Os autores gostariam de agradecer Elliot Abattoir em Chesterfield por fornecer olhos suínos. Os anéis de vidro foram feitos com base em nosso projeto pelo soprador de vidro Dan Jackson do Departamento de Química da Universidade de Sheffield. Os autores gostariam de agradecer ao Conselho de Pesquisa Médica (MR/S004688/1) pelo financiamento. Os autores também gostariam de agradecer à senhora deputada Shanali Dikwella pela ajuda técnica na preparação da córnea. Os autores gostariam de agradecer ao senhor deputado Jonathan Emery pela ajuda na formatação de imagens.

Coelhos do laboratório albino foram sacrificados em laboratório por outros trabalhos experimentais planejados sob protocolos aprovados pelo home office. Os olhos não eram necessários para uso experimental nesses estudos, por isso foram utilizados para este protocolo.
1. Esterilização
<ol><li>PASSO CRÍTICO: Desinfete todos os fórceps e tesouras de molho por 1h em 5% (v/v) solução de Distel em água destilada, limpe com escova, enxágue com água da torneira e esterilize em forno a 185 °C por um mínimo de 2h.</li>
	<li>Esterilize todos os outros vidros e reagentes autoclavando a 121 °C por 15 minutos ou prepare os reagentes de acordo com as instruções do fabricante. Realizar os seguintes procedimentos em um gabinete de segurança de microbiologia classe II.</li>
</ol>2. Coleta de amostras
<ol><li>Coleção de olhos suínos<ol>
<li>Grandes porcas brancas, uma cruz com um javali hampshire foi usada. Os animais foram atordoados com uma corrente elétrica e os olhos foram enucleados 2h depois no matadouro.</li>
		<li>PASSO CRÍTICO: Uma vez enucleado, transfira os olhos para o laboratório em uma solução salina tamponada de fosfato estéril (PBS) para evitar que eles sequem e processem-nos imediatamente após a chegada.</li>
	</ol>
</li>
	<li>Coleção de olhos de coelho<ol>
<li>Extireta as córneas e mande para o laboratório em PBS estéril.</li>
	</ol>
</li>
</ol>3. Preparação do botão corneoscleral
<ol><li>Use fórceps estéreis para segurar o tecido ao redor do globo ocular e transferi-lo para uma placa de Petri. Remova o tecido conjuntivo e muscular ao redor do globo ocular em uma placa de Petri usando lâmina de bisturi nº 15 e fórceps.</li>
	<li>Levante suavemente o globo ocular enquanto segura o nervo óptico com fórceps e transfira para um frasco de 0,5 L cheio de PBS estéril.</li>
	<li>Uma vez que todos os olhos estejam limpos do tecido circundante, mova-os usando fórceps estéreis para outro frasco de 0,5 L preenchido com 3% (v/v) iodo de povidona na PBS e deixe por 1 min.</li>
	<li>Transfira os globos oculares para outro frasco de 0,5 L com PBS estéril.</li>
	<li>Use fórceps para segurar o olho ainda em uma placa de Petri e fazer um corte perto da córnea com uma lâmina de bisturi no 10A.</li>
	<li>PASSO CRÍTICO: Segure a borda do corte e use a tesoura para extirar a córnea deixando cerca de 3 mm de esclera ao redor da córnea. Certifique-se de que a extremidade afiada da tesoura não perfura a íris ou o tecido coroidal e está no espaço supra-choroidal.</li>
	<li>Segure o botão córneacleral com fórceps e use outro par de fórceps ponta pontiagudas para separar suavemente o tecido úveal.</li>
	<li>Levante o botão corneoscleral do globo remanescente e enxágue-o brevemente em 1,5% (v/v) solução de iodo povidone em PBS em uma placa de 12 poços.</li>
	<li>Coloque o botão corneoscleral em outra placa de 12 poços cheia de PBS estéril.</li>
	<li>Depois de processar todos os olhos (recomendado máximo de 40 olhos em um lote), coloque cada botão corneoscleral em uma placa de Petri individual (34 mm de diâmetro) lado epitelial para cima e despeje em 3 mL de cultura média pré-aquecida a 37 °C. NOTA: A composição do meio de cultura é a seguinte: o meio modificado da Águia (DMEM) modificado de Dulbecco: Ham's [1:1] complementado com 5 μg∙mL-1 insulina e 10 ng∙mL-1 fator de crescimento epidérmico (EGF), 10 ng 10% (v/v) soro fetal da panturrilha (FCS), 100 U∙mL-1 penicilina, 100 U∙mL-1 estreptomicina e 2,5 μg∙mL-1 anfotericina B. Como etapa opcional, o meio pode ser complementado com 50 g∙L-1 dextran para evitar o inchaço da córnea excisada durante as etapas de incubação.</li>
	<li>Incubar a 37 °C em uma incubadora de cultura tecidificada.</li>
</ol>4. Manutenção dos botões corneosclerais
<ol><li>Após 24 horas, use técnica asséptica para remover a mídia e substituir por 3 mL de mídia de cultura pré-aquecida fresca contendo antibióticos. Mantenha os botões corneosclerais na mídia com antibióticos por 48 horas para desinfetar as córneas. Incubar a 37 °C em uma incubadora de cultura tecidificada.</li>
	<li>PASSO CRÍTICO: Após 48 horas, remova a mídia e enxágue córneas com 2 mL de PBS. Em seguida, mantenha os botões corneosclerais em mídia livre de antibióticos por um mínimo de dois ou idealmente três dias antes da infecção experimental, para remover antibióticos residuais do tecido.</li>
	<li>Incubar a 37 °C em uma incubadora de cultura tecidificada. Mude a mídia pelo menos mais uma vez dentro desses três dias. Descarte córneas se alguma turbidez se desenvolver no meio livre de antibióticos.</li>
</ol>5. Preparação de um inóculo
<ol><li>Despeje 10 mL de caldo LB em um frasco cônico de 50 mL com uma rolha de espuma.</li>
	<li>Transfira uma colônia de P. aeruginosa cepa PAO1 ou coe PA14 de uma placa de ágar fresco e incubar a 37 °C por 3-4 h até que as bactérias estejam em fase de registro médio.</li>
