Este trabajo fue financiado por becas de personal de la Wellcome Trust para Beate Kampmann (056.608, GRO77273).

1. Preparación de las acciones de BCG reportero lux micobacterias <ol><li> Crecer BCG lux (M. bovis BCG cepa Montreal transformadas con el plásmido lanzadera pSMT1) con agitación a 200 rpm hasta mediados de la fase logarítmica en caldo Middlebrook 7H9 que contenía 0,2% de glicerol, 0,05% entre 80 y 10% de enriquecimiento ADC. </li><li> Prepare 1 ml de una dilución de 1:10 de la BCG cultura lux en PBS estéril en un tubo luminómetro (900μl PBS + 100μl BCG cultura lux) por duplicado. </li><li> Carga del tubo que contiene N-decil aldehído (sustrato de la enzima luciferasa) en la parte posterior de la máquina y asegurarse de que la tapa esté asegurada y los tubos en su lugar. </li><li> Primer luminómetro inyector con el sustrato a través del programa principal, ajustado a inyectar 100 UCL x 3 en cada uno de 3 tubos de cebado vacío colocado en el luminómetro. </li><li> La carga de los 2 tubos en el luminómetro y tomar las lecturas a través de un programa conjunto de inyectar 100 UCL de sustrato en cada tubo y leer durante 20 segundos en intervalos de 1 seg. </li><li> Cuando la acción ha llegado a 1x10 8 RLU / ml a 2x10 8 RLU / ml (esto tarda unos 3-4 días de crecimiento), añadir un volumen igual de glicerol estéril al 30% a la cultura en un tubo de 50 ml de halcón y mezclar suavemente. </li><li> Alícuota de 1,5 ml los volúmenes de 2 ml en un recipiente etiquetado con tapón de rosca microtubo y almacenar a -80 ° C. </li></ol> 2. La determinación de valores de RLU / UFC correlación y el contenido de las alícuotas <ol><li> Añadir 15 ml de medio Middlebrook 7H9 cultura con un 10% suplemento ADC a un matraz Erlenmeyer de 200 ml con un tapón ventilado. </li><li> Añadir 15μl de higromicina y 30μl de 20% de Tween. </li><li> Saca una ampolla de la vacuna BCG lux del congelador y descongelar a temperatura ambiente (TA) en el gabinete de seguridad. Añadir el contenido del vial en el medio y apretar la tapa. </li><li> Incubar con agitación a 200 rpm a 37 ° C durante 4 días. </li><li> En cada día, conforman serie de 10 diluciones (1:10, 1:100, 1:1000, 1:10.000 y 1:100.000) para la determinación de UFC. </li><li> Establecer las mismas diluciones en paralelo y duplicado para determinar la luminiscencia (véase la parte 1.2 hasta 1.6). </li><li> Prepare dos de 3 compartimientos placas de agar Middlebrook 7H11 (1 l de medio líquido que contiene 0,5% de glicerol, 10% de suplemento OADC, 1 ml de Tween 20% y 1 ml de higromicina). </li><li> Placa de 100μl de cada dilución en un segmento de cada placa con crucetas por separado, la distribución del líquido por igual a través de la cámara individual. </li><li> Sellar cada placa con parafilm, colóquela en una bolsa de plástico estéril, selle con cinta de autoclave, y se incuba a 37 ° C durante 2 semanas con las tapas hacia abajo. </li><li> Inspeccione periódicamente hasta UFC aparecen (2-3 semanas). </li><li> Para contar las colonias, quitar las placas de la incubadora y el lugar en un contador de colonias. Calcular la media para cada dilución de las placas duplicadas. </li><li> Calcular la razón de RLU / UFC utilizando equivalente RLU y el recuento de UFC por cada dilución. La relación debe ser de entre 3 y 5 RLU / UFC. </li></ol> 3. Preparación de la vacuna BCG cultura lux para su inoculación en sangre total <ol><li> Añadir 15 ml de medio Middlebrook 7H9 cultura con un 10% suplemento ADC a un matraz Erlenmeyer de 200 ml con