Diese Arbeit wurde gefördert durch Gewährung PRB-009 CS und ein Promotionsstipendium an LC, aus dem Secretaría Nacional de für Wissenschaft und Technologie (SENACYT), Panama.

Alle Schritte des Protokolls, mit Ausnahme Zentrifugationen, sollte innerhalb einer Haube zu halten die Probe steril erfolgen. Ein. Late Stage Isolierung von P. falciparum-infizierten Erythrozyten Alle späten Stadien von Plasmodium-infizierten Erythrozyten kann mit dieser Methode getrennt werden, da Hemozoin, die Paramagnetismus überträgt der Parasit, ist eine gemeinsame Metabolit zur Gattung. Eine hohe Parasitämie (3-10%) in der Kultur wird empfohlen, bessere Ausbeuten bei diesem Protokoll zu erhalten, obwohl eine typische Kultur 2-4% Hämatokrit (Volumenprozentsatz von roten Blutkörperchen) und 1-8% Parasitämie (Prozentsatz der infizierten enthalten wird rote Blutzellen). Parasiten können kultiviert wie durch Trager und Jansen 1 beschrieben. <ol><li> Eine einfache, mehrteilige Installation muss für jeden Schizonten Verfahren zur Isolierung montiert werden. Dafür LS Säulen, ein Magnetic MidiMACS Separator und eine MACS MULTISTAND von Miltenyi Biotec (Abbildung 1 </strong&gt;) verwendet. Zuerst bringen Sie den Magnetabscheider der Metall-Ständer und dann die magnetischen Säule auf dem Separator. </li><li> In einem separaten Röhrchen (verwenden Sie 50 ml Schrägen überall), mischen 23,2 ml RPMI 1640 und 750 ul von 7,5% Natriumbicarbonat, um die Arbeit Lösung zu schaffen. Zusätzliche Röhren an Hand zum Verwerfen und Sammeln der Durchströmung des Parasiten Kultur. </li><li> 3 ml der Arbeit auf die Säule zu äquilibrieren, und entsorgen Sie die Flow-Through in ein Rohr. </li><li> 8 ml des P. falciparum Kultur auf die Säule, um sicherzustellen, dass, sobald der letzte Teil der Arbeit Lösung wurde aus der Säule durch die Kultur geschoben wird das Aufnahmerohr durch das Sammeln von ein bis Gewinnung des ungebundenen Kultur beginnen ersetzt. Dies ist notwendig, um zu vermeiden Verdünnen der Kultur nicht weiter. </li><li> Sobald die Strömung der Kultur durch die Säule hat aufgehört, 1 ml der Arbeit Lösung, um die verbliebenen Erythrozyten Push aus der Säule. </li><li> Waschschritt: Hinzufügen weitere 3 ml der Arbeit auf die Säule und sammelt das strömende Flüssigkeit in den &quot;Discard&quot; Rohr zur Vermeidung Verdünnen der Kultur als Durchströmung in dem anderen Rohr gespeichert. </li><li> Elution: Sobald die 3 ml durch die Säule passiert haben, lösen diese aus dem Stand und legen Sie sie auf einer 15-ml-Tube. </li><li> Wieder, 3 ml der Arbeit auf die Säule. Dieser Schritt eluiert die Erythrozyten mit späten Stadium Parasiten, die in den Wülsten der Säule eingefangen wurden infiziert. </li></ol> Hinweis: An dieser Stelle, und