Este trabajo fue financiado por el NIH RO1 AI079253 y el Departamento de Asuntos de Veteranos.

1. Modelos Animales <ol><li> Ratones: Use p47 phox-/ - ratones y la edad y el sexo combinado-ratones C57BL6/DBA. Obtener la aprobación para los experimentos de Cuidado de Animales institucional y el empleo. </li><li> Anestesia: Utilice un sistema de administración continua para inducir la anestesia con isoflurano. El sistema vaporizador (VetEquip) se llena con isoflurano (2-3%). Confirmar que los ratones son completamente anestesiado mediante la observación de la respiración, el movimiento y reflejo corneal en respuesta a estímulos externos. </li><li> Los procedimientos quirúrgicos: Frote el área quirúrgica (de sobremesa) con 70% de etanol. Use guantes estériles y una bata quirúrgica limpia y la máscara. Preparar los ratones por cortar el pelo y aplicar betadine alternando con 70% de etanol a la zona donde se realizará la incisión. Realizar la cirugía en una superficie estéril y utilizar instrumentos estériles. Monitor de ratones después de la operación hasta que despierto y moverse libremente. </li><li> Administración intratraqueal zymosan <ol><li> Administrar anestesiacomo se describe en 1,2. </li><li> Desinfectar área quirúrgica (cuello) con betadine y 70% de etanol y exponer la tráquea por disección quirúrgica. </li><li> Pierce tráquea con una aguja de calibre 27-e inyectar zymosan (St. Louis, MO) a una dosis de 1 mg / g usando un g 0,5 / l solución (volumen total de 50 l para un ratón de 25 g) disuelto en PBS estéril. </li><li> Cuello ratones Cerrar la herida con suturas de seda 5-0 estériles en condiciones asépticas. </li><li> Imagen después de 4 horas y 24 horas. </li></ol></li><li> Ligadura cecal y punción <ol><li> Administrar anestesia como se describe en 1.2. </li><li> Clip de pelo en el área quirúrgica (abdomen) y desinfectar con betadine y un 70% de etanol. </li><li> Lleve a cabo una laparotomía media e identificar el ciego. </li><li> Se liga el distal 50% de ciego expuesta con sutura de seda 4-0 y el ciego punción distal a la ligadura con una pasada de una aguja de calibre 21-. </li><li> Cierre la incisión con suturas de seda 4-0 estériles. </li><li> Imagen después de 4 horas y24 hr. </li></ol></li><li> Oral en tetracloruro de carbono (CCl 4) Administración <ol><li> Administrar anestesia como se describe en 1.2. </li><li> Administrar CCl 4 (2 mg / g, St. Louis, MO) disuelto en aceite de maíz por alimentación forzada oral. El procedimiento de administración de CCl4 se debe realizar en un área designada de acuerdo con IBC / EHS políticas. </li><li> Imagen después de 4 horas y 24 horas. </li></ol></li></ol> 2. La adquisición de una imagen de Bioluminescent <ol><li> Inicialice el IVIS 200 (Xenogen Corporation, Alameda, CA) Imaging System, en modo luminiscencia, y esperar a que el dispositivo de carga acoplada (CCD) temperatura de bloqueo. </li><li> Seleccione el tiempo de exposición, hurgar en la basura (medio) y F / Stop ajustes (8). </li><li> Ratones coloque en posición supina en la cámara de formación de imágenes en una etapa calentada para mantener la temperatura corporal normal. Administrar anestesia como se describe en 1,2 a través de cono de la nariz mientras que los ratones se encuentran en la cámara de formación de imágenes IVIS. </li><li> Administrar L-012<html> (20 mg / g) disuelto en PBS estéril (10 g / l) por vía intravenosa a través de retro-orbital de inyección en cada punto de tiempo seleccionado para la formación de imágenes. </li><li> Capturar imágenes que comienzan en 2 min después de la inyección L-012. Nos típicamente utilizan una exposición de 40 segundos. </li></ol> 3. Análisis de datos utilizando región de interés <ol><li> Analizar datos usando software Image Living V.4.2 (Xenogen Corporation, Alameda, CA). </li><li> Abra una imagen y seleccionar la medición de la Región de herramientas de Interés. </li><li> Seleccione la Región de Interés forma y tamaño sobre el pecho o / y el abdomen después de la superposición de una imagen digital pseudo-color (que representa la detección de fotones) en una imagen fotográfica del ratón. </li><li> Cuantificar la intensidad de la señal (flujo de fotones) de la región de interés. </li></ol> 4. Estadística <ol><li> Utilice el paquete de software estadístico para llevar a cabo análisis ANOVA de dos vías con Bonferroni post-pruebas en puntos de tiempo individuales. </li></ol>tle &quot;&gt; 5. Resultados Representante Zimosán es un pro-inflamatoria célula de levadura producto de la pared y un potente activador de la NADPH oxidasa 3. Anteriormente puso de manifiesto que zymosan intratraqueal induce una inflamación neutrofílica de pulmón