Os autores agradecem às comunidades na província de Yunnan, na República Popular da China e Agboville, Côte d&#39;Ivoire, para a sua participação nos estudos descritos acima. Estamos em dívida com as equipes de campo locais que tenham ajudado no teste e execução dos protocolos. Estamos particularmente gratos aos seguintes colegas da Tropical Suíço e Instituto de Saúde Pública: Yvette Endriss para fornecer as imagens microscópicas, Hanspeter Marti e sua equipe de laboratório de apoio e Krauth Stefanie por sua ajuda durante as filmagens de vídeo. Finalmente, agradecemos aos dois pareceristas anônimos para uma série de comentários úteis.

1. Kato-Katz Técnica <ol><li> Coloque um modelo Kato-Katz padrão com um orifício para a realização de 41,7 miligramas (mg) de fezes sobre uma lâmina de microscópio. </li><li> Colher 2-3 gramas (g) de uma amostra fecal fresco sobre um pedaço de jornal ou de folha de alumínio, e pressionar um pedaço de arame ou de malha de plástico por cima de peneira-lo. </li><li> Usando uma pequena espátula de plástico, raspar o material peneirado para fora da malha e encher completamente o orifício no molde Kato-Katz com ele. Para remover o excesso de material fecal, nivelar o conteúdo do orifício com a espátula. </li><li> Verticalmente remover o molde, sem perturbar o material fecal agora aderindo à lâmina de microscópio. O modelo e espátula podem ser limpos em água com detergente, e reutilizados. </li><li> Colocar uma folha de celofane, pré-embebidas em glicerina-malaquita solução verde durante pelo menos 24 horas (h), em relação à amostra fecal na lâmina de microscópio. </li><li> Para espalhar o material fecal em uma gota espessa, pressione suavemente a micro limpodeslizante alcance contra a lâmina de amostra, de maneira uniforme a distribuição do material dentro de um círculo com um diâmetro ligeiramente menor do que a largura da lâmina de microscópio. </li><li> Permitir o slide para limpar por 30-60 minutos (min), durante o qual os slides devem ser mantidos longe da luz solar direta. Em seguida, analisar sistematicamente o esfregaço espesso sob um microscópio de luz (40-100x ampliação). Contar o número de ovos STH e estratificar-los por espécie (ou seja, A.lumbricoides, T.trichiura, e ancilostomíase). De nota, ovos de helmintos outros (por exemplo, Schistosoma mansoni) podem também ser detectadas por este método e devem ser contados e registados separadamente. </li><li> Para obter uma estimativa normalizada da intensidade da infecção, convencionalmente expressa como o número de ovos por grama de fezes (OPG), multiplicando o número de ovos por 24 (24 x 41,7 mg ≈ 1 g). </li></ol> 2. Técnica de Baermann <ol><li> Montou um stand que pode segurar um copo funil e anexar um tubo de borracha, fechada sagacidadeha clipe, para a parte inferior do funil. </li><li> Coloque uma peneira ou um pedaço de tela de arame com a forma de um cone, no funil e uma camada com um pedaço de gaze. </li><li> Encha-se o funil com água da torneira e garantir que o conjunto de set-up não vaza. </li><li> Colher 20-30 g de fezes frescas para o meio da gaze e assegurar que ele está completamente submerso na água. Adicionar mais água se necessário. Dobre a gaze sobre a amostra. </li><li> A partir de baixo, a luz artificial brilhar no funil durante 2 h. </li><li> Drenar 50 mililitros (ml) da porção inferior da água no funil para um tubo de centrífuga por libertar suavemente o grampo do tubo de borracha. </li><li> Centrifugar a água recolhida a 600 xg (força centrífuga relativa) durante 5 min e com cuidado derramar o sobrenadante sem perturbar o sedimento. Reter as últimas gotas do tubo. </li><li> Re-suspender o sedimento e pipeta duas gotas da solução sobre uma lâmina de microscópio e examina a corrediçasob um microscópio de luz (ampliação de 40x para detecção e 100-400x de ampliação para confirmação espécies). Primeira fase de larvas (L1) de S. stercoralis estão vivos e movimentar ativamente sob o microscópio. </li></ol> 3. 20-m Shuttle Run Test <ol><li> Meça a 20-m percurso plano e reto correr e marcar com cones. </li><li> Ajudar o participante teste usar um monitor de freqüência cardíaca no pulso eo transmissor correspondente ao redor do tórax 18. Este equipamento permite a frequência cardíaca do participante a ser medido antes e depois do teste e, assim, controla se o participante tem exercido esforço máximo. </li><li> Instruir o participante a correr para trás e para frente no curso, seguindo o ritmo de sinais sonoros pré-gravados que delimitam 20 m de voltas. Começando com uma velocidade de funcionamento de 8,5 km por hora (km / h), a frequência dos sinais que indicam aumentos de 20 m por intervalos de um equivalente de 0,5 km / hr cada min. </li><li> Certifique