Este trabalho foi apoiado pelo financiamento da Fundação de Crohn e Colite da América, (CCFA; 3711) Prevenção de Câncer e Instituto de Pesquisa de Texas (CPRIT; RP160169) e UT Southwestern Medical Center dado a MHZ

Todos os experimentos aqui descritos foram realizados utilizando camundongos 6-8 semanas de idade do sexo masculino de tipo selvagem (C57BL6 / J) mantido em um free (SPF) instalação de patógenos específicos no Centro de Recursos Animal (ARC), UT Southwestern Medical Center. Todos os estudos foram aprovados pelo Comitê Institucional de Animal Care and Use (IACUC) e foram conduzidos de acordo com as diretrizes IACUC e do National Institutes of Health Guide para o Cuidado e Uso de Animais de Laboratório. 1. Recolha e Preparação do Colon <ol><li> Eutanásia ratos com asfixia com CO 2, seguida por deslocação cervical. </li><li> Spray de ratos com 70% de etanol e fixá-membros a uma placa prendendo mantendo lado o rato dorsal para baixo no tabuleiro. </li><li> Usando tesouras e pinças pré-esterilizados dissecando, fazer uma incisão na linha média no peritônio do abdômen. Abra o abdómen dobrando o peritônio com uma pinça. </li><li> Remover o intestino a partir da cavidade abdominal with dissecando pinça e tesouras. Separa-se o cólon a partir do intestino delgado através do corte na parte inferior do ceco e a outra extremidade no recto. </li><li> Colocar o cólon numa placa de Petri estéril contendo PBS gelado. Lavar o conteúdo do lúmen do cólon com PBS arrefecido em gelo, utilizando uma seringa de 20 ml que prende uma agulha G 20 ou uma agulha de gavagem oral até que as fezes no interior do lúmen do cólon é completamente removido. </li><li> Corte do cólon com uma tesoura longitudinalmente. Lavar o cólon, mediante agitação vigorosa em PBS arrefecido com gelo numa placa de Petri estéril. </li><li> Corte o tecido do cólon em pedaços de aproximadamente 1 cm de comprimento, utilizando um bisturi ou tesoura estéril. </li><li> Registar o peso das peças do cólon. NOTA: Todas as etapas na Seção 1 pode ser realizada dentro ou fora de uma cabine de segurança biológica. Uma vez que dois pontos estão prontos para a cultura, todas as etapas devem ser executadas dentro de uma cabine de segurança biológica para manter a esterilidade. </li></ol> 2. Colon Orguma Cultura <ol><li> Coloque um filtro celular (100 um) sobre uma placa de cultura celular de 6 poços. Transferir todas as peças do cólon recolhidos a partir de um rato para o filtro de células. </li><li> Adicionar 5 ml de meio DMEM / F12 contendo FBS a 5%, penicilina-estreptomicina (1x), e gentamicina (20 ug / ml). Completamente cobrir os pedaços do cólon com a mídia. </li><li> Incubar durante 2 horas a 37 ° C numa incubadora com 5% de CO 2 e 95% de ar. </li><li> Levante o filtro de células e aspirar a mídia. </li><li> Coloque o filtro de células de volta no bem e adicionar 5 ml meio fresco sem quaisquer antibióticos. Levante o filtro de células e aspirar a mídia. </li><li> Repetir o passo de lavagem acima (Passo 2.5) duas vezes mais (total de 3x) para remover todos os antibióticos residuais. </li><li> Transferir as peças do cólon a partir de um único filtro de células em um único poço de uma placa de cultura celular de 12 poços estéreis. </li><li> Adicionar meio DMEM / F12 contendo FBS a 5% (sem antibióticos). Ajuste o volume do meio com o weigHT das peças do cólon, por exemplo, 1 ml de meio por 100 mg de tecido. Incubar durante 12 h a 37 ° C numa incubadora de injecção de 5% de CO 2 e 95% de ar. </li><li> Recolhe-se o sobrenadante de cultura num tubo esterilizado de 1,5 mL. </li><li> Centrifuga-se a 12000 xg, a 4 ° C durante 5 min. Separa-se o sobrenadante para um novo tubo de 1,5 mL para uso nas bactérias matando ensaio e / ou outros ensaios imunológicos. O sobrenadante pode ser armazenado a -80 ° C até o ensaio. </li><li> Recolher as peças de cólon em um tubo para o isolamento de RNA. </li></ol> 3. E. coli Ensaio Killing <ol><li> Inocular E. coli em 5 mL de meio de Luria-Bertani (LB) num tubo de 15 ml e incuba-se a 37 ° C com agitação a 200 rpm durante a noite. Mantenha a tampa do tubo de cultura levemente solto. </li><li> Centrifuga-se o tubo de cultura bacteriana a 1.200 xg durante 10 min a 4 ° C. Remover o sobrenadante e ressuspender o sedimento de bactérias em 5 ml de PBS arrefecido com gelo. </li><li> Transferir 1 ml do Bactsuspensão erial em uma OD cuvete e medida a 600 nm. Use PBS como branco. </li><li> Calcula-se a unidade de formação de colónias (ufc) utilizando uma curva padrão predeterminada. Aqui, assumir que uma OD = 2 x 10 9 cfu / mL (aproximadamente). </li><li> Dilui-se a suspensão bacteriana para fazer a suspensão de 1 x 