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. 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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

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Ex vivo Expansion von Tumor-reaktiven T-Zellen mittels Bryostatin 1/Ionomycin und der Gemeinsamen gamma-Kette Zytokine Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Forschung

Immunologie und Infektion

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 ...

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 ...

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

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

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

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

Isolierung und Analyse von Brain-sequestered Leukozyten aus<em&gt; Plasmodium berghei</em&gt; ANKA-infizierte Mäuse

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

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

Automatische Trennung von<em&gt; C. elegans</em&gt; Variabel durch einen bakteriellen Erreger kolonisiert

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

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 ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

In-vivo-Bewertung der Nager Plasmodium Parasitämie und Merozoite Invasion durch Durchflusszytometrie

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

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 ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Biolumineszenz-Bildgebung zur Erkennung von Late Stage-Infektion der afrikanischen Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...