VK was funded by grants from the IUP Department of Biology and School of Graduate Studies and Research (Graduate Student Professional Development). CQD and TWS were supported by the IUP School of Graduate Studies and Research (Faculty Publication Costs/Incidental Research Expenses). We also thank members of the Diep laboratory for maintaining the zebrafish facility.

Los procedimientos descritos en este protocolo fue aprobado por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Indiana de Pennsylvania. 1. Los embriones Cosecha <ol><li> Mantener el pez cebra a 28,5 ° C, pH 7, conductividad entre 500-1.500 mS, y un ciclo luz / oscuridad de 14 horas de luz y 10 horas de oscuridad 24. Utilizar cepas de tipo silvestre, como tu, AB o híbrido TU / AB. Las diferentes cepas pueden responder de manera diferente al tratamiento químico 25. </li><li> Configurar el pescado para el apareamiento la noche antes de la recolección de huevos mediante la adición de agua del sistema de peces en un tanque de apareamiento. Añada peces machos y hembras en el tanque y separar los dos peces con un divisor. Cada par de pescado producirá una gama de 50-300 huevos. Para asegurarse de que se produzca una suficientes huevos, configurar 30 pares de peces. Por lo general, alrededor del 50% de los pares de pescado a la edad de apareamiento primer (6-9 meses de edad) va a producir huevos, lo que resulta hasta 200 huevos por pares y un máximo de hasta 3, 000 huevos para este experimento. </li><li> A la mañana siguiente, después de la luz se enciende, retire el divisor para iniciar el apareamiento. Compruebe los tanques de apareamiento para los huevos cada 15 minutos. </li><li> Una vez que los peces ponen huevos, cosechar todos los embriones utilizando un colador de té y combinarlos en un gran contenedor con tampón E3 (NaCl 5 mM, KCl 0,17 mM, 0,33 mM CaCl2, 0,33 mM de MgSO4). A 1,5 HPF, retirar y desechar los huevos no fertilizados con una pipeta de plástico bajo un microscopio de disección. Huevos no fecundados son opacos mientras que los huevos fertilizados son transparentes 34. </li><li> Transferencia de 50 embriones en una placa de 100 x 15 mm de Petri de vidrio que contiene 50 ml de tampón de E3 para cada condición de tratamiento. Se necesita un total de 33 platos para 11 condiciones de tratamiento y 3 repeticiones. </li></ol> 2. El tratamiento de los embriones <ol><li> Realizar los tratamientos en 2 horas después de la fecundación (HPF) 35. Incubar los embriones / larvas a 28,5 ° C y examinar las larvas a 120 HPF. Realizar por lo least tres réplicas de cada condición de tratamiento para el análisis estadístico. </li><li> Para 3 placas de Petri (cada uno con 50 embriones) en 2 hpf, eliminar el tampón E3 con una pipeta de transferencia y añadir 50 ml de etanol 300 mM diluido en tampón de E3. Cubrir las placas de Petri con Parafilm para minimizar la evaporación de etanol. <ol><li> Continuar la exposición de los embriones al etanol durante 22 horas. A continuación, eliminar el etanol con una pipeta de transferencia. Añadir 50 ml de tampón E3 y agitar el plato varias veces para lavar el etanol. Eliminar este tampón E3 con una pipeta de transferencia y repetir la etapa de lavado 2 veces más. </li></ol></li><li> Por 3 placas de Petri (cada uno con 50 embriones) en 2 HPF, eliminar el tampón E3 con una pipeta de transferencia y añadir 50 ml de tampón nuevo E3. </li><li> Para 9 placas de Petri (cada uno con 50 embriones) en 2 hpf, eliminar el tampón E3 con una pipeta de transferencia y añadir 50 ml de nitrito de sodio 1,000 mg / L disuelto en tampón