Trabalho nos laboratórios dos autores é suportado por concessões do Ciências Naturais e Pesquisa de Engenharia Council of Canada, os Institutos de Pesquisa em Saúde do Canadá, eo Canadá Research programa presidentes. JL é apoiado por uma bolsa de estudos do Groupe d&#39;Étude des Protéines Membranaires (GEPROM) através do financiamento do Fonds de Recherche en Santé du Québec.

1. Preliminar: cepas bacterianas e células epiteliais. Manipulações de células e bactérias são realizados de forma asséptica, debaixo de uma capela de fluxo laminar. <ol><li> Recém isolar a E. cepas de Escherichia 2787, 2787Δ Aida, e C600 de stocks glicerol em lisogenia Caldo (LB) ágar (triptona 1%, cloreto de sódio 0,5%, extrato de levedura 0,5%, ágar 1,5%) e crescer a 37 ° C. Para minimizar a variabilidade no ensaio, é aconselhável usar sempre cepas recém-banhado e para manter as cepas a 4 ° C em placas de Petri seladas apenas para um máximo de um par de semanas. </li><li> Cultura de células HEp-2 (ATCC CCL-23) em alta de glicose Modified Dulbecco Eagle Medium (DMEM) suplementado com 10% de soro bovino inactivado pelo calor (inativação de calor é realizada a 56 ° C por 30 min). Durante a cultura de rotina também adicionar 10 U / ml de penicilina e estreptomicina 10 mg / ml. </li><li> Crescer células Hep-2, a 37 ° C em uma célula incubadora com uma atmosfera contendo 5% CO 2. Use procedimentos padrão de cultura celular para manter as células, que são cultivadas em 75 cm 2 frascos e cada vez que eles atinjam subcultivadas confluência. Usamos nossas células cultivadas até atingirem 30-40 passagens e depois descartá-las. </li><li> Prepare um ensaio quando as células HEp-2 em um frasco de 75 cm 2 são quase atingindo confluência e, portanto, estão prontos para um ensaio de adesão. </li></ol> 2. DIA 1: Preparação do inóculo e as células epiteliais <ol><li> Lavar a células HEp-2 no frasco uma vez com fosfato de Dulbecco quente de solução salina tamponada (DPBS: 8 g / L NaCl, 0,2 g / L KCl, 0,2 g / L KH 2 PO 4, 0,21 g / L Na 2 HPO 4: 7H 2 O) </li><li> As células são incubadas com tripsina 0,05% - EDTA por 5 min antes de adicionar meio morno fresco completo. Depois de meio fresco é adicionado, as células são ressuspendidas pipetando cima e para baixo. </li><li> Centrifugar a suspensão celular a 2.000 rpm por 5 min e ressuspender o pelotas no DPBS e centrifugar novamente. </li><li> Ressuspender as células em meio fresco suplementado com 10% de soro, mas não contendo antibióticos, a uma concentração de 2x10 5 células / ml. </li><li> Mililitro uma semente de suspensão de células em três conjuntos de poços duplicado (um para cada cepa) no centro de uma placa de 24 poços e incubar a placa durante a noite em uma cela cultura incubadora. Nota: É importante que o soro ser cuidadosamente decomplemented e que os antibióticos são omitidos após esta fase. Não fazer isso pode matar a bactéria infectante. </li><li> Inocular uma colônia isolada de cada estirpe bacteriana (2787, 2787Δ Aida e C600) em 5 ml de caldo LB (triptona 1%, cloreto de sódio 0,5%, extrato de levedura 0,5%) e crescer durante a noite a 37 ° C com agitação vigorosa (180 rpm ). </li></ol> 3. DIA 2: Infecção das células <ol><li> Inspecionar as células HEp-2 em microscópio invertido para verificar que eles são pelo menos 90% confluentes e não contaminados. </li><li> Lavou as células com DPBS quente e, a cada poço, adicione 1 ml de meio fresco suplementado com 10% de soro, mas não contendo antibióticos. Também adicionar meio fresco a três poços sem células. Isso será usado para determinar o número total de bactérias no inóculo para cada cepa. </li><li> Medir a densidade óptica a 600 nm (OD 0,600 nm) das culturas bacterianas. Adicionar uma alíquota de cada cultura bacteriana a um conjunto de poços duplicados contendo células Hep-2 e para um bem, não contendo células. Geralmente, nós usamos um volume de cultura overnight correspondente a 10 6 unidades formadoras de colônia (UFC). Isso representa uma multiplicidade de infecção (MOI) de 5:1 (bactérias: células). Embora nós usar diretamente nossas culturas durante a noite, às vezes, é aconselhável centrífuga as bactérias e ressuspender-los em DPBS para evitar efeitos deletérios de moléculas secretadas presentes nas culturas durante a noite (como citotoxinas, por exemplo) Nota: O MOI pode variar entre 100:1 e 01:10. MOI maior variabilidade e maior rendimento de fundo e as bactérias tendem a se ater ao plástico da placa. Menor MOI também produz alta variabilidade. Uma vez que um MOI é escolhido, é imperativo para ser coerente e manter esta MOI. </li><li> Incubar as células infectadas na cultura de células incubadora durante 3 horas a 37 ° C com 5% de CO 2. Nota: Também é possível centrífuga brevemente a placa em baixa velocidade (por exemplo, 1.000 xg por 1-2 min), a fim de trazer todas as bactérias diretamente em contato com as células. Isto tem a vantagem adicional de sincronizar a infecção, e permitindo menor