Este trabalho foi financiado pelos Institutos Canadenses de Pesquisa em Saúde (MSH-95368, 89773 e MOP-MOP-106623 para CZ) e da Fundação Canadense para a Inovação. SCM é o destinatário de uma Associação CIHR / canadense de Gastroenterologia pós-doutorado. CZ é um investigador CIHR Novo.

1. Propagação ovos de Trichuris Muris <ol><li> Para gerar novos lotes de ovos de Trichuris Muris, 20-30 infectar camundongos imunodeficientes (ex. NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) ou 129S6/SvEvTac-Rag2 tm1Fwa (RAG2 - / -)) ou camundongos geneticamente suscetíveis ( por exemplo. AKR / J), que são de 6-8 semanas de idade com cerca de 300 ovos de Trichuris Muris por gavagem oral. </li><li> Depois de 32-35 dias sacrifício dos camundongos por asfixia CO 2. </li><li> Expor o lado ventral do mouse e molhar o abdômen com álcool 70%. </li><li> Forceps usando agarrar a pele abdominal e fazer uma pequena incisão com uma tesoura. Cortar a pele para expor o conteúdo abdominal. </li><li> Identificar o ceco eo intestino proximal grande, e removê-los. </li><li> Coloque o ceco e intestino grosso proximal em um prato de 10 centímetros de petri contendo Modified Dulbecco Eagle Medium (DMEM) com 500 U / ml de penicilina e estreptomicina 500 mg / ml (P / S). Corte o ceco aberta para expor a luz e os vermes. Utilizando uma pinça ceco lugar num copo de tampão fosfato (PBS) e agitar suavemente para remover as fezes. </li><li> Retorno ceco para placa de Petri e usando uma pinça curva suave (Roboz RS-5047) puxe para fora worms e colocá-los em um poço de uma placa de 6 bem contendo 5 ml de DMEM + P / S. </li><li> Repita esse processo para todos os ratos. </li><li> Coloque seis placa bem em um recipiente Tupperware com uma toalha de papel úmida para manter a humidade e incubar a 37 ° C por 4 horas. </li><li> Após 4 horas remover worms usando uma pinça curva suave e os dividiram em dois novos poços da placa contendo 6 bem DMEM 5mL + P / S. Colheita o conteúdo do poço original e colocar em um tubo falcon 15 ml. Tubo giratório a 3000 rpm e remover e sobrenadante reserva como antígeno 4 h (pode ser usado para reestimulação celular após PBS diálise, onde os rendimentos de proteínas típicos são 10-20 mg por 20 ratos) e ressuspender ovos contendo sedimento em água de torneira autoclavada. </li><li> Coloque a placa de 6 bem para trás a 37 ° C durante a noite. Remova e descarte a worms. Colher o conteúdo das cavidades em um tubo falcon e girar a 3000 rpm por 5 min. Remover o sobrenadante e reserva como antígeno durante a noite (pode ser usado para Trichuris Ag ELISAs isotipo específico de anticorpos após a PBS diálise, onde os rendimentos de proteínas típicos são 10-20 mg por 20 ratos) e ovos contendo ressuspender sedimento em água de torneira autoclavada. </li><li> Combine 4 horas e ovos noite e filtrar os ovos pela peneira 70μm para remover quaisquer restos de vermes em seguida, transferir para um balão de 75 cm 2. Manter frasco num lugar escuro coberto por papel alumínio por 6 semanas à temperatura ambiente (RT). </li><li> Lave os ovos em água de torneira autoclavada e ressuspender em 50 ovos viáveis ​​em 50 ul. Para fazer isso, os ovos centrífuga a 3000 rpm por 5 min e ressuspender em ~ 50ml água de torneira esterilizada. Remover 50 ovos mL e colocar em uma lâmina de vidro. Usando a ampliação de 40x contar o número de ovos embrionados viável na mL 50. Diluir os ovos para dar 50 ovos viáveis ​​em 50 ul. Para imagens de ovos viáveis ​​consulte o estudo de Kopper e Mansfield 16. </li></ol> 2. Infecção aguda experimental de camundongos com Trichuris Muris ovos (200 ovos / mouse) <ol><li> Ovos cuidadosamente ressuspender e remover volume suficiente para tratar o número de ratos X 2 (por exemplo, por 10 ratos tomar 200 mL x 10 ratos x 2 = 4 mls de ovos) e colocá-los em um tubo de 14 ml tampa snap. </li><li> Inverter o tubo de 14 ml tampa encaixe para misturar. Trazer até 200 mL de ovos em uma seringa ml em uma agulha de tamanho apropriado de alimentação (para os ratos com peso de 25 gramas que usamos calibre 18 1 1 / 2 &quot;agulhas). Em um scruff a mão do mouse e usando o outro lado administrar ovos por sonda oral . </li><li> Esperar 21 dias. </li></ol> 3. Pesca experimental <ol><li> No dia 21 sacrifício os ratos usando CO 2. </li><li> Coleta de sangue de ratos por punção cardíaca, em seringa não heparinizado, e armazenar a 4 ° C. No dia seguinte isolar soro e congelar a -20 ° C. Soro pode ser usado para a detecção de Trichuris específicos IgGs e IgE por ELISA. </li><li> Expor o lado ventral do mouse e molhar o abdômen com álcool 70%. </li><li> Forceps usando agarrar a pele abdominal e fazer uma pequena incisão com uma tesoura. Cortar a pele e membrana subjacente para expor o conteúdo abdominal. </li><li> Identificar o intestino delgado ceco e cólon. Agarre o ceco com uma pinça e retire da cavidade abdominal. Com outro par de fórceps empurrar intestino delgado para a direita expondo os linfonodos mesentéricos (MLNs). Remover o MLNs tomando