Este trabajo fue apoyado por la Fundación Nancy Lurie Marcas. Damos las gracias a la Dra. Nancy E. Kohl para la lectura crítica del manuscrito. Agradecemos Kin K. Wong por su ayuda en la preparación de este informe en video.

Nota: Todos los estudios con animales se realizaron de acuerdo con protocolos institucionales aprobados y las directrices de cuidado de animales. 1. Reactivos y Soluciones <ol><li> Solución de PMA para la inoculación sc: Preparar una solución madre de forbol 12-miristato 13-acetato (PMA) a 5 mg / ml en DMSO. Guarde la solución madre a -20 ° C. Antes de la inoculación, descongelar la solución madre y se diluye hasta 1 mg / ml de PMA en PBS. </li><li> Solución de LPS para la inoculación sc: Disolver lipopolisacárido (LPS de Salmonella enterica serotipo enteritidis) en PBS estéril a 1 mg / ml antes de la inoculación sc. </li><li> Solución de luminol para obtener imágenes de fase aguda: Disolver luminol (,4-ftalazinediona, sal de sodio de 5-amino-2 ,3-dihidro-1) en solución salina normal estéril (0,9% NaCl) a 10 mg / ml. La solución puede ser almacenada a -20 ° C antes de su uso. </li><li> Solución Lucigenin para obtener imágenes de la fase crónica: Disolver lucigenin (bis-N-metilacridinio nitrato) en solución salina normal estérila 2,5 mg / ml. La solución puede ser almacenada a -20 ° C antes de su uso. </li></ol> 2. Subcutánea PMA inflamación Modelo <ol><li> Anestesie NCr ratones desnudos en una cámara de inducción con isoflurano 1-2%. Confirmar anestesia general por la pérdida de movimiento y un ritmo respiratorio constante. Transferir a un animal a un cono de nariz suministrado con gas isoflurano-mezclado y mantener al animal bajo anestesia. </li><li> Utilice un alcohol isopropílico limpiar para limpiar y desinfectar el lugar de la inyección en el flanco izquierdo. </li><li> Utilizando una técnica estéril, inyectar 50 l de la solución de inoculación PMA (que contiene 50 g de PMA) en el espacio subcutáneo en el flanco izquierdo. Retire el exceso de líquido del sitio de la inyección con un algodón con alcohol isopropílico. Evite el uso de analgesia, ya que puede afectar a las respuestas inflamatorias. </li><li> Mueva el animal de nuevo a la jaula de la vivienda y seguir de cerca su recuperación de la anestesia. A fin de ayudar en la recuperación, use una almohadilla térmica para mantener el calor del animal.