Agradecemos a Peter Staeheli para a prestação de ratos B6.A2G-MX1 e Semra Özcelik para assessoria técnica. Este trabalho foi financiado pela Fundação Alemã de Pesquisa através SFB829 e KN536 / 16, eo Köln Fortune Programa / Faculdade de Medicina da Universidade de Colónia.

Declaração de ética. A preparação de folhas epidérmicas de animais sacrificados é realizado em estrita conformidade com as recomendações do Guia de Landesamt für Natur, Umwelt e Verbraucherschutz, Renânia do Norte-Vestfália (Alemanha). O estudo foi aprovado pelo LANUV NRW (Número 8.84-02.05.20.13.018). 1. Preparação de Instrumentos e Meios de Cultura <ol><li> Cultivar folhas epidérmicas em meio de Eagle modificado por Dulbecco (DMEM) / elevado teor de glucose contendo dipéptido glutamina, FCS a 10%, 100 IU / ml de penicilina, e 100 ug / ml de estreptomicina (a seguir referido como &quot;DMEM&quot;). </li><li> Para a preparação de folhas epidérmicas, dissolver cuidadosamente dispase II em pó (5 mg / ml) em PBS aquecido (até 37 ° C). Filtrar a solução através de um filtro de 0,22 um e usá-lo directamente para tratamento. </li><li> Para a preparação de toda monta epidérmica 13, preparar o bloqueio lustreer com 0,5% de leite em pó, 0,25% de gelatina de pele de peixe de água fria e 0,5% de Triton X-100 em TBS. Permitir substâncias para dissolver por incubação da mistura durante pelo menos 2 horas à temperatura ambiente num misturador rotativo. Também preparar 0,2% de Tween 20 em PBS como tampão de lavagem. Para a fixação, preparar 3,4% e 4% de formaldeído em PBS. </li><li> Para a imunocoloração de criocortes, preparar tampão de bloqueio com 5% de soro normal de cabra e 0,2% de Tween 20 em PBS. Para a fixação, preparar 0,5% de formaldeído em PBS. </li></ol> 2. Preparação de lâminas de epiderme da pele Murino <ol><li> Preparação da epiderme da pele recém-nascido de volta para os estudos de infecção <ol><li> Coloque um rato decapitados (1 a 3 dias após o nascimento) num prato de dissecação e cortar as extremidades e a cauda perto do tronco para permitir a remoção eficiente da pele do tronco completa. Durante a remoção das extremidades, para tentar manter os cortes tão pequena quanto possível de diâmetro. </li><li> Corrigir o tronco curvado com uma pinça finand mover suavemente tesoura ponta romba debaixo da pele do crânio de caudal ao longo das costas de separar a pele do tronco. Slit a pele ao longo das costas. </li><li> Para a análise FACS, o isolamento dos queratinócitos ou preparação de RNA, retire a pele completa do tronco, utilizando uma pinça para fixar o tecido e as tesouras ponta romba para empurrar o torso. Para a preparação de criosecções ou peças inteiras levar apenas pedaços de pele para trás. </li><li> Pique a pele para trás com um bisturi em 1-2 pedaços de aproximadamente 10 x 10 mm. Coloque as peças em um poço de uma placa de 6 poços com ~ 1 ml de PBS estéril. Certifique-se de que o lado dérmico virado para o fundo. </li><li> Substituir 1,5 ml de PBS por dispase II (5 mg / ml PBS) e incubar durante 30 min quer a 37 ° C (para as peças inteiras) ou O / N a 4 ° C. Lavar 3 vezes com PBS. Remova cuidadosamente a epiderme como uma folha intacta usando uma pinça para fixar a derme subjacente e os outros pinça para levantar a epiderme. Use um binóculo para dar este passo easy. Transferir a folha epidérmica em DMEM com o lado basal para o