Dominic Mills foi apoiado por uma Bloomsbury Faculdades PhD Studentship (2007-2010). Os autores gostariam de agradecer a ambos Ozan Gundogdu e Abdi Elmi para a sua assistência no desenvolvimento do modelo VDC.

1. Crescimento do IEC Monolayers em especial Filtros 0,4 mM <ol><li> Culturas células Caco-2 em meio essencial modificado por Dulbecco (DMEM) suplementado com 10% de soro (v / v) de vitela fetal, 100 U / ml de penicilina, 100 ug / ml de estreptomicina e 1% (v / v) de aminoácidos não essenciais em um cultura de tecido padrão incubadora a 37 ° C em 5% de CO 2 e 95% de ar. Semente de 4 x 10 5 células Caco-2 IECs por 0,4 um filtro de polarização e crescer para mais de 21 dias, passando os meios de comunicação a cada 2-3 dias. </li><li> Medir a TEER para confirmar o estado de polarização da monocamada utilizando um medidor de resistência de volt-ohm. Nos nossos estudos, o TEER de 21 dias as células Caco-2 cultivadas nesses filtros 0,4 mM é Ωcm 700-800 2. </li></ol> 2. Preparação de C. jejuni e Médio bacteriana para o Ensaio Coculturing <ol><li> 24 horas antes do ponto desejado do ensaio coculturing VDC partida, preparar um prato fresco agar sangue de C. jejuni e incubar a 37 ° C sob condições microaeróbias (85% de N 2, 5% de O2, e 10% de CO2) na atmosfera variável incubadora. </li><li> Ao mesmo tempo, pré-incubar 30 ml de caldo de brucela a 37 ° C sob condições microaeróbias numa incubadora de atmosfera variável. </li></ol> 3. Preparação e esterilização do meio Chambers VDC Antes do Ensaio <ol><li> Mergulhe completamente câmaras VDC meia, assim como os anéis e as fichas na solução de esterilização. </li><li> Usando um estéril seringa de 20 ml, lavar a solução de esterilização através das entradas de gás em ambas as câmaras meia. Não fazer isso pode resultar em contaminação das amostras durante coculturing. </li><li> Deixar os componentes imersos na solução de esterilização durante&gt; 1 h. </li><li> Lave todos os componentes com água estéril fresco, usando outra seringa de 20 ml estéril para lavar as entradas de gás. </li></ol> 4. Preparaçãodo inoculo bacteriano <ol><li> Colher metade de um prato de C. jejuni crescimento da placa de agar de sangue preparado no dia anterior em 1 ml de caldo de Brucella-incubadas. Isto deverá dar ~ 10 10 ufc / mL. </li><li> Determinar a densidade óptica da suspensão bacteriana a 600 nm para semiquantify unidades formadoras de colónias bacterianas (UFCs). </li><li> Ajuste a suspensão bacteriana para o nível desejado de inoculo em 4 ml do caldo de Brucella-incubadas. </li><li> Efectuar uma diluição em série a partir desta suspensão bacteriana definitivo seguido por plaqueamento das diluições apropriadas em placas de agar de sangue, em triplicado, em seguida, incubado a 37 ° C na atmosfera variável incubadora durante 48 horas para quantificar o número de CFUs bacterianas presentes no inoculo. </li></ol> 5. Configurando o VDC <ol><li> Coloque a metade inferior de uma câmara VDC plano para o banco. Montar um O-ring para a metade inferior da câmara. </li><li> Retire um filtro que transportam os IECs de tele transportador e lavar três vezes com 400 ul de PBS estéril. Coloque o filtro na metade câmara baixa, certificando-se o anel continua em vigor. </li><li> Delicadamente abaixar a metade câmara superior no lugar. Uma vez que as duas meias-câmaras são montados, apertar em conjunto, utilizando as braçadeiras de anel. Coloque a VCC horizontalmente no topo da bancada, com as duas aberturas voltadas para cima. </li><li> Adicionar 4 ml de inoculo bacteriano na metade câmara apical. </li><li> Adicionar 4 ml de meio de cultura celular para a metade câmara basolateral. </li><li> Coloque as peças finais nas aberturas de ambas as câmaras meia. </li></ol> 6. Colocar a VDC para o Manifold gás dentro da variável Atmosfera Incubadora <ol><li> Coloque a VCC na atmosfera variável incubadora em estreita proximidade com o colector de gás. </li><li> Abra o regulador de alimentação de gás ligados ao colector de gás. Ligar o tubo a partir do colector para dentro da entrada de gás na câmara de metade basolateral da VCC. Prenda a gas saída do tubo que conduz para fora da atmosfera variável incubadora para uma das saídas no final peça na metade câmara basolateral. </li><li> Feche o outro na saída da peça de extremidade na metade câmara basolateral. Abra o fluxo de gás no coletor de gás, tendo o cuidado para abri-lo muito lentamente para evitar o excesso de pressão do gás, pois isso pode levar à expulsão do meio basolateral. </li><li> O objetivo é um fluxo de 1 bolha a cada 2-5 segundos gás. Verifique periodicamente no fluxo de gás durante o curso do experimento e ajustar se necessário. </li></ol> 7. Desmontagem do VDC após Coculture <ol><li> Após o período de co-cultura apropriado, feche o regulador de gás ligado ao colector de gás. Feche o fluxo de gás para o VDC no coletor. Desligar a entrada do gás, a saída do gás, e a rolha do VCC. </li><li> Remover o VCC de uma incubadora de atmosfera variável. Remover o sobrenadante e armazenar apical e basolateral a -80 ° C para subsequenanálise t. </li><li> Remova os grampos anel e tomar cuidado para além do VCC. Retire o filtro a partir da metade da câmara e transferir para um 6-bem placa de cultura de células estéreis. Lavar as IECs três vezes com 400 ul de PBS estéril. </li><li> Por contagem do número total de bactérias que interagem, adicionar 400 ul de PBS estéril que continha 0,1% (v / v) de Triton X-100 para as IECs e incubar durante 20 min à temperatura ambiente para lisar o IEC. Efectuar uma diluição em série a partir desta ligado celular, seguido por plaqueamento das diluições apropriadas em placas de agar de sangue, em triplicado, em seguida, incubado a 37 ° C na atmosfera variável incubadora durante 48 horas para quantificar o número de interagir CFUs bacterianas. </li><li> Por contagem do número total de bactérias invasoras, adicionar 400 mL de meio de cultura celular DMEM contendo 150 ug / ml de gentamicina às IECs e incubar durante 2 horas numa incubadora de cultura de tecido padrão, a 37 ° C em 5% de CO 2 e 95% ar para matar as bactérias extracelulares,siga como para a etapa 7.4 acima. </li></ol> 8. Limpando o VDC após Coculture Lave o VDC com água estéril e loja para a próxima rodada de experimentos.

