Este trabajo fue apoyado por los Institutos Nacionales de Salud (NIH) AI091759 y la enfermedad de Crohn y Colitis Foundation of America y William Shelby Familia Modell Premio de Investigación Fundación (a MN). Esquistosomosis ratones infectados fueron suministrados por el Centro de Recursos del NIAID esquistosomosis (NIAID N01 Contrato A130026).

1. Depuradora Schistosoma mansoni Huevos <ol><li> Infect ratones Swiss-Webster con cercarias de esquistosomas, que es la etapa infecciosa en el ciclo de vida del Schistosoma 8. Por otra parte, obtener ratones infectados con esquistosomas del NIAID Centro de Recursos de la esquistosomiasis ( <a href="http://www.schisto-resource.org/" target="_blank">http://www.schisto-resource.org/~~V</a> ). Deja ratones para 6-7 semanas después de la infección, momento en el cual punto, los huevos están presentes en el hígado y puede ser recuperado como se describe a continuación (ver fig. 1). </li><li> La eutanasia de los ratones con el CO 2, y mojar con el 70% de etanol. Coloca el ratón sobre la espalda. Use unas pinzas y tijeras romas para cortar la piel y el peritoneo subyacente. Impuestos Especiales en el hígado, que se encuentra por debajo de la caja torácica y el diafragma. </li><li> Finamente picada hígados y añadir 20 ml por el hígado de la mezcla de digestión, compuesto de colagenasa / dispasa (0,5 mg / ml), penicilina (100 U / ml) y estreptomicina (100 ug / ml) en PBS. Ponga mixtoUre en un tubo Falcon de 50 ml y se incuba durante la noche en un agitador de 37 ° C. </li><li> Centrifugar mezcla a 400 xg / 3 min / 20 ° C. Verter suavemente el exceso de líquido y el tubo de llenado con PBS. Se centrifuga de nuevo y repita el lavado con PBS. Tras la centrifugación pasado, volver a suspender el pellet en 25 ml de PBS. </li><li> Cuele el precipitado resuspendido a través de un estándar de cocina metálico colador (utilice un émbolo de jeringa de 50 ml para triturar las piezas del hígado a través de en un vaso de precipitados de vidrio), seguido por hacer pasar la mezcla a través de un tamiz fino (utilizar 10 ml jeringa para empujar la mezcla a través del tamiz fino en un segundo vaso de vidrio). </li><li> Ponga la mezcla colada en un tubo de 50 ml Falcon. Se centrifuga a 400 xg / 5 min / 20 ° C. Vierta cuidadosamente el sobrenadante sin perder de pellets. Resuspender el sedimento en 3 ml de PBS. </li><li> Superposición mezclar suavemente sobre un Percoll 20%, 40 ml de gradiente 8 ml de Percoll y 32 ml de sacarosa 0,25 M (FW = 342,3 así 8,56 g sucrose/100 ml de H 2 0). </li><li> Centrifugar 10 min / 800 ug / 20 ° C. Pipetear fuera unad descartar capa gelatinosa parte superior de las células del hígado. Retire del huevo de pellets en la parte inferior con una pipeta de transferencia y poner en un tubo Falcon de 15 ml. </li><li> Lavado de pellets 3X en 10 ml de PBS / EDTA 1 mM / EGTA 1 mM utilizando la configuración de centrífuga: 3 min / 30 ug / 20 ° C. Resuspender en 500 l de PBS. </li><li> Superposición mezcla en un nuevo Percoll 25%, 10 ml de gradiente (2,5 ml de Percoll y 7,5 ml de sacarosa 0,25 M) utilizando la configuración de centrífuga: 10 min / 800 ug / 20 ° C. </li><li> Lavar 3X en 10 ml de PBS utilizando la configuración de centrífuga: 3 min / 30 ug / 20 ° C. </li><li> En los últimos 10 ml de lavado, remover el 100 l para contar los huevos. Los huevos se depositan rápidamente, por lo que el tubo Falcon deben mezclarse bien antes de extraer la muestra. Diluir la muestra de 100 l con 900 l de PBS. 100 L de la mezcla debe ser contado en forma de gotas sobre un portaobjetos de microscopio utilizando un microscopio de disección (dilución 1:10). De grueso calibre puntas de pipeta debe ser utilizado. </li><li> Resuspender los huevos a 50.000 huevos / ml en PBS. Los huevos se pueden almacenar durante varios meses a -80 ° C y se descongeló una o TWICe para su uso. Antes