Esta pesquisa foi financiada em partes por subvenções de NIH HL66299 TS, RB e SL, HL60024 de TS e HL136869 para MF.

Todos os procedimentos de animais foram revisados e aprovados pelo Comitê de cuidado Animal da Universidade do Sul do Alabama e institucional e foram realizados em conformidade com todas as regulamentações federais, estaduais e locais. Culturas primárias de rato pulmonar microvascular células endoteliais (PMVECs) foram obtidas da instalação de núcleo de cultura de célula Center da Universidade do Sul do Alabama para biologia do pulmão. As células foram preparadas usando os procedimentos descritos anteriormente16.
1. geração de sobrenadantes citotóxicos
Nota: Aqui, usamos dois diferentes cepas de p. aeruginosa: PA103, que tem um sistema de secreção do III tipo intacto capaz de transferir os exoenzymes ExoU e ExoT para células-alvo durante a infecção e ∆PcrV, que carece de um tipo sistema do secretion de III e é incapaz de transferir exoenzymes para células-alvo após a inoculação.
<ol><li>Raia de bactérias para a de placas de ágar Vogel-Bonner (VB)17 e crescer durante a noite a 37 ° C.<ol>
<li>Para preparar pratos, faça a seguinte solução estoque (sais de Vogel-Bonner; estoque de x 10): medir 2 g MgSO4, ácido cítrico de 20g (ácido livre), 100 g K2PO4e 35 g NaH2PO4. Adicione ddH2O de 1L e autoclave por 15 min com escape lento.</li>
		<li>Coloque 7,5 g ágar-ágar em 450ml ddH2O e autoclave como acima.</li>
		<li>Após a autoclavagem, Coloque ambas as soluções em um banho de água de 50 ° C para esfriar.</li>
		<li>Adicione 50 mL da solução de sal estoque de 10 x VB a agar.</li>
		<li>Adicionar carbenicilina a 400 µ g/mL (para as cepas de p. aeruginosa sendo usado) e misturar bem, agitando o frasco.</li>
		<li>Lugar 5 mL da solução de ágar de VB em pratos individuais cultura estéril, permitir que o agar para solidificar e em seguida armazenar a 4 ° C até o dia da semeadura bacterianas.</li>
		<li>No dia da semeadura bacterianas, pré-aquecer uma placa a 37 ° C e em seguida marcam as bactérias na placa utilizando uma ansa estéril. Lugar a placa em uma incubadora de 37 ° C durante a noite.</li>
	</ol>
</li>
	<li>Verificar a pureza PMVEC pela coloração positiva com fluorescente Griffonia simplicifolia lectina e coloração negativa com fluorescentes Helix pomatia lectina (um marcador para células endoteliais arteriais). Alíquotas células e congelamento em nitrogênio líquido.</li>
	<li>Para experiências, manter as células em modificado águia de Dulbecco médio (DMEM) suplementado com 10% de soro fetal bovino e desenvolvem-se células em uma incubadora de 37 ° C, contendo 5% de CO2. Use células em passagens 5-15.<ol>
<li>Para geração de sobrenadantes citotóxicos, placa de 2 x 106 células em pratos individuais 150cm e crescer por 3-4 dias até alcançar a confluência. Use duas placas por experiência (veja abaixo).</li>
		<li>Para análise dos sobrenadantes citotóxicos, células de placa 1 x 105 em poços individuais de um poço-6 prato e crescerem durante quatro dias até alcançar a confluência.</li>
	</ol>
</li>
	<li>Para produzir o sobrenadante, lavar um dos pratos de PMVECs dois 150cm da etapa 1.3.1 com tampão fosfato salino, trypsinize e depois contar as células (use um contador de célula automatizada e seguir o protocolo do fabricante; ver Tabela de materiais). Lavar o outro prato de PMVECs com Hank está equilibrado sal solução (HBSS) e então infectar com qualquer estirpe de p. aeruginosa em uma multiplicidade de infecção (MOI) de 20:1. Isto é conseguido da seguinte forma:<ol>