E3. Confirmar la concentración de la solución madre de nitrito de antemano utilizando unamodificación de la diazotación (método USEPA 354.1) 28 método espectrofotométrico. <ol><li> Continuará exponiendo 3 platos por 46 horas, 3 platos por 70 horas y 3 platos para 94 hr. Vuelva a colocar la solución de nitrito con una solución de nitrito de recién hecho todos los días. </li><li> Después de cada tiempo de exposición, retire el nitrito con una pipeta de transferencia. Añadir 50 ml de tampón E3 y agitar el plato varias veces para lavar el nitrito. Eliminar este tampón E3 con una pipeta de transferencia y repetir la etapa de lavado 2 veces más. </li></ol></li><li> Para 3 placas de Petri (cada uno con 50 embriones) en 2 hpf, eliminar el tampón E3 con una pipeta de transferencia y añadir 50 ml de nitrito de sodio 200 mg / L. Repetir esto con 400, 600, 800, y 1000 mg / L de nitrito de sodio. <ol><li> Continuar la exposición de los embriones a nitrito por 70 horas. Vuelva a colocar la solución de nitrito con una solución de nitrito de recién hecho todos los días. </li><li> Después del tiempo de exposición, retire el nitrito con una pipeta de transferencia. Añadir 50 ml de tampón E3 y agitar el plato variasveces para lavar el nitrito. Eliminar este tampón E3 con una pipeta de transferencia y repetir la etapa de lavado 2 veces más. </li></ol></li><li> Para 3 placas de Petri (cada uno con 50 embriones) en 2 hpf, eliminar el tampón E3 con una pipeta de transferencia y añadir 50 ml de nitrato de sodio 1,000 mg / L disuelto en tampón E3. Confirmar la concentración de la solución madre de nitrato de antemano utilizando una modificación de la reducción de cadmio (USEPA Método 353.3) método espectrofotométrico 28. <ol><li> Continuar la exposición de los embriones de nitrato de 70 hr. Vuelva a colocar la solución de nitrato de solución de nitrato de recién hecho todos los días. </li><li> Después del tiempo de exposición, retire el nitrato con una pipeta de transferencia. Añadir 50 ml de tampón E3 y agitar el plato varias veces para lavar el nitrato. Eliminar este tampón E3 con una pipeta de transferencia y repetir la etapa de lavado 2 veces más. </li></ol></li><li> Durante cada día de la exposición, contar el número de embriones / larvas muertas usando un microscopio estereoscópico. Los signos de la muerteincluir la falta de ritmo cardíaco y la circulación de la sangre, o la falta de motilidad después de 1 minuto de observación. Retire embriones / larvas muertas con una pipeta de transferencia para reducir la contaminación del tampón E3. </li><li> Cuando terminan los experimentos a 120 HPF, la eutanasia a las larvas. <ol><li> Eliminar el tampón E3 con una pipeta de transferencia. A continuación, añadir 50 ml de 0,2% de MS-222 (tamponada a pH 7) y esperar 10 minutos. </li><li> Retire el MS-222 con una pipeta de transferencia. Añadir 50 ml de tampón E3 y agitar para lavar el MS-222. </li><li> Eliminar el tampón de E3 con una pipeta de transferencia y añadir 20 ml de 4% de paraformaldehído (PFA) para fijar las larvas. Hacer remolinos en el plato varias veces. Usar una pipeta de transferencia para transferir las larvas en un vial de vidrio a lo largo con suficiente PFA para llenar el vial. Almacenar los viales en el refrigerador durante la noche (O / N). </li></ol></li><li> Tome fotos de larvas fija utilizando un estereoscopio con una cámara digital. Utilice un aumento de 30x, ISO 200, y 200 ms tiempo de exposición. Orientar las larvas de manera que el anterior a la left y el dorsal es a la parte superior del campo. </li></ol>

