This work was supported by NIH grants to T.W. (R01AI072060 and R01AI099123). G. Xie was supported by a Sealy Center for Vaccine Development Predoctoral Fellowship. We thank Dr. Richard Flavell (Howard Hughes Medical Institute, Yale University School of Medicine, New Haven) and Dr. Shizuo Akira (Osaka University, Japan) for providing the MyD88&#8211;/&#8211; mice and Dr. Y Cong (UTMB, Galveston) for providing OTII transgenic mice.

Todos os experimentos com animais foram aprovados pelo Comitê de Cuidados e Uso Animal da Universidade de Texas Medical Branch. 1. Isolamento de DCs de não-infectados e infectados pelo WNV Mice <ol><li> Idade e sexo partida 6-10 semanas de idade, do tipo selvagem C57BL / 6 (B6) e MyD88 - / - ratos. Inocular por via intraperitoneal (ip) com 500 placas formando unidade (UFP) de WNV NS4B- P38G mutante. No dia 3 de pós-infecção, eutanásia MyD88 B6 e - / - ratos com CO 2. Use camundongos não infectados de ambos os grupos como controles. Use três murganhos de cada grupo. </li><li> Pele molhada no lado esquerdo do rato sacrificados com etanol 70% e cortar a pele ao longo do lado esquerdo do mouse, a meio caminho entre as pernas dianteiras e traseiras. Cortar abrir a cavidade do corpo. Remover o baço, utilizando a pinça. Inserir o baço em uma placa de Petri com meio RPMI. </li><li> Isolar DCs esplénicas utilizando esferas magnéticas anti-CD11c segundoas instruções do fabricante. </li></ol> 2. Purificação e marcação das células T de OTII Transgenic Mice <ol><li> Colheita um baço de um rato OTII ingénuos como descrito no passo 1.2 acima e homogeneizar o baço entre as extremidades foscas de duas lâminas. Transferir a suspensão celular para um tubo de 15 ml, adicionar meio RPMI até 14 ml. Deixe a suspensão sentar por 5 min e 13 ml de transferir-lo para um novo tubo de 15 ml. </li><li> Isolar células T CD4 + de baço utilizando um kit de isolamento de células T CD4 + de acordo com as instruções do fabricante. </li><li> Transferência de células para um tubo cónico de 50 ml, lavar duas vezes com PBS. Ressuspender as células em PBS com 0,5% de albumina de soro bovino (BSA) a 1 x 10 6 culas por ml, e adicionar 0,5 umol / ml de CFSE. Incubar a 37 ° C durante 10 minutos, ao abrigo da luz. </li><li> Adicionar 5 ml de meio completo frio (RPMI-1640 com 10% de soro fetal bovino inactivado pelo calor, 1/100 volantibióticos / antimicóticos, 1/100 vol de L-glutamina e 1 / 1.000 vol de 1000 x 2-mercaptoetanol). </li><li> Incubar em gelo durante 5 min. Lave as células uma vez com PBS, corrigir 0,2 x 10 6 células CD4 + T rotulados em 2% PFA e adquirir em um citômetro de fluxo. Utilize essa amostra para determinar o nível basal de CFSE. Re-suspender o resto de células marcadas em meio completo. </li></ol> 3. Co-cultura OTII T células com DCs de infectados pelo WNV Mice <ol><li> Cultura de 2 x 10 5 células purificadas de ratos OTII T CD4 +, sozinho ou com as DCs purificadas de tipo selvagem ou MyD88 - / - murganhos (2 x 10 4) em uma placa de 24 poços com ou sem OVA 323-339 resíduo (1 g / ml) em 1 ml de meio completo por poço. </li><li> Incubar as células a 37 ° C durante 5 dias. Células colheita, lavar duas vezes em tampão FACS (PBS com 1% FBS) e fixar em 0,25 ml de 2% PFA. Vortex imediatamente. </li></ol> 4. Citometria de Fluxo de Análise In Vitro &lt;/ Em&gt; T celular Priming <ol><li> Para aquisição, clique duas vezes no ícone do software de citometria de fluxo. Para uma parcela densidade linear FSC-A vs. linear SSC-A, selecionar canais 0 a 200.000 em FSC-A e 0 a 200.000 em SSC-A. Em seguida, crie um portão em células vivas (P2; ver Figura 1A). </li><li> Para analisar a intensidade de fluorescência de CFSE, abra o histograma do canal FL1 e adquirir 20.000 eventos sobre a população fechado. Configurar um marcador em amostras de células cultivadas OTII sozinho (Figura 1A). </li><li> Adquirir amostras de células OTII co-cultivadas com DC do tipo selvagem (Figura 1B) ou MyD88 - / - DC (Figura 1C) de um modo semelhante. </li></ol> 5. Isolamento e Estimulação de Esplenócitos para Cytokine Ensaios <ol><li> Infect B6 e MyD88 - / - ratos com mutante WNV NS4B-P38G, utilizando o mesmo procedimento como descrito no step 1.1. No dia 30 pós-infecção, re-desafio sobreviver camundongos com 2.000 UFP do tipo selvagem WNV ip estirpe Nos dias 8 e 21 de infecção WNV pós-primária ou no dia 4 após a