Todos los espectros de RMN utiliza como ejemplos ilustrativos se derivan de un estudio publicado anteriormente 7, que fue apoyado financieramente por Nestlé.

1. La colonización de animales libres de gérmenes y la recogida de muestras <ol><li> Eliminar animales libres de gérmenes de aisladores y de la casa en una sala de cría en jaulas convencionales equipados con filtro delante de los animales convencionales que servirán como controles (Figura 1). </li><li> Mezclar medio de la camada (3 días) tomado de la caja de control convencional con la materia fecal de los animales libres de gérmenes. Siempre mantenga un tercio de la basura convencional sucia cada vez que hay que renovar con el fin de mantener un nivel de bacterias (mantener al menos durante 3 días). </li><li> Recoger la orina en un microtubo de 1,5 ml por el manejo del ratón sobre el tubo y ayudar a la micción por un suave masaje en el intestino. Complemento congelar inmediatamente en nitrógeno líquido. Almacene al menos a -40 ° C hasta su análisis de RMN. Un volumen mínimo de 20 l se requiere para la adquisición de 5 mm de la sonda de RMN, pero se recomienda el uso de 30 l para mejorar la calidad de los perfiles metabólicos. </li><li> Los animales deben be la eutanasia sin el uso de algún tipo de anestesia a fin de evitar resonancias de RMN de confusión debido a un metabolismo hepático de los compuestos anestésicos (por ejemplo, el uso dislocación cervical seguido por la confirmación de la muerte por desangramiento) </li></ol> 2. Recomendación para la recolección de la biopsia hepática <ol><li> No use ningún producto que contenga alcohol para evitar la contaminación. Lave las herramientas con solución de agua o solución salina. </li><li> No perfore la vesícula biliar. En caso de fuga de bilis, lavar los tejidos inmediatamente con agua o solución salina. </li><li> Recoger las biopsias de hígado (alrededor de 15-50 mg) en el lóbulo izquierdo, como se muestra en la Figura 2. Para las biopsias reproducible, recoger constantemente en el centro del lóbulo izquierdo evitando las zonas periféricas donde el tejido es más delgada. </li><li> Ajustar las biopsias de congelación en nitrógeno líquido inmediatamente y almacenarlos a -80 ° C hasta el análisis de RMN. </li></ol> 3. 1H NMR adquisición de orina microvolumen </p&gt; <ol><li> Prepare el fosfato de sodio 0,2 M tampón en D2O (99,8%), pH 7,4, que contenía 1 mM 3 - (trimetilsilil) de ácido propiónico-d 4 (TSP). </li><li> Mezclar 30 l de orina con 30 l de tampón fosfato de sodio. </li><li> Transferencia de 50 l de solución mixta en 1,7 mm tubo de RMN capilar (Figura 3 (2)) con una jeringa de vidrio de 50 l equipado con una aguja de metal (OD 0.5 mm). Tenga cuidado para evitar las burbujas. </li><li> Ajuste el adaptador capilar (Figura 3) (3) en la parte superior de la muestra de orina que contiene capilares y colocarlo en un microtubo de RMN de 2,5 mm por 5 mm de RMN de la sonda (Figura 3) (1). El uso de esta combinación de tubos como un tubo de RMN regular de 5 mm para la adquisición del espectro. </li><li> Utilice la barra de extracción (Figura 3) (4) para eliminar capilar de 2,5 mm tubo de RMN, atornillando el adaptador capilar con cuidado para sacarlo. </li></ol> . 4 1 H HR RMN MAS de tejido biopsia del hígado: la preparación de muestras <ol><li> MAS componentes del rotor y las herramientasdescritos en la Figura 4. </li><li> Inserte una biopsia (alrededor de 15-50 mg) en el rotor de circonio (Figura 4) (1) y llenar el resto del volumen con puro O D 2 de RMN de bloqueo. Tenga cuidado de no hacer que las burbujas, porque esto altera la calidad de las cuñas posteriores y la calidad de la adquisición de datos. </li><li> Introduzca 50 L espaciador de teflón (Figura 4) (2) utilizando el tornillo cilíndrico (Figura 4) (5). Desenroscar y calibrar usando el medidor de profundidad en el lado corto (Figura 4 (8)). En este paso, es importante prestar una atención específica a la muestra porque parte de ella puede filtrarse a través del orificio del espaciador. Si este es