya, M., Takeuchi, N., Kondo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+histamine+receptor+subtypes+in+human+inferior+turbinates.">Immunohistochemical localization of histamine receptor subtypes in human inferior turbinates.</a> Annals of Otology, Rhinology &amp; Laryngology. 113 (7), 552-557 (2004).</li><li>Ramsdell, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Trypan+Blue+to+demonstrate+the+immediate+skin+reaction+in+rabbits+and+guinea+pigs.">The use of Trypan Blue to demonstrate the immediate skin reaction in rabbits and guinea pigs.</a> The Journal of Immunology. 15 (4), 305-311 (1928).</li><li>Chase, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+sensitization+of+animals+with+simple+chemical+compounds:+X.+Antibodies+inducing+immediate-type+skin+reactions.">Studies on the sensitization of animals with simple chemical compounds: X. Antibodies inducing immediate-type skin reactions.</a> The Journal of Experimental Medicine. 86 (6), 489-514 (1947).</li><li>Goose, J., Blair, A. M. J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+cutaneous+anaphylaxis+in+the+rat,+induced+with+two+homologous+reagin-like+antibodies+and+its+specific+inhibition+with+disodium+cromoglycate.">Passive cutaneous anaphylaxis in the rat, induced with two homologous reagin-like antibodies and its specific inhibition with disodium cromoglycate.</a> Immunology. 16, 749-760 (1969).</li><li>Ovary, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+cutaneous+anaphylaxis+in+the+mouse.">Passive cutaneous anaphylaxis in the mouse.</a> The Journal of Immunology. 81 (4), 355-357 (1958).</li><li>Ovary, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+reactions+in+the+skin+of+experimental+animals+provoked+by+antibody-antigen+interactoin.">Immediate reactions in the skin of experimental animals provoked by antibody-antigen interactoin.</a> Progress in Allergy. 5, 459-508 (1958).</li><li>Akiyama, 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=Quantitiative+evaluation+of+passive+cutaneous+anaphylaxis+(PCA)+using+a+hand-held+spectrophotometer.">Quantitiative evaluation of passive cutaneous anaphylaxis (PCA) using a hand-held spectrophotometer.</a> Biological &amp; Pharmaceutical Bulletin. 19 (8), 1112-1114 (1996).</li><li>Teshima, R., 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=Simple+spectrophotometric+analysis+of+passive+and+active+ear+cutaneous+anaphylaxis+in+the+mouse.">Simple spectrophotometric analysis of passive and active ear cutaneous anaphylaxis in the mouse.</a> Toxicology Letters. 95 (2), 109-115 (1998).</li></ol>

The authors have nothing to disclose.

Numerosos marcadores de la enfermedad alérgica se utilizan para evaluar la solidez de las respuestas alérgicas tras la exposición de alérgenos, incluyendo cambios en los niveles de histamina circulantes, la producción de citocinas Th2, y el reclutamiento de células en el líquido broncoalveolar en el ajuste de la impugnación de las vías respiratorias. Mientras seguimiento de los cambios en estos parámetros es importante para el estudio de las reacciones alérgicas, marcadores biológicos no siempre se correlaci...

