Esta investigación fue financiada en parte por subvenciones de NIH HL66299 y TS, RB, SL, HL60024 a TS y HL136869 a MF.

Todos los procedimientos animales fueron revisados y aprobados por el Comité de cuidado del Animal de la Universidad de Alabama del sur e institucional y se realizaron conforme a las regulaciones federales, estatales y locales. Cultivos primarios de células endoteliales microvasculares pulmonar rata (PMVECs) se obtuvieron del fondo para el núcleo de la célula cultura en el centro de la Universidad de Alabama del sur para la biología del pulmón. Las células fueron preparadas usando procedimientos previamente descritos16.
1. generación de sobrenadantes citotóxicos
Nota: Aquí, utilizamos dos cepas diferentes de p. aeruginosa: PA103, que cuenta con un sistema de secreción de tipo intacto III capaces de transferir las exoenzimas ExoU y ExoT a células diana durante la infección y ∆PcrV, que carece de un tipo de sistema de secreción III y es incapaz de transferir exoenzimas a células de destino después de la inoculación.
<ol><li>Bacterias en de placas de agar de Vogel-Bonner (VB)17 la raya y crecer durante la noche a 37 ° C.<ol>
<li>Para preparar platos, hacer la siguiente solución (sales de Vogel-Bonner, stock de x 10): medir 2 g de MgSO4, 20 g ácido cítrico (ácido libre), 100 g de K2PO4y 35 g NaH2PO4. Añadir ddH2O a 1 L y autoclave durante 15 min con escape lento.</li>
		<li>Coloque el agar 7,5 g en 450 mL ddH2O y autoclave como el anterior.</li>
		<li>Después de autoclavar, coloque ambas soluciones en un baño de agua de 50 ° C para enfriar.</li>
		<li>Añadir 50 mL de la solución de sal común de 10 x VB en el agar.</li>
		<li>Añadir carbenicilina a 400 μg/mL (para las cepas de p. aeruginosa se utiliza) y mezclar bien agitando el frasco.</li>
		<li>Lugar 5 mL de la solución de agar VB en platos de cultivo estériles individuales, permitir que el agar solidifique y luego almacenar a 4 ° C hasta el día de la siembra bacteriana.</li>
		<li>En el día de la siembra bacteriana, caliente previamente las placas a 37 ° C y luego la raya las bacterias en la placa con un asa estéril. Coloque el plato en una incubadora de 37 ° C durante la noche.</li>
	</ol>
</li>
	<li>Verificar la pureza PMVEC por la coloración positiva con fluorescente Griffonia simplicifolia lectinas y tinción negativa con fluorescentes lectina de Helix pomatia (un marcador de células endoteliales arteriales). Células de alícuotas y congelación en nitrógeno líquido.</li>
	<li>Para los experimentos, mantener las células en el medio modificado Eagle de Dulbecco (DMEM) suplementado con suero bovino fetal 10% y crecen las células en una incubadora de 37 ° C con 5% CO2. Utilizar células en pasajes 5-15.<ol>
<li>Para la generación de sobrenadantes citotóxicos, placa de 2 x 106 células en platos individuales de 150 cm y crecer durante 3-4 días hasta alcanza la confluencia. Utilice dos placas por experimento (véase abajo).</li>
		<li>Para el análisis de sobrenadantes citotóxicos, placa de 1 x 105 células en pocillos individuales de un pozo de 6 plato y creciendo durante cuatro días hasta alcanza la confluencia.</li>
	</ol>
</li>
	<li>Para producir el sobrenadante, lavar uno de los platos de dos de 150 cm de PMVECs de paso 1.3.1 con solución salina con tampón fosfato, trypsinize y luego contar las células (utiliza un contador de células automatizado y seguir el protocolo del fabricante; véase Tabla de materiales). Lavar el otro plato de PMVECs con de Hank equilibrada solución de sal (HBSS) y luego infectar con cualquier cepa de p. aeruginosa en una multiplicidad de infección (MOI) de 20:1. Esto se logra como sigue:<ol>
<li>Para p. aeruginosa, OD540 0.25 = 2 x 108 UFC/mL. Para preparar esta disolución, añadir 1 mL HBSS a una cubeta desechable de 1 mL. Raspar las suficientes bacterias de la placa que se preparó en el paso 1.1 hasta un OD540 de 0,25 se obtiene cuando se mide con un espectrofotómetro. El siguiente paso le proporcionará un cálculo de cuánto se necesitará las bacterias.</li>
		<li>Una vez que se determina el número de PMVECs en la placa de cultivo (paso 1.4 anterior), determinar el número apropiado de bacterias en el plato de segunda, no tripsinizaron cultura que contiene las PMVECs. Por ejemplo, si se determina que el plato de cultura de PMVECs contiene 1.3 x 107 células, entonces preparar 1.3 x 107 células x 20 bacterias/celular x 1 mL/de 2 x 108 UFC = bacterias 1,3 mL.</li>
		<li>Diluir el 1,3 mL de bacterias en HBSS hasta un volumen final de 20 mL, para que toda la superficie de un plato de 150 cm puede ser cubierto con el líquido, luego añadir las bacterias diluidas a la placa de PMVECs.</li>
		<li>Incubar los PMVECs inoculados con la bacteria a 37 ° C en un incubador 5% CO2 para 4-5 h, hasta que las lagunas se observan formando en la monocapa de células cuando la placa se observa microscópicamente (ver figura 1 para el nivel adecuado de formación del boquete).</li>
		<li>Recoger el sobrenadante y luego centrifugar por 10 min a 2.000 x g en una centrífuga de mesa para eliminar los desechos celulares. Nota: Cualquier centrífuga de mesa capaz de generar g suficiente fuerza para que sedimenten las células unlysed y células grandes fragmentos se pueden utilizar para este paso.</li>
		<li>Vierta el sobrenadante en una jeringa que tiene un filtro de 0.2 μm unido a su extremo, entonces pasar el sobrenadante a través del filtro para eliminar las bacterias.</li>