ªao participante começa um intervalo de 20 m novo a cada vez que um sinal é emitido e termina-lo antes do próximo sinal chega. </li><li> Pare o participante se ele / ela não consegue acompanhar o ritmo de dois períodos consecutivos de 20 m de intervalos. </li><li> Gravar a maior velocidade de execução indicado para o último intervalo que o participante completou totalmente, juntamente com a idade e sexo do participante. </li><li> Verificar que a taxa cardíaca atingida no final do ensaio é a taxa da criança cardíaca máxima 19. Se esta taxa coração não for atingido, a criança pode não ter exercido o esforço máximo e, portanto, o teste deve ser repetido. </li><li> Estimativa da capacidade aeróbica máxima (VO2 max) do participante, usando uma equação colocada por Léger e colegas de trabalho: VO 2 max = 31,025 + 3,238 * velocidade (em km / h) - 3,248 * idade (em anos) + 0,1536 velocidade * (em km / h) * idade (em anos). </li></ol> 4. Resultados representativos Em termos deDiagnóstico STH com a técnica de Kato-Katz, as características únicas dos ovos de helmintos diferentes permitem a identificação da maioria das espécies de 20. Os ovos de A.lumbricoides (aproximadamente 45-75 micrómetros (um) de comprimento e 35-50 uM de largura, a figura 1) tem paredes mais grossas do que os ovos de casca de T.trichiura (Figura 2) e os ancilostomídeos (Figura 3). Um ovo fertilizado de A.lumbricoides (Figura 1) possui um revestimento exterior albuminosa, o que faz parecer dourar, enquanto que um óvulo fertilizado não carece deste revestimento exterior e é ligeiramente alongado e maior global. Os ovos de T.trichiura (cerca de 50 m de comprimento e 22 ^ M de largura, a figura 2) são alongadas e têm diferentes tampões polares. Ancilóstomo ovos (aproximadamente 64-76 m de comprimento e 36-40 uM de largura, a figura 3), por outro lado, tem uma parede muito fina, seguido de um anel em torno de um claro conjunto denso de célulasno centro do ovo. S.stercoralis larvas L 1 (Figura 4) são de aproximadamente 180-380 m de comprimento e tem uma cavidade bucal muito curto. Um distinto primórdio genital (realçado com uma seta azul na Figura 4) pode ser observado e a cauda das larvas (realçado com uma seta vermelha na Figura 4) tem uma ponta afiada. Como o verdadeiro impacto de infecções STH sobre a aptidão física das crianças ainda está sendo debatido 13,14,15, não há resultado representativo para o seu impacto neste momento. Interpretação dos resultados de lançadeira 20 m de execução de teste pode ser realizada de várias maneiras. A aptidão física de crianças podem ser comparados entre indivíduos infectados e não-infectados, que são semelhantes em termos de idade e sexo, outras condições de saúde potencialmente relevantes, status socioeconômico e hábitos culturais. Isto pode ser feito de um estudo transversal, onde o 13,14,15STH estado de infecção e de aptidão física de crianças infectadas e não infectadas são avaliados em um ponto determinado momento. A força das provas será maior quando na sequência de um design ensaio clínico randomizado, onde a aptidão física de crianças em idade escolar é avaliado antes e depois de anti-helmíntico / placebo tratamento, permitindo tanto para a avaliação de curto e longo prazo. Pode-se ainda tentar identificar uma relação dose-resposta entre a intensidade possível de infecções STH e de aptidão física e comparar diferentes níveis de multiparasitismo 1 com a aptidão física. No trabalho apresentado por Yap e colaboradores 15, aptidão física, como descrito pelo VO 2 max, foi encontrado para ser significativamente reduzido em crianças infectadas com T.trichiura em comparação com crianças que não estavam infectadas com T.trichiura (Figura 5) . <img alt="Figura 1" src="/files/ftp_upload/3966/3966fig1.jpg" /> Figu<html> re 1. imagens microscópicas de ovos fertilizados A.lumbricoides: (A) e não-fertilizado (B) com a ampliação de 500x. Barra de escala = 50 um. <img alt="Figura 2" src="/files/ftp_upload/3966/3966fig2.jpg" /> Figura 2. Imagem microscópica de um ovo T.trichiura sob ampliação de 500x. Barra de escala = 50 um. <img alt="Figura 3" src="/files/ftp_upload/3966/3966fig3.jpg" /> Figura 3. Imagem microscópica de um ovo de ancilóstomo sob a ampliação de 500x. Barra de escala = 50 um. <img alt="Figura 4" src="/files/ftp_upload/3966/3966fig4.jpg" /> Figura 4. Imagem microscópica de um L S.stercoralis uma larva com a ampliação de 250x. As setas indicam a cauda (vermelho) e primórdio genital (azul). Barra de escala = 100 pm. d/3966/3966fig5.jpg &quot;/&gt; Figura 5. Gráfico mostrando o nível de aptidão física de crianças infectadas com T.trichiura em comparação com o nível de aptidão física de crianças não infectadas com o T. trichiura.