Hipersensibilidad de tipo I está mediada por reticulación inducida por el antígeno de la IgE en la superficie de mastocitos y basófilos. Esto da como resultado la desgranulación celular y la liberación de mediadores vasoactivos y proinflamatorios tales como histamina, triptasa, y factor activador de plaquetas 2. Después de la liberación de mediadores preformados durante la degranulación de los mastocitos sintetizan y liberan prostaglandinas y leucotrienos, que aumentan aún más la permeabilidad vascular 3. La respuesta clínica inicial se produce rápidamente y se conoce como una &quot;reacción inmediata&quot;. En la piel, una respuesta de roncha y eritema es fácilmente visible a pocos minutos de la exposición al antígeno. Dependiendo de la dosis del desafío, es posible observar una &quot;respuesta de fase tardía&quot; unos pocos horas más tarde. Hinchazón de fase tardía se debe a edema localizado y el reclutamiento de leucocitos en los tejidos 2. La histamina, generalmente considerado como el principal mediador de participar enrespuestas alérgicas inmediatas, actúa sobre los receptores de histamina 1 (HR1) expresó en los buques y los receptores de histamina 2 (HR2) expresado en el músculo liso. El efecto combinado aumenta el flujo sanguíneo y la permeabilidad vascular en el sitio de la inflamación 4. Una variedad de modelos animales de la alergia se han desarrollado con el fin de estudiar los mecanismos implicados en la inflamación alérgica, incluyendo modelos de asma alérgica, anafilaxis sistémica y anafilaxia locales. La administración del colorante por vía intravenosa se ​​ha utilizado para medir las respuestas alérgicas localizadas en modelos animales durante casi un siglo, con publicaciones que describe esta técnica que se remonta a la década de 1920 5. Los conejos y conejillos de Indias fueron los primeros modelos animales utilizados para probar las reacciones de hipersensibilidad inmediatas, y las respuestas más sensibles se encuentran generalmente en el oído 5,6. El ensayo fue posteriormente validada para su uso en ratas y ratones 7 8. Históricamente, losuna variedad de métodos experimentales se han utilizado, incluyendo la inyección del antígeno antes de la inyección de colorante, la inyección de colorante antes de la inyección del antígeno, y la inyección simultánea de tinte y antígeno. La administración del colorante por vía intravenosa como un medio para la medición de las respuestas alérgicas es un ensayo versátil ya que puede ser utilizado para medir activo, pasivo, y revertir reacciones pasivas 5,9. Numerosos colorantes se han utilizado para evaluar las respuestas alérgicas, incluyendo azul de tripano, pontamina cielo azul, azul de Evans, Geigy Azul 536, y tinta de la India 5,6,9. Una solución de 0,5% azul de Evans es actualmente el colorante estándar utilizado para medir las respuestas alérgicas en la piel. La respuesta anafiláctica a desafío es transitoria; intensidad máxima se alcanza de 10 - 15 min de la inyección de colorante, y no hay reacción es visible si tinte se administra más de 30 min después de la exposición, independientemente de las especies animales utilizadas 9. La cuantificación de la extravasación de colorante era originalLY obtiene midiendo el tamaño de la roncha como se indica por el colorante azul 7-9. Además, los recuentos de mastocitos desgranulados se pueden cuantificar mediante la escisión de tejido de la piel en el sitio de la reacción y la tinción con azul de toluidina 7. Degranulación de los mastocitos se utiliza a menudo como un marcador para cutánea, las respuestas alérgicas mediadas por IgE, como mastocitos son la principal población celular local que expresa el receptor de IgE de alta afinidad Fc RI. Se desarrollaron técnicas espectrofotométricas para la medición de la extravasación de colorante en el tejido de la anafilaxia cutánea pasiva (PCA) en la rata y el ratón 10 11 en los años 1990. El siguiente protocolo de ensayo de la anafilaxia local fue adaptado de Kojima et al. 1, y utiliza la ovoalbúmina de huevo de pollo (OVA) como antígeno para provocar respuestas alérgicas. Sin embargo, los antígenos distintos de OVA se pueden usar si se desea. El ensayo se utiliza colorante azul de Evans para monitorear cambios en permeabilit vasculary que se producen debido a mastocitos IgE entrecruzamiento y la liberación de histamina.

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:225px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BALB/c Mice</td>
			<td style="width:167px;">The Jackson Laboratory</td>
			<td style="width:135px;">651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">PBS pH 7.4</td>
			<td style="width:167px;">Quality Biologic</td>
			<td style="width:135px;">114-058-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ovalbumin</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">A5503-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Imject Alum</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:135px;">77161</td>
			<td style="width:225px;">Mix thoroughly before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Evans Blue Dye</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">E2129</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Formamide</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">295876</td>
			<td style="width:225px;">99%+ Spectrophotometric grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Isoflurane, USP</td>
			<td style="width:167px;">Phoenix</td>
			<td style="width:135px;">NDC 57319-474-06</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1cc Insulin syringes</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">329654</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">3/10 cc Insulin syringe with 31G needle</td>
			<td style="width:167px;">Terumo</td>
			<td style="width:135px;">NDC 100861</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27G Needles</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">305109</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Forceps</td>
			<td style="width:167px;">F.S.T.</td>
			<td style="width:135px;">11000-12</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Surgical scissors</td>
			<td style="width:167px;">F.S.T.</td>
			<td style="width:135px;">14070-12</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">5ml Polystyrene round-bottom tubes</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:135px;">352058</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1.5ml Microcentrifuge tubes</td>
			<td style="width:167px;">Medical Supply Partners</td>
			<td style="width:135px;">15-1151</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">15ml Conical tubes</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:135px;">352097</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Flat-bottom 96 well plate</td>
			<td style="width:167px;">Costar</td>
			<td style="width:135px;">3590</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Scotch tape</td>
			<td style="width:167px;"></td>
			<td style="width:135px;"></td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">RC2 Rodent Anesthesia System</td>
			<td style="width:167px;">VetEquip</td>
			<td style="width:135px;">922100</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Vortex Genie 2</td>
			<td style="width:167px;">Scientific Industries</td>
			<td style="width:135px;">SI-0236</td>
			<td style="width:225px;">Model G560 with 3 inch platform</td>
		</tr>
	</tbody>
</table>