<ol>
	<li>Mills, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+Campylobacter+jejuni+invasion+of+intestinal+epithelial+cells+under+low-oxygen+coculture+conditions+that+reflect+the+in+vivo+environment.">Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.</a> Infect. Immun. 80, 1690-1698 (2012).</li><li>Marteyn, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Shigella+virulence+in+response+to+available+oxygen+in+vivo.">Modulation of Shigella virulence in response to available oxygen in vivo.</a> Nature. 465, 355-358 (2010).</li><li>Dorrell, N., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+century+of+Campylobacter+research:+recent+advances,+new+opportunities+and+old+problems.">The second century of Campylobacter research: recent advances, new opportunities and old problems.</a> Curr. Opin. Infect. Dis. 20, 514-518 (2007).</li><li>Young, K. T., Davis, L. M., Dirita, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Campylobacter+jejuni:+molecular+biology+and+pathogenesis.">Campylobacter jejuni: molecular biology and pathogenesis.</a> Nat. Rev. Microbiol. 5, 665-679 (2007).</li><li>Hu, L., Kopecko, D. J., Nachamkin, I., Szymanski, C. M., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17.">Chapter 17.</a> Campylobacter. , 297-313 (2008).</li><li>Everest, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+Caco-2+cells+as+a+model+for+enteric+invasion+by+Campylobacter+jejuni+and+C.+coli.">Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli.</a> J. Med. Microbiol. 37, 319-325 (1992).</li><li>Konkel, M. E., Hayes, S. F., Joens, L. A., Cieplak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+the+internalization+and+intracellular+survival+of+Campylobacter+jejuni+in+human+epithelial+cell+cultures.">Characteristics of the internalization and intracellular survival of Campylobacter jejuni in human epithelial cell cultures.</a> Microb. Pathog. 13, 357-370 (1992).</li><li>Monteville, M. R., Konkel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-facilitated+invasion+of+T84+eukaryotic+cells+by+Campylobacter+jejuni+occurs+preferentially+at+the+basolateral+cell+surface.">Fibronectin-facilitated invasion of T84 eukaryotic cells by Campylobacter jejuni occurs preferentially at the basolateral cell surface.</a> Infect. Immun. 70, 6665-6671 (2002).</li><li>Friis, L. M., Pin, C., Pearson, B. M., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+culture+methods+for+investigating+Campylobacter+invasion+mechanisms.">In vitro cell culture methods for investigating Campylobacter invasion mechanisms.</a> J. Microbiol. Methods. 61, 145-160 (2005).</li><li>Schuller, S., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerobic+conditions+enhance+type+III+secretion+and+adherence+of+enterohaemorrhagic+Escherichia+coli+to+polarized+human+intestinal+epithelial+cells.">Microaerobic conditions enhance type III secretion and adherence of enterohaemorrhagic Escherichia coli to polarized human intestinal epithelial cells.</a> Environ. Microbiol. 12, 2426-2435 (2010).</li><li>Cottet, S., Corthesy-Theulaz, I., Spertini, F., Corthesy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerophilic+conditions+permit+to+mimic+in+vitro+events+occurring+during+in+vivo+Helicobacter+pylori+infection+and+to+identify+Rho/Ras-associated+proteins+in+cellular+signaling.">Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.</a> J. Biol. Chem. 277, 33978-33986 (2002).</li></ol>

