Este proyecto fue financiado por los fondos de la Case Western Reserve University de inicio prevista para E. Jolly. Caracoles infectados fueron proporcionados por el Centro de Recursos esquistosoma, NIH-NIAID (NIAID Contrato No. HHSN272201000009I). También queremos agradecer a Chris King y Ronald Blanton de apoyo en la obtención de los caracoles infectados por esquistosomas y útil para los debates.

Las medidas de seguridad: Cercarias esquistosoma puede penetrar directamente en la piel sin las necesidades de un corte o los poros y causa la esquistosomiasis enfermedades crónicas. S. mansoni es un nivel de bioseguridad 2 organismo, por lo tanto, el equipo de protección personal y las medidas de seguridad se deben seguir cuando se trabaja con cercarias infecciosas. La mayoría de los equipos necesarios para crecer y transformar la esquistosomiasis es estándar, con algunas excepciones clave pocas indica en la tabla al final del protocolo. Todo el trabajo de cercarias se debe hacer en una habitación aislada. Schistosomules deben ser cultivadas en una campana estéril, aprobado bioseguridad. Dos capas de nitrilo o guantes de látex deben ser usados, así como una bata de laboratorio, resistente al agua un par de zapatos, pantalones sin agujeros o rasgones, y protectores a prueba de agua la manga para las muñecas y los brazos. En el caso de un derrame accidental, pulverizar inmediatamente el área contaminada con etanol al 70% o 10% de lejía, frotar vigorosamente si en su persona. Esto será suficiente para desactivar el parásito, pero todas las posibles infecciones deben ser informados y tomados en serio. Aunque schistosomules no son netamente infeccioso, las directrices institucionales varían, y debe ser consultado para determinar si BSL2 precauciones son necesarias durante las etapas de la vida no infecciosas. Desinfecte todas las superficies que entran en contacto con cualquier sustancia potencialmente infecciosos después de que el experimento se lleva a cabo y deseche los residuos en un contenedor de riesgo biológico aprobado. NOTA: la cubierta contenedores caracol 1 a 2 días antes de su vertimiento y protegerlo de la luz 1. La recolección de las cercarias caracol <ol><li> Llene un vaso limpio, libre de 500 ml de detergentes medio llena con agua destilada equilibrarse a temperatura ambiente. Si se trabaja con los caracoles huésped infectado con miracidios en fechas diferentes, utilizar un vaso para cada fecha de la infección (Esto es especialmente importante si los caracoles serán reutilizados para futuros experimentos). El tamaño del vaso también puede ser menor dependiendo de la experiencia del usuario. </li><li> Recoge los huéspedes infectados caracol que desea arrojar y depositar con cuidado en el vaso con un pez de acuario estándar de red y las pinzas de uso general, según sea necesario. </li><li> Colocar en un vaso de precipitados de emergencia para derrames bandeja de aproximadamente 8 a 12 pulgadas por debajo de una fuente de luz incandescente. Deje reposar durante 1-2 horas a la luz. Cercarias salir del caracol cuando se expone a la luz brillante. </li><li> Cuidadosamente filtra los caracoles de la mezcla de cercarias. Tome las precauciones de seguridad adicionales, como una mezcla es altamente infecciosa. Recuerde, cercarias directamente pueden penetrar la piel humana! </li><li> Vuelva a colocar la mezcla en la fuente de luz durante unos 15 minutos, permitiendo que cualquier material de desecho de partículas se asienten. </li><li> Transferencia de cercarias en un erlenmeyer limpio usando una pipeta, evitando la suciedad en el fondo del vaso. Colocar el matraz aproximadamente a un ángulo de 45 ° sobre el hielo en la oscuridad durante 45 - 60 minutos, lo que permite cercarias para establecerse en una esquina. </li><li> Utilice una pipeta de 50 mL para eliminar 75 a 90% del sobrenadante y depositarlo en el vaso que contiene lejía al 10% o 70% de