<li>Para p. aeruginosa, OD540 0,25 = 2 x 108 CFUs/mL. Para preparar esta diluição, adicione 1 mL HBSS para uma cubeta descartável de 1ml. Raspe bastante bactérias do prato que foi preparada no passo 1.1 até uma OD540 de 0,25 é alcançado quando medido utilizando um espectrofotômetro. O próximo passo irá fornecer um cálculo de quanto serão necessário bactérias.</li>
		<li>Uma vez que o número de PMVECs a placa de cultura é determinado (etapa 1.4 acima), determinar o número apropriado de bactérias a ser adicionado ao prato em segundo lugar, não-trypsinized de cultura contendo os PMVECs. Por exemplo, se for determinado que a placa de cultura de PMVECs contém 1,3 x 107 células e, em seguida, preparar a 1,3 x 107 células x 20 bactérias/celular x 1 mL/2 x 108 CFUs = bactérias 1,3 mL.</li>
		<li>Dilua a 1,3 mL de bactérias em HBSS até um volume final de 20 mL, para que toda a superfície de um prato de 150 cm pode ser coberto com fluido e, em seguida, adicionar as bactérias diluídas para a placa de PMVECs.</li>
		<li>Incube as PMVECs inoculadas com as bactérias a 37 ° C numa incubadora 5% CO2 para 4-5 h, até que as lacunas podem ser vistas formando em monocamada a célula quando a placa é observada microscopicamente (ver Figura 1 para um nível adequado de formação de lacuna).</li>
		<li>Recolher o sobrenadante e, em seguida, centrifugar durante 10 min a 2.000 x g em uma centrífuga de mesa para remover restos celulares. Nota: Qualquer centrífuga de mesa capaz de gerar suficiente g force para células unlysed e células grandes fragmentos podem ser usados para esta etapa de Pelotas.</li>
		<li>Despeje o líquido sobrenadante em uma seringa que tem um filtro de 0,2 µm conectado à sua extremidade, então passar o sobrenadante através do filtro para remover as bactérias.</li>
		<li>Use uma alíquota do sobrenadante estéril para teste de citotoxicidade (ver 2.1) e congelar o resto do sobrenadante a-80 ° C para uso futuro.</li>
	</ol>
</li>
</ol>2. análise de sobrenadantes citotóxicos
<ol><li>Ensaio de citotoxicidade<ol>
<li>Cresce PMVECs em pratos 6-poços até a confluência. Poços de lavar uma vez com HBSS, em seguida, adicionar 1,5 mL do sobrenadante filtro esterilizado (colhido ou células inoculadas por PA103 ou ∆PcrV) para poços individuais. Adicione HBSS estéril para outro bem como controlo negativo.</li>
		<li>Coloque a placa para a incubadora de CO2 para 21-24 h e, em seguida, observar para matar a célula/citotoxidade (ver próximo passo). Use sobrenadantes que exibem citotoxicidade para novas experiências e descartar aqueles que não mostram a matança de célula.</li>
		<li>Quantificação de morte celular<ol>
<li>Liberação de lactato desidrogenase (LDH) medida de células mortas, usando Kits de ensaio disponíveis comercialmente LDH e do fabricante recomendado procedimentos.</li>
			<li>Alternativamente, quantificar a matança de célula ao microscópio utilizando o software ImageJ. Por isso, gravar imagens microscópicas e áreas da placa de cultura contendo células intactas e, em seguida, quantificar regiões faltam células (indicativas de morte celular em um monolayer) usando uma macro personalizada do ImageJ (National Institutes of Health) (ver arquivo de codificação complementar ). Se desejado, realize as etapas macro manualmente da seguinte forma:<ol>
