Agradecemos a Francisco Candal, CDC Oficina de Transferencia de Tecnología, Georgia Atlanta para HBEC-5i células. Este trabajo fue financiado por el Wellcome Trust (4 años las becas para estudios de doctorado a la CA y la Beca Superior en Ciencias Biomédicas Básicas de JAR, número de concesión 084226).

Recomendaciones generales Microvascular cerebro humano las células endoteliales (HBEC-5i) la cultura se ha descrito anteriormente en 6, 8. P. los parásitos P. falciparum fueron cultivadas como en 9. Ambos HBEC-5i y P. falciparum parásito culturas deben mantenerse en condiciones estériles en todo momento. Todos los reactivos deben ser pre-calentado a 37 ° C. Se recomienda comprobar regularmente la contaminación por micoplasma 10 por PCR (Minevera Biolabs, las instrucciones del fabricante siguiente). El protocolo se resume en la figura 1. 1. De las células endoteliales cultivo de rutina <ol><li> Preparar los reactivos necesarios. </li></ol><table border="1"><tbody><tr><td> Medio para preparar </td><td> Reactivos </td><td> Cantidad </td></tr><tr><td> &quot;Incompleta DMEM&quot; </td><td> DMEM-F12 de Ham </td><td> 500 ml </td></tr><tr><td> Ynbsp; </td><td> L-glutamina 200 mM </td><td> 5 ml </td></tr><tr><td></td><td> Penicilina / estreptomicina 100X </td><td> 5 ml </td></tr><tr><td></td><td> NaOH 1M </td><td> 1,3 ml (ajustar el pH a 7.4) </td></tr><tr><td> &quot;DMEM completo&quot; </td><td> &quot;Incompleta DMEM&quot; </td><td> 450 ml </td></tr><tr><td></td><td> Suero bovino fetal inactivado por calor </td><td> 50ml </td></tr><tr><td></td><td> De las células endoteliales suplemento de crecimiento </td><td> 5 ml </td></tr></tbody></table> <ol start="2"><li> Cultura HBEC-5i en un respiradero de 25cm 2 frasco con 10 ml de medio DMEM completo en una incubadora a 37 ° C con 5% de CO 2. </li><li> Las células de paso cuando se hacen confluentes. Eliminar el medio de edad por succión y lavar dos veces con medio DMEM incompleto o cultivo de tejidos de grado PBS (Ca 2 + y Mg 2 + libre) pre-calentado a 37 ° C. </li><li> Añadir 1 ml de pre-calentado TratePSIN-EDTA (0,025% de tripsina, 0,5 mM EDTA) en forma de remolino para cubrir la totalidad del frasco y se incuba durante ~ 2 min a 37 ° C. </li><li> Revisar bajo microscopio invertido al menos el 90% de las células se han separado. Si es necesario, suavemente golpee el fondo del frasco para desprender las células adherentes. Añadir 10 ml de medio DMEM completo para bloquear las células tripsina y la transferencia en un tubo cónico de 15 ml. Centrifugar durante 4 min a 300 g, a temperatura ambiente (TA). </li><li> Descartar el sobrenadante y resuspender el precipitado con 10 ml de medio DMEM completo. Pipeta la solución de arriba y abajo para volver a suspender completamente las células. </li><li> Agregar 1 o 2 ml de la suspensión de células en un frasco de cultivo nuevo y añadir 8 ml de medio fresco para mantener la cultura. Evaluar el crecimiento de la cultura todos los días bajo un microscopio invertido y el cambio medio de cada 2 o 3 días antes de que se vuelve amarillo. </li></ol> 2. Preparación de las células endoteliales para una selección <ol><li> Dos días antes del día de la selectio<html> n, añade fibronectina diluido en PBS estéril (2 mg / cm 2) en una (o más) 60 mm placa de Petri. Incubar la placa durante 5 a 20 min a 37 ° C, extraer la solución de fibronectina, que pueden ser almacenadas a 4 ° C durante un mes y volver a utilizarse una vez. </li><li> Las células de paso como se describe en las secciones 1.3. a 1,5. Asumiendo el 100% de células confluentes se separaron y se resuspendieron en 10 ml de medio DMEM completo (sección 1.6., Volver a suspender con un volumen equivalente de medio si la confluencia es menor, por ejemplo, volver a suspender con 8 ml si la confluencia fue del 80%), añadir 1,5 ml de la suspensión las células a la placa recubierta de fibronectina Petri y otro 1,5 ml de medio DMEM completo. </li><li> Coloque el plato de Petri sembradas en la incubadora. Nota: Lo ideal sería que la confluencia será de alrededor de 90% en el momento de la selección de dos días más tarde. </li><li> Opcional. Para activar HBEC-5i, agregue el TNF (Factor de Necrosis Tumoral) de citoquinas a una concentración final de 50μg/ml 24 horas antes de la selección. </li></ol>e_title &quot;&gt; 3. Plasmodium falciparum cultivo de rutina <ol><li> Preparar los reactivos necesarios (ver tabla aquí debajo). Prepare los glóbulos rojos (GR) por la separación de sangre total (grupo O +) por el