Este trabajo fue apoyado por el TransRegio Sonderforschungsbereich 19 (SFB/TR19, C2) de la Deutsche Forschungsgemeinschaft (DFG), el Ministerio Federal de Economía y Tecnología proyecto 2727801MD0 ZIM-KF y el Centro de Competencia de Innovación - la respuesta inmune humoral en Enfermedades Cardiovasculares ( ZIK-caminata, BMBF FKZ 03Z2CN12) del Ministerio Federal de Educación e Investigación de Alemania. Vivienda y experimentos con animales se realizaron de conformidad con las recomendaciones de la Sociedad de Ciencia Animal de Laboratorio (Gesellschaft für Versuchstierkunde, GV-SOLAS) y la Federación Europea de Ciencia Animal de Laboratorio (Asociación FELASA).

1. IgG aislamiento de muestras de pacientes <ol><li> Preparar mini-inmunoadsorción columnas de relleno 3 ml de anti-IgG sepharose por 2 ml de plasma EDTA paciente en una columna vacía Econo Pac. IgG aislamiento requiere al menos 2 ml de plasma del paciente. </li><li> Colocar un filtro en la parte superior de la sefarosa anti-IgG, corte la punta inferior de la columna y pulse el filtro para alcanzar una velocidad de flujo de aproximadamente 1 gota / seg. Que la solución de preservación correr fuera de la columna. </li><li> Lavar la columna con 3 volúmenes de NaCl al 0,9% por volumen de plasma. </li><li> Pipetear el plasma en la columna. Cuando el plasma ha completamente sumergido en la sefarosa anti-IgG, se lava con el mismo volumen de 0,9% de NaCl. </li><li> Pipetear un plasma de volumen de HCl 0,2 M glicina, pH 2,8 en la columna y dejar que sumerja en la sefarosa. Añadir otro volumen de glicina HCl en la columna. Recoger el flujo a través de un tubo de 15 ml y ajustar el pH a 7,3 con 0,5 M Tris-HCl, pH 8,0. </li><li> Para regenerar la columna, Se lava con 3 volúmenes de HCl plasma glicina. Después de un lavado final con 2 volúmenes de NaCl al 0,9%, la columna se puede utilizar para la siguiente muestra de plasma. Alternativamente, la columna puede ser llenado con solución de preservación y se almacenó a 4 - 8 ° C durante un máximo de 4 semanas. </li><li> Para el uso en los cardiomiocitos, la fracción recogida IgG tiene que ser dializado frente a tampón experimental (EB). Para la preparación de EB, añadir 117 mM NaCl, 2,8 mM KCl, 0,6 mM de MgCl 2, 1,2 mM KH 2 PO 4, 1,2 mM CaCl 2, 10 mM de HEPES y 20 mM de glucosa a 1 l de agua destilada en un agitador magnético y ajustar el pH a 7,3. </li><li> Llene la fracción IgG a un tubo de éster de celulosa membrana de diálisis con un corte de peso molecular de 100 kDa. Poner el tubo en L EB 1 y se agita lentamente con un agitador magnético a 4 ° C. Después de 8 horas, transferir el tubo de diálisis en 3 L de EB fresco y dializar para otro hr 20 a 4 º C. </li><li> Después de la diálisis, el calor de la muestra durante 30 min a 57 ° C en un baño de agua a inactivatfactores e complemento. Determinar la concentración total de IgG y preparar alícuotas que contienen 330 mg cada IgG. Cuando se añade a la muestra para la medición de los cardiomiocitos (paso 4,3), llenar las alícuotas de 1 ml con EB. Alícuotas diluidas se pueden almacenar a -20 ° C hasta su uso posterior. </li></ol> 2. Aislamiento de cardiomiocitos de rata <ol><li> Para la preparación de Ca 2 +-libre tampón de aislamiento (IB), añadir 110 mM NaCl, 2,6 mM KCl, 1,2 mM MgSO 4, 1,2 mM KH 2 PO 4, 20 mM HEPES y 11 mM de glucosa a 1 l de agua destilada usando un magnética agitador. Ajustar el pH a 7,4 a temperatura ambiente y se filtra a través de un filtro de 0,2 micras superior botella para la esterilización. </li><li> Completa 80 ml de IB en el depósito superior de un sistema de perfusión Langendorff termostatizada. Ajustar la configuración del termostato para mantener una temperatura de amortiguamiento de 37 ° C y airear buffer con% O 95 2/5% CO 2 durante 30 min. </li><li> Pesar aproximadamente 10.000 - 12.000 U de collagENasa tipo II, 175 mg de BSA libre de ácidos grasos y se disuelven en 20 ml que contenían IB 22,5 l de una solución madre 100 mM de CaCl 2. Controlar el pH del tampón de aislamiento en el depósito superior y ajuste si es necesario. </li><li> Anestesiar una rata Wistar de 175 a 200 g con 375 mg / kg de tiopental peso corporal. Añadir 2500 UI de heparina a la solución de tiopental. Después de toracotomía, extirpar el corazón con cuidado con un arco aórtico intactos y la transfiere en helado 0,9% NaCl. Limpiar el corazón de la sangre y el tejido circundante y la transferencia a fresco 0,9% de NaCl. Exponer la aorta, abrir el reductor de flujo del sistema de Langendorff para obtener una velocidad de