el caso, entonces parte de la biopsia se destruye y el peso de la muestra ya no es confiable. Lo que es necesario volver a empezar desde el principio la preparación de la muestra. </li><li> Coloque el pasador THEAD (Figura 4) (3) y el tornillo suavemente con el destornillador (Figura 4 (6)). Secar el agua residual con un pedazo de tejido. </li><li> Colocar la tapa (figura 4) (4) en elparte superior del rotor y la inserta en el empacador rotor (Figura 4 (6)). Presione firmemente hasta que la tapa está en su lugar. No debe haber ningún espacio entre el rotor y la tapa. </li><li> Marca la mitad de la parte inferior del rotor con un rotulador negro para permitir la detección óptica de la velocidad de rotación. </li><li> Coloque el rotor en el interior del espectrómetro de RMN y comenzar a girar a 5 kHz. Adquirir un espectro de RMN de H con CPMG secuencia de pulsos 13 de acuerdo con las directrices del fabricante. </li><li> El uso de resonancia α glucosa anomérico a 5,22 ppm (doblete) para calibrar los espectros de RMN. </li><li> Para descomprimir el rotor, proceder mediante la eliminación de la tapa con el removedor de la tapa (Figura 4 (9)). Desenroscar y quitar elementos thead pin espaciador de teflón con el tornillo cilíndrico. Lavar exhaustivamente con agua y detergente. </li></ol> 5. Resultados representante La actividad microbiana del intestino pueden ser monitoreados utilizando un perfil metabólico urinario. Un gran número de microorganismos urinariosco-metabolitos identificados por RMN-1H han sido descritos en la literatura 7,14-17. Estos co-metabolitos microbianos son particularmente útiles para vigilar el proceso de colonización, ya que proporcionan una manera rápida y no invasiva para estimar que el ecosistema de reciente creación es estable. Figura 5A ilustra claramente la aparición de microbios intestinales co-metabolitos sobre el proceso de colonización. Esta cifra muestra un perfil metabólico urinario obtenerse por el procedimiento descrito en el paso 2 para un animal colonizó 20 días usando el procedimiento descrito en el Paso 1. Este animal no excretar sulfato indoxyl y cantidades muy pequeñas de phenylacetylglycine (PAG) y p-cresol sulfato en el estado libre de gérmenes (día 0-azul). Como la colonización avanza, estos tres marcadores del metabolismo de proteínas por la flora intestinal aumentan considerablemente para llegar a un equilibrio en el día 20 (rojo). Esto es particularmente fácil de controlar para un grupo de animales, como se ilustra en la Figura 5B con el PAGresonancia. Este diagrama se obtiene integrando el área bajo la resonancia con fondo gris en la Figura 5 (δ 7.40-7.43), que corresponde a una resonancia específica (triplete) de PAG de un grupo de 7 animales. 1 H de alta resolución angular Magic Spinning (MAS HR) espectroscopía de RMN es una técnica no destructiva que permite la adquisición rápida y reproducible de los perfiles metabólicos de cualquier tipo de biopsia 18. En este protocolo, se utilizó esta técnica de gran alcance para obtener un perfil metabólico hepático de los ratones antes de 2 (azul) y después (rojo) la colonización (Figura 6). Esta figura ilustra bien la información que se puede derivar de un MAS RMN basados ​​en el perfil metabólico. Numerosos aminoácidos, así como los metabolitos derivados del metabolismo energético, como la glucosa, glucógeno, lactato, triglicéridos, (D)-3-hidroxibutirato y nicotinurate puede ser visualizada. Estos perfiles contienen información relevante para el estrés oxidativo (es decir, un ascórbicocid, el glutatión), el metabolismo de nucleótidos (es decir, inosina, uridina) y el metabolismo de metilamina (es decir, la colina, trimetilamina N-óxido). En este ejemplo, es muy claro que la libre de gérmenes del ratón muestra casi ningún glucógeno y cantidades muy bajas de glucosa y triglicéridos, se publicó anteriormente 7. <img src="/files/ftp_upload/3642/3642fig1.jpg" alt="Figura 1."