Damos las gracias a Peter Staeheli para proporcionar ratones B6.A2G-MX1 y Semra Özcelik de asesoramiento técnico. Este trabajo fue apoyado por la Fundación Alemana de Investigación a través SFB829 y KN536 / 16, y el Köln fortuna Programa / Facultad de Medicina de la Universidad de Colonia.

Declaración de Ética. La preparación de láminas epidérmicas de los animales sacrificados se lleva a cabo en estricta conformidad con las recomendaciones de la Guía de Landesamt für Natur, Umwelt y Verbraucherschutz, Renania del Norte-Westfalia (Alemania). El estudio fue aprobado por LANUV NRW (Número 8.84-02.05.20.13.018). 1. Preparación de los instrumentos y medios de cultivo <ol><li> Cultivar láminas epidérmicas en medio de Eagle modificado por Dulbecco (DMEM) / alto contenido de glucosa que contiene dipéptido glutamina, 10% de FCS, 100 IU / ml de penicilina, y 100 mg / ml de estreptomicina (denominado en lo sucesivo &quot;DMEM&quot;). </li><li> Para la preparación de láminas epidérmicas, disolver cuidadosamente dispasa II polvo (5 mg / ml) en PBS climatizada (hasta 37 ° C). Se filtra la solución a través de un filtro de 0,22 micras y utilizarlo directamente para el tratamiento. </li><li> Para la preparación de toda epidérmica monta 13, el bloqueo de buff prepararer con 0,5% de leche en polvo, 0,25% de gelatina de piel de pescado de agua fría y 0,5% de Triton X-100 en TBS. Permitir que las sustancias se disuelvan mediante la incubación de la mezcla durante al menos 2 horas a RT en un mezclador giratorio. También hay que preparar 0,2% de Tween 20 en PBS como tampón de lavado. Para la fijación, preparar 3,4% y 4% de formaldehído en PBS. </li><li> Para la inmunotinción de criosecciones, preparar tampón de bloqueo con 5% de suero normal de cabra y 0.2% Tween 20 en PBS. Para la fijación, preparar 0,5% de formaldehído en PBS. </li></ol> 2. Preparación de láminas epidérmicas de la piel murina <ol><li> Preparación de la epidermis de la piel de la espalda del recién nacido para los estudios de infección <ol><li> Coloque un ratón decapitado (1 a 3 días después del nacimiento) en un plato de disección y cortar las extremidades y la cola cerca del torso para permitir la eliminación eficaz de la piel contra el torso completo. Durante la eliminación de las extremidades, trate de mantener los recortes lo más pequeña posible de diámetro. </li><li> Fijar el torso con curva pinzas finas unnd mueva suavemente tijeras de punta roma debajo de la piel de craneal a caudal a lo largo de la espalda para separar la piel del torso. Corte la piel a lo largo de la parte posterior. </li><li> Para el análisis FACS, el aislamiento de los queratinocitos o preparación de ARN, pelar la piel completa del torso utilizando pinzas para fijar el tejido y las tijeras de punta roma para empujar el torso. Para la preparación de cryosections o montajes enteros tomar sólo piezas de la piel de la espalda. </li><li> Picar la piel de la espalda con un bisturí en 1-2 trozos de aproximadamente 10 x 10 mm. Ponga los pedazos en un pocillo de una placa de 6 pocillos con ~ 1 ml de PBS estéril. Asegúrese de que el lado dérmico se enfrenta a la parte inferior. </li><li> Reemplace PBS por 1,5 ml de dispasa II (5 mg / ml de PBS) y se incuba durante cualquiera de 30 minutos a 37 ° C (para montajes enteros) o O / N a 4 ° C. Lavar 3 veces con PBS. Retire suavemente la epidermis como una hoja intacta usando uno pinzas para fijar la dermis subyacente y las otras pinzas para levantar la