10 5 CFU / mL. </li><li> Transferir o sobrenadante de cultura do cólon órgão (500 ul / poço) recolhida na secção 2.10 em poços duplicados de uma placa de cultura celular de 24 poços. </li><li> Adiciona-se 10 uL de cultura de E. coli (1000 cfu) para um poço contendo 500 uL do cólon sobrenadante da cultura de órgãos. Deixar o outro bem sem qualquer inoculação de E. coli para confirmar que o sobrenadante de cultura do cólon órgão não contém qualquer contaminação. </li><li> Incubar o mesmo número de bactérias (1000 cfu) no meio de cultura (DMEM / F12 + 5% de FBS) sem quaisquer antibióticos como um controlo. </li><li> Incubar a 37 ° C durante 1 h. </li><li> Transferir 50 uL de cada amostra gota-wise em placas de ágar MacConkey. Incubar as placas de ágar de MacConkey a 37 ° C durante a noite. </li><li> Contar o número de colónias e calcular a CFU / ml. </li></ol> 4. O efeito de fatores extrínsecos e intrínsecos no cólon Antimicrobianos respostas de defesa do hospedeiro NOTA: O ensaio de morte antimicrobiano, tal como descrito aqui pode ser adoptada para examinar o efeito de padrões de agentes patogénicos associados moleculares (PAMPs) e citocinas sobre a actividade bactericida de sobrenadante de cultura de órgãos. Um exemplo de tal experiência usando IL-1β e IL-18 é descrita abaixo. <ol><li> Recolha dois pontos de camundongos como descrito na Seção 1. </li><li> Coloque os dois pontos em uma toalha de papel estéril. Corte do cólon longitudinalmente em três partes com uma tesoura (Figura 2). </li><li> Lave cada parte do cólon em PBS gelado como descrito na Seção 1. </li><li> Corte cada parte do cólon em pedaços pequenos e pesam de forma asséptica. Transferir as peças de cada partedo cólon em três filtros celulares separadas (100 um) colocadas em três poços de uma placa de cultura de células de 6 poços (Figura 2). </li><li> Adicionar 2 ml de meio DMEM / F12 contendo FBS a 5%, penicilina-estreptomicina (1x), e gentamicina (20 ug / ml). </li><li> Incubar durante 2 horas a 37 ° C numa incubadora de injecção de 5% de CO 2 e 95% de ar. </li><li> Lavam-se as peças do cólon tal como descrito no 2,4-2,6. Transferir as peças de cólon de um único filtro de células em um único poço de uma placa de cultura celular de 12 poços estéreis. Os três poços contendo três partes do cólon a partir de um único rato deve ser designado como não tratada, a IL-1β, e IL-18 (Figura 2). </li><li> Adicionar meio DMEM / F12 contendo FBS a 5% (sem antibióticos). Ajustar o volume do meio com o peso das peças do cólon, por exemplo, 1 mL de meio por 100 mg de tecido. </li><li> Estimular a cultura de órgãos cólon com IL-1β (20 ng / ml) ou IL-18 (20 ng / mL) durante 12 h. Os dois pontos não tratados<html> cultura de órgãos serve como controlo. </li><li> Após 12 h de incubação a 37 ° C numa incubadora de injecção de 5% de CO 2 e 95% de ar, recolher o sobrenadante da cultura em um tubo de 1,5 ml estéril. </li><li> Transferir 500 uL de sobrenadante de cultura em poços duplicados de uma placa de 24 poços. </li><li> Inocular E. coli (1.000 ufc) no cólon órgão sobrenadante de cultura, conforme descrito na Seção 3. Para cada condição, inocular E. coli em um único poço. A outra cavidade contendo sobrenadante de cultura de órgãos mesmo sem E. coli vai servir como um controlo para a contaminação bacteriana. </li><li> Incubar a mesma quantidade de bactérias em meios de cultura sem nenhum antibiótico como um controlo. </li><li> Incubar a 37 ° C durante 1 h. </li><li> Coloque 50 mL de cada amostra gota a gota, em placas de ágar MacConkey. Incubar as placas de agar MacConkey para a noite a 37 ° C. </li><li> Contar o número de colónias e calcular o CFU / mL (Figura 4). </li></ol>e_title &quot;&gt; 5. Medição da expressão de genes antimicrobianos <ol><li> Após incubação durante a noite (12 h) de cultura de órgão do cólon tal como descrito nas secções 2 e 4, lavar as peças do cólon com 2 ml de PBS arrefecido com gelo (2x). </li><li> Recolher as peças do cólon em um tubo livre com tampa de rosca 2 mL RNase / DNase. Colocar os tubos em gelo. </li><li> Adicionar 1 ml de reagente Trizol comercial e lisar os grânulos da matriz para dentro do tubo. </li><li> Lise do tecido utilizando um homogeneizador de tecido automatizada. </li><li> Recolhe-se o lisado de tecido para um novo tubo de microcentrifugação. </li><li> Isolar RNA usando o protocolo padrão. </li><li> Medir a concentração de ARN. </li><li> Diluir apropriadamente ARN com água desionizada e utilizar 500 ng de ARN para sintetizar ADNc. </li><li> Usar o cDNA para a análise em tempo real de RT-PCR dos genes alvo antimicrobianas, citocinas, quimiocinas, e outros genes de interesse. </li></ol>