infecção secundária, eutanásia ratos com CO 2 para a coleção de baços . NOTA: Nós usamos 2-3 ratos por grupo para cada ponto de tempo. </li><li> Adicione uma suspensão de células isoladas de esplenócitos como acima descrito no passo 1.2 e passo 2.2. Contagem de células utilizando um hemocitómetro e re-suspender-los em 10 ml de meio completo. </li><li> Dilui-se as células com meio completo a 2,5 x 10 6 células / ml. Adicionar 1 ml de esplenócitos em um tubo de micro-centrifugação de 1,5 ml. </li><li> Para simular as células T CD8 +, diluir NS4B e E péptidos específicos de WNV (SSVWNATTA e IALTFLAV) a 1 mg / ml em DMSO. Para a estimulação de células T CD4 +, diluir NS3 e E péptidos específicos de WNV (RRWCFDGPRTNTILE e PVGRLVTVNPFVSVA) a 1 mg / ml em DMSO. Adicionar 10 ul de péptidos para as células followed por 1 ml de Brefeldin A solução. Use células sem tratamento como controlos de péptidos. Misturar as células. </li><li> Faça dois furos com uma agulha 18 G na tampa do tubo. Incubar as células a 37 ° C durante 5 h. </li></ol> 6. intracelular Cytokine Coloração <ol><li> Transferência de células para um novo tubo de micro-centrífuga. Gire células por 5 min a 300-400 xg e deitar fora o sobrenadante. Adicionar 1 ml de tampão FACS, rotação 5 min a 300-400 xg e suspender novamente as células por pipetagem com 120 ul FACS buffer. </li><li> Adicionar 2 mL de Fc bloqueador de células e incubar à temperatura ambiente durante 10 min. Adicionar 1 mL de tampão de FACS para cada tubo. Rotação 5 min a 300-400 x g. </li><li> Re-suspender as células em 300 ul FACS tampão e dividi-los em três tubos (cerca de 0,8 x 10 6 células por tubo). Como mostrado na Tabela 1, reservam-se o primeiro tubo de cada condição de cultura para rato IgG-PE de coloração. Para o segundo tubo, adicionar 3 mL de anti-CD4 APC para CD4 células peptídeos-tratados, 3 μl de anti-CD8 FITC às células tratadas com péptidos ou CD8 ambos os anticorpos a células sem tratamento com péptidos. Use o terceiro tubo para a coloração de compensação. Para cada condição de cultura, utilize três tubos de células marcadas com APC conjugada CD4, CD8 conjugado com FITC ou anticorpos marcados pe- CD3 (3 mL por tubo), como controles de compensação para FL1, FL2 e canais FL4. </li><li> Células Vortex brevemente. Deixar em gelo durante 20 min e proteger da luz. Adicionar 1,4 ml de tampão de FACS. Gire 5 min a 300-400 xg e deitar fora o sobrenadante. </li><li> Agita-se a pelete de células perturbar (ou brevemente vórtice). Re-suspender pellet celular em 250 ul fixação / permeabilização solutionby pipetagem. Incubar à temperatura ambiente durante 20 min e proteger da luz. </li><li> Adicionar 1,2 ml de tampão de FACS. Rotação 5 min a 300-400 xg e ressuspender as células em 300 ul de tampão de FACS. NOTA: Neste ponto, as células podem ser transferidas para um laboratório BL2 para processamento adicional ou armazenada num frigorífico e protegida da luz durante up a três dias. </li><li> Adicionar 500 ul de tampão de FACS. Rotação 5 min a 300-400 xg e ressuspender as células em 500 ul de tampão de permeabilização / lavagem 1x. Rotação 5 min a 300-400 x g. </li><li> Re-suspender as células em 100 l 1x Perm / Wash, adicionar 3,5 ul anti-IFN-y-PE ao tubo adicionou anteriormente com anticorpos para CD4 e / ou marcadores de células T CD8 em passo 6,3 e 3,5 l rato IgG-PE no tubo reservados para IgG de controlo, durante 25 min em gelo e proteger da luz. Lave as células, adicionando 1,4 ml de tampão de permeabilização / lavagem 1x. Rotação 5 min a 300-400 x g. Repita a etapa de lavagem, adicionando 1,4 ml 1x tampão permeabilização / lavagem. </li><li> Gire 5 min a 300-400 xg e decantar o sobrenadante. Lavar as células por adição de 1,4 mL de tampão de FACS e re-suspensão em 400 ul de tampão de FACS para aquisição final. </li></ol> 7. Análise de Citometria de Fluxo Coloração de citocinas intracelulares <ol><li> Use as mesmas configurações de aquisição como no passo 4.1. Create um portão em células vivas (P2; ver Figura 2A). Ajustar as tensões utilizando os tubos de compensação para cada grupo. </li><li> Abertas dois novos gráficos de pontos, uma para visualizar os dados recolhidos para FL1 vs. FL2 (CD8 vs. IFN-γ), e o outro é para exibir os dados recolhidos para FL4 vs. FL2 (CD4 vs. IFN-γ). Adquirir 50.000 eventos para a população fechado de cada amostra. </li></ol>