		<li>Utilizar una alícuota del sobrenadante estéril a prueba de citotoxicidad (ver 2.1) y congelar el resto del sobrenadante a-80 ° C para su uso futuro.</li>
	</ol>
</li>
</ol>2. Análisis de sobrenadantes citotóxicos
<ol><li>Ensayo de citotoxicidad<ol>
<li>Crecer PMVECs en pozo 6 platos hasta confluencia. Pozos de lavado una vez con HBSS, luego añadir 1,5 mL de sobrenadante esterilizado por filtro (recogido de cualquier células inoculadas PA103 o ∆PcrV) a pocillos individuales. Añadir HBSS estéril a otro así como control negativo.</li>
		<li>Coloque la placa en la incubadora de CO2 por 21-24 h, y luego observar para matar/citotoxicidad celular (ver siguiente paso). Utilice sobrenadantes que exhiben citotoxicidad para más experimentación y descartar aquellos que no muestran la muerte de la célula.</li>
		<li>Cuantificación de la matanza de la célula<ol>
<li>Liberación de lactato deshidrogenasa (LDH) medida de las células muertas utilizando comercialmente disponibles Kits de análisis de LDH y de fabricante procedimientos recomendados.</li>
			<li>Por otra parte, cuantificar muerte celular microscópico utilizando el software ImageJ. Para esto, grabar imágenes microscópicas y las áreas de la placa de cultivo que contiene las células intactas, luego cuantificar las regiones que carecen de células (indicativas de muerte celular en una monocapa) utilizando una macro personalizada de ImageJ (institutos nacionales de salud) (véase archivos de codificación complementarios ). Si lo desea, realizar los pasos de la macro manualmente como sigue:<ol>
<li>Entrada de imágenes (en formato RGB o Tiff) en la macro y ajustar el contraste de los píxeles 15% saturado. Duplicar imágenes ajustar contraste y realizar el comando "quitar fondo" para obtener una imagen de alto contraste de célula y espacio dentro del campo de visión.</li>
				<li>Reste esta imagen de la imagen original y la imagen resultante con la imagen original utilizando la calculadora de imagen "Y" la función se combinan. Convertir la imagen resultante a negro (gaps) y máscara blanca (células) con la función de umbral (mínimo 0, máximo 5) y utilizar la función "binario erosionar" para eliminar el ruido de la imagen.</li>
				<li>Medir la relación del negro al blanco píxeles de la imagen resultante utilizando la medida de "fracción de área". Parcela y expresar áreas fraccionarias para cada punto de tiempo de tratamiento como por ciento del área de separación máxima.</li>
				<li>Medios de calcular y comparar utilizando unidireccional ANOVA con prueba post-hoc de Tukey, con valores de p menor de 0,05 considerado significativo.</li>
			</ol>
</li>
		</ol>
</li>
		<li>Utilice el análisis de immunoblot para establecer la presencia de amiloide, tau oligomérica y Aß en sobrenadantes de cultivo. Realizar el análisis de immunoblot usando procedimientos estándar10,11,12, excepto para concentración de sobrenadantes de cultivo por lo menos 10 veces antes de la electroforesis.<ol>
<li>Para concentrar el sobrenadante, coloque 1-2 mL de sobrenadante en una unidad de filtro de centrifugación cuya membrana tiene un corte de peso molecular de 10 kDa. Luego, coloque la unidad de filtro en una centrífuga de mesa y centrifugar a 2.000 x g hasta el grado necesario de concentración es logra (normalmente 45-60 min).</li>
			<li>Determinar la cantidad de proteína en el sobrenadante concentrado11 y cargar cantidades iguales de muestras en los pocillos del gel.</li>
			<li>Correr los geles, las proteínas de transferencia a nitrocelulosa, luego probe borrones con A11 del anticuerpo anti-amiloide, anticuerpos de anti-oligomeric tau T22 o anticuerpo de anti-Aß MOAB2 seguida de anticuerpos secundarios apropiados. Desarrollar el Blot usando procedimientos estándar de la quimioluminescencia.</li>
		</ol>
</li>
		<li>Para hacer un thioflavin T ensayo, usar fluorescencia de tioflavina T (ThT) para cuantificar amiloides en sobrenadantes. Para estas mediciones, utilizar sobrenadante esterilizado por filtro.<ol>
<li>Prepare una solución (x 50) de tioflavina T suspendiendo tioflavina T (ThT) de 8 mg en solución salina 10 mL tamponada fosfato (PBS). Después de mezclar, filtrar la solución a través de un filtro de 0,22 μm para eliminar partículas.</li>
			<li>Para las medidas, añadir 20 μl del stock de ThT a 1 mL de PBS en una cubeta del espectrofotómetro 1 mL. Coloque la muestra diluida en un espectrofluorímetro.</li>
			<li>Medir la emisión de fluorescencia basal utilizando excitación nm 425 y exploración de la emisión de fluorescencia de 450-575 nm en 2 incrementos de nm.</li>
			<li>Realizar un time-lapse análisis mediante excitación nm 425 y 482 emisión nm, con los datos adquiridos cada 0.2 s 60 s.</li>
			<li>La inicial 20 s del time-lapse análisis medidas de fluorescencia en la cubeta en blanco. A los 20 s, pausar el análisis y añadir 10 μl del sobrenadante esterilizado por filtro a la cubeta. La cubeta la mezcla por inversión y luego vuelva a colocar el espectrofluorímetro.</li>
			<li>Reanudar el análisis basado en el tiempo, adquirir el final 40 s de datos.</li>
			<li>Al finalizar el scan Time-lapse, realizar un análisis de espectro de emisión de fluorescencia final con ajustes idénticos, como se detalla en 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>