<ol>
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The authors have nothing to disclose.

El método aquí descrito demuestra la utilidad de pez cebra para evaluar el potencial teratogénico de nitrito y nitrato. En comparación con otros vertebrados, el pez cebra tiene ventajas que incluyen alta fecundidad, la fertilización externa, transparencia óptica, y un rápido desarrollo. Mutantes disponibles que carecen de pigmentación (por ejemplo, el pez cebra Casper 36) también ayudan a mejorar la visibilidad de los órganos internos. También es fácil de generar pez cebra transgénico con genes i...

Teratogénesis es un proceso que altera el desarrollo normal de un embrión o feto, causando anomalías estructurales y funcionales permanentes, retraso del crecimiento, o aborto involuntario en casos graves 1. Puede ser causada por ciertos agentes naturales (teratógenos), que interfieren con el desarrollo embrionario de múltiples maneras 2. Durante el desarrollo fetal humano, se han reportado teratógenos común, tales como la radiación, agentes infecciosos, metales tóxicos y productos químicos orgánicos para causar defectos en los pliegues epicánticos (el pliegue de piel en el párpado superior) y clinodactilia (dedo curvado o en el pie) a través de los errores morfogenéticos 1. La comprensión del mecanismo molecular de teratogénesis es el primer paso hacia el desarrollo de tratamiento y prevención. Varios modelos de vertebrados tales como la rana de uñas africana (Xenopus laevis) y el pez cebra (Danio rerio) se han utilizado para determinar las vías moleculares afectadas por teratOgens. Estudios anteriores han utilizado el pez cebra como modelo para la epidemiología, toxicología y teratogénesis 3-7. Scholz et al. considerado el pez cebra como un &quot;estándar de oro&quot; para la evaluación de la toxicidad ambiental. Esto se debe, en parte, a la transparencia del embrión de pez cebra, que permite a los investigadores visualizar el defecto de desarrollo, ya que se produce 8. Aproximadamente el 70% de los genes humanos tienen ortólogos en el pez cebra, por lo que el pez cebra un modelo vertebrado deseable para el estudio de los defectos humanos 9. Algunos estudios epidemiológicos han sugerido que el nitrato y el nitrito, comúnmente presentes en los alimentos agrícolas y agua, están asociados con defectos de nacimiento y abortos espontáneos 10,11, mientras que otros estudios no apoyan esta asociación 12. El nitrato (NO 3 -) y nitrito (NO 2 -) están presentes de forma natural en el suelo y el agua. Ellos son una fuente de nitrógeno para las plantas y son una parte de la nciclo itrogen 13. Los alimentos como las judías verdes, zanahorias, calabaza, espinaca, remolacha y de las granjas que utilizan fertilizantes con alto contenido de nitrato han aumentado significativamente los niveles de nitrato y nitrito 7. La leche de vacas alimentadas con alimentos altos de nitratos y peces en agua de alta nitrato (principalmente de la salida del suelo 30) puede conducir a los seres humanos que consumen grandes cantidades de nitratos y nitritos 14. El nitrato y el nitrito también se utilizan comúnmente en la conservación de alimentos, lo que aumenta drásticamente la cantidad ingerida por los seres humanos 12. Los niveles óptimos de nitrato y nitrito desempeñan papeles fundamentales en los procesos fisiológicos como la homeostasis vascular y la función, la neurotransmisión y los mecanismos de defensa del huésped inmunológicos 13-15. Sin embargo, la exposición a altos niveles de nitrato y nitrito puede dar lugar a efectos adversos, especialmente en lactantes y niños de 16. nitrato ingerido se convierte después en nitrito en la cavidad oral por la microflora y en THe tracto gastrointestinal por la microflora intestinal 17. El nitrato pone bebés en un alto riesgo para el síndrome del bebé azul por la oxidación de la hemoglobina en metahemoglobina, deteriorando la hemoglobina de su capacidad de oxígeno 18 que lleva. Esto da como resultado el color azul de la piel que se extiende a los tejidos periféricos en los casos más graves. La oxigenación de los tejidos inhibido resultados en otros síntomas, más severa que conduce a coma y muerte 19,20. Síntomas similares se observan en los bebés y adultos en concentraciones más altas de nitrato de 21. Los niveles elevados de metahemoglobina en los adultos debido a los resultados de intoxicación de nitritos en la cianosis, dolor de cabeza, trastornos de la respiración 31, y la muerte si no se trata, debido a complicaciones relacionadas con la hipoxia tejido vital 32,33. El nitrato ingerido en los niveles superiores también puede resultar en diversas complicaciones de salud. La diabetes infantil, la diarrea recurrente, e infecciones recurrentes del tracto respiratorioen los niños se han relacionado con una alta ingesta de nitrato de 11,17,22. La exposición crónica a un alto nivel de nitrato se asocia con la micción y la hemorragia del bazo. La exposición a altas dosis aguda de nitratos puede conducir a una amplia gama de condiciones médicas como dolor abdominal, debilidad muscular, sangre en las heces y la orina, desmayos, y la muerte 11. La exposición prenatal a nitrato en niveles altos se ha asociado con defectos del tubo neural y músculo-esquelético 11. Un informe reciente mostró que el tratamiento con nitrito de embriones de pez cebra condujo a la yema edema salida, malformaciones craneofaciales y axial, y nadar noninflation vejiga 5. En este estudio, se demuestra un método para el tratamiento de embriones de pez cebra con nitrato y nitrito para determinar su potencial teratogénico. Los embriones fueron expuestos a nitrito a diferentes concentraciones y diferentes periodos de tiempo. El etanol se utiliza como un control positivo, ya que es un teratógeno establecido 23. Our método mostró que ambas altas concentraciones y tiempos de exposición largos a nitrito eran perjudiciales para la supervivencia y dieron lugar a diversos fenotipos, que van desde leve (edema) a graves defectos de desarrollo (brutos). Por lo tanto, el pez cebra es un modelo útil para explorar directamente los posibles efectos teratogénicos de nitrato y nitrito en embriones para complementar estudios epidemiológicos.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">DREL/2010 instrument</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">26700-03</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Ethanol</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">KIMAX glass Petri Dish</td>
			<td style="width:185px;">VWR</td>
			<td style="width:104px;">89001-244</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">MS-222</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E10521</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">NitraVer 5 Nitrate Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14034-46</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">NitriVer 3 Nitrite Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14065-99</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Parafilm</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">3-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Paraformaldehyde</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">S6E stereomicroscope</td>
			<td style="width:185px;">Leica</td>
			<td style="width:104px;">10446294</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Sodium nitrate</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S343</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Sodium nitrite</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S347</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">Transfer pipets</td>
			<td style="width:185px;">Laboratory Products Sales</td>
			<td style="width:104px;">L320072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Glass vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">03-338B</td>
			<td></td>
		</tr>
	</tbody>
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zebrafish as a model to assess the teratogenic potential of nitrite