un tapón ventilado. </li><li> Añadir 15μl de higromicina y 30μl de 20% de Tween. </li><li> Saca una ampolla de la vacuna BCG lux del congelador y descongelar a temperatura ambiente en el gabinete de seguridad. Añadir el contenido del vial en el medio y apretar la tapa. </li><li> Incubar con agitación a 200 rpm a 37 ° C durante 2 a 4 días. </li><li> Prepare 1 ml de una dilución de 1:10 de la BCG cultura lux en PBS estéril en un tubo luminómetro (900μl PBS + 100μl BCG cultura lux) por duplicado. </li><li> Primer luminómetro con el sustrato (como se describió anteriormente), suavemente vórtice de los 2 tubos y carga en el luminómetro y tomar las lecturas (como se describió anteriormente). </li><li> Use esta lectura para diluir por la cultura en PBS para dar un equivalente de 7x10 6 RLU (esto le dará un inóculo de aproximadamente 2x10 5 UFC / ml de sangre y una relación de BCG a los monocitos de aproximadamente 1:1, asumiendo un recuento de monocitos de de 2x10 5 4x10 5 de monocitos por ml de sangre). </li></ol> 4. Preparación de sangre entera <ol><li> Tomar de 3 a 5 ml de sangre venosa en un tubo con heparina sin conservantes. </li><li> La transferencia de la sangre a un tubo de 50 ml halcón y diluir con un volumen igual de RPMI 1640 con HEPES 25 mM de glutamina y (no penicilina / estreptomicina). </li><li> 900μl alícuota de sangre diluida por triplicado en tubos estériles bijou para cada momento (6 tubos en total: 3. 0hrs de 96 horas y 3 para Establecer tubos adicionales para los puntos de tiempo adicional, si es necesario. </li><li> 900μl alícuota del medio Middlebrooks cultura 7H9 con ADC de 10% de suplemento higromicina + 50μg/ml (el mismo medio que utiliza para la cultura de la BCG lux) por duplicado para los controles de crecimiento. </li><li> Agregar 100μl de los gases de BCG lux a cada tubo de sangre, y para el control de un crecimiento del 2los tubos. </li><li> Mezclar bien y colocar el 3 bijous para el 96 h de duración punto y el 2, controla el crecimiento de su lado en la incubadora oscilante a 37 ° C, 20 rev / min (CO 2 no es obligatorio). </li></ol> 5. Luminiscencia de medición (a 0hr y 96 horas) <ol><li> Centrifugar los 3 bijous a 2000 g durante 10 minutos. </li><li> Retire con cuidado 300μl de sobrenadante sin perturbar el precipitado y el lugar de 2 ml con tapa de rosca microtubos para congelar a -80 ° C para el análisis posterior de citocinas. </li><li> Agregar 300μl de PBS a cada uno de bijou para reemplazar el volumen de sobrenadante retirado. </li><li> Aspirar el contenido de cada joya en el correspondiente tubo Falcon de 50ml y añadir 8 ml de agua destilada a cada tubo. Iniciar un temporizador de 10 minutos. Nota: Este es un paso sensible al tiempo, y la incubación no debe haber más de 10 minutos, a partir de cuando las primeras células entran en contacto con el agua. </li><li> Enjuague cada bijou con 2 ml de agua destilada y agitar durante 5 segundos antes de inflexión en el correspondiente tubo Falcon. </li><li> Al final de la incubación de 10 minutos, centrifugar los tubos falcon en 2000 g durante 10 minutos. </li><li> Decantar el sobrenadante en Surfanios desinfectante. </li><li> Añadir algunas perlas de vidrio para cada pastilla y agitar. </li><li> Añadir 1 ml de PBS estéril a cada tubo y agitar. </li><li> En los tubos de luminómetro, hacer diluciones 1:10 de cada muestra por duplicado con PBS (900μl PBS + 100μl de la muestra) y 96 h en el momento de punto. Hacer las mismas diluciones de cada uno de los controles de