<li>Imagens (formato RGB ou Tiff) de entrada para a macro e ajustar o contraste para 15% saturada pixels. Duplique o contraste ajustado imagens e executar o comando "subtrair o plano de fundo" para obter uma imagem de alto contraste da área tanto a célula e a lacuna dentro do campo de visão.</li>
				<li>Subtrair a esta imagem da imagem original e combinar com a imagem original, usando a calculadora de imagem "E" função, a imagem resultante. Converta a imagem resultante em preto (lacunas) e máscara branca (células) com a função de limite (mínimo de 0, máximo de 5) e usar a função "corroer a binário" para remover o ruído da imagem.</li>
				<li>Medir a proporção de preto para brancos pixels dentro da imagem resultante usando a medição de "fração de área". Plotar e expressar áreas fracionárias para cada ponto de tempo de tratamento como por cento da área de abertura máxima.</li>
				<li>Meios de calcular e comparar usando ANOVA One-Way com teste post hoc de Tukey, com valores de p inferior a 0,05 considerado significativo.</li>
			</ol>
</li>
		</ol>
</li>
		<li>Use a análise de immunoblot para estabelecer a presença de amiloide e tau oligoméricas Aβ em sobrenadantes de cultura. Realizar análise de immunoblot usando11,os procedimentos padrão10,12, exceto concentrando sobrenadantes de cultura pelo menos 10 vezes antes da electroforese.<ol>
<li>Para concentrar-se o sobrenadante, coloque 1 a 2 mL do sobrenadante para uma unidade de filtro de centrifugação cuja membrana tem um peso molecular de corte de 10 kDa. Em seguida, coloque a unidade de filtro em uma centrífuga de mesa superior e centrifugar x 2.000 g até o necessário grau de concentração é alcançado (geralmente de 45-60 min).</li>
			<li>Determinar a quantidade de proteína na concentrada sobrenadante11 e carregar quantidades iguais de amostras em poços individuais do gel.</li>
			<li>Executar géis, transferir as proteínas para nitrocelulose e sondar os borrões com anticorpo antiamilóide de A11, anticorpo antioligoméricas tau de T22 ou anticorpo anti-Aβ de MOAB2 seguido por anticorpos secundários apropriados. Desenvolva os borrões usando procedimentos padrão de quimioluminescência.</li>
		</ol>
</li>
		<li>Para fazer um ensaio de T de thioflavin, use thioflavin T fluorescência (ThT) quantificar amiloides em sobrenadantes. Para as medições, use filtro esterilizado sobrenadantes.<ol>
<li>Prepare uma solução de stock (50x) de thioflavin T, suspendendo a 8 mg thioflavin T (ThT) em soro fisiológico 10ml tamponado fosfato (PBS). Após a mistura, filtre a solução através de um filtro de 0,22 µm para remover partículas.</li>
			<li>Para medições, adicione 20 µ l de estoque ThT a 1 mL de PBS em um fluorímetro de 1 mL. Coloca a amostra diluída em um spectrofluorimeter.</li>
			<li>Medir a emissão de fluorescência de base usando 425 excitação nm e a emissão de fluorescência de 450-575 nm em incrementos de 2 nm de digitalização.</li>
			<li>Executar uma verificação de lapso de tempo usando 425 excitação nm e emissão de 482 nm, com dados adquiridos a cada 0.2 s por 60 s.</li>
			<li>O inicial 20 s do scan Time-Lapse mede a fluorescência na célula em branco. A 20 s, pausar o scan e adicionar 10 µ l do sobrenadante filtro esterilizados para a cubeta. Misture a cubeta por inversão e coloque para o spectrofluorimeter.</li>
			<li>Retomar a verificação baseada em tempo, adquirindo o final 40 s de dados.</li>
			<li>Após a conclusão da digitalização lapso de tempo, execute uma varredura de espectro de emissão de fluorescência final usando configurações idênticas, conforme detalhado em 2.1.5.3.</li>
		</ol>
</li>
	</ol>
</li>
</ol>

