Este trabalho foi apoiado pelo Inovador Jovem Biotechnologist Award BT/BI/12/040/2005 e concessão BT/PR13289/BRB/10/745/2009 do Departamento de Biotecnologia, Governo da Índia e os fundos centrais do Centro de Impressões Digitais de DNA e Diagnóstico, Hyderabad. MNR e GB são os destinatários de júnior e sênior de pesquisa Bolsas de Estudo do Conselho de Pesquisa Científica e Industrial para a busca de um doutorado da Universidade Manipal. SB é o destinatário de Júnior e Sociedade do Departamento de Biotecnologia de Pesquisa Sênior para a busca de um doutorado da Universidade Manipal.

Recomenda-se a realizar C. experimentos de infecção glabrata em um laboratório com nível de contenção de biossegurança 2 (BSL-2). <ol><li> Preparação de células THP-1 monocamada de macrófagos. </li><li> Preparação de C. suspensão de células glabrata. </li><li> A infecção de células THP-1 com os macrófagos C. células glabrata. </li><li> A medição da taxa de fagocitose e replicação intracelular através do ensaio de formação de colónias unidade. </li><li> Monitoramento da replicação intracelular por microscopia confocal a laser. </li></ol> 1. Preparação de células THP-1 macrófago monocamada <ol><li> THP-1 é uma linha celular monocítica humana derivada a partir do sangue periférico de um menino de 1 ano de idade, sofrendo de leucemia monocítica aguda 14. THP-1, uma linha de células em suspensão, é habitualmente mantida a 37 ° C em humidade a 5% de CO 2 em meio de cultura RPMI-1640 suplementado com 10% FBS (soro fetal bovino). O tratamento com forbol 12-myristate 13-acetato (PMA, um éster de forbol) resulta na diferenciação terminal de células THP-1 monocitárias em macrófagos. É importante ressaltar que o tratamento PMA não afeta a viabilidade das células e células diferenciadas não se dividem. </li><li> Semente de aproximadamente 1,5 x 10 6 monócitos THP-1 em placas de 100 mm de cultura em meio RPMI-1640 completo (meio RPMI-1640 suplementado com 10% de soro, 2 mM de glutamina e antibióticos 1x, 10 mL / prato) e deixar crescer as células a 37 ° C em 5% de CO 2 durante 2 dias. </li><li> Uma vez que as células THP-1 chegaram a uma densidade de 8 x 10 5 células / ml, transferir a placa de cultura para a cultura de células de fluxo laminar vertical e agitar suavemente para ressuspender as células que se estabeleceram. </li><li> Pipetar a suspensão de células para fora, colocar num tubo de 15 ml estéril e centrifugar a 1000 rpm (130 x g) durante 4 min. Elimine o sobrenadante e suspender de THP-1 sedimento celular em 5 ml, pré-aquecido, de meio completo RPMI-1640 fresco. </li><li> Enumerar as células com um hemocitômetro e diluir o cell suspensão para uma densidade final de 10 6 células / ml com meio completo pré-aquecido RPMI-1640 </li><li> Adicionar 1 ml de estoque de 160 mM PMA (12-miristato de solução de 13-acetato de forbol preparada em dimetil-sulfóxido) para 10 ml de THP-1 de suspensão celular (concentração final 16 nM) e misture bem. Repartir 1 ml de suspensão celular em cada poço de uma placa de cultura de tecidos de 24 cavidades e incubar placa na incubadora mantida a 37 ° C com 5% de CO 2 durante 12 horas. </li><li> Remover o meio velho, adicionar 1 ml de