Los autores agradecen al Dr. Le Trinh y el Dr. Tobin generosamente para proporcionar la α-catenina: línea de peces citrino y Bill Jackman para permitirnos hacer el rodaje en su laboratorio. Los autores reconocen las fuentes de financiación Institutos Nacionales de la Salud (Becas 5P20RR016463, 8P20GM103423 y R15AI094406) y USDA (Proyecto # ME0-H-1-00517-13). Este manuscrito se publica como principal Agricultura y Silvicultura Experimento Estación publicación número 3371.

NOTA: Todos los protocolos de atención de pez cebra y los experimentos se realizaron de acuerdo con las directrices del NIH bajo Institucional Cuidado de Animales y el empleo Comisión (IACUC) A2012-11-03 protocolo. 1. El pez cebra Cría a 4 días después de la fecundación <ol><li> Recoger AB pez cebra, o cualquier otro líneas transgénicas, dentro de la primera después de la fertilización 3 hr, como se muestra en otro vídeo 34. </li><li> Incubar 120 huevos en unos 15 cm placas de Petri que contenían 150 ml de medio E3 (NaCl 5 mM KCl; mM 0,17; 0,33 mM CaCl 2; 0,33 mM MgCl 2, 2 mM HEPES, pH 6,8) con azul de metileno (0,3 mg / ml final concentración) en una incubadora a 33 ° con 12/12 de luz / oscuridad fotoperíodo. </li><li> Reemplace los medios de comunicación después de 6 horas con el E3 + PTU (1-fenil-2-tiourea, / ml concentración final de 10 mg) y retirar los huevos muertos. </li><li> Incubar durante 4 días a 33 ° C, el intercambio de los medios de comunicación con frescos E3 + PTU después de 2 días. </li></ol> 2. Enproyección de micro-aguja Preparación <ol><li> Tire capilar de borosilicato (OD 1,2 mm; ID 0,69 mm) en un micro-aguja usando un extractor de aguja con los siguientes ajustes (550 Heat; 0 Tire; 130 Velocity; 110 Time). NOTA: Los ajustes variarán de filamento a filamento para obtener micro-agujas de forma similar (ver Figura 1) y tendrá que ser reajustado cada vez que se instala un nuevo filamento. </li><li> Llenar un micro-aguja con 3 l de 0,4 micras filtrada-solución salina tamponada con fosfato (PBS; NaCl 137 mM; KCl 2,7 mM; 10 mM Na 2 HPO 4; 1.8 mM KH 2 PO 4; pH 7,4) usando un microloader pipeta 20 l punta. </li><li> Configure la micro-aguja en un soporte micropipeta en un micromanipulador conectada a un sistema de inyección a presión. Ajuste la presión de inyección a 30 PSI y la duración del pulso en 30 ms. </li><li> El uso de una placa de Petri con agua destilada, baje la micro-aguja hasta 1/5 de la aguja está en el agua. Sujete el wi micro-agujath pinzas finas justo por debajo del nivel del agua. Traiga la micro-aguja fuera del agua y empujar el pedal varias veces hasta que aparezca un bolo. </li><li> Medir el diámetro del bolo mediante la reducción de la micro-aguja en un hemocitómetro en capas con una gota de aceite de inmersión tipo A. Ajustar la duración del impulso para obtener el volumen deseado (ver Tabla 1). </li><li> Establecer la contrapresión para el bolo se mantenga estable en aceite (bajar la presión si el bolo aumenta el volumen y aumentar la contrapresión si el volumen del bolo disminuye). Registre la duración del pulso para cada micro-aguja. </li><li> Retire la PBS de la micro-aguja aspirando usando una punta de pipeta microloader y expulsar el sobrante PBS colocándolo de nuevo en el inyector y presionar el interruptor de pulso continuo. Reserve cada micro-aguja. </li></ol> 3. Candida Preparación <ol><li> Streak Candida albicans transforman con codones optimizados dTomato, GFP, BFP, EOS o RemotoRojo de una madre congelado (-80 ° C de valores en 25% de glicerol) usando una clavija de madera estéril en un peptine dextrosa de levadura (YPD) placa de agar (10 g de extracto / L de levadura, 20 g / L de peptona, 20 g / l de dextrosa , agar / L 20 g; autoclave y se vierte 25 ml en placas de Petri) y se incuba durante la noche a 30 ° C. </li><li> Elige una colonia usando una clavija de madera estéril e inocular a 5 ml de líquido YPD (10 g de extracto / L