Me gustaría dar las gracias a CL Richards-Zawacki ayuda conceptualizar el diseño experimental y la asistencia con materiales, A. Pessier ayuda el establecimiento de un régimen de tratamiento y análisis de la necropsia, MWH Chatfield, MJ Robak, K. Tasker y D. Lenger ayuda en el laboratorio y apoyo y M. McFadden y P. Harlow en el Zoológico de Taronga para el uso de su espacio durante el rodaje. El proyecto fue financiado en parte por el Departamento de Vida Silvestre y Pesca de Louisiana a través de la subvención de la Comisión de Educación Ambiental de Luisiana otorgado a LA Brannelly y por Life Technologies.

Aprobación ética: Este estudio y sus métodos fueron aprobadas por Animal uso y cuidado Comité de la Universidad de Tulane (Protocolo n º 0.407.). 1. Las pruebas para detectar la infección por Bd <ol><li> Hisopado para Bd <ol><li> Animales hisopos con un hisopo de algodón estéril en cinco ocasiones en la zona ventral, cinco veces en cada muslo, cada lado, y cada extremidad por un total de 45 golpes. Swabbing es un método no invasivo de muestreo para Bd en la piel de los anfibios. </li><li> Gire suavemente el hisopo durante y entre golpes para asegurar la mayor cantidad de ADN se recoge en el bastoncillo. </li><li> Hisopos tienda en 1,5 ml microtubos a -20 ° C. </li></ol></li><li> Extracción de ADN y el ensayo qPCR <ol><li> Extraer el ADN genómico a partir de los hisopos utilizando un kit de aislamiento de ADN disponibles comercialmente, de acuerdo con las instrucciones para el tejido animal. </li><li> Analizar el ADN extraído utilizando cuantitativa en tiempo real PCR (qPCR) después Boyle <em&gt; et al 17, con las siguientes modificaciones.: </li><li> Diluir las muestras de extracción de ADN a 1:10 con agua doblemente desionizada. </li><li> Añadir 0,7 l de albúmina de suero bovino (BSA) para evitar la inhibición de la PCR. </li><li> Incluir un control positivo interno en cada muestra para asegurar inhibición de la PCR no estaba afectando los resultados. </li><li> Corra las muestras con un control positivo y negativo y una serie de normas de dilución para estimar zoosporas (infección) de carga. </li></ol></li></ol> 2. Inoculación <ol><li> Cultura Bd <ol><li> Preparar caldo de triptona mediante la adición de 10 g de triptona al 1 L de ósmosis inversa o agua desionizada, en autoclave (121 ° C durante 40 min) y se dejó enfriar. Una vez enfriado a 23 ° C, añadir 1 ml de penicilina-estreptomicina. </li><li> Crece la tensión Bd (JEL411 de J. Longcore, aislado de Guabal, Panamá de un Phyllomedusa lemur) en un caldo triptona durante siete días a 23 ° C. </li></ol></li><li> Transfiera la broth cultura de triptona y placas de agar. <ol><li> Para hacer las placas, añadir 10 g de triptona y 10 g de agar a 1 L de ósmosis inversa o agua desionizada y se autoclave (121 ° C durante 40 min). </li><li> Una vez que la mezcla en autoclave se enfríe lo suficiente para tocar, pero antes de que se congela, verter en placas de Petri, 1/4-1/3 completo, en una campana de flujo laminar. </li><li> Cuando el agar se ha solidificado y enfriado completamente, añadir 0,5 ml de caldo Bd a cada placa y se extendió uniformemente alrededor de la placa. Deje que se seque. </li><li> Una vez que las placas se inoculan con zoosporas Bd, placas de sellado con Parafilm. </li><li> Permitir placas se incuban a 23 ° C durante cinco a siete días. Compruebe zoosporas movimiento para asegurar la viabilidad antes de la inoculación. </li></ol></li><li> Placas de inundación para la solución de la inoculación de zoosporas <ol><li> Después de los cinco a siete días de incubación y crecimiento Bd, inundar cada placa con 