Damos las gracias a la Dra. Diane Baronas-Lowell para la lectura crítica del manuscrito. Este trabajo fue apoyado por un Charles E. Schmidt Colegio FAU del Fondo Semilla de Ciencia y una Beca de Envejecimiento de la Fundación Médica Ellison a KJ

1. Placas XLD (xilosa lisina desoxicolato) Agar Agar XLD es un medio de crecimiento selectivo para Salmonella, que aparece como colonias negras en placas de agar XLD. Sin embargo, si no hay problemas de contaminación, una placa regular de LB puede ser sustituido. <ol><li> Pesar 5,5 g de agar XLD y resuspender en 5 ml de agua desionizada. </li><li> Mezclar bien hasta que todo el agar está mojado. Añadir 95 ml de agua desionizada hasta que todos queden grumos y el medio se volvieron a suspender por completo. </li><li> Hervir el medio para disolver por completo (no en autoclave). </li><li> Enfriar el medio a temperatura ambiente a 50 ° C. </li><li> Verter 25 ml de agar de cada 95 x 15 mm (diámetro x altura) de la placa (placas selladas con Parafilm pueden almacenarse a 4 ° C durante un máximo de 1 mes). </li></ol> 2. Nematodo Medio de Crecimiento (NGM) de alimentación RNAi placas Preparación de C. placas elegans NGM se ha descrito anteriormente 19. Aquí un procedimiento se describe brevemente para añadir el antibiótico ampicilina y el ARNi inductor químico isopropil β-D-1-tiogalactopiranósido (IPTG) en los medios de comunicación NGM para hacer las placas de alimentación de RNAi. <ol><li> Disolver 3 g de NaCl y 2,5 g de Bacto peptona en 1 L de agua desionizada. </li><li> Añadir 17 g de agar Bacto en los medios de comunicación. </li><li> Autoclave los medios de comunicación durante 45 minutos y enfriar el medio a 50 ° C en un baño de agua. </li><li> Añadir las siguientes soluciones: 1 ml de colesterol (5 mg / ml en etanol al 95%), 1 ml de 1 M de CaCl2, 1 ml de 1 M MgSO4, y 25 ml de 1 M de tampón de fosfato de potasio (pH 6,0). Mezclar bien. </li><li> Añadir 1 ml de 1 M de IPTG y 500 l de ampicilina (100 mg / ml en agua estéril). </li><li> Mezcle bien la solución y verter en placas de Petri de 60 x 15 mm (diámetro x altura) utilizando una pipeta de Ayuda y 25 ml de pipeta serológica siguientes procedimientos estériles. Llene cada plato con 12 ml de agar. Las placas se pueden almacenar a 4 ° C durante hasta 1 mes. </li></ol> El gen esencial autofagia BEC-1 se utiliza como un ejemplo para examinar la función de un gen de acogida en la defensa contra la infección por Salmonella. Los procedimientos experimentales se ilustran en la Figura 1 y la Tabla 1. El protocolo para la preparación de los animales tratados con ARNi para la infección sigue, con el día de cada paso experimentales dados en paréntesis. <ol><li> Inocular bec-1 RNAi alimentación y el control de vector vacío L4440 alimentación RNAi bacterias mediante la colocación de un copo de -80 ° C las bacterias congeladas en 2 ml de medio LB suplementado con 100 mg / ml de ampicilina (Día 1). Repita este paso una vez a la semana durante todo el experimento de tener bacterias RNAi frescas. Guarde la cultura en el 4 ° C refrigerador cuando no se utiliza. </li><li> Seed 100 ml de cultivo bacteriano durante la noche en placas de RNAi RNAi. Prepare de tres bec-1 RNAi y el control de tres v vacíaector placas de RNAi. Incubar