	<li>Transfira a cultura das bactérias para um tubo de 50 mL e centrífuga a 3.000 x g por 5 min. Remova o supernatante e suspenda a pelota da célula na PBS.</li>
	<li>Repita o passo 5.3 mais duas vezes para lavar as células. Suspenda a pelota de célula em PBS e ajuste a densidade óptica em 600 nm para aproximadamente 0,6 usando PBS estéril como um branco.</li>
</ol>6. Infectando o botão corneoscleral
<ol><li>Remova a mídia da placa de Petri e enxágue córneas duas vezes com 1 mL de PBS estéril.</li>
	<li>Aperte suavemente as fórceps enquanto segura a córnea no meio. Use um bisturi de 10A para fazer quatro cortes – dois verticais, dois horizontais – na seção central do botão corneoscleral através da camada epitelial até o estroma subjacente.</li>
	<li>Coloque um molde de vidro estéril em uma placa de 6 poços com a parte larga para cima e coloque a córnea no meio do molde de vidro, lado do epitélio voltado para baixo. Faça o corte bem no centro da parte inferior do molde de vidro.</li>
	<li>PASSO CRÍTICO: Despeje 1 mL de 1% (w/v) ágar de ponto de fusão baixo dissolvido em DMEM para encher completamente o molde de vidro com córnea.</li>
	<li>Deixe o ágar definir e, em seguida, inverter o molde de vidro para que o epitélio córnea esteja voltado para cima.</li>
	<li>Pipeta 15 μL da cultura bacteriana com OD600nm = 0,6 (para P. aeruginosa isso equivale a aproximadamente 1 x 107 unidades de formação de colônias (CFU) em 15 μL) diretamente em uma área cortada e, em seguida, adicionar 85 μL de PBS ao topo para manter o moist epitélio da córnea. Diluir a cultura bacteriana restante e a placa no ágar para contar unidades de formação de colônias para o inóculo.</li>
	<li>Adicione 1 mL de DMEM sem antibióticos no fundo de cada poço com o molde de vidro. Incubar a placa de 6 poços com os botões corneosclera infectados em uma incubadora umidificada a 37 °C com 5% de CO2 por até 24 h.</li>
	<li>Configure córnea de controle não infectada ao lado de todos os experimentos. Para configurar o controle não infectado, substitua os 15 μL de cultura bacteriana na etapa 6.6 por PBS estéril.</li>
</ol>7. Homogeneização da córnea para colher as bactérias
<ol><li>Descarte o meio DMEM da parte inferior da placa de 6 poços e adicione 1 mL de PBS estéril para enxaguar a parte inferior do poço.</li>
	<li>Remova o PBS suavemente por pipetação sem tocar na parte central do botão corneoscleral. Remova o anel de vidro usando fórceps estéreis e coloque-o no Distel de 5%.</li>
	<li>Enxágue suavemente a parte superior do botão corneoscleral com 1 mL de PBS duas vezes [opcional].</li>
	<li>Segure a borda do botão corneoscleral com fórceps finos e desprende-o do ágar por baixo.</li>
	<li>Transfira a córnea para um tubo de 50 mL cheio de gelo frio de 1-2 mL de PBS.</li>
	<li>Use um homogeneizador de ponta fina para ver o topo da córnea infectada. O tecido não precisa ser completamente liquidizado. O homogeneizador ajuda a separar bactérias do epitélio córnea e da área de corte.</li>
	<li>Vórtice a córnea em PBS por alguns segundos para misturar o conteúdo.</li>
	<li>Adicione 20 μL do homogeneado a 180 μL de PBS e realize diluições seriais em uma placa de 96 poços.</li>
	<li>Diluir a suspensão em 10-4 e 10-5 diluição e pipeta 10 μL do homogeneato diluído com bactérias em uma placa de ágar de sangue. Incubar a placa por 8 horas e contar o número de UFC. Ao testar o efeito de antimicrobianos, o fator de diluição apropriado deve ser chegado experimentalmente.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug-Resistant+Pseudomonas+aeruginosa+Keratitis+Risk+Factors,+Clinical+Characteristics,+and+Outcomes.">Multidrug-Resistant Pseudomonas aeruginosa Keratitis Risk Factors, Clinical Characteristics, and Outcomes.</a> Ophthalmology. 122 (10), 2110-2114 (2015).</li><li>Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratitis.">Keratitis.</a> Bioscience Reports. 21 (4), 419-444 (2001).</li><li>Sharma, G., 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=Pseudomonas+aeruginosa+biofilm:+Potential+therapeutic+targets.">Pseudomonas aeruginosa biofilm: Potential therapeutic targets.</a> Biologicals. 42 (1), 1-7 (2014).</li><li>Ersoy, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+transmission+from+contact+lenses+to+porcine+corneas:+An+ex+vivo+study.">Bacterial transmission from contact lenses to porcine corneas: An ex vivo study.</a> Investigative Ophthalmology &amp; Visual Science. 46 (6), 2042-2046 (2005).</li><li>Duggal, N., 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=Zinc+oxide+tetrapods+inhibit+herpes+simplex+virus+infection+of+cultured+corneas.">Zinc oxide tetrapods inhibit herpes simplex virus infection of cultured corneas.</a> Molecular Vision. 23, 26-38 (2017).</li><li>Brothers, K., 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=Bacterial+Impediment+of+Corneal+Cell+Migration.">Bacterial Impediment of Corneal Cell Migration.</a> Investigative Ophthalmology &amp; Visual Science. 56 (7), (2015).</li><li>Alekseev, O., Tran, A. 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O principal motor por trás do desenvolvimento deste modelo de ceratite usando córnea porcina ex vivo é fornecer aos pesquisadores que desenvolvem novos antimicrobianos com um modelo in vitro representativo para determinar com mais precisão a eficácia antimicrobiana nos estágios pré-clínicos. Isso fornecerá aos pesquisadores envolvidos no desenvolvimento de novos antimicrobianos maior controle sobre o design e formulação de medicamentos nos estágios pré-clínicos, aumentará o sucesso em ensaios clínicos, re...

