Expressamos a nossa gratidão para as crianças e famílias do estudo coorte COPSAC2010 por todo seu apoio e compromisso. Reconhecemos e apreciamos os esforços exclusivos da equipa de investigação de COPSAC e a ajuda técnica do técnico Lisbeth Buus Rosholm, da Universidade Técnica da Dinamarca, para a medição de citocinas e quimiocinas.

Os estudos foram conduzidos em conformidade com os princípios orientadores da declaração de Helsinque. Aprovação do Comitê de ética para Copenhaga (KF 01-289/96 para COPSAC2000 ) e H-B-2008-093 para COPSAC2010e Agência Dinamarquesa de proteção de dados foram obtidas, e foi obtido consentimento escrito de ambos os pais de cada assunto antes de inscrição.
1. experimental Setup
<ol>
	<li>Use folhas de papel de filtro (folhas de poliéster-hidroxilados fibrosas, consulte a Tabela de materiais)6,7.<ol>
		<li>Corte em tiras de 3 x 15 mm em tamanho para uma criança de 1 mês de idade e em forma de L para crianças mais velhas e adultos (5 x 20 x 10 mm (braço curto da L)).</li>
	</ol>
	</li>
	<li>Inserir um pedaço de papel de filtro em cada narina da criança usando fórceps. Coloque o papel de filtro na parte anterior do corneto inferior (aproximadamente 1 cm dentro da narina para neonatos e 1,5 cm para todas as outras idades).<ol>
		<li>Coloque recém-nascidos em uma cama e dar-lhes água com açúcar para o conforto. Nota: Para crianças de todas as outras idades, a amostragem é realizada mais facilmente com o sujeito sentado em uma cadeira.</li>
		<li>Para crianças mais velhas, inserir o longo braço do papel de filtro em forma de L em cada narina.</li>
	</ol>
	</li>
	<li>Aplica um grampo do nariz ao redor das narinas para minimizar o desconforto e evitar a perda acidental do papel filtro.</li>
	<li>Remova os papéis de filtro depois de 2 min de absorção.</li>
	<li>Coloque os papéis de filtro em tubos etiquetados e congelá-los imediatamente a-80 ° C.</li>
	<li>Tome nota de quaisquer sintomas de infecção das vias respiratórias, mostrado pela criança no dia de amostragem.</li>
	<li>Grave qualquer espirro, choro persistente ou epistaxe que ocorre durante a 2 min da amostragem.</li>
</ol>
2. quantificação das vias aéreas imune-mediadores
<ol>
	<li>Tome até 10 amostras aleatórias do freezer. Os números de identificação de registro. Mantenha as amostras no gelo durante todo o processo de trabalho.</li>
	<li>Após o descongelamento no gelo, mergulhe os papéis de filtro (por assunto) de ambas as narinas em 300 µ l de preparada na hora do buffer (veja a Tabela de materiais) contendo um tablet completo protease inibidor (consulte a Tabela de materiais) por 25 mL de tampão.<ol>
		<li>Ajuste o volume da reserva conforme o tamanho do papel filtro.<ol>
			<li>Use 300 µ l de tampão para papéis de filtro 3 x 15 mm de tamanho.</li>
			<li>Se apenas um papel de filtro está disponível, use metade do volume de reserva (150 µ l).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transferi as amostras para um agitador de placa (400 rpm) 5 min. Use um timer.</li>
	<li>Transferir os papéis de filtro húmidos e buffer de ensaio para a Copa de um filtro de tubo de acetato de celulose (tamanho de poros de 0,22 µm) colocado dentro microcentrifuga tubo (ver a Tabela de materiais).</li>
	<li>Centrifugar a 16.000 x g por 5 min em uma centrífuga de refrigeração (a 4 ° C) para obter o extrato nasal filtrado dentro do tubo.</li>
	<li>Retire a taça e manter os tubos em gelo enquanto alíquotas do extrato nasal nos poços das placas de armazenamento de baixa proteína de ligação (consulte a Tabela de materiais).</li>
	<li>Loja a-80 ° C até análise.</li>
	<li>Determinar as concentrações de citocinas e quimiocinas nos extratos nasais usando um sistema de alta sensibilidade, com base em electrochemiluminesce matriz multiplexado (consulte a Tabela de materiais). Nota: Um ensaio de 2 citocina humana 10-plex TH1/TH, 9-plex chemokine ensaio e single-plex IL-17A, TGF-β1 e TSLP foram realizados aqui.<ol>
		<li>Para os ensaios, incubar os extratos nasais durante a noite a 4 ° C. Realizar as medições pelo fabricante padrão protocolo (consulte a Tabela de materiais). Nota: Os imunoensaios (consulte a Tabela de materiais) geralmente têm uma alta gama dinâmica de medição que varia entre 1 &#38; 10.000 pg/mL, mas pode ser para alguns ensaios, 100.000 pg/mL. Isto significa que todas as amostras podem ser executadas na mesma diluição, limitando assim a influência de diferentes diluições em indivíduos saudáveis e doentes, como é o caso em outros imunoensaios semelhantes. O limite inferior de detecção para todas as citocinas foi 1 pg/mL ou menos e para quimiocinas, variou entre 1 &#38; 50 pg/mL. TSLP não era detectável em 98% das amostras em 1 mês de idade.</li>
	</ol>
	</li>
</ol>
3. estatísticas
<ol>
	<li>Log-transformar os dados antes da análise para obter resíduos normalmente distribuídos dos níveis de citocinas e chemokine.</li>
	<li>Se as amostras são analisadas em mais de um lote, corrigi os dados de citocinas e chemokine para centrar batch-wise sobre os dados de log-transformadas.<ol>
		<li>Incorpore a média global e o antitransformação log de para obter dados em unidades de medida do original. Nota: Para cada conjunto de análises feito, considere ajustar as variáveis que afetam o nível de citocinas e quimiocinas. Isto poderia ser influências maternas (por exemplo, o consumo de álcool e fumar durante a gravidez), aleitamento materno, eventos adversos durante a amostragem, irmãos, animais de estimação no lar, etc.</li>
	</ol>
	</li>
</ol>

