Este trabajo fue financiado por los Institutos Canadienses de Investigación en Salud (MSH-95368, 89773 y MOP-MOP-106623 a la República Checa) y la Fundación Canadiense para la Innovación. SCM es el destinatario de una Asociación de CIHR / canadiense de la comunión Gastroenterología postdoctoral. CZ es un investigador de Nueva CIHR.

1. Propagación de los huevos de Trichuris muris <ol><li> Para generar nuevos lotes de huevos de Trichuris muris, 20-30 infectar ratones inmunodeficientes (por ejemplo, NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) o 129S6/SvEvTac-Rag2 tm1Fwa (RAG2 - / -)) o ratones genéticamente susceptibles ( por ejemplo. AKR / J), que es de 6-8 semanas de edad con aproximadamente 300 huevos de Trichuris muris mediante una sonda nasogástrica. </li><li> Después de 32-35 días el sacrificio de los ratones por asfixia de CO 2. </li><li> Exponer el lado ventral del ratón y húmedo en el abdomen con un 70% de etanol. </li><li> Pinzas de agarre utilizando la piel del abdomen y hacer una pequeña incisión con unas tijeras. Cortar la piel para exponer el contenido abdominal. </li><li> Identificar el ciego y el intestino grueso proximal, y eliminarlos. </li><li> Coloque el ciego y el intestino grueso proximal en una placa de Petri de 10 cm que contiene Dulbecco modificado por Eagle (DMEM) con 500 U / ml de penicilina y 500 mg / ml de estreptomicina (P / S). Cortar el ciego abierta para exponer la luz y los gusanos. Utilizando unas pinzas ciego colocarlos en un vaso de tampón fosfato salino (PBS) y agite suavemente para eliminar las heces. </li><li> Volver al ciego placa de Petri y el uso de pinzas curvas suaves (Roboz RS-5047) tire suavemente de gusanos y colocarlos en un pozo de una placa de 6 pocillos que contienen 5 ml de DMEM + P / S. </li><li> Repita este proceso para todos los ratones. </li><li> Colocar 6 pocillos en un recipiente Tupperware con una toalla de papel húmeda para mantener la humedad y se incuba a 37 ° C durante 4 horas. </li><li> Después de 4 horas eliminar los gusanos utilizando pinzas curvas suaves y los dividieron en dos nuevos pozos de la placa de 6 pocillos que contienen 5 ml de DMEM + P / S. Cosecha el contenido del pozo original y colocarla en un tubo Falcon de 15 ml. Girar el tubo a 3000 rpm y eliminar el sobrenadante de reserva como el antígeno de 4 horas (se puede utilizar para re-estimulación de células después de la PBS de diálisis, donde los rendimientos son típicas de proteínas 10-20 mg por cada 20 ratones) y resuspender que contengan huevo sedimento en el agua del grifo en autoclave. </li><li> Coloque la placa de 6 y de nuevo a 37 ° C durante la noche. Remoción y eliminación de los gusanos. Cosecha de los contenidos de los pozos en un tubo Falcon y giran a 3000 rpm durante 5 min. Aspirar el sobrenadante y la reserva como antígeno durante la noche (se puede utilizar para Trichuris Ag. ELISA específico isotipo de anticuerpos después de la PBS de diálisis, donde los rendimientos son típicas de proteínas 10-20 mg por cada 20 ratones) y que contiene huevo volver a suspender el pellet en el agua del grifo en autoclave. </li><li> Combine 4 horas y los huevos durante la noche y el filtro de los huevos a través del tamiz 70μm para eliminar los gusanos de sobra a continuación, transferir a un matraz de 75 cm 2. Mantenga matraz en un sitio oscuro cubierto de lámina por 6 semanas a temperatura ambiente, (RT). </li><li> Lavar los huevos en el agua del grifo en autoclave y volver a suspender a 50 huevos viables en 50 microlitros. Para ello, los huevos centrífuga a 3000 rpm durante 5 min y se resuspenden en 50 ml de agua de la llave ~ estéril. Retire 50 huevos l y el lugar en un portaobjetos de vidrio. Usando el aumento de 40x contar el número de huevos embrionados viables en el l 50. Diluir los huevos para dar 50 huevos viables en 50 microlitros. Para las imágenes de huevos viables por favor vea el estudio de Kopper y Mansfield 16. </li></ol> 2. La infección aguda de los ratones experimentales con Trichuris muris huevos (200 huevos / ratón) <ol><li> Huevos bien volver a suspender y eliminar un volumen suficiente para tratar el número de ratones X 2 (por ejemplo, de 10 ratones tener 200 x l 10 ratones x 2 = 4 ml de huevos) y colocarlos en un tubo de tapa de 14 ml fácil. </li><li> Invierta el tubo de 14 ml snap tapa para mezclar. Lleva hasta 200 l de huevos en jeringa de 1 ml en una aguja de tamaño de la alimentación adecuada (para los ratones un peso de 25 gramos que utiliza calibre 18 1 1 / 2 &quot;agujas). En pescuezo por un lado, el ratón y con la otra mano administrar los huevos mediante una sonda nasogástrica . </li><li> Esperar 21 días. </li></ol> 3. De pesca experimental <ol><li> El día 21 el sacrificio de los ratones con CO 2. </li><li> Recoger la sangre de los ratones mediante punción cardíaca, en una jeringa no heparinizada, y se almacenan a 4 ° C. Al día siguiente aislar el suero y congelar a -20 ° C. Suero puede ser utilizado para la detección de IgG específica Trichuris e IgE por ELISA. </li><li> Exponer el lado ventral del ratón y húmedo en el abdomen con un 70% de etanol. </li><li> Pinzas de agarre utilizando la piel del abdomen y hacer una pequeña incisión con unas tijeras. Cortar la piel y la membrana subyacente para exponer el contenido abdominal. </li><li> Identificar el intestino delgado, el ciego y el colon. Coge el ciego con unas pinzas y extraer de la cavidad abdominal. Con otro par de pinzas de empujar el intestino delgado hacia la