las placas a 37 ° C durante la noche (Día 2). </li><li> Retire las placas de RNAi de la 37 ° C incubadora y deje que se enfríen a temperatura ambiente en el banquillo. Recoge bien alimentados L4 tipo salvaje hermafroditas N2 y transferirlos a Bec-1 RNAi y controlan placas RNAi vector vacío. Coloque tres gusanos por placa, en placas por triplicado. El mismo día, se preparan platos de RNAi como se describe en el paso 3.2 (día 3). </li><li> Incubar las placas de RNAi con gusanos en la incubadora de 20 ° C para 36 a 40 hy de transferencia de los gusanos a las placas de RNAi correspondientes frescos preparados en el paso 3.3. Después de gusanos se transfieren, incubar las placas en el 20 ° C incubadora durante 64 horas (día 4). </li></ol> 4. Preparar para la infección por Salmonella <ol><li> Salmonella racha de -80 ° C madre congelada en 1 placa de agar XLD e incubar la placa a 37 ° C durante la noche (el día 5 </stRong&gt;). </li><li> Elige un bien aislada de Salmonella negro colonia e inocular en 2 ml de medio LB a 37 ° C con agitación durante la noche (Día 6). </li><li> Seed 80 ml ​​Salmonella cultivo de una noche en 1 C. elegans 60 x 15 mm (diámetro x altura) placa de agar NGM y preparar 6 placas en total. Las placas se incuban a temperatura ambiente durante 6 horas. El cultivo bacteriano debe secarse y formar un césped en la placa (día 7). </li></ol> 5. Worms tratados con RNAi Infect con Salmonella <ol><li> Traslado bec-1 RNAi tratados y control de vectores hermafroditas L4 N2 vacías RNAi tratados (la progenie de gusanos creados en el paso 3) para placas de Salmonella. Coloque 40 gusanos por placa en 3 placas para cada grupo. Incubar las placas de gusanos a 20 ° C durante 48 horas (día 7). </li><li> Prepare un juego de placas de RNAi frescas tal como se describe en los pasos 3.1 y 3.2 (Día 7 y <strong&gt; día 8). </li><li> Después de 48 horas de infección, transferir gusanos infectados por Salmonella en las correspondientes placas de RNAi preparados en la etapa 5.2 y se incuba a 20 ° C (día 9). </li></ol> 6. Ensayo de supervivencia <ol><li> Puntuación de la supervivencia de los gusanos diario y transferir gusanos correspondientes placas de RNAi frescas durante la época de puesta de huevos. Prepare un conjunto de placas de RNAi frescas antes de cada transferencia de gusano como se describe en los pasos 3.1 y 3.2. Toque el cuerpo del gusano (cabeza, parte media y cola) suavemente con un alambre de platino final aplanado. Un gusano se califica como muerto si no se observa el movimiento del cuerpo del gusano. </li><li> Puntuación de la supervivencia de los gusanos al día o cada dos días, y la transferencia de los gusanos de las placas correspondientes RNAi frescas dos veces a la semana después de que los gusanos dejan de poner huevos. </li><li> Después de todos los gusanos mueren, agrupar los datos de supervivencia de placas por triplicado como un conjunto de datos. Introduzca los datos de supervivencia de cada grupo en el software estadístico apropiado como GraphPad Prismo para generar curvas de supervivencia y para llevar a cabo el análisis de supervivencia de Kaplan-Meier. Todo el experimento se repite al menos una vez para confirmar la conclusión. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