pré-aquecido de RPMI-1640 completo e permitir que as células recuperar durante 12 horas a 37 ° C em 5% de CO 2. </li><li> Observar células sob um microscópio invertido para garantir que células THP-1 monocítica ovais em suspensão foram diferenciadas em macrófagos achatados, em forma de fuso e aderentes. Estas células diferenciadas estão agora prontos para conduzir C. estudos de infecção glabrata. </li></ol> 2. Preparação de C. glabrata &amp; #Suspensão celular; 160 C. glabrata estirpe selvagem Bg2 será utilizado para infectar células THP-1 macrófagos. C. glabrata células são rotineiramente cultivadas em meio YPD líquido (extracto de levedura (1%), peptona (2%), dextrose (2%)) médio. Meio YPD sólida é preparada por adição de 2% de agar antes da autoclavagem médio. <ol><li> Para preparar uma cultura de C. glabrata, escolhe-se uma única colónia da placa de agar com um lacete estéril, descartável e inocular em 10 ml de meio YPD líquido e incubar durante 14-16 horas, com agitação (200 rpm) a 30 ° C. </li><li> Centrífuga esta cultura (1 ml) a 4000 rpm (1800 xg) durante 5 min, em cima de uma mesa de microcentrífuga, lavar as células três vezes com PBS (solução salina tamponada com fosfato, pH 7,4) estéril e suspender sedimento celular em 1 ml de PBS. </li><li> Medida OD600 de C. suspensão de células glabrata e dilui-se com um volume adequado de PBS estéril para obter uma densidade de 2 x 10 6 células / ml (DO600= 0,1). Em alternativa, a densidade de células desejada pode ser conseguida por meio da contagem de células de levedura usando um hemocitómetro. </li></ol> 3. A infecção de células THP-1 de macrófagos C. Células glabrata <ol><li> Adicionar 50 ul C. suspensão de células glabrata THP-1 nos macrófagos (10 6 células semeadas por poço de uma placa de cultura de 24 poços) para obter um MOI (multiplicidade de infecção) de 0,1 e Incubar a placa a 37 ° C com 5% de CO 2. </li><li> Para determinar as contagens de levedura viáveis, diluir 50 ul C. glabrata de células em suspensão de 100 vezes em PBS estéril e placa 100 uL de meio sólido de YPD. Incubar as placas a 30 ° C, e contar manualmente o número de colónias de levedura que aparecem após 1-2 dias. Esses números serão, doravante, ser referido como 0 hr C. glabrata UFC (unidades formadoras de colônia). </li><li> Depois de 2 horas coculturing de células THP-1 com C. células glabrata, meio de cultura de descarte invertendo placa de 24 poços de cultura de tecidos ina reservatório e lavar cuidadosamente três vezes com PBS estéril pré-aquecido para remover C. extracelular nonphagocytosed células glabrata. Lavagens gentil com PBS são absolutamente essenciais para realizar a remoção completa de todo o fermento extracelular, sem causar qualquer dano ao THP-1 macrófagos monocamada. </li></ol> 4. Medição de fagocitose e Taxa de replicação intracelular via Colony Forming Assay Unit <ol><li> Lyse C. glabrata infectadas THP-1, macrófagos, em 1 ml de H 2 O estéril durante 2 min, raspe para remover os detritos de macrófagos do poço e recolher o lisado celular num tubo de microcentrífuga. Verificação microscópica da lise dos macrófagos completo é fundamental para a recuperação de todas as leveduras internalizadas. </li><li> Prepara-se