Os autores gostariam de agradecer ao Dr. James (Zhijian) Chen (UT Southwestern, Biologia Molecular) para fornecimento de reagentes essenciais, incluindo os Mavs - / - ratos, e Ren Dr. Sun (Universidade da Califórnia-Los Angeles, Farmacologia e Medicina Molecular ) para proporcionar o cromossoma bacteriano artificial de γHV68 para este estudo.

1. Infecção mouse com γHV68 <ol><li> Ratinhos ninhada de seis a oito semanas de idade, género, combinados, (8-12 ratos / grupo) foram utilizados para a infecção viral. Permitir ratos para se aclimatar ao longo de quatro dias inteiros (96 horas) após a expedição. </li><li> Passos de protocolo utilizando vírus deve ser realizado numa câmara de segurança biológica de nível 2 (BSL2) usando o padrão de BSL2 precauções. </li><li> Prepare suspensão viral (40 a 1 x 10 5 unidades formadoras de placas [PFU]) de γHV68 em 30 ul de PBS estéril, por ratinho imediatamente antes da experiência. Manter a suspensão viral em gelo. </li><li> Preparar a solução de cetamina / xilazina (1,5 mg de cetamina e 0,15 mg xylazine/20 g de peso corporal, com 100 ul / ratinho) para sedação mouse. Certifique-se de que os ratos foram sedados através da realização de um aperto do dedo do pé. </li><li> Sedar ratos com 1,5 mg de cetamina e xilazina 0,15 mg por 20 mg de peso corporal (100 ul / ratinho) por injecção intra-peritoneal. </li><li> Entregar suspensão viral (30 ^ l / ratinho) por via intranasal, em uma, gota a gotaforma, para uma das narinas dos ratos sedados. </li><li> Lay ratos, por um lado, durante 5 - 10 min para facilitar a entrega nas vias aéreas do vírus no pulmão. </li><li> Camundongos lugar de volta em camundongos gaiola e monitor até que tenham recuperado totalmente de sedação. </li><li> Em vários dias pós-infecção, sacrificar animais por asfixia CO 2 e colheita, os pulmões depois de garantir a morte / perda de consciência profunda. Pulmões colocar em estéreis de 1,5 ml com tampa de rosca tubos contendo 500 ul de 1,0 milímetros de zircônia / sílica contas. Manter os tubos em gelo. Armazenar as amostras a -80 ° C se não for de prosseguir para o próximo passo no mesmo dia. O baço ou de tecido do fígado pode ser recolhido no momento para a análise de latência viral, dependendo do período experimental. </li><li> Adicionar 1 ml de frio DMEM isento de soro para dentro do tubo e homogeneizar os pulmões por pérola em parada 30 segundos. Relaxar tubos em gelo durante pelo menos 1 min. Repita este processo uma vez. </li><li> Centrifugar os lisados ​​de pulmão a 16.000 rcf a 4 ° C durante 1 min e usar supernatant para determinar título viral por um teste de placas usando uma monocamada NIH3T3 ou BHK21 (ver secção 5 para mais detalhes). </li></ol> 2. Determinar γHV68 cinética de várias etapas de crescimento de fibroblastos embrionários de ratinho <ol><li> Crescer fibroblastos do tipo selvagem embrionárias de rato (MEFs) e os deficientes em um gene hospedeiro para sub-confluentes de densidade (aproximadamente 80%), antes de células em placas. </li><li> Dividir MEFs em placa de 24 poços a 10.000 células / poço para um baixo multipilicity de infecção (MOI) no dia anterior à infecção. As experiências são normalmente realizados em triplicata e a uma MOI entre 0,001 - 0,05 é normalmente utilizado. </li><li> Prepare γHV68 suspensão contendo a quantidade desejada de virus (0,5 ml por cavidade). </li><li> Remover médio e cobrir MEFs com 0,5 ml de suspensão por cavidade γHV68. </li><li> Incubar a placa de cultura de tecidos incubadora, rocha a cada 30 min, e permitir a incubação prosseguir durante 2 h. </li><li> Remova a suspensão viral, e cobrir as células com 0,5 ml c frescoomplete meio DMEM contendo 8% de soro fetal bovino. </li><li> Em vários dias após a infecção, a colheita do meio e as células em tubos de centrífuga estéreis, 1,5 ml. Congelar imediatamente os tubos a -80 ° C. </li><li> Libertação de γHV68 MEFs por congelamento a -80 ° C, descongelação a 37 ° C Banho de água e centrifugação. Três ciclos de congelação e descongelação é geralmente aplicado às amostras. </li><li> Determine título viral por um teste de placas usando uma