epidermis. Utilice un binocular para hacer este paso easy. La transferencia de la hoja epidérmica en DMEM con su lado basal hacia la parte inferior. Utilice las hojas de inmediato para experimentos de infección. NOTA: Utilice la preparación de la epidermis de la piel del recién nacido principalmente para el análisis FACS, el aislamiento de los queratinocitos o preparación de ARN. Debido a su fragilidad, la preparación de hojas infectadas de cryosections o montajes enteros es menos adecuado. </li></ol></li><li> Preparación de la epidermis de la piel de la cola de adultos para estudios de infección <ol><li> La eutanasia a los ratones a la edad de 1 a 3 meses por dislocación cervical. Tome la cola en lugar de piel de la espalda ya que los folículos del pelo largo incrustados en la parte posterior dificultan la separación de láminas epidérmicas intactas. Cortar la cola de los ratones sacrificados y raja longitudinalmente con un bisturí. </li><li> Despegar la piel del hueso y cortar con un bisturí en trozos de unos 5 x 5 mm. Ponga hasta 4 piezas en un solo bien y proceder a la incubación en dispasa II como se describe en 2.1.5 NOTA: El uso del tai adultosl piel para preparar cryosections o montajes enteros. Como alternativa a la utilización inmediata, las colas pueden ser almacenados o transportados por aproximadamente 24 horas a 4 ° C sin perder eficiencia de infección significativo. Si sale cruz se mantienen hasta 48 horas, láminas epidérmicas podrían apenas infectadas con HSV-1. </li></ol></li><li> La disociación de la epidermis para el análisis FACS NOTA: La detección de la superficie de la proteína de expresión por análisis FACS requiere técnicas de disociación de células apropiadas que no dañan la superficie celular y la proteína de interés. <ol><li> Incubar láminas epidérmicas recién nacido en una placa de 6 pocillos con la parte basal de 1,5 ml / solución de disociación celular bien recombinante basado en tripsina durante 15 min a TA y durante 15 min a 37 ° C. </li><li> Detener la incubación mediante la adición de 3 ml de DMEM. Disociar queratinocitos utilizando una curva pinzas finas para mover suavemente la epidermis de la placa de 6 pocillos para que los queratinocitos se disuelven. Recoger la suspensión celular y repita este paso 3 vecessiempre utilizando DMEM fresco. NOTA: Este procedimiento de disociación es adecuada para detectar nectin-1 en la superficie de los queratinocitos. </li><li> Alternativamente, se incuban las láminas epidérmicas en solución de disociación celular libre de enzimas (CDS) durante 15 min a RT y 15 min a 37 ° C. Incubar otros 15 minutos a temperatura ambiente para eliminar suavemente los queratinocitos pipeteando CDS arriba y hacia abajo. </li><li> Recoger la suspensión de células y repetir el paso de lavado 3 veces utilizando DMEM fresco. NOTA: Esta disociación es adecuada para detectar la expresión superficial de Hvem. La desventaja de la solución CDS es que menos células se disocian con una solución de disociación celular a base de tripsina recombinante. Además de la superficie expresión, expresión nuclear o citoplásmica tal como la expresión ICP0 se puede analizar por FACS después de la disociación de las láminas epidérmicas infectados con solución de disociación celular a base de tripsina recombinante. </li></ol></li><li> La disociación de la epidermis para aislar queratinocitos o exARN del tracto <ol><li> Ponga láminas epidérmicas recién nacidos en una placa de 6 pocillos con el lado