goteo de 2-3/sec y adjuntar el corazón a la cánula. </li><li> Perfundir el corazón durante un máximo de 3 min con una velocidad de flujo de 1 gota / seg para lavar la sangre restante. Empieza a circular el IB en el sistema de Langendorff. Eliminar 20 ml de tampón desde el depósito superior, llenar en la solución de colagenasa y vuelva a llenar como tampón tanto como sea necesario para llegar a 80 ml. Circular el c<html> ollagenase solución para otro 27 min. Tasa de flujo debe ser controlado periódicamente y se mantuvo a 1 gota / segundo utilizando el reductor de flujo. </li><li> Separe el corazón de la cánula, limpio de aurículas y aorta y cortar en trozos pequeños. Deje que la solución de colagenasa a ejecutar en el depósito inferior, baje el volumen a un máximo de 40 ml y aún más digerir los ventrículos picadas durante 10 - 15 minutos con aireación continua con un 95% de O 2/5% CO 2. Después, se filtra la suspensión celular a través de una gasa de tamaño de malla de 200 micras en un tubo de 50 ml. </li><li> Restaurar la Ca 2 + contenido de las células en 3 pasos: centrifugar la suspensión celular a 43 xg durante 2 min a temperatura ambiente y se resuspenden las células en 10 ml que contenían IB 200 mM de CaCl 2. Centrifugar de nuevo y resuspender las células en 10 ml de IB con 500 mM de CaCl 2. Después de una centrifugación final resuspender cardiomiocitos en EB filtrado estéril (ver 1,8) a una densidad de 50.000 células / ml. </li></ol>e_title &quot;&gt; 3. Fura-2 tinción de cardiomiocitos <ol><li> Cubra un cubreobjetos 4 bien calibrada con laminina mg 10 por pocillo y espere hasta que esté completamente seca. </li><li> Llenar 1 ml de la suspensión de cardiomiocitos en cada pocillo utilizando una micropipeta con punta de corte. Permitir que las células se adhieran durante 1 hora a temperatura ambiente. </li><li> Diluir 10 l de la 1 mg / ml Fura-2 AM solución madre en 5 ml EB. Mantener la solución de tinción en la oscuridad. </li><li> Despegar las células de amortiguamiento y no unida de que el cubreobjetos y la pipeta 0,5 ml de la solución de tinción en cada pocillo. Incuban las células durante 10 min bajo agitación moderada. A continuación, retire la solución colorante, lavar las células una vez con 1 ml EB y finalmente llenar EB 0,5 ml en cada pocillo. Evite la luz directa durante el proceso de tinción y siempre mantener las células teñidas en la oscuridad. </li></ol> 4. Grabación de acortamiento celular y Ca 2 + transitorios <ol><li> Coloque el cubreobjetos cámaras en una inversa m fluorescenciaicroscope conectado a un miocito calcio y sistema de grabación de la contractilidad. Coloque un electrodo de encargo con una afluencia y el tubo de flujo de salida en el primer pocillo. Cardiomiocitos Superfuse con 1 ml / min EB y comenzar la estimulación eléctrica (20 V, 1 Hz, 5 ms de duración). Permitir que las células de adaptación a la estimulación por alrededor de 2 min. </li><li> Elija una barra de cardiomiocitos en forma intacta con estrías claras y contracción constante. Posición de la celda en el centro del campo visual y abrir el canal de la cámara. Orientar la celda horizontalmente dentro del área de vídeo mediante la rotación del adaptador de encuadre celular y moviendo la platina del microscopio. </li><li> Ajuste el borde detectar elementos de control del área de vídeo (Figura 2), abrir el canal fluorescente y registro 10 a 15 contracciones para obtener el acortamiento celular inicial y Ca 2 + transitorios. </li><li> Cierre el canal de luz microscopio para evitar la decoloración de la fluorescencia de la mancha. Cambie el flujo a través de EB a la muestraderivación con una válvula de 3-vías y cardiomiocitos superfuse con 1 ml de IgG paciente. Parar la bomba justo antes de aire alcanza el pozo. </li><li> Abrir el canal de luz y grabar otro 10 a 15 contracciones para obtener la respuesta aguda de células. Haga una pausa la grabación y cerrar el canal de luz de nuevo. Repita la grabación después de 2 y 5 min. </li><li> Sacar el electrodo del pozo. Limpiar bien los tubos con EB y continuar con el próximo pozo del cubreobjetos cámaras. </li><li> Analizar el acortamiento celular y Ca 2 + transitorios con el software IonWizard utilizando el &quot;Edge-Length/Length&quot; y el &quot;Fura 2-numérico resta / Ratio&quot; de la ventana, respectivamente. Calcular el porcentaje de cambio desde el acortamiento celular inicial y Ca 2 + transitorios de la altura del pico se refiere a la línea de base (BL% pico h) para el agudo, el 2 min y la respuesta de 5 min. </li></ol>