etanol, teniendo cuidado de no dejar que las gotas de agua que contienen cercarias por goteo de la pipeta. </li><li> Agitar el matraz Erlenmeyer que contiene cercarias suavemente para volver a suspender las cercarias en la solución. Use una pipeta para transferir la muestra a la estéril tubos de 50 ml cónicos. Coloque los tubos de nuevo en hielo durante 20 minutos en la oscuridad. Cercarias se depositarán en el fondo del tubo en la oscuridad. </li></ol> 2. Transformación manual de cercarias al schistosomules Antes de empezar: Asegúrese RPMI 1640 completo (RPMI, el 5% de suero fetal bovino, 1X penicilina / estreptomicina) y calentar a 37 º C. (Es muy importante que el suero fetal bovino utilizado ha sido inactivada por incubación a 56 ° C durante 1 hora como esquistosomas pueden ser sensibles al complemento). <ol><li> En una campana de riesgo biológico, eliminar el sobrenadante a un 5 a 10 ml de solución en cada uno cónico de 50 ml con una pipeta o un dispositivo de vacío. </li><li> Adjunte una de calibre 22 de doble punta, luer lok emulsionante aguja a una jeringa de tamaño adecuado. </li><li> Dibujar lentamente la mezcla en la jeringa. Recoger cercarias de todos los tubos de 50 ml en una jeringa. Esté atento de goteo. </li><li> Con cuidado, gira la jeringa sobre manera que el extremo aguja separada está para arriba. Añada otra jeringa a este lado de la aguja emulsionante. </li><li> Pase la mezcla a través de la aguja de un total de 40 veces (20 veces de ida y vuelta) para transformar las cercarias. </li><li> La posición de la jeringa verticalmente de manera que la mezcla está contenida en la jeringa y la parte inferior de la jeringa superior está vacía. Retire la jeringa encima y desinfección en el 70% de etanol. </li><li> Fuerte caída de la mezcla a gota en un recipiente limpio de alta amurallada plato 100mm x 50mm pyrex o cristalizador. Desinfectar agujas y jeringas en un 70% de etanol. </li><li> Añadir 37 ° C medio RPMI 1640 completo de manera que elparte inferior de la placa de Petri está totalmente cubierta, como si el vertido de un plato de caldo de Luria. </li><li> Hágalo girar suavemente para recoger schistosomules en el centro de la placa de Petri. Evitar salpicaduras. Nota: Puede ser útil para apagar la luz de campana para dar este paso para visualizar más adecuadamente la schistosomules en el centro. </li><li> Inmediatamente depósito schistosomules concentrado en un nuevo máximo de 12 y placa de cultivo celular y mediante un gotero estéril. </li><li> Repetir 2.9 y 2.10 hasta schistosomules ya no están presentes en el centro del plato (aproximadamente 10 veces). Depósito de cada uno posterior recogida en un lugar bien fresco. </li><li> Llene cada uno bien hasta la mitad (alrededor de 1,5 ml) precalentado con medio RPMI completo. </li><li> Visualizar en un microscopio invertido para verificar la presencia de schistosomules y la ausencia de cercarias. </li><li> Crecimiento de la schistosomules en una incubadora de CO 2 fijado a 37 ° C y 5% de CO 2. </li></ol> 3. Los resultados representativos: Transferencia pendiente de schistosomules transformado en medios de cultivo, la visualización en una brillante invertido microscopio de campo (ver tabla de equipos para un ejemplo) debería dar una imagen similar a la Figura 1, en el que sólo schistosomules transformado están presentes en el pozo. Los contaminantes potenciales podrían incluir cercarias intacto, cortó las colas de cercarias, u otros desechos. Si no se dispone de microscopio invertido, un microscopio estándar o incluso un alcance disección será suficiente para visualizar el schistosomules. Tenga en cuenta que este procedimiento no siempre es transformar el 100% de cercarias. <img src="/files/ftp_upload/3191/3191fig1.jpg" alt="Figura 1"/> Figura 1: Transformada schistosomulum <img src="/files/ftp_upload/3191/3191fig2.jpg" alt="Figura 2"/> Figura 2: Ruptura de las colas y Cercariae

