Damos las gracias a Jessica Jones, Clark Erica, Ducar Constanza, Donna Smith, Lewis Roy, Apedaile Lily, Wiesner Joanne, Adams Devin, McBain Corey y Voght Stephen por su ayuda en el desarrollo de este método, manuscrito y video. Este trabajo fue financiado por la Fundación Bill y Melinda Gates CAVD subvención 38.645 (MJM) y los Institutos Nacionales de Salud subvenciones UM1 AI068618 y AI069481 U01 (MJM). EA-N. es apoyado por el NIH subvención T32 AI007140. Damos las gracias a la James B. Pendleton Charitable Trust por su donación generosa equipo.

Nota: Para proteger los anticuerpos conjugados con fluorocromo de la luz, realice todos los pasos en un gabinete de bioseguridad con la luz apagada. 1. Tinción de anticuerpos Panel Preparación <ol><li> El panel de tinción de anticuerpos se pueden encontrar en la Tabla 1. La concentración de anticuerpo debe ser definido por titulación con sangre entera y usando el equipo de citometría de flujo mismo y los procedimientos que se utilizarán para adquirir las muestras teñidas fenotipificación. </li><li> Una vez los títulos de tinción apropiados se determinan, se combinan todos los anticuerpos en una única mezcla en un tubo de bloqueo de la tapa. Añadir tampón de flujo de lavado (PBS de Dulbecco con 2% inactivado por calor suero fetal bovino) para llevar el volumen total a 100 l. Escala de la mezcla para el número de muestras que se están teñidas. Esta mezcla se puede almacenar a 4 ° C durante hasta ocho semanas. </li></ol> 2. La tinción <ol><li> Si la sangre recogida es para ser utilizado para otros fines en anunciocondición para este ensayo, establecer una alícuota a un lado mientras más sensibles al tiempo los procedimientos se realizan en la sangre restante. La alícuota se pueden almacenar a temperatura ambiente durante hasta 4 horas después de la punción venosa, sin pérdida significativa de células. </li><li> Compruebe que hay un intacta pellet talón en la parte inferior del tubo Trucount y etiqueta el tubo para identificar la muestra que se tiñeron. En los ensayos clínicos HVTN, el Laboratorio de Sistema de Gestión de Datos (Frontier Science y Technology Research Foundation; Amherst, NY) se utiliza para etiquetar y rastrear muestras de colores. </li><li> Anote los números de lote y fechas de caducidad de todos los reactivos. Anote el número de cuenta tubo Trucount talón proporcionado por el fabricante de la bolsa de tubos, asegúrese de que el número de lote en la bolsa coincide con el número de lote del tubo. </li><li> Utilizar Invertir pipeteado para pipetear exactamente 100 l de sangre total en el tubo Trucount, justo encima del retenedor metálico. Evitar manchas de sangre por el lado del tubo. </li><li&gt; Uso normal (hacia adelante) técnica de pipeteo, pipeta 100 l del panel de anticuerpos tinción mixta (Ver Tabla 1) en el tubo Trucount. Tapar el tubo y agitar a baja velocidad durante aproximadamente 15 segundos para mezclar. Inspeccione visualmente el tubo para asegurar que el gránulo cordón esté completamente disuelto. </li><li> Incubar el tubo Trucount durante 15 minutos a temperatura ambiente (15-30 ° C) en la oscuridad. </li><li> Si es necesario, se diluye una alícuota de 10 × solución de lisis FACS a 1X utilizando DIH 2 O. Añadir 900 l 1 x solución de lisis FACS para el tubo. </li><li> Tapar el tubo y agitar cuidadosamente a baja velocidad durante aproximadamente 15 segundos para mezclar. Empuje la tapa hacia abajo firmemente en la posición de bloqueo en el tubo y la junta con película de laboratorio. </li><li> Almacenar el tubo a -65 ° C a -95 ° C hasta que la muestra está lista para su envío al laboratorio central de análisis o para el análisis en casa. Las muestras son estables en esta etapa durante al menos cuatro semanas. Si va a enviar o ensayar inmediatatamente, este paso puede ser omitido. </li></ol> 3. Envío Nota: Las siguientes instrucciones utilizan un sistema de envío aislados de Saf-T-Pak, Inc. diseñado específicamente para envío categoría B biológicos sustancias exentas de acuerdo con International Air y Transport Association (IATA). Si el análisis de las muestras en la misma ubicación que la tinción ocurrió, vaya a la sección 4. <ol><li> Las muestras pueden ser enviado inmediatamente después de la tinción o una vez que se congelan a -65 ° C a -95 ° C. Envuelva cada tubo completamente en papel de aluminio y coloque en el cuadro de muestra de colores. Coloque la caja espécimen manchado el interior de una bolsa de polietileno a prueba de fugas con materiales absorbentes. </li><li> Coloque la prueba de filtración bolsa de polietileno y el contenido en una bolsa de Tyvek y sello con tan poco aire como sea posible en la bolsa. </li><li> Coloque el paquete espécimen (muestra dentro de un embalaje secundario) dentro de la caja marrón interior. </li><li> B Coloque el interior marrónbuey en el interior del pecho de espuma de poliestireno, fijándola en la muesca para evitar que se desplacen. </li><li> Llene el pecho de espuma de poliestireno con hielo seco (aproximadamente 8 kg) y coloque la tapa firmemente en el pecho. </li><li> Asegure con cinta adhesiva la caja de envío y de la nave como de Sustancias biológicas, Categoría B (UN3373) con el hielo seco adecuado (UN1845) del producto, siga IATA PI-650 instrucciones. </li><li> Tras la recepción, las muestras se almacenaron a -65 ° C a -95 ° C hasta que se analizaron. </li></ol> 4. Descongelación y análisis de citometría de flujo <ol><li> Retirar la muestra teñida del congelador se descongele a temperatura