derecha exponer los nódulos linfáticos mesentéricos (mLNs). Retire la mLNs teniendo cuidado de no cortar los intestinos y el lugar en un falcon de 15 ml que contiene 2 ml de los medios de comunicación. Si lo desea estos pueden ser procesados ​​y volvieron a estimular in vitro (4x10 6 / ml en 1 ml en placas de 24 pocillos con 1 mg / ml αCD3/CD28 o con 50μg/ml 4h Trichuris Ag) durante 72 horas para el análisis de citoquinas por ELISA y f intracelularcitometría de baja. </li><li> Cortar el ciego y el colon. </li><li> Con unas pinzas, expulsar partículas fecales de colon distal y el lugar en un tubo de 2 ml Axygen. Congelar a -20 ° C. Bolitas fecales pueden ser analizados por Western blot para la expresión de la resistina, como la molécula de β (β-RELM). </li><li> Retire la punta del ciego (~ 0,5 cm). Para eliminar las heces mantener la punta del ciego con unas pinzas y enjuague con PBS utilizando una jeringa de 3 ml y aguja de calibre 22 en una placa de Petri. Lugar en paraformaldehído al 4% fresco en un tubo de 5 ml tapa Falcon fácil. Almacenar a 4 ° C. Estos pueden ser incluidos en parafina, se cortaron y montaron en portaobjetos para su análisis histológico. </li><li> Extraer un pedazo de colon proximal (aproximadamente 1 cm) para el ARN. Ponga la muestra en 2 ml tubo Axygen con 500 l RNAlater. Almacenar a 4 ° C. Estas muestras pueden ser analizadas para la expresión del ARNm de qPCR. </li><li> Coloque el resto del ciego en una placa de Petri etiquetados y se congelan a -20 ° C. </li></ol> 4. La enumeración de los gusanos Trichuris muris en el ciego <ol><li> Quitar el ciego del congelador y colóquela en un plato de Petri que contiene ~ 5 ml de agua. </li><li> El uso de tijeras, corte ciego para exponer la luz. </li><li> Mantenga el ciego con unas pinzas y agite vigorosamente el ciego para eliminar la mayoría de las heces. </li><li> Transferencia a otro ciego placa de Petri que contiene el agua y el uso de pinzas curvas suaves raspe suavemente la IEC el tejido subyacente. </li><li> Transferencia de ciego a una nueva placa de Petri que contiene agua y raspar el tejido restante vigorosamente con fórceps, extracción del tejido en trozos pequeños. </li><li> Utilizar un microscopio de disección para contar todos los gusanos en los 3 de los platos de Petri. Como encontrar un gusano de sacarlo de la placa para asegurarse de que el gusano lo mismo no se cuenta dos veces. Si los gusanos son dañados (es decir, roto por la mitad) sólo cuentan los extremos gruesos o finos para obtener una cifra exacta. </li></ol> 5. Resultados representante El modelo de Trichuris muris infección permite a los investigadores para cuantificar la carga parasitaria y la respuesta inmune sistémica, así como para examinar histológicamente la respuesta inflamatoria en el intestino ciego distal. Cuando la luz del ciego cosechado se expone, gusanos Trichuris se pueden enumerar usando un microscopio de disección (Figura 1-B). Parámetros de inmunidad a Trichuris también incluyen isotipo de anticuerpos IgG1 cambiar a (asociado con la inmunidad de tipo Th2) en los animales resistentes, y IgG2a (asociados con la inmunidad de tipo Th1) en animales susceptibles (fig. 1C). Expresión de la copa de células específicas de la proteína resistin-como la molécula de β (β-RELM) se asocia con un aclaramiento de Trichuris y es detectable por análisis de Western blot (Figura 2). La porción distal del intestino ciego se puede teñir con ácido periódico de Schiff (PAS) para evaluar las respuestas de las células caliciformes y el grado de inflamación intestinal en respuesta a la infección (Figura 3). <img src="/files/ftp_upload/2774/2774fig1.jpg" alt="Figura 1"/> Figura 1. La cuantificación de la inmunidad de Trichuris muris. (A) Resistente (B6) expulsar a los ratones Trichuris muris a los 21 días post-infección. Por el contrario, susceptibles (AKR / J) de ratones con infección crónica por Trichuris, lo que resulta en el establecimiento de los parásitos. Cada barra representa la media ± SEM de 4-8 animales por grupo. (B) Los gusanos se cuentan utilizando un microscopio de disección. El panel superior muestra los gusanos en el aislamiento, el panel inferior muestra los gusanos en la presencia de la IEC y las heces. (C) Trichuris específicos de los títulos de IgG2a se determinó por ELISA de suero diluido (1:2 dilución, a partir de las 1:20 de dilución y terminando a las 1:2560) en placas recubiertas con antígeno excretor Trichuris (O / N Ag, 5 mg / ml). Susceptibles AKR / J ratones tienen niveles notablemente más altos de Trichuris IgG2a específicos en suero de ratones B6 resistente. <img src="/files/ftp_upload/2774/2774fig2.jpg" alt="Figura 2"/> Figura 2. Liquidación de Trichuris muris se asocia con la expresión de la copa de células específicas de la proteína β-RELM 7. RELM-β de producción pueden ser detectados a partir de partículas fecales de Trichuris infectadas resistentes (B6), pero no susceptibles (AKR / J) en ratones mediante el análisis de Western blot. Cada carril es representante de un solo animal, (N) = ingenuo, (Tm) = 21 días Trichuris ratones infectados. <img src="/files/ftp_upload/2774/2774fig3.jpg" alt="Figura 3"/> Figura 3. El examen histológico de la infección por Trichuris ciego siguientes distal. Secciones 7.5 micras de la parte distal ciego teñidos con PAS. Hiperplasia de células caliciformes y la producción de moco son evidentes en resistencia (B6), pero no susceptibles (AKR / J) ratones después de la infección por Trichuris.