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No hay conflictos de interés declarado.

El método que se presenta ofrece una forma adecuada para detectar autoanticuerpos funcionales cardíacos efectivos en pacientes con MCD de origen incierto. En comparación con otros métodos, por ejemplo, la detección de anticuerpos funcionales activos contra la β1-adrenoceptor por su impacto en los niveles de AMPc 6, nuestro método es independiente de un epítopo específico. Por supuesto, hay otros ensayos de epítopos independientes descritos en la literatura, es decir, la tasa de rec...

<table border="1"><tbody><tr><td> Nombre del reactivo / equipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Immunosorba preservación solución </td><td> Fresenius Medical Care </td><td> 903609211 </td><td></td></tr><tr><td> Colagenasa de tipo 2 </td><td> Sistema Celular </td><td> LS004176 </td><td></td></tr><tr><td> BSA libre de ácidos grasos </td><td> Sigma-Aldrich </td><td> A6003 </td><td></td></tr><tr><td> La laminina </td><td> Sigma-Aldrich </td><td> L2020 </td><td></td></tr><tr><td> Fura-2 AM </td><td> Sigma-Aldrich </td><td> F0888 </td><td> 1 mg / ml en DMSO </td></tr><tr><td> Econo Pac Cromatografía Columnas </td><td> BioRad </td><td> 732-1010 </td><td></td></tr><tr><td> Spectra / Por Biotech éster de celulosa Diálisis Membrane </td><td> Spectrumlabs </td><td> 131417 </td><td></td></tr><tr><td> 4 bien compartimentado cubreobjetos </td><td> Nunc </td><td> 155383 </td><td></td></tr><tr><td> Calcio miocitos y sistema de grabación contractilidad </td><td> IonOptix </td><td></td><td></td></tr><tr><td> IonWizard 6,0 ​​software de análisis </td><td> IonOptix </td><td></td><td></td></tr></tbody></table>