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V., 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=Neonatal+Cytokine+Profile+in+the+Airway+Mucosal+Lining+Fluid+Is+Skewed+by+Maternal+Atopy.">Neonatal Cytokine Profile in the Airway Mucosal Lining Fluid Is Skewed by Maternal Atopy.</a> Am J Respir Crit Care Med. 185 (3), 275-280 (2012).</li><li>Chawes, B. L., 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=Effect+of+Vitamin+D3+Supplementation+During+Pregnancy+on+Risk+of+Persistent+Wheeze+in+the+Offspring:+A+Randomized+Clinical+Trial.">Effect of Vitamin D3 Supplementation During Pregnancy on Risk of Persistent Wheeze in the Offspring: A Randomized Clinical Trial.</a> JAMA. 315 (4), 353-361 (2016).</li><li>Bischoff, A. L., 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=Altered+Response+to+A(H1N1)pnd09+Vaccination+in+Pregnant+Women:+A+Single+Blinded+Randomized+Controlled+Trial.">Altered Response to A(H1N1)pnd09 Vaccination in Pregnant Women: A Single Blinded Randomized Controlled Trial.</a> PloS One. 8 (4), 56700 (2013).</li><li>Wolsk, H. M., 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=Picornavirus-Induced+Airway+Mucosa+Immune+Profile+in+Asymptomatic+Neonates.">Picornavirus-Induced Airway Mucosa Immune Profile in Asymptomatic Neonates.</a> J Infect Dis. 213 (8), 1262-1270 (2016).</li><li>Wolsk, H. M., Chawes, B. L., F&#248;lsgaard, N. V., Rasmussen, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+technique+for+recovery+of+cytokines+from+inflammatory+sites+in+situ.">Development of a new technique for recovery of cytokines from inflammatory sites in situ.</a> J Immunol Methods. 155 (1), 25-29 (1992).</li><li>Ishizaka, 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=New+bronchoscopic+microsample+probe+to+measure+the+biochemical+constituents+in+epithelial+lining+fluid+of+patients+with+acute+respiratory+distress+syndrome.">New bronchoscopic microsample probe to measure the biochemical constituents in epithelial lining fluid of patients with acute respiratory distress syndrome.</a> Crit Care Med. 29 (4), 896-898 (2001).</li><li>Ishizaka, 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=Elevation+of+KL-6,+a+lung+epithelial+cell+marker,+in+plasma+and+epithelial+lining+fluid+in+acute+respiratory+distress+syndrome.">Elevation of KL-6, a lung epithelial cell marker, in plasma and epithelial lining fluid in acute respiratory distress syndrome.</a> Am J Physiol Lung Cell Mol Physiol. 286 (6), 1088-1094 (2004).</li><li>Nakano, Y., 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=Endothelin-1+level+in+epithelial+lining+fluid+of+patients+with+acute+respiratory+distress+syndrome.">Endothelin-1 level in epithelial lining fluid of patients with acute respiratory distress syndrome.</a> Respirology. 12 (5), 740-743 (2007).</li><li>Komaki, Y., 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=Cytokine-mediated+xanthine+oxidase+upregulation+in+chronic+obstructive+pulmonary+disease's+airways.">Cytokine-mediated xanthine oxidase upregulation in chronic obstructive pulmonary disease's airways.</a> Pulm Pharmacol Ther. 18 (4), 297-302 (2005).</li><li>Moeller, A., Franklin, P., Hall, G. 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Todos os autores não declaram nenhum conflito de interesses potencial, percebido ou real em relação ao conteúdo deste manuscrito. Agências de fomento não tinha qualquer papel na concepção e execução do estudo; a recolha, gestão e interpretação dos dados; ou a preparação, revisão e aprovação do manuscrito. Nenhuma empresa farmacêutica estava envolvida no estudo.