ambiente en la oscuridad antes de recoger en el citómetro de flujo. Si la recopilación de datos en más de una muestra, estandarizar el proceso para todos los tubos mediante el escalonamiento descongelación manera que los tubos no están sentados a temperatura ambiente durante más de 1 hora cada uno. </li><li> Las muestras deben ser obtenidas usando un citómetro de flujo láser cuatro equipado con filtros adecuados, tales como el BD LSRII. Utilice la calibración citómetro estándar y métodos de fluorescencia de compensación para la recolección de datos 4. </li></ol> Nota: No establezca umbrales de dispersión hacia adelante o al lado de dispersión durante la recogida 5. Perlas Trucount puede caer por debajo del valor de umbral más bajo posible para estos parámetros causantes de un subconjunto de perlas no tenerse en cuenta durante el análisis. Si es requerido por el instrumento, para establecer un umbral más bajo posible establecer el umbral Am Cian canal. Debido a que los leucocitos CD45 + teñidas con el panel estará Am Cian positivo, y los granos también Trucount fluorescencia en el canal Am Cian, esto debería permitir a todos los datos relevantes para ser debidamente recogidos. <ol start="3"><li> Vortex el tubo de muestra durante 5 segundos antes de cargar en el citómetro de flujo. Cuentas Trucount fluorescente altamente en muchos canales. Puerta de las cuentas durante la recolección mediante la búsqueda de la población que es muy positivo para la doble-PE Cy5 y APC (la colo dosrs que distinguen más fácilmente las perlas de las células puede variar dependiendo de la instrumentación). Seleccione la puerta de perla como la puerta de detener y registrar datos hasta por lo menos 20.000 cuentas se adquieren. </li><li> Analizar los datos con el software adecuado, como FlowJo (Treestar; Ashland, OR). Figura 1 se muestra el esquema de puerta utilizado para el análisis de las diferentes poblaciones de leucocitos de un empate de sangre del control representativo. </li></ol> 5. Cálculos Trucount <ol><li> Cada tubo contiene Trucount una pastilla liofilizada de perlas fluorescentes. Después de la adición de líquido al tubo y la agitación en vórtex, las perlas deben ser distribuidas por igual en toda la muestra. El número de los granos en la pastilla varía ligeramente según el número de lote y se puede encontrar en la bolsa de almacenamiento para los tubos. </li><li> Puerta de las cuentas Trucount y poblaciones de células, como se muestra en la Figura 1 para determinar el recuento de eventos para cada población. Comparando el número de eventos in de la puerta de vidrio hasta el número total de bolas originalmente en el tubo le permitirá determinar la proporción de la muestra recogidos, que entonces se puede utilizar para determinar la concentración absoluta (es decir, las células / l) para cada población. La siguiente ecuación se puede usar para este propósito: la concentración de la célula (# células / sangre entera l) = [# de población acontecimientos (eventos / # / # grano granos totales en pellet)] / 100 mu l. </li></ol> 6. Los resultados representativos <img src="/files/ftp_upload/4302/4302fig1.jpg" alt="Figura 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4302/4302fig1highres.jpg" /> Figura 1. Gating esquema utilizado para el análisis de grandes poblaciones de leucocitos que muestran datos representativos de un voluntario sano. A) perlas Trucount (i) están cerradas y excluido de las células. Granulocitos (ii) se delinean y lympohcytes y monocitos se dividen en 3 popluations: CD14linfocitos negativos (iii), todas las células CD14 negativas (iv) y que no son linfocitos (v). B) CD14 linfocitos negativos son gated para distinguir células T CD4 + (vi), células CD8 + T (vii), células B (viii), células CD56 brillantes NK (ix), CD56 dim células NK (x), y CD56 células NK negativos (xi). C) Todas las células CD14 negativas están cerradas para distinguir mieloide (xii) y (xiii plasmacitoides) células dendríticas. D) No lympocytes están cerradas para distinguir no clásicos (xiv), intermedio (XV) y clásico (xvi) los monocitos. <a href="http://www.jove.com/files/ftp_upload/4302/4302fig1large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . Subconjuntos de células más específicas que no se muestran en la Figura 1 (por ejemplo, las células NKT o neutrófilos) también se pueden distinguir mediante el panel se presentan, y el esquema de puerta puede ser ampliado o modificado para satisfacer las necesidades específicas de estudio. Algunos pasos de activación periódica mostrados son únicas a este método. Cabe destacar que una puerta de la inclusión y lapuerta de exclusión se dibujan alrededor de las perlas Trucount y se coloca en la parte superior de uno al otro, uno a la puerta de las perlas para el recuento, y la otra para excluir las perlas del análisis celular (Figura 1A). Además, debido a los linfocitos, monocitos y granulocitos no son como se distingue fácilmente en la sangre entera como lo son en las células mononucleares de sangre periférica por dispersión frontal y dispersión lateral, gating estas células utilizando expresión CD45 y la dispersión lateral es a menudo necesario (Figura 1A). Granulocitos contaminantes (círculo) que no pudieron ser separados de linfocitos y monocitos utilizando CD45 y dispersión lateral son distinguibles en algunas parcelas por su alta expresión de CD16 (Figura 1C y Figura 1D). El número de granulocitos contaminantes es generalmente pequeño, y que no interfieran con gating celular de monocitos y NK.

