Desejamos agradecer à Johnson &amp; Johnson, à TB Alliance, à Astra Zeneca, à Rib-X e à Trius Therapeutics pela prestação de bedaquilina, PA-824 (pré-tonsanida), AZD5847, radezolid e tedizolid, respectivamente. Brendan Prideaux, Matthew Zimmerman, Stephen Juzwin, Emma Rey-Jurado, Nancy Ruel, Leyan Li e Danielle Weiner forneceram suporte com análise MALDI, métodos bioanalíticos, preparação do substituto caseu, síntese química e isolamento de caseum de coelho. Este trabalho foi realizado com financiamento da Fundação Bill e Melinda Gates, o prêmio # OPP1044966 e OPP1024050 para V. Dartois, NIH Shared Instrumentation Grant S10OD018072, bem como o financiamento conjunto da Fundação Bill e Melinda Gates e Wellcome Trust para um Centro de Excelência Para Lead Optimization para Doenças do Mundo em Desenvolvimento a P. Wyatt.

Todos os estudos com animais foram realizados de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório dos Institutos Nacionais de Saúde com a aprovação do Comitê Institucional animal Cuidado e Uso do NIAID (NIH), Bethesda, MD. Todos os estudos envolvendo o M. tuberculosis foram realizados num laboratório com nível de segurança biológica de contenção 3 (BSL-3). Modelo Infecção 1. Coelho e Coleção caseum <ol><li> Infect de coelhos Nova Zelândia brancos com M. tuberculosis usando um sistema de exposição de aerossóis só de nariz, como descrito anteriormente. 12, 13 permitir que a infecção progride para 12-16 semanas. Sedar os coelhos com 35 mg / kg de cetamina e 5 mg / kg de xilazina por via intramuscular, sacrificar os coelhos, com 0,22 ml / kg de pentobarbital de sódio por via intravenosa e a fenitoína de sódio e prosseguir com as necropsias. </li><li> Usando pinças e um bisturi, remove pulmões do ca peitoVidade. A partir de cada lobo pulmonar, dissecar cavidades individuais e grandes granulomas necróticos usando um bisturi. Cuidadosamente raspar fora caseu da cavidade e granuloma paredes. Pesar, gravar e armazenar amostras em tubos de 2 mL com tampa roscada a -20 ° C até que esteja pronto para uso. </li><li> Irradia-se as amostras de caseu infecciosas a 3 MegaRad em gelo seco para torná-las não infecciosas e seguras para utilização num laboratório BSL-2. </li></ol> 2. Geração In Vitro de Substituto Caseum de células THP-1 <ol><li> Crescer monócitos THP-1 em meio RPMI 1640 (L-glutamina 2 mM e soro fetal bovino a 10%) em frascos de cultura de células T175 (80 mL / balão). Incubar os frascos numa atmosfera de CO2 a 5% a 37 ° C durante 3-4 dias. </li><li> Centrifugar a cultura de um balão T175 em dois tubos cônicos de 50 mL a 150 xg por 5 min. Descartar o sobrenadante e suspender o sedimento em 10 mL de meio RPMI 1640. </li><li> Pipetar 5 μL desta cultura em um tubo de 1,5 mL contendo 45 μL de tazul rypan. Mistura-se cuidadosamente por pipetagem. Transferir 10 uL para um hemocitómetro e contar o número de monócitos THP-1 viáveis ​​(sem corante), utilizando um microscópio óptico (ampliação de 10X). Calcular o número de células viáveis ​​por ml de cultura. Diluir com meio RPMI para a densidade final de 1,25 x 10 6 culas / mL. </li><li> Carregar 40 ml da cultura em uma placa de cultura de células grandes (50 x 10 6 culas / placa). Adicionar 40 uL de 100 uM de PMA (forbol 12-myristate13-etilo preparado em etanol) e permitir que as células a aderir durante a noite na incubadora. NOTA: A concentração final de PMA é de 100 nm. </li><li> Dilui-se o ácido puro oleico (OA) (0,89 g / mL) em etanol para a concentração de 0,1 M (ou seja 31,7 mL de OA em 968,3 mL de etanol). Dilui-se esta solução em meio fresco RPMI pré-aquecido a uma concentração de 10 mM. Dilui-se esta suspensão de OA a 0,4 mM (concentração final de trabalho), em meio RPMI pré-aquecido a 37 ° C. </li><li> Remova a mídia e não existentesDas células de cultura de células e adicionar suavemente 40 mL de OA 0,4 mM aos macrófagos THP-1 (THP-M). Incubar a 37 ° C na incubadora durante a noite. </li><li> Use um microscópio de luz com uma ampliação de 40x para confirmar visualmente a presença de numerosas inclusões de corpo lipídico em cada THP-M. Remover todo o meio RPMI das placas de cultura de células e lavar cuidadosamente as células aderentes duas vezes com solução salina tamponada com fosfato (PBS) utilizando uma pipeta