basal hacia el fondo del plato, que contiene 1,5 ml / solución de disociación celular a base de tripsina bien recombinante. Incubar durante 30 min a TA. Añadir 3 ml de DMEM y disociar los queratinocitos utilizando un curvadas unas pinzas finas para mover suavemente la epidermis en el plato. </li><li> Recoger la suspensión celular y repita este paso 3 veces con DMEM fresco. Utilice esta suspensión de células para extraer el ARN o a los queratinocitos murinos cultivo primario. </li></ol></li></ol> 3. La infección de las láminas epidérmicas con HSV-1 <ol><li> Preparar una solución de HSV-1 cepa en peso 17 11 en no menos de 500 l DMEM y reemplazar el medio por la solución de virus en la que las láminas epidérmicas están flotando. Este punto de tiempo se define como tiempo 0 12. Efectuar la infección de una hoja epidérmica en un pocillo de una placa de 24 pocillos a 37 ° C. El cálculo de la concentración de virus se basa en el número estimadode las células en la capa basal epidérmica por hoja (aproximadamente 2 x 10 5 células por 5 x 5 mm hoja). </li><li> Infectar con HSV-1 a aproximadamente 100 unidades formadoras de placa (UFP) / célula y reemplazar el medio que contiene el virus por DMEM a 1 hr pi NOTA: En caso de láminas epidérmicas de la piel del recién nacido, tenga en cuenta que las hojas comienzan a disociarse durante la incubación. </li></ol> 4. Visualización de las células infectadas <ol><li> Epidérmico Whole Mounts 13 <ol><li> Fijar láminas epidérmicas con 3,4% de formaldehído o bien durante 2 horas a temperatura ambiente o O / N a 4 ° C. Lavar dos veces con PBS y bloquear con 0,5% de leche en polvo, 0,25% de gelatina de piel de pescado de agua fría y 0,5% de Triton X-100 en TBS durante 1 h a RT 14. </li><li> Incubar las hojas de O / N con el ratón anti-ICP0 (anticuerpo monoclonal 11060) 15 diluido 1:60 en tampón de bloqueo a temperatura ambiente. Para visualizar el red de filamentos intermedios se incuba simultáneamente con policlonal de conejo anti-queratina ratón 14 (100 ng / ml). </li><li> Lavar 4 a 5 veces con PBS-Tween 20 0,2% durante 4 horas a RT. Incubar O / N con AF488-conjugado anti-IgG de ratón (1 mg / ml), IgG anti-conejo-AF555 conjugado (1 mg / ml), y DAPI (4 &#39;, 6-diamidino-2-fenilindol) (100 ng / ml) en tampón de bloqueo a temperatura ambiente. </li><li> Lavar con PBS-Tween 20 0,2% durante 4 horas a RT. Láminas epidérmicas de montaje con su parte basal en la parte superior de un portaobjetos, incrustar en medio de montaje fluorescente y cubren con cubreobjetos. </li></ol></li><li> Epidérmico criosecciones <ol><li> Láminas epidérmicas adultos fueron fijadas en formaldehído al 4% en PBS durante 20 min a TA y se lavaron 2 veces con PBS. Hojas fijas Insertar en el tejido medio de congelación y congelación en nitrógeno líquido. Cortar 8-10 micras secciones con un criomicrotomo. </li><li> Fijar las secciones de nuevo con 0,5% de formaldehído en PBS durante 10 min a TA, se lava 2 veces con PBS, bloque con 5% de suero normal de cabra y 0.2% Tween 20 en PBS durante 30 min a TA, y luego manchar durante 60 min con mouse anti-ICP0 (anticuerpo monoclonal 11060) 15 diluido 1:60 en tampón de bloqueo, seguido de lavado 3 veces con el tampón de bloqueo. </li><li> A continuación, se incuba con AF488-conjugado anti-IgG de ratón (1 mg / ml) y DAPI (100 ng / ml) en tampón de bloqueo durante 45 min a TA y lavar 3 veces. Insertar secciones en medio de montaje fluorescente y cubrir con cubreobjetos. </li></ol></li></ol>