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Os autores não têm nada a revelar.

WNV é um patógeno BL3. Devido às normas de segurança, testes imunológicos com amostras infectadas com WNV muitas vezes são limitadas pela disponibilidade de equipamentos no BL3 instalações ou mais longo e tedioso para executar. Em um recente estudo, foram utilizados dois métodos baseados em citometria de fluxo para estudar as respostas imunes mediadas por células durante a infecção WNV 5. Em ambos os ensaios, as células infectadas com WNV foram tratados com 1-2% PFA directamente ou com uma soluç...

Vírus do Nilo Ocidental (WNV), um neurotrópico, plus-sensoriamento flavivírus, é uma ameaça emergente de saúde pública. Actualmente, não há vacinas foram aprovados para uso humano 1. Uma estirpe atenuada WNV, o qual possui uma substituição no P38G não estrutural (NS) da proteína 4B, é conhecida por induzir sem letalidade em ratinhos mas citocinas maior inatas e respostas de células T em ratinhos de tipo selvagem do que o WNV NY99 estirpe 2. Os ratinhos imunizados com o mutante NS4B-P38G foram todos protegidos a partir de um desafio letal com secundária de tipo selvagem WNV. Isto sugere que o mutante de NS4B-P38G tem características apropriadas para um candidato a vacina ideal. Os mecanismos pelos quais o mutante NS4B-P38G induz a imunidade adaptativa protetor de alta não são claramente entendidos ainda. Receptores do tipo Toll (TLRs) que reconhecem padrões moleculares associados a agentes patogénicos, desempenham um papel essencial na iniciação da imunidade inata de infecção viral. A via de sinalização TLR núcleo utiliza mielóide resposta diferenciação primária gene 88 (MyD88) como o adaptador primário 3,4. Em um estudo recente, a sinalização MyD88 foi mostrado desempenhar um papel importante no desenvolvimento da imunidade mediada por células durante a infecção mutante WNV NS4B- P38G em ratinhos 5. As células dendríticas (CDs) são uma das células apresentadoras de antigénio que apresentam mais importantes a capacidade única para iniciar respostas de células T primárias durante a infecção viral 6,7. As células T CD4 + e CD8 + ambos contribuem para a imunidade protetora de longa duração e são importantes para a sobrevivência de acolhimento na sequência de tipo selvagem infecção WNV 8,9. Dois ensaios imunológicos foram utilizados neste estudo para avaliar as funções destas células nos mutantes de camundongos infectados com NS4B-P38G. Em primeiro lugar, um ensaio in vitro priming células T foi utilizado para comparar a capacidade apresentadora de antigénio de DCs infectadas com WNV de tipo selvagem e MyD88 - / - ratos. Para aumentar a sensibilidade do ensaio, naïve CD4 &lt;sup&gt; + células T foram isoladas a partir de ratinhos transgénicos, que expressam OTII um específico Vα2 / Vβ5 TCR para a ovalbumina de galinha (OVA), péptido 323 - 339. DCs de camundongos infectados WNV foram purificados, e co-cultivados com carboxifluoresceína succinimidil páscoa (CFSE marcado com células) na presença de OVA péptido T CD4 +. Após 5 dias de co-cultura, as células foram recolhidas e fixadas com paraformaldeído (PFA) e analisadas por citometria de fluxo. Os ensaios de proliferação foram tradicionalmente realizada através da incorporação de 5-bromo-2&#39;-desoxiuridina (BrdU) ou timina deoxyriboside tritiada (3HTdR) 10. No entanto, estes ensaios são ou radioativo e / ou que necessitam de equipamentos especiais a nível de biossegurança (BL) 3 instalações, onde os estudos são conduzidos WNV. A análise de citometria de fluxo da proliferação de linfócitos por redução a metade de série da intensidade de fluorescência do corante vital CFSE tornou-se mais comumente utilizado em ensaios imunológicos, como o corante é mais estavelmentee uniformemente incorporado nas células, detectado facilmente por citometria de fluxo, e é não radioactiva 11. O ensaio também tem a capacidade de avaliar o número de divisões celulares. Uma grande vantagem da utilização deste teste em estudos