je nach der Menge der Parasiten erforderlich, starten eine neue Runde / s für die Sammlung, natürlich in den Grenzen der Parasitämie in der ursprünglichen Kultur. Die Spalte können so lange wiederverwendet werden, da der Strom einen stationären Zustand aufrechterhält. <ol start="9"><li> Konzentration der gesammelten Parasiten: zentrifugieren 15 ml Röhrchen mit dem Eluat für 5 min bei 150 × g bei Raumtemperatureine dunkle Pellets aus befallenen Erythrozyten zu bilden. </li><li> Entfernen Sie den Überstand, wobei genügend Flüssigkeit, um eine sehr kleine Probe nehmen und bestimmen die Konzentration und Gesamtertrag durch Mikroskopie (siehe Schritt 2 unten). </li><li> In diesem Moment, wenn die Isolierung und Erfassung von späten Stadium Parasiten zufriedenstellend durchgeführt wird, Inkubieren der Kultur gesammelt wieder mit frischen Medien. </li><li> Verdünnen Sie die gesammelten Parasiten im Volumen und die Lösung für weitere Versuche notwendig. </li></ol> 2. Reinheit und Ausbeute Analysis <ol><li> Überprüfen der Konzentration von Parasiten unter Verwendung eines Hämozytometers und Zählen unter einem Lichtmikroskop erzielt. Zu zählen, fügen Sie 10 ul der Mischung aus den gesammelten Parasiten und den Medien unter dem Deckglas des Hämozytometer. Zähle die Anzahl der Parasiten in den vier Quadranten des Hämozytometer und teilen die Gesamtzahl von 4. Multiplizieren Sie diese Zahl durch 10 4, um die Konzentration von infizierten E zu erhaltenrythrocytes pro ml enthält. </li></ol> Anmerkungen: a) Die Konzentration der Mischung wird durch den Benutzer im Moment des Resuspendieren des Pellets entschieden werden, indem mehr oder weniger Medium. Ein Vorschlag Arbeitskonzentration beträgt 5,5 X 10 4 schizontes pro ul (dies wird in der Regel durch Zugabe von 100-300 ul zu den gesammelten Parasiten erreicht). Wenn 20 ul dieser Konzentration auf ein Endvolumen von 100 ul Medienmix und Erythrozyten, eine Parasitämie von 1% in einer typischen 4% Hämatokrit Kultur zugegeben wird erhalten wird. b) Zählen unter dem Hämozytometer muss schnell sein, da die Erythrozyten lysiert, wenn die Probe trocken im Inneren der Kammer zu beginnen. Infizierten Erythrozyten, die die Mehrheit sein sollte, zeigen einen dunklen Fleck im Inneren, die sie aus dem nicht-infizierten. <ol start="2"><li> Führen Sie eine Dünnschicht-Giemsa-Färbung, um die Reinheit der Sammlung durch die Fixierung der Folien sehr schnell in 10% Methanol, Trocknen und Eintauchen t beurteilen,Saum in einer 20% Lösung von Giemsa mit destilliertem Wasser verdünnt Fleck. Flecken auf den Objektträger für 10 min, nach dem sie von der Luft getrocknet werden kann und unter dem Mikroskop untersucht. A Parasitämie deutlich über 70% erreichbar sein. </li></ol>