<ol>
	<li>Koelle, D. M., Corey, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex:+insights+on+pathogenesis+and+possible+vaccines.">Herpes simplex: insights on pathogenesis and possible vaccines.</a> Annu. Rev. Med. 59, 381-395 (2013).</li><li>Roizman, B., Knipe, D. M., Whitley, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+Viruses.">Herpes simplex Viruses.</a> Fields Virology. , 1823-1897 (2013).</li><li>Syrj&#228;nen, S., Mikola, H., Nyk&#228;nen, M., Hukkanen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+establishment+of+lytic+and+nonproductive+infection+by+herpes+simplex+virus+type+1+in+three-dimensional+keratinocyte+culture.">In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture.</a> J. Virol. 70, 6524-6528 (1996).</li><li>Visalli, R. J., Courtney, R. J., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+and+replication+of+herpes+simplex+virus+type+1+in+an+organotypic+epithelial+culture+system.">Infection and replication of herpes simplex virus type 1 in an organotypic epithelial culture system.</a> Virology. 230, 236-243 (1997).</li><li>Hukkanen, V., Mikola, H., Nyk&#228;nen, M., Syrj&#228;nen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+infection+has+two+separate+modes+of+spread+in+three-dimensional+keratinocyte+culture.">Herpes simplex virus type 1 infection has two separate modes of spread in three-dimensional keratinocyte culture.</a> J. Gen. Virol. 80, 2149-2155 (1999).</li><li>Gescher, K., 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=Inhibition+of+viral+adsorption+and+penetration+by+an+aqueous+extract+from+Rhododendron+ferrugineum+L.+as+antiviral+principle+against+herpes+simplex+virus+type-1.">Inhibition of viral adsorption and penetration by an aqueous extract from Rhododendron ferrugineum L. as antiviral principle against herpes simplex virus type-1.</a> Antiviral Res. 82, 408-413 (2010).</li><li>Petermann, P., Haase, I., Knebel-M&#246;rsdorf, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Rac1+and+Cdc42+signaling+during+early+herpes+simplex+virus+type+1+infection+of+keratinocytes.">Impact of Rac1 and Cdc42 signaling during early herpes simplex virus type 1 infection of keratinocytes.</a> J. Virol. 83, 9759-9772 (2009).</li><li>Everett, R. D., Maul, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSV-1+IE+protein+Vmw110+causes+redistribution+of+PML.">HSV-1 IE protein Vmw110 causes redistribution of PML.</a> EMBO J. 13, 5062-5069 (1994).</li><li>Chrostek, A., 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=Rac1+is+crucial+for+hair+follicle+integrity+but+is+not+essential+for+maintenance+of+the+epidermis.">Rac1 is crucial for hair follicle integrity but is not essential for maintenance of the epidermis.</a> Mol. Cell. Biol. 26, 6957-6970 (2006).</li><li>Petermann, P., 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=Entry+mechanisms+of+Herpes+Simplex+Virus+Type+1+into+murine+epidermis:+Involvement+of+nectin-1+and+HVEM+as+cellular+receptors.">Entry mechanisms of Herpes Simplex Virus Type 1 into murine epidermis: Involvement of nectin-1 and HVEM as cellular receptors.</a> J. Virol. 89, 262-274 (2015).</li><li>McGeoch, D. J., 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=The+complete+DNA+sequence+of+the+long+unique+region+in+the+genome+of+herpes+simplex+virus+type+1.">The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1.</a> J. Gen. Virol. 69, 1531-1574 (1988).</li><li>Schelhaas, M., Jansen, M., Haase, I., Knebel-M&#246;rsdorf, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+exhibits+a+tropism+for+basal+entry+in+polarized+epithelial+cells.">Herpes simplex virus type 1 exhibits a tropism for basal entry in polarized epithelial cells.</a> J. Gen. Virol. 84, 2473-2484 (2003).