Agradecemos ao Dr. Diane Baronas-Lowell para a leitura crítica do manuscrito. Este trabalho foi apoiado por uma FAU Charles E. Schmidt Faculdade de Ciências Grant Semente e uma bolsa Envelhecimento da Fundação Médica Ellison para KJ

1. Placas XLD (xilose lisina desoxicolato) Agar Ágar XLD é um meio de crescimento seletivo para Salmonella, que aparece como colônias pretas nas placas de ágar XLD. No entanto, se não existem preocupações de contaminação, uma placa LB regular pode ser substituído. <ol><li> Pesar 5,5 g de ágar XLD e ressuspender em 5 ml de água deionizada. </li><li> Misture bem até que todo o agar é molhado. Adicionar 95 ml de água deionizada até que todos os pedaços se foram e o meio é completamente em suspensão. </li><li> Ferva o meio para dissolver completamente (não autoclave). </li><li> Arrefece-se o meio à temperatura ambiente e 50 ° C. </li><li> Verter 25 mL de agar em cada um 95 x 15 mm (diâmetro x altura) da placa (placas seladas com Parafilm pode ser armazenado a 4 ° C durante até 1 mês). </li></ol> 2. Nematode Crescimento Médio (NGM) RNAi Alimentando Plates Preparação de C. elegans placas NGM foi descrito anteriormente 19. Aqui um procedimento é descrito resumidamente a adicionar o antibiótico ampicilina e a RNAi indutor químico isopropílico β-D-1-tiogalactopiranósido (IPTG) para o meio de NGM para fazer as placas de alimentação de RNAi. <ol><li> Dissolve-se 3 g de NaCl e 2,5 g de Bacto peptona em 1 L de água desionizada. </li><li> Adicione 17 g de ágar Bacto na mídia. </li><li> Autoclave a media durante 45 min e arrefecer a mídia para 50 ° C em um banho de água. </li><li> Adicionar as seguintes soluções: 1 ml de colesterol (5 mg / ml em etanol a 95%), 1 mL de 1 M de CaCl2, 1 ml 1 M MgSO4, e tampão de fosfato de potássio 25 ml 1 M (pH 6,0). Misture bem. </li><li> Adicionar 1 ml de 1 M de IPTG e 500 ul de ampicilina (100 mg / ml em água esterilizada). </li><li> Misturar a solução bem e despeje em 60 x 15 mm (diâmetro x altura) placas de Petri usando uma pipeta Aid e 25 ml de pipeta sorológica seguintes procedimentos estéreis. Encha cada placa com 12 ml de ágar. As placas podem ser armazenadas a 4 ° C durante até 1 mês. </li></ol> O gene essencial autofagia bec-1 é utilizado como um exemplo para examinar a função de um gene hospedeiro na defesa contra a infecção por Salmonella. Os procedimentos experimentais são ilustradas na Figura 1 e Tabela 1. O protocolo para a preparação de animais tratados em RNAi para infecção segue, com o dia de cada passo experimental dado entre parênteses. <ol><li> Inocular bec-1 RNAi alimentação e controlar o vector vazio L4440 RNAi bactérias alimentação pela colocação de um floco de -80 ° C bactérias congeladas em 2 ml de meio LB suplementado com 100 mg / ml de ampicilina (Dia 1). Repita este passo uma vez por semana, durante todo o experimento ter bactérias RNAi frescos. Armazenar a cultura no frigorífico a 4 ° C quando não usado. </li><li> Semente 100 ml de cultura bacteriana durante a noite RNAi em placas de RNAi. Prepare três bec-1 RNAi e três controle vazio vEctor placas de RNAi. Incubar as placas a 37 ° C durante a noite (dia 2). </li><li> Remova as placas de RNAi da 37 ° C incubadora e deixe esfriar até a temperatura ambiente no banco. Pegue bem alimentados L4 tipo selvagem hermafroditas N2 e transferi-los para bec-1 RNAi e controlar placas RNAi vetores vazios. Coloque três vermes por placa, em placas triplicadas. No mesmo dia, para preparar as placas de RNAi, como descrito no passo 3.2 (Dia 3). </li><li> Incubar as placas de RNAi com vermes na incubadora a 20 ° C durante 36-40 hr e de transferência de vermes para placas frescas de RNAi correspondentes preparados no passo 3.3. Depois de vermes são transferidos, incubar as placas no 20 ° C incubadora por 64 horas (dia 4). </li></ol> 4. Prepare Salmonella para a Infecção <ol><li> Streak Salmonella -80 ° C lote congelado em 1 placa de ágar XLD e incubar a placa a 37 ° C durante a noite (dia 5 </strong&gt;). </li><li> Escolha bem isolado preto colónia de Salmonella e inocula-lo em 2 ml de meio LB a 37 ° C com agitação durante a noite (dia 6). </li><li> Semente de 80 ml ​​de cultura de uma noite de Salmonella em 1 de C. elegans 60 x 15 mm (diâmetro x altura) placa de ágar NGM e preparar 6 pratos no total. Incubar as placas à temperatura ambiente durante 6 horas. A cultura bacteriana deve secar e formar um relvado sobre a placa (dia 7). </li></ol> 5. Worms tratados com RNAi Infect com Salmonella <ol><li> Transferir bec-1 e controle de vetores vazios tratados com RNAi hermafroditas L4 N2 (progênie de minhocas criadas no passo 3) para placas de Salmonella tratados com RNAi. Colocar 40 vermes por placa em 3 placas para cada grupo. Incubar as placas sem-fim, a 20 ° C durante 48 h (Dia 7). </li><li> Prepare um conjunto de placas de RNAi frescos como descrito nos passos 3.1 e 3.2 (dia 7 e <strong&gt; Dia 8). </li><li> Após 48 horas de infecção, transferir vermes infectadas com Salmonella às placas correspondentes RNAi preparados na etapa 5.2 e incubar a 20 ° C (dia 9). </li></ol> 6. Ensaio de sobrevivência <ol><li> Marque a sobrevivência dos vermes diária e transferir worms para frescas correspondentes placas de RNAi durante o tempo de postura. Preparar um conjunto de novas placas de RNAi, antes de cada transferência de sem-fim, tal como descrito nas etapas 3.1 e 3.2. Toque o corpo verme (cabeça, parte intermediária e cauda) delicadamente com um fio de platina achatado-end. Um worm é marcado como morto se nenhum movimento do corpo verme é observada. </li><li> Marque a sobrevivência dos vermes diárias ou em dias alternados, e transferir para vermes frescas correspondentes placas de RNAi duas vezes por semana depois de vermes parar de pôr ovos. </li><li> Depois de todos os vermes morrem, reunir os dados de sobrevivência de placas triplicadas como um conjunto de dados. Introduza os dados de sobrevivência de cada grupo em software estatístico adequado, como GraphPad PRISM para gerar curvas de sobrevivência e para realizar a análise de sobrevida de Kaplan-Meier. Toda a experiência foi repetida pelo menos uma vez para confirmar a conclusão. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