serológica de 50 mL. NOTA: Os corpos lipídicos aparecem como estruturas esféricas pequenas e claras no citoplasma do THP-M. </li><li> Adicionar 40 mL de ácido etilenodiaminotetraacético 5 mM (EDTA) em PBS a cada placa. Incubar durante 15 min a 37 ° C. </li><li> Desmonte os macrófagos espumosos (FM) pipetando repetidamente para cima e para baixo sobre a superfície da placa inteira usando uma pipeta serológica de 10 mL. Transferir a suspensão celular para um tubo cônico de 50 mL e girar para baixo a 150 xg por 5 min. </li><li> Ressuspender o sedimento celular em 10 mL de PBS (terceira lavagem com PBS) aNd de transferência para um pré-pesados ​​15 mL mL tubos cônicos. Girar novamente para 150 xg durante 5 min. Aspirar cuidadosamente o sobrenadante utilizando uma pipeta serológica e descartar. </li><li> Submeter as pastilhas de FM a 3 ciclos de congelamento-descongelamento para lisar as células e incubá-las a 75 ° C durante 20-30 min para desnaturar as proteínas na matriz. Armazenar os grânulos a -20 ° C até estar pronto para uso. </li></ol> 3. Ensaio de Diálise de Equilíbrio Rápido (RED) <ol><li> Preparar soluções-mãe 10 mM de todos os compostos de teste em dimetilsulfóxido (DMSO). Diluir a soluções de trabalho de 500 μM em DMSO antes de cada ensaio. </li><li> Pesar o tubo contendo a pastilha substituta de caseu. Subtrair o peso do tubo vazio para derivar o peso do grânulo sozinho. Adicionar 2-3 esferas metálicas por tubo e, utilizando um homogeneizador de tecidos a 1200 golpes / minuto durante 1 minuto, interromper o caseu ou a matriz substituta em PBS (1: 9 p / v) para se obter a suspensão diluída 10x de cada matriz. </li><li> Spike 6,5 μL dos 500ΜM do composto de teste em 643,5 μL de homogeneizado para se obter a concentração final de 5 μM (≤ 1% de DMSO) e vórtex. </li><li> Coloque as inserções VERMELHAS na placa de base. Adicionar 200 μL da matriz droga-cravada na câmara doadora (anel vermelho) de cada inserto VERMELHO e 350 μL de PBS em cada câmara receptora. Preparar 3 insertos para cada composto de teste (amostras em triplicado). Selar a placa com um vedante de placa adesiva e incubar a 37 ° C no termomixador a 200 rpm (1 xg) durante 4 h. </li><li> Após a incubação, misturar suavemente o conteúdo das câmaras doador e receptor por pipetagem para cima e para baixo 2-3 vezes. Pipetar para fora alíquotas de 20 μL de homogeneizado das câmaras de dador e adicionar 20 μL de PBS limpo num tubo de 1,5 mL (1: 1). Do mesmo modo, pipetar para fora alíquotas de 20 μL de amostras de PBS das câmaras receptoras e adicionar 20 μL de homogeneizado limpo (correspondência de matriz). 8 NOTA: A correspondência de matriz elimina aEd para 2 curvas de calibração separadas (em homogeneizado e PBS) a serem feitas para análise quantitativa. O conteúdo da câmara doadora pode sedimentar ao longo do tempo. Misture suavemente o conteúdo por pipetagem antes de remover as alíquotas. </li></ol> 4. Quantificação de LC-MS e Análise de Dados <ol><li> Adicionar 160 μL de metanol: acetonitrilo 1: 1 contendo diclofenac 500 ng / mL ou verapamil 10 ng / mL (padrão interno) a cada tubo e vortex para precipitar as proteínas. Centrifugar a 10 000 xg durante 5 min para sedimentar o precipitado e transferir os sobrenadantes para placas de poço profundo de 96 poços para análise de cromatografia líquida-espectrometria de massa (LCMS). 7 </li><li> Construir curvas de calibração de 1-1000 nM para cada composto de teste, mantendo a mesma composição de matriz que as amostras acima. Quantificar a concentração do composto de teste em amostras das câmaras doadora e receptora utilizando um método LC-MS. </li><li> Calcular a fracção não ligada ( f u ) oF o fármaco em matriz diluída usando a equação 1. Calcule o f u em matriz não diluída usando a equação 2 ( D = fator de diluição de 10). 14 <img alt="Equação 1" src="/files/ftp_upload/55559/55559eq1.jpg" /> <img alt="Equação 2" src="/files/ftp_upload/55559/55559eq2.jpg" /></li><li> Verificar a recuperação (balanço de massa) de cada composto utilizando a equação 3 para identificar compostos com estabilidade / metabolismo / questões de ligação não específicas. NOTA: Recuperação normalmente cai entre 70% e 130%. 15 <img alt="Equação 3" src="/files/ftp_upload/55559/55559eq3.jpg" /></li></ol>

