Los autores agradecen a las comunidades de la provincia de Yunnan, República Popular de China y Agboville, Côte d&#39;Ivoire, por su participación en los estudios descritos anteriormente. Estamos en deuda con los equipos de campo locales que colaboraron en las pruebas y la ejecución de los protocolos. Estamos especialmente agradecidos a los siguientes colegas de la Swiss Tropical y Salud Pública Instituto: Yvette Endriss para proporcionar las imágenes microscópicas, Hanspeter Martí y su equipo de apoyo de laboratorio y Krauth Stefanie por su ayuda durante la filmación de video. Por último, agradecemos a los dos árbitros anónimos por una serie de observaciones útiles.

1. Kato-Katz Técnica <ol><li> Coloque un nivel de Kato-Katz plantilla con un agujero para la celebración de 41,7 miligramos (mg) de materia fecal en un portaobjetos de microscopio. </li><li> Scoop 2-3 gramos (g) de una muestra fecal fresca sobre un trozo de papel de periódico o papel de aluminio, y presiona un trozo de alambre o malla de plástico en la parte superior a lo tamiz. </li><li> Usando una espátula de plástico pequeño, raspe el material tamizado fuera de la malla y llenar completamente el orificio de la plantilla de Kato-Katz con él. Para eliminar el exceso de material fecal, nivelar el contenido del agujero con la espátula. </li><li> Verticalmente eliminar la plantilla sin alterar el material fecal ahora adherirse al portaobjetos. La plantilla y la espátula se pueden lavar en agua con detergente, y volver a utilizar. </li><li> Colocar un trozo de celofán, pre-empapados en solución verde malaquita glicerina durante al menos 24 horas (h), sobre la muestra fecal en el portaobjetos de microscopio. </li><li> Para difundir el material fecal en una gota gruesa, presione suavemente un paño limpio microdiapositiva alcance contra el portaobjetos de la muestra, la distribución uniforme del material dentro de un círculo de un diámetro ligeramente menor que la anchura del portaobjetos de microscopio. </li><li> Permitir la diapositiva para borrar por 30-60 minutos (min), durante los cuales las diapositivas deben mantenerse lejos de la luz solar directa. A continuación, examinar sistemáticamente la gota gruesa con un microscopio óptico (40-100x aumentos). Cuente el número de huevos STH y estratificar ellos por especie (es decir, A. lumbricoides, T.trichiura y anquilostoma). De nota, los huevos de helmintos otros (por ejemplo, Schistosoma mansoni) también pueden ser detectados por este método y se cuentan y se registran por separado. </li><li> Para obtener una estimación normalizada de la intensidad de la infección, expresada convencionalmente como número de huevos por gramo de heces (EPG), multiplicar el recuento de huevos por 24 (24 x 41,7 mg ≈ 1 g). </li></ol> 2. Técnica de Baermann <ol><li> Establecer un soporte que puede sostener un vaso embudo y coloque un tubo de goma, cerró el ingenioclip de ha, a la parte inferior del embudo. </li><li> Coloque un tamiz o una pieza de malla de alambre en forma de cono, en el embudo y la capa con un trozo de gasa médica. </li><li> Llenar el embudo con agua del grifo y asegurarse de que la forma de organización no se escape. </li><li> Scoop 20-30 g de heces frescas en el medio de la gasa médica y asegurarse de que está totalmente sumergida en agua. Añadir más agua si es necesario. Doble la gasa médica sobre la muestra. </li><li> Desde abajo, brilla la luz artificial en el embudo durante 2 horas. </li><li> Escurrir 50 mililitros (ml) de la parte inferior del agua en el embudo en un tubo de centrífuga por liberando suavemente la pinza en el tubo de goma. </li><li> Centrifugar el agua recogida a 600 xg (fuerza centrífuga relativa) durante 5 min y verter cuidadosamente el sobrenadante sin perturbar el sedimento. Conserve las últimas gotas en el tubo. </li><li> Se resuspende el sedimento y la pipeta de 2 gotas de la solución sobre un portaobjetos de microscopio y examinar la diapositivabajo un microscopio óptico (aumento de 40x para la detección y amplificación 100-400x para confirmación de especie). Primera etapa de larvas (L1) de S. stercoralis están vivos y se mueven activamente en el microscopio. </li></ol> 3. 20-m de traslado Test Run <ol><li> Medir un curso de 20 metros de longitud, plano y recto con la marca de conos. </li><li> Ayude al participante en la prueba usar un monitor de ritmo cardíaco en la muñeca y el correspondiente transmisor en el pecho 18. Este equipo permite que el ritmo cardíaco del participante para ser medidos antes y después de la prueba y por lo tanto controla si el participante ha ejercido un esfuerzo máximo. </li><li> Instruya a los participantes a ir y venir en el curso, siguiendo el ritmo de las señales de sonido pre-grabados que delimitan 20-m vueltas. A partir de una velocidad de funcionamiento de 8,5 kilómetros por hora (km / h), la frecuencia de las señales que indican 20-m aumenta los intervalos de un equivalente de 