<ol>
	<li>Jensen, J. B., Trager, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+in+culture:+use+of+outdated+erythrocytes+and+description+of+the+candle+jar+method.">Plasmodium falciparum in culture: use of outdated erythrocytes and description of the candle jar method.</a> J. Parasitology. 63 (5), 883-886 (1977).</li><li>Guzman, I. Y., Francis, S. E., Oksman, A., Smith, C. E., Duffin, K. L., Goldberg, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+and+specificity+of+the+Plasmodium+falciparum+hemoglobin+degradation+pathway.">Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway.</a> J. Clin. Invest. 93, 1602-1608 (1994).</li><li>Rosenthal, P. J., Meshnick, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemoglobin+catabolism+and+iron+utilization+by+malaria+parasites.">Hemoglobin catabolism and iron utilization by malaria parasites.</a> Mol. Biochem. Parasitol. 83 (2), 131-139 (1996).</li><li>Fitch, C. D., Chevli, R., Kanjananggulpan, P., Dutta, P., Chevli, K., Chou, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+ferriprotoporphyrin+IX+is+a+lytic+agent.">Intracellular ferriprotoporphyrin IX is a lytic agent.</a> Blood. 62 (6), 1165-1168 (1983).</li><li>Hebbel, R. P., Eaton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathobiology+of+heme+interaction+with+the+erythrocyte+membrane.">Pathobiology of heme interaction with the erythrocyte membrane.</a> Semin. Hematol. 26 (2), 136-149 (1989).</li><li>Egan, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemozoin+formation.">Haemozoin formation.</a> Mol. Biochem. Parasitol. 157 (2), 127-136 (2008).</li><li>Hempelmann, E., Marques, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+malaria+pigment+from+Plasmodium+falciparum.">Analysis of malaria pigment from Plasmodium falciparum.</a> J. Pharmacol. Toxicol. Methods. 32 (1), 25-30 (1994).</li><li>Fitch, C. D., Kanjananggulpan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+ferriprotoporphyrin+IX+in+malaria+pigment.">The state of ferriprotoporphyrin IX in malaria pigment.</a> J. Biol. Chem. 262 (32), 15552-15555 (1987).</li><li>Moore, L. R., Fujioka, H., Williams, P. S., Chalmers, J. J., Grimberg, B., Zimmerman, P. A., Zborowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemoglobin+degradation+in+malaria-infected+erythrocytes+determined+from+live+cell+magnetophoresis.">Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis.</a> FASEB J. 20 (6), 747-749 (2006).</li><li>Spadafora, C., Gerena, L., Kopydlowski, K. M. <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+the+in+vitro+invasive+capabilities+of+Plasmodium+falciparum+schizonts+isolated+by+Percoll+gradient+or+using+magnetic+based+separation.">Comparison of the in vitro invasive capabilities of Plasmodium falciparum schizonts isolated by Percoll gradient or using magnetic based separation.</a> Malaria J. 10, 96 (2011).</li><li>Jackson, K. E., Spielmann, T., Hanssen, E., Adisa, A., Separovic, F., Dixon, M. W., Trenholme, K. R., Hawthorne, P. L., Gardiner, D. L., Gilberger, T., Tilley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+permeabilization+of+the+host+cell+membrane+of+Plasmodium+falciparum-infected+red+blood+cells+with+streptolysin+O+and+equinatoxin+II.">Selective permeabilization of the host cell membrane of Plasmodium falciparum-infected red blood cells with streptolysin O and equinatoxin II.</a> Biochem. J. 403, 167-175 (2007).</li><li>Goodyer, I. D., Johnson, J., Eisenthal, R., Hayes, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+mature-stage+Plasmodium+falciparum+by+gelatine+flotation.">Purification of mature-stage Plasmodium falciparum by gelatine flotation.</a> Ann. Trop. Med. Parasitol. 88 (2), 209-211 (1994).</li><li>Pasvol, G., Wilson, R. J., Smalley, M. E., Brown, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+viable+schizont-infected+red+cells+of+Plasmodium+falciparum+from+human+blood.">Separation of viable schizont-infected red cells of Plasmodium falciparum from human blood.</a> Ann. Trop. Med. Parasitol. 72, 87-88 (1978).</li><li>Pertoft, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+cells+and+subcellular+particles+with+Percoll.">Fractionation of cells and subcellular particles with Percoll.</a> J. Biochem. Biophys. Methods. 44 (1-2), 1-30 (2000).</li><li>Paul, F., Roath, S., Melville, D., Warhurst, D. C., Osisanya, J. O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+malaria-infected+erythrocytes+from+whole+blood:+use+of+a+selective+high-gradient+magnetic+separation+technique.">Separation of malaria-infected erythrocytes from whole blood: use of a selective high-gradient magnetic separation technique.</a> The Lancet. 318, 70-71 (1981).</li><li>Trang, D. T., Huy, N. T., Kariu, T., Tajima, K., Kamei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+concentration+of+malarial+parasite-infected+red+blood+cells+and+removal+of+contaminating+white+blood+cells.">One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells.</a> Malar. J. 3, 7 (2004).</li><li>Nillni, E. A., Londner, M. V., Spira, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+separation+of+uninfected+erythrocytes+from+those+infected+with+Plasmodium+berghei+and+for+isolation+of+artificially+released+parasites.">A simple method for separation of uninfected erythrocytes from those infected with Plasmodium berghei and for isolation of artificially released parasites.</a> Z. Parasitenkd. 64, 279-284 (1981).</li></ol>