measurement of antibody effects on cellular function of isolated cardiomyocytes

La cardiomiopatía dilatada (DCM) es una de las principales causas de la insuficiencia cardíaca en los adultos más jóvenes 1. Aunque la disposición genética y exposición a sustancias tóxicas se conocen las causas de esta enfermedad en aproximadamente un tercio de los pacientes, el origen de DCM sigue siendo en gran medida poco clara. En un número importante de estos pacientes, los autoanticuerpos dirigidos contra epítopos cardíacas se ha detectado y se sospecha que juegan un papel central en la aparición y progresión de la enfermedad de 2,3. La importancia de autoanticuerpos cardiacas está subrayado por una mejoría hemodinámica en pacientes DCM después de la eliminación de autoanticuerpos por inmunoadsorción 3-5. Una variedad de antígenos específicos ya se han identificado 2,3 y anticuerpos contra estos objetivos se pueden detectar mediante inmunoensayos. Sin embargo, estos ensayos no pueden discriminar entre la estimulación (y, por tanto funcionalmente eficaz) y el bloqueo de los autoanticuerpos. Existen cada vez más evidence que esta distinción es crucial 6,7. También se puede suponer que los objetivos para un número de anticuerpos cardiotrópicos están aún sin identificar y por lo tanto no se puede detectar por inmunoensayos. Por lo tanto, se estableció un método para la detección de anticuerpos funcionalmente activos cardiotrópicos, independientemente de su antígeno respectivo. El fondo para el método es la alta homología generalmente observado para las regiones funcionales de las proteínas cardiacas en mamíferos entre 8,9. Esto sugiere que los anticuerpos dirigidos contra los antígenos cardíacos humanos se reacciona de forma cruzada con las células diana no humanos, lo que permite las pruebas de IgG de pacientes con MCD en cardiomiocitos de ratas adultas. Nuestro método consta de 3 etapas: primero, la IgG es aislada del plasma del paciente utilizando sefarosa acoplados anti-IgG de anticuerpos obtenidos a partir de columnas de inmunoadsorción (PlasmaSelect, Teterow, Alemania). Segundo, los cardiomiocitos adultos son aislados por perfusión de colagenasa en un aparato de perfusión de Langendorff utilizando aprotocolo modificado a partir de trabajos previos 10,11. Los cardiomiocitos obtenidos se adjuntan a la laminina cubreobjetos recubiertos de cámara y se tiñeron con Fura-2, un tinte fluorescente de calcio selectivo que puede ser fácilmente llevado en la célula para observar el calcio intracelular (Ca 2 +) contenidos 12. En el último paso, el efecto de la paciente IgG en el acortamiento celular y Ca 2 + transitorios de cardiomiocitos de campo estimuladas se monitoriza en línea utilizando una cálcico comercial miocitos y contractilidad sistema de monitorización (IonOptix, Milton, MA, EE.UU.), conectado a un fluorescente típica inversa microscopio.

Dos ejemplos para la medición de inotropıa celular estimulada en campo cardiomiocitos adultos (Figura 2) utilizando un miocitos Ca 2 + y sistema de grabación de la contractilidad se dan a continuación. Figura 3 da una impresión de una medición de control, mientras que en la figura 4 el efecto de los anticuerpos cardiodepressive de un paciente con DCM se muestra. En ambos ejemplos, el acortamiento celular inicial y Ca 2 ...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

El método presentado ofrece una manera de detectar autoanticuerpos funcionales cardiotrópicos eficaces en el plasma de pacientes con miocardiopatía dilatada, con independencia del antígeno específico, mediante el análisis del impacto de la inmunoglobulina paciente aislado en el acortamiento celular y los transitorios de calcio intracelular en los cardiomiocitos de rata aislados.

Medición de los efectos de anticuerpos en la función celular de cardiomiocitos aislados

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

measurement-antibody-effects-on-cellular-function-isolated

Research

JoVE Journal

Immunology and Infection

11.8K vistas.  University Medicine Greifswald. El método presentado ofrece una manera de detectar autoanticuerpos funcionales cardiotrópicos eficaces en el plasma de pacientes con miocardiopatía dilatada, con independencia del antígeno específico, mediante el análisis del impacto de la inmunoglobulina paciente aislado en el acortamiento celular y los transitorios de calcio intracelular en los cardiomiocitos de rata aislados.

Video: Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Medición de la citosólica de Ca 2 + En aislados linfáticos contráctil

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

Investigación

Inmunología e Infección

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Un ensayo de microplacas para evaluar los efectos químicos sobre RBL-2H3 mástil degranulación: Efectos de Triclosan y sin uso de un disolvente orgánico

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Los métodos para la detección cuantitativa de anticuerpos inducida por la activación del complemento en los glóbulos rojos

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

El análisis de los efectos de las células del estroma en el reclutamiento de leucocitos de Flujo

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Análisis de citometría de flujo de partículas unida a Bet v 1 de alérgenos en el MP10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

La identificación de un epítopo lineal de células B reconocido por anticuerpos monoclonales péptido Escaneo-asistida

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Prueba de tolerancia a glucosa intraperitoneal, medición de función pulmonar y la fijación del pulmón para estudiar el impacto de la obesidad y metabolismo deteriorado de resultados pulmonares

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Evaluación de los efectos de drogas basados en anticuerpos en médula ósea murina y activación de los macrófagos peritoneales

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Un método para la medición de la función de la glándula salival en ratones

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

Construcción de fagos sintético muestra fabulosa biblioteca con diversidad a medida

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...