We thank Dr Jamil Jubrail for reading the manuscript. This work was supported by grants from CNRS, Inserm, Universit&#233; Paris Descartes, Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM DEQ20130326518 including a doctoral fellowship for GLB) and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales (ANRS, including a post-doctoral fellowship for CD) to FN. A. Dumas was supported by doctoral fellowships from Universit&#233; Paris Descartes and Sidaction.

Das Protokoll muss in strikter Übereinstimmung mit nationalen und internationalen Gesetzen und örtlichen Vorschriften durchgeführt. Blut von gesunden Spendern, die ihre Zustimmung Blut für Forschungszwecke zu spenden gab wurde von Bluttransfusionszentren erhalten, mit denen die Institutionen Vereinbarung unterzeichnet haben. Besondere Schutzmaßnahmen getroffen werden müssen, wenn menschliches Blut verwendet wird. Experimente mit HIV-1 muss in einem Biosicherheitsstufe 3 oder 2 (BSL-3 oder 2) Labor nach den örtlichen Vorschriften durchgeführt werden. 1. Herstellung von Human-Monozyten abgeleiteten Makrophagen (hMDMs) durch Dichtegradientenzentrifugation und Auswahl durch Adhesion <ol><li> Beginnen Sie mit frischem Blut von gesunden Spendern (9 ml). Verdünne das gesamte Volumen von frischem Blut mit sterilem 1x phosphatgepufferter Kochsalzlösung (PBS) ohne Ca 2+ und Mg 2+ ein Endvolumen von 70 ml zu erhalten , und sanft das verdünnte Blut in zwei 50 ml konische Röhrchen (35 ml pro Röhrchen) hinzuzufügen , oben auf 15 ml eineutral, stark verzweigten, hoher Masse, hydrophile Polysaccharid in Lösung bereits in jedem Röhrchen. </li><li> Zentrifuge beide Rohre von Blut bei 537 xg für 20 min bei 20 ° C ohne Bremse. Dann sammeln Sie die peripheren mononukleären Blutzellen (PBMCs) in den bewölkten Zelle Ring an der Schnittstelle enthalten sind, und übertragen sie in eine neue 50 - ml - Röhrchen mit 15 ml 1x PBS ohne Ca 2+ und Mg 2+. </li><li> Zentrifuge die Zellen bei 218 · g für 5 min bei 20 ° C und das Pellet in 45 ml 1x PBS ohne Ca 2+ und Mg 2+. </li><li> Zentrifuge die Zellen bei 218 · g für 5 min bei 20 ° C, Resuspendieren des Pellets in 10 ml 1x PBS ohne Ca 2+ und Mg 2+ und die Zellen zählen , indem zu einer Endverdünnung von 1/200 in Trypan Blau verdünnt. </li><li> Zentrifugieren der Zellen bei 218 xg für 5 min bei 20 ° C und das Pellet in RPMI (Roswell Park Memorial Institute) 1640-Medium, ergänzt mit 2 mM L-Glutamin und 100 ug / ml Penicillin-Streptomycin 7 x 10 6 PBMCs in 2 ml Medium in jeder Vertiefung einer 6 - Well-Platte pro Vertiefung zu haben. </li><li> Inkubiere die Platten bei 37 ° C mit 5% CO 2 für 2 Std. Nach 2 Stunden wird die Monozyten an den Kunststoff eingehalten. </li><li> Damit frisch isolierten Monozyten in hMDMs zu unterscheiden, aspirieren das Medium und ersetzen Sie es mit 2 ml hMDM Medium (RPMI 1640, 10% decomplemented Fetale Kälberserum (FCS), 2 mM L-Glutamin und 1% Penicillin-Streptomycin), ergänzt mit rekombinantes humanes M-CSF (RHM-CSF) in einer Endkonzentration von 10 ng / ml. </li><li> Inkubieren der Zellen bei 37 ° C mit 5% CO 2 für 11 Tage (Abbildung 1). </li><li> Am Tag 11 das Medium abgenommen und gewaschen werden 2 mal mit 2 ml kaltem hMDM Medium pro Vertiefung. Weiter waschen jeder Vertiefung 2 mal mit 1 ml kaltem 1x PBS. </li><li> Zum Abnehmen ausdifferenzierten hMDMs, waschen Sie sich gut 1 mal mit 1 ml kaltem 1x PBS mit 2 mM ethylenediaminetetraacetatic Säure (EDTA) und die Zellen in 2 ml kaltem 1x PBS mit 2 mM EDTA pro Vertiefung für 15-60 min bei 4 ° C inkubieren. HINWEIS: Alternative Methoden die Zellen existieren (zB Trypsin oder Trypsin-ähnliche Aktivitäten) zu lösen , die gute Ergebnisse 11 geben könnte. </li><li> Nach Ablösung (wird von sanft bis Pipettieren und nach unten in das Bohrloch), um die Zellen zu sammeln und sie in einem 50-ml-Röhrchen auf Eis 10 ml kaltem hMDM Medium enthält. </li><li> Zentrifuge die Zellen bei 218 · g für 5 min bei 20 ° C, das Pellet in 10 ml kaltem hMDM Medium und die Zellen zählen, indem 1/20 in Trypan Blau verdünnt. </li><li> Seed die Zellen bei 1 x 10 6 hMDMs pro 35 mm Mikroskopie Glasbodenschale und Inkubation der Platten bei 37 ° C mit 5% CO 2 für 1 Tag. </li></ol> 2. Herstellung und Quantifizierung von HIV-1-Virus Stocks HINWEIS: NLR4.3 HIV-1 Gag-iGFP (Green Fluorescent Protein) trägt eine R5-trop Umschlag, Geschenk von M. Schindler 10 wird verwendet , Makrophagen zu infizieren und infizierte Zellen in Echtzeit zu sehen. <ol><li> Produzieren viralen Bestände durch Transfektion von humanen embryonalen Nieren 293T Zellen (2 x 10 6 in 100 - mm - Schale) mit 6 ug des entsprechenden provirale DNA Kommerzielle Transfektionsreagenz verwenden. </li><li> Quantifizierung der Infektiosität der Virusstämme mit den Indikatorzellen HeLa TZM-bl (das ß-Galactosidase-Gen unter der Kontrolle des HIV-1 LTR trägt) unter Verwendung von seriellen Verdünnungen der viralen durch eine ß-Galactosidase Färbung der Zellen und das Zählen gefolgt Bestände der blauen Zellen 12. </li></ol> 3. Die Infektion von hMDMs mit HIV-1 <ol><li> Füge das Virus bei einer Multiplizität der Infektion (MOI) von 0,02 bis 0,03 in 1 ml Medium zu hMDM hMDMs (plattierten Zellen in 1,13). Um zu steuern , Brunnen hinzufügen nur 1 ml hMDM Medium und Inkubation das Geschirr bei 37 ° C mit 5% CO 2 für 2 Tage (Abbildung 1). </li><li> Am Tag 2 waschen Sie die Zellen mit hMDM medium 3 Mal und 1 ml frisches hMDM Medium pro Schale. Inkubieren der Zellen bei 37 ° C mit 5% CO 2 für 6 Tage (Abbildung 1). </li></ol> 4. Opsonisierung von roten Blutkörperchen vom Schaf <ol><li> Zur Herstellung von 7 x 10 6 pro Schale SRBCs, wäscht den SRBCs zweimal in 100 ul Lösung , die 0,1% Rinderserumalbumin (BSA) in 1x PBS mit Zentrifugation bei 600 xg für 4 min enthält. </li><li> Resuspendieren der gewaschenen SRBCs in 500 ul 1x PBS / BSA 0,1% mit Kaninchen-IgG anti-SRBCs bei einer sub-agglutinierende Konzentration pro 5 ul SRBCs und Inkubation für 30 min mit Rotation bei RT. HINWEIS: Um die Unter agglutinierend Konzentration von anti-SRBCs IgG, bereiten serielle Verdünnungen von IgG (Stammkonzentration bei 13,1 mg / ml) von 1/50 bis 1 / 25.600 in 20 &amp; mgr; l in einer 96-Well-Platte bestimmen. In 2 x 10 6 SRBCs in 20 &amp; mgr; l in jede Vertiefung und stellte den Teller in einem dunklen Raum während mehrerer Stunden. Der Teil agglutinierende Konzentration ist dieVerdünnung der gut, kurz bevor die gut mit Agglutination (IgG + SRBCs ein Netzwerk bilden). </li><li> Nach der Drehung Zentrifugation der IgG-opsonisierter-SRBCs bei 600 × g für 4 min und Waschen mit 100 ul 1x PBS / BSA 0,1% mit Zentrifugation bei 600 × g für 4 min. </li><li> Resuspendieren der IgG-opsonisiertes-SRBCs in vorgewärmten Phenolrot-freiem RPMI-Medium mit 2 mM L-Glutamin und 1% Penicillin-Streptomycin (1 ml / Schale). </li></ol> 5. Live Cell Videomikroskopie Phagozytose Assay <ol><li> Verwenden eines konfokalen Abbildungssystems , wie beispielsweise einem Spinnplattensystem mit einer Heizkammer ausgerüstet bei 37 ° C mit CO 2 mit Wasser für die Befeuchtung durch eine Flasche vorbei. </li><li> Schalten Sie die Heizkammer vor dem Experiment das Mikroskoptisch zu haben, bei 37 ° C vor dem Beginn des Phagocytose-Assay. Schalten Sie das Mikroskop und Computer, und laden Sie die Bildbearbeitungssoftware. </li><li> Optimieren