Wir haben nichts zu offenbaren.

In-vitro-Kulturen des Malaria-Parasiten P. falciparum Parasitämie weisen eine begrenzte, mit mehr als der Hälfte der nicht infizierten Erythrozyten auf dem höchsten Punkt der Proliferation Kultur. Für die meisten Forschungs-Experimenten ist es wünschenswert, dass es nur mit den infizierten Erythrozyten. Zu diesem Zweck ist ein Trennverfahren notwendig, die Kultur nach der Infektion zu unterteilen. Nützliche Verfahren umfassen die Verwendung von Streptolysin O zu permeabilisieren und lysieren die ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> RPMI 1640 Hepes Geändert </td><td> Sigma-Aldrich </td><td> R4130 </td><td> Ergänzt mit 10% menschlichem Serum, 2% Glucose und 0,2% Natriumbicarbonat </td></tr><tr><td> MidiMACS Separator </td><td> MACS Miltenyi Biotec </td><td> 130-042-302 </td><td></td></tr><tr><td> MACS MULTISTAND </td><td> MACS Miltenyi Biotec </td><td> 130-042-303 </td><td></td></tr><tr><td> LS Columns </td><td> MACS Miltenyi Biotec </td><td> 130-042-401 </td><td></td></tr><tr><td> Hämazytometer </td><td> GRAFCO </td><td> GRAFCO Neubauer Kammer </td><td> Kann durch viele andere Lieferanten gefunden werden </td></tr></tbody></table>

separation of plasmodium falciparum late stage-infected erythrocytes by magnetic means

Im Gegensatz zu anderen Plasmodium-Arten, P. falciparum kann im Labor, die ihre Studie 1 erleichtert kultiviert werden. Während die Parasitämie erreicht die ≈ 40%-Grenze erreichen kann, die Ermittler in der Regel hält der Anteil bei rund 10%. In vielen Fällen ist es notwendig, die Parasiten-haltigen roten Blutkörperchen (Erythrozyten) von den infizierten diejenigen, zu isolieren, um die Kultur zu bereichern und fahren Sie mit einem gegebenen Experiment. Wenn P. falciparum infiziert den Erythrozyten, verschlechtert sich die Parasiten und speist von Hämoglobin 2, 3. Allerdings muss der Parasit mit einem sehr giftig eisenhaltigen Häm-Einheit 4, 5 umzugehen. Der Parasit entzieht seiner Toxizität durch die Umwandlung der Häm in einem inerten Kristall Polymer namens haemozoin 6, 7. Diese eisenhaltigen Molekül in seiner Nahrungsvakuole gespeichert und das Metall in sich hat einen oxidativen Zustand, der sich von dem in Häm 8 unterscheidet. Die Eisen-III von Eisen in der Häm-ozoin verleiht es eine paramagnetische Eigenschaft fehlt in infizierten Erythrozyten. Da die eindringenden Parasiten Reife erreicht, der Inhalt haemozoin erhöht auch 9, die noch Paramagnetismus schenkt den letzten Stadien von P. falciparum Inneren der Erythrozyten. Basierend auf dieser paramagnetische Eigenschaft, die neuesten Stadien von P. falciparum infizierten Erythrozyten-Zellen können durch Passieren der Kultur über eine Säule mit magnetischen Kügelchen abgetrennt werden. Diese Perlen werden magnetisch, wenn die Spalten enthält sie auf einem Magnethalter gestellt werden. Infizierten Erythrozyten, die aufgrund ihrer Paramagnetismus, werden dann im Inneren der Säule eingefangen werden, während die Flow-Through enthält, zum größten Teil nicht infizierten Erythrozyten und solchen mit frühen Stadien des Parasiten. Hier beschreiben wir die Methodik, um die Bevölkerung der späten Phase Parasiten mit magnetischen Säulen, die gute Parasiten Lebensfähigkeit 10 hält bereichern.Nach Durchführung dieses Verfahrens kann das ungebundene Kultur in einen Inkubator zurückgegeben werden, damit die verbleibenden Parasiten weiter wachsen.