del uso, inspeccionar bajo un microscopio para asegurar que los huevos son todavía intacta (ver fig. 1). </li></ol> 2. La sensibilización por vía intraperitoneal con S. mansoni Huevos: Día 0 <ol><li> Prepare los huevos a 5.000 individuos/100 l de PBS en un tubo de 5 ml snapcap. Para la inyección, estiman los huevos un 50% más de lo necesario. </li><li> Coloque una aguja de calibre 23 y una 3/4inch jeringa de 1 ml con 100 l de suspensión de huevos por ratón. Huevos resolver, por lo que la aguja de carga con los huevos inmediatamente antes de usar. </li><li> Roca jeringa de un lado a otro para mezclar los huevos antes de cada inyección, y se inyecta por vía intraperitoneal 100 L de la suspensión por ratón. </li></ol> 3. Retro-orbital desafío por vía intravenosa con S. mansoni Huevos: Día 14 <ol><li> Prepare los huevos que el anterior en 5.000 individuos/100 l. </li><li> Anestesie ratones en una cámara especializada con isoflurano (4%), o por vía intraperitoneal con xilacina (10 mg / kg) y ketamina (120 mg / kg). La profundidad de Anesthesia está determinada por pinzamiento de la almohadilla plantar y la garantía de respuesta física. </li><li> Después de la carga de la jeringa de 1 ml y aguja de calibre 23 3/4inch con huevos, utilizar pinzas para doblar la aguja en un ángulo de 90 ° con el bisel hacia abajo en el ángulo. Asegúrese de que no hay burbujas. </li><li> Coloque el ratón sobre un lado. Retirar la piel para que los ojos sobresalgan, e inyectar 100 l en los vasos detrás del globo ocular en un ángulo de 45 ° a la nariz. </li><li> Retire la aguja y aplicar una ligera presión para controlar el sangrado. </li></ol> Todos estos procedimientos deben llevarse a cabo con cuidado, y los ratones deben ser monitoreados hasta que se recuperen de la anestesia. Estos protocolos fueron aprobados por la Universidad de Pennsylvania Cuidado de Animales institucional y el empleo Comisión (IACUC). 4. Experimental de cosecha: Día 22 <ol><li> La eutanasia de los ratones con el CO 2. </li><li> Del ratón mojado con etanol al 70%, y el lugar en la parte posterior. </li><li> Usando contundente FORCeps, sujete el abdomen del ratón y hacer una pequeña incisión en la piel. Retire con cuidado la piel con unas tijeras y exponer la caja torácica y el peritoneo. </li><li> Cortar el peritoneo. Cortar la aorta abdominal para recuperar la sangre con una pipeta Pasteur. Guarde en el hielo. </li><li> Mueva el hígado para exponer el diafragma. Con unas tijeras finas para cortar el diafragma. Con cuidado, corte abierto la caja torácica para exponer el tejido pulmonar. </li><li> Recuperar los ganglios linfáticos que drenan el paratímico de pulmón con unas pinzas finas. Estos están metidos debajo de la caja torácica, a ambos lados del timo 9. Tienda en hielo en 1 ml de medios estériles (DMEM suplementado con inactivado por calor 10% de suero fetal de ternero, 100 U / ml de penicilina, 100 mg / mL de estreptomicina, 2 mM de L-glutamina, 50 mM 2-mercaptoetanol, todos disponibles de Invitrogen). </li><li> Para recuperar el BAL, e inflar los pulmones para su análisis histológico, la intubación endotraqueal se debe realizar. Exponer la tráquea moviendo las glándulas salivales a un lado, y placing unas pinzas finas bajo la tráquea. Con unas tijeras finas, cortar un agujero en el centro de la tráquea e insertar el tubo en los pulmones. Asegure la tubería mediante la vinculación con hilo quirúrgico. </li><li> Adjuntar PBS-lleno de jeringa de 1 ml al tubo e inflar los pulmones con 1 ml de PBS. Con cuidado, lavado recuperar BAL con la jeringa. Guarde en el hielo. </li><li> Coloque 4% paraformaldehído (PFA) en PBS-jeringa precargada de 1 ml a los tubos y se inflan los pulmones. </li><li> Retire el tubo y apriete el hilo quirúrgico para evitar la filtración de la PFA. </li><li> Diseccionar cuidadosamente el tejido pulmonar y el lugar en