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The authors have no competing financial interests.

As células do epitélio intestinal são muito sensíveis em termos das suas necessidades de crescimento e, por conseguinte, difícil de cultura. As células epiteliais isoladas por tratamento com EDTA não sobrevivem no meio de cultura celular convencionais, tais como DMEM 8. Portanto, estudos de interação patógeno-hospedeiro usando cripta isolados ou células epiteliais primárias são muito desafiador. Recentemente, Sato et ai. descrito um sistema de cultura organï¿½de cripta, que é mui...

O intestino representa um sistema dinâmico, que actua como uma barreira para os microrganismos comensais, luta contra os agentes patogénicos invasores, e regula a composição microbiana 1. As células epiteliais do intestino, que consiste em enterócitos, células caliciformes, células de Paneth e células enteroendócrinas, são as principais populações de células que fornecem respostas de defesa do hospedeiro contra a microbiota intestinal. As células caliciformes produzir mucinas que criam uma zona desmilitarizada na parte superior da camada epitelial 2. As células de Paneth e enterócitos produzem peptídeos antimicrobianos, citocinas e espécies de oxigênio e nitrogênio reativos que constituem respostas de defesa do hospedeiro antimicrobianos e contribuir para a definição da composição microbiana intestinal 3, 4. Em adição às células epiteliais, as células do sistema imunológico, incluindo macrófagos, células dendríticas, neutrófilos, células assassinas naturais, os linfócitos e inna<html> células linfóides te na lâmina própria e submucosa desempenhar um papel crítico na resposta de defesa do hospedeiro antimicrobianos intestinais pela produção de citocinas, quimiocinas e outros mediadores 5 - 7. A fim de compreender como o sistema imune da mucosa regula microbiota e fornece protecção contra a infecção microbiana, é importante considerar a interação complexa entre as populações de células heterogéneas do intestino. No entanto, um modelo in vitro que engloba todas as características do intestino não está disponível. Portanto, os estudos moleculares sobre a interação patógeno-hospedeiro no intestino são altamente desafiador. Ao longo dos últimos anos, vários sistemas modelo que mimetizam aspectos da mucosa intestinal tem sido desenvolvido para investigar os processos patofisiológicos envolvidos em doenças inflamatórias do intestino (IBD) e outras desordens gastrointestinais 8 -= &quot;xref&quot;&gt; 14. linhas de células epiteliais intestinais imortalizadas são muitas vezes usadas para estudar as respostas específicas de células epiteliais. No entanto, por causa da expressão diferencial de genes e função de células imortalizadas, os dados obtidos a partir de células usando esses muitas vezes não coincidir com os observados em estudos in vivo. Cripta intestinal organï¿½de cultura surgiu recentemente como um potencial instrumento para avaliar a resposta do epitélio intestinal a diferentes estímulos 13. Neste sistema, as células estaminais criptas são deixadas a crescer e a desenvolver uma estrutura organï¿½de 3D. Enquanto o sistema de cultura organï¿½de é muito útil para estudar muitos aspectos do epitélio intestinal, ele não imitam o complexo interacção de células do sistema imunológico, células epiteliais e produtos microbianos. A cultura ex vivo do tecido intestinal oferece uma melhor representação in vivo de respostas de defesa do hospedeiro. Neste método, uma parte do intestino é cultivada numa placa de cultura de células with meios adequados que permitem que os diferentes tipos de populações de células no intestino para ser metabolicamente activa durante pelo menos 48 h. Assim, uma cultura ex vivo do órgão pode ser utilizado para medir a expressão de genes antimicrobianos e as respostas de defesa do hospedeiro do intestino a um estímulo específico. Os