Agradecemos Wambsganss Nadine por su excelente asistencia técnica. Este trabajo fue apoyado por una beca de la Deutsche Forschungsgemeinschaft (GL599/1-1) y en parte por una subvención del Fondo de Innovación FRONTIER (Universidad de Heidelberg) al CAG

1. Protocolo <ol><li> Prepare Buffers la siguiente manera: <ol><li> Preparar tampón para aislar PBMC: &quot;Tampón de lavado&quot; = 0,02% de EDTA en PBS (uso 0,5 M EDTA). </li><li> Preparar tampón para el aislamiento de monocitos: &quot;tampón MACS aclarado&quot; = 0,5% de BSA (250 mg) + 2 mM de EDTA (200 l) + PBS (50 ml). Desgasifica la memoria intermedia. </li><li> Preparar tampón de tinción FACS y almacenamiento de las células: &quot;tampón FACS&quot; = 10% de FCS en PBS y &quot;tampón de fijación&quot; = 1% de PFA en PBS. </li></ol></li><li> Dibuja 30 ml de sangre entera de una vena del antebrazo. Usa EDTA como anticoagulante. </li><li> Aislar PBMC (Histopaque) de la siguiente manera: <ol><li> Preparar dos tubos estériles de 50 ml (sin poli-estireno). </li><li> Diluir la sangre desde el paso 2 con PBS 1:01. </li><li> Añadir 25 ml Histopaque + 25 ml de sangre entera / PBS por tubo. Asegúrese de que Histopaque y la sangre no se mezclan. </li><li> Centrifugar a 400 xg durante 30 min a TA, sin freno. </li><li> Aspirar plasma y descarte. </li><li> Aspirar interfaz opaca y pipeta en limpio 50 ml bañerae. </li><li> Añadir 20 ml de 0,02% de EDTA en PBS. </li><li> Centrifugar a 250 xg durante 5 min a 4 ° C. </li><li> Desechar el sobrenadante. </li><li> Añadir 10 ml de 0,02% de EDTA en PBS. </li></ol></li><li> Contar las células como sigue: <ol><li> Mezclar bien con un vórtex durante 10 s. </li><li> Tomar 200 l de suspensión celular + 300 l 0,02% de EDTA en PBS + 500 l de azul de tripano (200-500 células/10 plazas, de lo contrario cambiar el factor de dilución). </li></ol></li><li> Realizar el aislamiento negativo de los monocitos de la siguiente manera: <ol><li> Centrifugar las células obtenidas en la etapa 3.10 durante 10 min, 120 x g. Desechar el sobrenadante. </li><li> Lavar 2x sedimento celular mediante la adición de 10 ml de PBS + 0,02% de EDTA y se centrifuga durante 10 min, 120 x g. </li><li> Añadir 9 ml de agua estéril durante 3 segundos, añadir 1 ml de PBS 10X. </li><li> Lavar una vez más con PBS + 0,02% de EDTA y se centrifuga 10 min, 120 x g. </li><li> Contar durante la centrifugación. </li><li> Diluir en tampón EasySep a 5 x 10 7 / ml. </li><li> Añadir 50 l / ml polla enriquecimiento de monocitoscola. </li><li> Incubar durante 10 min a 4 ° C. </li><li> Cuentas Vortex durante 30 segundos. </li><li> Añadir 50 l / ml de perlas. </li><li> Incubar durante 5 min a 4 ° C. </li><li> Llenar con tampón EasySep para completar el volumen de 2,5 ml. La solución ahora aparece de color marrón. </li><li> Poner en imán. </li><li> Esperar 2,5 min a TA. Durante este tiempo, las células no monocıticas vinculados a la perla magnética se trasladarán a la pared del tubo y se adhieren allí. </li><li> Vierta tampón con monocitos no vinculantes en el tubo estéril fresco. Esta solución parece blanquecino. </li><li> Lavar una vez con PBS + 0,02% de EDTA y se centrifuga durante 10 min, 120 x g. </li></ol></li><li> Células de la placa en una placa de plástico o placa de paredes múltiples a una densidad de 0,5 x 10 6 / cm 2. Añadir 1 ml de medio de cultivo / x 10 6 células 1. </li><li> Células de cultivo en condiciones de interés para 6-14 días. </li><li> Cambie los medios después de 3 días, sustituyendo el 50% de los medios de comunicación por medio fresco (ver Tabla 1 para más detalles). </li><li>Seis días después, las células de la cosecha para el aislamiento de ARNm, citometría de flujo, transferencia Western, etc. </li></ol>