Com a técnica apresentada aqui, éramos capazes de determinar na vivo superior-via aérea da mucosa imune perfil em crianças desde o início como 1 mês de idade, que não foi feito anteriormente. Observamos que a presença de bactérias específicas das vias respiratórias e picornaviruses7,11, bem como outros pré e perinatais exposições, foram espelhadas no perfil imune dos recém-nascidos das vias respiratórias. Além disso, usamos esses dados d...

O fluido da mucosa do nariz torna-se a parte líquida do sistema superior-das vias respiratórias. Consiste de uma matriz complexa de mediadores derivado a interação entre o epitélio e as células imunitárias que compõem a primeira linha de defesa contra a invasão de microorganismos. A mucosa nasal é facilmente acessível, e há uma forte relação funcional e imunológica entre o nariz e os brônquios1. Este compartimento é de especial interesse em relação às doenças das vias respiratórias que são comuns na infância, como asma e rinite alérgica, mas também a uma gama de outras doenças respiratórias mais prevalentes mais tarde na vida.
Aqui, descrevemos a implementação de um método para amostra intacta da mucosa revestimento fluido da cavidade nasal usando uma técnica não-invasiva, baseada em papel de filtro, bem como um procedimento de extração subsequente, usada para eluir analitos baseados em proteína dos papéis de filtro antes da sua quantificação. Esta técnica por exemplo, pode ser usada para obter na vivo assinaturas imunes de indivíduos saudáveis e indivíduos com várias doenças respiratórias. Além disso, é possível examinar as exposições de importância para uma assinatura específica imune e avaliar se é um preditor ou mediador do desenvolvimento posterior da doença.
Fluido de mucosa anteriormente tiver sido obtido por lavagem nasal2, que muitas vezes é precedida por um teste de desafio nasal, onde um alérgeno é introduzido em níveis elevados para estimular uma resposta inflamatória3,4. No entanto, a técnica de lavagem nasal não é viável em crianças pequenas e introduz um factor de diluição desconhecido, que confunde os resultados, como o mediador diluído níveis podem cair abaixo do limite de detecção do ensaio5. Além disso, devido ao fator de diluição desconhecido, as analito medida respostas dos testes desafio nasal não são comparáveis entre indivíduos, limitando assim a utilidade da técnica de lavagem nasal em uma coorte de configuração. Finalmente, desafio de alérgeno só é aplicável em indivíduos sensibilizados e outros desafios, como o desafio de histamina, não são fisiologicamente relevantes, potencialmente causando um efeito de teto na liberação de mediador. Estes problemas são contornados na técnica apresentada em papel de filtro para recolha de fluidos de mucosa, onde a individual secreção de fluidos e níveis de analito são apenas fatores que influenciam a variação inter-individual.
Durante o procedimento de extração, analitos são eluídos dos papéis de filtro após a adição de volumes idênticos de buffer para todas as amostras. Isso favorece semelhante ex vivo diluição das amostras. Uma solução tampão de solução salina isotônica baseada em albumina é usado para a etapa de extração; permite a extração de mediadores à base de proteínas e estabiliza as proteínas para limitar a desnaturação durante o congelamento subsequente de proteínas eluted antes da quantificação. Para evitar a degradação proteica durante a fase de extração, um coquetel de inibidores de protease é adicionado para o buffer de extração.