Sie die Imaging-Einstellungen wie Scangeschwindigkeit, magnificatIon, Auflösung usw. mindestens eine Zelle pro Feld zu haben und Bild eine jede Minute zwischen 60 bis 120 min - Rahmen. HINWEIS: Hier wird die Probe ein Rahmen jede Minute während 60 min mit 63X Objektiv abgebildet wird. </li><li> Setzen Sie die Druckschale auf dem Mikroskoptisch. Stellen Sie den Fokus und die Lage zu finden, nur eine ganze HIV-1-infizierten Makrophagen in das Feld ein. Verwenden Sie geeignete Anregungs- / Emissions-Einstellungen auf der Basis des verwendeten Abbildungssystems und der Sonde. Fügen Sie ein helles Feld (BF) Kanal phagosomes (Abbildung 1II) zu beobachten. Optimieren das Aussehen der verschiedenen Kanalbilder, indem der Prozentsatz von durchgelassenem Licht und die Belichtungszeit eingestellt wird. HINWEIS: Hier NLR4.3 HIV-1 Gag-iGFP war begeistert , eine 491 - nm - Laser mit 50 ms Zeitbelichtung mit 20% der Laser (Abbildung 1 i) verwendet wird . </li><li> Nehmen Sie die Bildschale und 1 ml SRBC - Suspension bei 7 x 10 6 SRBCs / ml in die Schale (Abbildung 1). </li><li> Zentrifuge bei 500 × g für2 min bei RT Phagozytose, notieren Sie die Zeit am Ende dieser Zentrifugation und Rück die Schale auf die Bühne zu synchronisieren. </li><li> Optimierung des Fokus und capture GFP und BF Bilder in Z-Stapel (über die Dicke der Zelle mit einem Schritt-Abstand von 0,3 um - in der Regel 20-Ebenen) mindestens 1 Stunde jede Minute für. Speichern Sie die Zeitraffer-Video im nativen Dateiformat des verwendeten Abbildungssystems. HINWEIS: Hier Zeitraffer-Filme im nativen Format gespeichert wurden, * .STK Dateien. </li></ol> 6. Die Analyse der Zeitraffer-Filme <ol><li> Für Videobearbeitung, klicken Sie auf das Dropdown-Menü &quot;Apps&quot; und auf der Registerkarte &quot;Bewertungs Multi Dimensional Data&quot; in der Video-Editing-Software. <ol><li> Um die Datei zu öffnen, klicken Sie auf &quot;Select Base-Datei&quot; und dann auf &quot;Select Directory&quot;. In der &quot;Datensätze&quot; Feld, wählen Sie den Erwerb (in .ND-Format) zu analysieren, und klicken Sie auf &quot;Ansicht&quot;. Die Daten werden in einer Tabelle mit der Zeit in der Spalte ein repräsentiertd Z-Plan in der Leitung. </li><li> Um die Infektion in einer Z-Projektion darstellen (Abbildung 2, links Platten), wählen Sie 491 nm Wellenlänge im &quot;Wavelengths&quot; und klicken Sie auf den &quot;Z Projektion&quot; Tab mit &quot;allen Ebenen&quot;. </li><li> Um eine zeitliche Abfolge analysieren, BF Wellenlänge im &quot;Wavelengths&quot; ein und wählen Sie die optimale Ebene , auf der Z-Achse wählen zu externen SRBCs unterscheiden (Abbildung 2, roten Pfeilspitze), interne SRBCs (Abbildung 2, roter Kreis) und den Kern ( Abbildung 2, blauer Kreis). </li><li> Um die Videomontagen speichern, klicken Sie auf &quot;Auswahl [X]&quot; und dann auf &quot;Load Image (s)&quot;. Schließlich die geladenen Bilder in .tif-Format speichern und Software als nächstes auf ImageJ öffnen. </li></ol></li><li> Verwenden Sie das Plugin &quot;Manuelle Spur&quot; auf der ImageJ Software die Position des Kerns und die verschiedenen phagosomes in BF Kanal (Abbildung 3) beobachtet zu messen. <ol><li> Download das &quot;Manual-Tracking&quot; Plugin auf der ImageJ Website. Auf ImageJ, öffnen Sie das Plugin und die Bildfolge analysiert werden (Abbildung 3, Schritt 1 und 2). </li><li> Geben Sie die Einstellungen wie &quot;Time Interval&quot; , die zwischen benachbarten Rahmen Höhe der Zeit darstellt und die &quot;x / y - Kalibrierung&quot; , die den Abstand pro Pixel darstellt (Abbildung 3, Schritt 3). HINWEIS: Hier speichern Sie die Bildfolge