de levadura, 20 g / L de peptona, 20 g / l de dextrosa, autoclave y asépticamente alícuotas en 5 ml en cultivo de vidrio de 16 x 150 mm tubos). </li><li> Incubar durante la noche a 30 ° C en un tambor de rodillo a 60 rpm. </li><li> Recoge 1 ml de C. cultura albicans y la transferencia en un tubo de centrífuga estéril 1,7 ml. </li><li> Centrifugar a 5000 xg durante un par de segundos. Vacíe el sobrenadante y añadir 1 ml de PBS estéril, mezclar bien. Repita esto dos veces. </li><li> Contar las colonias utilizando un hemocitómetro diluyendo el 1 ml agotadas 1: 1000 en PBS estéril. </li></ol> 4. La inyección de Pez Cebra enla vejiga natatoria <ol><li> Calentar un plato de inyección (2% de agarosa) durante 30 min en una incubadora a 33 °. </li><li> En el momento deseado, retirar 100 de los 150 ml de E3 + PTU desde el plato de 15 cm de Petri que contiene el pescado para inyectar usando una pipeta de 25 ml sin aspiración de cualquier pez cebra. </li><li> Anestesie 4 dpf pez cebra mediante la adición de 2 ml de un / ml tamponada tricaína metano sulfonato de stock 4 mg (TR) a los 50 ml restantes medios (concentración final de 200 mg / ml) y esperar 15 min. </li><li> Bajo un microscopio de disección, seleccionar peces con una vejiga natatoria inflada. Los peces también se puede examinar en ese momento para fenotipo homogéneo, tales como la distribución de los neutrófilos en mpo: línea de GFP usando un microscopio de disección de epifluorescencia. </li><li> Diluir la C. Stock albicans a partir de su concentración original (paso 3.8) a 1,5 x 10 7 unidades formadoras de colonias por ml (UFC / ml) se forman en PBS. </li><li> Transfiera 30 pez cebra en el plato de la inyección con un tubo de transferencia de plásticotte y eliminar el exceso de medios de comunicación. </li><li> Con una herramienta de hilos de pesca (línea de pesca de diámetro 0,012 pulgadas súper pegada en un capilar de borosilicato), haga 3 líneas de 10 peces, sosteniendo el plato de inyección vertical y orientar a los peces verticalmente. </li><li> Retire el exceso de medios de comunicación. </li><li> Agite bien en vórtex el tubo con 1,5 x 10 7 ufc / ml C. albicans. </li><li> Llene una micro-aguja con 3 l de C. albicans utilizando una nueva punta de pipeta microloader y ajustar la duración del pulso en el valor adecuado (véase el paso 2.9). </li><li> Gire el plato de inyección para formar un ángulo de 20 ° entre la micro-aguja y la cabeza del pez, apuntando hacia la parte posterior de la vejiga natatoria (Figura 3A). </li><li> Empuje la micro-aguja en la vejiga natatoria, asegurándose de que el orificio de la aguja está en la luz, y presione el pedal una vez. </li><li> Repita el procedimiento para cada pez y transferir a un plato de recuperación (25 ml E3 + PTU) inundando el plato con un E3d vaciarlo en el plato de recuperación. </li><li> Transferencia otros 30 peces para el plato de la inyección. </li><li> Repetir la inyección con un nuevo micro-aguja llena de fondo vórtex C. albicans. </li><li> Finalizar inyectando el control de PBS, si es necesario, utilizando un plato de inyección diferente para evitar la contaminación y la transferencia a un plato de recuperación independiente (paso 4,10). </li></ol> 5. El cribado del Pescado para inóculo deseado <ol><li> Preparar 40 ml de 0,4% bajo punto de fusión solución de agarosa (LMA) mediante la adición de 160 mg de bajo punto de fusión de agarosa a 40 ml de E3, hervir en el microondas hasta que se disuelva, y enfriamiento a 37 ° C. Añadir 400 l de TR (concentración final de 200 mg / ml) una vez enfriado. </li><li> Después de 30 min de recuperación, anestesiar el pescado tal como se describe (paso 4.3 utilizando sólo 1 ml de TR en 25 ml E3 + PTU, concentración final 200 g / ml). </li><li> Transferencia de 30 peces (con tan poco como sea posible los medios de comunicación) a 2 ml de LMA en un barco de peso. </li><li> Aspirar peces individuales con