tempo de incubação (tão pouco quanto 15-30 min). </li><li> Remova a mídia a partir de células infectadas e lavar as células 3 vezes com DPBS quente. Nesta etapa, as bactérias aderentes geralmente pode ser visto com um microscópio normal e rotineiramente realizar esta verificação. </li><li> Para lisar as células e retirar o bac aderiutérios, adicione 100 ml de 1% Triton X-100 a cada poço contendo as células. Detergentes podem ser utilizados outros (como saponina por exemplo). </li><li> Incubar as células por 10 min em temperatura ambiente e em seguida, adicione 900 mL de meio LB. </li><li> Suavemente homogeneizar as suspensões por repetidas up-and-down pipetagem. Os poços sem células contendo o inóculo são igualmente suavemente homogeneizado. Nota: Algumas bactérias podem ser muito sensíveis ao detergente, nesse caso, destacamos as células epiteliais através da incubação com tripsina 0,05% - EDTA por 20 min a 37 ° C. Células juntamente com as bactérias aderiram também pode ser raspado dos poços com uma pipeta. </li><li> Prepare série de 10 diluições das suspensões de bactérias aderidas e inóculo utilizando caldo LB e 100 mL placa de 3 diluições (geralmente as diluições 1:1.000, 1:10.000 e 1:100.000) em ágar LB e incubar durante a 37 ° C . </li></ol> 4. DIA 3: ufc contagem e apresentação dos dados. <ol><li> Contagem de colônias nas placas e calcular o número de ufc de bactérias aderidas e do inóculo pela média de cada série de diluições. Placas somente com entre 10-300 colônias devem ser contadas. </li></ol> 5. Resultados representativos: A seguinte tabela mostra os resultados típicos da ufc contagem de bactérias aderidas e inóculo de 3 experimentos realizados em dias diferentes: <img src="/files/ftp_upload/2783/2783table1.jpg" alt="Tabela 1" /> Os resultados podem ser reportados diretamente como aderiu ufc, como mostrado na Figura 1A. Uma vez que os tamanhos de inóculo pode variar entre linhagens por causa das diferenças nas taxas de crescimento ou erros de pipetagem, muitas vezes é aconselhável para relatar os resultados em percentagem de bactérias aderidas. A porcentagem de bactérias aderidas pode ser calculado dividindo o número de ufc de bactérias aderidas pelo número de ufc do inóculo, como mostrado na Figura 1B. Ao realizar uma medida repetidas ANOVA e Bonferroni pós-testes para comparar todas as colunas da Figura 1B, podemos ver que existem diferenças significativas (p &lt;0,05) entre 2787 e 2787Δ Aida, bem como entre 2787 e C600, mas não houve diferença significativa entre 2787Δ Aida e C600. <img src="/files/ftp_upload/2783/2783fig1.jpg" alt="Figura 1"/> Figura 1. Representação dos resultados, ufc de bactérias aderidas (A) ou a porcentagem de bactérias aderidas (B) O percentual de adesão observada é freqüentemente muito dependente do set-up experimental (e, em menor medida, no experimentador). Especialmente importantes são os MOI e do número de lavagens, de modo que o percentual não deve ser interpretada literalmente. As bactérias no inóculo às vezes pode crescer muito mais rapidamente do que as bactérias na presença de células, desviando o tamanho do inóculo, em comparação com o número real total de bactérias em poços com células. Para aliviar este problema, recomenda-se: (i) usar os poços com células para determinar o inóculo: células HEp-2 são semeadas em um poço adicional e infectados. No final do ensaio, em vez de descartar o sobrenadante e lavar roupa, 100 ul de 10% Triton X-100 é adicionado para o bem. Lisar as células e os lisado contendo bactérias aderidas e não aderidas é suavemente homogeneizada por repetidas up-and-down pipetagem;. Ou (ii) recolher os sobrenadantes de células infectadas no final do ensaio, bem como os sobrenadantes dos as lavagens DPBS e determinar o número de ufc nos sobrenadantes reunidos. O resultado será o número de ufc de bactérias não aderiu. A porcentagem de bactérias aderidas pode então ser calculada dividindo o número de ufc de bactérias aderidas pela adição do número de ufc de bactérias aderidas e não aderidas. Para ilustrar a robustez do ensaio, podemos comparar este protocolo com o ensaio realizado com S4074, uma cepa patogênica adesivo suína de Actinobacillus pleuropneumoniae 7, incubados com as células de uma linhagem de células recém-criada traqueal leitão, nptr 8. Além das diferenças nos meios de comunicação necessários para o crescimento das bactérias e as células, a única diferença é que as bactérias usadas para a infecção são de uma cultura em crescimento exponencial, e não de uma cultura em fase estacionária durante a noite. Com A. pleuropneumoniae é também muito importante respeitar um MOI de 10:1 e um máximo de 3 horas depois da infecção, caso contrário, a secreção de toxinas irá causar a morte celular e viés nos resultados. Além disso, esta cepa de A. pleuropneumoniae pode facilmente aderir ao plástico por isso é importante o uso de células confluentes e verifique visualmente que as células não são sloughing off.