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> N Engl J Med. 346, 1212-1220 (2002).</li><li>Steinmann, P., Keiser, J., Bos, R., Tanner, M., Utzinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis+and+water+resources+development:+systematic+review,+meta-analysis,+and+estimates+of+people+at+risk.">Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk.</a> Lancet Infect Dis. 6, 411-425 (2006).</li><li>Savioli, L., Albonico, M., Engels, D., Montresor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis.">Progress in the prevention and control of schistosomiasis and soil-transmitted helminthiasis.</a> Parasitol Int. 53, 103-113 (2004).</li><li>King, C. H., Dickman, K., Tisch, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reassessment+of+the+cost+of+chronic+helmintic+infection:+a+meta-analysis+of+disability-related+outcomes+in+endemic+schistosomiasis.">Reassessment of the cost of chronic helmintic infection: a meta-analysis of disability-related outcomes in endemic schistosomiasis.</a> Lancet. 365, 1561-159 (2005).</li><li>Haas, W., Grabe, K., Geis, C., Pach, T., Stoll, K., Fuchs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+and+invasion+of+human+skin+by+Schistosoma+mansoni+cercariae:+the+key-role+of+L-arginine.+Parasitology.">Recognition and invasion of human skin by Schistosoma mansoni cercariae: the key-role of L-arginine. Parasitology.</a> 124, 153-167 (2002).</li><li>Grabe, K., Haas, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigation+within+host+tissues:+cercariae+orientate+towards+dark+after+penetration.">Navigation within host tissues: cercariae orientate towards dark after penetration.</a> Parasitol Res. 93, 111-113 (2004).</li><li>Shiff, C. J., Cmelik, S. H., Ley, H. E., Kriel, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+human+skin+lipids+on+the+cercarial+penetration+responses+of+Schistosoma+haematobium+and+Schistosoma+mansoni.">The influence of human skin lipids on the cercarial penetration responses of Schistosoma haematobium and Schistosoma mansoni.</a> J Parasitol. 58, 476-480 (1972).</li><li>Saladin, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+cercarial+responses+to+irradiance+changes.">Schistosoma mansoni: cercarial responses to irradiance changes.</a> J Parasitol. 68, 120-124 (1982).</li><li>Chai, M., McManus, D. P., McInnes, R., Moertel, L., Tran, M., Loukas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+lung+schistosomula,+in+vitro+cultured+schistosomula+and+adult+Schistosoma+japonicum.">Transcriptome profiling of lung schistosomula, in vitro cultured schistosomula and adult Schistosoma japonicum.</a> Cell Mol Life Sci. 63, 919-929 (2006).</li><li>Gobert, G. N., Tran, M. H., Moertel, L., Mulvenna, J., Jones, M. K., McManus, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+changes+in+Schistosoma+mansoni+during+early+schistosomula+development+and+in+the+presence+of+erythrocytes.">Transcriptional changes in Schistosoma mansoni during early schistosomula development and in the presence of erythrocytes.</a> PLoS Negl Trop Dis. 4, (2011).</li><li>Basch, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+Schistosoma+mansoni+in+vitro.+I.+Establishment+of+cultures+from+cercariae+and+development+until+pairing.">Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing.</a> J Parasitol. 67, 179-185 (1981).</li><li>Yoshino, T. P. &. a. m. p. ;. a. m. p., Laursen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Schistosoma+mansoni+daughter+sporocysts+from+mother+sporocysts+maintained+in+synxenic+culture+with+Biomphalaria+glabrata+embryonic+(Bge)+cells.">Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells.</a> J Parasitol. 81, 714-722 (1995).</li><li>Colley, D. G., Wikel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+simplified+method+for+the+production+of+schistosomules.">Schistosoma mansoni: simplified method for the production of schistosomules.</a> Exp Parasitol. 35, 44-51 (1974).</li><li>Lazdins, J. K., Stein, M. J., David, J. R., Sher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+rapid+isolation+and+purification+of+schistosomula+of+different+developmental+stages+by+centrifugation+on+discontinuous+density+gradients+of+Percoll.">Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll.</a> Exp Parasitol. 53, 39-44 (1982).</li><li>Cousin, C. E., Stirewalt, M. A., Dorsey, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+ultrastructure+of+early+transformation+of+skin-+and+shear-pressure-derived+schistosomules.">Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules.</a> Exp Parasitol. 51, 341-365 (1981).</li><li>Lewis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Curr Protoc Immunol. Chapter 19, 1-1 (2001).</li></ol>

No hay conflictos de interés declarado.