<ol>
	<li>Brancati, F. L., Chow, J. W., Wagener, M. M., Vacarello, S. J., Yu, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+pneumonia+really+the+old+man's+friend?+Two-year+prognosis+after+community-acquired+pneumonia.">Is pneumonia really the old man's friend? Two-year prognosis after community-acquired pneumonia.</a> Lancet. 342 (8862), 30-33 (1993).</li><li>Hedlund, J. U., Ortqvist, A. B., Kalin, M. E., Granath, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+of+importance+for+the+long-term+prognosis+after+hospital+treated+pneumonia.">Factors of importance for the long-term prognosis after hospital treated pneumonia.</a> Thorax. 48 (8), 785-789 (1993).</li><li>Meier, C. R., Jick, S. S., Derby, L. E., Vasilakis, C., Jick, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+tract+infections+and+risk+of+first-time+acute+myocardial+infarction.">Acute respiratory tract infections and risk of first-time acute myocardial infarction.</a> Lancet. 351 (9114), 1467-1471 (1998).</li><li>Waterer, G. W., Kessler, L. A., Wunderink, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium-term+survival+after+hospitalization+with+community-acquired+pneumonia.">Medium-term survival after hospitalization with community-acquired pneumonia.</a> Am J Resp Crit Care Med. 169 (8), 910-914 (2003).</li><li>Yende, S., 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=Inflammatory+markers+at+hospital+discharge+predict+subsequent+mortality+after+pneumonia+and+sepsis.">Inflammatory markers at hospital discharge predict subsequent mortality after pneumonia and sepsis.</a> Am J Resp Crit Care Med. 177 (11), 1242-1247 (2008).</li><li>Corrales-Medina, V. F., 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=Association+between+hospitalization+for+pneumonia+and+subsequent+risk+of+cardiovascular+disease.">Association between hospitalization for pneumonia and subsequent risk of cardiovascular disease.</a> J Am Med Assoc. 313 (3), 264-274 (2015).</li><li>Frank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+exoenzyme+S+regulon+of+Pseudomonas+aeruginosa.">The exoenzyme S regulon of Pseudomonas aeruginosa.</a> Mol Microbiol. 26 (4), 621-629 (1997).</li><li>Engel, J., Balachandran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Pseudomonas+aeruginosa+type+III+effectors+in+disease.">Role of Pseudomonas aeruginosa type III effectors in disease.</a> Curr Opin Microbiol. 12 (1), 61-66 (2009).</li><li>Ochoa, C. D., Alexeyev, M., Pastukh, V., Balczon, R., Stevens, T. <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+exotoxin+Y+is+a+promiscuous+cyclase+that+increases+endothelial+tau+phosphorylation+and+permeability.">Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability.</a> J. Biol. Chem. 287 (30), 25407-25418 (2012).</li><li>Balczon, R., 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+exotoxin+Y-mediated+tau+hyperphosphorylation+impairs+microtubule+assembly+in+pulmonary+microvascular+endothelial+cells.">Pseudomonas aeruginosa exotoxin Y-mediated tau hyperphosphorylation impairs microtubule assembly in pulmonary microvascular endothelial cells.</a> PLoS One. 8, e74343 (2013).</li><li>Morrow, K. A., 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+exoenzymes+U+and+Y+induce+a+transmissible+endothelial+proteinopathy.">Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy.</a> Am J Physiol Lung Cell Molec Physiol. 310 (4), L337-L353 (2016).</li><li>Balczon, R., 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+infection+liberates+transmissible,+cytotoxic+prion+amyloids.">Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids.</a> FASEB Journal. 31 (7), 2785-2796 (2017).</li><li>Stevens, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Pseudomonas+aeruginosa+exoenzyme+Y+impairs+endothelial+cell+proliferation+and+vascular+repair+following+lung+injury.">The Pseudomonas aeruginosa exoenzyme Y impairs endothelial cell proliferation and vascular repair following lung injury.</a> Am J Physiol Lung Cell Molec Physiol. 306 (10), L915-L924 (2014).</li><li>Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creutzfeldt-Jakob+disease+and+scrapie+prions.">Creutzfeldt-Jakob disease and scrapie prions.</a> Alzheimer Dis Assoc Disord. 3 (1-2), 52-78 (1989).</li><li>Hunter, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrapie:+Uncertainties,+biology,+and+molecular+approaches.">Scrapie: Uncertainties, biology, and molecular approaches.</a> Biochem. Biophys. Acta. 1772 (6), 619-629 (2007).</li><li>King, J., 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=Structural+and+functional+characteristics+of+lung+macro-+and+microvascular+endothelial+cell+phenotypes.">Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes.</a> Microvasc Res. 67 (2), 139-151 (2004).</li><li>Marmont, L. S., 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=Oligomeric+lipoprotein+PelC+guides+Pel+polysaccharide+export+across+the+outer+membrane+of+Pseudomonas+aeruginosa.">Oligomeric lipoprotein PelC guides Pel polysaccharide export across the outer membrane of Pseudomonas aeruginosa.</a> Proc Natl Acad Sci USA. 114 (11), 2892-2897 (2017).</li></ol>