In Abbildung 2 wird das Kulturmedium durch den magnetischen Spalte gezeigt, vor (A) und nach dem Verfahren (B). Ein bis zwei infizierten Erythrozyten werden in der Regel auf einem 100-facher Vergrößerung Feld gesehen, wie in 2 gezeigt, wobei die Pfeile zeigen auf infizierten Erythrozyten in 2A. In einem typischen Verfahren, beginnend mit einem Kulturmedium mit 5% Parasitämie (2A), die Leistung dieses Verfahrens erzeu...

Watch this Scientific Journal Video about Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means at JoVE.com

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

Die paramagnetischen Eigenschaften des Hämozoins werden verwendet, um späten Stadien der Isolierung<em&gt; Plasmodium falciparum</em&gt; Infiziert rote Blutkörperchen wachsen in der Kultur. Die Methode ist einfach und schnell und hat keinen Einfluss auf die nachfolgenden invasiven Fähigkeiten der Parasiten.

Trennung<em&gt; Plasmodium falciparum</em&gt; Late Stage-infizierten Erythrozyten durch magnetische Mittel

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

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Research

JoVE Journal

Immunology and Infection

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

13.8K Aufrufe.  Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP). Die paramagnetischen Eigenschaften des Hämozoins werden verwendet, um späten Stadien der Isolierung Plasmodium falciparum Infiziert rote Blutkörperchen wachsen in der Kultur. Die Methode ist einfach und schnell und hat keinen Einfluss auf die nachfolgenden invasiven Fähigkeiten der Parasiten.

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

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

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

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

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Auswahl der Plasmodium falciparum Parasiten für Cytoadhesion zu Human Brain Endothelial Cells

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

Forschung

Immunologie und Infektion

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 ...

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

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

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

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

Ein einfaches Protokoll für Thrombozyten-vermittelte Verklumpung<em&gt; Plasmodium falciparum</em&gt;-Infizierten Erythrozyten in einer Ressource Schlechte Einstellung

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

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0&#8211;1%. 10 &#181;L of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

Erkennung und Quantifizierung von Plasmodium Falciparum in wässrigen roten Blutkörperchen durch gedämpft totale Reflexion Infrarotspektroskopie und Multivariate Datenanalyse

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total ...

Chemie

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

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

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

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

High Yield Reinigung von<em&gt; Plasmodium falciparum</em&gt; Merozoiten Zur Verwendung in opsonierende Antikörper-Assays

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

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

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

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

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

Methoden, um die regulatorische Rolle kleiner RNAs und Ribosomal Belegung von Untersuchen<em&gt; Plasmodium falciparum</em

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

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Auswertung der extrazellulären Vesikel Funktion im Malaria-Infektion

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Analyse von Einzel-Zell-Gene Transkription durch RNA-Fluorescent<em&gt; In Situ</em&gt; Hybridisierung (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Biologie

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Messung natürlich erworbener Phagozytose-induzierender Antikörper gegen Plasmodium falciparum Parasiten durch einen Flow Cytometry-based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

In vitro Untersuchung der Abtötung von Plasmodium-infizierten roten Blutkörperchen durch zytotoxische Lymphozyten

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

Trennung

Zusammenfassung

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Ein

Ein einfaches Protokoll für Thrombozyten-vermittelte Verklumpung

Erkennung und Quantifizierung von Plasmodium Falciparum in wässrigen roten Blutkörperchen durch gedämpft totale Reflexion Infrarotspektroskopie und Multivariate Datenanalyse

High Yield Reinigung von

Methoden, um die regulatorische Rolle kleiner RNAs und Ribosomal Belegung von Untersuchen

Auswertung der extrazellulären Vesikel Funktion im Malaria-Infektion

Analyse von Einzel-Zell-Gene Transkription durch RNA-Fluorescent

Messung natürlich erworbener Phagozytose-induzierender Antikörper gegen Plasmodium falciparum Parasiten durch einen Flow Cytometry-based Assay

In vitro Untersuchung der Abtötung von Plasmodium-infizierten roten Blutkörperchen durch zytotoxische Lymphozyten