As infecções corneais são importantes causas de cegueira e ocorrem em proporções epidêmicas em países de baixa e média renda. A etiologia da doença varia de região para região, mas as bactérias são responsáveis pela grande maioria desses casos. Pseudomonas aeruginosa é um patógeno importante que causa uma doença rapidamente progressiva. Em muitos casos, os pacientes ficam com cicatrizes estromais, astigmatismo irregular, necessitam de transplante ou, no pior dos casos, perdem um olho1,2.
A ceratite bacteriana causada pela P. aeruginosa é uma infecção ocular difícil de tratar particularmente devido ao crescente surgimento de cepas resistentes antimicrobianas de P. aeruginosa. Na última década, tornou-se evidente que testar e desenvolver novos tratamentos para infecções córneas, em geral, e aqueles causados por Pseudomonas sp., em particular, são essenciais para combater a tendência atual de resistência a antibióticos3.
Para testar a eficácia de novos tratamentos para infecções córneas, os métodos microbiológicos in vitro convencionais são um substituto ruim devido à diferença na fisiologia bacteriana durante a cultura laboratorial e durante infecções in vivo, bem como devido à falta da interface hospedeira4,5. Os modelos in vivo animal, no entanto, são caros, demorados, só podem entregar um pequeno número de réplicas e levantar preocupações sobre o bem-estar animal.
Neste artigo, demonstramos um modelo simples e reprodutível organotípico ex vivo porcina de ceratite que pode ser usado para testar vários tratamentos para infecções agudas e crônicas. Usamos P. aeruginosa para este experimento, mas o modelo também funciona bem com outras bactérias, e organismos como fungos e leveduras que causam ceratite.