</li></ol> 3. Subcutánea LPS inflamación Modelo <ol><li> Anestesie ratones C57BL/6J con 1-2% isoflurano en una cámara de inducción. Una vez que se establece la anestesia general, la transferencia de un animal a un cono de la nariz suministrado con gas isoflurano y mantener al animal bajo anestesia. </li><li> Utilice un alcohol isopropílico para limpiar la zona de la inyección en la pata izquierda. </li><li> Utilizando una técnica estéril, inyectar 50 l de la solución de inoculación de LPS (que contiene 50 g de LPS) en la almohadilla de la pata izquierda. Retire el exceso de líquido del sitio de la inyección con un algodón con alcohol isopropílico. Evite el uso de analgesia, ya que puede afectar a las respuestas inflamatorias. </li><li> Mueva el animal de nuevo a la jaula de la vivienda y seguir de cerca su recuperación de la anestesia. Use una almohadilla térmica para mantener caliente el animal durante la recuperación. </li></ol> 4. Bioluminiscencia de imágenes de la inflamación <ol><li> Se anestesia al animal en una cámara de inducción con isoflurano 1-2% de gas mezclado. Confirm anestesia general por la pérdida de movimiento y un ritmo respiratorio constante. </li><li> Mientras que el animal es todavía bajo anestesia, por vía intraperitoneal (ip) inyectar la solución de luminol (10 mg / ml), o la solución de lucigenina (2,5 mg / ml) para obtener imágenes de la inflamación aguda o crónica, respectivamente. La dosis final es 100 mg / kg para el luminol y 25 mg / kg para lucigenina. Una dosis más baja de lucigenina (10-15 mg / kg) se puede utilizar para evitar la posible toxicidad de algunas cepas de ratón. Los signos de toxicidad incluyen falta de aliento y dificultad respiratoria. La cepa C57BL/6J tiene menor lucigenin tolerabilidad que la cepa nude NCR. </li><li> Transferir el animal en la cámara de formación de imágenes de un sistema de imágenes de bioluminiscencia. </li><li> Realizar imágenes de bioluminiscencia secuencial a intervalos de 1 min. Cada paso de imagen está compuesto por 1 min tiempo de adquisición, f / stop = 1, hurgar en la basura = 16 y 0 segundos de retardo. </li><li> En el panel de adquisición de imágenes, para permitir secuencial de imágenes de varios pasos, haga clic en la secuencia de la creación button. Proporcionar suficientes medidas de imagen en el perfil de adquisición para determinar la producción de luminiscencia máxima (normalmente 15 pasos de un minuto será suficiente). La sección de formación de imágenes 15-min permite tiempo suficiente para la absorción de sustrato y la circulación sistémica. </li><li> Retire el animal de la cámara de imágenes y moverlo de nuevo a la jaula de la vivienda. A fin de ayudar en la recuperación, use una almohadilla térmica para mantener el calor del animal. </li><li> Durante el análisis post-adquisición, utilizar el paquete de software de imágenes para el cálculo de pico de la señal bioluminiscente total a través de las regiones estandarizados de interés (ROI). Las imágenes se presentan como resplandor en fotones / s / cm 2 / sr con un umbral mínimo y máximo indicados. Los datos cuantitativos se presentan como flujo total de fotones por segundo por ROI. </li></ol>