uma diluição de 100 vezes do lisado em PBS estéril, placa de 100 ul de meio sólido de YPD e incubar as placas a 30 ° C. </li><li> Após 1-2 dias, a contagem manual do número de C. glabrata colônias que apreceu em meio YPD, multiplique com o fator de diluição (2 horas UFC) e calcular a taxa de fagocitose, que se refere à percentagem de C. glabrata células que são ingeridas por THP-1, macrófagos, após co-incubação de 2 horas com a seguinte fórmula. Fagocitose% = [(UFCs em 2 h) / (CFU em 0 h)] X 100 </li><li> Para medir a taxa de replicação intracelular de C. glabrata células THP-1 em macrófagos, recolher levedura intracelular em diferentes momentos pós ie infecção. 4, 6, 8, 10, 12, e 24 horas, repetindo os passos 3,3-4,3. </li><li> Calcule vezes sobrevivência / replicação de C. glabrata em células THP-1, macrófagos, dividindo o total de UFC em qualquer ponto no tempo, com os de 2 horas (de levedura fagocitado). </li></ol> 5. Monitoramento de replicação intracelular Usando confocal Laser Microscopia <ol><li> Seguindo os passos descritos na secção 1, as sementes tratadas com PMA 5 x 10 5 células THP-1 em cada poço de dois lâminas de câmara de quatro poços e incuba-se a 37 ° C com 5% de CO 2 durante 12 horas. </li><li> Substituir meio velho pré-aquecido com meio RPMI-1640 completo e permitir que as células se recuperar a partir do tratamento de PMA durante 12 horas. </li><li> Prepare GFP (proteína fluorescente verde)-marcado C. suspensão de células glabrata, conforme descrito na Seção 2 e infectar a THP-1 macrófagos para um MOI de 1. Se C. glabrata estirpes expressando GFP não está disponível, as células de levedura marcado com FITC (isotiocianato de fluoresceína) pode ser utilizado para monitorar eventos precoces pós infecção viz. taxa de fagocitose e maturação phagolysosomal em 2 horas. </li><li> Incubar durante 2 horas a 37 ° C com 5% de CO 2, inverte-los cuidadosamente para descartar meio e lavar 3x com estéril, pré-aquecido PBS. </li><li> Adicionar 500 mL pré-aquecido RPMI-1640 completo medihum para cada câmara de uma corrediça e incube-a 37 ° C com 5% de CO 2 durante 22 horas. </li><li> Para corrigir 2 horas C. glabrata infectadas THP-1, macrófagos, adicionar 500 mL de 3,7% de formaldeído (em PBS) a cada uma das câmaras da outra corrediça e incubar-la à temperatura ambiente durante 20 min no escuro. </li><li> Lavar deslizar três vezes com PBS, adicionar 500 ul de Triton-X (0,7%), e incuba-se à temperatura ambiente durante 5 min no escuro. </li><li> Lavar 3x slides com PBS, remova câmara de cultura de slides e ar seque-o por 3-5 min no escuro. </li><li> Monte cuidadosamente uma lamela utilizando meio Vectashield montagem contendo DAPI (4 &#39;,6-diamidino-2-phenylindole) no slide evitando a formação de bolhas. Remova suavemente o excesso de líquido com Kimwipe e selar bordas lamela com unha polonês. Slides loja no escuro a 4 ° C até o uso. </li><li> Repita os passos 5,4 e 5,6-5,9 para processar células THP-1 24 horas após a infecção. </li><li> Células de imagem usando um confo de varredura a lasermicroscópio cal (objetiva 60X de imersão em óleo, a excitação a 405 nm e 488 nm para DAPI e GFP mancha, respectivamente). </li></ol>