un tubo de 50 ml con 5 ml Falcon 4% PFA. </li></ol> 5. Preparación de tejidos recuperados <ol><li> Suero: Después del almacenamiento en hielo durante h 30 min a 2 coagulación de la sangre, suero, se recupera de la sangre por centrifugación (13000 xg / 10 min / 4 ° C). El suero se almacenó a -20 ° C y puede ser utilizado para ELISA de citoquinas o S. mansoni huevos antígenos específicos de isotipo IgG ELISA <sup&gt; 6, 10. </li><li> Los nodos linfáticos: suspensiones de células individuales se preparan y los recuentos de células utilizado para evaluar la magnitud de la respuesta inmune. Para examinar la polarización de las células Th2, las células se vuelven a estimulado con S. mansoni antígeno de huevos durante 72 horas. Tinción de citoquinas intracelulares pueden ser examinadas por citometría de flujo, y los sobrenadantes se puede recuperar y se almacenó a -20 ° C para ELISA de citocinas Th2 (IL-4, IL-5, IL-13) 6. </li><li> BAL: Las células se sedimentan (400 ug / 5 min / 4 ° C). BAL líquido se recupera y se almacenó a -20 ° C para el análisis de las citocinas Th2 por ELISA. Recuentos de células BAL se utilizan como una lectura de inflamación de las vías. Preparaciones cytocentrifuge de 10.000-100.000 células (500 rpm / 5 min / 4 ° C), seguido por tinción Diffquik permitirá enumeración de infiltrado inflamatorio 4, 6. Alternativamente, las células BAL puede ser examinado por citometría de flujo 6. </li><li> El tejido pulmonar: El tejido pulmonar es almacenamientonoche a la mañana ed a 4 ° C. Tejido fijado es incluido en parafina, seccionadas y montadas sobre portaobjetos para el análisis histológico e inmunofluorescencia. </li></ol> 6. Los resultados representativos Este protocolo de todos los detalles de los pasos necesarios para (i) preparar huevos de Schistosoma mansoni purificada para su uso in vivo (Fig. 1), (ii) sensibilizar y desafiar a los ratones con estos huevos y (iii) recuperar los órganos para el examen de la respuesta inflamatoria de los pulmones. Este modelo in vivo de forma reproducible impulsa una potente respuesta inflamatoria de los pulmones Th2. Esto se muestra por inflamación de las vías y eosinofilia como se visualizó por los recuentos de células BAL y preparados cytocentrifuge (Fig. 2). H &amp; E-manchado secciones de pulmón puede ser examinado para el huevo inducida por granulomas (Figura 3a), y la tinción de inmunofluorescencia de secciones de pulmón permite la visualización de los macrófagos activados alternativamente y Th2 inducida por citoquinas, tales como los genes Resisestaño molécula similar a (RELM) α (Fig. 3b). La respuesta antígeno-específica CD4 citocinas Th2 es examinado por ELISA para la IL-5, IL-4 e IL-13 de antígeno-estimulados células de ganglios linfáticos paratímico (fig. 4). <img alt="Figura 1" src="/files/ftp_upload/3905/3905fig1.jpg" /> Figura 1 Schistosoma mansoni modelo de inyección de huevo imágenes representativas de los hígados granulomatosas, características de alta huevo S.mansoni-cargas y de los huevos S.mansoni se indica -.. De tamaño medio (L) 120 m (al W) 50 micras. <img alt="Figura 2" src="/files/ftp_upload/3905/3905fig2.jpg" /> Figura 2. S. mansoni inyección de huevos impulsa inflamación de las vías. A. BAL número de células (x10 5) de ingenuos (N) o S.mansoni (SM) de huevo ratones inyectados. * P &lt;0,05. B. Preparación citocentrífuga de células BAL. Mac, los macrófagos; Eos, eosinófilos, los nódulos linfáticos, los linfocitos. Bar, 10 micras. <img alt="Figura 3" src="/files/ftp_upload/3905/3905fig3.jpg" /> Figura 3. La observación de granulomas pulmonares y macrófagos activados alternativamente rodean el óvulo S.mansoni. A. Granuloma pulmonar que rodea el huevo Sm (negro flecha) se visualizó en H &amp; E-manchado secciones de pulmón. B. La tinción de inmunofluorescencia para la RELMα (verde), receptor de manosa (rojo) y DAPI (azul) revela los macrófagos activados alternativamente (flecha blanca) en el granuloma que rodea al óvulo autofluorescente Sm (flecha negro). Bar, 50 micras. <img alt="Figura 4" src="/files/ftp_upload/3905/3905fig4.jpg" /> Figura 4. S.mansoni huevo antígeno específico de la respuesta de citoquinas Th2. Drenaje paratímico células de nódulos linfáticos de los ratones no tratados previamente (N) o Sm huevo inyectado fueron estimulados con SM &lt;/ Em&gt; antígeno huevo durante 72 horas, seguido por ELISA de los sobrenadantes para la IL-4, IL-5 e IL-13. Escala, ng / ml. * P &lt;0,05.