crecimiento. </li><li> Suavemente en el Vórtex y la carga en el luminómetro y tomar las lecturas (como se describió anteriormente) Nota: Las lecturas entre triplicado de la misma muestra deben estar dentro del 15% de uno a otro. </li></ol> 6. Los resultados representativos: Es importante la utilización de bacterias en fase logarítmica de crecimiento para toda la sangre de los ensayos de lux, es decir, creció más de 48-72 horas antes de la inoculación de las muestras, como la actividad metabólica no es óptima, si bien en línea recta de la nevera o en fase estacionaria. El crecimiento antes debe estandarizarse para una serie de experimentos a 48 o 72 horas para evitar la variabilidad. La figura 1 muestra la curva de crecimiento de una cultura de representante de BCG lux. El tiempo de duplicación es de aproximadamente 24 horas hasta que se alcanza la fase estacionaria. <img alt="Figura 1" src="/files/ftp_upload/3332/3332fig1.jpg" /> Figura 1. Representante de la curva de crecimiento de BCG lux durante 96 horas en medio 7H9 y la correlación de RLU y UFC. Aunque los valores de RLU siempre se correlaciona con la UFC, el número de RLU que corresponde a una sola UFC puede variar dependiendo de las acciones, por lo que es una buena práctica para utilizar el stock congelado misma a través de una serie de experimentos para garantizar una multiplicidad de infección constante ( MOI). La Tabla 1 muestra un ejemplo de la fecha de primas en el momento de la inoculación (T 0) y en 96 horas. Los ratios de crecimiento se calculan utilizando la fórmula T 96 / T 0, pero los intervalos de tiempo de otros, por supuesto, también puede ser medido. Es, sin embargo, no se recomienda el uso de las culturas más allá de 96 horas, como la muerte celular significativa. <img alt="Tabla 1" src="/files/ftp_upload/3332/3332table1.jpg" /> Tabla 1. Ejemplo de los datos en bruto en el momento de la inoculación (T0) y en 96 horas (T96) y se calcularon los cocientes de crecimiento de 3 donantes adultos. Dependiendo de las respuestas antígeno-específicas de la memoria disponible y el recuento de neutrófilos, posiblemente, también, las tasas de crecimiento varían entre los individuos, como se muestra en la Figura 2 en la sangre de los niños con y sin infección por el VIH. En promedio, los niños pequeños tienen mayores tasas de crecimiento que los adultos, prueba de la tuberculina (TST) + personas han tienen menores tasas de crecimiento que las personas han TST-, y los pacientes con VIH tienen altas tasas de crecimiento debido a la deficiencia de la población de células T CD4, uno de los principales mediadores de la respuesta inmune celular a la micobacteria. <img alt="Figura 2" src="/files/ftp_upload/3332/3332fig2.jpg" /> Figura 2 variabilidad entre los donantes:. Ratios de crecimiento de T 0 frente a 96 horas por un grupo de pacientes, dependiendo de la situación subyacente de VIH. Reproducibilidad de los ratios de crecimiento durante un período de tiempo se muestra en la figura 3, que resume los resultados de 64 donantes sangrado dos veces en un período de 12 meses y de un solo donante sangrado varias veces para los experimentos de control. Entre las posibles causas de la variabilidad podría haber cambios en la sensibilidad de micobacterias o la variabilidad en los niveles de citocinas de acogida, como se observa en muchos bioensayos. <img alt="Figura 3" src="/files/ftp_upload/3332/3332fig3.jpg" /> Figura 3. (A) solo donante en 12 ocasiones en 12 meses. (B) varios (64) donantes en 2 ocasiones más de 12 meses.