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Os três protocolos descritos neste documento foram testados e executados na parte sudoeste da província de Yunnan, na República Popular da China entre os membros da minoria étnica Bulang 21 e em diferentes partes da África 6,13,14. Há vários modelos disponíveis para a técnica de Kato-Katz. Cada orifício de tamanho distinto-se administrar uma quantidade diferente de material fecal. Para o cálculo da GEP, pelo que é necessário conhecer a unidade do molde, de ...

Nota: Os materiais nesta lista são genéricas e podem ser obtidos a partir de fontes diferentes. <table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Kato-Katz kit [400 modelos de plástico com um furo de 6 milímetros (mm) (diâmetro) em um modelo de 1,5 mm de espessura, 400 espátula de plástico, um papel de 20 m de tela de nylon de 80 malhas, um papel de 20 m de celofane hidrofílico, 34 um de espessura.] </td><td> Vestergaard Frandsen </td><td></td><td> Por favor, note que a solução verde malaquita glicerina não está incluído neste kit mas pode ser comprado a partir de qualquer fornecedor de produtos químicos. </td></tr><tr><td> Funil de vidro [cerca de 8 centímetros (cm) de largura e 6 cm de profundidade] </td><td></td><td></td><td> O funil de vidro para a técnica de Baermann deve ser capaz de reter cerca de 60 mL de água. Thtamanho e da mangueira de borracha e gaze é dependente do tipo de funil de vidro utilizado. </td></tr><tr><td> Microscópio biológico </td><td> Olimpo </td><td> CX21LED/CX21 </td><td></td></tr><tr><td> Centrifugador </td><td> Xangai Medical Instruments Group </td><td> 80 - 2T </td><td></td></tr><tr><td> Pré-gravados sinais </td><td> Bitworks design </td><td> Equipe Bleep Test Versão 1.3.1 </td><td> aplicativo para iPhone </td></tr><tr><td> Monitor de freqüência cardíaca </td><td> POLAR </td><td> FT1 </td><td> Assista e transmissor </td></tr></tbody></table>

determining soil-transmitted helminth infection status and physical fitness of school-aged children