<table>
	<tbody>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>SLS</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar 12 well plate</td>
			<td>Greiner Bio-One</td>
			<td>665180</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma</td>
			<td>31425-100mg-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Distel</td>
			<td>Fisher Scientific</td>
			<td>12899357</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM + glutamax</td>
			<td>SLS</td>
			<td>D0819</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual Oven Incubator</td>
			<td>SLS</td>
			<td>OVe1020</td>
			<td>Sterilising oven</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>SLS</td>
			<td>E5036-200UG</td>
			<td></td>
		</tr>
		<tr>
			<td>F12 HAM</td>
			<td>Sigma</td>
			<td>N4888</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal calf serum</td>
			<td>Labtech International</td>
			<td>CA-115/500</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>15307805</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld homogeniser 220</td>
			<td>Fisher Scientific</td>
			<td>15575809</td>
			<td>Homogeniser</td>
		</tr>
		<tr>
			<td>Heracell VIOS 160i</td>
			<td>Thermo Scientific</td>
			<td>15373212</td>
			<td>Tissue culture incubator</td>
		</tr>
		<tr>
			<td>Heraeus Megafuge 16R</td>
			<td>VWR</td>
			<td>521-2242</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Insulin, recombinant Human</td>
			<td>SLS</td>
			<td>91077C-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Sigma</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>Multitron</td>
			<td>Infors</td>
			<td>Not appplicable</td>
			<td>Bacterial incubator</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>SLS</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>SLS</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific</td>
			<td>12664785</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 35x10mm CytoOne</td>
			<td>Starlab</td>
			<td>CC7672-3340</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone iodine</td>
			<td>Weldricks pharmacy</td>
			<td>2122828</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe 2020</td>
			<td>Fisher Scientific</td>
			<td>1284804</td>
			<td>Class II microbiology safety cabinet</td>
		</tr>
		<tr>
			<td>Scalpel blade number 15</td>
			<td>Fisher Scientific</td>
			<td>O305</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Swann Morton</td>
			<td>Fisher Scientific</td>
			<td>11849002</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing a porcine ex vivo cornea model for studying drug treatments against bacterial keratitis