C. elegans é um simples organismo modelo genético que se alimenta de bactérias como fonte de nutrientes. Assim, é fácil de substituir a sua comida bacteriana normal com um patógeno intestinal para investigar as interações entre C. elegans e o agente patogénico escolhido. Nisto um protocolo é descrito combinar infecção por Salmonella e C. elegans RNAi alimentando tratamento para examinar o papel dos genes do hospedeiro na defesa contra a infecção por Salmonella. ...

O solo de vida livre Caenorhabditis elegans nematóide é um organismo modelo simples e geneticamente tratável usada para estudar muitas questões biológicas. C. elegans dominantemente existe como hermafroditas auto-fertilização. Os machos são gerados de forma espontânea por não-disjunção do cromossomo X durante a gametogênese 1,2. Na presença de alimento abundante, C. elegans desenvolver continuamente através de quatro fases larval ao adulto. A temperatura também influencia C. desenvolvimento elegans; desenvolvimento mais rápido é observada a temperaturas mais elevadas. No laboratório, C. elegans é cultivada a uma temperatura normal de 20 ° C em placas de agar com semeado bactéria Escherichia coli (estirpe OP50) como alimento de 1,2. Na última década, C. elegans tem emergido como um organismo de invertebrados para estudar as interações patógeno-hospedeiro 3-5. Na natureza, C. elegans come bactérias como nutrient 1,2 fonte. Sua comida laboratório bacteriana normal, OP50, pode ser facilmente substituído por outros agentes patogénicos para examinar as interações entre C. elegans e qualquer agente patogénico escolhido. Sob estas condições, o intestino é o principal local da infecção. De facto, uma grande variedade de agentes patogénicos bacterianos tem sido mostrado para infectar letalmente C. elegans 3-5. A bactéria Salmonella gram-negativo é um patógeno gastrointestinal que causa doenças de origem alimentar humana em todo o mundo 6,7. C. elegans é um bom anfitrião modelo para Salmonella typhimurium como esta bactéria replica e apresenta infecções intestinais persistentes 8-10. C. elegans foi utilizado para identificar novas e anteriormente conhecida virulência de Salmonella factores 11. Curiosamente, a C. sistema imunitário elegans limita com sucesso a replicação de Salmonella. Foi relatado anteriormente que inibidoition de genes autofagia torna o aumento da replicação Salmonella em C. elegans, resultando em morte precoce de vermes infectados 10. Macroautofagia (aqui referido como autofagia) é um processo dinâmico, que envolve o rearranjo de membranas intracelulares para sequestrar citoplasma e organelas para entrega do lisossoma para degradação 12. Autophagy foi reportado para limitar a replicação de Salmonella em C. elegans e em mamíferos 10,13. O C. elegans genoma foi o primeiro genoma eucarionte multicelular seqüenciado; ele é sensível ao tratamento 14-16 RNAi. Além disso, o RNAi pode ser administrado de forma eficaz, sujeitando vermes ingerir bactérias contendo o ARN de cadeia dupla do gene alvo, conhecido como RNAi alimentação 16,17. Genoma bibliotecas inteiras de alimentação RNAi foram gerados por RNAi do genoma triagem 16,18. Nisto, uma infecção pro Salmonellatocolo está acoplado com o RNAi alimentação para permitir o teste C. elegans genes de interesse para a sua capacidade de proteger contra infecção 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