una pipeta de transferencia y la placa a los peces con 1 gotas de LMA en una placa de imagen de 96 pocillos, utilizando sólo los 10 x 6 pozos centrales. </li><li> Repita hasta que todos los peces a la pantalla se cargan a la placa de imagen. </li><li> Deje que los medios de comunicación se solidifican a temperatura ambiente durante 5 min. </li><li> Coloque el pescado en su lado, asegurándose de que estén en contacto con el fondo de cristal. </li><li> Usando el objetivo de 20X en una epifluorescencia / microscopio confocal invertido y el filtro adecuado, registrar el número de C. células de levadura albicans en vejiga natatoria de cada pez. </li><li> Seleccione el pescado con 15-25 células de levadura en la vejiga natatoria (o inóculo se desea), la eutanasia de peces no seleccionado (800 g / ml de TR en E3) y volver peces seleccionado a un plato profundo de Petri con 50 ml de E3 + PTU por la adición de 3 gotas de E3 a los pocillos de placas de imágenes seleccionados y aspirar el pescado con una pipeta de transferencia de plástico. </li><li> Incubar a 33 ° C durante la duración deseada. </li></ol> 6. Imaging the Fish Post-screening <ol><li> En el momento deseado, retirar 25 de los 50 ml de medio E3 + PTU de la placa de Petri profunda que contiene el pescado a la imagen. </li><li> Anestesiar el pescado tal como se describe (paso 4.3 utilizando sólo 1 ml de TR en 25 ml E3 + PTU restante, concentración final de 200 mg / ml). </li><li> Placa de los peces en una placa de imagen de 96 pocillos y la posición apropiada (pasos 5.3 a 5.7). </li><li> Imagen de microscopía de barrido confocal utilizando los láseres apropiados y filtros de emisión. En primer lugar se centran en los peces usando el objetivo 4X bajo contraste de interferencia diferencial (DIC) y adquirir imágenes de la región de interés utilizando el objetivo 20X con el láser confocal en el modo de exploración con 2 velocidades microsegundos / pixel y 1024 x 600 píxeles relación de aspecto. Adquirir Z-pilas con 1 m de tamaño de paso, aplicando el modo de filtro Kalman de 3 marcos. </li><li> Regreso a un plato hondo de Petri con 50 ml E3 + PTU o en individual pozos (24 pocillos plato cultura, 3 ml E3 + PTU) y se incuba a 33 ° C. </li></ol> 7. Disección de la vejiga natatoria <ol><li> Llene una jeringa de 10 ml con grasa de alto vacío. </li><li> Preparar una LMA 2% (paso 5.3 con 160 mg de bajo punto de fusión de agarosa en 8 ml de E3). </li><li> En el momento deseado, anestesiar el pescado (paso 5.2). </li><li> Transferencia de 10 peces a un tubo de 1,7 ml de centrífuga con 1 ml de E3 y la eutanasia mediante la adición de 200 l de 4 mg / ml tricaína (concentración final de 800 mg / ml) al tubo de centrífuga e incubando durante 15 min a temperatura ambiente. </li><li> Preparar un portaobjetos de vidrio de formación de imágenes mediante la colocación de 4 gotas de grasa de vacío en una plaza, 1 cm de distancia en una MicroSlide (75 x 25 mm, Figura 2). </li><li> Coloque 2 gotas de 2% LMA en el centro de la plaza. </li><li> Coloque un pescado en un portaobjetos de vidrio separada bajo un microscopio de disección y verifique que el corazón ha dejado de latir, eliminar el exceso de agua. </li><li&gt; Con ayuda de pinzas finas diseccionar los vejiga natatoria por mantener la cabeza en una posición fija con las pinzas izquierda y tirando hacia abajo el intestino anterior con las pinzas derecha (Figura 4A). </li><li> Recoge la vejiga natatoria por el conducto neumático con las pinzas izquierda, que lo separa del tracto digestivo, y colocarlo en el centro de la LMA 2% antes de que solidifique. </li><li> Colocar un cubre (18 x 18 mm) en la parte superior de la lámina de vidrio de imágenes y empujarlo hasta que toque la grasa de vacío, así como la LMA (Figura 2). </li><li> Imagen dentro de los 10 min de la disección como se describe en el paso 6.4. Repita el paso 07.07 a 07.11 con los otros peces. </li></ol>