Ao desenvolver novos antimicrobianos, o sucesso dos ensaios em animais depende da extrapolação precisa da eficácia antimicrobiana de testes in vitro a infecções em animais in vivo. Os testes in vitro existentes tipicamente superestimam a eficácia antimicrobiana, pois a presença de tecido hospedeiro como barreira de difusão não é contabilizada. Para superar esse gargalo, desenvolvemos um modelo de ceratite bacteriana ex vivo porcina usando Pseudomonas aeruginosa como organismo prototipado. Este artigo descreve a preparação da córnea suína e o protocolo para o estabelecimento da infecção. Moldes de vidro sob medida permitem a configuração direta da córnea para estudos de infecção. O modelo imita a infecção in vivo, pois a proliferação bacteriana depende da capacidade da bactéria de danificar o tecido córnea. O estabelecimento da infecção é verificado como um aumento no número de unidades formadoras de colônias avaliadas por meio de placas viáveis. Os resultados demonstram que a infecção pode ser estabelecida de forma altamente reprodutível nas córneas ex vivo usando o método descrito aqui. O modelo pode ser estendido no futuro para imitar ceratite causada por microrganismos diferentes de P. aeruginosa. O objetivo final do modelo é investigar o efeito da quimioterapia antimicrobiana no progresso da infecção bacteriana em um cenário mais representativo de infecções in vivo. Ao fazê-lo, o modelo descrito aqui reduzirá o uso de animais para testes, melhorará as taxas de sucesso em ensaios clínicos e, finalmente, permitirá a tradução rápida de novos antimicrobianos para a clínica.

O design dos moldes de vidro é uma ideia inovadora e original, sendo que o uso nos permitiu configurar o modelo de forma consistente com problemas mínimos/sem contaminação. Os moldes foram preparados por um soprador de vidro na Universidade de Sheffield com base em um design (Figura 1A). A configuração experimental mantém a forma convexa da córnea e mantém bactérias no topo do epitélio onde ocorre a infecção(Figura 1B).
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Watch this Scientific Journal Video about Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis at JoVE.com

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

Este artigo descreve um protocolo passo a passo para criar um modelo de ceratite porcina ex vivo de ceratite bacteriana. Pseudomonas aeruginosa é usado como organismo prototípico. Este modelo inovador imita a infecção in vivo, pois a proliferação bacteriana depende da capacidade da bactéria de danificar o tecido córnea.

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 ...

establishing-porcine-ex-vivo-cornea-model-for-studying-drug

Research

JoVE Journal

Immunology and Infection

5.1K visualizações.  University of Sheffield. Este artigo descreve um protocolo passo a passo para criar um modelo de ceratite porcina ex vivo de ceratite bacteriana. Pseudomonas aeruginosa é usado como organismo prototípico. Este modelo inovador imita a infecção in vivo, pois a proliferação bacteriana depende da capacidade da bactéria de danificar o tecido córnea.

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

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Ceratite herpética recorrente Estroma em camundongos, um modelo para estudar HSK Humanos

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Imunologia e Infecção

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Imagem bacteriana Bioluminescent<em&gt; Em Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

Ensaios para a identificação de novos antivirais contra a febre catarral ovina vírus

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Um<em&gt; Ex vivo</em&gt; Modelo de um Ataque de T-Cell dirigiu-Oligodendrocyte em Agudos fatias de cérebro

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

De alto rendimento quantitativo em tempo real de RT-PCR Ensaio para Determinar Perfis de Expressão do tipo I e III do interferão subtipos

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

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

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

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

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

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

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

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Um ensaio Compatível de alta capacidade para avaliar droga Eficácia contra macrófagos passadas<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Ensaio de matar Opsonophagocytic para avaliar as respostas imunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Multi-scale Analysis of Bacterial Growth Under Stress Treatments

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to characterize the response of bacterial cells to stress-inducing treatments such as exposure to antibiotics or other antimicrobial compounds, irradiation, non-physiological pH, temperature, or salt concentration. Different stress treatments might disturb different cellular processes, including cell division, DNA replication, protein synthesis, membrane integrity, or cell cycle regulation. These effects are usually associated with specific phenotypes at the cellular scale. Therefore, understanding the extent and causality of stress-induced growth or viability deficiencies requires a careful analysis of several parameters, both at the single-cell and at the population levels. The experimental strategy presented here combines traditional optical density monitoring and plating assays with single-cell analysis techniques such as flow cytometry and real time microscopy imaging in live cells. This multiscale framework allows a time-resolved description of the impact of stress conditions on the fate of a bacterial population.

This protocol allows a time-resolved description of bacterial growth under stress conditions at the single-cell and the cell population levels.

Análise em multiescala do crescimento bacteriano tratamentos de estresse

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.