paso a través de un filtro de los leucocitos (véase &quot;Métodos de Investigación de Paludismo&quot;, la publicación 11 de la malaria en general el cultivo de parásitos). Lavar dos veces al RBC por centrifugación a 400 g durante 5 minutos y resuspender con 10 ml de RPMI incompleto. Mantenga lava RBC a 4 ° C en medio incompleto el 50% de hematocrito. </li></ol><table border="1"><tbody><tr><td> Medio para preparar </td><td> Reactivos </td><td> Cantidad </td></tr><tr><td> &quot;Incompleta RPMI&quot; </td><td> RPMI 1640 (con bicarbonato) </td><td> 500 ml </td></tr><tr><td></td><td> Hepes 1M </td><td> 12.5ml </td></tr><tr><td></td><td> Glucosa al 20% </td><td> 5 ml </td></tr><tr><td></td&gt; <td> L-glutamina 200 mM </td><td> 5 ml </td></tr><tr><td></td><td> Gentamicina 50mg/ml </td><td> 250μl </td></tr><tr><td></td><td> NaOH 1M </td><td> 0,7 ml (ajustar el pH a 7.2) </td></tr><tr><td> &quot;RPMI completo&quot; </td><td> &quot;Incompleta RPMI&quot; </td><td> 450 ml </td></tr><tr><td></td><td> Humano agrupado (no inmunes) de suero </td><td> 50ml </td></tr></tbody></table> <ol start="2"><li> Cultivo de P. falciparum infectados RBC con medio RPMI completo en el 2% de hematocrito y se incuba a 37 ° C con el 3% de CO 2, 1% O 2, y 96% N 2. Realizar frotis de Giemsa 11 todos los días para evaluar el grado de desarrollo de los parásitos (Figura 2). </li><li> Regularmente (aproximadamente una vez por semana) sincronizar la cultura por el tratamiento de sorbitol 12. El día antes de la selección del ensayo, el objetivo de una cultura de anillo etapa en 5% de parasitemia o más (lo ideal es por lo menos 10%). </li></ol> 4. Selección de P. falciparum de cytoadhesion a las células endoteliales <ol><li> En el día de la prueba, el cultivo de parásitos deben estar en fase de trofozoito pigmentados (Figura 2) (idealmente parasitemia 10%), mientras que HBEC-5i la cultura debe ser de 50 a 100% de confluencia (idealmente 90%). 30μl volumen celular de cultivo de parásitos que se necesita por HBEC-5i placa de Petri. </li><li> Lavar dos veces los parásitos por centrifugación (500 g por 5 min) 1,5 ml de cultivo de parásitos. Desechar el sobrenadante y resuspender con 10 ml de recién hecho, caliente, medio DMEM incompleto. Repetir el lavado por segunda vez. Resuspender el 30μl volumen celular con 1,5 ml de incompleta con BSA al 1% DMEM. </li><li> Lavar el plato recubierto HBEC-5i Petri doble aspiración de mediano y agregar 3 ml de DMEM incompleto. </li><li> Añadir la solución de los parásitos al plato HBEC-5i y se incuba a 37 ° C durante 75 min. Resuspender los parásitos en dos ocasiones (después de 30 y 60 minutos) durante la incubaciónmoviendo suavemente el plato en las cuatro direcciones, así como las agujas del reloj y en sentido contrario. </li><li> Después de la incubación, lavar el plato cinco veces por medio de aspiración, utilizando una pipeta de plástico Pasteur para añadir 3 ml de medio DMEM incompleto cálido y suave balanceo. Si los platos se están usando, guárdelas en una superficie caliente, como un frasco lleno de agua a 37 ° C. </li><li> Revise el plato bajo un microscopio invertido. Si muchos no infectada RBC son todavía visibles, hacer más lavados como se describió anteriormente. Manejar la placa de Petri con mucho cuidado para evitar cualquier riesgo de contaminación. </li><li> Quitar medio del plato y añadir 3 ml de agua tibia medio RPMI completo con un volumen de 40μl hematocrito de uRBC fresco. </li><li> Coloque el plato en una cámara hermética de incubación, gas, durante 3 minutos y colocar la cámara en una incubadora a 37 ° C durante la noche. </li><li> Al día siguiente, la cosecha de los parásitos mediante el lavado con medio RPMI incompleto, de un modo similar al descrito en la sección 4.4. pero con más fuerza. Keep todos los medio de comunicación utilizado (que contiene resuspendido RBC) en un tubo cónico de 15 ml. Revisar bajo microscopio invertido que todos los glóbulos rojos se han eliminado de la antena. </li><li> Se precipitan los parásitos, descartar el sobrenadante, resuspender en 5 ml de medio RPMI completo y colocar la mezcla en un frasco de cultivo normal. </li></ol> 5. Resultados representante Parásitos no seleccionados muestran un bajo nivel de la unión a HBEC-5i (Figura 3). Así, después de la primera ronda de selección, muy pocos parásitos serán cosechados y puede tomar hasta un mes de cultivo para llegar a una parasitemia suficiente para la segunda ronda de selección. Después de cada ronda de selección, los parásitos