<ol>
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Os autores declaram que não têm interesses financeiros concorrentes.

Sistema imune inata desempenha um papel importante no controle de infecções causadas por fungos oportunistas. Os macrófagos contribuem para antifúngicos defesa por ingestão e destruição do patógeno. Assim, a elucidação de fatores que são necessários para a sobrevivência e / ou neutralizar as funções antimicrobianas de macrófagos permitirão uma melhor compreensão das estratégias de virulência de fungos. Neste contexto, nós estabelecemos um sistema in vitro de modelo de cultura celular, utili...

Espécies de Candida são a principal causa de infecções fúngicas invasivas risco de vida em pacientes imunocomprometidos 1. Candida glabrata, um patógeno nosocomial emergente, é o segundo ou terceiro mais frequentemente isoladas espécies de Candida de pacientes unidade de terapia intensiva, dependendo da localização geográfica 1-3. Filogeneticamente, C. glabrata, uma levedura de brotamento haplóides, é mais estreitamente relacionado com os Saccharomyces não modelo levedura patogênica cerevisiae do que patogênica Candida spp. incluindo C. albicans 4. Coerente com isso, C. glabrata carece de algumas características de virulência de fungos-chave, incluindo o acasalamento, a actividade proteolítica secretada e plasticidade morfológica 4-5. Embora C. O glabrata não formam hifas, pode sobreviver e replicar-se em macrófagos de ratinho e humanos 6-8 sugerindo que desenvolveu mecanismos de patogénese únicas. Não existe informação suficiente sobre as estratégias que C. glabrata emprega para sobreviver ambiente intracelular de macrófagos pobres em nutrientes e neutralizar respostas do hospedeiro não oxidativos montados por células do sistema imunológico do hospedeiro 5 oxidativo e. Um sistema modelo de macrófagos pertinente é um pré-requisito para delinear a interação de C. glabrata com células fagocíticas do hospedeiro através de abordagens de genômica e proteômica funcional. As células mononucleares do sangue periférico (PBMC) e os macrófagos derivados da medula óssea (BMDMs) de origem humana e de murino, respectivamente, têm sido anteriormente utilizados para estudar a interacção de C. glabrata com células do sistema imunológico do hospedeiro 7,9. No entanto, a dificuldade na obtenção de PBMC e BMDMs, a sua duração de vida limitada e variação intrínseca entre diferentes doadores de mamíferos restringir a utilização destas células como sistemas modelo versáteis. Aqui, descrevemos um método para o estabelecimento de um sistema in vitro para estudar a intracellulcomportamento de ar C. células glabrata em macrófagos derivados da linha celular monocítica humana THP-1. O objetivo geral deste protocolo foi a promulgar um sistema modelo de cultura de células simples, barato, rápido e reprodutível, que pode ser facilmente manipulado para estudar diferentes aspectos da interação patógeno-hospedeiro. Células THP-1 foram anteriormente usadas para decifrar a resposta imune do hospedeiro contra uma vasta gama de agentes patogénicos, incluindo as bactérias, vírus e fungos 10-12. Monocíticas THP-1, as células são fáceis de manter e podem ser diferenciados, após tratamento do éster de forbol, para os macrófagos, que imitam os macrófagos derivados de monócitos de ser humano e expressam marcadores de macrófagos adequados 13. As principais vantagens do THP-1, do sistema modelo de macrófagos são a facilidade de utilização e a falta de equipamento sofisticado requisito. O protocolo aqui apresentado é facilmente adaptável para estudar a interação de outros fungos patógenos humanos com hosAs células T do sistema imunológico. O processo actual pode também ser empregue para identificar os factores de virulência para o agente patogénico de interesse, utilizando telas de mutantes de elevada capacidade. Esta prova de conceito foi exemplificado pelo uso bem sucedido de THP-1 sistema de modelo de cultura para identificar um conjunto de 56 genes que são necessários para a sobrevivência de C. glabrata em macrófagos humanos 8.