WNV é que a fixação das células infectadas com 1-2% PFA poderia inactivar WNV 12, que vai permitir a aquisição de amostras com um citómetro de fluxo em um laboratório BL2. Em seguida, um processo de coloração de citocinas intracelulares modificado (ICS) foi usado para estudar o papel da sinalização MyD88 na regulação de respostas de células T WNV específicos em NS4B-P38G ratos infectados com mutantes. Neste ensaio, os esplenócitos isolados de ratinhos infectados foram tratados in vitro com péptidos específicos WNV. Brefeldina A foi adicionada para reter as citocinas no interior da célula. Após 5 horas de incubação, as células foram colhidas, lavadas e coradas para subconjuntos de células T. As células foram depois fixadas em PFA, permeabilizadas e coradas para o interferão (IFN) -γ e analisadas por citometria de fluxo.Como com outro ensaio baseado em citometria de fluxo, uma vez que as células são tratadas com tampão de fixação e permeabilização contendo PFA, amostras infectadas podem ser transferidas para um laboratório BL2 para posterior processamento e análise. Em vários estudos publicados, temos usado ICS para medir T funções efetoras de células em camundongos infectados com WNV 13,14. Embora esteja bem estabelecida, uma desvantagem principal deste ensaio é que o processo é muito lento e pode ser mais demorado quando realizada dentro das instalações BL3. Aqui, um método de ICS baseado no tubo de micro-centrifugação foi demonstrado ser mais viável, mais fácil proceder e menos demoradas quando realizada dentro de um laboratório BL3.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:153px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">RPMI 1640</td>
			<td style="width:153px;">Invitrogen</td>
			<td align="right" style="width:119px;">11875</td>
			<td>warm up at 37C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD11c magnetic beads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-001</td>
			<td>follow the manufacturer&#8217;s instructions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD4 magnetic beads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-248</td>
			<td>follow the manufacturer&#8217;s instructions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFSE</td>
			<td style="width:153px;">Invitrogen</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OVA residue 323-339</td>
			<td>Genscript Corporation</td>
			<td>RP10610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Peptides</td>
			<td style="width:153px;">Proimmune</td>
			<td>PC0AD-D</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Brefeldin A solution</td>
			<td style="width:153px;">BD Bioscience</td>
			<td align="right">555029</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:216px;">Mouse Fc Blocker</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>14-0161-85</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">APC-conjugated CD4&#160;</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>17-0041-81</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">FITC-conjugated CD8&#160;</td>
			<td style="width:153px;">e- bioscience</td>
			<td>11-0081-82</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Fixation/Permeabilization Solution</td>
			<td style="width:153px;">BD- Bioscience</td>
			<td align="right" style="width:119px;">554722</td>
			<td></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">Permeabilization/wash buffer</td>
			<td style="width:153px;">BD- Bioscience</td>
			<td align="right">554723</td>
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			<td height="58" style="height:58px;">anti-IFNg-PE&#160;</td>
			<td style="width:153px;">e-Bioscience</td>
			<td>12-7311-82</td>
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		<tr height="58">
			<td height="58" style="height:58px;width:216px;">Accuri flow cytometer</td>
			<td style="width:153px;">BD Bioscience</td>
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in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection