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Lesões necróticas pulmonares e cavidades em pacientes infectados com tuberculose contêm subpopulações de bactérias que são recalcitrantes ao tratamento medicamentoso. Os núcleos caseosos destas estruturas são particularmente responsáveis ​​por abrigar estes persisters em um ambiente extracelular. Acredita-se que uma distribuição favorável de agentes anti-bacterianos nestes locais remotos seja um determinante importante da eficácia do fármaco contra a tuberculose. Antes da validação deste protocolo, ...

O tratamento da doença pulmonar tuberculosa requer distribuição eficaz de fármacos em diferentes tipos de lesões. As lesões necróticas e as cavidades contêm centros caseosos que abrigam subpopulações de bactérias tolerantes a drogas ou &quot;persistentes&quot;. 1 , 2 A doença cavitária está associada a taxas de cura inferiores e mau prognóstico. 3 , 4 Estudos anteriores mostraram, usando técnicas quantitativas e de imagem, que a capacidade de penetrar o caseu varia significativamente de uma classe de droga para outra. 5 , 6 Estes métodos, no entanto, exigem o uso de modelos de infecção animal que são lentos e tediosos. Foi concebido um ensaio in vitro que mede a ligação do fármaco ao caseu ex vivo . Verificou-se que esta ligação estava inversamente correlacionada com a penetração do fármaco em granulomas caseosos e, por conseguinte, éusado como uma ferramenta de previsão. 7 Diálise de equilíbrio é considerado como o método padrão de ouro para estudos de ligação de proteína de plasma. O dispositivo rápido equilíbrio de diálise (VERMELHO) proporciona um sistema rápido, fácil de usar e fiáveis ​​para a realização de tais ensaios. 8 O dispositivo é constituído por dois componentes: de utilização única, descartáveis que consistem inserções de 2 câmaras separadas por um cilindro vertical de membrana semi-permeável; e placas de base reutilizáveis ​​que pode armazenar até 48 inserções de cada vez. A membrana de diise tem um 8 kDa de peso molecular de corte (MWCO) que é ideal para estudos de ligação a drogas macromolécula. A razão de área para volume elevada superfície do compartimento da membrana de diálise permite uma rápida e equilibração. Ambos os insertos e a placa de base foram validados para o mínimo de ligação não específica. A combinação do dispositivo VERMELHO com técnicas bioanalíticas fornece estimativas precisas sobre as fracções não ligadas de medicamentos em pLasma 8 , 9 Embora originalmente concebido para medir a ligação às proteínas plasmáticas, o dispositivo RED foi utilizado em vários estudos de ligação de tecidos utilizando homogeneizados. 10 , 11 Neste protocolo, medimos a ligação do fármaco ao caseu, os detritos necróticos excisados ​​das lesões necróticas e cavidades de coelhos infectados com tuberculose. A natureza acelular e não vascular do material caseoso facilita a homogeneização numa suspensão homogénea que é compatível com o ensaio. Dado que o caseu é tedioso para produzir e difícil de encontrar, o protocolo também foi validado para uso com uma matriz substituta que é preparada a partir de macrófagos espumosos. Os macrófagos derivados de monócitos THP-1 são induzidos com ácido oleico para acumular múltiplos corpos lipídicos que lhes conferem a sua aparência &quot;espumosa&quot;. Estas células carregadas de lípidos são colhidas eProcessado para produzir uma matriz que usamos como um substituto de caseu. Este estudo mostrou que a ligação do fármaco a esta matriz substituta se correlaciona bem com a ligação ao caseu, imitando eficazmente o processo in vivo que impede a penetração do fármaco no núcleo caseoso de granulomas e cavidades.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">New Zealand White Rabbits</td>
			<td style="width:203px;">Covance</td>
			<td style="width:147px;">-</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">HN878 Mycobacterium tuberculosis</td>
			<td style="width:203px;">BEI Resources</td>
			<td style="width:147px;">NR-13647&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Ketathesia (Ketamine) 100mg/mL C3N</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:147px;">56344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Anased (Xylazine) 100mg/mL</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:147px;">33198</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Euthasol (pentobarbital sodium and phenytoin sodium) Solution&#160;</td>