mit einem Zeitintervall von 1 Minute zwischen jedem Rahmen und x / y Kalibrierung von 0,205 &amp; mgr; m , da die 63X - Zoom und eine Kamera mit Pixelgröße von 6,45 x 6,45 &amp; mgr; m 2 verwendet werden. </li><li> Um die Verfolgung zu starten, klicken Sie auf &quot;Add Track&quot; (Abbildung 3, Schritt 4) und klicken Sie auf ein SRBC Zentrum beim ersten Mal , wenn er internalisiert wird. Das nächste Bild erscheint automatisch. </li><li> Weiter auf der SRBC - Center in allen Frames klicken verschiedenen Positionen während der Zeit zu haben (Abbildung 3, Schritt 6 im roten Kasten). <ol><li> Starten Sie den Kern zu verfolgen seine Position durch einen Klick auf das Zentrum in allen Frames zu haben. Spur folgen die phagosomes (durch seine Mitte zu klicken) nacheinander zu der Zeit (Rahmen) ihrer Verinnerlichung, unterscheidet sich in Abhängigkeit von SRBCs. Der Einfachheit halber SRBC auf BF - Kanal zu sehen, verwenden Sie die &quot;Helligkeit und Kontrast&quot; Fenster während der Tracking (Abbildung 3, Schritt 5). </li></ol></li><li> Zwischen jedem SRBC - Tracking, klicken Sie auf &quot;Add Track&quot; (Abbildung 3, Schritt 4) eine neue Spur zu haben. Die Anzahl der Tracking wird in der zweiten Spalte &quot;Track n °&quot; der Ergebnistabelle (Abbildung 3, Schritt 6) geändert werden. </li><li> Speichern der Daten in einer Tabelle mit dem nächsten Schritt der Analyse fortzusetzen. </li></ol></li><li> Verwenden , Tabellenkalkulationssoftware , die zurückgelegte Strecke von Phagosomen enthält SRBCs Richtung Kern und der Geschwindigkeit der Phagosomen während der ersten 5 min nach der Internalisierung von SRBCs zu berechnen (FiAbbildung 4). <ol><li> Öffnen Sie die Tabelle Tabelle und eine neue Tabellenkalkulationsdatei. Transfer in diese neue Datei nur die folgenden Parameter, die Zeit, die x- und y- Koordinate des Kerns und alle SRBCs (4A). </li><li> Um den Abstand zwischen SRBCs berechnen und den Kern nur mit ihren Koordinaten betrachten den Kern und einen SRBC in einem XY-Koordinatenebene (4B). HINWEIS: Der Abstand zwischen zwei Punkten ist die Länge der Strecke, die sie verbinden. In der Ebene wird der Abstand zwischen SRBC und dem Kern durch den Satz des Pythagoras gegeben. <ol><li> Wenn c (der Abstand zwischen dem Kern und dem SRBC) , um die Länge der Hypotenuse und A und b die Längen der beiden anderen Seiten bezeichnen exprimieren Pythagoras &#39;Theorem als pythagoreischen Gleichung: <img alt="Gleichung 1" src="/files/ftp_upload/54568/54568eq1.jpg" /> HINWEIS: In der gleichen Zeit auf der orthonormal, der horizontale Abstanda (x - x Kern SRBC) und der vertikale Abstand b (y -y Kern SRBC). </li><li> So berechnen Sie den Abstand zwischen dem Kern und dem SRBC (4A, lila Box) von: <img alt="Gleichung 2" src="/files/ftp_upload/54568/54568eq2.jpg" /> HINWEIS: Multiplizieren der Wert, der Abstand (in Pixeln) von x / y-Kalibrierung den Abstand in &amp; mgr; m zu haben. Hier wird die x / y-Kalibrierung ist 0,205 &amp; mgr; m. </li><li> Zu jedem Zeitpunkt, zu subtrahieren , den Abstand zwischen dem Kern und dem SRBC auf den Anfangsabstand (4C). </li><li> Zeichnen Sie die Messung gegen die Zeit für den ersten 5 min. Tragen Sie eine lineare Trendlinie (5A) auf dem Grundstück und bestimmen die Steigung der linearen Trend-Linie, die die phagosome Geschwindigkeit in Richtung des Kerns während der ersten 5 Minuten nach der SRBC Internalisierung darstellt. </li><li> Collate die Daten den Durchschnitt und die statistischen Fehler von zu berechnendie Geschwindigkeitswerte und sie in geeigneter Form zeichnen die entsprechende Software (5B) verwendet wird . </li></ol></li></ol></li></ol>