fundo. Use as folhas imediatamente para experimentos de infecção. NOTA: Utilize a preparação da epiderme da pele recém-nascido, principalmente para análise FACS, o isolamento dos queratinócitos ou preparação de RNA. Por causa da sua fragilidade, a preparação de folhas infectadas para criossecções ou peças inteiras é menos adequado. </li></ol></li><li> Preparação da epiderme de pele da cauda de adulto para estudos de infecção <ol><li> Eutanásia dos ratos com a idade de 1 a 3 meses, por deslocamento cervical. Tome cauda em vez de pele para trás uma vez que os folículos pilosos longo embutidos na parte traseira dificultar a separação de folhas epidérmicas intactas. Corte a cauda de camundongos sacrificados e cortou-o no sentido do comprimento com um bisturi. </li><li> Retire a pele do osso e pique-o com um bisturi em pedaços de cerca de 5 x 5 mm. Coloque até 4 peças em um bem e prosseguir com a incubação em dispase II como descrito em 2.1.5 NOTA: Use tai adultol pele para preparar criocortes ou peças inteiras. Como alternativa à utilização imediata, caudas pode ser armazenado ou transportado para, aproximadamente, 24 h a 4 ° C sem perda significativa de eficiência de infecção. Se caudas são mantidos até 48 horas, folhas epidérmicas poderia ser mal infectados com HSV-1. </li></ol></li><li> A dissociação da epiderme, para a análise por FACS Nota: A detecção da expressão da proteína de superfície por análise de FACS requer técnicas de dissociação de células adequadas que não prejudiquem a superfície da célula e a proteína de interesse. <ol><li> Incubar lâminas de epiderme recém-nascidos em uma placa de 6 poços, com o lado basal em 1,5 mL / poço recombinante solução de dissociação de células de tripsina à base durante 15 min à TA e durante 15 min a 37 ° C. </li><li> Parar a incubação pela adição de 3 ml de DMEM. Dissociar queratinócitos utilizando uma pinça fina curvadas para mover suavemente a epiderme na placa de 6 poços de modo a que os queratinócitos se dissolvem. Recolha a suspensão de células e repetir este passo 3 vezessempre usando DMEM fresco. NOTA: Este procedimento de dissociação é adequado para detectar Nectin-1 na superfície de queratinócitos. </li><li> Alternativamente, as folhas epidérmicas incubar em solução de dissociação de células sem enzima (CDS) durante 15 min à temperatura ambiente e 15 min a 37 ° C. Incubar mais 15 minutos à temperatura ambiente para limpar suavemente os queratinócitos pipetando CDS cima e para baixo. </li><li> Recolha a suspensão de células e repita o passo de lavagem 3 vezes com DMEM fresco. NOTA: Esta dissociação é adequado para detectar a expressão de superfície de HVEM. A desvantagem da solução CDS é que menos células são dissociadas com uma solução de dissociação de células recombinante baseado em tripsina. Além disso a superfície de expressão, expressão citoplasmática ou nuclear, tais como expressão ICP0 podem ser analisados ​​por SCAF após a dissociação das folhas epidérmicas infectadas com o recombinante solução de dissociação de células de tripsina à base. </li></ol></li><li> A dissociação da epiderme para isolar queratinócitos ou exRNA trato <ol><li> Coloque folhas epidérmicas recém-nascidos em uma placa de 6 poços, com o lado basal para o fundo do prato que contém 1,5 ml / poço solução de dissociação de células recombinante baseado em tripsina. Incubar durante 30 min