<table border="0" cellpadding="0" cellspacing="0" width="596">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="39">
			<td height="39" style="height:40px;width:163px;">THP-1</td>
			<td style="width:105px;">American Type Culture Collection</td>
			<td style="width:85px;">TIB 202</td>
			<td style="width:243px;">Human acute monocytic leukemia cell line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">RPMI-1640</td>
			<td style="width:105px;">Hyclone</td>
			<td style="width:85px;">SH30096.01</td>
			<td style="width:243px;">For maintaining THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Phorbol 12-myristate 13-acetate</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td style="width:85px;">P 8139</td>
			<td style="width:243px;">Caution: Hazardous</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">YPD&#160;</td>
			<td style="width:105px;">BD-Difco</td>
			<td style="width:85px;">242710</td>
			<td style="width:243px;">For growing Candida glabrata cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Formaldehyde</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td style="width:85px;">F8775</td>
			<td style="width:243px;">For fixation of C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Phosphate buffered saline (PBS)</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl,&#160; pH 7.4) for washes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Saline-sodium citrate (SSC)&#160;</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (3 M NaCl, 0.3 M sodium citrate for 20x concentration) for washes</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Prehybridization buffer</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (50% formamide, 5x Denhardt&#8217;s solution, 5x SSC, 1% SDS) for hybridization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">VECTASHIELD mounting medium</td>
			<td style="width:105px;">Vector Labs</td>
			<td style="width:85px;">H-1200</td>
			<td style="width:243px;">For mounting slides for confocal microscopy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">32P-labeled &#945;-dCTP</td>
			<td style="width:105px;">JONAKI-BARC</td>
			<td style="width:85px;">LCP-102</td>
			<td style="width:243px;">For radiolabeling of signature tags</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">100 mm tissue culture dishes</td>
			<td style="width:105px;">Corning</td>
			<td style="width:85px;">430167</td>
			<td style="width:243px;">To culture THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">24-well tissue culture plate</td>
			<td style="width:105px;">Corning</td>
			<td style="width:85px;">3527</td>
			<td style="width:243px;">To perform C. glabrata infection studies in THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">4-chamber tissue culture-treated glass slide</td>
			<td style="width:105px;">BD Falcon</td>
			<td style="width:85px;">REF354104</td>
			<td style="width:243px;">To image C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Hemocytometer</td>
			<td style="width:105px;">Rohem India</td>
			<td style="width:85px;"></td>
			<td style="width:243px;">For enumeration of cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Table top microcentrifuge</td>
			<td style="width:105px;">Beckman Coulter</td>
			<td style="width:85px;">Microfuge 18</td>
			<td style="width:243px;">For spinning down cells in microtubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Table top centrifuge</td>
			<td style="width:105px;">Remi</td>
			<td style="width:85px;">R-8C</td>
			<td style="width:243px;">For spinning down cells in 15 ml tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Spectrophotometer</td>
			<td style="width:105px;">Amersham Biosciences</td>
			<td style="width:85px;">Ultraspec 10</td>
			<td style="width:243px;">To monitor absorbance of yeast cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Plate incubator</td>
			<td style="width:105px;">Labtech</td>
			<td style="width:85px;">Refrigerated</td>
			<td style="width:243px;">To grow C. glabrata cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Shaker incubator</td>
			<td style="width:105px;">New Brunswick</td>
			<td style="width:85px;">Innova 43</td>
			<td style="width:243px;">To grow C. glabrata cells</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Water jacketed CO2&#160; incubator</td>
			<td style="width:105px;">Thermo Electron Corporation</td>
			<td style="width:85px;">Forma series 2</td>
			<td style="width:243px;">To culture THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Confocal microscope</td>
			<td style="width:105px;">Carl Ziess</td>
			<td style="width:85px;">Ziess LSM 510 meta&#160;</td>
			<td style="width:243px;">To observe C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Compound microscope</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:85px;">CKX 41</td>
			<td style="width:243px;">To observe C. glabrata and THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">PCR machine</td>
			<td style="width:105px;">BioRad&#160;</td>
			<td style="width:85px;">DNA Engine</td>
			<td style="width:243px;">To amplify unique tags from input and output genomic DNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Hybridization oven</td>
			<td style="width:105px;">Labnet</td>
			<td style="width:85px;">Problot 12S</td>
			<td style="width:243px;">For hybridization&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">PhosphorImager</td>
			<td style="width:105px;">Fujifilm</td>
			<td style="width:85px;">FLA-9000</td>
			<td style="width:243px;">For scanning hybridized membranes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Thermomixer</td>
			<td style="width:105px;">Eppendorf</td>
			<td style="width:85px;">Thermomixer Comfort</td>
			<td style="width:243px;">For denaturation of radiolabeled signature tags</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:163px;">Gel documentation unit</td>
			<td style="width:105px;">Alphainnontech</td>
			<td style="width:85px;">Alphaimager</td>
			<td style="width:243px;">To visualize ethidium bromide-stained DNA in agarose gels</td>
		</tr>
	</tbody>
</table>

establishment of an in vitro system to study intracellular behavior of candida glabrata in human thp-1 macrophages