</li><li>Braun, K. M., Niemann, C., Jensen, U. B., Sundberg, J. P., Silva-Vargas, V., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+stem+cell+proliferation+and+lineage+commitment:+visualisation+of+label-retaining+cells+in+wholemounts+of+mouse+epidermis.">Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis.</a> Development. 130, 5241-5255 (2003).</li><li>Rahn, E., Petermann, P., Hsu, M. J., Rixon, F. J., Knebel-M&#246;rsdorf, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+pathways+of+herpes+simplex+virus+type+1+into+human+keratinocytes+are+dynamin-+and+cholesterol-dependent.">Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent.</a> PLoS One. 6, e25464 (2011).</li><li>Everett, R. D., Cross, A., Orr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+truncated+form+of+herpes+simplex+virus+type+1+immediate-early+protein+Vmw110+is+expressed+in+a+cell+type+dependent+manner.">A truncated form of herpes simplex virus type 1 immediate-early protein Vmw110 is expressed in a cell type dependent manner.</a> Virology. 197, 751-756 (1993).</li><li>Horisberger, M. A., Staeheli, P., Haller, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+induces+a+unique+protein+in+mouse+cells+bearing+a+gene+for+resistance+to+influenza+virus.">Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus.</a> Proc. Natl. Acad. Sci. USA. 80, 1910-1914 (1983).</li><li>Christian, A. E., Haynes, M. P., Phillips, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+cyclodextrins+for+manipulating+cellular+cholesterol+content.">Use of cyclodextrins for manipulating cellular cholesterol content.</a> J. Lipid Res. 38, 2264-2272 (1997).</li><li>Ilangumaran, S., Hoessli, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cholesterol+depletion+by+cyclodextrin+on+the+sphingolipid+microdomains+of+the+plasma+membrane.">Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane.</a> Biochem. J. 335, 433-440 (1998).</li><li>Yancey, P. G., 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=Cellular+cholesterol+efflux+mediated+by+cyclodextrins:+Demonstration+of+kinetic+pools+and+mechanism+of+efflux.">Cellular cholesterol efflux mediated by cyclodextrins: Demonstration of kinetic pools and mechanism of efflux.</a> J. Biol. Chem. 271, 16026-16034 (1996).</li><li>Montgomery, R. I., Warner, M. S., Lum, B. J., Spear, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus-1+entry+into+cells+mediated+by+a+novel+member+of+the+TNF/NGF+receptor+family.">Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family.</a> Cell. 87, 427-436 (1996).</li><li>Geraghty, R. J., Krummenacher, C., Cohen, G. H., Eisenberg, R. J., Spear, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+alphaherpesviruses+mediated+by+poliovirus+receptor-related+protein+1+and+poliovirus+receptor.">Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.</a> Science. 280, 1618-1620 (1998).</li><li>Taylor, J. M., Lin, E., Susmarski, N., Yoon, M., Zago, A., Ware, C. F., Pfeffer, K., Miyoshi, J., Takai, Y., Spear, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+entry+receptors+for+herpes+simplex+virus+and+their+roles+in+disease.">Alternative entry receptors for herpes simplex virus and their roles in disease.</a> Cell Host Microbe. 2, 19-28 (2007).</li><li>Barron, M. J., Brookes, S. J., Draper, C. E., Garrod, D., Kirkham, J., Shore, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+adhesion+molecule+nectin-1+is+critical+for+normal+enamel+formation+in+mice.">The cell adhesion molecule nectin-1 is critical for normal enamel formation in mice.</a> Hum. Mol. Genet. 17, 3509-3520 (2008).</li><li>Wang, Y., Subudhi, S. K., Anders, R. A., Lo, J., Sun, Y., Blink, S., Wang, Y., Liu, X., Mink, K., Degrandi, D., Pfeffer, K., Fu, Y. X. <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+herpesvirus+entry+mediator+as+a+negative+regulator+of+T+cell-mediated+responses.">The role of herpesvirus entry mediator as a negative regulator of T cell-mediated responses.</a> J. Clin. Invest. 115, 711-717 (2005).</li></ol>