0,5 km / h cada min. </li><li> Asegúrese de then el participante inicia un nuevo intervalo de 20-m cada vez que se hace sonar una señal y termina antes de que la señal llegue el siguiente. </li><li> Detenga el participante si él / ella no puede seguir el ritmo de dos períodos consecutivos de 20-m intervalos. </li><li> Registrar la más alta velocidad de funcionamiento indicado para el último intervalo el cual el participante completado totalmente, junto con la edad y el sexo del participante. </li><li> Compruebe que la frecuencia cardíaca obtenidos al final de la prueba es la tasa de niño cardíaca máxima 19. Si la frecuencia cardíaca no se alcanza, el niño podría no haber ejercido un esfuerzo máximo, y por lo tanto la prueba debe repetirse. </li><li> Estimar la capacidad aeróbica máxima (VO 2 máx) del participante, mediante una ecuación planteada por Léger y compañeros de trabajo: VO 2 max = 31.025 + 3.238 * Velocidad (km / h) - 3,248 * edad (en años) + 0,1536 * Velocidad (km / hr) * Edad (en años). </li></ol> 4. Los resultados representativos En términos deDiagnóstico STH con la técnica de Kato-Katz, características únicas de los huevos de helmintos diferentes permiten la identificación de la mayoría de las especies 20. Los huevos de A. lumbricoides (aproximadamente 45-75 micrómetros (micras) de largo y 35-50 micras, Figura 1) tienen paredes más gruesas de concha que los huevos de T.trichiura (Figura 2) y anquilostomas (Figura 3). Un óvulo fertilizado de A. lumbricoides (Figura 1) tiene una capa externa albuminosa, que hace que se vea marrón dorado, mientras que un huevo no fertilizado carece de esta capa externa y es ligeramente alargado y grande en general. Los huevos de T.trichiura (aproximadamente 50 micras de largo y de ancho 22 m, Figura 2) son alargados y tienen distintos tapones polares. Anquilostomiasis huevos (aproximadamente 64-76 micras de largo y de ancho 36-40 micras, Figura 3), por otra parte, tienen una pared muy delgada, seguido por un claro anillo alrededor de un grupo denso de célulasen el centro del huevo. S.stercoralis L 1 larvas (Figura 4) son de aproximadamente 180-380 micras de largo y tienen una cavidad bucal muy corto. Un primordio genital diferenciado (resaltado con una flecha azul en la Figura 4) puede ser observado y la cola de las larvas (resaltado con una flecha roja en la Figura 4) tiene un extremo afilado. Como el verdadero impacto de las infecciones STH sobre la aptitud física de los niños es aún objeto de debate 13,14,15, no hay un resultado representativo de su impacto en este punto. Interpretación de 20-m resultados de la prueba de enlace que van puede realizarse de varias maneras. La condición física de los niños pueden ser comparados entre los sujetos infectados y no infectados, que son similares en cuanto a edad y sexo, otros problemas de salud potencialmente relevantes, nivel socioeconómico y hábitos culturales. Esto se puede hacer en un estudio transversal 13,14,15 donde laEstado de infección por geohelmintos y la aptitud física de los niños infectados y no infectados son evaluados en un momento particular. La fuerza de las pruebas será mayor cuando se sigue un diseño de ensayo controlado aleatorio en el que se evaluó la condición física de los niños en edad escolar antes y después del tratamiento antihelmíntico / placebo, lo que permite la evaluación tanto a corto como a largo plazo. Uno puede además tratar de identificar una posible relación dosis-respuesta entre la intensidad de las infecciones por geohelmintos y la aptitud física y comparar diferentes niveles de multiparasitismo con un buen estado físico. En el trabajo presentado por Yap y compañeros de trabajo-15, buen estado físico, según lo representado por el VO 2 máx, se encontró que se redujo significativamente en los niños infectados con T.trichiura comparación con los niños que no estaban infectadas con T.trichiura (Figura 5) . <img alt="Figura 1" src="/files/ftp_upload/3966/3966fig1.jpg" /> Figu<html> re 1. Imágenes microscópicas de los huevos de A. lumbricoides: fecundado (A) y no fertilizados (B) con aumento 500x. Barra de escala = 50 micras. <img alt="Figura 2" src="/files/ftp_upload/3966/3966fig2.jpg" /> Figura 2. Imagen microscópica de un huevo T.trichiura bajo un aumento de 500x. Barra de escala = 50 m. <img alt="Figura 3" src="/files/ftp_upload/3966/3966fig3.jpg" /> Figura 3. Imagen microscópica de un huevo de anquilostoma bajo un aumento de 500x. Barra de escala = 50 micras. <img alt="Figura 4" src="/files/ftp_upload/3966/3966fig4.jpg" /> Figura 4. Imagen microscópica de un S.stercoralis L 1 larva bajo un aumento de 250x. Las flechas indican la cola (rojo) y el primordio genital (azul). Barra de escala = 100 micras. d/3966/3966fig5.jpg &quot;/&gt; Figura 5. Gráfico que muestra el nivel de aptitud física de los niños infectados con T.trichiura en comparación con el nivel de aptitud física de los niños no infectados con T. trichiura.