Varios posibles problemas podrían surgir durante el proceso de transformación, sin embargo, este protocolo está diseñado para evitar la mayoría de estos problemas. Un peligro potencial es la obstrucción de la aguja emulsionantes, que se presenta generalmente debido a los escombros sobrantes de la cosecha en el paso 1.3. Asegúrese de permitir todas las partículas que se asientan en el paso 1.5 y la transferencia de las cercarias con cuidado en el paso 1.6 para evitar este problema. Además, como se ha descrito antes, trabajando dentro de una cabina de seguridad biológica, la utilización de equipos de protección personal (incluso protectores para la cara) y evitar una presión excesiva sobre las jeringas es aconsejable. Las colas cercarias pueden ser visualizados en los medios de comunicación, lo cual es indicativo de la separación de los pobres durante el paso 2.9. Las colas muestran un movimiento similar al espasmo y tienen una forma distinta (ver Figura 2), y thusly se distinguen fácilmente de las schistosomules. Las colas pueden ser removidos por el lavado de la schistosomules de más de medio RPMI completo que permite la schistosomules a un acuerdo. Las colas se quedan flotando y se puede aspirar fuera. Si cercarias intacto están presentes en los medios de comunicación, la transformación en el paso 2.5 no se ha ejecutado correctamente. Una imagen de una cercaria intacta se muestra en la Figura 3b de referencia. Si se produce este problema, asegúrese de que la aguja emulsionante que se utiliza es del tamaño adecuado. Además, el número de veces que se pasa la mezcla cercarias través de la aguja se puede aumentar, aunque el 40 veces suele ser mucho más que suficiente para alcanzar el 100% de corte de las colas de cercarias. Transformación de la cercaria en schistosomules fue descrita por primera vez en 1974 por Colley y Wikel (13). Desde entonces, la preparación de schistosomules se ha hecho utilizando la estrategia de corte con una aguja y una jeringa como se describe en este protocolo, o utilizando un enfoque de agitación y se separa con gradientes de Percoll (14, 15) o girando como se demuestra en este trabajo. Estas dos técnicas para la preparación de schistosomules están muy bien descritas por Fred Lewis (16) y más información para el cultivo de más de schistosomules de crecimiento a largo plazo se puede encontrar en el NIAID recursos esquistosoma Center ( <a href="http://www.schisto-resource.org/">www.schisto-resource.org</a> ).

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> 22g x 2-7/8 &quot;de aguja emulsionante </td><td> Fisher Scientific </td><td> 14 a 825-17G </td></tr><tr><td> RPMI 1640 1X (500ml) </td><td> Invitrogen </td><td> 11835030 </td></tr><tr><td> De suero fetal bovino (FBS) inactivado por calor </td><td> Invitrogen </td><td> 10082139 </td></tr><tr><td> Penicilina / estreptomicina (100 veces) </td><td> Invitrogen </td><td> 15070063 </td></tr><tr><td> Cristalizador (100 mm) </td><td> Fisher Scientific </td><td> 08-741D </td></tr><tr><td> Pinzas de uso general </td><td> Fisher Scientific </td><td> 10-316C </td></tr><tr><td> Petco Acuario neto de camarones en salmuera </td><td> Petco </td><td> SKU: 1190954 </td></tr></tbody></table>

cercarial transformation and in vitro cultivation of schistosoma mansoni schistosomules