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	<li>Flannagan, R. S., Jaumouille, V., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+phagocytosis.">The cell biology of phagocytosis.</a> Annu. Rev. Pathol. 7, 61-98 (2012).</li><li>Niedergang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Cell Biology. 2, 751-757 (2016).</li><li>Blocker, A., Griffiths, G., Olivo, J. C., Hyman, A. A., Severin, F. 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S., Cosio, G., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+mechanisms+of+phagocytes+and+bacterial+evasion+strategies.">Antimicrobial mechanisms of phagocytes and bacterial evasion strategies.</a> Nat Rev Microbiol. 7, 355-366 (2009).</li><li>Feasey, N. A., Dougan, G., Kingsley, R. A., Heyderman, R. S., Gordon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+non-typhoidal+salmonella+disease:+an+emerging+and+neglected+tropical+disease+in+Africa.">Invasive non-typhoidal salmonella disease: an emerging and neglected tropical disease in Africa.</a> Lancet. 379, 2489-2499 (2012).</li><li>Mazzolini, 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=Inhibition+of+phagocytosis+in+HIV-1-infected+macrophages+relies+on+Nef-dependent+alteration+of+focal+delivery+of+recycling+compartments.">Inhibition of phagocytosis in HIV-1-infected macrophages relies on Nef-dependent alteration of focal delivery of recycling compartments.</a> Blood. 115, 4226-4236 (2010).</li><li>Dumas, 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=The+HIV-1+protein+Vpr+impairs+phagosome+maturation+by+controlling+microtubule-dependent+trafficking.">The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking.</a> J Cell Biol. 211, 359-372 (2015).</li><li>Greenberg, S., el Khoury, J., Kaplan, E., Silverstein, S. 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The authors have nothing to disclose.