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No hay conflictos de interés declarado.

En este informe se presenta un método basado en perlas para enumerar las poblaciones de leucocitos en sangre entera fresca por citometría de flujo y cubrir los parámetros necesarios para su uso en un ensayo clínico multicéntrico con el análisis de muestras centralizado. Este método se basa en el protocolo y optimiza BD Trucount y permite su uso confiable en un entorno ensayo clínico multicéntrico. El ensayo de tinción es simple y toma aproximadamente 45 minutos para llevar a cabo, por lo que es factible para l...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Trucount absolutos Tubos de conteo </td><td> BD Biosciences </td><td> 340334 </td></tr><tr><td> FACS 10X Solución de lisis </td><td> BD Biosciences </td><td> 349202 </td></tr><tr><td> Categoría B &amp; Sistema de Envío Quito, con aislamiento </td><td> Saf-T-Pak </td><td> STP-320 </td></tr><tr><td> CD45 AmCyan anticuerpo monoclonal </td><td> BD Biosciences </td><td> 339192 </td></tr><tr><td> CD3 FITC anticuerpo monoclonal </td><td> BD Biosciences </td><td> 349201 </td></tr><tr><td> CD8-PerCP Cy 5,5 anticuerpo monoclonal </td><td> BD Biosciences </td><td> 341051 </td></tr><tr><td> CD4 Alexa Fluor 700 Antibo monoclonaldy </td><td> BD Biosciences </td><td> 557922 </td></tr><tr><td> HLA-DR ECD anticuerpo monoclonal </td><td> Beckman Coulter </td><td> IM3636 </td></tr><tr><td> V450 CD14 anticuerpo monoclonal </td><td> BD Biosciences </td><td> 560349 </td></tr><tr><td> CD19 PE anticuerpo monoclonal </td><td> BD Biosciences </td><td> 555413 </td></tr><tr><td> CD16 APC-H7 anticuerpo monoclonal </td><td> BD Biosciences </td><td> 560195 </td></tr><tr><td> CD56 PE-Cy7 anticuerpo monoclonal </td><td> BD Biosciences </td><td> 335791 </td></tr><tr><td> CD11c APC anticuerpo monoclonal </td><td> BD Biosciences </td><td> 559877 </td></tr><tr><td> CD123 PE-Cy5 anticuerpo monoclonal </td><td> BD Biosciences </td><td> 551065 </td></tr></tbody></table>

enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay

Crioconservación de leucocitos de sangre periférica se utiliza ampliamente para conservar las células para las evaluaciones de la respuesta inmune en los ensayos clínicos y ofrece muchas ventajas para la facilidad y la normalización de las evaluaciones inmunológicas, pero los efectos perjudiciales de este proceso se han observado en algunos subconjuntos de células, tales como los granulocitos, células B , y las células dendríticas 1-3. Ensayo de leucocitos frescas da una imagen más precisa del estado in vivo de las células, pero a menudo es difícil de realizar en el contexto de ensayos clínicos grandes. Los ensayos de células frescas dependen de compromisos voluntarios y los plazos, y si consume tiempo, su aplicación puede ser poco práctico debido a las horas de trabajo necesarias del personal de laboratorio. Además, cuando los ensayos se llevan a cabo en varios centros, laboratorios con los recursos y la capacitación necesarios para llevar a cabo los ensayos no pueden estar situados en la proximidad suficiente a los sitios clínicos. Para abordar estas cuestiones, tenemos que desarrorrollado un panel de anticuerpos 11-tinción de color que se puede utilizar con tubos de Trucount (Becton Dickinson, San Jose, CA) a fenotipo y enumerar las principales poblaciones de leucocitos en la sangre periférica, con un rendimiento más robusto de células de tipo información específica de ensayos tales como un Recuento sanguíneo completo (CSC) o ensayos comercialmente disponibles con paneles diseñados para tubos Trucount que manchan por sólo unos pocos tipos de células. El procedimiento de tinción es simple, requiere sólo 100 l de sangre entera fresca, y tarda aproximadamente 45 minutos, por lo que es factible para el estándar de procesamiento de la sangre para llevar a cabo los laboratorios. Es una adaptación de la hoja BD Trucount tubo técnica de datos ( <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">versión 8/2010</a> ). El cóctel de anticuerpos de tinción puede ser preparado de antemano a granel en un laboratorio central de ensayo y se envían a los laboratorios de transformación en el lugar. Tubos manchados puede ser fijadoy se congelan para su envío al laboratorio de ensayo central para el análisis multicolor citometría de flujo. Los datos generados a partir de este panel de tinción se puede utilizar para rastrear los cambios en las concentraciones de leucocitos en el tiempo en relación con la intervención y fácilmente podrían ser desarrolladas para evaluar los estados de activación de tipos de células específicas de interés. En este informe, demuestran el procedimiento utilizado por los técnicos de laboratorio de procesamiento de sangre para realizar la tinción de sangre entera fresca y los pasos para analizar estas muestras teñidas en un laboratorio de ensayo de soporte central un ensayo clínico multicéntrico. Los detalles del video del procedimiento, ya que se realiza en el contexto de un proyecto de ensayo clínico de la sangre en la Red de Ensayos de Vacunas contra el VIH (HVTN).

Watch this Scientific Journal Video about Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay at JoVE.com

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

En este informe, demuestran los pasos de tinción y análisis de un ensayo de fenotipo realiza en la sangre entera fresca para enumerar las principales poblaciones de leucocitos innatos y adaptativos. Hacemos hincapié en las consideraciones para llevar a cabo estos procedimientos en el contexto de un ensayo clínico multicéntrico.

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 ...

enumeration-major-peripheral-blood-leukocyte-populations-for

Research

JoVE Journal

Immunology and Infection

14.9K vistas.  Fred Hutchinson Cancer Research Center. En este informe, demuestran los pasos de tinción y análisis de un ensayo de fenotipo realiza en la sangre entera fresca para enumerar las principales poblaciones de leucocitos innatos y adaptativos. Hacemos hincapié en las consideraciones para llevar a cabo estos procedimientos en el contexto de un ensayo clínico multicéntrico.

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

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

El agotamiento de las poblaciones de células específicas por el agotamiento del complemento

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Investigación

Inmunología e Infección

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

La purificación de la población de células específicas de clasificación celular activado por fluorescencia (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

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 ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansión de la sangre periférica humana de células T γδ con zoledronato

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (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 titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

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

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of 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 ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

Evaluación rápida y específica de la peroxidasa Actividad de halogenación en muestras de sangre enriquecida en leucocitos

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

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 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Análisis unicelular de Immunophenotype y producción de citoquinas en sangre periférica por citometría de masas

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...