Um sistema de modelo de cultura de células, se um mímico de perto as condições ambientais do hospedeiro, pode servir como uma alternativa barata, reprodutível e facilmente manipulável para sistemas de modelo animal para o estudo de um passo específico de uma infecção patogénica microbiana. Uma linha celular monocítica humana THP-1, o qual, após tratamento do éster de forbol, subdivide-se em macrófagos, anteriormente tem sido utilizado para estudar as estratégias de virulência de vários agentes patogénicos intracelulares, incluindo o Mycobacterium tuberculosis. Aqui, discutimos um protocolo para adoptar um modelo in vitro de cultura de células sistema usando THP-1 macrófagos para delinear a interação de um oportunista levedura Candida glabrata patógeno humano com células fagocíticas host. Este sistema modelo é simples, rápido, passíveis de telas de mutantes de alto rendimento, e não requer equipamentos sofisticados. A THP-1 experiência típica a infecção dos macrófagos leva aproximadamente 24 horas, com um adicional de 24-48 horas para permitir intracelular recuperadolevedura de crescer em meio rico para formadoras de colônia análise de viabilidade com base em unidade. Como outros sistemas in vitro do modelo, uma possível limitação dessa abordagem é a dificuldade em extrapolar os resultados obtidos para um circuito de célula imunológica altamente complexo existente no hospedeiro humano. No entanto, apesar disso, o protocolo atual é muito útil para elucidar as estratégias que um fungo pode empregar para evitar / neutralizar resposta antimicrobiana e sobreviver, adaptar e proliferar no ambiente pobre em nutrientes das células do sistema imunológico do hospedeiro.

Analisa Infecção tratados com PMA THP-1 macrófagos com C. tipo selvagem glabrata (em peso), as células revelaram que as células wt foram fagocitados por macrófagos, a uma taxa de 55-65%, após co-incubação de 2 horas. Além disso, C. glabrata células foram capazes de resistir a morte por THP-1 macrófagos e passou por uma moderada 5 - para aumento de 7 vezes no UFC após 24 horas de coculturing com THP-1 macrófagos 8. Replicação intracelular de células de tipo ...

Watch this Scientific Journal Video about Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages at JoVE.com

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

O presente artigo descreve um protocolo para estabelecer um sistema modelo de cultura de células in vitro para estudar a interação de um patógeno humano intracelular facultativo de fungos Candida glabrata com macrófagos humanos, que será uma ferramenta útil para aumentar o nosso conhecimento dos mecanismos de virulência de fungos.

Estabelecimento de um<em&gt; In vitro</em&gt; Sistema para estudar o comportamento intracelular de<em&gt; Candida glabrata</em&gt; Em humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

establishment-an-vitro-system-to-study-intracellular-behavior-candida

Research

JoVE Journal

Immunology and Infection

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

11.5K visualizações.  Centre for DNA Fingerprinting and Diagnostics, Andhra Pradesh, India.  O presente artigo descreve um protocolo para estabelecer um sistema modelo de cultura de células in vitro para estudar a interação de um patógeno humano intracelular facultativo de fungos Candida glabrata com macrófagos humanos, que será uma ferramenta útil para aumentar o nosso conhecimento dos mecanismos de virulência de fungos. 

Vídeo: Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac modificação do sistema Transposon de células T primárias Humanos

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Imunologia e Infecção

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Um<em&gt; In vitro</em&gt; Modelo para estudar a heterogeneidade de diferenciação de macrófagos humanos e Polarização

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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 ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Estudo da fagolisossomo Biogenesis em macrófagos ao vivo

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

A transfecção altamente eficiente de THP-1 por macrófagos humanos Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Aplicação de nanopartículas fluorescentes para estudar Reformas do Sistema Endo-lysosomal por bactérias intracelulares

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

Aplicando um sistema de expressão induzível para estudar interferência de fatores de virulência bacteriana com Sinalização Intracelular

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento de Salmonella typhimurium-contendo Phagosomes de macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Transfecção altamente eficiente de macrófagos primários com mRNA Transcrito in vitro

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...