más robusto y pro-inflamatorios en la producción de citoquinas p47 phox-/ - en comparación con ratones de tipo salvaje 1. En este sentido, nuestro objetivo fue comparar la generación de ROS en los pulmones de tipo salvaje y p47 phox-/ - ratones después de la exposición zymosan. A las 4 horas después de la administración intratraqueal zymosan, se observó un aumento significativo de la emisión de fotones sobre el pecho en ratones de tipo salvaje en comparación con el valor basal, así como un aumento en la emisión de fotones en comparación con el tipo salvaje p47 phox-/ - ratones en 4 h y 24 h. En contraste, las señales de bioluminiscencia en p47 phox-/ - ratones se mantuvieron en niveles de línea de base en 4 horas después del tratamiento zymosan (Fig.1 AB). Por lo tanto, la NADPH oxidasa parece be la fuente principal de la generación de ROS en los pulmones después de la administración zymosan. A continuación, se evaluó la producción de ROS en el modelo de sepsis inducida por CLP. La sepsis es un síndrome potencialmente mortal asociada con lesión de órganos diana, incluyendo la lesión pulmonar aguda (LPA) y síndrome de distrés respiratorio agudo (SDRA) 4, 5. ROS mediada por lesión ha sido considerado como un factor importante en la sepsis inducida por disfunción multiorgánica. Se encontró que ROS se incrementaron significativamente en el pecho (4 hr) y sobre el abdomen (24 hr) después de CLP en ratones de tipo salvaje en comparación con el valor basal. Sin embargo, los niveles de ROS en p47 phox-/ - ratones fueron similares a los niveles basales en ambos puntos de tiempo (figura 2 AC). Estos resultados muestran que la generación de ROS en el modelo de sepsis inducida por CLP ratón es NADPH oxidasa dependiente. Finalmente, se utilizaron imágenes de bioluminiscencia para detectar la producción de ROS en un CCl 4-inducida por daño hepático modol. CCl 4 causa la necrosis hepatocelular, y se ha utilizado para modelar tanto daño hepático agudo y fibrosis hepática 6. En contraste con los modelos descritos anteriormente de la inflamación y la lesión, se encontró que la producción de ROS en el abdomen fue modestamente (aproximadamente 35%) se incrementó sobre la línea base en p47 phox-/ - ratones en 4 h después de la administración de CCl 4. Sin embargo, la magnitud de CCl 4-inducida por la generación de ROS fue significativamente mayor en los ratones de tipo salvaje que en p47 phox-/ - ratones (Fig. 3 AB). Estos datos sugieren que tanto la generación de ROS p47 phox que contiene NADPH oxidasa dependiente e independiente-ocurren en aguda CCl 4-inducida por la lesión hepática. <img alt="Figura 1" src="/files/ftp_upload/3925/3925fig1.jpg" /> Figura 1. ROS producción en los pulmones después del tratamiento zymosan. A) Representante bioluminescence imágenes de la producción de ROS. Tenga en cuenta que además del pecho, la luminiscencia es detectable sobre el abdomen en tanto de tipo salvaje y p47 phox-/ - ratones y luminiscencia abdominal no se modifica por el tratamiento zymosan. B) fotones conteos del pecho de tipo salvaje y p47 phox-/ - ratones después de una sola inyección intratraqueal (IT) de zymosan. Imágenes de bioluminiscencia se realizó al inicio, 4 h, y 24 h. La emisión de luz desde la región de interés sobre el pecho se identificó utilizando la indicada pseudo-color escala. Los resultados se presentan como media ± SE, n = 6-9 por grupo, * = p &lt;0,05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig1large.jpg" target="_blank">Haga clic aquí para agrandar la figura</a> . <img alt="Figura 2" src="/files/ftp_upload/3925/3925fig2.jpg" /> Figura 2. Detección de ROS tras CLP. A) Representante imágenes de bioluminiscencia de producción de ROS, BFotones) cuenta desde el pecho y C) los recuentos de fotones desde el abdomen de tipo salvaje y p47 phox-/ - ratones al inicio del estudio, 4 h, y 24 h después de CLP. Los resultados se presentan como media ± SE, n = 4-5 por grupo, * = p &lt;0,05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig2large.jpg" target="_blank">Haga clic aquí para agrandar la figura</a> . <img alt="Figura 3" src="/files/ftp_upload/3925/3925fig3.jpg" /> Figura 3. Producción de ROS en el abdomen después de CCl 4-inducida por la lesión hepática. Una imagen) bioluminiscencia Representante de la producción de ROS y B) cuenta fotones del abdomen de tipo salvaje y p47 phox-/ - ratones al inicio del estudio, 4 horas, y 24 horas después de la CCL 4 tratamiento bucal. Los resultados se presentan como media ± SE;. N = 4-5 por grupo, * = p &lt;0,05 <a href="http://www.jove.com/files/ftp_upload/3925/3925fig3large.jpg" target="_blank">Haga clic aquí para agrandar la figura</a> </a&gt;.