à TA. Adicionar 3 ml de DMEM e dissociar queratinócitos utilizando uma pinça fina curvadas para mover suavemente a epiderme do prato. </li><li> Recolha a suspensão de células e repetir este passo 3 vezes usando fresco DMEM. Utilizar esta suspensão de células para extrair ARN ou aos queratinócitos murinos cultura primária. </li></ol></li></ol> 3. A infecção das lâminas epidérmicas com HSV-1 <ol><li> Prepara-se uma solução de HSV-1 estirpe 17 11 em peso, em não menos de 500 ul de DMEM e substituir o meio por a solução de vírus em que as folhas epidérmicas são flutuante. Este ponto de tempo é definido como o tempo 0 12. Execute infecção de uma folha epidérmica em um poço de uma placa de 24 poços a 37 ° C. O cálculo do título do vírus é baseada no número estimadode células na camada basal da epiderme por folha (de aproximadamente 2 x 10 5 células por folha de 5 x 5 mm). </li><li> Infect com o HSV-1 em cerca de 100 unidades formadoras de placas (PFU) / célula e substituir o meio contendo vírus por DMEM a 1 hr pi NOTA: No caso de folhas epidérmicas de pele recém-nascido, tenha em mente que as folhas começam a dissociar durante a incubação. </li></ol> 4. Visualização de células infectadas <ol><li> Epidérmica Whole Mounts 13 <ol><li> Fix folhas epidérmicas com 3,4% de formaldeído, quer durante 2 h à TA ou O / N a 4 ° C. Lavar duas vezes com PBS e bloquear com 0,5% de leite em pó, 0,25% de gelatina de pele de peixe de água fria e 0,5% de Triton X-100 em TBS durante 1 hora à TA 14. </li><li> Incubar as folhas de O / N com murganho anti-ICP0 (anticorpo monoclonal 11060) 15 diluída 1:60 em tampão de bloqueio à temperatura ambiente. Para a visualização da rede intermédia de filamentos simultaneamente incubar com anticorpo policlonal de coelho anti-rato queratina 14 (100 ng / mL). </li><li> Lavar 4 a 5 vezes com PBS-Tween 20 a 0,2% durante 4 horas à temperatura ambiente. Incubar O / N com AF488-conjugado anti-IgG de ratinho (1 mg / ml), AF555-conjugado anti-IgG de coelho (1 ug / ml), e DAPI (4 &#39;, 6-diamidino-2-fenilindol) (100 ng / ml) em tampão de bloqueio à temperatura ambiente. </li><li> Lavar com PBS-0,2% de Tween 20, durante 4 h à TA. Folhas epidérmicas Monte com o seu lado basal em cima de uma lâmina de amostra, incorporar no meio de montagem fluorescente e cobrir com lamelas. </li></ol></li><li> Epidérmica Cortes congelados <ol><li> Folhas epidérmicas mediana foram fixadas em formaldeído a 4% em PBS durante 20 min à temperatura ambiente e lavou-se 2 vezes com PBS. Folhas fixas Incorporar em tecido de congelação médio e congelamento em nitrogênio líquido. Corte 8-10 mm seções com um cryomicrotome. </li><li> Fixar as secções mais uma vez com 0,5% de formaldeído em PBS durante 10 min à temperatura ambiente, lava-se 2 vezes com PBS, bloco com 5% de soro normal de cabra e 0,2% de Tween 20 em PBS durante 30 min à temperatura ambiente, e em seguida corar durante 60 min com mouse anti-ICP0 (anticorpo monoclonal 11060) 15 diluída 1:60 em tampão de bloqueio, seguido por lavagem 3 vezes com tampão de bloqueio. </li><li> Em seguida incubar com anti-IgG de ratinho conjugado com AF488 (1 ug / ml) e DAPI (100 ng / ml) em tampão de bloqueio durante 45 minutos à temperatura ambiente e lava-se 3 vezes. Seções incorporar em meio de montagem fluorescente e cobrir com lamelas. </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.