			<td style="width:203px;">Virbac</td>
			<td style="width:147px;">710101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">THP-1 monocytic cell line</td>
			<td style="width:203px;">ATCC</td>
			<td style="width:147px;">ATCC TIB-202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">175cm&#178; TC-Treated Flask (T175)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>T-3400-175</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 media w/o glutamine</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>MT-15-040-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyclone Fetal Bovine Serum, Gamma irradiated&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>SH3091003IR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyclone L-glutamine, 200mM</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>SH3003401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellstar TC dish, 145x20mm, vented</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>T-2881-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phorbol 12-myristate13-acetate (PMA)&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>BP685-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:203px;">Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oleic acid&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>ICN15178101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED Device Reusable Base Plate</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>PI-89811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED Device Inserts, 50/box&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>PI-89809</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED insert removal tool&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>89812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adhesive plate seal</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>08-408-240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH7.4, 10 X 500 mL (Gibco)&#160;</td>
			<td style="width:203px;">Life Technologies&#160;</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">DMSO</td>
			<td style="width:203px;">Sigma</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Acetonitrile</td>
			<td style="width:203px;">Sigma</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Methanol</td>
			<td style="width:203px;">Sigma</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Verapamil hydrochloride</td>
			<td style="width:203px;">Sigma</td>
			<td>V4629</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Diclofenac sodium salt</td>
			<td style="width:203px;">Sigma</td>
			<td>93484</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>15-250-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Ethanol, 200 proof</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">2010 Geno/Grinder</td>
			<td style="width:203px;">SPEX SamplePrep</td>
			<td style="width:147px;">2010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Bead Mill Homogenizer Accessory, Metal Bulk Beads</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>15-340-158</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">484R Cobalt 60 Irradiator</td>
			<td style="width:203px;">JL Shepard</td>
			<td style="width:147px;">7810-484-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">INCYTO&#160;C-Chip Disposable Hemacytometers</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:147px;">22-600-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Upright Light Microscope</td>
			<td style="width:203px;">Leica</td>
			<td style="width:147px;">DM1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Binary Liquid Chromatography system</td>
			<td style="width:203px;">Agilent</td>
			<td style="width:147px;">1260</td>
			<td>Multi-compenent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Mass spectrometer</td>
			<td style="width:203px;">AB Sciex</td>
			<td>4000</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro caseum binding assay that predicts drug penetration in tuberculosis lesions