The authors declare that they have no competing financial interests.

Cuando las hojas epidérmicas de la piel del adulto de ratones C57BL / 6 están infectadas con HSV-1 a aproximadamente 100 UFP / célula, se observa infección en casi todas las células de la capa basal en la epidermis interfolicular mientras que las dosis de virus más bajos se correlacionan con células menos infectados y una más lenta el progreso de la infección. En general, los folículos pilosos muestran un número variable de células infectadas; mientras que la mayoría de los más pequeños queratinocitos que...

El virus del herpes simple (VHS) puede causar una serie de enfermedades en los seres humanos a partir de lesiones mucocutáneas sin complicaciones leves a las infecciones que amenazan la vida. HSV tipo 1 (HSV-1) está predominantemente asociado con las infecciones orofaciales y la encefalitis, mientras que HSV tipo 2 (HSV-2) más probable causa infecciones genitales 1. Si bien hay un avance significativo en la comprensión de cómo el VHS entra en las células en cultivo, se inicia la infección y produce progenie viral, sabemos poco sobre la vía viral invasión (s) en el tejido a nivel celular 2. Para los estudios de la piel HSV o infecciones de las mucosas, ratones, conejos y cobayas se han utilizado como modelos animales. Infección de la piel fue establecida por la inyección intradérmica o por el rascado de la piel en la presencia de virus, y desarrollo de la enfermedad se correlacionó con la producción de virus. Estos métodos ayudaron a entender varios aspectos de la patogénesis de la enfermedad, y se utilizan para evaluar fármacos antivirales. Para estudiar la infección por HSV en el tissuNivel E, se han aplicado modelos de piel humana organotípicos. A medida que la tasa de infección está restringida de estas culturas balsa, sólo un número limitado de estudios de investigación de la infección, propagación viral y los efectos de los componentes antivirales han sido publicados 3-6. Para caracterizar determinantes celulares que juegan un papel en la infección por HSV-1 en el epitelio intacto, hemos establecido un protocolo para estudios ex vivo de infección de la epidermis murinos 7. Se preparó a partir de la piel recién nacidos o de las colas de ratones adultos. Desde HSV-1 no podía infectar muestras de piel completas, que fueron sumergidos en un medio que contiene el virus, separamos la epidermis de la dermis mediante el tratamiento dispasa II. Después de flotación de las hojas epidérmicas en un medio que contiene el virus, las células infectadas se pueden visualizar en la capa basal de la epidermis en las varias horas después de la infección (pi) 7. Para visualizar el inicio de la infección en las células individuales antes de la replicación viral de unand producción de virus, se tiñeron con un anticuerpo contra la proteína de las células infectadas 0 (ICP0), que es una de las primeras proteínas expresadas durante HSV-1 infección. La localización celular de ICP0 pasa a través de fases distintas durante la infección temprana. Mientras ICP0 está presente en focos nucleares durante una etapa temprana de la expresión génica viral, relocalización de ICP0 al citoplasma indica una fase posterior de la infección 8. Se utilizó el ensayo ex vivo infección de láminas epidérmicas de diferentes modelos de ratón para probar el papel potencial de diversos factores celulares durante la infección. Para hacer frente al impacto de Rac1 como un regulador clave de la dinámica de la actina, estamos infectados la epidermis de ratones con una deleción-queratinocitos específica del gen Rac1 9. Este modelo nos permitió estudiar las consecuencias de la deficiencia de Rac1 en la eficiencia de HSV-1 infección en capas de queratinocitos epidérmicos. La comparación con la epidermis infectados de camada de control rerevelado ninguna diferencia significativa, lo que indica que la ausencia de Rac1 no tuvo efecto sobre la iniciación de la infección en la capa basal de la epidermis 7. El uso de otros modelos de ratón nos ha permitido abordar la cual los receptores celulares median entrada en la epidermis. La infección de láminas epidérmicas de cualquiera nectin-1- o ratones deficientes en Hvem con HSV-1 reveló que la entrada viral inicial en el tejido depende fuertemente de la presencia de nectin-1 10. Además, nuestros resultados demuestran que Hvem también puede servir como receptor en la epidermis murinos, aunque menos eficiente que nectin-1 10. Para hacer frente a la distribución espacial de las células infectadas en las capas epidérmicas, visualizamos expresión ICP0 en secciones de tejido y los montajes enteros epidérmico (Figura 1). En cryosections de piel completa, no hay células que expresan ICP0 se detectan (Figura 1). En contraste, cryosections de láminas epidérmicas demuestran citoplásmicaICP0 expresión en la capa basal ya a las 3 horas pi (Figura 1). En épocas posteriores, virus se propague a suprabasal capas se puede visualizar. La distribución espacial de las células infectadas en la capa basal se puede seguir fácilmente en preparaciones completas epidérmico (Figura 1). Tras la infección con HSV-1 en 100 PFU / célula, aproximadamente el 50% de los queratinocitos basales de la epidermis interfolicular muestran expresión ICP0 en 1,5 hr pi En este punto de tiempo de las células infectadas expresan más ICP0 nuclear. La relocalización de ICP0 al citoplasma que indica una etapa posterior de la expresión génica temprana está presente en casi todas las células en 3 pi hr (Figura 1). Estos modos de visualización de las células infectadas ex vivo sobre HSV-1 infección proporcionan un poderoso ensayo para estudiar el efecto de los inhibidores o de componentes celulares mutados eliminados / en la entrada viral y la propagación en el tejido.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/high glucose/GlutaMAX</td>
			<td>Life Technologies</td>
			<td align="right">31966047</td>
			<td>needed for cultivation of epidermal sheets</td>
		</tr>
		<tr>
			<td>dispase II powder</td>
			<td>Roche</td>
			<td align="right">4942078001</td>
			<td>has to be solved in heated PBS</td>
		</tr>
		<tr>
			<td>enzyme-free cell dissociation solution</td>
			<td>Sigma</td>
			<td>C5914</td>
			<td>needed for very gentle dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>TrypLE select cell dissociation solution</td>
			<td>Life Technologies</td>
			<td>12563-029</td>
			<td>needed for dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>chelex 100 resin</td>
			<td>Bio-Rad</td>
			<td>142-2832</td>
			<td>needed for chelation of polyvalent metal ions from the fetal calf serum</td>
		</tr>
		<tr>
			<td>gelatin from cold water fish skin&#160;</td>
			<td>Sigma</td>
			<td>G7765</td>
			<td>needed for minimization of non-specific antibody binding</td>
		</tr>
		<tr>
			<td>Keratin 14 Polyclonal Antibody (AF64) (conc.: 1 mg/ml)</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td>used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis</td>
		</tr>
		<tr>
			<td>Mouse IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 488 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-11029</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 555 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-21429</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (conc.: 0.1 mg/ml)</td>
			<td>Sigma</td>
			<td align="right">36670</td>
			<td>used to counterstain the nucleus</td>
		</tr>
	</tbody>
</table>