<ol>
	<li>Cheng, S. H., Walker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+increased+anti-mycobacterial+activity+in+peripheral+blood+monocytes+after+BCG+vaccination+in+British+school+children.">Demonstration of increased anti-mycobacterial activity in peripheral blood monocytes after BCG vaccination in British school children.</a> Clin Exp Immunol. 74, 20-205 (1988).</li><li>Cheng, S. H., Walker, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+antimycobacterial+activity+before+and+after+Mycobacterium+bovis+BCG+vaccination+in+Chingleput,+India,+and+London,+United+Kingdom.">Monocyte antimycobacterial activity before and after Mycobacterium bovis BCG vaccination in Chingleput, India, and London, United Kingdom.</a> Infect Immun. 61, 4501-4503 (1993).</li><li>Silver, R. F., Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+virulence+of+Mycobacterium+tuberculosis+within+human+monocytes:+virulence+correlates+with+intracellular+growth+and+induction+of+tumor+necrosis+factor+alpha+but+not+with+evasion+of+lymphocyte-dependent+monocyte+effector+functions.">Expression of virulence of Mycobacterium tuberculosis within human monocytes: virulence correlates with intracellular growth and induction of tumor necrosis factor alpha but not with evasion of lymphocyte-dependent monocyte effector functions.</a> Infect Immun. 66, 1190-1199 (1998).</li><li>Hoft, D. F., Worku, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+relationships+between+immune-mediated+inhibition+of+mycobacterial+growth+and+other+potential+surrogate+markers+of+protective+Mycobacterium+tuberculosis+immunity.">Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity.</a> J Infect Dis. 186, 1448-1457 (2002).</li><li>Worku, S., Hoft, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+control+and+antigen-specific+T+cells+on+intracellular+mycobacterial+growth.">Differential effects of control and antigen-specific T cells on intracellular mycobacterial growth.</a> Infect Immun. 71, 1763-1773 (2003).</li><li>Kampmann, B., Gaora, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+human+antimycobacterial+immunity+using+recombinant+reporter+mycobacteria.">Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria.</a> J Infect Dis. 182, 895-901 (2000).</li><li>Cheon, S. H., Kampmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bactericidal+activity+in+whole+blood+as+a+potential+surrogate+marker+of+immunity+after+vaccination+against+tuberculosis.">Bactericidal activity in whole blood as a potential surrogate marker of immunity after vaccination against tuberculosis.</a> Clin Diagn Lab Immunol. 9, 901-907 (2002).</li><li>Janulionis, E., Sofer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+activity+of+orally+administered+clofazimine+against+intracellular+Mycobacterium+tuberculosis+in+whole-blood+culture.">Lack of activity of orally administered clofazimine against intracellular Mycobacterium tuberculosis in whole-blood culture.</a> Antimicrob Agents Chemother. 48, 3133-3135 (2004).</li><li>Kampmann, B., Tena, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+human+in+vitro+system+for+evaluating+antimycobacterial+vaccines.">Novel human in vitro system for evaluating antimycobacterial vaccines.</a> Infect Immun. 72, 6401-6407 (2004).</li><li>Kampmann, B., Tena-Coki, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+antimycobacterial+immune+responses+in+HIV-infected+children+receiving+HAART.">Reconstitution of antimycobacterial immune responses in HIV-infected children receiving HAART.</a> Aids. 20, 1011-1018 .</li><li>Martineau, A. R., Wilkinson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+dose+of+vitamin+D+enhances+immunity+to+mycobacteria.">A single dose of vitamin D enhances immunity to mycobacteria.</a> Am J Respir Crit Care Med. 176, 208-213 (2007).</li></ol>