monocamada NIH3T3 ou BHK21 (ver secção 5 para mais detalhes). </li><li> Leia título viral e enredo γHV68 curva de crescimento multi-passo em MEFs. </li></ol> 3. Dissecção molecular de γHV68 replicação lítica em fibroblastos de rato embrionárias <ol><li> Realize infecção viral como descrito nos passos 2.1 a 2.6 do capítulo 2. </li><li> Em vários dias após a infecção, desprezar o sobrenadante. Enxaguar as células com PBS frio e células Tripsinizar. Pellet células por centrifugação a 1000 rcf em temperatura ambiente por 1 min. Descartar o sobrenadante ecélulas armazenar a -80 ° C. </li><li> Extrair DNA total (hospedeiro e do genoma viral) e o ARN total de acordo com métodos previamente publicados 5,6. </li><li> Realizar PCR em tempo real utilizando o ADN total e iniciadores específicos para transcrições virais líticas, tais como RTA (ORF50), ORF57 e ORF60. Determinar a quantidade relativa de genoma γHV68 intracelular em referência a um gene housekeeping hospedeiro (por exemplo, β-actina). </li><li> Para remover a contaminação de ADN genómico, o tratamento com RNase-free DNase é crítica para a preparação de cDNA de RNA total e de (dT) iniciador oligo 11-19. Consulte a referência 5 para obter mais detalhes sobre a extração de RNA incluindo o tratamento com RNase DNase e RT-PCR. Realizar PCR em tempo real utilizando iniciadores de ADNc e como acima descrito para determinar a quantidade relativa de transcritos virais em referência a esse gene housekeeping de acolhimento. </li></ol> 4. Gerando recombinante usando γHV68 cromossomo bacteriano artificial O metodod descrito aqui é utilizada para introduzir mutações num gene γHV68 que está envolvido na interacção hospedeiro-vírus. <ol><li> Preparar um fragmento de DNA (cerca de 1,5 kb) de sequência de tipo selvagem ou uma sequência de transporte mutações desejadas na região central por PCR. </li><li> Prepare cromossoma artificial bacteriano (BAC), que contém uma inserção de transposão 7 em torno do local da mutação, que inactiva o gene especificamente gene de interesse, por midi escala de purificação (OriGene). Loja BAC DNA a 4 ° C (para evitar o congelamento / descongelamento de BAC de ADN). </li><li> Transfectar ADN BAC e produto de PCR contendo as mutações desejadas do gene de interesse em células (por exemplo, células NIH3T3 ou BHK21), que são altamente permissivo para a replicação γHV68 lítica, com Lipofectamine 2000 (Invitrogen). </li><li> Manter divisão de células até que o efeito citopático (CPE), mostra-se na monocamada. Recolher-vírus contendo o sobrenadante e, se necessário, amplificar o vírus em células NIH3T3 ou BHK21. </li><li> Infectar células NIH3T3 comrecombinante do vírus por centrifugação a 1800 rpm, 30 ° C durante 30 min. </li><li> Colheita γHV68 infectadas células NIH3T3 e preparar circularizado genoma viral utilizando o protocolo de Hirt de 8,9. </li><li> Transformar células de Electro-MAX DH10B competentes (Invitrogen) por electroporao e tela de colónias que são a resistência ao cloranfenicol (Cm), mas sensível à canamicina (Kan) (Cm-resistente gene está no backbone BAC, enquanto Kan-resistente gene está no transposon inserção). </li><li> O crescimento de colônias Cm R Kan S em meio contendo chlorophenicol e preparar BAC DNA com a purificação ou mini-midi-escala (OriGene). </li><li> Verificar a mutação desejada no gene alvo por PCR, utilizando iniciadores específicos para as regiões de flanco do local de mutação e a sequenciação. </li><li> Confirmar nenhum rearranjo cromossómico em BAC por digestão com enzimas de restrição seleccionadas e pulso campo de eletroforese em gel. </li><li> Transfectar BAC recombinante em células NIH 3T3 ou BHK21 com Lipofectamine2000 (Invitrogen) para preparar γHV68 recombinante. </li><li> Mantenha passagens em células até CPE mostra-se na monocamada. Recolher-vírus contendo o sobrenadante e amplificar γHV68 recombinante em células de NIH3T3 ou BHK21. </li><li> Determinar titulação do vírus recombinante viral e caracterizar as propriedades de crescimento ex vivo e in vivo, tal como descrito nas secções 1 e 2. </li></ol> 5. Determinar título virai por um