<ol>
	<li>Gordon, S., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+and+macrophage+heterogeneity.">Monocyte and macrophage heterogeneity.</a> Nat. Rev. Immunol. 5, 953-964 (2005).</li><li>Gordon, S., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+plasticity+of+mononuclear+phagocytes.">Diversity and plasticity of mononuclear phagocytes.</a> European Journal of Immunology. 41, 2470-2472 (2011).</li><li>Martinez, F. O., Sica, A., Mantovani, A., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+and+polarization.">Macrophage activation and polarization.</a> Front. Biosci. 13, 453-461 (2008).</li><li>Ingersoll, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+gene+expression+profiles+between+human+and+mouse+monocyte+subsets.">Comparison of gene expression profiles between human and mouse monocyte subsets.</a> Blood. 115, 10-19 (2010).</li><li>Gleissner, C. A., Sanders, J. M., Nadler, J., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+aldose+reductase+during+foam+cell+formation+as+possible+link+among+diabetes,+hyperlipidemia,+and+atherosclerosis.">Upregulation of aldose reductase during foam cell formation as possible link among diabetes, hyperlipidemia, and atherosclerosis.</a> Arterioscler. Thromb. Vasc. Biol. 28, 1137-1143 (2008).</li><li>Gleissner, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL4+downregulates+the+atheroprotective+hemoglobin+receptor+CD163+in+human+macrophages.">CXCL4 downregulates the atheroprotective hemoglobin receptor CD163 in human macrophages.</a> Circ. Res. , (2009).</li><li>Gleissner, C. A., Shaked, I., Little, K. M., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXC+chemokine+ligand+4+induces+a+unique+transcriptome+in+monocyte-derived+macrophages.">CXC chemokine ligand 4 induces a unique transcriptome in monocyte-derived macrophages.</a> J. Immunol. 184, 4810-4818 (2010).</li><li>Martinez, F. O., Gordon, S., Locati, M., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+the+human+monocyte-to-macrophage+differentiation+and+polarization:+New+molecules+and+patterns+of+gene+expression.">Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: New molecules and patterns of gene expression.</a> J. Immunol. 177, 7303-7311 (2006).</li><li>Verreck, F. A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+il-23-producing+type+1+macrophages+promote+but+il-10-producing+type+2+macrophages+subvert+immunity+to+(myco)bacteria.">Human il-23-producing type 1 macrophages promote but il-10-producing type 2 macrophages subvert immunity to (myco)bacteria.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 4560-4565 (2004).</li><li>Rey-Giraud, F., Hafner, M., Ries, 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=In+vitro+generation+of+monocyte-derived+macrophages+under+serum-free+conditions+improves+their+tumor+promoting+functions.">In vitro generation of monocyte-derived macrophages under serum-free conditions improves their tumor promoting functions.</a> PLoS One. 7, e42656 (2012).</li><li>Boyle, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+intraplaque+hemorrhage+evokes+a+novel+atheroprotective+macrophage+phenotype.">Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype.</a> American Journal of Pathology. 174, 1097-1108 (2009).</li><li>Cho, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+dendritic+cell-like+phenotype+in+macrophages+during+foam+cell+formation.">Induction of dendritic cell-like phenotype in macrophages during foam cell formation.</a> Physiol Genomics. 29, 149-160 (2007).</li><li>Vogt, G., Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+human+macrophages+with+enhanced+antimycobacterial+activity.">In vitro differentiation of human macrophages with enhanced antimycobacterial activity.</a> The Journal of Clinical Investigation. 121, 3889-3901 (2011).</li><li>Mikita, T., Porter, G., Lawn, R. M., Shiffman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidized+low+density+lipoprotein+exposure+alters+the+transcriptional+response+of+macrophages+to+inflammatory+stimulus.">Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus.</a> Journal of Biological Chemistry. 276, 45729-45739 (2001).</li><li>Qin, Z. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+THP-1+cells+as+a+model+for+mimicking+the+function+and+regulation+of+monocytes+and+macrophages+in+the+vasculature.">The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature.</a> Atherosclerosis. 221, 2-11 (2012).</li><li>Daigneault, M., Preston, J. A., Marriott, H. M., Whyte, M. K. B., Dockrell, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+of+markers+of+macrophage+differentiation+in+pma-stimulated+THP-1+cells+and+monocyte-derived+macrophages.">The identification of markers of macrophage differentiation in pma-stimulated THP-1 cells and monocyte-derived macrophages.</a> PLoS One. 5, e8668 (2010).</li><li>Wahl, L. M., Wahl, S. M., Smythies, L. E., Smith, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+monocyte+populations.">Isolation of human monocyte populations.</a> Current Protocols in Immunology. , (2001).</li><li>Waldo, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+human+macrophages+in+culture+and+in+atherosclerotic+plaques.">Heterogeneity of human macrophages in culture and in atherosclerotic plaques.</a> Am. J. Pathol. 172, 1112-1126 (2008).</li><li>Mart&#237;n-Fuentes, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+variation+of+scavenger+receptor+expression+in+human+macrophages+with+oxidized+low-density+lipoprotein+is+associated+with+a+differential+inflammatory+response.">Individual variation of scavenger receptor expression in human macrophages with oxidized low-density lipoprotein is associated with a differential inflammatory response.</a> The Journal of Immunology. 179, 3242-3248 (2007).</li></ol>