No hay conflictos de interés declarado.

Ensayos bactericidas en el contexto de la investigación vacunas contra la tuberculosis están bien establecidos en modelos animales, donde se aplican a la pantalla de vacunas potencialmente prometedores. La reducción de la carga bacteriana en las funciones de varios órganos como la principal lectura de la inmunogenicidad. La primera generación de humanos ensayos bactericidas fueron diseñados utilizando los macrófagos solo, infectadas con el M. tuberculosis. UFC sirvió como la...

<table border="1"><tbody><tr><td> Reactivo </td><td> Empresa </td><td> No gato </td></tr><tr><td> BCG lux </td><td> Varios </td><td> n / a </td></tr><tr><td> Middlebrook 7H11 agar </td><td> Becton Dickinson </td><td> 283810 </td></tr><tr><td> Caldo de Middlebrook 7H9 </td><td> BD Biosciences </td><td> 271310 (500g) </td></tr><tr><td> Middlebrook ADC enriquecimiento </td><td> BD Biosciences </td><td> 212352 </td></tr><tr><td> Middlebrook OADC suplemento </td><td> Becton Dickinson </td><td> 212240 </td></tr><tr><td> Tween 80 </td><td> Sigma Aldrich </td><td> 274364 </td></tr><tr><td> Glicerol </td><td> Sigma Aldrich </td><td> G5150 </td></tr><tr><td> Higromicina B </td><td> Roche </td><td> 10843555001 (20 ml) </td></tr><tr><td> N-decil aldehído </td><td> Sigma </td><td> D7384-100G </td></tr><tr><td> Etanol (&gt; 99,7%) </td><td> VWR </td><td> 101077 </td></tr><tr><td> PBS estéril </td><td> En Casa </td><td> n / a </td></tr><tr><td> RPMI-1640 con L-Glutamina, HEPES 25 mM </td><td> Sigma </td><td> R0883 </td></tr><tr><td> El agua destilada estéril </td><td> En Casa </td><td> n / a </td></tr><tr><td colspan="3"> Equipo </td></tr><tr><td> Tubo luminómetro (puerto inyectable obligatorio) </td><td> Berthold AutoLumat LB953 o de un solo tubo de Sirius luminómetro </td><td></td></tr><tr><td> Tubos luminómetro, 12 x 75 mm, pyrex sin montura </td><td> Corning de VWR </td><td> 99445-12 </td></tr><tr><td> Erlenmeyer con tapones ventilados, estéril 250 ml de policarbonato </td><td> Corning a través de Fisher Scientific </td><td> 2150329 </td></tr><tr><td> BD Vacutainer tubos de extracción de sangre (heparina sin conservantes) </td><td> BD </td><td> 367676 </td></tr><tr><td> 50 ml Falcon tubos cónicos </td><td> BD </td><td> 352077 </td></tr><tr><td> 7ml Sterilin muestra de polipropileno bijou tubos </td><td> VWR </td><td> 99445-12 </td></tr><tr><td> Sterilin Universal de los contenedores (30 ml) </td><td> Laboratorio de Análisis LTD </td><td> 128C </td></tr><tr><td> Cuentas de vidrio de 2 mm de diámetro </td><td> SigmaAldrich </td><td> Z143928 </td></tr><tr><td> 10 ml pipetas y pipetas niño </td><td> cualquier </td><td></td></tr><tr><td> 1000μl y 200μl pipetas y puntas con filtro </td><td> cualquier </td><td></td></tr><tr><td colspan="3"> Equipo de laboratorio: </td></tr><tr><td> Clase II gabinete de seguridad del gabinete </td><td></td><td></td></tr><tr><td> Refrigerador (4 ° C) </td><td></td><td></td></tr><tr><td> Centrífugo </td><td></td><td></td></tr><tr><td> Mecedora agitador de plataforma (180 0) para mezclar los tubos </td><td></td><td></td></tr><tr><td> Agitador incubadora a 37 ° C para crecer cultivos bacterianos </td><td></td><td></td></tr></tbody></table>

a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)