Los altos niveles de nitrato en el medio ambiente pueden causar defectos congénitos o abortos involuntarios en los seres humanos. Presumiblemente, esto es debido a la conversión de nitrato a nitrito por las bacterias intestinales y salivales. Sin embargo, en otros estudios de mamíferos, los altos niveles de nitrito no causan defectos de nacimiento, aunque pueden conducir a resultados reproductivos pobres. Por lo tanto, el potencial teratogénico de nitrito no está claro. Sería útil disponer de un sistema modelo de vertebrados para evaluar fácilmente efectos teratogénicos de nitrito o cualquier otro producto químico de interés. Aquí, se demuestra la utilidad de pez cebra (Danio rerio) para detectar compuestos de toxicidad y defectos embrionarios. embriones de pez cebra son fertilizados externamente y tienen un desarrollo rápido, por lo que un buen modelo para los estudios teratogénicos. Se demuestra que el aumento del tiempo de exposición a nitrito afecta negativamente a la supervivencia. El aumento de la concentración de nitrito también afecta negativamente a la supervivencia, mientras que el nitrato no lo hace. Para tha embrionest sobrevivir a la exposición nitrito, varios defectos pueden ocurrir, incluyendo pericárdico y la yema de un edema salida, nadar noninflation vejiga, y la malformación craneofacial. Nuestros resultados indican que el pez cebra es un sistema conveniente para estudiar el potencial teratogénico de nitrito. Este enfoque puede ser fácilmente adaptado para probar otros productos químicos por sus efectos sobre el desarrollo de vertebrados temprano.

La exposición a etanol 300 mM durante 22 h no tenía ningún efecto sobre la supervivencia (datos no mostrados), consistente con informes anteriores 5,23,26. Esto se esperaba, ya que el etanol es un teratógeno conocido y sirvió como un control positivo. Fenotipos observados se incluyen edema pericárdico, noninflation vejiga natatoria (Figura 1), defectos craneofaciales, y retraso en el desarrollo (datos no mostrados). <p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite at JoVE.com

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

La exposición a teratógenos puede causar defectos de nacimiento. El pez cebra son útiles para determinar el potencial teratogénico de productos químicos. Se demuestra la utilidad de pez cebra mediante la exposición de los embriones a diferentes niveles de nitrito y también en diferentes momentos de exposición. Se demuestra que el nitrito puede ser tóxico y causar graves defectos de desarrollo.

El pez cebra como modelo para evaluar el potencial teratogénico de nitrito

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

zebrafish-as-a-model-to-assess-the-teratogenic-potential-of-nitrite

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9.4K vistas.  Indiana University of Pennsylvania.  La exposición a teratógenos puede causar defectos de nacimiento. El pez cebra son útiles para determinar el potencial teratogénico de productos químicos. Se demuestra la utilidad de pez cebra mediante la exposición de los embriones a diferentes niveles de nitrito y también en diferentes momentos de exposición. Se demuestra que el nitrito puede ser tóxico y causar graves defectos de desarrollo. 

Video: Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Investigación

Inmunología e Infección

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Uso del ratón humanizado BLT como un modelo de células madre del tumor Terapia génica basada en

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

El insecto<em&gt; Galleria mellonella</em&gt; Como un modelo de infección poderosa para investigar la patogénesis bacteriana

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Cuantificación de la respuesta estallido respiratorio como un indicador de la salud inmune innata en el pez cebra

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

El uso de<em&gt; Galleria mellonella</em&gt; Como organismo modelo para estudiar<em&gt; Legionella pneumophila</em&gt; Infección

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

La ligadura y punción cecal inducida por sepsis como un modelo para estudiar la autofagia en ratones

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imagen no invasiva de la respuesta inmune innata en un modelo de larvas de pez cebra de<em&gt; Streptococcus iniae</em&gt; Infección

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Retroviral transducción de células T colaboradoras como un enfoque genético para estudiar mecanismos que controlan su diferenciación y función

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Sobreexpresión y Purificación de Humanos<em&gt; Cis</em&gt; -preniltransferasa en<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

La cucaracha que silba como un modelo alternativo de animales no mamíferos para investigar la eficacia de la droga, virulencia y patogenia de Madagascar

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

El pez cebra como modelo para evaluar el potencial teratogénico de nitrito

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