<ol>
	<li>Kline, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesins+in+host-microbe+interactions.">Bacterial adhesins in host-microbe interactions.</a> Cell Host Microbe. 5, 580-580 (2009).</li><li>Cossart, P., Boquet, P., Normark, S., Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+microbiology+emerging.">Cellular microbiology emerging.</a> Science. 271, 315-315 (1996).</li><li>Elsinghorst, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+invasion+by+gentamicin+resistance.">Measurement of invasion by gentamicin resistance.</a> Methods Enzymol. 236, 405-405 (1994).</li><li>Pizarro-Cerda, J., Lecuit, M., Cossart, P., Sansonetti, P., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cellular Microbiology. , 161-161 (2002).</li><li>Srivastava, A., Isberg, R. R., Sansonetti, P., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cellular Microbiology. , 179-179 (2002).</li><li>Benz, I., Schmidt, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+an+adhesin+(AIDA-I)+involved+in+diffuse+adherence+of+enteropathogenic+Escherichia+coli.">Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.</a> Infect Immun. 57, 1506-1506 (1989).</li><li>Charbonneau, M. E., Berthiaume, F., Mourez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+processing+is+not+essential+for+multiple+functions+of+the+Escherichia+coli+autotransporter+adhesin+involved+in+diffuse+adherence+(AIDA-I).">Proteolytic processing is not essential for multiple functions of the Escherichia coli autotransporter adhesin involved in diffuse adherence (AIDA-I).</a> J Bacteriol. 188, 8504-8504 (2006).</li><li>Jacques, M., Paradis, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesin-receptor+interactions+in+Pasteurellaceae.">Adhesin-receptor interactions in Pasteurellaceae.</a> FEMS Microbiol Rev. 22, 45-45 (1998).</li><li>Auger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+of+Actinobacillus+pleuropneumoniae+with+porcine+lung+and+tracheal+epithelial+cells.">Host-pathogen interactions of Actinobacillus pleuropneumoniae with porcine lung and tracheal epithelial cells.</a> Infect Immun. 77, 1426-1426 (2009).</li><li>Jacques, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lipo-oligosaccharides+and+lipopolysaccharides+in+bacterial+adherence.">Role of lipo-oligosaccharides and lipopolysaccharides in bacterial adherence.</a> Trends Microbiol. 4, 408-408 (1996).</li><li>Nataro, J. P., Kaper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diarrheagenic+Escherichia+coli.">Diarrheagenic Escherichia coli.</a> Clin Microbiol Rev. 11, 142-142 (1998).</li><li>Jallat, C., Darfeuille-Michaud, A., Rich, C., Joly, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+clinical+isolates+of+diarrhoeogenic+Escherichia+coli:+diffusely+adhering+E.+coli+strains+with+multiple+adhesive+factors.">Survey of clinical isolates of diarrhoeogenic Escherichia coli: diffusely adhering E. coli strains with multiple adhesive factors.</a> Res Microbiol. 145, 621-621 (1994).</li></ol>

Não há conflitos de interesse declarados.