Ensayos funcionales han desempeñado un papel clave en la medición de la inmunogenicidad de una vacuna administrada. Esto se expresa convencionalmente como suero títulos bactericidas. Los estudios de títulos bactericidas séricos en respuesta a las vacunas infantiles nos han permitido desarrollar y validar niveles de corte de la respuesta inmunitaria protectora y como corte-offs son de uso rutinario. No hay tales pruebas se han llevado adelante en la evaluación de rutina de las vacunas que inducen principalmente inmunidad mediada por células en forma de respuestas de células T efectoras, tales como vacunas contra la tuberculosis. En el modelo animal, el rendimiento de un candidato a vacuna dada es evaluada en forma rutinaria en los ensayos normalizados bactericida, y todos los actuales nueva vacuna contra la tuberculosis, los candidatos han sido sometidos a este paso en su evaluación antes de la fase 1 de ensayos humanos. La evaluación de la inmunogenicidad y por lo tanto, la probabilidad de eficacia protectora de la novela de vacunas contra la tuberculosis idealmente deben ser sometidos a un similar paso a paso la evaluación de los modelos humanos ahora, incluyendo las mediciones en los ensayos de bactericidas. Ensayos bactericidas en el contexto de la investigación sobre la tuberculosis vacuna ya están bien establecidos en los modelos animales, donde se aplican a la pantalla de vacunas potencialmente prometedores. La reducción de la carga bacteriana en las funciones de varios órganos como la principal lectura de la inmunogenicidad. Sin embargo, estos ensayos no han sido incorporados en los ensayos clínicos de nuevas vacunas contra la tuberculosis hasta la fecha. Aunque todavía hay incertidumbre sobre los mecanismos exactos que llevan a la muerte de las micobacterias interior de los macrófagos humanos, la interacción de los macrófagos y las células T con micobacterias es claramente necesario. El ensayo se describe en este documento representa una generación nueva de ensayos bactericidas que permite a los estudios de estos componentes celulares claves con todos los demás factores humorales y celulares presentes en la sangre entera sin hacer suposiciones acerca de su contribución individual relativa. El ensayo descrito por nuestro grupo utiliza pequeños volúmenes de sangre total y ya ha sido empleado en los estudios de adultos y niños en las zonas endémicas de TB-configuración. Hemos demostrado la inmunogenicidad de la vacuna BCG, un mayor crecimiento de las micobacterias en pacientes VIH-positivos, así como el efecto de la terapia anti-retroviral y la vitamina D en la supervivencia de micobacterias in vitro. Aquí un resumen de la metodología, y presentar nuestros datos de reproducibilidad utilizando este modelo relativamente simple, de bajo costo y el campo de usar. Nota: Las definiciones / abreviaturas BCG lux = M. bovis BCG, cepa de Montreal, transformada con pSMT1 plásmido lanzadera que lleva los genes luxAB de Vibrio harveyi, bajo el control de la micobacteria GroEL (hsp60) promotor. UFC = unidades formadoras de colonias (una medida de la viabilidad de micobacterias).

Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

Se describe un método alternativo para la enumeración de las micobacterias<em&gt; In vitro</em&gt;, Que utiliza el reportero de genes etiquetados micobacterias en lugar de unidades formadoras de colonias (UFC). &quot;Survival&quot; de los organismos así como el anfitrión de respuesta se miden los marcadores simultáneamente, proporcionando un bajo costo, un sistema versátil y funcional para el estudio de las interacciones hospedante / patógeno en el contexto de la tuberculosis.

Un ensayo funcional de sangre completa para medir la viabilidad de las micobacterias, usando Reporter-Gene Tagged BCG o M. tuberculosis (BCG Lux / M. Tb Lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

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A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

13.8K vistas.  Imperial College London. Se describe un método alternativo para la enumeración de las micobacterias In vitro, Que utiliza el reportero de genes etiquetados micobacterias en lugar de unidades formadoras de colonias (UFC). "Survival" de los organismos así como el anfitrión de respuesta se miden los marcadores simultáneamente, proporcionando un bajo costo, un sistema versátil y funcional para el estudio de las interacciones hospedante / patógeno en el contexto de la t...

Video: A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansión, purificación y evaluación funcional de la sangre periférica humana células NK

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Investigación

Inmunología e Infección

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

La enumeración de las principales poblaciones de leucocitos de sangre periférica en ensayos clínicos multicéntricos utilizando un ensayo de sangre entera Fenotipado

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Antígenos funcional protegido glóbulos rojos por la membrana injerto de compactos Poligliceroles hiperramificados

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Uso de la superposición de ensayo para medir cualitativamente la producción bacteriana y sensibilidad hacia neumocócica bacteriocinas

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Una citometría de flujo simple método para medir la captación de glucosa y la expresión del transportador de la glucosa por monocitos subpoblaciones en sangre entera

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

La eficacia protectora y la respuesta inmune pulmonar tras la administración subcutánea e intranasal BCG en ratones

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

El uso de una monocapa de monocitos Ensayo para evaluar la fagocitosis mediada por el receptor Fc gamma

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Expresión de antígenos exógenos en la vacuna de BCG de Mycobacterium bovis por genética no decoración de superficie con el sistema avidina-biotina

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

Optimizado de interferón-gamma ELISpot análisis a las respuestas de la célula de T de medida en el modelo de conejillo de Indias después de la vacunación

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...