5 ml de agua del grifo de edad y permitir que las zoosporas entren los medios de comunicación durante 10 minutos. </li><li> Vierta la suspe zoosporasnsion en un recipiente portátil. </li><li> Calcule la concentración de zoosporas con un hemocitómetro. </li><li> Diluir la mezcla a una concentración de 1 x 10 6 por 3 ml con agua del grifo edad. </li></ol></li><li> Se inoculan los animales <ol><li> En individuales 50 ml contenedores, inocular cada animal mediante el vertido de 3 ml de mezcla de inóculo sobre la zona ventral. Permita inóculo adicional gotee en la base del recipiente de la inoculación. </li><li> Cada animal se mantendrá en el recipiente de la inoculación durante 24 horas para asegurar la infección. </li><li> Después de 24 horas de la inoculación, los animales volver a terrarios desinfectados individuales. </li><li> Permitir la infección para construir durante dos semanas. Confirme la infección con hisopo y qPCR resultados, y comenzar el tratamiento. </li><li> Para los controles Bd-negativas, inundaciones Bd-libre de las placas de agar con 5 ml de edades comprendidas agua del grifo, y la inoculación simulada a los animales con los mismos métodos que el anterior. </li></ol></li></ol> 3. Régimen de tratamiento <ol><li> Prepare baño de tratamiento <ol><li> Añadir 2,50 ml itraconazol acuosa (1% solución oral de Sporanox) a 1 Solución de L Anfibios Ringer (0.0025% itraconazol - la concentración de tratamiento eficaz más baja). <ol><li> Anfibio de solución de Ringer receta: 1 L de ósmosis inversa o agua desionizada, 6,6 g de cloruro de sodio, 0,15 g de cloruro de potasio, 0,15 g de cloruro de calcio, y 0,2 g de bicarbonato de sodio. Solución autoclave (121 ° C durante 40 min) y dejar enfriar a temperatura ambiente. </li><li> Por concentración de tratamiento itraconazol 0,01%, añadir itraconazol acuosa 10,00 ml (1% Sporanox Solución oral) a la Solución 1 L Anfibios de Ringer </li><li> Por concentración de tratamiento itraconazol 0,005%, añadir 5,00 ml itraconazol acuosa (1% Sporanox Solución oral) a la Solución 1 L Anfibios de Ringer. </li></ol></li><li> Para los controles Bd-positivos y negativos-Bd, bañar animales en solución de Ringer Anfibios sin itraconazol. </li></ol></li><li> Tratamiento &lt;ol&gt; <li> Coloque anfibios individuales en desinfectados 50 ml de contenedores con 35 ml de la solución de tratamiento. </li><li> Asegurarse de animales están totalmente cubiertos por cinco minutos por mover el recipiente. </li><li> Animales Retorno a terrarios desinfectados. Repita durante seis días. </li></ol></li><li> Asegurar el éxito del tratamiento <ol><li> Animales de ensayo para la infección Bd en el tratamiento de día comience. </li><li> Animales de ensayo para la infección Bd una semana después de acabados de tratamiento, y una vez a la semana durante cuatro semanas para asegurar que la infección haya desaparecido. </li></ol></li></ol> 4. Bd como Biohazard <ol><li> Desinfectar semanal terrarios individual. <ol><li> Bañe los contenedores terrarios y tratamiento en un 10% de lejía baño solución. </li><li> Enjuague cada contenedor blanqueado dos veces con agua corriente. </li><li> Permitir a los contenedores que se seque durante 24 horas antes de la reutilización. </li></ol></li><li> Use guantes de nitrilo limpios al manipular animales para evitar la contaminación cruzadade Bd patógeno. Cambie los guantes entre el manejo de cada animal. </li><li> Desinfecte todos los residuos líquidos antes de su eliminación por lo que la solución al 10% de lejía. </li><li> Autoclave todos los residuos sólidos que se puedan estar expuestos a BD antes de su eliminación. </li></ol>