Nenhum relatório.

Aqui, simples em vitro métodos estão descritos que permitem a demonstração da geração de amiloides citotóxicas durante a infecção com uma organismo causador de pneumonia. Estes métodos incluem um ensaio de citotoxicidade de cultura de células, immunoblotting, quantificação de célula usando um novo método microscópico e ThT vinculação a matar. Análises dos agentes citotóxicos têm demonstrado que eles são amiloide na natureza (Figura 2 e <stron...

Pacientes que sobrevivem a pneumonia tem elevadas taxas de mortalidade nos meses seguintes hospital descarga1,2,3,4,5,6. Na maioria dos casos, a morte ocorre por algum tipo de falha final-órgão, incluindo renais, pulmonares, cardíacas, ou eventos de fígado, bem como derrame5,6. A razão para a elevada taxa de mortalidade nesta população de pacientes nunca foi estabelecida.
Pneumonia é classificada como também sendo adquirida na Comunidade ou hospitalar (nosocomial) e agentes que podem causar pneumonia incluem bactérias, vírus, fungos e substâncias químicas. Uma das principais causas de pneumonia nosocomial é a bactéria Pseudomonas aeruginosa. P. aeruginosa é um organismo Gram-negativo que usa um sistema de secreção do tipo III para transferir várias moléculas efetoras, denominadas exoenzymes, diretamente para o citoplasma das células de destino7,8. Durante a infecção de células endoteliais pulmonares, os exoenzymes alvo várias proteínas intracelulares, incluindo um formulário endotelial do microtubule-associada da proteína tau9,10,11,12 , levando à desagregação de barreira endotelial, resultando em edema pulmonar grave, diminuição da função pulmonar e, muitas vezes, morte.
Como dito anteriormente, pacientes que sobrevivem a pneumonia inicial elevaram taxas de mortalidade nos primeiros 12 meses após a alta hospitalar. Um mecanismo potencial para explicar esse fenômeno é que algum tipo de toxina long-lived é gerado durante a infecção inicial que conduz ao pobre resultado a longo prazo. Duas observações suportam esta possibilidade. Em primeiro lugar, culturas pulmonares células endoteliais, que são tratadas inicialmente com p. aeruginosa falharem a proliferar por até uma semana depois que as bactérias são mortas por antibióticos13. Segundo, long-lived priões e agentes com características de príon foram demonstrados em várias doenças humanas e animais, particularmente doenças associadas com o sistema nervoso de14,15.
Nunca têm sido descritos métodos para analisar o potencial de produção de agentes citotóxicos long-lived durante a infecção pulmonar. Aqui uma série de ensaios simples em vitro são descritos que podem ser usados para investigar cytotoxin produção e actividade após infecção usando um comum agente causando uma pneumonia, p. aeruginosa. Estes ensaios devem ser facilmente adaptávelas para investigar indução cytotoxin possível após infecção usando outros agentes que causam a pneumonia, e os sobrenadantes são gerados também devem ser útil para investigar os efeitos dos citotoxinas no todo órgãos ou animais. Finalmente, os ensaios que são descritos aqui provavelmente será adaptávelas para testar os fluidos biológicos humanos e animais para a produção de citotoxinas durante uma pneumonia.

<table><tbody><tr><td>Rabbit anti beta amyloid</td><td>Thermo</td><td>71-5800</td><td></td></tr><tr><td>A11 amyloid oligomer antibody</td><td>StressMarq</td><td>SPC-506D</td><td></td></tr><tr><td>T22 anti-tau oligomer antibody</td><td>EMD Millipore</td><td>ABN454</td><td></td></tr><tr><td>Thioflavin T</td><td>Sigma Aldrich</td><td>T3516</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025-092</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>0.22 micron syringe filters</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>HRP Goat antirabbit IgG</td><td>Abcam</td><td>ab6721-1</td><td></td></tr><tr><td>Strains PA103 and &amp;Delta;PcrV</td><td></td><td></td><td>These strains of P. aeruginosa were obtained from Dr. Dara Frank, University of Wisconsin Medical College, Milwaukee, WI</td></tr><tr><td>FITC Griffonia lectin</td><td>Sigma Aldrich</td><td>L9381</td><td></td></tr><tr><td>TRITC Helix pomatia lectin</td><td>Sigma Aldrich</td><td>L1261</td><td></td></tr><tr><td>Agar</td><td>Fisher</td><td>BP1423-500</td><td></td></tr><tr><td>0.22 micron nitrocellulose</td><td>BioRad</td><td>162-0112</td><td></td></tr><tr><td>Type 2 collagenase</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone</td><td>SH30898.03IH</td><td></td></tr><tr><td>PenStrep</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>Carbenicillin Disodium salt</td><td>Sigma&amp;nbsp;</td><td>C1389</td><td></td></tr><tr><td>Microcetrifugation concentrators- 10,000 MW cut-off</td><td>Millipore</td><td>UFC801008</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma&amp;nbsp;</td><td>795496-500G</td><td></td></tr><tr><td>Magnesium phosphate heptahydrate</td><td>MP Biomedicals</td><td>MP021914221</td><td></td></tr><tr><td>Citric Acid</td><td>G Biosciences</td><td>50-103-5801</td><td></td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Sigma</td><td>S9506-500G</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10277</td><td></td></tr></tbody></table>