The authors have nothing to disclose.

Avances y limitaciones del modelo de enfermedad microinyección vejiga natatoria El modelo que se presenta aquí es una extensión del modelo de inmersión candidiasis mucosa se ​​describe en Gratacap et al (2013).; que añade las ventajas de un tiempo de infección controlada, una dosis infección altamente reproducible, y por lo tanto mejora de la eficiencia. Se demuestra aquí nuevos métodos que permitan la documentación temporal no invasiva de la dinámica de la infección en...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name</td>
			<td style="width:259px;">Company</td>
			<td style="width:176px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL tubes</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deep Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>89107-632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>VWR Scientific</td>
			<td>90000-726</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>VWR Scientific</td>
			<td>90000-264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>VWR Scientific</td>
			<td>90000-760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine tweezers (Dumont Dumoxel #5)</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wooden Dowels</td>
			<td>VWR Scientific</td>
			<td>10805-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Melt Agarose</td>
			<td>VWR Scientific</td>
			<td>12001-722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming Brown Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate capillary</td>
			<td>Sutter Instruments</td>
			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPPI-3 Injection system</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPPI-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Back Pressure Unit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>BPU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Holder kit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPIP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foot Switch</td>
			<td>Applied Scientific Instrumentation</td>
			<td>FSW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic Base</td>
			<td>Applied Scientific Instrumentation</td>
			<td>Magnetic Base</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine methane sulfonate</td>
			<td>Western Chemical Inc.</td>
			<td>MS-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scope</td>
			<td>Olympus</td>
			<td colspan="2">SZ61 top SZX-ILLB2-100 base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Olympus</td>
			<td colspan="2">IX-81 with FV-1000 laser scanning confocal system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20x microscope objective</td>
			<td>Olympus</td>
			<td>UPlanSApo 20x/0.75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roller drum</td>
			<td>New Brunswick Scientific</td>
			<td>TC-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass culture tubes (16 x 150 mm)</td>
			<td>VWR Scientific</td>
			<td>60825-435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>VWR Scientific</td>
			<td>BDH4534-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>VWR Scientific</td>
			<td>BDH4532-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>VWR Scientific</td>
			<td>BDH0246-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (Corning)</td>
			<td>VWR Scientific</td>
			<td>BDH4520-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Children clay (Play-Doh)</td>
			<td>Hasbro</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td>VWR Scientific</td>
			<td>VW6276-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTU</td>
			<td>Sigma</td>
			<td>P7629-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer (Hausser scientific)</td>
			<td>VWR Scientific</td>
			<td>15170-172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type A immersion oil</td>
			<td>Blue Marble Products</td>
			<td>51935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genie</td>
			<td>VWR Scientific</td>
			<td>14216-184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose (Lonza)</td>
			<td>VWR Scientific</td>
			<td>12001-870</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fishing wire</td>
			<td>Stren</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well imaging plate (Sensoplate)</td>
			<td>Greiner Bio-One</td>
			<td>655892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease (Dow Corning)</td>
			<td>VWR Scientific</td>
			<td>59344-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microslide (25 x 75 mm)</td>
			<td>VWR Scientific</td>
			<td>48300-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips (18 x 18 mm), No 1.5</td>
			<td>VWR Scientific</td>
			<td>48366-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 cm Petri dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>32-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol (EMD chemicals)</td>
			<td>VWR Scientific</td>
			<td>EMGX0185-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well culture dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weight boats (8.9 cm)</td>
			<td>VWR Scientific</td>
			<td>89106-766</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling mucosal candidiasis in larval zebrafish by swimbladder injection