Não temos nada a divulgar.

A utilização de métodos de cultura de células in vitro para estudar as interacções dos agentes patogénicos bacterianos com células hospedeiras é uma técnica amplamente utilizada em muitos laboratórios de investigação. No entanto, como estes ensaios de cultura de células são realizadas em condições aeróbias, estes modelos in vitro não pode representar exactamente o ambiente in vivo nas quais as interacções hospedeiro-agente patogénico a ter lugar. A amplamente uti...

As interacções dos agentes patogénicos bacterianos com células hospedeiras têm sido investigados extensivamente usando métodos de cultura de células in vitro. Usando estes ensaios de cultura celular, a adesão bacteriana a células hospedeiras, a identificação de receptores de células hospedeiras, células do hospedeiro vias de sinalização e invasão bacteriana de células hospedeiras têm sido estudadas em pormenor, resultando em muitas observações importantes. No entanto, tais ensaios de cultura de células são realizadas em condições aeróbias, que podem não ser representativas do ambiente in vivo. Uma grande limitação de modelos in vitro utilizados para estudar as infecções gastrointestinais é que as condições de cultura, incluindo níveis elevados de oxigénio, geralmente favorece a sobrevivência de células eucarióticas. No entanto as condições no lúmen intestinal será quase anaeróbias. Patógenos entéricos em um ambiente muito baixa de oxigênio expressar genes de virulência cuja expressão mudanças em condições aeróbias 2. Como tal, os dados obtidos usando o padrão de cultura de células modelos may dar uma indicação imprecisa de interações bacterianas com células hospedeiras. Campylobacter jejuni é o principal agente causador de gastroenterite aguda bacteriana em todo o mundo, com sintomas que vão desde diarréia leve a enterite inflamatória grave. A maioria dos C. jejuni infecções resultam em gastroenterite simples, no entanto C. jejuni é também o agente infeccioso mais comumente identificados em neuropatias periféricas, incluindo a síndrome de Guillain-Barré (GBS). No Reino Unido, estima-se que existem mais de 500000 casos de enterite causadas por C. jejuni infecção a cada ano, com um custo previsto para a economia do Reino Unido de £ 580 milhões. No mundo em desenvolvimento, C. jejuni é a principal causa de mortalidade entre as crianças. Apesar da importância inquestionável da infecção por Campylobacter e décadas de pesquisa, incluindo a análise baseada em genômica completa, C. jejuni patogênese ainda é mal understood, em contraste com outros agentes patogénicos entéricos, tais como Salmonella, Escherichia coli, Shigella, e Vibrio cholerae. A falta de um modelo animal de pequeno conveniente é uma das principais razões para esta 3. Também a amplamente utilizados em modelos in vitro de infecção são mais apropriado para estudar microaerofílica C. jejuni, do que para outros agentes patogénicos entéricos que são anaeróbios facultativos. Enquanto C. jejuni é reconhecido como um agente patogénico invasor, os mecanismos de C. jejuni invasão das células epiteliais intestinais (IECs) ainda não são claras 4,5. C. invasão jejuni demonstrou ser dependente quer microfilamentos, os microtúbulos, uma combinação de ambos, nem 5. A confusão nesta área é mais provavelmente devido ao uso de inadequado em condições de ensaio in vitro. Um número de diferentes ensaios de cultura de células foram utilizadas para investigar as interacções de C. jejunicom as células hospedeiras. Caco-2, 6, 7 INT 407, T84 e 8 linhas de células têm sido usados ​​para estudar as capacidades de adesão e invasão de diferente C. jejuni. No entanto, os níveis de adesão bacteriana e invasão por C. jejuni com IECs são drasticamente mais baixos do que para outros patógenos entéricos 9. O coculturing de C. jejuni com IECs é normalmente realizado em uma incubadora de CO 2 