Parásitos esquistosoma son los agentes causantes de la esquistosomiasis, una enfermedad crónica debilitante que afecta a más de 200 millones de personas a nivel mundial y ocupa el segundo lugar de la malaria entre las enfermedades parasitarias en términos de salud pública y el impacto socio-económico (1-4). Parásitos esquistosoma son gusanos trematodos con un ciclo de vida complejo intercambio entre una vida parasitaria en los ejércitos de moluscos y mamíferos, con intervención de nado libre etapas. En pocas palabras, libre cercarias infectar a un huésped mamífero, penetrando la piel con la ayuda de proteasas secretadas, tiempo durante el cual las cercarias perder sus colas, transformándose en schistosomules. El schistosomules ahora deben evadir el sistema inmune del huésped, desarrollar un intestino para la digestión de las células rojas de la sangre, y aunque la migración de los pulmones y la circulación portal en ruta hacia su destino final en el sistema porta hepática y, finalmente, las venas mesentéricas (por S. mansoni) en par de gusanos machos y hembras y aparearse, produciendo cientos de huevos cada día. Algunos de los huevos son excretados del cuerpo en el agua dulce, donde los huevos se transforman en miracidios que nadan libremente (5-10). Los miracidios infectar a especies concretas de caracol y se transforman en esporocistos madre e hija, que a su vez, producen cercarias infectantes, completando el ciclo de vida. Por desgracia, todo el ciclo de vida de esquistosomas no pueden ser cultivados in vitro, pero cercarias infecciosas se pueden transformar en schistosomules, y el schistosomules pueden ser cultivadas durante semanas para el análisis del desarrollo de esquistosoma en la vitro o el análisis de microarrays. En este protocolo, se presenta una descripción visual de la transformación de cercarias y cultivo in vitro de schistosomules. Nos despojamos de cercarias infecciosas del caracol Biomphalaria glabrata de acogida y de forma manual los transforman en schistosomules separando la cola con un emulsionante aguja de doble punta. El in vitro y técnicas de transformación de cercarias schistosomules cultura describe evitar el uso de un huésped mamífero, lo que simplifica la visualización de los esquistosomas y facilita la recolección de los parásitos para el análisis experimental. Transformación in vitro y técnicas de cultivo de los esquistosomas se han hecho durante años (11, 12), pero no existen protocolos visuales han sido desarrollados que están disponibles para toda la comunidad.

Watch this Scientific Journal Video about Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules at JoVE.com

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Se describe un<em&gt; In vitro</em&gt; Método para esquistosómula cultivo del parásito platelminto<em&gt; Schistosoma mansoni</em&gt;, A través de la recolección y transformación de cercarias infecciosas del caracol de agua dulce huésped intermedio glabrata Biomphalaria.

Transformación y cercarias In vitro El cultivo de la Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

cercarial-transformation-vitro-cultivation-schistosoma-mansoni

Research

JoVE Journal

Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

17.9K vistas.  Case Western Reserve University. Se describe un In vitro Método para esquistosómula cultivo del parásito platelminto Schistosoma mansoni, A través de la recolección y transformación de cercarias infecciosas del caracol de agua dulce huésped intermedio glabrata Biomphalaria.

Video: Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

En uncoating in vitro del VIH-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Investigación

Inmunología e Infección

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

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

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

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

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

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

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Cuantitativo<em&gt; In vitro</em&gt; Ensayo para medir la adhesión de neutrófilos al Activado Primaria células endoteliales microvasculares humanas en condiciones estáticas

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

El cultivo de<em&gt; Heligmosomoides polygyrus:</em&gt; Un inmunomodulador nematodo parásito y sus productos secretados

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Transformación biolística de un fluorescente Tagged gen en el oportunista por hongos patógenos<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Transformación de la levadura probiótica y su recuperación de tejidos gastrointestinales inmune después de alimentación forzada oral en ratones

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Métodos in vitro para comparar la unión de diana y la inducción de CDC entre anticuerpos terapéuticos: Aplicaciones en análisis de biosimilitud

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Aislamiento y caracterización de las vesículas extracelulares de adultos Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

Análisis de interacciones entre endobióticos y microbiota de tripa humana utilizando sistemas de fermentación de bañoin in vitro

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Transfección altamente eficiente de macrófagos primarios con ARNm transcrito in Vitro

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...