A erradicação da tuberculose requer regimes medicamentosos que possam penetrar as múltiplas camadas de lesões pulmonares complexas. A distribuição de fármacos nos núcleos caseosos de cavidades e lesões é especialmente crucial porque abrigam subpopulações de bactérias tolerantes a fármacos também vulgarmente referidas como persisters. Os métodos existentes para a medição da penetração de fármaco em lesões de tuberculose envolvem costosos e demorados estudos farmacocinéticos in vivo acoplados a técnicas bioanalíticas ou de imagem. A medida in vitro da ligação do fármaco às macromoléculas de caseu foi proposta como uma alternativa a essas técnicas, uma vez que esta ligação dificulta a difusão passiva de moléculas de fármaco através do caseu. A diálise de equilíbrio rápido é um sistema rápido e confiável para realizar estudos de ligação a proteínas plasmáticas e tecidos. Neste protocolo, usamos um dispositivo de diálise de equilíbrio rápido (RED) para medir a ligação do fármaco a homogenatos de caseu que é excised das lesões e as cavidades de coelhos infectados com tuberculose. O protocolo também descreve como gerar uma matriz de substituição de lípido carregado THP-1 macrófagos para usar em lugar de caseum. Este ensaio de ligação caseum / substituto é uma ferramenta importante na descoberta de drogas tuberculose e pode ser adaptado para ajudar o estudo da distribuição de drogas em lesões ou abcessos causados ​​por outras doenças.

Usando este protocolo, testámos centenas de compostos de desenvolvimento de tuberculose para a sua eficiência previsto no penetrante caseum. A Figura 1 visualiza os conceitos básicos do ensaio VERMELHO. A membrana de diálise das inserções VERMELHOS permite para pequenas moléculas não ligadas a difundir a partir do dador bem ao receptor bem como, finalmente, atingir um equilíbrio entre os dois compartimentos. As pequenas moléculas que são ligadas a macromolécu...

Watch this Scientific Journal Video about An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions at JoVE.com

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Aqui descrevemos um método de diálise de equilíbrio rápido (RED) para medir a ligação do fármaco ao caseu de lesões e cavidades de tuberculose pulmonar. O protocolo também é usado com uma matriz espumosa derivada de macrófagos que é um substituto efetivo do caseu.

A<em&gt; In Vitro</em&gt; Caseum Ensaio de Ligação ao que prevê a penetração da droga na tuberculose Lesões

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

an-vitro-caseum-binding-assay-that-predicts-drug-penetration

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Unit 1

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Nonlinear Pharmacokinetics

Introduction to Respiratory System Drugs

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11.6K visualizações.  Rutgers.  Aqui descrevemos um método de diálise de equilíbrio rápido (RED) para medir a ligação do fármaco ao caseu de lesões e cavidades de tuberculose pulmonar. O protocolo também é usado com uma matriz espumosa derivada de macrófagos que é um substituto efetivo do caseu. 

Vídeo: An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

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

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Facilitar Drug Discovery: um ensaio automatizado de alta Inflamação conteúdo em Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

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

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

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

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

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

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

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Um modelo experimental para estudar a Tuberculose-Malária Coinfection sobre Transmissão Natural de Mycobacterium tuberculosis E Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Investigando os efeitos dos probióticos sobre pneumocócica Colonização Usando um<em&gt; In Vitro</em&gt; Adesão Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Demonstrando um multi-resistente<em&gt; Mycobacterium tuberculosis</em&gt; Ampliação Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Um método de microdiluição em caldo de referência para Dalbavancin<em&gt; In Vitro</em&gt; Teste à Susceptibilidade de bactérias que crescem aeròbia

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Estabelecimento de um programa de configuração de alta capacidade para a triagem de pequenas moléculas que modulam c-di-GMP Sinalização<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

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 ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Os métodos in vitro para Comparando meta vinculativa e CDC indução Entre anticorpos terapêuticos: Aplicações em Análise Biosimilarity

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...