más y más se unen a las células endoteliales y las selecciones se pueden repetir a intervalos de tiempo más corto. Después de 4-5 rondas de selección, de alta vinculante poblaciones de parásitos se obtienen (Figura 3). En nuestras manos, la unión de HB3-HBEC a HBEC-5i o TNF activa HBEC-5i fue de simnivel de ILAR (datos no mostrados). El protocolo descrito aquí se ha probado con 4 P. falciparum cepas: HB3, IT/FCR3, 3D7 y Dd2 (Tabla 1). Sólo este último no se ha demostrado capaz de unirse a HBEC-5i, incluso después de 5 rondas de selección. HB3 parásitos también fueron seleccionados para cytoadherence a dérmicos humanos y pulmonar células endoteliales microvasculares (HDMEC y HPMEC). Después de cuatro rondas de selección, utilizando el método descrito aquí, una población de alto vinculante se obtuvo en ambos HDMEC y HPMEC (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/3122/3122fig1.jpg" /> Figura 1. Descripción general del proceso de selección. <img alt="Figura 2" src="/files/ftp_upload/3122/3122fig2.jpg" /> Figura 2. Etapas de desarrollo de P. falciparum infectados glóbulos rojos. Frotis de Giemsa visualizaron bajo microscopio con un aumento de 1000X. <img alt="Figura 3" src="/files/ftp_upload/3122/3122fig3.jpg" /> Figura 3. Un ejemplo típico de los parásitos unión a HBEC-5i. (A) La capa azul es HBEC-5i fija con glutaraldehído y se tiñeron con Giemsa, visualizaron bajo microscopio con un aumento de 1000X. Fotografías fueron tomadas después de uRBC y iRBC consolidados fueron arrasadas. El panel izquierdo muestra una sola P. falciparum HB3 (no seleccionado) parásito obligado a HBEC-5i. En el panel derecho de los parásitos HB3-HBEC había sido seleccionado por 5 rondas. (B) Los datos representan el promedio de dos experimentos independientes, cada uno realizado por duplicado. El número de parásitos obligados por las células endoteliales se había contado. <img alt="Tabla 1" src="/files/ftp_upload/3122/3122table1.jpg" /> Tabla 1. Resumen de Plasmodium falciparum cepas que se han seleccionado con éxito por la unión a HBEC-5i. Tenga en cuenta que HB3 también fue seleccionado en el TNF-activa HBCE-5i. Después de 5 rondas de selección, la cepa Dd2 no mostró ningún aumento en la unión a HBEC-5i en comparación con el no seleccionado-Dd2. <img alt="Figura 4" src="/files/ftp_upload/3122/3122fig4.jpg" /> Figura 4. La unión de P. falciparum HB3 parásitos dérmicos (HDMEC) y pulmonar (HPMEC) las células endoteliales, antes y después de cuatro rondas de selección. pesar de que el medio de cultivo para HDMEC y HPMEC difiere ligeramente (ver instrucciones del proveedor), el protocolo utilizado para la selección es idéntico al igual que con HBEC-5i. Las imágenes tomadas con una ampliación de 400X. <table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> DMEM-F12 de Ham </td><td> Sigma </td><td> D6421 </td><td> Por medio DMEM completo </td></tr><tr><td> L-glutamina 200 mM </td><td>GIBCO </td><td> 25030 </td><td> Por medio DMEM y RPMI completo </td></tr><tr><td> Penicilina / estreptomicina 100X (10.000 unidades / ml y 10mg/ml) </td><td> ScienCell </td><td> 0503 </td><td> Por medio DMEM completo </td></tr><tr><td> Suero bovino fetal inactivado por calor </td><td> ScienCell </td><td> 0025 </td><td> Por medio DMEM completo </td></tr><tr><td> Tripsina-EDTA (0,025% de tripsina, 0,5 mM EDTA) </td><td> ScienCell </td><td> 0103 </td><td></td></tr><tr><td> De las células endoteliales suplemento de crecimiento </td><td> ScienCell </td><td> 1052 </td><td> Por medio DMEM completo </td></tr><tr><td> El cultivo de tejidos tratados 60 mm X 15 mm cajas de Petri </td><td> BD </td><td> 353002 </td><td></td></tr><tr><td> La fibronectina humana </td><td> Millipore </td><td> FC010 </td><td> El uso de 2 mg / cm 2 </td></tr><tr><td> TNF </td><td> I +D Systems </td><td> 210-TA </td><td> opcional, el uso de 50 mg / ml </td></tr><tr><td> RPMI 1640 </td><td> Lonza </td><td> BE12-167F </td><td> Por medio RPMI completo </td></tr><tr><td> Gentamicina 50mg/ml </td><td> Lonza </td><td> 17-518Z </td><td> Por medio RPMI completo </td></tr><tr><td> Hepes 1M </td><td> Lonza </td><td> Be17-737E </td><td> Por medio RPMI completo </td></tr><tr><td> HDMEC </td><td> ScienCell </td><td> 2000 </td><td> La línea celular primaria </td></tr><tr><td> HPMEC </td><td> ScienCell </td><td> 3000 </td><td> La línea celular primaria </td></tr><tr><td> HBEC-5i </td><td></td><td></td><td> Se obtiene de Francisco Candal (fcandal@cdc.gov) </td></tr></tbody></table> Tabla 2. Materiales