Quando as folhas epidérmicas da pele de um adulto de ratinhos C57BL / 6 são infectadas com HSV-1 em cerca de 100 PFU / célula, observa-se infecção em quase todas as células da camada basal da epiderme interfoliculares enquanto que doses de vírus mais baixos correlacionam-se com as células menos infectados e um mais lento o progresso da infecção. Em geral, os folículos de cabelo mostram um número variável de células infectadas; enquanto a maioria dos queratinócitos bastante pequenas que revestem os folícu...

Herpes simplex vírus (HSV) pode causar uma série de doenças em humanos de lesões mucocutâneas sem complicações leves a infecções potencialmente fatais. HSV tipo 1 (HSV-1) é predominantemente associada com infecções orofacial e encefalites, enquanto o HSV tipo 2 (HSV-2), mais provavelmente provoca infecções genitais 1. Embora haja progressos significativos na compreensão de como HSV entra nas células em cultura, inicia a infecção e produz progênie viral, sabemos pouco sobre a via de invasão viral (s) no tecido no nível celular 2. Para os estudos de HSV pele ou infecções das mucosas, ratinhos, coelhos e porquinhos da índia foram utilizados como modelos animais. Infecção da pele foi estabelecida por injecção intradérmica ou por raspagem da pele, na presença de vírus, e o desenvolvimento da doença foi correlacionada com a produção de vírus. Estes métodos ajudou a compreender vários aspectos da patogénese da doença, e são usados ​​para avaliar as drogas antivirais. Para estudar a infecção por HSV na tissunível e, organotípicas modelos de pele humana foram aplicados. À medida que a taxa de infecção é restrita nestas culturas jangadas, apenas um número limitado de estudos que investigam a infecção, propagação virai e os efeitos dos componentes antivirais foram publicados 3-6. A fim de caracterizar determinantes celulares que desempenham um papel durante a infecção por HSV-1 no epitélio intacto, foi estabelecido um protocolo ex vivo para estudos de infecção de epiderme 7 de murídeo. A pele foi preparada a partir de recém-nascidos ou da cauda de camundongos adultos. Uma vez que o HSV-1 não pode infectar amostras de pele completos, que foram submersas em meio contendo vírus, que separada da epiderme da derme por tratamento com dispase II. Após flutuação das folhas epidérmicas em meio contendo vírus, as células infectadas podem ser visualizados na camada basal da epiderme vários momentos após a infecção em (PI) 7. Para visualizar a iniciação da infecção em células individuais antes da replicação de um vírusND produção de vírus, é corada com um anticorpo contra a proteína de célula infectada 0 (ICP0), que é uma das primeiras proteínas expressas durante a infecção por HSV-1. A localização celular de ICP0 passa por fases distintas durante a infecção inicial. Enquanto ICP0 está presente em focos nucleares durante a fase inicial da expressão de genes virais, relocalização de ICP0 para o citoplasma indica uma fase posterior da infecção 8. Utilizou-se o ensaio ex vivo infecção de folhas epidérmicas a partir de diferentes modelos de ratinho para testar o papel potencial de vários factores celulares durante a infecção. Para lidar com o impacto da Rac1 como um regulador chave da dinâmica de actina, que infectou os epiderme de camundongos portadores de deleção específicas de queratinócitos do gene rac1 9. Este modelo permitiu-nos estudar as consequências da deficiência Rac1 na eficiência de infecção por HSV-1 em camadas epidérmicas de queratinócitos. A comparação com epiderme infectadas da mesma ninhada de controle revealed nenhuma diferença significativa, indicando que a falta de Rac1 não teve nenhum efeito sobre o início da infecção na camada basal da epiderme 7. O uso de mais modelos de ratos nos permitiu abordar quais os receptores celulares mediar entrada para a epiderme. Infectar folhas epidérmicas a partir de qualquer Nectin-1- ou murganhos deficientes em HVEM com o HSV-1 revelou que a entrada viral inicial no tecido depende fortemente da presença de Nectin-1 10. Além disso, os nossos resultados demonstram que o HVEM pode também servir como receptor de epiderme em murinos, embora de forma menos eficiente do que Nectin-1 10. Para abordar a distribuição espacial de células infectadas nas camadas epidérmicas, que ICP0 visualizar a expressão em secções de tecido e as peças inteiras da epiderme (Figura 1). Em criocortes de pele completa, não há células que expressam ICP0 são detectados (Figura 1). Em contraste, criocortes de folhas epidérmicas demonstrar citoplasmáticaICP0 expressão na camada basal às 3 horas já pi (Figura 1). Em tempos mais recentes, a propagação viral camadas suprabasais pode ser visualizado. A distribuição espacial de células infectadas na camada basal pode ser facilmente seguido de pele epidérmicos em montagens inteiras (Figura 1). Após a infecção com HSV-1 a 100 UFP / célula, aproximadamente 50% dos queratinócitos basais interfoliculares na epiderme mostram expressão ICP0 em 1,5 h pi Neste ponto de tempo a maioria das células infectadas expressam ICP0 nuclear. A relocalização da ICP0 para o citoplasma, indicando uma fase posterior de expressão do gene precoce está presente em quase todas as células a 3 hr pi (Figura 1). Estes modos de visualizar as células infectadas ex vivo após a infecção por HSV-1 proporcionam um ensaio poderoso para estudar o efeito de inibidores ou de excluídos / componentes celulares mutadas para a entrada virai e disseminação no tecido.