Um vírus atenuado do Nilo Ocidental (WNV), um não-estrutural (NS) 4B-P38G mutante, citocina induzida maior inata e respostas de células T do que o de tipo selvagem em ratinhos WNV. Recentemente, factor de diferenciação mielóide 88 (MyD88) de sinalização foi mostrado ser importante para o escorvamento inicial das células T e o desenvolvimento de células T de memória durante a infecção mutante WNV NS4B-P38G. Neste estudo, dois fluir métodos baseados em citometria - in vitro de células T e um ensaio de priming citocina coloração intracelular (ICS) - foram utilizados para avaliar as células dendríticas (DC) e funções de células T. Na célula T priming ensaio, a proliferação celular foi analisada por citometria de fluxo após a co-cultura de DC a partir de ambos os grupos de ratinhos com éster de succinimidilo de carboxifluoresceína (CFSE) - rotulados células T CD4 + de ratinhos transgénicos OTII. Esta abordagem proporcionou uma determinação exacta da percentagem de proliferação de células T CD4 + com melhorou significativamente a sensibilidade global do que a tradiçãoai ensaia com reagentes radioactivos. Um sistema de tubo de microcentrifugadora foi utilizado em ambos os procedimentos de cultura de células e de coloração de citocinas do protocolo ICS. Comparado com o tradicional sistema baseado em placa de cultura de tecido, este procedimento modificado foi mais fácil para se apresentar no nível de biossegurança (BL) 3 instalações. Além disso, as células infectadas foram tratadas com WNV- paraformaldeído em ambos os ensaios, o que permitiu uma análise mais aprofundada fora das unidades de BL3. Em geral, estes ensaios imunológicos in vitro pode ser utilizado para avaliar a eficiência mediada por respostas imunes celulares durante infecção WNV.

No ensaio de células T de escorva, as células T CD4 + foram cultivadas CFSE rotulados com DCs purificadas a partir do tipo selvagem NS4B-P38G infectadas com mutante e MyD88 - / - ratos na presença ou ausência de péptidos OVA. As células T cultivadas marcadas por si só, com ou sem OVA durante 5 dias, foram utilizadas como controlos negativos. Como mostrado na Figura 1A, as células T totais foram fechado para a análise da intensidade de fluorescência no canal FL1. O marcado...

Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Vitro Análise do mediada por Myd88 Cellular resposta imune a vírus do Nilo Ocidental Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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Research

JoVE Journal

Immunology and Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

8.1K visualizações.  The University of Texas Medical Branch. Two flow cytometry-based methods – an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Vídeo: In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

Fluxo multicolor de Citometria de Análises de resposta imune celular em macacos rhesus

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Imunologia e Infecção

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sete Passos para Stellate Células

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Uma Introdução à Vespas parasitas de<em&gt; Drosophila</em&gt; E Resposta Imune a antiparasitários

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

PRP como uma nova abordagem para prevenir a infecção: Preparação e<em&gt; In vitro</em&gt; Propriedades antimicrobianas do PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Uma<em&gt; In vitro</em&gt; Modelo para estudar respostas imunes de sangue periférico humano células mononucleares para Vírus Respiratório Sincicial Humano Infecção

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Usando RNA-interferência para investigar a imunitário inatas de resposta em macrófagos

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Um<em&gt; In Vitro</em&gt; Modelo para medição de respostas imunes a Malária no Contexto do HIV co-infecção

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Uma abordagem simples e eficiente para construir vectores de vírus mutante Vaccinia

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Um modelo do rato para avaliar a resposta imune inata à infecção de Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...