cuidado para não cortar os intestinos e coloque em uma falcon 15 ml contendo 2 ml de mídia. Se desejado estes podem ser processados ​​e restimulated in vitro (4x10 6 / ml em 1 ml em 24 placas bem com um αCD3/CD28 mcg / ml ou com 50μg/ml 4h Trichuris Ag) por 72 horas para análise de citocinas por ELISA e f intracelularcitometria de baixo. </li><li> Cortar o ceco e cólon. </li><li> Utilizando uma pinça, empurrar para fora pelotas fecais do cólon distal e coloque em um tubo de 2 ml Axygen. Congelar a -20 ° C. Fezes podem ser analisadas por western blot para expressão de resistina-like molécula de β-(RELM β). </li><li> Retire a ponta do ceco (~ 0,5 cm). Para remover fezes segurar a ponta ceco com uma pinça e lavar com PBS usando uma seringa de 3 ml e agulha calibre 22 em uma placa de Petri. Lugar em paraformaldeído a 4% fresco em um Falcon de 5 ml tubo tampa snap. Armazenar a 4 ° C. Estes podem ser incluídos em parafina, seccionado e montado em lâminas para análise histológica. </li><li> Retire um pedaço de cólon proximal (~ 1 cm) de RNA. Coloque amostra em dois tubos com 500 ml Axygen RNAlater mL. Armazenar a 4 ° C. Essas amostras podem ser analisados ​​para a expressão de mRNA por qPCR. </li><li> Coloque o restante do ceco em uma placa de Petri etiquetadas e congeladas a -20 ° C. </li></ol> 4. Enumeração de Trichuris Muris worms no ceco <ol><li> Retire do freezer ceco e coloque em um prato de petri contendo ~ ml de água 5. </li><li> Com uma tesoura, corte aberto ceco para expor o lúmen. </li><li> Segure o ceco com uma pinça e agite vigorosamente o ceco para remover a maioria das fezes. </li><li> Ceco transferência para outra placa de petri contendo água e usando uma pinça curva suave delicadamente raspar o IECs o tecido subjacente. </li><li> Ceco transferência para uma nova placa de petri contendo água e raspar vigorosamente tecido remanescente com uma pinça, rasgando o tecido em pedaços pequenos. </li><li> Use um microscópio de dissecação para contar todas os vermes em todas as 3 da placas de Petri. Como você encontrar um verme removê-la do prato para garantir o worm mesmo não é contado duas vezes. Se worms estão danificados (isto é quebrado pela metade) contagem só termina grossas ou finas para obter uma contagem precisa. </li></ol> 5. Resultados representante O modelo de infecção Trichuris Muris permite aos pesquisadores quantificar carga parasitária e resposta imune sistêmica, bem como para analisar histologicamente a resposta inflamatória dentro do ceco distal. Quando o lúmen do ceco colhida é exposto, worms Trichuris podem ser enumerados usando um microscópio de dissecação (Figura 1A-B). Parâmetros de imunidade para Trichuris também incluem isotipo de anticorpos mudar para IgG1 (associado com a imunidade do tipo Th2) em animais resistentes e IgG2a (associado com tipo de imunidade Th1) em animais suscetíveis (Figura 1C). Expressão da taça de células específicas de proteínas resistina-like molécula de β-(RELM β-) está associada com a depuração de Trichuris e é detectável por análise de Western blot (Figura 2). A porção distal do ceco pode ser manchada com Periodic Acid-Schiff (PAS) para avaliar as respostas de células caliciformes e da extensão da inflamação intestinal em resposta à infecção (Figura 3). <img src="/files/ftp_upload/2774/2774fig1.jpg" alt="Figura 1"/> Figura 1. Quantificação de imunidade para Trichuris Muris. (A) Resistente (B6) ratos expulsar Muris Trichuris ao dia 21 pós-infecção. Pelo contrário, suscetíveis (AKR / J) camundongos cronicamente infectados com Trichuris, resultando no estabelecimento do parasita. Cada barra representa a média ± SEM de 4-8 animais por grupo. (B) Worms são contados através de um microscópio de dissecação. Painel superior mostra worms em isolamento, o painel inferior mostra worms na presença de IECs e fezes. (C) títulos Trichuris IgG2a específicos são determinados por ELISA de soro diluído (1:2 diluição, a partir de diluição de 1:20 e terminando às 1:2560) em placas revestidas com antígeno de excreção e Trichuris (O / N Ag, 5 mg / ml). Suscetíveis AKR / J ratos têm níveis mais elevados de Trichuris nomeadamente específicos IgG2a soro do que resistente B6 camundongos. <img src="/files/ftp_upload/2774/2774fig2.jpg" alt="Figura 2"/> Figura 2. Apuramento de Trichuris Muris está associada a expressão da taça de células específicas de proteínas RELM-β 7. RELM-β de produção podem ser detectados a partir de pelotas fecais de Trichuris infectadas resistentes (B6), mas não suscetíveis (AKR / J) ratos por análise de western blot. Cada pista é representante de um único animal, (N) = Naive, (Tm) = 21 dias Trichuris camundongos infectados. <img src="/files/ftp_upload/2774/2774fig3.jpg" alt="Figura 3"/> Figura 3. Exame histológico da infecção distal Trichuris ceco seguinte. Seções 5-7 mM do ceco distal corados com PAS. Hiperplasia de células caliciformes e produção de muco são evidentes em resistente (B6), mas não suscetíveis (AKR / J) ratos após a infecção Trichuris.