investigadores têm vindo a utilizar o sistema de cultura ex vivo de órgãos para estudar as respostas de defesa do hospedeiro contra a infecção microbiana no intestino 15-21. Nós recentemente adoptado o sistema de cultura de órgãos para estudar o papel da inflammasome nas respostas de defesa do hospedeiro antimicrobianos em dois pontos do rato 22. O inflamassoma é uma plataforma molecular para a activação de caspase-1, o que é necessário para a produção de IL-1β amadurecido e IL-18. Mostrámos que a IL-1β e IL-18 induzem péptidos antimicrobianos que matam eficazmente pathobionts comensais, tais como E. coli </em&gt;. Esta observação foi consistente com aumento da carga E. coli em dois pontos do rato inflammasome defeituoso 22. Este sistema, por conseguinte, pode ser utilizado para estudar o papel de receptores de reconhecimento de padrões (PRRs) e outras moléculas imunitárias inatas em respostas de defesa do hospedeiro antimicrobianos intestinais, bem como patogénese de desordens intestinais, tais como doença inflamatória do intestino (IBD) e cancro colorrectal (CRC). Existem mais de 200 genes de susceptibilidade, IBD e mutações em muitos destes genes estão associados com a composição microbiana alterada no intestino. É de grande importância clínica para determinar o mecanismo preciso através do qual os genes de susceptibilidade IBD-regular a flora intestinal. O objectivo geral do presente método é a introdução de um protocolo básico de cultura de órgãos ex vivo do cólon e demonstrar como este método de cultura pode ser usado para estudar as respostas de defesa do hospedeiro antimicrobianos do intestino.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/ F12</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline, modified, w/o Calcium chloride &#38; Magnesium chloride</td>
			<td>Sigma</td>
			<td>5634</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS, heat inactivated</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin solution</td>
			<td>Sigma</td>
			<td>G1272</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-1b recombinan</td>
			<td>Reprokine</td>
			<td>RKP10749</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-18 recombinant</td>
			<td>Reprokine</td>
			<td>RKP70380</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Luria-Bertani Broth&#160;</td>
			<td>BD Bioscience</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Difco Dehydrated Culture Media: MacConkey Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0075-17-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Uded to measure RNA concentration</td>
		</tr>
		<tr>
			<td>UV/Vis Spectrophotometer</td>
			<td>BECKMAN</td>
			<td>DU 530</td>
			<td>Used to determine E. coli count</td>
		</tr>
		<tr>
			<td>iScript RT Supermix, 100 rxns</td>
			<td>Bio-Rad</td>
			<td>1708841</td>
			<td></td>
		</tr>
		<tr>
			<td>iTaq Univer SYBR Green Supermix&#160;</td>
			<td>Bio-Rad</td>
			<td>1725125&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing Matrix S (1/8&#34;), 2 mL Tube</td>
			<td>MP Biomedicals</td>
			<td>116925500</td>
			<td>Used to homgenize colon organ for RNA isolation</td>
		</tr>
		<tr>
			<td>FastPrep-24 5G System</td>
			<td>Bio-Rad</td>
			<td>116005500</td>
			<td></td>
		</tr>
		<tr>
			<td>100x15 Petri Dish</td>
			<td>Falcon</td>
			<td>5687</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate 6well ps TC CS100, Cellstar, 6w, tc, F-bottom (Flat), w/lid, sterile</td>
			<td>Cellstar</td>
			<td>5085</td>
			<td></td>
		</tr>
		<tr>
			<td>100 micron cell strainer</td>
			<td>Falcon</td>
			<td>5698</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21R Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ST40R Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Scientific orbital shaker</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the ex vivo colon organ culture and its use in antimicrobial host defense studies