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El autor declara no los intereses financieros de la competencia.

El uso de este protocolo hemos sido capaces de reducir la dosis de tratamiento y el tiempo de tratamiento itraconazol para Bd en anfibios con seguridad y eficacia. La dosis de tratamiento más bajo y el tiempo (0.0025% por seis días) fue de un tratamiento exitoso para I. infección subclínica nebulifer. La eficacia de administrar una dosis más baja y el tiempo de tratamiento demuestran la necesidad de ensayos clínicos de tratamiento para tratamientos de la infección de Bd, como a...

Los anfibios están actualmente experimentando el mayor declive de la biodiversidad de todos los taxones de vertebrados 1. La quitridiomicosis, una enfermedad de la piel causada por los patógenos fúngicos Batrachochytrium dendrobatidis (Bd chytrid), es una de las principales causas de estos descensos dramáticos 2 Bd es el peor patógeno en el expediente para causar la pérdida de biodiversidad:. Infecta a más de 600 especies de anfibios y tiene causado la disminución de más de 300 especies, de las cuales por lo menos 200 especies se han convertido en grave peligro de extinción o extintas 2,3. El hongo quítrido no es sede específica y no está claro cuáles son las cualidades de la máquina permite la infección Bd progresar causando una enfermedad grave. La quitridiomicosis causa la muerte mediante la interrupción de los canales iónicos en la piel, dando como resultado la pérdida de electrolitos e insuficiencia cardíaca 4. También, Bd. sobrenadante (que no contiene zoosporas Bd) provoca una respuesta inmune en renacuajos 5 leading investigadores creen Bd produce un subproducto tóxico 6. Debido a que muchas especies están disminuyendo rápidamente, las colonias de aseguramiento de la cautividad y los programas de cría son importantes medidas de conservación, y pueden ser la única tabla de salvación para algunas especies. El establecimiento y mantenimiento de la enfermedad colonias libres es un reto para las instalaciones de gestión de anfibios. Los métodos quimioterapéuticos actuales para el tratamiento de infecciones Bd, aunque es eficaz, pueden ser perjudiciales para los animales que son tratados, y ningún método de tratamiento único ha sido un éxito en todas estas especies y clases de edad de anfibios 7. Se han realizado pocos ensayos clínicos de tratamientos para la infección Bd. 7. Los tres métodos más utilizados del tratamiento son la terapia de calor, el cloranfenicol y el itraconazol. La terapia de calor es una opción de tratamiento nonchemotherapeutic que parece tener pocos efectos secundarios, pueden ser utilizados en múltiples etapas de la vida, y es generalmentealiado eficaz. En un ensayo de tratamiento, Chatfield y Richards-Zawacki 8 encontraron que el 96% de los animales tratados se había despejado las infecciones después de ser alojados a 30 ° C durante 10 días. Aunque este ensayo no fue del 100% de éxito, proporciona una línea de base para que otros puedan evaluar la terapia de calor para su propia especie. La terapia de calor, aunque es eficaz para muchas especies, es poco práctico para muchas especies alpinas que no puede permanecer a altas temperaturas durante un período prolongado de tiempo. Cloranfenicol, un antibacteriano, se ha utilizado con éxito para tratar Bd en muchas especies, e inhibe el crecimiento de hongos in vitro. Como un antibacteriano, el cloranfenicol no ataca el hongo específicamente, pero se ha sabido para causar la apoptosis en células eucariotas 9. Se ha especulado que la terapia de cloranfenicol provoca la apoptosis en la piel, causando la muerte de esporangios intracelular, aunque el mecanismo de acción exacto es desconocido. Aunque ningún estudio clínico ha sido realizado, y por lo tantola dosis y el tiempo de tratamiento no han sido evaluados, los protocolos de tratamiento actuales suelen requerir la inmersión de dos a cuatro semanas 10,11, por lo que este tratamiento poco práctico para los anfibios terrestres. El cloranfenicol también ha sido restringido en algunos países para uso veterinario, ya que la exposición humana se ha asociado con una forma rara de anemia aplásica 12. El itraconazol, un antifúngico azol, es el más utilizado para el tratamiento de la infección Bd en anfibios en cautiverio. El protocolo más utilizado es el animal de baño en una solución de itraconazol 0,01% durante cinco minutos al día durante 11 días consecutivos. Este protocolo se deriva de las normas utilizadas para la administración de los mamíferos, y, si bien es efectivo para los anfibios, que no se basa en los ensayos clínicos en los anfibios 11,13. Este protocolo puede causar efectos secundarios negativos en ciertas especies, especialmente en el género Rana y en renacuajos y metamorfos últimos 11.Renacuajo tratamiento ha sido exitoso el uso de una concentración mucho más baja (0,0005%) de itraconazol, pero se asoció con despigmentación 14. Esta misma concentración se probó en metamorfos recientes y se encontró que no tener éxito en el tratamiento de la infección 15. Debido a que existe el riesgo de efectos secundarios negativos con el tratamiento itraconazol, algunos médicos han utilizado la mitad de la concentración habitual (0,005%) durante 11 días consecutivos, y se encontró que esto tenga éxito en la curación de las infecciones Bd 16. El presente estudio es un resumen de un ensayo de tratamiento clínico utilizando itraconazol para tratar la infección Bd. 15. El protocolo describe un intento de encontrar la dosis y tiempo de tratamiento más bajo que es seguro y efectivo para el tratamiento de animales infectados con Bd itraconazol. Los animales utilizados en este estudio fueron los últimos metamorfos de sapos costa del golfo, Incilius nebulifer.