Diese Technik hat mehrere wichtige Schritte. Zum einen ist die Herstellung von hMDMs und ihre Infektion durch HIV-1 wichtig, da der Prozentsatz der Infektion Spender abhängt. Bemerkenswert ist , haben wir beschlossen , Makrophagen zu verwenden , die in vitro vor der Infektion nicht polarisiert sind, weil der Status der Makrophagen durch das Virus in vivo angetroffen möglicherweise nicht gut , so weit charakterisiert. Wir überprüften die Expression verschiedener Oberflächenmarker, was darauf hinwei...

Macrophages play a major role in the innate immune system and in homeostasis. They are professional phagocytes that internalize and eliminate pathogens and debris by a process called phagocytosis 1,2. The phagosome, the closed compartment that forms after the engulfment of particulate material, undergoes a series of fusion and fission events with endocytic compartments, leading to a degradative compartment called the phagolysosome. This compartment has an acidic pH, due to the acquisition of proton-pumping v-ATPases, contains hydrolytic enzymes and is enriched in lysosomal-associated membrane proteins (LAMPs). The maturation of phagosomes is accompanied by their migration on microtubules 3,4 towards the cell center to reach a perinuclear location where lysosomes are accumulated.
Many pathogens have been reported to hijack phagosome maturation, including bacteria with intracellular lifestyles that modify the vacuolar environment where they reside 5. The Human Immunodeficiency Virus (HIV)-1 targets macrophages in addition to T cells. As macrophages are resistant to the cytopathic effects of the virus, unlike T cells, they can be considered as a reservoir for the virus. In addition, macrophages infected with HIV-1 show defective phagocytic functions and contribute to the emergence of opportunistic diseases. In particular, severe invasive non-typhoidal Salmonella disease caused by Salmonella Typhimurium ST313 has been prevalent for the last three decades in sub-Saharan African children or adults infected with HIV 6. It has been estimated that the risk of developing tuberculosis is more than 20 times greater in people living with HIV than among those without HIV infection. 
For all these reasons, it is important to better define the molecular mechanisms underlying the phagocytic defects in HIV-infected macrophages. We have shown that the uptake of particulate material, opsonized particles, bacteria or fungi, was inhibited in HIV-infected macrophages 7. Given that this inhibition is partial, we then set out to analyze the fate of the internalized phagosomes in HIV infected human macrophages 8. Because phagosome maturation is tightly connected with migration to the cell center and fusion with lysosomes, a defect in phagosomal maturation can be due to modifications of the trafficking modalities in the infected cell. The method described here uses IgG-opsonized Sheep Red Blood Cells (IgG-SRBCs) as a model to target receptor-mediated phagocytosis and in particular receptors for the Fc portion of immunoglobulins (FcR). These particles are easier to image in bright field (BF) than latex beads because extracellular and intracellular SRBCs show different refraction properties 9. To measure the velocity of phagosomes moving towards the nucleus in HIV-infected macrophages, we used a fluorescent virus 10 and set up a simple manual tracking method that is described here. The method does not require advanced programming and simply uses ImageJ. It is amenable to adherent cells and any type of particle or pathogen that can be visualized with bright field microscopy or with fluorescent imaging.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Falcon 100mm TC-Treated Cell Culture Dish</td>
			<td style="width:124px;">Corning</td>
			<td style="width:111px;">353003</td>
			<td style="width:245px;">For viral production</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Glass Bottom Dishes 35 mm uncoated 1.5</td>
			<td style="width:124px;">MatTek corporation</td>
			<td style="width:111px;">P35G-1.5-14-C Case</td>
			<td style="width:245px;">For acquisition</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Falcon Tissue Culture Plates 6-well</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">Corning. Inc. 353934</td>
			<td style="width:245px;">For human monocyte-derived macrophages</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Ficoll-Plaque PLUS</td>
			<td style="width:124px;">Dominique Dutscher</td>
			<td style="width:111px;">17-1440-03</td>
			<td style="width:245px;">a neutral, highly branched, high-mass, hydrophilic polysaccharide in solution for density centrifugation</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">DPBS, no calcium, no magnesium</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">14190-094</td>
			<td style="width:245px;">Room temperature</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) 1X, liquid (High Glucose)</td>
			<td style="width:124px;">GIBCO, Molecular probes</td>
			<td style="width:111px;">31966-021</td>
			<td style="width:245px;">Conserved at 4&#176;C ; for HEK cells culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">RPMI 1640 medium GLUTAMAX&#160; Supplement</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:111px;">61870-010</td>
			<td style="width:245px;">Conserved at 4&#176;C; for hMDMs culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">F&#339;tal Calf Serum (FCS)&#160;</td>
			<td style="width:124px;">Eurobio</td>
			<td style="width:111px;">CVFSVF0001</td>
			<td style="width:245px;">Conserved at -20&#176;C ; decomplemented</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">15140-122</td>
			<td style="width:245px;">Conserved at -20&#176;C ; for hMDMs culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">RPMI 1640 medium, no phenol red (10x500 ml)</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:111px;">11835-105</td>
			<td style="width:245px;">Warm in 37&#176;C water bath before use ; for phagocytosis assay</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">FuGENE6 Transfection Reagent</td>
			<td style="width:124px;">Promega</td>
			<td style="width:111px;">E2692</td>
			<td style="width:245px;">Conserved at 4&#176;C ; for viral production</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Sheep red blood cells (SRBCs)</td>
			<td style="width:124px;">Eurobio</td>
			<td style="width:111px;">DSGMTN00-0Q</td>
			<td style="width:245px;">Conserved in Alsever buffer at 4&#176;C before use</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Anti-sheep red blood cells IgG</td>
			<td style="width:124px;">MP Biomedicals</td>
			<td style="width:111px;">55806</td>
			<td style="width:245px;">Conserved at 4&#176;C</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Bovine Serum Albumin heat shock fraction, pH 7, &#8805;98%&#160;</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:111px;">A7906</td>
			<td style="width:245px;">Conserved at -20&#176;C</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Inverted microscope DMI600</td>
			<td style="width:124px;">Leica</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Confocal Spinning Disk Unit&#160; CSU-X1M1</td>
			<td style="width:124px;">Yokogawa</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">491 nm 50mW laser</td>
			<td style="width:124px;">COBOLT CALYPSO</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">HCX PL APO CS Objectif</td>
			<td style="width:124px;">Leica</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">Objective lens ; Magnification 100x ; Numerical aperture 1.40 ; Immersion oil</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">CoolSnap HQ2 (FireWire) Camera</td>
			<td style="width:124px;">Photometrics</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">Pixel size 6.45 x 6.45 &#181;m ; Definition 1392 x 1040 ; Encoding the image in 14 Bit</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Metamorph 7.7.5 software</td>
			<td style="width:124px;">Molecular Devices</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">For the control of the microscope</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">GraphPad Prism software</td>
			<td style="width:124px;"></td>
			<td style="width:111px;"></td>
			<td style="width:245px;">For the statistics analysis</td>
		</tr>
	</tbody>
</table>