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Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela The University of British Columbia.

Este protocolo detalhes um alto padrão dose aguda Trichuris Muris infecção que pode ser modificado conforme exigido pelo investigador. Por exemplo, ratos podem ser sacrificados e os tecidos colhidas em dias diferentes. Para determinar que os ratos com sucesso estabelecida carga parasitária total que pode ser sacrificada no dia 14, altura em que todos os camundongos deve levar uma carga de aproximadamente 200 worms. Ratos também podem ser infectados por 32 dias, onde qualquer worm detectado terá atingido m...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Animais Agulhas Alimentação (18 x 1 ½ &quot;) </td><td> Popper </td><td> 7912 </td><td></td></tr><tr><td> Pinça curva suave </td><td> Roboz </td><td> RS-5047 </td><td></td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11965 </td><td></td></tr><tr><td> NOD.Cg-Prkdc scid Il2r gtm1Wjl / SzJ (NSG) </td><td> Jackson Laboratories </td><td> 005557 </td><td> Estes são os ratos que usamos, no entanto, qualquer camundongos imunodeficientes ou linhagem suscetível deve funcionar. </td></tr><tr><td> RNAlater </td><td> Qiagen </td><td> 76104 </td><td></td></tr><tr><td> 2 tubos de ml </td><td> Axygen </td><td> MCT-200-C </td><td></td></tr><tr><td> Tubos de 15 ml </td><td> Falcão </td><td> 352096 </td><td></td></tr><tr><td> 6 placas bem </td><td> Falcão </td><td> 353046 </td><td></td></tr><tr><td> Paraformaldeído </td><td> Electron Microscopy Ciência </td><td> 15710 </td><td></td></tr><tr><td> α-RELMβ anticorpos </td><td> PeproTech Inc </td><td> 0694270Rb </td><td></td></tr></tbody></table>