<ol>
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No tenemos nada que revelar.

El sello distintivo de la malaria cerebral es la retención de P. falciparum iRBC dentro del cerebro microvasculatura 2, 3. Sin embargo, en cultivos in vitro de P. falciparum sólo poco a cytoadhere HBEC-5i, un modelo para el endotelio microvascular cerebro humano. Aquí hemos desarrollado un ensayo sencillo para enriquecer a una población de la unión a HBEC-5i, uno más &quot;in-vivo como&quot; fenotipo. Tres de cada 4 P. cepas de P. falciparum se han seleccionado con éxito utilizando este método. Además, HB3 también fue seleccionado en HDMEC y HPMEC, lo que indica que este protocolo se puede utilizar para los parásitos y diversos tipos de células endoteliales. Diferentes hipótesis pueden explicar la falta de unión con la cepa Dd2. La más probable es que el hecho de que esta línea parásito sin botón, lo que impide la profunda cytoadherence 13, 14. Se recomienda el cultivo de parásitos seleccionados junto a la selección de proproceso para proporcionar un control de comparación. Esto permitirá, por ejemplo, comparar el transcriptoma de los parásitos vinculantes y no vinculantes, con la esperanza de descubrir los candidatos parásito ligando. TNF es una citocina encontrado a un alto nivel en los pacientes con paludismo cerebral y ha sido mostrar a inducir la expresión de proteínas de la superficie muchos de HBEC-5i (Claessens et al, en preparación y 8, 15, 16). En este caso, la cantidad de iRBC obligado fue similar en condiciones normales HBEC-5i en comparación con el activo HBEC-5i. Este &quot;modelo de secuestro&quot;, también se puede utilizar para estudiar la interacción molecular entre el iRBC y las células endoteliales, así como el efecto de la supuesta cytoadherence anti-drogas. En este caso, se recomienda placas HBEC-5i en pequeños pozos, tales como la &quot;cámara de diapositivas de 8 pocillos&quot; (BD 354 628) o &quot;CultureWell&quot; (Sigma C7735-20EA).