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis:+report+of+a+WHO+expert+committee.">Prevention and control of schistosomiasis and soil-transmitted helminthiasis: report of a WHO expert committee.</a> WHO Technical Report Series 912. , (2002).</li><li>Pearce, E. J., MacDonald, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunobiology+of+schistosomiasis.">The immunobiology of schistosomiasis.</a> Nat. Rev. Immunol. 2, 499-511 (2002).</li><li>Sandler, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+profiles+during+acute+pathogen-induced+pulmonary+inflammation+reveal+divergent+roles+for+Th1+and+th2+responses+in+tissue+repair.">Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and th2 responses in tissue repair.</a> J. Immunol. 171, 3655-3667 (2003).</li><li>Perrigoue, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-31-IL-31R+interactions+negatively+regulate+type+2+inflammation+in+the+lung.">IL-31-IL-31R interactions negatively regulate type 2 inflammation in the lung.</a> J. Exp. Med. 204, 481-487 (2007).</li><li>Sandor, M., Weinstock, J. V., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulomas+in+schistosome+and+mycobacterial+infections:+a+model+of+local+immune+responses.">Granulomas in schistosome and mycobacterial infections: a model of local immune responses.</a> Trends Immunol. 24, 44-52 (2003).</li><li>Nair, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatively+activated+macrophage-derived+RELM-{alpha}+is+a+negative+regulator+of+type+2+inflammation+in+the+lung.">Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung.</a> J. Exp. Med. 206, 937-952 (2009).</li><li>Dalton, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+of+schistosome+eggs+and+miracidia+free+of+contaminating+host+tissues.">A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues.</a> Parasitology. 115, 29-32 (1997).</li><li>Lewis, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+laboratory+maintenance+of+Schistosoma+mansoni,+with+observations+on+three+schistosome/snail+host+combinations.">Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations.</a> J. Parasitol. 72, 813-829 (1986).</li><li>Tilney, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+lymphatic+drainage+in+the+adult+laboratory+rat.">Patterns of lymphatic drainage in the adult laboratory rat.</a> J. Anat. 109 (Pt. 3), 369-383 (1971).</li><li>Lewis, F., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Current protocols in immunology. Chapter 19, Unit 19 (2001).</li><li>Kaplan, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th2+cells+are+required+for+the+Schistosoma+mansoni+egg-induced+granulomatous+response.">Th2 cells are required for the Schistosoma mansoni egg-induced granulomatous response.</a> J. Immunol. 160, 1850-1856 (1998).</li><li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrotic+disease+and+the+T(H)1/T(H)2+paradigm.">Fibrotic disease and the T(H)1/T(H)2 paradigm.</a> Nat. Rev. Immunol. 4, 583-594 (2004).</li><li>Cheever, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+hepatic+fibrosis+and+granuloma+size+among+mouse+strains+infected+with+Schistosoma+mansoni.">Variation of hepatic fibrosis and granuloma size among mouse strains infected with Schistosoma mansoni.</a> Am. J. Trop. Med. Hyg. 37, 85-97 (1987).</li></ol>