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No hay conflictos de interés declarado.

&quot;Tiempo real&quot; medición de las especies reactivas de oxígeno (ROS) en los animales vivos se puede lograr mediante el uso de sondas fluorescentes y quimioluminiscentes. Mientras que las sondas fluorescentes sufren de tener débiles señal-a-ruido 12, se describe la técnica de imagen es más sensible para la detección de la emisión de luz después de una reacción química de ROS con el sustrato luminol basado en L-012. Como todas las técnicas de formación de imágenes bioluminiscentes, esta met...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> El número de catálogo </td><td> Comentarios </td></tr><tr><td> L-012 </td><td> Wako Chemicals EE.UU., Inc. </td><td> 120-04891 </td><td></td></tr><tr><td> Zymosan </td><td> Sigma, St. Louis, MO </td><td> Z4250 </td><td></td></tr><tr><td> tetracloruro de carbono </td><td> Sigma, St. Louis, MO </td><td> 289116 </td><td></td></tr></tbody></table>

bioluminescence imaging of nadph oxidase activity in different animal models

La NADPH oxidasa es una enzima crítica que media la defensa del huésped antibacteriana y antifúngica. Además de su papel en la defensa del huésped antimicrobiana, la NADPH oxidasa tiene funciones críticas de señalización que modulan la respuesta inflamatoria 1. Por lo tanto, el desarrollo de un método para medir en &quot;tiempo real&quot; la cinética de la NADPH oxidasa derivada de la generación de ROS se espera que sea una valiosa herramienta de investigación para comprender los mecanismos pertinentes para la defensa del huésped, la inflamación y lesiones. La enfermedad granulomatosa crónica (EGC) es un trastorno hereditario de la NADPH oxidasa se caracteriza por infecciones graves y la inflamación excesiva. La activación de la NADPH oxidasa de los fagocitos requiere la translocación de sus subunidades citosólicas (p47 phox, p67 phox y p40 phox) y RAC a un flavocitocromo unida a la membrana (compuesto de una gp91 phox y p22 phox heterodímero). Pérdidade mutaciones de función en cualquiera de estos componentes de la NADPH oxidasa resultado en la EGC. Similar a los pacientes con EGC, gp91 phox los ratones deficientes en p47 y los ratones deficientes tienen phox-defectuoso actividad de la NADPH oxidasa de los fagocitos y deterioro de la defensa de acogida 13, 14. Además de los fagocitos, que contienen los componentes de la NADPH oxidasa descritos anteriormente, una variedad de otros tipos de células expresan diferentes isoformas de la NADPH oxidasa. Aquí se describe un método para cuantificar la producción de ROS en ratones vivos y para delinear la contribución de NADPH oxidasa para la generación de ROS en modelos de inflamación y lesiones. Este método se basa en el ROS reaccionar con L-012 (un análogo de luminol) para emitir luminiscencia que se registra mediante un dispositivo de carga acoplada (CCD). En la descripción original de la sonda L-012, L-012-dependiente quimioluminiscencia fue completamente abolida por la superóxido dismutasa, que indica que el principal ROS detectado en esta reacción fue superóxido de anión 15. Estudios posteriores han demostrado que la L-012 puede detectar otros radicales libres, incluyendo especies reactivas de nitrógeno 16, 17. Kielland et al. 17 mostraron que la aplicación tópica de acetato de forbol miristato, un potente activador de la NADPH oxidasa, llevado a NADPH oxidasa dependiente de la generación de ROS que podría ser detectado en ratones utilizando la sonda luminiscente L-012. En este modelo, se mostró que la luminiscencia L-012-dependiente fue abolida en p47 phox de ratones deficientes. Hemos comparado la generación de ROS en ratones de tipo salvaje y NADPH oxidasa deficiente en p47 phox-/ - ratones 2 en los tres modelos siguientes: 1) la administración intratraqueal de zymosan, una célula fúngica pro-inflamatoria pared producto derivado que puede activar la NADPH oxidasa; 2) ligadura cecal y punción (CLP), un modelo de sepsis