Los autores no tienen que revelar cualquier conflicto de intereses.

Macrófagos derivados de monocitos representan el tipo de célula clave del sistema inmune innato. Juegan un papel importante en muchas enfermedades inflamatorias, incluyendo la aterosclerosis o el cáncer 2. Por lo tanto, el estudio de la biología de los macrófagos es esencial para aumentar nuestro conocimiento sobre la fisiopatología de muchas enfermedades y permitir el desarrollo de terapias novedosas. Muchos estudios se aplican modelos de ratón que sobreexpresan o carecen ...

Macrófagos derivados de monocitos representan un importante componente celular del sistema inmune innato y contribuyen a muchos procesos inflamatorios agudos o crónicos 1. Los macrófagos juegan un papel importante en muchas enfermedades inflamatorias como la aterosclerosis o el cáncer 2. Los macrófagos muestran un alto grado de plasticidad y son capaces de asumir diferentes fenotipos dependiendo de la micromilieu locales 3. Por lo tanto, el estudio de la diferenciación de los macrófagos y la heterogeneidad es esencial para aumentar nuestro conocimiento de la fisiopatología de muchas enfermedades y para permitir la identificación de nuevas dianas terapéuticas y el desarrollo de terapias novedosas. En muchos casos, los modelos murinos se utilizan para investigar la fisiopatología de las enfermedades específicas. Sin embargo, el estudio de la biología de los macrófagos utilizando modelos de ratón se acompaña de varios defectos: (1) La proporción de los números de subconjuntos de leucocitos (por ejemplo, monocitos y granulocitos) en peripheral sangre de los ratones o seres humanos difiere significativamente sugiriendo diferentes funciones de los monocitos en la fisiopatología murino y humano. (2) Existen diferencias sustanciales en la expresión génica entre los monocitos de sangre periférica murinos y humanos sugieren diferencias sustanciales en su función durante la salud y la enfermedad 4. (3) Un número de marcadores que se utilizan para identificar los monocitos murinos y macrófagos (F4/80, LyC, etc.) No existe en las células mieloides humanas, por lo que la transferencia de los resultados en modelos de ratón a la situación humana bastante difícil. Por lo tanto, con el fin de aumentar nuestra comprensión de la diferenciación de los macrófagos y la heterogeneidad en la enfermedad humana, tenemos que hacer uso de modelos de trabajo con los macrófagos humanos. Por lo tanto, aquí describimos un modelo de generación de macrófagos primaria humana que es fácil de usar y permite el estudio de los macrófagos derivados de los monocitos humanos in vitro en diversas condiciones resultantes en diferentes macrófagos potipos vascularización. En varios estudios, hemos utilizado el modelo in vitro de los macrófagos derivados de los monocitos humanos primarios para analizar la biología de los macrófagos y su potencial relevancia para la aterosclerosis humana 5-7. A pesar de que no sustituir completamente a los experimentos con animales o tejidos humanos obtenidos post mortem, el modelo descrito aquí permite la identificación y validación de los mecanismos de la enfermedad y dianas terapéuticas que pueden ser altamente relevantes para diversas enfermedades humanas.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube (sterile)</td>
			<td>Fisher</td>
			<td>055398</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS (1X), liquid (no calcium or magnesium)</td>
			<td>Invitrogen</td>
			<td>14190-250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>T9285</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-8806</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EasySep Human Monocyte Enrichment Kit</td>
			<td>StemCell Technologies</td>
			<td>19059</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fisher</td>
			<td>352008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Macrophage-SFM (1X)</td>
			<td>Invitrogen</td>
			<td>12065-074</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P-4458</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nutridoma-SP</td>
			<td>Roche</td>
			<td>11011375001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>human M-CSF 10 &mu;g</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell Culture Plates 6-well</td>
			<td>Fisher</td>
			<td>07-200-80</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an in vitro model to study heterogeneity of human macrophage differentiation and polarization