Solo transmitidas por helmintos (STH) infecções são comuns. Na verdade, mais de 1 bilhão de pessoas são afetadas, principalmente no mundo em desenvolvimento, onde a pobreza prevalece e comportamento de higiene, abastecimento de água e saneamento são muitas vezes 1,2 deficiente. Ascaris lumbricoides, Trichuris trichiura, e as duas espécies de ancilostomídeos, Ancylostoma duodenale e Necator americanus, são as mais prevalentes STHS 3. O fardo global estimado devido à ancilostomíase doença, ascaridíase e tricuríase é 22,1, 10,5 e 6,4 milhões anos de vida ajustados por incapacidade (AVAI), respectivamente 4. Além disso, um número estimado de 30-100000000 pessoas estão infectadas com Strongyloides stercoralis, as espécies mais negligenciadas STH de importância mundial que possivelmente também causa um impacto na saúde pública considerável 5,6. Várias espécies de infecções (por exemplo, STHS diferentes abrigadas em um único indivíduo) são comuns, e infecções foram lin<html> ked a menor produtividade e perspectivas assim econômico dos países em desenvolvimento 1,3. Para o diagnóstico de STHS comuns, a Organização Mundial de Saúde (OMS) recomenda a técnica de Kato-Katz 7,8, que é um método relativamente simples para determinar a prevalência e intensidade de tais infecções. Ela facilita a detecção de ovos do parasita que indivíduos infectados passam em suas fezes. No que diz respeito ao diagnóstico de S.stercoralis, não existe actualmente uma ferramenta simples e preciso disponível. A técnica de Baermann é o método mais amplamente utilizado para o diagnóstico. O princípio por trás da técnica de Baermann é que as larvas S.stercoralis ativo migrar para fora de uma amostra fecal fresca iluminado como as larvas são phototactic 9. Ela exige materiais de laboratório menos sofisticados e é menos demorado do que cultura e métodos imunológicos 5. t &quot;&gt; morbidades associadas gama infecções STH de sintomas agudos, mas comum, como dor abdominal, diarréia e prurido, a sintomas crônicos, como anemia, sub-e desnutrição e déficit cognitivo 10. Uma vez que os sintomas são geralmente inespecíficos e sutil, que muitas vezes passam despercebidos, são considerados uma condição normal de indivíduos afetados, ou são tratadas como sintomas de outras doenças que podem ser mais comuns em um dado contexto. Portanto, é concebível que o verdadeiro peso de infecções STH é subestimado por avaliação ferramentas contando com auto-declarados sinais e sintomas, como é geralmente o caso em inquéritos de base populacional. No final de 1980 e início de 1990, Stephenson e seus colegas destacaram a possibilidade de infecções STH redução da aptidão física de meninos com idades entre 6-12 anos 11,12. Esta linha de investigação científica ganhou novo impulso recentemente 13,14,15. O jogador de 20 metros (m) de teste shuttle run foi desenvolvido umd validado por 16. Léger et al e é utilizado mundialmente para medir a capacidade aeróbica de crianças de 17. O teste é fácil de padronizar e pode ser feita sempre que uma de 20 m de longo curso e plano em execução e uma fonte de áudio estão disponíveis, tornando seu uso atraente em recursos limitados configurações 13. Para facilitar e padronizar as tentativas de avaliar se as infecções STH ter um efeito sobre a aptidão física de crianças em idade escolar, apresentamos metodologias que diagnosticar infecções STH ou aptidão física medida que são simples de executar e ainda, fornecer resultados precisos e reprodutíveis. Isso vai ajudar a gerar novas evidências sobre o impacto na saúde de infecções STH.

Watch this Scientific Journal Video about Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children at JoVE.com

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

A infecção crônica por helmintos solo-transmissíveis (STHS) provoca má absorção, baixa estatura, e desperdiçando na criança em crescimento. Assim, é plausível que estas infecções também reduzir a aptidão física das crianças. Aqui, visualizamos duas técnicas para o diagnóstico de STHS e os 20 metros de teste shuttle run para avaliar aptidão física das crianças.

Determinação do solo transmitida Estado de infecção por helmintos e Aptidão Física de crianças em idade escolar

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

determining-soil-transmitted-helminth-infection-status-physical

Research

JoVE Journal

Immunology and Infection

23.1K visualizações.  Swiss Tropical and Public Health Institute, Basel, Switzerland. A infecção crônica por helmintos solo-transmissíveis (STHS) provoca má absorção, baixa estatura, e desperdiçando na criança em crescimento. Assim, é plausível que estas infecções também reduzir a aptidão física das crianças. Aqui, visualizamos duas técnicas para o diagnóstico de STHS e os 20 metros de teste shuttle run para avaliar aptidão física das crianças.

Vídeo: Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Determinação da atividade fagocítica de amostras de anticorpos Clínica

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Imunologia e Infecção

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Usando Ovos de<em&gt; Schistosoma mansoni</em&gt; Como um<em&gt; Em vivo</em&gt; Modelo de Helmintos induzida por inflamação pulmonar

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Análise da maturação das células dendríticas e migração pulmonar durante a inflamação alérgica das vias aéreas

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Fluorescência métodos de microscopia para determinar a viabilidade de bactérias em associação com células de mamíferos

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Extração de proteínas e 2-D métodos Eletroforese em Gel de<em&gt; Burkholderia</em&gt; Espécies

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Crescimento Pairwise competição ensaio para determinar a replicação de Fitness de Imunodeficiência vírus humanos

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

Determinar a supressão imune Sistema contra CNS Proteção para farmacológicos intervenções na desmielinização auto-imune

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizando os Efeitos de escarro no desenvolvimento de biofilmes usando um modelo Chambered lamela

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Protocolos para investigar a distribuição no tecido hospedeiro, o modo de transmissão e o efeito na aptidão do hospedeiro de um Densovírus na lagarta do algodoeiro

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

Elevado-throughput sequenciamento paralela à medida Fitness de Leptospira interrogans Transposon inserção mutantes durante a Golden sírio Hamster infecção

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...