methods for detecting cytotoxic amyloids following infection of pulmonary endothelial cells by pseudomonas aeruginosa

Pacientes que sobrevivem a pneumonia tem elevadas taxas de mortalidade nos meses após a alta hospitalar. Isso tem sido a hipótese de que a infecção do tecido pulmonar durante uma pneumonia resulta na produção de citotoxinas long-lived que podem levar ao fracasso subsequente fim órgão. Nós desenvolvemos em vitro ensaios para testar a hipótese que citotoxinas são produzidas durante a infecção pulmonar. As células endoteliais pulmonar isolado de rato e a bactéria Pseudomonas aeruginosa são usados como sistemas modelo, e a produção de cytoxins após a infecção das células endoteliais por bactérias é demonstrada usando cultura de células, seguida por quantificação directa utilizando ensaios de lactato desidrogenase e um novo método microscópico utilizando tecnologia ImageJ. A natureza amiloide destas citotoxinas foi demonstrada por ensaios de thioflavin T obrigatórios e immunoblotting e immunodepletion usando anticorpo antiamilóide de A11. Novas análises usando immunoblotting demonstraram que tau oligoméricas e Aβ foram produzidos e liberados por células endoteliais após infecção por p. aeruginosa. Esses métodos devem ser facilmente adaptávelas para análises de amostras clínicas humanas.

Um ensaio simples em vitro foi desenvolvido para ensaio para a presença de citotoxinas em sobrenadantes de células infectadas com a bactéria p. aeruginosa. Basicamente, meio de cultura de células infectadas é coletado 4 h após a adição de bactérias, as bactérias são removidas pela esterilização do filtro do sobrenadante da cultura, e em seguida o sobrenadante estéril é adicionado a uma nova população de células. As células são então observadas 21-24 ...

Watch this Scientific Journal Video about Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa at JoVE.com

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Métodos para detecção de citotóxicos amiloides seguir infecção de pulmonar células endoteliais por Pseudomonas aeruginosa

Métodos simples são descritos para demonstrar a produção de amiloides citotóxicos após infecção do endotélio pulmonar por Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

methods-for-detecting-cytotoxic-amyloids-following-infection

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

5.5K visualizações.  University of South Alabama. Métodos simples são descritos para demonstrar a produção de amiloides citotóxicos após infecção do endotélio pulmonar por Pseudomonas aeruginosa.

Vídeo: Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

preparação e<em&gt; In Vivo</em&gt; O uso de uma sonda baseado em atividades para<em&gt; N</em&gt; Amidase Ácido -acylethanolamine

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Bioquímica

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Medidas de ruptura vacuolar única célula causadas por patógenos intracelulares

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Imunologia e Infecção

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Pseudomonas aeruginosa Lesão Pulmonar Induzida Modelo

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Infecção pneumocócica de células endotélias humanas primárias em fluxo constante

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Cultura de Variante de Pequena Colônia de Pseudomonas aeruginosa e Quantitação de seu Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. aeruginosa Co-Cultura 3D infectada de células epiteliais e macrófagos bronquiais na interface ar-líquida para avaliação pré-clínico de anti-infecciosos

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Estudando efeitos da fumaça do cigarro na infecção por pseudomonas em células epiteliais pulmonares

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Visualização de Pseudomonas aeruginosa no Escarro de Pacientes com Fibrose Cística

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

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 ...

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

Detecção automatizada e de alto rendimento da adesão bacteriana às células hospedeiras

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...