ensaio de placas fágicas <ol><li> Crescer células NIH3T3 para sub-confluentes de densidade (aproximadamente 80%), antes de células em placas. </li><li> Dividir NIH3T3 em placas de 24 poços a 20.000 células / poço, no dia antes da infecção. </li><li> Preparar 10 vezes em série os sobrenadantes virais diluídas com meio DMEM contendo 8% de soro de vitelo recém-nascido (NCS). </li><li> Remover médio e cobrir células NIH3T3 com 0,5 ml de suspensão γHV68. </li><li> Incubar a placa de cultura de tecidos incubadora, agitar a cada 30 min, e permitir a incubação prosseguirdurante 2 h. </li><li> Remova a suspensão viral e células de cobertura com 0,5 ml meio DMEM contendo 2% de NCS e metilcelulose a 0,75% (Sigma). </li><li> Incubar a placa de cultura de tecidos incubadora. Contar as placas no dia 6 pós-infecção. A coloração da monocamada, por exemplo, com violeta de cristal, pode facilitar a contagem de placa. </li><li> Calcula-se o título viral nos lisados ​​de tecido não diluídas ou lisados ​​celulares utilizando a seguinte fórmula: Titer (PFU / ml) = D x N x 1000 ul / ml ÷ V ul. N, a média do número de placas a uma diluição adequada, D, diluições (por exemplo, 5, 10, 100 .....) V (pi), volume de sobrenadante diluído em série por poço. </li></ol> 6. Resultados representativos: Três figuras representativas são mostrados aqui, incluindo γHV68 replicação lítica no pulmão do tipo selvagem e Mavs - / - 10 rato, γHV68 fenótipos replicação líticas em fibroblastos de rato embrionárias (MEF), e recombinformiga γHV68 realização mutações nos sítios de fosforilação que são modulados pela IKKβ MAV-dependente. Estes três experimentos comprobatórios constituem um esquema para definir as funções dos componentes inatos imunes em γHV68 replicação lítica in vivo e ex vivo. <img alt="Figura 1" src="/files/ftp_upload/3140/3140fig1.jpg" /> Figura 1 γHV68 cargas nos pulmões dos Mavs + / + e - Mavs. / - Ratos. A) duas vias de sinalização principal inata jusante do Mavs. Os relés de adaptador Mavs molécula de sinalização citosólicas de RIG-I-like receptors para activar NFkB e factores de interferão reguladoras (IRFs) que, por sua vez, se-regular a expressão de genes de citoquinas pró-inflamatórias e interferões. B) Mavs + / + e - / - Mavs ratinhos foram infectados com 40 PFU intranasalmente γHV68 e cargas virais no pulmão em pontos de tempo indicados foram deter<html> minada por um ensaio de placas utilizando células NIH3T3 monocamada. Cada símbolo representa um rato. <img alt="Figura 2" src="/files/ftp_upload/3140/3140fig2.jpg" /> Figura 2. ΓHV68 cinética de replicação lítica em fibroblastos de rato embrionários (MEFs). A replicação litica de γHV68 em Mavs + / + e - Mavs / - MEFs foi avaliada por curvas de várias etapas de crescimento (A) e quantitativa de PCR em tempo real (B). Para ambos os experimentos, igual número de MEFs e quantidade de γHV68 foram utilizados para a infecção viral, a uma multiplicidade de infecção (MOI) de 0,01. (A) As células e os sobrenadantes foram colhidos em intervalos de tempo indicados e sujeitos a um ensaio de placas para determinar os títulos virais. (B) de ARN total foi extraído a partir de γHV68 infectado MEFs e analisadas por quantitativo em tempo real, PCR com primers específicos para determinados transcritos líticas (RTA, ORF57 e ORF60). ig3.jpg &quot;/&gt; Figura 3. As cinéticas de replicação litica de recombinantes γHV68 transportando mutações dentro do domínio de transactivação RTA que abolirem fosforilação por IKKγ. (A) curvas de várias etapas de crescimento de vírus de tipo selvagem recombinante (γHV68.wt) e vírus mutante (γHV68.mut) em Mavs + / + e - Mavs / - células MEFs (MOI = 0,01). MEFs foram infectadas com γHV68 a um MOI de 0,01. As células e os sobrenadantes foram colhidos em intervalos de tempo indicados e os títulos virais foram determinados por um ensaio de placas fágicas utilizando NIH 3T3 monocamada. (B) mRNA γHV68 nível RTA γHV68 em células NIH3T3 infectadas (MOI = 0,01). A 30 h após a infecção, o RNA total foi extraído a partir de células infectadas com γHV68 NIH 3T3 e analisadas por quantitativa PCR em tempo real.