/> Figura 1. Descripción general del protocolo de la colonización. Animales libres de gérmenes y convencionales están alojados en jaulas equipadas con filtros de lado a lado y sus camadas se intercambian para permitir la colonización progresiva de la flora intestinal convencional (1). La actividad microbiana del intestino se controla mediante un perfil de RMN basado metabólico (2-3). El metabolismo hepático es evaluado por 1 H RMN MAS de recursos humanos basada en el perfil metabólico (4-5). <img src="/files/ftp_upload/3642/3642fig2.jpg" alt="Figura 2."/> Figura 2. Ratón vivor anatomía. El hígado se muestra como la parte plana del órgano frente a la mesa. Para las biopsias reproducible, se recomienda recoger muestras siempre desde el centro del lóbulo izquierdo según lo indicado por el rectángulo de trazos. <img src="/files/ftp_upload/3642/3642fig3.jpg" alt="Figura 3."/> Figura 3 1,7 mm kit RMN capilar para trabajar con microvolúmenes clave:.. 1: 2,5 mm microtubo RMN, 2: 1,7 mm tubo de RMN capilar, 3: adaptador capilar, 4: Extracción de la varilla. <img src="/files/ftp_upload/3642/3642fig4.jpg" alt="Figura 4."/> . MAS Figura 4 rotor equipos clave: 1.: Rotor MAS, 2: 50 l espaciador de teflón, 3: Thead pines, 4: gorra, 5: tornillos cilíndricos, 6: destornillador, 7: rotor envasador, 8: medidor de profundidad. <img src="/files/ftp_upload/3642/3642fig5.jpg" alt="Figura 5."/> Figura 5. Evolución de los perfiles metabólico urinario durante colonization. <ol class="ualpha"><li> Zoom en la región aromática del espectro de entre 6.8 a 7.8 ppm, donde co-metabolitos microbianos pueden ser visualizados. 1 H RMN se deriva de una sola persona en el día 0 (azul), 4 (verde), 15 (naranja) y 20 (rojo) post-colonización. La zona gris corresponde a la zona que se ha integrado para hacer el diagrama de la B. Principales: 1-MeHistamine: 1-metilhistamina; indoxil-S: El sulfato de indoxyl;:; Su p-cresol-S Histidina: sulfato de p-cresol, PAG : Phenylacetylglycine. </li><li> Concentración de PAG promedio durante la colonización (n = 7). La t de Student se utilizó la prueba para comparar la diferencia en la concentración de PAG en diferentes puntos de tiempo: a: p &lt;comparación con el día 0 0,05; b: p &lt;0,01 en comparación con los 10 días. </li></ol><img src="/files/ftp_upload/3642/3642fig6.jpg" alt="Figura 6."/> Figura 6. Típicos 600 MHz 1 H HR espectros de RMN MAS de las biopsias hepáticas derivadas de libres de gérmenes (azul) y ex-libre de gérmenes (red) los ratones. Protones en negrita son los responsables de la resonancia de triglicéridos clave:. 3-HB: 3-hidroxibutirato, GSH: glutatión reducido, TG: triglicéridos, OTMA: trimetilamina-N-óxido.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+partial+least+squares+models+and+related+chemometric+approaches+in+metabonomics+and+metabolic+phenotyping.">The evolution of partial least squares models and related chemometric approaches in metabonomics and metabolic phenotyping.</a> Journal of Chemometrics. 24, 636-649 (2010).</li><li>Merrifield, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metabolic+system-wide+characterisation+of+the+pig:+a+model+for+human+physiology.">A metabolic system-wide characterisation of the pig: a model for human physiology.</a> Mol. Biosyst. , (2011).</li><li>Tugnoli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+human+gastric+mucosa+by+HR-MAS+magnetic+resonance+spectroscopy.">Molecular characterization of human gastric mucosa by HR-MAS magnetic resonance spectroscopy.</a> International Journal of Molecular Medicine. 14, 1065-1071 (2004).</li><li>Sitter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+HR+MAS+MR+spectroscopic+profiles+of+breast+cancer+tissue+with+clinical+parameters.">Comparison of HR MAS MR spectroscopic profiles of breast cancer tissue with clinical parameters.</a> NMR Biomed. 19, 30-40 (2006).</li><li>Beckonert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiling,+metabolomic+and+metabonomic+procedures+for+NMR+spectroscopy+of+urine,+plasma,+serum+and+tissue+extracts.">Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.</a> Nat. Protoc. 2, 2692-2703 (2007).</li></ol>