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Los autores declaran no tener ningún conflicto de interés en este estudio.

En este informe, se demuestra un método para obtener imágenes de bioluminiscencia no invasivo de la inflamación en los animales vivos. Tomando ventaja de dos sustratos luminiscente, el luminol y lucigenina, el método puede distinguir diferentes fases de la inflamación. Bioluminiscencia Luminol se asocia con los neutrófilos en la fase aguda de la inflamación, mientras que la bioluminiscencia lucigenina está mediada por los macrófagos en la fase crónica. Relativamente pequeño (PM = 177,16 g / mol) y no cargado ...

La inflamación es una respuesta biológica altamente regulado que participan en una variedad de enfermedades humanas, incluyendo la infección microbiana 1, cicatrización de la herida 2, 3 la diabetes, el cáncer de 4, 5 cardiovasculares, neurodegenerativas 6, 7 y enfermedades autoinmunes. Inflamación de tejidos requiere la coordinación adecuada de diversas células inmunes con el fin de lograr la autorización patógeno, la reparación de tejidos, y la resolución de la enfermedad. Los neutrófilos y los macrófagos son mediadores inmunes clave de la inflamación del tejido. En la fase aguda de la inflamación, los neutrófilos son los primeros en responder a diversos estímulos nocivos y daños en los tejidos 8. Los neutrófilos extravasación rápidamente de la circulación para el sitio de la lesión, donde las células inactivan los microbios invasores mediante la liberación de gránulos anti-microbianos y la fagocitosis. Durante la fagocitosis, neutrófilos fagocitan los microbios invasores en fagosomas, dentro de la cual las células producen niveles elevados de superóxidoide (O 2 · -). Superóxido phagosomal es la fuente principal de muchas especies reactivas de oxígeno (ROS aguas abajo). Por ejemplo, se puede dismutado superóxido a peróxido de hidrógeno (H 2 O 2) por dismutación espontánea o por la superóxido dismutasa (SOD) 9, 10. En los neutrófilos, la mieloperoxidasa (MPO) convierte más peróxido de hidrógeno a ácido hipocloroso antimicrobiana (HOCl) 11. Como las respuestas inflamatorias siguen, los monocitos circulantes migran gradualmente en el sitio de la lesión y se diferencian en macrófagos maduros 2, cuya función fagocítica ayudar a eliminar patógenos inactivados y restos celulares. Además, como un regulador clave en la última fase de la inflamación, los macrófagos promueve la reparación del tejido mediante la producción de citocinas antiinflamatorias y 12 mediante la generación de ROS extracelular a un nivel inferior 9. El ROS generado en esta etapa posterior a regular la remodelación de tejidos, formación de nuevos vasos, y reepithelialización 13. Fagocito NADPH oxidasa (Phox) es la fuente primaria de la producción de superóxido en neutrófilos y macrófagos 9. Phox es un complejo multi-subunidad cuyo montaje es estrictamente regulados 9. La holoenzima contiene varias subunidades reguladoras citosólicas (p67 phox, p47 phox, p40 phox y RAC) y una membrana heterodímero citocromo b 558 (compuesto por subunidades CYBA y CYBB). Citocromo b 558 es el núcleo de reacción dentro de la cual la subunidad CYBB (también conocido como p91 phox y NOX2) lleva a cabo la reacción en cadena redox primaria 9. Curiosamente, sus lugares de concentración son diferentes entre los neutrófilos y los macrófagos. En los neutrófilos en reposo, el citocromo b 558 es sobre todo presente en la membrana de los gránulos de almacenamiento intracelular 14. Durante la fagocitosis, neutrófilos montar la holoenzymes en fagosomas 9, donde también están presentes altos niveles de actividad de MPO. El neutrófilo Phox consume rápidamente oxígeno y ejerce su poder microbicida por la producción de ROS, un fenómeno denominado el estallido respiratorio 11. En contraste, los macrófagos tienen un nivel más bajo de expresión de MPO y el citocromo b 558 se encuentra principalmente en la membrana de plasma 15, 16. Por lo tanto los neutrófilos producen altos niveles de superóxido para la actividad anti-microbiana, mientras que los macrófagos generan menos superóxido para las funciones de regulación 15. Dado que la inflamación es un intrincado proceso in vivo, los métodos de imagen no invasivas específicos para las diferentes fases de la inflamación serían permiten la evaluación cuantitativa y longitudinal de modelos de enfermedad. Uso de estudios mecanísticos, hemos demostrado previamente el uso de dos agentes quimioluminiscentes, luminol (5-amino-2 ,3-dihidro-1 ,4-ftalazinediona) y lucigenina (bis-N-metilacridinio nitrato), Para formación de imágenes no invasiva de las etapas agudas y tardías (crónica) de la inflamación, respectivamente 17. Luminol permite la visualización de la actividad de MPO de neutrófilos en la fase aguda de la inflamación 18-20, mientras que la bioluminiscencia lucigenina se puede utilizar para evaluar la actividad de los macrófagos en asociación con la fase tardía o la inflamación crónica 17. En este manuscrito, se utilizaron dos modelos experimentales de inflamación (sc PMA y sc LPS) para demostrar estas técnicas de imagen.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>P8139</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide from Salmonella enterica serotype enteritidis</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>L2012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>A4685</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lucigenin (bis-N-methylacridinium nitrate)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>M8010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS Spectrum imaging system with Living Imaging 4.2 software package</td>
			<td>Caliper LS/Perkin Elmer, Hopkinton, MA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vivo imaging method to distinguish acute and chronic inflammation