ex vivo infection of murine epidermis with herpes simplex virus type 1

Para entrar en su huésped humano, virus herpes simplex tipo 1 (HSV-1) debe superar la barrera de la mucosa superficies, la piel o córnea. HSV-1 Objetivos queratinocitos durante la entrada inicial y establece una infección primaria en el epitelio, que es seguido por la infección latente de las neuronas. Después de la reactivación, los virus pueden ser evidentes en los sitios de mucocutáneas que aparecen como vesículas en la piel o úlceras de la mucosa. Cómo HSV-1 invade la piel o la mucosa y llega a sus receptores es poco conocido. Para investigar la ruta invasión de HSV-1 en el tejido epidérmico en el nivel celular, se estableció un modelo ex vivo de la infección epidermis murino, que representa el sitio de la infección primaria y recurrente en la piel. El ensayo incluye la preparación de la piel murina. La epidermis se separa de la dermis mediante el tratamiento dispasa II. Después de que las láminas epidérmicas que flota en el medio que contiene el virus, el tejido es fija y la infección puede ser visualizado en diversos momentos después de la infección portinción de las células infectadas con un anticuerpo contra el HSV-1 ICP0 proteína inmediata temprana. -Expresión de ICP0 células se puede observar en la capa basal de queratinocitos ya en 1,5 hr después de la infección. Con tiempos más largos de infección, las células infectadas se detectan en las capas suprabasales, lo que indica que la infección no se limita a los queratinocitos basales, pero el virus se propaga a otras capas en el tejido. El uso de láminas epidérmicas de varios modelos de ratón, el protocolo de la infección permite la determinación de la implicación de los componentes celulares que contribuyen a HSV-1 invasión en el tejido. Además, el ensayo es adecuado para poner a prueba inhibidores en el tejido que interfieren con los pasos iniciales de entrada, propagación de célula a célula y la producción de virus. Aquí se describe el protocolo ex vivo infección en detalle y presentamos nuestros resultados utilizando ratones nectin-1- o Hvem deficiente.

El reto del método es para preparar láminas epidérmicas en el que HSV-1 puede penetrar desde la capa basal. El paso crítico es la separación de la epidermis de la dermis mediante el tratamiento dispasa II, que, dependiendo de la cepa de ratón, necesita ser adaptado. La concentración de dispasa II puede variar de 2,5 a 5 mg / ml, y el tiempo de incubación de 20 a 45 min. La tinción de la proteína de filamentos intermedios de queratina 14 permite fácilmente predecir si la capa epidérmica basal puede estar infe...

Watch this Scientific Journal Video about Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1 at JoVE.com

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Ex Vivo Infección de murino Epidermis con virus herpes simplex tipo 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

ex-vivo-infection-of-murine-epidermis-with-herpes-simplex-virus-type-1

Research

JoVE Journal

Immunology and Infection

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

11.1K vistas.  University of Cologne. The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Video: Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Ex vivo de imágenes de células T en los ganglios linfáticos murino Rebanadas con campo amplio y microscopios confocal

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Investigación

Inmunología e Infección

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Un sistema de neuronas cultivo primario para el estudio de la latencia del virus del herpes simple y Reactivación

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Citometría de flujo El aislamiento de células primarias murino tipo II alveolares epiteliales de Estudios funcionales y moleculares

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Trans-vivo hipersensibilidad de tipo retardada Ensayo para Antigen Reglamento Específico

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Evaluación de la eficacia y la toxicidad de los ARN del VIH-1 Orientación de la producción para uso en terapia génica o de drogas

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Ex Vivo Infección de la Mucosa Genital tejido linfoide humano y mujer con Virus de inmunodeficiencia humana 1 y Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Un modelo de cultivo primario de células Bursal de pollo Ex Vivo para estudiar la patogenesia de la enfermedad Bursal infecciosa Virus

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Análisis temporal de la translocación Nuclear-a-citoplásmico de una proteína del Virus 1 del Herpes simple por inmunofluorescencia microscopia Confocal

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Un modelo murino avanzado para esteatohepatitis no alcohólica en asociación con diabetes tipo 2

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Prueba IN Vitro ELISA para evaluar la potencia de la vacuna contra la rabia

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...