selection of plasmodium falciparum parasites for cytoadhesion to human brain endothelial cells

La mayoría de las muertes por malaria humana es causada por el Plasmodium estadio en sangre los parásitos P. falciparum. La malaria cerebral, la complicación más mortal de la enfermedad, se caracteriza por una acumulación de Plasmodium falciparum los glóbulos rojos infectados (iRBC) en la etapa de trofozoíto pigmentadas en la microvasculatura del cerebro 2-4. Esta obstrucción microvascular (secuestro) conduce a la acidosis, la hipoxia y perjudiciales citoquinas inflamatorias (revisado en 5). El secuestro también se encuentra en la mayoría de los tejidos microvascular del cuerpo humano 2, 3. El mecanismo por el cual iRBC adhieren a las paredes de los vasos sanguíneos aún es poco conocido. La línea inmortalizada Humanos microvascular cerebral de células endoteliales (HBEC-5i) ha sido utilizado como un modelo in vitro de la barrera sangre-cerebro 6. Sin embargo, el Plasmodium falciparum iRBC adjuntar sólo poco a HBEC-5i in vitro, a diferencia de la densa sequestrationes que se produce en los casos de malaria cerebral. Por lo tanto, desarrolló un ensayo panorámico para seleccionar (enriquecer) varias P. falciparum cepas para la adhesión a HBEC-5i para obtener poblaciones de alto vinculante parásitos, más representativo de lo que ocurre in vivo. Una muestra de un cultivo de parásitos (mezcla de iRBC y no infectados RBC) en la fase de trofozoito pigmentadas se lava y se incubaron en una capa de HBEC-5i cultivadas en una placa de Petri. Después de la incubación, el plato está cuidadosamente lavados de uRBC iRBC y no consolidados. Fresco uRBC se añaden a la iRBC pocas condiciones para HBEC-5i y se incubaron durante la noche. Como los parásitos esquizonte etapa explosión, merozoitos reinvade RBC y los parásitos en el estadio de anillo se cosechan al día siguiente. Los parásitos se cultivan hasta que se obtiene material suficiente (generalmente de 2 a 4 semanas) y una nueva ronda de selección se puede realizar. Dependiendo de la P. falciparum cepa, de 4 a 7 rondas de selección son necesarios para obtener una poblacióndonde la mayoría de los parásitos se unen a HBEC-5i. El fenotipo de la unión se pierde progresivamente a las pocas semanas, lo que indica un cambio en la expresión del gen del antígeno de superficie variante, por lo tanto la selección regular de HBEC-5i es necesaria para mantener el fenotipo. En resumen, hemos desarrollado un ensayo de selección de la representación P. falciparum parásitos más &quot;adhesivo malaria cerebral&quot; fenotipo. Hemos sido capaces de seleccionar 3 de cada 4 P. falciparum cepas de HBEC-5i. Este ensayo también ha sido utilizado con éxito para seleccionar los parásitos de la unión a las células endoteliales humanas dérmicas y pulmonares. Es importante destacar que este método puede ser usado para seleccionar tejidos específicos poblaciones de parásitos a fin de identificar ligandos candidato parásito para la unión al endotelio cerebral. Por otra parte, este ensayo puede ser utilizado para detectar el supuesto secuestro de medicamentos anti-7.