<table border="0" cellpadding="0" cellspacing="0" width="503">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">0.1% Aqueous Itraconazole, Sporonox</td>
			<td style="width:147px;">Janssen Pharmaceutica Inc.</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">S640-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">KCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP366-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CaCl2</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP510-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaHCO3</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP328-500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Sterile cotton swabs</td>
			<td style="width:147px;">Medical and Wire Equipment</td>
			<td style="width:177px;">MW113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">1.5 ml micro tubes</td>
			<td style="width:147px;">Sarstedt</td>
			<td style="width:177px;">73.703.217</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Qiagen DNEasy Blood and Tissue Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:177px;">69581</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">7500 Real-time PCR</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4351104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">VIC IPC</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4308323</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">BSA</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">AM2618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Agar</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">30391023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Tryptone</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">Q10029</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Penicillin-streptomycin</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Petri Dish</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:177px;">CLS430589</td>
		</tr>
	</tbody>
</table>

reduced itraconazole concentration and durations are successful in treating batrachochytrium dendrobatidis infection in amphibians

Los anfibios están experimentando el mayor descenso de toda clase de vertebrados y la principal causa de este descenso es un hongo patógeno Batrachochytrium dendrobatidis (Bd), que causa la enfermedad quitridiomicosis. Colonias de aseguramiento en cautiverio son importantes en todo el mundo para las especies de anfibios amenazadas y pueden ser la única tabla de salvación para los de amenaza crítica de extinción. El mantenimiento de colonias gratuitas de enfermedades es una prioridad de los directivos en cautividad, no se han identificado aún los tratamientos seguros y eficaces para todas las especies y entre las etapas de vida. El tratamiento quimioterapéutico más utilizado es el itraconazol, aunque la dosis utilizada puede ser perjudicial para algunos individuos y especies. Se realizó un ensayo de tratamiento clínico para evaluar si una dosis más baja y más seguro, pero eficaz de itraconazol se pudo encontrar para curar infecciones de BD. Hemos encontrado que mediante la reducción de la concentración de tratamiento desde 0,01 hasta 0,0025% y la reducción de la duración del tratamiento de 11-6 Días de 5 mínimo baños, ranas podrían curarse de la infección Bd con menos efectos secundarios y menos mortalidad asociada al tratamiento.

Tratamiento itraconazol fue considerada exitosa si todos los animales estaban Bd-negativo cuatro semanas después de finalizado el tratamiento. Antes del tratamiento se inició, dos semanas después de la inoculación, los animales inoculados con Bd fueron infectadas, con una carga de zoosporas media de 1,121.7 (± 17,34), mientras que cuatro semanas después de finalizado el tratamiento, los animales de control Bd-positivos tenían una media de carga de zoosporas de 16,765.12 ( ± 14643.08). ...

Watch this Scientific Journal Video about Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians at JoVE.com

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Infección por Batrachochytrium dendrobatidis en los anfibios puede ser eliminado utilizando una concentración reducida (0,0025%) y menor duración (baños de cinco minutos durante seis días) de itraconazol que la que se utiliza generalmente. Se observaron menos mortalidad y menos efectos secundarios negativos utilizando este protocolo de tratamiento modificado.

Disminución de la concentración de itraconazol y Duración del éxito en el tratamiento de<em&gt; Batrachochytrium dendrobatidis</em&gt; La infección en anfibios

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium ...

reduced-itraconazole-concentration-durations-are-successful-treating

Research

JoVE Journal

Immunology and Infection

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

11.5K vistas.  James Cook University.  Infección por Batrachochytrium dendrobatidis en los anfibios puede ser eliminado utilizando una concentración reducida (0,0025%) y menor duración (baños de cinco minutos durante seis días) de itraconazol que la que se utiliza generalmente. Se observaron menos mortalidad y menos efectos secundarios negativos utilizando este protocolo de tratamiento modificado. 

Video: Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualización de virus del dengue a través de Alexa Fluor etiquetado

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Investigación

Inmunología e Infección

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de un medio artificial de esputo para probar la eficacia de antibióticos contra la<em&gt; Pseudomonas aeruginosa</em&gt; En condiciones más pertinentes a la fibrosis quística pulmonar

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia cultivo del Sistema Nervioso Central Neonatal y Adultos

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

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 ...

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.

Aislamiento de neutrófilos y análisis para determinar su papel en el linfoma de células sensibles a los agentes terapéuticos

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 ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Análisis de análogos de ácido iophenoxic en pequeña mangosta india (Herpestes Auropunctatus) Sera para su uso como un marcador biológico de vacunación de rabia oral

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Infección recurrente del tracto urinario por Escherichia coli desencadenada por la exposición de la vejiga de Gardnerella vaginalis en ratones

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Separación de subpoblaciones de células inmunitarias en muestras de sangre periférica de niños con mononucleosis infecciosa

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. 
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

Estandarización de la transferencia a través de laboratorios entre citómetros de flujo para la detección de linfocitos en niños vacunados con encefalitis japonesa

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...