A implementação de técnicas que permitem a quantificação do intacta, na vivo-mediadores imunes gerados em locais da mucosa é de extrema importância. Primeiro, o site da mucosa torna-se o maior órgão imunológico no corpo. Em segundo lugar, a localização nasal é o principal local de exposição no ar e está firmemente conectada ao compartimento imunológicas respiratórias dos pulmões1. Em terceiro lugar, a possibilidade de levantamento deste órgão importante com uma técnica não-invasiva abre a possibilidade de fornecer uma infinidade de informações sobre o eixo importante interação micróbio imune em relação à saúde e doença nas vias aéreas. Em quarto lugar, existem muitas outras possíveis aplicações desta técnica, tais como estudar alterações imunológicas locais em experimentações randomized, controladas de drogas e micronutrientes.
Inicialmente implementamos a técnica nos estudos prospectivos Copenhague sobre asma em coorte de2000 (COPSAC2000) de infância, onde determinamos o perfil imunológico do fluido em crianças de 7 anos de idade com rinite alérgica versus controles saudáveis13mucosa. Posteriormente, com êxito aplicamos esta técnica à coorte longitudinal de2010 COPSAC e imune perfis das vias respiratórias avaliados em 1 mês, 2 anos e 6 anos de idade e em instâncias de sintomas respiratórios agudos. Resultados de neonatos a 1 mês de idade demonstraram importantes associações entre a assinatura imune e a exposição ambiental do início da vida7,8,9,10,11,12.

<table border="0" cellpadding="0" cellspacing="0" width="1688">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Fibrous hydroxylatedpolyester sheets</td>
			<td style="width:103px;">Accuwik Ultra&#160;</td>
			<td style="width:119px;">SPR0730</td>
			<td style="width:1084px;">Filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milliplex Assay Buffer&#160;</td>
			<td>Millipore</td>
			<td>L-AB</td>
			<td>Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">low-protein binding storage plates&#160;</td>
			<td>Thermo Scientific</td>
			<td>CLS8161</td>
			<td>Plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor</td>
			<td style="width:103px;">Roche</td>
			<td align="right" style="width:119px;">11873580001</td>
			<td>complete EDTA-free Protease Inhibitor Cocktail</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Reader of multi-spot plates</td>
			<td style="width:103px;">Mesoscale</td>
			<td style="width:119px;">NA</td>
			<td>Sector imager 6000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Assays&#160;</td>
			<td style="width:103px;">Mesoscale</td>
			<td style="width:119px;"></td>
			<td>Human 10-plex TH1/TH2 cytokine assay and 9-plex chemokine assay, and singleplex IL-17A, TGF-&#946;1and TSLP. A description can be found online on www.mesoscale.com</td>
		</tr>
	</tbody>
</table>

noninvasive sampling of mucosal lining fluid for the quantification of in vivo upper airway immune-mediator levels