Watch this Scientific Journal Video about Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells at JoVE.com

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Un<em&gt; In vitro</em&gt; Modelo para el secuestro de la malaria cerebral se describe<sup&gt; 1</sup&gt;.<em&gt; Plasmodium falciparum</em&gt; Los glóbulos rojos infectados son seleccionados para unirse al inmortalizado células del cerebro humano endoteliales microvasculares. Los parásitos seleccionados muestran un fenotipo distinto. El proceso de selección pueden ser aplicados utilizando diversos<em&gt; P. falciparum</em&gt; Cepas y líneas de células endoteliales.

Selección de Plasmodium falciparum Los parásitos de cytoadhesion a las células cerebrales endoteliales humanas

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

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Research

JoVE Journal

Immunology and Infection

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

19.0K vistas.  University of Edinburgh. Un In vitro Modelo para el secuestro de la malaria cerebral se describe 1. Plasmodium falciparum Los glóbulos rojos infectados son seleccionados para unirse al inmortalizado células del cerebro humano endoteliales microvasculares. Los parásitos seleccionados muestran un fenotipo distinto. El proceso de selección pueden ser aplicados utilizando diversos P. falciparum Cepas y líneas de células endoteliales....

Video: Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Un<em&gt; In vitro</em&gt; Co-infección modelo para estudiar<em&gt; Plasmodium falciparum</em&gt;-VIH-1 en las interacciones humano primario de monocitos derivados de las células inmunitarias

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Investigación

Inmunología e Infección

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Un protocolo simple para el agrupamiento de plaquetas mediada por<em&gt; Plasmodium falciparum</emEritrocitos&gt; por el VIH en un recurso pobre ajuste

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Aislamiento y análisis de Brain-secuestrados Los leucocitos de<em&gt; Plasmodium berghei</em&gt; ANKA de ratones infectados

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Separación de los<em&gt; Plasmodium falciparum</em&gt; Late Stage-eritrocitos infectados por medios magnéticos

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

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 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Aislamiento de células dendríticas mieloides y células epiteliales de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Alto Rendimiento Purificación de<em&gt; Plasmodium falciparum</em&gt; Los merozoitos Para uso en opsonizante Antibody Ensayos

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

Métodos para investigar el papel de Regulación de pequeños ARN ribosomal y ocupación de<em&gt; Plasmodium falciparum</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

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

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

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

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

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

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Infección por neumococo de células endoteliales humanas primarias en flujo constante

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...