No tenemos nada que revelar.

En este protocolo, que describe un procedimiento de colonización progresiva en un entorno abierto para seguir investigando el impacto de la microbiota intestinal en el metabolismo hepático evaluado por 1 H RMN de perfiles de recursos humanos del MAS intacta biopsia. Varios métodos de colonización han sido descritos en la literatura. Los métodos más comunes para colonizar los animales con una microbiota definida son una sonda nasogástrica o beber agua contaminada 19,20. Inoculación fecal tam...

Tabla de reactivos y equipos específicos: <table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> 2,5 mm microtubo </td><td> Nueva Era </td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1,7 mm tubo capilar </td><td> Sigma-Aldrich </td><td> NORS175001 </td><td></td></tr><tr><td> Adaptador capilar </td><td> Nueva Era </td><td> NE-325-5/1.7 </td><td></td></tr><tr><td> La extracción de la barra </td><td> Nueva Era </td><td> NE-341-5 </td><td></td></tr><tr><td> HR-MAS rotor BL4 con 50 l esférica de teflón kit de espaciadores </td><td> Bruker </td><td> HZ07213 </td><td></td></tr><tr><td> Kit de herramientas para 50 inserciones l </td><td> Bruker </td><td> B2950 </td><td></td></tr><tr><td> Avance III 600MHz RMN </td><td> Bruker </td><td></td><td></td></tr><tr><td> 1 H HR MAS RMN sólidos sonda </td><td> Bruker </td><td></td><td></td></tr><tr><td> Óxido de deuterio 99,9% </td><td> Sigma-Aldrich </td><td> 530867-1L </td><td></td></tr><tr><td> 3 - (trimetilsilil) propiónico ácido-d 4 (TSP) </td><td> Sigma-Aldrich </td><td> 269913 </td><td></td></tr></tbody></table>

assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1h nmr spectroscopy

Es bien sabido que las bacterias intestinales contribuyen significativamente a la homeostasis de acogida, proporcionando una serie de beneficios tales como la protección inmunológica y la síntesis de la vitamina. También proveen el host con una considerable cantidad de nutrientes, por lo que este ecosistema un órgano metabólico esencial. En el contexto de la creciente evidencia de la relación entre la flora intestinal y el síndrome metabólico, la comprensión de la interacción metabólica entre el huésped y su microbiota intestinal se está convirtiendo en un reto importante de la biología moderna. 1-4 Colonización (también conocido como proceso de normalización) designa a la creación de micro-organismos en un antiguo libre de gérmenes de origen animal. Si bien es un proceso natural que ocurre al nacer, que también se utiliza en adultos libres de gérmenes animales de controlar el ecosistema floral intestinal y más determinar su impacto sobre el metabolismo de acogida. Un procedimiento común para controlar el proceso de colonización es utilizar el método de alimentación forzada con una single o una mezcla de microorganismos. Este método da como resultado una colonización muy rápida y presenta la desventaja de ser muy estresante 5. Por tanto, es útil para minimizar el estrés y para obtener un proceso de colonización más lenta para observar poco a poco el impacto de la creación de bacterias en el metabolismo de acogida. En este artículo, se describe un procedimiento para evaluar la modificación del metabolismo hepático durante el proceso de colonización gradual mediante una técnica no destructiva del metabolismo de perfiles. Proponemos para controlar la colonización microbiana intestinal mediante la evaluación de la actividad metabólica microbiana intestinal refleja en la excreción urinaria de los metabolitos microbianos co-por un perfil de RMN basado metabólico. Esto permite una apreciación de la estabilidad de la actividad microbiana intestinal más allá del establecimiento estable del ecosistema microbiano intestinal suele evaluarse mediante el control de bacterias fecales por DGGE (electroforesis en gel de gradiente desnaturalizante). 6 Elcolonización se lleva a cabo en un ambiente abierto convencional y se inicia con una arena sucia contaminada con animales convencionales, que servirá como control. Los roedores son animales coprófagos, lo que garantiza una colonización homogénea a lo descrito previamente 7. Perfiles metabólicos hepáticos se mide directamente a partir de una biopsia de hígado intacto con una H de alta resolución Magic Angle Spinning espectroscopía de RMN. Esta técnica semi-cuantitativa ofrece una forma rápida de evaluar, sin dañar la estructura celular, los principales metabolitos, como los triglicéridos, la glucosa y el glucógeno con el fin de estimar más la compleja interacción entre el proceso de colonización y el metabolismo hepático 70-10. Este método también se puede aplicar a cualquier tejido de la biopsia 11,12.

Watch this Scientific Journal Video about Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy at JoVE.com

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

El procedimiento se describe la colonización progresiva de seguir evaluando su impacto sobre el metabolismo hepático de acogida. La colonización se controla de manera no invasiva mediante la evaluación de la excreción urinaria de los metabolitos microbianos co-mediante RMN basados ​​en los perfiles metabólicos, mientras que el metabolismo hepático es evaluado por alto ángulo Resolución Magic Spinning (MAS HR) de perfiles de RMN de la biopsia intactas.

La evaluación de alteraciones hepáticas metabólicas durante la colonización progresiva de la libre de gérmenes de ratón por 1 H espectroscopía de RMN

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

assessing-hepatic-metabolic-changes-during-progressive-colonization

Research

JoVE Journal

Immunology and Infection

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

13.0K vistas.  The University of Reading. El procedimiento se describe la colonización progresiva de seguir evaluando su impacto sobre el metabolismo hepático de acogida. La colonización se controla de manera no invasiva mediante la evaluación de la excreción urinaria de los metabolitos microbianos co-mediante RMN basados ​​en los perfiles metabólicos, mientras que el metabolismo hepático es evaluado por alto ángulo Resolución Magic Spinning (MAS HR) de perfiles de RMN de la biopsia intactas.

Video: Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

La cuantificación de la colonización por hongos, esporogenesis y producción de micotoxinas mediante bioensayos del núcleo

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Investigación

Inmunología e Infección

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

La colonización de Euprymna scolopes Squid por Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

La detección de microARNs en la microglía por PCR en tiempo real en el SNC normal y durante la neuroinflamación

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caracterización De Las Respuestas Inflamatorias Durante La Colonización Intranasal Con Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

La evaluación de la actividad anti-fúngica de aislados macrófagos alveolares por microscopía confocal

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Evaluación De La Invasión Bacteriana De Las Células Cardíacas En Cultivo Y Colonización Del Corazón En Ratones Infectados Usando Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

La recolección selectiva de leucocitos marginando-hepáticas

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonización vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Difusión molecular en el plasma membranas de linfocitos primarios medidos por espectroscopia de correlación de fluorescencia

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

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 ...