Este protocolo descreve a amostragem não-invasiva de fluido de mucosa das vias aéreas superiores não perturbadas. Ele também detalha o procedimento de extracção utilizado antes da análise dos mediadores imunes em eluídos fluidos para o estudo da via aérea tópica imune assinatura, sem a necessidade de procedimentos de estimulação (frequentemente usados por outras técnicas). O fluido de mucosa é amostrado em uma tira de papel de filtro colocado na parte anterior do corneto e esquerdo por 2 min de absorção inferior. Analitos são eluídos dos papéis de filtro, e os eluídos extraídos à base de proteínas são analisados por um imunoensaio baseado em eletroquimioluminescência, permitindo a quantificação de alta sensibilidade de analitos de baixo e de alto nível na mesma amostra. Medimos os níveis na vivo de 20 mediadores imunes pré-selecionadas relacionados com imunológico específico sinalização de caminhos na mucosa das vias aéreas superiores, mas a técnica não está limitada a esse painel específico ou local de amostragem. A técnica foi primeiramente implementada em crianças de 7 anos desde os estudos prospectivos de Copenhaga sobre asma em coorte de2000 (COPSAC2000) de infância com rinite alérgica. Posteriormente foi usado no COPSAC2010 nascimento coorte longitudinal, amostrada em 1 mês, 2 anos e 6 anos de idade e em instâncias de sintomas respiratórios agudos. Estamos com sucesso obtidas e analisadas amostras de 620 (89%) de 700 crianças de 1 mês de idade; algumas amostras estavam abaixo do limite de detecção do ensaio (relatado como a mediana (intervalo inter-quartil (IQR)). O número de amostras abaixo do limite de detecção (ou seja, de 0 até o ponto de ajuste para o limite inferior de detecção) para cada mediador foi 29 (7.25-119,5). Esta técnica permite a quantificação do vivo em vias aéreas da mucosa imune perfil desde o nascimento, pode ser aplicada no sentido longitudinal e pode ser aplicada a estudos sobre o efeito da genética e exposições ambientais iniciais de vida, fisiopatologia, endotyping e monitoramento de doenças respiratórias e desenvolvimento e avaliação de novas terapêuticas.

Baseline Characteristics of the Airway Immune Profiles: 
Complete data on upper airway mucosal immune mediator levels at 1 month of age was obtained in 620 (89%) of the 700 children enrolled in the COPSAC2010 cohort. Ten neonates were enrolled before the technique was established, and 19 did not attend the 1-month visit. Additionally, 47 samples were excluded because they were extracted and measured in another laboratory used in a pilot study, and 4 sample...

Watch this Scientific Journal Video about Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels at JoVE.com

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Este protocolo descreve uma técnica não-invasiva para a amostragem de líquido imperturbado mucosa das vias aéreas superiores. Ele pode ser usado para realizar a quantificação dos níveis na vivo de mediadores de proteína, tais como citocinas e quimiocinas, em indivíduos de todas as idades.

Amostragem não-invasiva de mucosa líquido para a quantificação dos níveis de imune-mediador na Vivo das vias aéreas superiores

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

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Immunology and Infection

Unit 1

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Upper Respiratory Tract Infections

SCIENTISTS IN ACTION

10.1K visualizações.  University of Copenhagen. Este protocolo descreve uma técnica não-invasiva para a amostragem de líquido imperturbado mucosa das vias aéreas superiores. Ele pode ser usado para realizar a quantificação dos níveis na vivo de mediadores de proteína, tais como citocinas e quimiocinas, em indivíduos de todas as idades.

Vídeo: Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Ensaio de cocultura para avaliar migração trans epitelial de neutrófilo induzida por patógenos

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

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. ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

A administração intranasal de recombinantes Vacinas contra Influenza em quimérico rato Modelos para estudar a imunidade mucosal

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Um método rápido para processamento de sangue para aumentar o rendimento dos níveis plasmáticos de peptídeos no sangue humano

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Administração intra-traqueal de<em&gt; Haemophilus influenzae</em&gt; No mouse modelos para o Estudo de inflamação das vias aéreas

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Swab método de amostragem para a detecção do norovírus Humano em Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Formação de biofilmes induzida por sais biliares em patógenos entéricos: técnicas para identificação e quantificação

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Quantification of Atherosclerosis in Mice

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

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

Quantificação da aterosclerose em camundongos

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

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Isolamento de células mononucleares tonsiláres para estudar respostas imunes inatas ex vivo em um tecido linfoide mucosal humano

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.

Produção de anticorpos monoclonais contra aminopeptidase N no epitélio da mucosa intestinal suína

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response ...