No hay conflictos de interés declarado.

Aquí, un modelo que emplea Schistosoma mansoni huevos para inducir inflamación pulmonar tipo 2 se describe. Los rasgos característicos de este modelo incluyen la potente respuesta inmune tipo Th2, inflamación de las vías y la formación de granulomas pulmonares. Desde estos parámetros dependen de las respuestas de células CD4 + Th2 11, este es un modelo útil para investigar los efectos de las supresiones específicas de proteínas o de linaje específico-en respuestas de las células Th2. Aqu...

<table border="1"><tbody><tr><td> Reactivo </td><td> Tamaño / Descripción </td><td> Empresa </td><td> Catálogo # </td></tr><tr><td> Colagenasa / dispasa </td><td> 500 mg </td><td> Roche Ciencias Aplicadas </td><td> 11 097 113 001 </td></tr><tr><td> Diff Quik Stain Hema 3 </td><td> Empacar </td><td> Fisher Scientific Co. LLC </td><td> 122-911 </td></tr><tr><td> DMEM </td><td> Líquido </td><td> Gibco </td><td> 11965 </td></tr><tr><td> Falcon 50 ml </td><td> Empacar </td><td> Halcón </td><td> 352070 </td></tr><tr><td> Intramedic PE Tubo </td><td> 0,58 mm </td><td> Becton Dickinson and Co. </td><td> 427410 </td></tr><tr><td> El isoflurano </td><td> 250 ml </td><td> Abad de Sanidad Animal </td><td> 52600405 </td></tr><tr><td> La ketamina </td><td> 100 mg / ml </td><td> Fort Dodge, de Sanidad Animal </td&gt; <td> N / A </td></tr><tr><td> Anticuerpo del receptor de manosa </td><td> Biotina </td><td> AbD Serotec </td><td> MCA2235B </td></tr><tr><td> Filtro metálico </td><td> Colador de cocina estándar </td><td> Objetivo </td><td> Laurentiis </td></tr><tr><td> Paraformaldehído </td><td> 16% </td><td> Electron Microsc. Ciencias de la </td><td> 15710 </td></tr><tr><td> La penicilina / estreptomicina </td><td> 100x </td><td> Gibco </td><td> 15070 </td></tr><tr><td> Percoll </td><td> 1 Litro </td><td> GE Healthcare Biosciences </td><td> 17 01 0891 </td></tr><tr><td> Anticuerpos RELMα </td><td> 50 ug </td><td> Peprotech </td><td> 500-P214 </td></tr><tr><td> Hilo quirúrgico </td><td> 2,0 USP 100 yardas </td><td> Surgical Corp. Especialidades </td><td> SP118 </td></tr><tr><td> Aguja de la jeringa </td><td> 23g, 3/4 pulgada </td><td> BD Vacutainer laboratorio. Med. </td><td>305143 </td></tr><tr><td> EE.UU. estándar de pruebas tamiz </td><td> 150 micras, 0,0059 &quot; </td><td> WS Tyler, Inc. </td><td> ASTME-11, Especificaciones. N º 100 </td></tr><tr><td> Xilacina </td><td> 20 mg / ml </td><td> Mayordomo </td><td> N / A </td></tr></tbody></table>

using eggs from schistosoma mansoni as an in vivo model of helminth-induced lung inflammation