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Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por la Universidad de British Columbia.

Este protocolo detalla un estándar de alta dosis aguda de la infección por Trichuris muris que pueden ser modificados según sea necesario por el investigador. Por ejemplo, los ratones pueden ser sacrificados y tejidos recolectados en días diferentes. Para determinar que los ratones han establecido con éxito la carga del gusano completo que puede ser sacrificado en el día 14, momento en el que todos los ratones que llevan una carga de aproximadamente 200 gusanos. Los ratones también pueden ser infectados ...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Agujas de alimentación animal (18 x 1 ½ &quot;) </td><td> Corchete </td><td> 7912 </td><td></td></tr><tr><td> Suave pinzas curvas </td><td> Roboz </td><td> RS-5047 </td><td></td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11965 </td><td></td></tr><tr><td> NOD.Cg-Prkdc scid IL2R gtm1Wjl / SzJ (NSG) </td><td> Jackson Laboratories </td><td> 005557 </td><td> Estos son los ratones que hemos utilizado, sin embargo, cualquier ratones inmunodeficientes o cepa susceptible debería funcionar. </td></tr><tr><td> RNAlater </td><td> Qiagen </td><td> 76104 </td><td></td></tr><tr><td> Tubos de 2 ml </td><td> Axygen </td><td> MCT-200-C </td><td></td></tr><tr><td> Tubos de 15 ml </td><td> Halcón </td><td> 352096 </td><td></td></tr><tr><td> 6 placas </td><td> Halcón </td><td> 353046 </td><td></td></tr><tr><td> Paraformaldehído </td><td> Microscopía electrónica de la Ciencia </td><td> 15710 </td><td></td></tr><tr><td> α-RELMβ anticuerpos </td><td> PeproTech Inc </td><td> 0694270Rb </td><td></td></tr></tbody></table>