C. elegans es un simple organismo modelo genético que come bacterias como fuente de nutrientes. Por lo tanto, es fácil de sustituir su alimentaria bacteriana normal con un patógeno intestinal para investigar las interacciones entre C. elegans y el patógeno elegido. En la presente memoria se describe un protocolo para combinar la infección por Salmonella y C. elegans alimentación RNAi tratamiento para examinar el papel de los genes del huésped en la defensa contra la infección ...

La vida libre de nematodos Caenorhabditis elegans suelo es un simple y manejable de forma genética organismo modelo utilizado para estudiar muchas cuestiones biológicas. C. elegans existe predominantemente como hermafroditas autógamas. Los varones son generados espontáneamente por la no disyunción del cromosoma X durante 1,2 gametogénesis. En la presencia de abundante comida, C. elegans desarrollar continuamente a través de cuatro estadios larvarios a adulto. La temperatura también influye en C. desarrollo elegans; desarrollo más rápido se observó a temperaturas más altas. En el laboratorio, C. elegans se cultiva a una temperatura estándar de 20 ° C en placas de agar con cabeza de serie bacteria Escherichia coli (cepa OP50) como alimento 1,2. En la última década, C. elegans se ha convertido en un organismo invertebrado para estudiar las interacciones huésped-patógeno 3-5. En la naturaleza, C. elegans come bacterias como su nutrien1,2 t fuente. Su comida normal de laboratorio bacteriana, OP50, puede ser fácilmente sustituido con otros patógenos para examinar las interacciones entre C. elegans y cualquier patógeno elegido. En estas condiciones, el intestino es el sitio primario de la infección. De hecho, se ha demostrado una amplia gama de patógenos bacterianos para infectar letalmente C. elegans 3-5. La bacteria Salmonella gramnegativos es un patógeno gastrointestinal que causa la enfermedad transmitida por los alimentos humanos en todo el mundo 6,7. C. elegans es un buen modelo de acogida de Salmonella typhimurium ya que esta bacteria se replica y exhibe las infecciones intestinales persistentes 8-10. C. elegans ha sido utilizado para identificar tanto novedoso y anteriormente conocido virulencia de Salmonella factores 11. Curiosamente, la C. sistema inmune elegans limita con éxito la replicación de Salmonella. Se ha informado anteriormente de que no habilition de genes autofagia hace que el aumento de la replicación de Salmonella en C. elegans, lo que resulta en la muerte temprana de los gusanos infectados 10. Macroautofagia (aquí referido como la autofagia) es un proceso dinámico que implica la reorganización de las membranas subcelulares para secuestrar citoplasma y orgánulos para la entrega a los lisosomas para degradación 12. La autofagia ha sido reportado para limitar la replicación de Salmonella en C. elegans y en los mamíferos 10,13. El C. elegans genoma fue el primer genoma eucariota multicelular secuenciado; que es sensible a RNAi tratamiento 14-16. Por otra parte, el ARNi puede ser administrado eficazmente sometiendo gusanos de ingerir las bacterias que contienen el ARN de doble cadena del gen diana, conocido como alimentación RNAi 16,17. Genoma bibliotecas de alimentación RNAi enteras han sido generados para la selección de ARNi de todo el genoma 16,18. En la presente memoria, una infección de Salmonella Proprotocolo es, junto con alimentación RNAi para permitir pruebas de C. genes elegans de interés por su capacidad de proteger contra la infección por Salmonella.

<table><tbody><tr><td>LB Broth</td><td>Fisher</td><td>BP9723-500</td><td></td></tr><tr><td>XLD agar</td><td>EMD Chemicals</td><td>1.05287.0500</td><td></td></tr><tr><td>Bacto Agar</td><td>Fisher</td><td>DF0140-01-0</td><td></td></tr><tr><td>Peptone</td><td>Fisher</td><td>BP1420-500</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher</td><td>S671-500</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher</td><td>C69-500</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Fisher</td><td>M65-500</td><td></td></tr><tr><td>IPTG</td><td>Gold Biotechnology</td><td>12481C50</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>C8667-25G</td><td></td></tr><tr><td>Ampicillin</td><td>Fisher</td><td>BP1760-25</td><td></td></tr><tr><td>&lt;em&gt;Salmonella typhimurium&lt;/em&gt;</td><td>ATCC</td><td>ATCC14028</td><td></td></tr><tr><td>Petri Dish 95 x 15 mm</td><td>Fisher</td><td>FB0875714G</td><td></td></tr><tr><td>Petri Dish 60 x 15 mm&amp;nbsp;</td><td>Fisher</td><td>08-757-13A</td><td></td></tr><tr><td>Falcon Serological pipet</td><td>Fisher</td><td>13-668-2</td><td></td></tr><tr><td>Falcon Express Pipet-Aid</td><td>Fisher</td><td>13-675-42</td><td></td></tr><tr><td>MaxQ6000 shaking incubator&amp;nbsp;</td><td>Thermo Scientific</td><td>SHKE6000-7</td><td></td></tr><tr><td>Incubator</td><td>Percival</td><td>I-36DL</td><td></td></tr></tbody></table>

a protocol to infect caenorhabditis elegans with salmonella typhimurium

En la última década, C. elegans se ha convertido en un organismo invertebrado para estudiar las interacciones entre los hosts y patógenos, incluyendo la defensa del huésped contra bacterias gram-negativas bacteria Salmonella typhimurium. Salmonella establece una infección persistente en el intestino de C. elegans y resulta en la muerte temprana de los animales infectados. Un número de mecanismos de la inmunidad se ha identificado en C. elegans para defenderse de las infecciones por Salmonella. La autofagia, una vía de degradación lisosomal conservadas evolutivamente, se ha demostrado que limitar la replicación de Salmonella en C. elegans y en los mamíferos. Aquí, un protocolo se describe para infectar C. elegans con Salmonella typhimurium, en la que los gusanos están expuestos a la Salmonella por un tiempo limitado, similar a la infección por Salmonella en los seres humanos. infección por Salmonella acorta significativamente la vida útil de C. elegans &lt;/ Em&gt;. Usando el gen de la autofagia esencial BEC-1 como un ejemplo, se combinaron este método de infección con C. elegans alimentación RNAi enfoque y mostró este protocolo puede ser utilizado para examinar la función de C. genes del huésped elegans en la defensa contra la infección por Salmonella. Desde C. elegans todo el genoma RNAi bibliotecas están disponibles, este protocolo permite a la pantalla exhaustivamente para C. genes elegans que protegen contra la Salmonella y otros patógenos intestinales utilizando RNAi bibliotecas de todo el genoma.