sob condições próximas a níveis de oxigénio atmosférico, necessária para a sobrevivência das IECs. C. expressão gênica jejuni será muito diferente no ambiente de baixo oxigênio do lúmen intestinal em relação às condições atmosféricas de oxigênio. A utilização de um sistema de câmara de difusão vertical (VCC), foi desenvolvido o qual permite a co-cultura de bactérias e células hospedeiras diferentes meios e sob condições de gás 1,10,11. Este sistema mimetiza as condições presentes no intestino humano, onde as bactérias irão ser under condições de muito baixa tecido, enquanto o oxigênio será fornecido com o oxigênio da corrente sanguínea. As monocamadas de IEC polarizadas crescidas em filtros especiais 0,4 mM foram colocados num VCC criar um compartimento apical e basolateral, que foram preenchidos individualmente com caldo bacteriano e meio de cultura de células, respectivamente (Figura 1). O VDC foi colocado na atmosfera variável incubadora contendo 85% de N 2, 5% de O2, e 10% de CO 2, a 37 ° C, o que representa condições óptimas para C. jejuni. O compartimento apical foi deixada em aberto e exposto à atmosfera microaeróbia dentro da atmosfera variável incubadora, enquanto que o compartimento basolateral selado foi fornecido com oxigénio por administração constante de 5% de CO 2 / S mistura gasosa de 95% 2, com um tubo de saída da prevenção da acumulação de pressão . Caco-2, a sobrevivência das células e integridade monocamada sob estas condições foram confirmadas através da monitorização do elemento transepitelialresistência ctrical (TEER) através da monocamada durante 24 h, para demonstrar a separação de sobrevivência de células Caco-2 em monocamada de células e física dos compartimentos apical e basolateral sob baixas condições de oxigénio no compartimento apical. O TEER de monocamadas em VDCs mantidos na incubadora de atmosfera variável (condições microaeróbias) e um padrão de cultura de células incubadora de CO 2 (condições aeróbias) não apresentaram diferenças significativas, indicando integridade da monocamada de células em diferentes condições atmosféricas 1. Sob condições microaeróbicas, junções apertadas permaneceu presente e uniformemente distribuída entre as bordas das células, com um padrão de coloração occludin semelhantes às células mantidas sob condições aeróbicas 1. As interações de C. jejuni com células Caco-2 e T84 células na VCC foram investigados avaliando interacção bacteriana (adesão e invasão) e invasão. Dois C. diferente jejuni tipo selvagem strains foram usados ​​1. C. jejuni 11168H hypermotile é um derivado da estirpe NCTC11168 sequência original. A tensão 11168H mostra níveis muito mais elevados de colonização em um modelo de colonização garota e, assim, é considerada uma cepa adequada de usar para estudos de interação patógeno-hospedeiro. 81-176 é isolar um ser humano e é uma das estirpes laboratoriais amplamente estudados mais invasivos. C. jejuni foram adicionados ao compartimento apical de uma VCC, quer sob condições microaeróbias ou aeróbico. Observou-se taxas mais elevadas para a interação e invasão foram registradas para C. jejuni em condições microaeróbicas 1. O aumento C. jejuni interacções não eram devidas a um aumento no número de bactérias em condições microaeróbias 1. Estes dados apoiam a hipótese de que o ambiente de baixo teor de oxigénio no compartimento apical do VDC aumenta as interacções de bactérias hospedeiras, e indica que as propriedades invasivas de C. jejuni são incraliviou sob estas condições. Este foi o primeiro relato da utilização do modelo de VCC a estudar um agente patogénico bacteriano invasiva e realça a importância de efectuar ensaios de infecção in vitro sob condições que imitar mais de perto a situação in vivo. O VDC modelo pode ser usado para estudar as interacções hospedeiro-agente patogénico para muitas espécies de bactérias diferentes.