phagosome migration and velocity measured in live primary human macrophages infected with hiv-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

FcR-vermittelte Phagozytose durch HIV-1-infizierten und nicht-infizierten hMDMs wird hier beschrieben unter Verwendung von IgG-opsonisiertes SRBCs als Modellzielen (Abbildung 1). Die kritischen Schritte dieses Protokolls sind die Herstellung von hMDMs und die Infektion mit HIV-1. Tatsächlich variiert die Ausbeute und die Qualität der differenzierten Makrophagen unter den Gebern sowie die Infektionsrate mit einem Wirkungsgrad im Bereich von 10-40%. Darüber hinaus ist d...

Watch this Scientific Journal Video about Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 at JoVE.com

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Phagosom Migration und Geschwindigkeit gemessen in Live-primären humanen Makrophagen mit HIV-1 infizierten

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

phagosome-migration-velocity-measured-live-primary-human-macrophages

Research

JoVE Journal

Immunology and Infection

8.1K Aufrufe.  Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité. We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes. 

Serie: Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Genotypische Inference von HIV-1 Tropismus mit Populations-basierte Sequenzierung von V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Forschung

Immunologie und Infektion

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Herstellung und Verwendung von HIV-1 infizierten primären CD4 + T-Zellen als Zielzellen in der natürlichen Killerzellen Zytotoxische Assays

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Verstärkung und Quantifizierung von HIV-1 RNA in HIV-infizierten Personen mit Viruslast unter der Nachweisgrenze von Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

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.

In vitro Uncoating von HIV-1-Kerne

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 ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Imaging von HIV-1 Envelope-induzierte virologische Synapse und Signalisierung auf Synthetic Lipiddoppelschichten

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

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.

Ein<em&gt; In-vitro-</em&gt; Co-Infektion Modell zum Studium<em&gt; Plasmodium falciparum</em&gt; HIV-1-Wechselwirkungen in primären menschlichen Monozyten abgeleitete Immunzellen

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

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

Neue Tools erweitern regulatorischen T-Zellen von HIV-1-infizierten Personen

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Studium der Biogenese Phagolysosom im Live-Makrophagen

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Beurteilung der Innate Sensing von HIV-1-infizierten CD4<sup&gt; +</sup&gt; T-Zellen durch Plasmazytoide Dendritische Zellen unter Verwendung eines<em&gt; Ex-vivo-</em&gt; Co-Kultursystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

Messung von<em&gt; In Vitro</em&gt; Integration Aktivität von HIV-1 Preintegration Komplexe

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...