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Em resposta a uma infecção viral, os MAV-dependentes imunes inatas vias de sinalização são activados para promover a produção de citocinas inflamatórias antivirais 10-14. Usando γHV68 murina como um modelo para o vírus herpes humano oncogênico sarcoma de Kaposi associado e vírus Epstein-Barr 3,4, descobrimos que γHV68 usurpa a via de MAV-IKKβ a promover a replicação viral via lítica de activação da transcrição 5. Empregando MEFs geneticamente modificado e técnicas d...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668-019 </td></tr><tr><td> Electro-MAX células DH10B competentes </td><td> Invitrogen </td><td> 18290-015 </td></tr><tr><td> Metilcelulose </td><td> Sigma </td><td> M0512 </td></tr><tr><td> POWERPREP HP Sistema Miniprep plasmídeo </td><td> OriGene </td><td> NP100004 </td></tr><tr><td> POWERPREP HP Sistema Midiprep plasmídeo </td><td> OriGene </td><td> NP100006 </td></tr></tbody></table>

dissecting host-virus interaction in lytic replication of a model herpesvirus

Em resposta a uma infecção viral, um hospedeiro desenvolve várias respostas de defesa, tais como a activação imune inata vias de sinalização que conduzem à produção de citocina 1,2 antiviral. A fim de colonizar o hospedeiro, os vírus são obrigatório para evadir respostas do hospedeiro anti-virais e manipular as vias de sinalização. Desvendando a interação hospedeiro-vírus vai lançar luz sobre o desenvolvimento de novas estratégias terapêuticas contra a infecção viral. Murino γHV68 está intimamente relacionado com o herpesvírus humano oncogênico sarcoma de Kaposi associado e Epsten-Barr 3,4. γHV68 infecção em ratos de laboratório fornece um modelo animal tratável pequeno para examinar todo o curso da resposta do hospedeiro e infecção viral in vivo, que não estão disponíveis para o herpesvírus humanos. Neste protocolo, apresentamos um painel de métodos para caracterização fenotípica e dissecção molecular de componentes do hospedeiro sinalização em γHV68 replic líticoation in vivo e ex vivo. A disponibilidade das estirpes de rato geneticamente modificados permite a interrogação dos papéis das vias de sinalização do hospedeiro durante a infecção aguda γHV68 in vivo. Além disso, os fibroblastos de rato embrionários (MEFs) isoladas a partir destas estirpes de murganhos deficientes podem ser usados ​​para dissecar ainda mais as funções destas moléculas durante γHV68 vivo lítica de replicação ex. Usando ensaios de biologia molecular e virológicos, podemos identificar o mecanismo molecular de hospedeiro-vírus e identificar interacções hospedeiro e genes virais essenciais para a replicação virai lítica. Finalmente, um cromossoma artificial bacteriano sistema (BAC), facilita a introdução de mutações no factor de viral (s) que, especificamente, interromper a interacção hospedeiro-vírus. Recombinante γHV68 realização destas mutações podem ser usadas para recapitular os fenótipos de γHV68 replicação lítica em MEFs deficientes em hospedeiro chave sinalização componentes.Este protocolo oferece uma excelente estratégia para interrogar interação patógeno-hospedeiro em vários níveis de intervenção in vivo e ex vivo. Recentemente, descobrimos que γHV68 usurpa uma via de sinalização imune inata a promover a replicação viral lítica 5. Especificamente, γHV68 de infecção ativa o novo IKKβ quinase imunológico e IKKβ ativada fosforila o mestre fator de transcrição viral, replicação e transativador (RTA), para promover a ativação da transcrição viral. Ao fazê-lo, de forma eficiente γHV68 casais sua ativação transcricional para sediar ativação imune inata, facilitando assim a transcrição e replicação lítico. Este estudo fornece um excelente exemplo que pode ser aplicado a outros vírus de interrogar hospedeiro-vírus interacção.