La inflamación es un aspecto fundamental de muchas enfermedades humanas. En este informe de vídeo, se demuestra las técnicas de imagen no invasivas bioluminiscencia que distinguen a la inflamación aguda y crónica en modelos de ratón. Con daños en los tejidos o la invasión de patógenos, los neutrófilos son la primera línea de defensa, jugando un papel importante en la mediación de la respuesta inflamatoria aguda. A medida que avanza la reacción inflamatoria, los monocitos circulantes migran gradualmente en el sitio de la lesión y se diferencian en macrófagos maduros, que median la inflamación crónica y promover la reparación del tejido mediante la eliminación de restos de tejido y la producción de citocinas antiinflamatorias. La inyección intraperitoneal de luminol (,4-ftalazinediona, sal de sodio de 5-amino-2 ,3-dihidro-1) permite la detección de la inflamación aguda en gran parte mediada por neutrófilos infiltrantes de tejidos. Luminol reacciona específicamente con el superóxido generado dentro de los fagosomas de neutrófilos desde bioluminiscencia resulta de una mieloperoxidasa (MPO) reacción mediada. Lucigenin (bis-N-metilacridinio nitrato) también reacciona con el superóxido con el fin de generar bioluminiscencia. Sin embargo, lucigenin bioluminiscencia es independiente de MPO y se basa únicamente en la NADPH oxidasa de fagocitos (Phox) en los macrófagos durante la inflamación crónica. Juntos, el luminol y lucigenina permiten la visualización no invasiva y la evaluación longitudinal de diferentes poblaciones fagocitos a través de fases inflamatorias tanto agudas como crónicas. Teniendo en cuenta el importante papel de la inflamación en una variedad de enfermedades humanas, creemos que este método de formación de imágenes no invasiva puede ayudar a investigar los papeles diferenciales de los neutrófilos y macrófagos en una variedad de condiciones patológicas.

Se realizó imágenes de bioluminiscencia longitudinal para evaluar la inflamación aguda y crónica en modelos experimentales de inflamación. Forbol 12-miristato 13-acetato (PMA) es un agonista de la proteína quinasa C potente (PKC) que activa Phox para la producción de anión superóxido y desencadena intensas respuestas inflamatorias agudas 21. Inyección subcutánea de 50 mg de PMA en ratones desnudos NCr causó irritación de la piel rápido y la inflamación aguda en los sitios de inyección 18,...

Watch this Scientific Journal Video about In vivo Imaging Method to Distinguish Acute and Chronic Inflammation at JoVE.com

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Se describe un método de imagen no invasiva para distinguir las fases inflamatorias. El suministro sistémico de luminol revela zonas de inflamación aguda que dependen de la actividad MPO en neutrófilos. En contraste, la inyección de lucigenina permite la visualización de la inflamación crónica dependiente de la actividad Phox en los macrófagos.

In vivo método de imagen para distinguir la inflamación aguda y crónica

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

in-vivo-imaging-method-to-distinguish-acute-and-chronic-inflammation

Research

JoVE Journal

Immunology and Infection

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

20.0K vistas.  Harvard Medical School. Se describe un método de imagen no invasiva para distinguir las fases inflamatorias. El suministro sistémico de luminol revela zonas de inflamación aguda que dependen de la actividad MPO en neutrófilos. En contraste, la inyección de lucigenina permite la visualización de la inflamación crónica dependiente de la actividad Phox en los macrófagos.

Video: In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

De imagen no invasivas de Homing leucocitos y Migración In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Investigación

Inmunología e Infección

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Con óvulos de<em&gt; Schistosoma mansoni</em&gt; Como un<em&gt; En vivo</em&gt; Modelo de la inflamación pulmonar inducida por helmintos

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Imágenes bacteriana Bioluminiscente<em&gt; En Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Un modelo de ratón para Patógenos inducida inflamación crónica en los sitios locales y sistémicos

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Un método optimizado para aislar y expandir Invariante Natural Killer células T de ratón Bazo

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Un método no invasivo y técnicamente no intensivos para la inducción y Fenotipificación Experimental de neumonía bacteriana en ratones

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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 ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murina de linfocitos Etiquetado por<sup&gt; 64</sup&gt; Cu-Anticuerpo objetivo de receptores de<em&gt; En Vivo</em&gt; Tráfico de células mediante PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Un método eficiente y Simple para establecer líneas de células T de pacientes con infección crónica activa Virus Epstein - Barr y NK

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

Seguimiento en Vivo a largo plazo de la dinámica de la célula inflamatoria dentro de pupas de Drosophila

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...