Ninguno para el informe.

Aquí, simple en vitro métodos están descritos que permiten la demostración de la generación de amiloides citotóxicos durante la infección con una pulmonía que organismo. Estos métodos incluyen ensayos de citotoxicidad en cultivo celular, immunoblotting, cuantificación de célula matar utilizando un novedoso método microscópico y enlace de ThT. Análisis de los agentes citotóxicos han demostrado que son amiloide en la naturaleza (figura 2 y <strong cla...

Los pacientes que sobreviven la neumonía han elevado las tasas de mortalidad en los meses siguientes hospital descarga1,2,3,4,5,6. En la mayoría de los casos, la muerte se produce por algún tipo de insuficiencia de órgano, incluyendo renal, pulmonar, cardiaca, o hígados eventos como la carrera5,6. Nunca se ha establecido la razón de la elevada tasa de mortalidad en esta población de pacientes.
La neumonía se clasifica como siendo ya sea adquirida en la comunidad o adquirida en el hospital (nosocomial) y los agentes causantes de neumonía incluyen bacterias, virus, hongos y productos químicos. Una de las principales causas de neumonía nosocomial es la bacteria Pseudomonas aeruginosa. P. aeruginosa es un organismo Gram-negativo que utiliza un sistema de secreción tipo III para transferir varias moléculas efectoras, como exoenzimas, directamente en el citoplasma de las células de destino7,8. Durante la infección de células endoteliales pulmonares, las exoenzimas objetivo diversas proteínas intracelulares, incluyendo una forma endotelial de la proteína asociada a microtúbulos tau9,10,11,12 , conduce a la ruptura de la barrera endotelial dando lugar a edema pulmonar severo, disminución de la función pulmonar y, a menudo, la muerte.
Como se indicó anteriormente, los pacientes que sobreviven la neumonía inicial han elevado las tasas de mortalidad en los primeros 12 meses después del alta hospitalaria. Un mecanismo potencial para explicar este fenómeno es que se genera algún tipo de toxina duradera durante la infección inicial que conduce a pobres resultados a largo plazo. Dos observaciones apoyan esta posibilidad. En primer lugar, cultivadas pulmonar las células endoteliales que son tratadas inicialmente con p. aeruginosa no proliferan por hasta una semana después de que las bacterias se destruyen con antibióticos13. Priones en segundo lugar, larga vida y agentes con las características del prión se han demostrado en varias enfermedades humanas y animales, particularmente las enfermedades asociadas con el sistema nervioso14,15.
Nunca se han descrito métodos para examinar el potencial de producción de agentes citotóxicos duraderos durante la infección pulmonar. Aquí se describen una serie de ensayos simples en vitro que puede utilizarse para investigar producción de citotoxina y actividad después de la infección con un agente que causa común de neumonía, p. aeruginosa. Estos ensayos deben ser fácilmente adaptables para investigar la inducción de la citotoxina posible después de la infección con otros agentes causantes de neumonía, y los sobrenadantes que se generan también deberían ser útiles para investigar efectos de citotoxinas en todo órganos o animales. Por último, los ensayos que se describen aquí es muy probable que será adaptables a prueba de fluidos biológicos humanos y animales para la producción de citotoxinas en neumonía.