Watch this Scientific Journal Video about Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus at JoVE.com

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

Nós descrevemos um protocolo para identificar papéis fundamentais de moléculas de sinalização de acolhimento na replicação lítica de um herpesvírus modelo, gama herpesvírus 68 (γHV68). Utilizando linhagens de camundongos geneticamente modificados e fibroblastos embrionárias para γHV68 replicação lítica, o protocolo permite tanto caracterização fenotípica e molecular de interrogatório vírus-hospedeiro interações em replicação lítica viral.

Dissecando interação hospedeiro-vírus em replicação lítica de um herpesvírus modelo

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

dissecting-host-virus-interaction-lytic-replication-model

Research

JoVE Journal

Immunology and Infection

10.8K visualizações.  UT Southwestern Medical Center. Nós descrevemos um protocolo para identificar papéis fundamentais de moléculas de sinalização de acolhimento na replicação lítica de um herpesvírus modelo, gama herpesvírus 68 (γHV68). Utilizando linhagens de camundongos geneticamente modificados e fibroblastos embrionárias para γHV68 replicação lítica, o protocolo permite tanto caracterização fenotípica e molecular de interrogatório vírus-hospedeiro interações em replicação lítica viral.

Vídeo: Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Identificação de genes desregulados Induzida pelo Herpesvirus sarcoma de Kaposi-associado (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Imunologia e Infecção

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Dissecando imunitário inatas Sinalização em Evasion Viral da produção de citocinas

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

De alta capacidade: Ensaio para Fenótipo<em&gt; Salmonella enterica</em&gt; Typhimurium Association, invasão e replicação em macrófagos

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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

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

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

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

Estudar comunidades microbianas<em&gt; In Vivo</em&gt;: Um modelo de interação mediada por anfitrião entre<em&gt; Candida Albicans</em&gt; E<em&gt; Pseudomonas aeruginosa</em&gt; Nas Airways

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

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis como um organismo modelo para a Avaliação da Interação de bactérias anaeróbias com células do hospedeiro

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. 
Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. 
This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

Desenvolvimento e validação de um método PCR quantitativa para equídeo herpesvírus 2 Diagnostics em fluidos respiratórios

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

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

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

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

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

A<em&gt; In Vitro</em&gt; Modelo da barreira sangue-cérebro que usa espectroscopia de impedância: Um foco na interação celular T Cell-endotelial

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

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Citometria de fluxo e assassinas naturais celular Lytic Actividade em sangue total humano

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Dissecando a proteínas multi sinalização complexos pela purificação da afinidade de complementação Bimolecular (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.