<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 introduzir o seu hospedeiro humano, vírus herpes simplex tipo 1 (HSV-1) tem de ultrapassar a barreira de superfícies mucosas, pele e córnea. HSV-1 alvos os queratinócitos durante a entrada inicial e estabelece uma infecção primária no epitélio, que é seguida por uma infecção latente de neurónios. Após a reativação, o vírus pode tornar-se evidente em locais mucocutâneas que aparecem como vesículas ou úlceras cutâneas mucosas. Como o HSV-1 invade pele ou mucosa e atinge os seus receptores é mal compreendida. Para investigar a via de invasão de HSV-1 no tecido epidérmico, ao nível celular, estabelecemos um modelo ex vivo de infecção epiderme murino, que representa o local da infecção primária e recorrente da pele. O ensaio inclui a preparação de pele de murino. A epiderme é separada da derme por tratamento com dispase II. Depois de as folhas epidérmicas flutuante em meio contendo vírus, o tecido é fixa e a infecção pode ser visualizado em vários momentos após a infecção pelacoloração de células infectadas com um anticorpo contra o HSV-1 de proteína precoce imediata ICP0. ICP0 células que expressam podem ser observados na camada basal dos queratinócitos já em 1,5 h após a infecção. Com vezes mais longo da infecção, as células infectadas são detectados nas camadas suprabasais, indicando que a infecção não se limita aos queratinócitos basais, mas o vírus propaga-se para outras camadas no tecido. Utilizando folhas epidérmicas de vários modelos de ratinho, o protocolo de infecção permite a determinação do envolvimento de componentes celulares que contribuem para o HSV-1 no tecido invasão. Além disso, o ensaio é adequado para testar inibidores em tecidos que interferem com os passos iniciais de entrada, disseminação célula-a-célula e produção de vírus. Aqui, descrevemos o protocolo ex vivo infecção em detalhes e apresentar os nossos resultados utilizando camundongos Nectin-1- ou HVEM deficiente.

O desafio do método é o de preparar as folhas epidérmicas em que o HSV-1 pode penetrar a partir da camada basal. O passo crítico é a separação da epiderme da derme por tratamento com dispase II, que, dependendo da estirpe de ratinho, deve ser adaptado. A concentração de dispase II pode variar de 2.5 a 5 mg / ml, e o tempo de incubação de 20 a 45 min. A coloração da proteína de filamento intermediário queratina 14 permite prever prontamente se a camada basal da epiderme pode ser infectado ou se irá mostra...

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 Infecção de Murino Epiderme com vírus 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 visualizações.  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.

Vídeo: 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 imagens de células T em fatias Node murino linfáticos com Widefield e microscópios confocal

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

Imunologia e Infecção

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.

Um sistema de cultura primária Neuron para o Estudo da Herpes Simplex Virus latência e reativação

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.

Isolamento de citometria de fluxo de células primárias Murino Tipo II epiteliais alveolares de Estudos funcionais e Molecular

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 Atraso Tipo Ensaio Hipersensibilidade para o Antigen Regulamento 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.

Avaliação da eficácia e toxicidade de RNAs Segmentação HIV-1 Produção para uso em Gene ou Quimioterapia

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 Infecção da Mucosa Genital humano tecido linfoide e fêmea com vírus de imunodeficiência humana 1 e 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.

Um modelo de cultura primária de células bolsas serosas Ex Vivo frango para estudar a patogênese de doenças infecciosas bolsas serosas vírus

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.

Análise temporal de translocação Nuclear e citoplasmática de uma Herpes Simplex vírus 1 proteína pela microscopia Confocal imunofluorescência

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.

Um modelo murino avançado para esteatohepatite não alcoólica em associação com 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.

Teste in vitro ELISA para avaliar a potência da vacina antirrábica

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