Macrófagos derivados de monocitos representan un tipo de célula importante del sistema inmune innato. Los modelos de ratón que estudian la biología de los macrófagos sufren de las diferencias fenotípicas y funcionales entre los macrófagos derivados de monocitos murinos y humanos. Por lo tanto, aquí describimos un modelo in vitro para generar y estudiar los macrófagos humanos primarios. En pocas palabras, después de centrifugación en gradiente de densidad de sangre periférica extraída de una vena del antebrazo, los monocitos se aíslan a partir de células mononucleares de sangre periférica mediante el aislamiento del grano magnético negativo. Estos monocitos se cultivaron a continuación durante seis días en condiciones específicas para inducir diferentes tipos de diferenciación de los macrófagos o de polarización. El modelo es fácil de usar y evita los problemas causados ​​por las diferencias de cada especie, entre el ratón y el hombre. Por otra parte, está más cerca de las condiciones in vivo que el uso de líneas celulares inmortalizadas. En conclusión, el modelo descrito aquí es adecuado para estudiar macrófagos dee biología, identificar mecanismos de la enfermedad y nuevas dianas terapéuticas. A pesar de que no sustituir completamente a los experimentos con animales o tejidos humanos obtenidos post mortem, el modelo descrito aquí permite la identificación y validación de los mecanismos de la enfermedad y dianas terapéuticas que pueden ser altamente relevantes para diversas enfermedades humanas.

Utilizando el protocolo descrito anteriormente, que habitualmente obtenemos 25.1 sangre x 10 6 ± 2,2 x 10 6 monocytes/100 ml (media ± error estándar de 26 experimentos independientes, Figura 1A). Pureza de monocitos tal como se determina por tinción de citometría de flujo para CD14 es habitualmente mayor que 95% (97,1 ± 0,4%, media ± error estándar de 3 experimentos independientes, la Figura 1B). La viabilidad celular de los monocitos recién aislados como ...

Watch this Scientific Journal Video about An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization at JoVE.com

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Macrófagos derivados de monocitos son células importantes del sistema inmune innato. A continuación, describimos un fácil de utilizar<em&gt; In vitro</em&gt; Modelo para generar estas células. Uso de centrifugación en gradiente, el aislamiento negativa de la microesfera y las condiciones específicas de cultivo de células, los macrófagos derivados de los monocitos se pueden generar para los estudios fenotípicos y funcionales.

Un<em&gt; In vitro</em&gt; Modelo para estudiar la heterogeneidad de diferenciación de los macrófagos humanos y polarización

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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Research

JoVE Journal

Immunology and Infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

18.6K vistas.  University of Heidelberg. Macrófagos derivados de monocitos son células importantes del sistema inmune innato. A continuación, describimos un fácil de utilizar In vitro Modelo para generar estas células. Uso de centrifugación en gradiente, el aislamiento negativa de la microesfera y las condiciones específicas de cultivo de células, los macrófagos derivados de los monocitos se pueden generar para los estudios fenotípicos y funcionales.

Video: An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

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

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Investigación de la polarización de macrófagos utilizando la médula ósea macrófagos derivados

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Establecimiento de un<em&gt; In vitro</em&gt; Sistema para estudiar el comportamiento intracelular de<em&gt; Candida glabrata</em&gt; En humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In vitro e in vivo modelo para estudiar la adhesión bacteriana a la pared del recipiente en condiciones de flujo

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Un<em&gt; In Vitro</em&gt; Modelo para medir la respuesta inmune a la malaria en el contexto del VIH Co-infección

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Análisis de linfocitos extravasación El uso de una<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro humano

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

Fabricación de superficies de cristal de calidad óptica para estudiar la fusión de macrófagos

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

In Vitro Diferenciación del Factor del Granulocyte-macrófago-estimulante de colonias (GM-CSF) de ratón-producción de linfocitos de T (THGM)

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response.
Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology.
Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall.
Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls.
This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis.

Un estudio de Immunohistopathologic a perfil el folato del Receptor Beta macrófago y microambiente inmunológico Vascular en Arteritis de células gigantes

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...