measuring local anaphylaxis in mice

Las respuestas alérgicas son el resultado de la activación de los mastocitos y basófilos y la posterior liberación de mediadores vasoactivos y proinflamatorios. La exposición a un alérgeno en un individuo sensibilizado puede dar lugar a síntomas clínicos que varían desde leves hasta anafilaxia eritema que amenazan la vida. En el laboratorio, se han desarrollado diversos modelos animales para comprender los mecanismos que impulsan respuestas alérgicas. En este documento, se describe un método detallado para medir los cambios en la permeabilidad vascular para cuantificar las respuestas alérgicas localizadas. El ensayo de la anafilaxia local fue reportado por primera vez en la década de 1920, y ha sido adaptada de la técnica publicada por Kojima et al. en 2007 1. En este ensayo, los ratones sensibilizados a OVA son desafiados en el oído izquierdo con vehículo y en la oreja derecha con OVA. Esto es seguido por una inyección intravenosa de colorante azul de Evans. Diez minutos después de la inyección de azul de Evans, el animal es sacrificado y el tinte que se ha extravasado enpara los oídos se extrae durante la noche en formamida. La absorbancia del colorante extraído se cuantificó luego con un espectrofotómetro. Este método da con fiabilidad una manifestación visual y cuantificable de una respuesta alérgica local.

Los animales que han sido sometidos a la prueba con éxito tendrán la piel y los ojos que aparecen de color azul. PBS animales sensibilizados no deben reaccionar a PBS o desafío OVA, por lo tanto ambos oídos deben permanecer blanco (Figura 1A). En los animales sensibilizados OVA, el oído recibir el desafío PBS (izquierda) debe ser completamente blanco o ligeramente azul en forma localizada en el sitio de inyección. El oído recibir el desafío OVA (derecha) debe convertirse en progresivamente más...

Watch this Scientific Journal Video about Measuring Local Anaphylaxis in Mice at JoVE.com

Measuring Local Anaphylaxis in Mice

Las respuestas alérgicas, caracterizados por la activación de los mastocitos y basófilos, están impulsados ​​por la reticulación de IgE y liberación de mediadores proinflamatorios. Una evaluación cuantitativa de las respuestas alérgicas se puede lograr mediante el uso de colorante azul de Evans para monitorear los cambios en la permeabilidad vascular después de la provocación con alergeno.

Medición anafilaxia local en ratones

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

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JoVE Journal

Immunology and Infection

19.1K vistas.  Uniformed Services University of the Health Sciences.  Las respuestas alérgicas, caracterizados por la activación de los mastocitos y basófilos, están impulsados ​​por la reticulación de IgE y liberación de mediadores proinflamatorios. Una evaluación cuantitativa de las respuestas alérgicas se puede lograr mediante el uso de colorante azul de Evans para monitorear los cambios en la permeabilidad vascular después de la provocación con alergeno. 

Video: Measuring Local Anaphylaxis in Mice

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medición de la actividad calpaína en células fijadas y vivir por citometría de flujo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Investigación

Inmunología e Infección

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

La medición de la carga bacteriana y la respuesta inmune en ratones infectados con Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Measurement of &amp;#947;HV68 Infection in Mice

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

La medición de γHV68 infección en los ratones

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

La medición de granulocitos y de monocitos fagocitosis y la actividad de la explosión oxidativa en la sangre humana

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Medición de la deformabilidad y la heterogeneidad de glóbulos rojos en sangre por Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Caracterización de las respuestas de isotipo de inmunoglobulina dependiente del timo y timo-independiente en ratones usando el análisis enzima-ligado del inmunosorbente

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

Medición de la temperatura del cuerpo de ratón con termómetro infrarrojo pasiva Anafilaxia sistémica y evaluación de la alergia alimentaria

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

Cuantificación de la aterosclerosis en ratones

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Inmunoterapia adoptiva de células iNKT en ratones RA inducidos por glucosa-6-fosfato (G6PI)

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Medición de la fagocitosis de Aspergillus fumigatus Conidia por leucocitos humanos utilizando citometría de flujo

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...