Defensa temprana contra los agentes patógenos de la mucosa consiste tanto en una barrera epitelial y células inmunes innatas. La inmunocompetencia de ambos, y su intercomunicación, son de suma importancia para la protección contra las infecciones. Las interacciones de las células inmunes innatas epiteliales y con un patógeno están mejor investigados en vivo, donde el comportamiento complejo se desarrolla en el tiempo y el espacio. Sin embargo, los modelos existentes no permiten una fácil imágenes espacio-temporal de la batalla con los patógenos a nivel de la mucosa. El modelo desarrollado aquí crea una infección de las mucosas por inyección directa del patógeno fúngico, Candida albicans, en la vejiga natatoria del pez cebra juvenil. La infección resultante permite imágenes de alta resolución de comportamiento de la célula epitelial y inmune innata en todo el desarrollo de la enfermedad de la mucosa. La versatilidad de este método permite la interrogación del huésped a la sonda la secuencia detallada de los eventos inmunológicos que conducen a phreclutamiento agocyte y examinar el papel de los tipos particulares de células y vías moleculares en la protección. Además, el comportamiento del patógeno como una función de ataque inmune se pueden obtener imágenes de forma simultánea mediante el uso de la proteína fluorescente que expresan C. albicans. El aumento de la resolución espacial de la interacción huésped-patógeno también es posible usando la técnica de disección vejiga natatoria rápido descrito. El modelo de infección de la mucosa se describe aquí es sencillo y altamente reproducible, por lo que es una valiosa herramienta para el estudio de la candidiasis mucosa. Este sistema también puede ser ampliamente traducible a otros patógenos de la mucosa tales como microbios micobacterianas, bacterianas o virales que normalmente infectan a través de las superficies epiteliales.

La microinyección en la vejiga natatoria posterior El método experimental presentado aquí describe la inyección de una dosis constante de C. células de levadura albicans en la vejiga natatoria de 4 dpf pez cebra. El trabajo previo con el modelo de inmersión sugiere que la respuesta inmune a la vejiga natatoria C. albicans es similar a la candidiasis mucosa de mamíferos 32. Aquí se demuestra un método de infección modificado que es más sencillo, r...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Modelado de las mucosas Candidiasis en larvas de pez cebra por inyección de Swimbladder

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

Research

JoVE Journal

Immunology and Infection

10.1K vistas.  University of Maine. In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Video: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

La imagen no invasiva de la candidiasis diseminada en larvas de pez cebra

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Investigación

Inmunología e Infección

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Ensayo de cocultivo para evaluar la migración transepitelial de neutrófilos inducida por patógenos

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

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 ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Muestreo no invasivo de revestimiento mucoso líquido para la cuantificación In Vivo de la vía aérea superior mediador inmune niveles

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Formación de biopelículas inducida por sales biliares en patógenos entéricos: técnicas para la identificación y cuantificación

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modelado de la Tuberculosis de Mycobacterium marinum infección pez cebra adulto

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

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

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

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

El modelo de infección de Waxworm Galleria mellonella para la Candidiasis diseminada

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

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Inducción de lesión pulmonar de ratón por inyección endotraqueal de bleomicina

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Dirigirse a los medicamentos a los macrófagos de peces cebra sellos larvarios mediante la inyección de liposomas con drogas

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Aislamiento de células mononucleares tonitarias para estudiar las respuestas inmunitarias innatas ex vivo en un tejido linfático mucoso humano

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...