O intestino tem uma arquitectura de estruturas cripta repetitivas que consistem em diferentes tipos de células epiteliais, lâmina própria contendo células do sistema imunológico, e estroma. Todas estas células heterogéneas contribuem para a homeostase intestinal e participar na defesa do hospedeiro antimicrobiana. Portanto, a identificação de um modelo substituto para estudar a resposta imune e atividade antimicrobiana do intestino em um ambiente in vitro é extremamente desafiador. Os estudos in vitro utilizando linhas de células epiteliais do intestino imortalizadas ou cripta mesmo primária organï¿½de cultura não representam a fisiologia exata do intestino normal e seu microambiente. Aqui, nós discutimos um método de cultura de tecido do cólon do rato numa placa de cultura e como este sistema de cultura ex vivo de órgãos pode ser implementado em estudos relacionados com as respostas de defesa do hospedeiro antimicrobianos. Em experiências representativas, que mostrou que dois pontos em cultura de órgão expressar peptídeos antimicrobianos em response de IL-1β e IL-18. Além disso, as moléculas efectoras antimicrobianos produzidos pelos tecidos do cólon em cultura de órgãos a matar eficientemente Escherichia coli in vitro. Esta abordagem, portanto, pode ser utilizada para dissecar o papel de padrões moleculares pathogen- e associada ao perigo e os seus receptores celulares na regulação de respostas imunes inatas intestinais e as respostas de defesa do hospedeiro antimicrobianos.

Uma imagem representativa de dois pontos em cultura de órgãos é mostrado na Figura 1. As peças do cólon na cultura permanecem metabolicamente e fisiologicamente ativa. Eles responder de forma eficiente aos estímulos exógenas adicionadas ao meio de cultura. Um fluxo da preparação do tecido do cólon para a cultura ex vivo e estimulação com estímulos exógenos, por exemplo, IL-1β e IL-18 de trabalho esquemática, é mostrado na Figura...

Watch this Scientific Journal Video about The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies at JoVE.com

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

o<em&gt; Ex Vivo</em&gt; Órgão Cultura Colon e seu uso em Antimicrobial host Estudos de Defesa

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

the-ex-vivo-colon-organ-culture-its-use-antimicrobial-host-defense

Research

JoVE Journal

Immunology and Infection

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

13.9K visualizações.  UT Southwestern Medical Center. The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine. 

Vídeo: The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

In vivo de imagens de parasitas Leishmania transgênicas em um host ao vivo

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Imunologia e Infecção

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Utilização do Modelo EpiAirway para caracterizar a longo prazo Interações Hospedeiro-Patógeno

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

o<em&gt; Ex Vivo</em&gt; Cultura e Reconhecimento de Padrões Receptor Stimulation of Mouse Intestinal Organóides

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

Culturas placentária e Decidual órgão humano para estudar Infecções na interface materno-fetal

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

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

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

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

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

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

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

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Um modelo de cultura primária de células bolsas serosas Ex Vivo frango para estudar a patogênese de doenças infecciosas bolsas serosas vírus

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

Otimização da Técnica de Manguito para Transplante de Coração de Murine

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

Estabelecendo um modelo de córnea porcina ex vivo para estudar tratamentos medicamentosos contra ceratite bacteriana

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Um ensaio ex vivo para estudar candida albicans Hórgênese Háfal no Trato Gastrointestinal

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

Teste de Eficácia de Antibióticos em um Modelo Ex vivo de Pseudomonas aeruginosa e Staphylococcus aureus Biofilms no Pulmão da Fibrose Cística

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

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