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Speciality vertical diffusion system for use with Snapwell inserts</td>
			<td>Harvard Apparatus</td>
			<td>66-0001</td>
			<td>Manifold &#38; six Snapwell chambers</td>
		</tr>
		<tr>
			<td>Caps</td>
			<td>Harvard Apparatus</td>
			<td>66-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>O-rings</td>
			<td>Harvard Apparatus</td>
			<td>66-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamps</td>
			<td>Harvard Apparatus</td>
			<td>66-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Snapwell filters (pore size 0.4 &#956;m)</td>
			<td>Corning Costar</td>
			<td>3407</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell ERS-2 Volt-Ohm resistance meter</td>
			<td>Millipore</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>WPA Lightwave II spectrophotometer</td>
			<td>Biochrom</td>
			<td>80-3003-72</td>
			<td></td>
		</tr>
	</tbody>
</table>

enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model

As interacções dos agentes patogénicos bacterianos com células hospedeiras têm sido investigados extensivamente usando métodos de cultura de células in vitro. No entanto, como estes ensaios de cultura de células são realizadas em condições aeróbias, estes modelos in vitro não pode representar exactamente o ambiente in vivo nas quais as interacções hospedeiro-agente patogénico a ter lugar. Nós desenvolvemos um modelo in vitro de infecção que permite a co-cultura de bactérias e células hospedeiras diferentes meios e sob condições de gás. A Câmara de difusão vertical (VDC) modelo imita as condições do intestino humano, onde as bactérias estarão em condições de muito baixo do tecido, enquanto o oxigênio será fornecido com o oxigênio do sangue. Colocação de células (IEC) monocamadas epiteliais intestinais polarizadas crescidas em Snapwell insere um VCC cria compartimentos separados apical e basolateral. O compartimento basolateral é preenchido com meio de cultura de células, selado e perfundido com oxigénio wHilst o compartimento apical é enchido com caldo, mantida aberta e incubados sob condições microaeróbias. Tanto as células Caco-2 e T84 IECs pode ser mantida na VCC sob estas condições sem quaisquer efeitos nocivos aparentes na sobrevivência celular ou a integridade monocamada. Coculturing experimentos realizados com diferentes C. as estirpes de tipo selvagem jejuni e diferentes linhas IEC no modelo VCC com condições microaeróbias no compartimento apical reprodutivelmente resultar num aumento do número de interagir (quase 10 vezes) e bactérias intracelulares (quase 100 vezes) em comparação com as condições de cultura aeróbias 1. O ambiente criado no modelo VCC imita mais de perto o ambiente encontrado por C. jejuni no intestino humano e destaca a importância da realização de ensaios de infecção in vitro em condições que mais de perto imitar a realidade em vivo. Propomos que o uso do modelo VDC permite novas interpretações das interações apostarween agentes patogénicos bacterianos e as células hospedeiras.

Coculturing experimentos realizados com um C. jejuni estirpe de tipo selvagem e IECs no modelo VCC com condições microaeróbias no compartimento apical demonstraram um aumento no número de interagir (quase 10 vezes) e bactérias intracelulares (quase 100 vezes) em comparação com as condições de cultura aeróbias em um tempo maneira dependente da 1. Esta observação foi reprodutível usando duas C. diferente jejuni do tipo selvagem (11168H e 81-176) e duas linhas IEC diferent...

Watch this Scientific Journal Video about Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model at JoVE.com

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

Realizando experimentos de cultura celular para investigar a adesão bacteriana e invasão em condições aeróbias é geralmente representativa do<em&gt; In vivo</em&gt; Ambiente. Um modelo de câmara de difusão vertical permite estudo das interações do patógeno humano<em&gt; Campylobacter jejuni</em&gt; Com células epiteliais intestinais em mais<em&gt; In vivo</em&gt; Condições semelhantes, resultando na invasão bacteriana melhorada.

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 ...

enteric-bacterial-invasion-intestinal-epithelial-cells-vitro-is

Research

JoVE Journal

Immunology and Infection

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

13.7K visualizações.  London School of Hygiene & Tropical Medicine. Realizando experimentos de cultura celular para investigar a adesão bacteriana e invasão em condições aeróbias é geralmente representativa do In vivo Ambiente. Um modelo de câmara de difusão vertical permite estudo das interações do patógeno humano Campylobacter jejuni Com células epiteliais intestinais em mais In vivo Condições semelhantes, resultando na invasão bacteriana melhorada.

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

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

In vitro a formação de biofilme em um slide Câmara de 8 poços

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Imunologia e Infecção

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Usando a luciferase Infecções bacterianas Imagem em camundongos

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Invasão das células humanas por um agente patogénico

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

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 ...

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 ...

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. 
We found the optimum fatty acid concentrations to be between 1-5 ng/&mu;l, and that for cholera toxin to be &lt; 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

Determinação de ácidos graxos tolerável e Concentrações toxina da cólera Usando Humanos células epiteliais intestinais e BALB / c mouse macrófagos

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

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 ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Avaliação da invasão bacteriana de células cardíacas na cultura e colonização cardíaca em camundongos infectados usando listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Análise de imunofluorescência da formação de grânulos de estresse após o desafio bacteriano de células de mamífero

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Um do elevado-throughput Shigella-ensaio bactericida específico

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...