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

Trichuris Muris é um patógeno natural de ratos e é biologicamente e antigenicamente semelhante a espécies de Trichuris que infectam seres humanos e animais 1. Ovos infectantes são dadas por gavagem oral, escotilha no intestino delgado distal, invadem as células epiteliais intestinais (CIEM) que revestem as criptas do ceco e cólon proximal e sobre a maturação dos vermes liberam os ovos no ambiente 1. Este modelo é uma ferramenta poderosa para analisar os fatores que a ativação de células CD4 controle + T helper (Th), bem como alterações no epitélio intestinal. A resposta imune que ocorre em cepas resistentes puras, tais como C57BL / 6 e BALB / c, é caracterizada por citocinas Th2 polarizada (IL-4, IL-5 e IL-13) e expulsão de vermes enquanto Th1 associado citocinas (IL -12, IL-18, IFN-γ) promover infecções crônicas em AKR geneticamente suscetíveis / mice J 2-6. Citocinas Th2 promover mudanças fisiológicas no microambiente intestinal incluindo rotatividade de IECs, a diferenciação de células caliciformes, recrutamento e alterações na permeabilidade epitelial e contração do músculo liso, as quais têm sido implicados na expulsão verme 15/07. Aqui detalhamos um protocolo para a propagação de ovos de Trichuris Muris que pode ser usado em experimentos posteriores. Nós também fornecemos uma colheita de amostra experimental com sugestões para análise pós-infecção. Globalmente, este protocolo irá fornecer aos pesquisadores as ferramentas básicas para realizar uma Trichuris Muris modelo de infecção do mouse que podem ser utilizados para tratar questões relativas à propensão Th no trato gastrointestinal, bem como as funções efectoras imunes de IECs.

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

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

Trichuris Muris infecção intestinal é um modelo de imunidade Th2 onde os ratos resistentes ao gerar uma resposta protetora Th2 e camundongos suscetíveis de gerar uma resposta Th1 patológico.

Trichuris Muris Infecção: Um Modelo de imunidade do tipo 2 e inflamação no intestino

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

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

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Immunology and Infection

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

16.2K visualizações.  University of British Columbia. Trichuris Muris infecção intestinal é um modelo de imunidade Th2 onde os ratos resistentes ao gerar uma resposta protetora Th2 e camundongos suscetíveis de gerar uma resposta Th1 patológico.

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

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

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

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

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

Capsular sorotipagem de<em&gt; Streptococcus pneumoniae</em&gt; Por aglutinação em látex

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

Imunologia e Infecção

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

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

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

Isolamento, caracterização e exploração funcional da célula de rede gengival Imune

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

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

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

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

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

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

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

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

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

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

Aplicando Transferência de ressonância de fluorescência Energia (FRET) para examinar Eficiência Effector translocação pela<em&gt; Burnetii Coxiella</em&gt; Durante siRNA silenciamento

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

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

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

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

Um método não-invasivo e tecnicamente não-intensivo para indução e fenotipagem of Experimental pneumonia bacteriana em ratos

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

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

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

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

Rato pulmão Slices para Visualize células vivas pulmonar dendríticas precisão de corte

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

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

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

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

-Fluorescência baseado Neuraminidase Ensaio de Inibição Para avaliar a susceptibilidade do vírus influenza A de neuraminidase Inibidor de Classe Antivirais

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

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

Medição de alto rendimento da membrana plasmática, efectuou a eficiência em células de mamíferos

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

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

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

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

Efeitos de gosto sinalização Abolishment de proteína na inflamação do intestino em um modelo de Mouse de doença inflamatória intestinal

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

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

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

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

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

O modelo de infecção Waxworm Galleria mellonella para candidíase disseminada

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