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No hay conflictos de interés declarado.

Los tres protocolos descritos en este documento han sido probados y se ejecuta en la parte sur-occidental de la provincia de Yunnan, República Popular de China entre los miembros de la minoría étnica Bulang 21 y en diferentes partes de África 6,13,14. Hay varias plantillas disponibles para la técnica de Kato-Katz. Cada agujero de tamaño distinto entregará una cantidad distinta de la materia fecal. Para el cálculo de las EPG, por lo que es necesario conocer la un...

Nota: El material de esta lista son genéricos y se puede obtener de diferentes fuentes. <table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Kato-Katz kit [400 plantillas de plástico con un agujero de milímetro 6 (mm) (diámetro) en una plantilla de 1,5 mm de espesor; 400 espátula de plástico, un papel de 20-m de la pantalla de nylon de tamaño de malla 80, un papel de 20-m de celofán hidrófilo, 34 m de espesor.] </td><td> Vestergaard Frandsen </td><td></td><td> Tenga en cuenta que la solución de glicerina verde malaquita no está incluido en este kit, pero se pueden comprar en cualquier proveedor de productos químicos. </td></tr><tr><td> Embudo de vidrio [unos 8 centímetros (cm) de ancho y 6 de profundidad cm] </td><td></td><td></td><td> El embudo de vidrio para la técnica de Baermann debe ser capaz de mantener aproximadamente 60 ml de agua. Thtamaño e de la manguera de goma y la gasa es dependiente del tipo de embudo de vidrio utilizado. </td></tr><tr><td> Microscopio biológico </td><td> Olimpo </td><td> CX21LED/CX21 </td><td></td></tr><tr><td> Centrífugo </td><td> Shanghai Medical Instruments Group </td><td> 80 - 2T </td><td></td></tr><tr><td> Pre-grabados señales </td><td> Bitworks Diseño </td><td> Equipo de Rayos Prueba Versión 1.3.1 </td><td> aplicación para iPhone </td></tr><tr><td> Pulsómetro </td><td> POLAR </td><td> FT1 </td><td> Reloj y el transmisor </td></tr></tbody></table>