A 20 ° C, la esperanza de vida media de tipo salvaje gusanos N2 es de 17 días (Figura 2A y Tabla 2). Infección por Salmonella disminuye significativamente la esperanza de vida media de los gusanos N2 a 10,5 días (p = 0,0002, prueba de log-rank) (Figura 2A ). Si una C elegans gen desempeña un papel importante en la defensa contra la infección por Salmonella, se prevé que su inhibición impartirá la susceptib...

Watch this Scientific Journal Video about A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium at JoVE.com

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans se ha convertido en un nuevo modelo genético para estudiar las interacciones huésped-patógeno. Aquí se describe un protocolo para infectar C. elegans con Salmonella typhimurium, junto con la técnica de RNAi interferencia de doble hebra para examinar el papel de los genes del huésped en la defensa contra la infección por Salmonella.

Un protocolo para infectar<em&gt; Caenorhabditis elegans</em&gt; Con<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

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Research

JoVE Journal

Immunology and Infection

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

11.5K vistas.  Florida Atlantic University.  C. elegans se ha convertido en un nuevo modelo genético para estudiar las interacciones huésped-patógeno. Aquí se describe un protocolo para infectar C. elegans con Salmonella typhimurium, junto con la técnica de RNAi interferencia de doble hebra para examinar el papel de los genes del huésped en la defensa contra la infección por Salmonella. 

Video: A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

El nematodo Caenorhabditis Elegans - un modelo versátil en Vivo para el estudio de las interacciones huésped-microbio

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Investigación

Genética

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

La separación automatizada de<em&gt; C. elegans</em&gt; Variablemente colonizada por un patógeno bacteriano

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

Inmunología e Infección

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Ensayo de alto rendimiento para Fenotipo<em&gt; Salmonella enterica</em&gt; Asociación Typhimurium, Invasión y replicación en macrófagos

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Ensayo de alto rendimiento para medir la ingesta de alimentos y la esperanza de vida de Caenorhabditis elegans

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Autor destacado: Explorando la relación entre la lipotoxicidad y la HFpEF 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Cuantificación de la ingesta de alimentos en Caenorhabditis elegans mediante la medición del aclaramiento bacteriano

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

Comportamiento

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium&#160;vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Zebrafish (Danio rerio) are becoming a widely-used vertebrate animal model for microbial colonization and pathogenesis. This protocol describes the use of the protozoan Paramecium caudatum as a vehicle for food-borne infection in zebrafish larvae. P. caudatum readily internalizes bacteria and get taken up by larval zebrafish through natural preying behavior.

Utilizando el protozoo Paramecium caudatum como un vehículo para las infecciones transmitidas por los alimentos en las larvas de pez cebra

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Infección crónica por salmonela fibrosis intestinal inducida

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm&#160;model.

Estudio de la inmunidad hereditaria en un modelo de Caenorhabditis elegans de infección por microsporidios

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

Caracterización de la peritonitis séptica inducida por Salmonella Typhimurium en ratones

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

Un protocolo para infectar

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Cuantificación de la ingesta de alimentos en Caenorhabditis elegans mediante la medición del aclaramiento bacteriano

Cuantificación de la ingesta de alimentos en Caenorhabditis elegans mediante la medición del aclaramiento bacteriano

Cuantificación de la ingesta de alimentos en Caenorhabditis elegans mediante la medición del aclaramiento bacteriano

Utilizando el protozoo Paramecium caudatum como un vehículo para las infecciones transmitidas por los alimentos en las larvas de pez cebra

Infección crónica por salmonela fibrosis intestinal inducida

Estudio de la inmunidad hereditaria en un modelo de Caenorhabditis elegans de infección por microsporidios

Caracterización de la peritonitis séptica inducida por Salmonella Typhimurium en ratones