<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

Los pacientes que sobreviven la neumonía han elevado las tasas de mortalidad en los meses después del alta hospitalaria. Se ha presumido que la infección del tejido pulmonar durante la neumonía los resultados en la producción de citotoxinas larga vida que puede llevar a insuficiencia del órgano de extremo posterior. Hemos desarrollado en vitro ensayos para probar la hipótesis que citotoxinas son producidos durante la infección pulmonar. Las células endoteliales pulmonares aislado de rata y la bacteria Pseudomonas aeruginosa se utilizan como sistemas modelo, y la producción de cytoxins después de la infección de las células endoteliales por la bacteria se demuestra mediante cultivo celular seguido por cuantificación directa mediante los análisis de lactato deshidrogenasa y un novedoso método microscópico utilizando tecnología de ImageJ. La naturaleza amiloide de estas citotoxinas fue demostrada por thioflavin T enlace ensayos y por immunoblotting y immunodepletion usando anticuerpo anti-amiloide de A11. Posteriores análisis mediante immunoblotting demostraron que los tau y Aβ se producida y liberada por las células endoteliales después de la infección por p. aeruginosa. Estos métodos deben ser fácilmente adaptables a los análisis de muestras clínicas humanas.

Un análisis simple en vitro ha sido desarrollado para analizar la presencia de citotoxinas en sobrenadantes de células infectadas con la bacteria p. aeruginosa. Básicamente, medio de cultivo de las células infectadas se recogen 4 h después de la adición de bacteria, las bacterias se eliminan por la esterilización del filtro de la cultura sobrenadante y luego se añade el sobrenadante estéril a una nueva población de células. Las células luego se observan 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 la detección de amiloides siguiente infección de pulmonar endotelial células citotóxicas por Pseudomonas aeruginosa

Se describen métodos sencillos para la demostración de la producción de amiloides citotóxicos después de la infección del endotelio 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 vistas.  University of South Alabama. Se describen métodos sencillos para la demostración de la producción de amiloides citotóxicos después de la infección del endotelio pulmonar por Pseudomonas aeruginosa.

Video: 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.

Preparación y<em&gt; En Vivo</em&gt; El uso de una sonda basada en la actividad de<em&gt; N</em&gt; -acylethanolamine Ácido amidasa

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 ...

Investigación

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.

Sola célula Medidas de Ruptura Vacuolar 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 ...

Inmunología e Infección

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 inducida por la lesión pulmonar 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.

Infección por neumococo de células endoteliales humanas primarias en flujo 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.

Cultivo de la Pequeña Colonia Variante de Pseudomonas aeruginosa y Cantidad de su Alginato

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-cultivo 3D infectado de células epiteliales bronquiales y macrófagos en la interfaz aire-líquido para la evaluación preclínica de los antiinfecciosos

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.

Estudio de los efectos del humo del cigarrillo en la infección por pseudomonas en células epiteliales 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.

Visualización de Pseudomonas aeruginosa en el esputo de pacientes con fibrosis quística

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.

Pruebas de Eficacia De Antibióticos En Un Modelo Ex vivo De Pseudomonas aeruginosa Y Biopelículas De Staphylococcus aureus En El Pulmón De Fibrosis Quística

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.

Detección automatizada y de alto rendimiento de la adherencia bacteriana a las células huésped

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