Parásitos Schistosoma son trematodos de la sangre que infecta a un estimado de 200 millones de personas en todo el mundo 1. En la infección crónica por Schistosoma, la patología severa, incluyendo la fibrosis hepática y esplenomegalia, es causada por la respuesta inmune a los huevos del parásito en lugar del propio parásito 2. Huevos de parásitos inducir una respuesta Th2 caracterizada por la producción de IL-4, IL-5 e IL-13, la activación alternativa de macrófagos y el reclutamiento de eosinófilos. A continuación, describimos la inyección de huevos de Schistosoma mansoni como un modelo para examinar los parásitos específicos de las respuestas de citocinas Th2 en el pulmón y los ganglios linfáticos de drenaje, la formación de granulomas pulmonares que rodean el huevo, y la inflamación de las vías respiratorias. Tras la sensibilización intraperitoneal y desafío intravenosa, S. huevos mansoni son transportados a los pulmones a través de las arterias pulmonares donde están atrapadas dentro del parénquima pulmonarpor granulomas compuestos por linfocitos, eosinófilos y macrófagos activados alternativamente 3-6. Asociado con formación de granulomas, inflamación en los espacios bronco-alveolares, la expansión de los ganglios linfáticos de drenaje y activación de células T CD4 puede ser observado. A continuación le detallamos el protocolo para el aislamiento de huevos de Schistosoma mansoni a partir de hígados infectados (modificado a partir de 7), la sensibilización y desafiar los ratones, y la recuperación de los órganos (lavado broncoalveolar (LBA), pulmón y ganglios linfáticos de drenaje) para su análisis. También se incluyen histológica representativa y los datos inmunológicos y sugerencias para el análisis inmunológico adicional. En general, este método proporciona un modelo in vivo para investigar helmintos inducidos por las respuestas inmunológicas en el pulmón, el cual es ampliamente aplicable al estudio de las enfermedades inflamatorias Th2, incluyendo la infección por helmintos, enfermedades fibróticas, la inflamación alérgica y el asma. Las ventajas de este modelo para el estudio de típicoe 2 inflamación en el pulmón incluyen la reproducibilidad de un potente respuesta Th2 inflamatoria en el pulmón y ganglios linfáticos de drenaje, la facilidad de evaluación de la inflamación mediante el examen histológico de los granulomas que rodean el óvulo, y la posibilidad de almacenamiento a largo plazo del parásito huevos.

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Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Huevos de Schistosoma mansoni son potentes estimuladores del tipo T helper 2 (Th2) la respuesta inmune, característico de la infección por el parásito, el asma y la inflamación alérgica. Este protocolo utiliza S. mansoni de inyección de huevos para generar un recuento de CD4 Th2 citoquinas inducida por la respuesta inflamatoria en el pulmón, caracterizada por la formación de un granuloma pulmonar alrededor del huevo, la eosinofilia y la activación de los macrófagos alternativa.

Con óvulos de<em&gt; Schistosoma mansoni</em&gt; Como un<em&gt; En vivo</em&gt; Modelo de la inflamación pulmonar inducida por helmintos

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

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Immunology and Infection

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

13.7K vistas.  University of Pennsylvania. Huevos de Schistosoma mansoni son potentes estimuladores del tipo T helper 2 (Th2) la respuesta inmune, característico de la infección por el parásito, el asma y la inflamación alérgica. Este protocolo utiliza S. mansoni de inyección de huevos para generar un recuento de CD4 Th2 citoquinas inducida por la respuesta inflamatoria en el pulmón, caracterizada por la formación de un granuloma pulmonar alrededor del huevo, la eosinofilia y la activaci?...

Video: Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Transformación y cercarias In vitro El cultivo de la Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Investigación

Inmunología e Infección

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

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

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

La inducción de la enfermedad de injerto contra huésped y<em&gt; En Vivo</em&gt; T Monitoreo Celular Utilizando un modelo murino MHC de concordancia

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo método de imagen para distinguir la inflamación aguda y crónica

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Una<em&gt; Ex vivo</em&gt; Modelo de un ataque de células T Oligodendrocyte dirigida en Rebanadas cerebral agudo

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Propagación del virus de la influenza en embrionados de pollo Huevos

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Un método optimizado para aislar y expandir Invariante Natural Killer células T de ratón Bazo

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

Sondas de detección óptica de novela bacterias específicas en el tejido de pulmón humano Ex Vivo por láser Confocal Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Evaluación de la citotóxica y efectos inmunomoduladores de sustancias en lonchas de pulmón humano de precisión de corte

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Aislamiento y caracterización de las vesículas extracelulares de adultos Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...