Na última década, C. elegans tem emergido como um organismo de invertebrados para estudar interações entre hospedeiros e patógenos, incluindo a defesa do hospedeiro contra bactérias gram-negativas bactéria Salmonella typhimurium. Salmonella estabelece infecção persistente no intestino de C. elegans e resulta na morte prematura dos animais infectados. Um número de mecanismos de imunidade foram identificados em C. elegans para se defender contra infecções por Salmonella. Autophagy, uma via de degradação lisossomal evolutivamente conservadas, foi mostrado para limitar a replicação de Salmonella em C. elegans e nos mamíferos. Aqui, é descrito um protocolo para infectar C. elegans com Salmonella typhimurium, em que os vermes são expostos a Salmonella por um tempo limitado, semelhante à infecção por Salmonella em humanos. infecção por Salmonella encurta significativamente o tempo de vida de C. elegans &lt;/ Em&gt;. Usando o gene essencial autofagia bec-1 como exemplo, que combinado este método de infecção com C. elegans ARNi alimentação abordagem e mostraram que este protocolo pode ser utilizado para examinar a função de C. genes do hospedeiro elegans em defesa contra a infecção por Salmonella. Desde C. elegans genoma bibliotecas inteiras RNAi estão disponíveis, este protocolo torna possível para a tela de forma abrangente para C. elegans genes que protegem contra Salmonella e outros patógenos intestinais usando bibliotecas RNAi genômicos.

A 20 ° C, o tempo de vida médio de tipo selvagem vermes N2 de 17 dias (Figura 2A e Tabela 2). Infecção por Salmonella diminui significativamente o tempo de vida médio de vermes N2 a 10,5 dias (p = 0,0002, teste de log-rank) (Figura 2A ). Se um C. elegans gene desempenha um papel importante na defesa contra a infecção por Salmonella, prevê-se que a sua inibição irá conferir susceptibilidade à infecção ...

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 tem emergido como um novo modelo genético para estudar as interações patógeno-hospedeiro. Aqui nós descrevemos um protocolo para infectar C. elegans com Salmonella typhimurium, juntamente com a técnica de interferência dupla vertente RNAi para examinar o papel dos genes do hospedeiro na defesa contra a infecção por Salmonella.

Um protocolo para infectar<em&gt; Caenorhabditis elegans</em&gt; Com<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 visualizações.  Florida Atlantic University.  C. elegans tem emergido como um novo modelo genético para estudar as interações patógeno-hospedeiro. Aqui nós descrevemos um protocolo para infectar C. elegans com Salmonella typhimurium, juntamente com a técnica de interferência dupla vertente RNAi para examinar o papel dos genes do hospedeiro na defesa contra a infecção por Salmonella. 

Vídeo: 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.

O nemátode Caenorhabditis Elegans - um modelo versátil na Vivo para estudar interações do anfitrião-micróbio

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

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.

A separação automática de<em&gt; C. elegans</em&gt; Variably Colonizada por um 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 ...

Imunologia e Infecção

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.

De alta capacidade: Ensaio para Fenótipo<em&gt; Salmonella enterica</em&gt; Typhimurium Association, invasão e replicação em macrófagos

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

Ensaio de alto rendimento para medir a ingestão de alimentos e a expectativa 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 em destaque: Explorando a relação entre lipotoxicidade e ICFEP 

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

Quantificando a ingestão de alimentos em Caenorhabditis elegans medindo a depuração bacteriana

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

Comportamento

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.

Usando o protozoário Paramecium caudatum como um veículo para infecções de origem alimentar em larvas de Zebrafish

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.

Infecção crônica por Salmonella induzida por fibrose intestinal

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.

Estudando imunidade herdada em um modelo de infecção por microsporidia

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.

Caracterizando Salmonella Typhimurium-induced Septic Peritonitis em camundongos

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 ...

Um protocolo para infectar

Resumo

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De alta capacidade: Ensaio para Fenótipo

Quantificando a ingestão de alimentos em Caenorhabditis elegans medindo a depuração bacteriana

Quantificando a ingestão de alimentos em Caenorhabditis elegans medindo a depuração bacteriana

Quantificando a ingestão de alimentos em Caenorhabditis elegans medindo a depuração bacteriana

Usando o protozoário Paramecium caudatum como um veículo para infecções de origem alimentar em larvas de Zebrafish

Infecção crônica por Salmonella induzida por fibrose intestinal

Estudando imunidade herdada em um modelo de infecção por microsporidia

Caracterizando Salmonella Typhimurium-induced Septic Peritonitis em camundongos