intraabdominal con la inflamación pulmonar aguda secundaria y lesiones, y 3) el tetracloruro de carbono orales(CCl 4), un modelo de lesión hepática dependiente de ROS. Estos modelos se seleccionaron específicamente para evaluar la NADPH oxidasa dependiente de la generación de ROS en el contexto de la inflamación no infecciosa, sepsis polimicrobiana y lesión inducida por la toxina del órgano, respectivamente. Comparación de bioluminiscencia en los ratones de tipo salvaje p47 phox a-/ - ratones nos permite delinear la contribución específica de ROS generados por p47 phox de la NADPH oxidasa que contiene a la señal bioluminiscente en estos modelos. Resultados de bioluminiscencia de formación de imágenes que demostraron el aumento de los niveles de ROS en ratones de tipo salvaje en comparación con p47 phox-/ - ratones indicó que la NADPH oxidasa es la fuente principal de la generación de ROS en respuesta a estímulos inflamatorios. Este método proporciona un método mínimamente invasivo para el &quot;tiempo real&quot; monitoreo de la generación de ROS durante la inflamación in vivo.

Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

La NADPH oxidasa es la mayor fuente de especies reactivas de oxígeno (ROS) en los fagocitos. Debido a la naturaleza efímera de ROS, es difícil de medir y controlar los niveles de ROS en los animales vivos. Un procedimiento mínimamente invasivo para la cuantificación de serie de ROS en ratones vivos se describe.

Bioluminiscencia de imágenes de la actividad NADPH oxidasa en diferentes modelos animales

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

bioluminescence-imaging-nadph-oxidase-activity-different-animal

Research

JoVE Journal

Immunology and Infection

16.1K vistas.  Vanderbilt University School of Medicine. La NADPH oxidasa es la mayor fuente de especies reactivas de oxígeno (ROS) en los fagocitos. Debido a la naturaleza efímera de ROS, es difícil de medir y controlar los niveles de ROS en los animales vivos. Un procedimiento mínimamente invasivo para la cuantificación de serie de ROS en ratones vivos se describe.

Video: Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

El diagnóstico de ecto y endoparásitos en ratas y ratones de laboratorio

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Investigación

Inmunología e Infección

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

La saliva, la glándula salival, y la Colección hemolinfa de las garrapatas Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Largo Plazo intravital Multifotónica de imágenes de microscopía de células inmunitarias en sanos y enfermos de hígado utilizando ratones CXCR6.Gfp Reporter

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Aislamiento y Trasplante de diferentes Aged murinos tímicos injertos.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Imágenes de bioluminiscencia para detectar la infección en fase tardía de la tripanosomiasis africana

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Análisis de citometría de flujo de las células asesinas naturales lítico Actividad en sangre entera humana

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

En Vivo Investigación de Terapia de luz azul antimicrobianos para multirresistente Acinetobacter baumannii infecciones de quemaduras El uso de bioluminiscencia Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

No invasiva<em&gt; En Vivo</emHome Idiomas Ingresar a Epistemonikos Búsqueda avanzada Fluorescencia de imágenes ópticas de la actividad MMP inflamatoria utilizando un agente de imagen fluorescente activable

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Imágenes en vivo de la actividad antifúngica por los neutrófilos humanos primarios y los monocitos en respuesta a las<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Detección de la colonización de Escherichia Coli enterohemorrágica en anfitrión murino no invasiva In Vivo el sistema de bioluminiscencia

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...