trichuris muris infection: a model of type 2 immunity and inflammation in the gut

Trichuris muris es un patógeno natural de los ratones y es biológicamente y antigénicamente similares a las especies de Trichuris que infectan a los seres humanos y animales 1. Los huevos infectantes se dan mediante una sonda nasogástrica, eclosionan en el intestino delgado distal, invaden las células epiteliales intestinales (IEC) que la línea de las criptas del ciego y el colon proximal y en la maduración de los huevos de los gusanos liberación en el medio ambiente 1. Este modelo es una herramienta poderosa para examinar los factores que las células CD4 + de control T helper (Th) activación de las células, así como cambios en el epitelio intestinal. La respuesta inmune que se produce en las cepas endogámicas, como C57BL / 6 y BALB / c, se caracteriza por las citocinas Th2 polarizadas (IL-4, IL-5 e IL-13) y la expulsión de los gusanos, mientras que Th1 asociada a las citocinas (IL -12, IL-18, IFN-γ) promover las infecciones crónicas en AKR genéticamente susceptibles / J ratones 2-6. Citocinas Th2 promover cambios fisiológicos en el microambiente intestinal incluyendo la rápida rotación de la IEC, la diferenciación de las células caliciformes, la contratación y cambios en la permeabilidad del epitelio y la contracción del músculo liso, que se han implicado en la expulsión del gusano 7.15. Aquí detallamos un protocolo para la propagación de los huevos de Trichuris muris que puede ser utilizado en experimentos posteriores. También proporcionamos una pesca experimental de la muestra con sugerencias para el análisis post-infección. En términos generales, este protocolo se proporcionará a los investigadores con las herramientas básicas para realizar una Trichuris muris ratón modelo de infección que puede ser utilizado para tratar las cuestiones relacionadas con proclividad Th en el tracto gastrointestinal, así como funciones efectoras inmunes de la IEC.

Watch this Scientific Journal Video about Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut at JoVE.com

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infección intestinal es un modelo de la inmunidad Th2 en ratones resistentes al generar una respuesta Th2 protección y ratones susceptibles de generar una respuesta Th1 patológicos.

Trichuris muris infección: un modelo de tipo 2 inmunidad y la inflamación en el intestino

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

trichuris-muris-infection-model-type-2-immunity-inflammation

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Immunology and Infection

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

16.2K vistas.  University of British Columbia. Trichuris muris infección intestinal es un modelo de la inmunidad Th2 en ratones resistentes al generar una respuesta Th2 protección y ratones susceptibles de generar una respuesta Th1 patológicos.

Video: Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsular Serotipificación de<em&gt; Streptococcus pneumoniae</em&gt; Por partículas de látex

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Investigación

Inmunología e Infección

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

El aislamiento, caracterización y examen funcional de la red celular gingival Inmune

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Aislamiento de neutrófilos y análisis para determinar su papel en el linfoma de células sensibles a los agentes terapéuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

La aplicación de fluorescencia la transferencia de energía de resonancia (FRET) para examinar la eficiencia de efector por translocación<em&gt; Coxiella burnetii</em&gt; Durante siRNA silenciamiento

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

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 ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Precisión de corte pulmón de ratón Rebanadas para visualizar las células en vivo pulmonar dendríticas

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

La neuraminidasa Ensayo de inhibición basado en la fluorescencia para evaluar la susceptibilidad de los virus de la influenza a El Inhibidor de la neuraminidasa clase de antivirales

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Efectos del gusto señalización abolición de proteína sobre la inflamación intestinal en un modelo de ratón de la enfermedad inflamatoria intestinal

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Establecimiento de la infección Viral y análisis de la interacción huésped-Virus en Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

El modelo de infección de Waxworm Galleria mellonella para la Candidiasis diseminada

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...