Este protocolo descreve um ensaio de adesão padrão de bactérias que podem ser modificados para estudar a invasão (por exemplo, utilizando gentamicina ensaio de proteção 3). A contagem de unidades formadoras de colônia permite quantificar, em comparação com abordagens contando com técnicas de visualização padrão sob um microscópio, como coloração Giemsa. Este último só dá uma visão qualitativa de adesão, mas muitas vezes é um complemento útil, pois pode diferenciar vários padrões de ad...

<table border="1"><tbody><tr><th> Reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> DMEM alta glicose </td><td> GIBCO-Invitrogen </td><td> 12430-054 </td></tr><tr><td> DDPBS </td><td> GIBCO-Invitrogen </td><td> 14190-144 </td></tr><tr><td> Serum Crescimento bovina </td><td> Hyclone </td><td> SH3054 </td></tr><tr><td> Penicilina / estreptomicina </td><td> GIBCO-Invitrogen </td><td> 15140-148 </td></tr><tr><td> 24 placas bem </td><td> Corning </td><td> 3337 </td></tr><tr><td> Triton X-100 </td><td> Fisher Scientific </td><td> BP151-100 </td></tr></tbody></table>

in vitro assay of bacterial adhesion onto mammalian epithelial cells

Para causar infecções, as bactérias precisam colonizar seu hospedeiro. Patógenos bacterianos expressar várias moléculas ou estruturas capazes de promover a ligação às células hospedeiras 1. Estas adesinas confiar em interações com os receptores da célula hospedeira superfície ou proteínas solúveis agindo como uma ponte entre bactérias e hospedeiro. Adesão é um primeiro passo crítico antes da invasão e / ou secreção de toxinas, por isso, é um evento-chave a ser estudado na patogênese bacteriana. Além disso, as bactérias aderiram muitas vezes induzem exquisitely aperfeiçoá-lo respostas celulares, os estudos que deram origem ao campo da 2 &#39;celular microbiologia. Ensaios robusta para adesão bacteriana em células hospedeiras e sua invasão, portanto, têm um papel fundamental em estudos de patogenia de bactérias e têm sido muito utilizados em muitos laboratórios pioneira 3,4. Estes ensaios estão agora praticado pela maioria dos laboratórios que trabalham com patogênese bacteriana. Aqui, descrevemos um teste de aderência normal ilustrando a contribuição de uma adesina específica. Usamos a cepa de Escherichia coli 2787 5, uma cepa patogênica humana expressando o autotransporter Adhesin Envolvidos na adesão difusa (AIDA). Como controle, utilizamos uma estirpe mutante sem o gene Aida, 2787Δ aida (F. e M. Berthiaume Mourez, não publicado), e uma cepa de laboratório comercial de E. coli, C600 (New England Biolabs). As bactérias são deixados para aderir às células do comumente usado HEp-2 linha celular humana epiteliais. Este ensaio tem sido menos extensivamente descrito anteriormente 6.

Watch this Scientific Journal Video about In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells at JoVE.com

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

Este protocolo é um ensaio de adesão bacteriana simples que consiste na contagem do número de unidades formadoras de colônia de bactérias que são aderidos em células em cultura. O ensaio é robusto, independente da adesina estudado, e inúmeras variações são usadas na maioria dos laboratórios que trabalham com patogênese bacteriana.

Ensaio in vitro da adesão bacteriana em mamíferos Células Epiteliais

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

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JoVE Journal

Immunology and Infection

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

25.2K visualizações.  Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire. Este protocolo é um ensaio de adesão bacteriana simples que consiste na contagem do número de unidades formadoras de colônia de bactérias que são aderidos em células em cultura. O ensaio é robusto, independente da adesina estudado, e inúmeras variações são usadas na maioria dos laboratórios que trabalham com patogênese bacteriana.

Vídeo: In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Aplicação de um rato ligado patch Peyer Assay alça intestinal para avaliar absorção pelas células bacterianas M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Imunologia e Infecção

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitativo<em&gt; In vitro</em&gt; Ensaio medir a adesão de neutrófilos ativados primárias células humanas endoteliais microvasculares em condições estáticas

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

Entérico invasão bacteriana de células epiteliais intestinais<em&gt; In Vitro</em&gt; É dramaticamente aumentada usando um Vertical Modelo câmara de difusão

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

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

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

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

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

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

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

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Fluxo de Neutrófilos Humanos Secção Ensaio de Adesão

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Estática Adesão Ensaio para o Estudo da integrina Ativação em Linfócitos T

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

A electroporação de efectores bacterianos funcionais em células de mamíferos

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In Vitro e In Vivo modelo para estudar a adesão bacteriana para o Vaso de parede em condições de escoamento

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Ensaio de Adesão Sob tensão de cisalhamento para o Estudo da Linfócitos T-Molécula de adesão de Interações

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...