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

Helmintos transmitidos por el suelo (STH), las infecciones son comunes. De hecho, más de 1 mil millones de personas se ven afectadas, principalmente en el mundo en desarrollo, donde predomina la pobreza y los hábitos de higiene, abastecimiento de agua y el saneamiento son a menudo deficientes 1,2. Ascaris lumbricoides, Trichuris trichiura, y las dos especies de anquilostomas, Ancylostoma duodenale y Necator americanus, son los más prevalentes STHS 3. La carga global estimada por anquilostomiasis, ascariasis, tricuriasis y es de 22,1, 10,5 y 6,4 millones ajustados por discapacidad años de vida (DALY), respectivamente 4. Por otra parte, se estima que 30 hasta 100 millones de personas están infectadas con Strongyloides stercoralis, la especie STH más desatendidas de importancia mundial que podría decirse que también causa un impacto considerable de salud pública 5,6. Múltiples especies infecciones (por ejemplo, STHS diferentes albergaron en un solo individuo) son comunes, y las infecciones han sido lin<html> ked a la productividad baja y las perspectivas tanto económica de los países en desarrollo 1,3. Para el diagnóstico de STHS comunes, la Organización Mundial de la Salud (OMS) recomienda la técnica de Kato-Katz 7,8, que es un método relativamente sencillo para determinar la prevalencia y la intensidad de estas infecciones. Se facilita la detección de huevos de parásitos que sujetos infectados pasan en sus heces. Con respecto al diagnóstico de S.stercoralis, actualmente no hay disponible herramienta simple y precisa. La técnica de Baermann es el método más utilizado para el diagnóstico. El principio detrás de la técnica de Baermann es que activa las larvas migran S.stercoralis de un sistema de iluminación de la muestra fecal fresca como las larvas son fototácticas 9. Requiere materiales de laboratorio menos sofisticados y menos tiempo que los métodos de cultivo e inmunológicas 5. t &quot;&gt; Las morbilidades asociadas con infecciones STH gama de síntomas agudos, pero comunes, como dolor abdominal, diarrea y prurito, síntomas crónicos, como la anemia, la desnutrición y la malnutrición y deterioro cognitivo 10. Dado que los síntomas suelen ser inespecíficos y sutil, que a menudo pasan desapercibidos, son considerados como una condición normal de los individuos afectados, o son tratados como síntomas de otras enfermedades que podrían ser más comunes en un entorno determinado. Por lo tanto, es concebible que la carga real de infecciones STH es subestimada por la evaluación herramientas de confiar en los autoproclamados signos y síntomas como suele ser el caso en las encuestas de población. A finales de 1980 y principios de 1990, Stephenson y sus colegas destacaron la posibilidad de infecciones STH bajar la condición física de los niños de 6-12 años 11,12. Esta línea de investigación científico adquirido un nuevo impulso recientemente 13,14,15. El 20-metros (m) PCN se desarrolló und validado por Léger et al. 16 y se utiliza en todo el mundo para medir la capacidad aeróbica de los niños 17. La prueba es fácil de estandarizar y se puede realizar siempre que sea un curso de 20 metros de longitud, largo y plano y una fuente de audio están disponibles, lo que hace atractivo su uso en entornos con recursos limitados 13. Para facilitar y estandarizar los intentos de evaluar si las infecciones STH tiene un efecto sobre la condición física de los niños en edad escolar, se presentan metodologías para diagnosticar infecciones STH o aptitud física medida que son fáciles de ejecutar y, sin embargo, proporcionan resultados exactos y reproducibles. Esto ayudará a generar nuevas evidencias sobre el impacto en la salud de las infecciones 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

La infección crónica por helmintos transmitidos por el suelo (STHS) provoca malabsorción, retraso del crecimiento y la emaciación en el niño en crecimiento. Por lo tanto, es plausible que estas infecciones también reducen la aptitud física de los niños. A continuación, visualizamos dos técnicas para el diagnóstico de STHS y la prueba de lanzadera de 20 metros de largo para la evaluación de la aptitud física de los niños.

La determinación de helmintos transmitidos por el suelo el estado de infección y estado físico de los niños en edad 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 vistas.  Swiss Tropical and Public Health Institute, Basel, Switzerland. La infección crónica por helmintos transmitidos por el suelo (STHS) provoca malabsorción, retraso del crecimiento y la emaciación en el niño en crecimiento. Por lo tanto, es plausible que estas infecciones también reducen la aptitud física de los niños. A continuación, visualizamos dos técnicas para el diagnóstico de STHS y la prueba de lanzadera de 20 metros de largo para la evaluación de la aptitud física de los niños.

Video: 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.

La determinación de la actividad fagocítica de los anticuerpos de muestras clínicas

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

Investigación

Inmunología e Infección

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.

Con óvulos de<em&gt; Schistosoma mansoni</em&gt; Como un<em&gt; En vivo</em&gt; Modelo de la inflamación pulmonar inducida por helmintos

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.

El análisis de la maduración pulmonar de células dendríticas y migración durante la inflamación de las vías alérgica

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.

Fluorescencia métodos de microscopía para determinar la viabilidad de las bacterias en asociación con las 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.

Extracción de proteínas totales y 2-D Métodos gel de electroforesis para<em&gt; Burkholderia</em&gt; Especies

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.

Crecimiento por parejas Ensayo de competición para determinar la aptitud de replicación de virus de inmunodeficiencia 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.

La determinación de supresión del sistema inmunológico frente a la protección del SNC para Intervenciones farmacológicas en la desmielinización autoinmune

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.

La visualización de los Efectos de esputo en el desarrollo del biofilm mediante un modelo de Septadas cubreobjetos

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.

Los protocolos para la investigación de la distribución original de los tejidos, modo de transmisión, y el efecto sobre la aptitud de un anfitrión Densovirus en el gusano de la cápsula del algodón

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.

Alto rendimiento de secuenciación paralelo a la idoneidad de la medida de Leptospira interrogans Transposon inserción mutantes durante oro sirios Hamster infección

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