Ringraziamo Francesco Candal, CDC Technology Transfer Office, Atlanta Georgia per HBEC-5i cellule. Questo lavoro è stato finanziato dal Wellcome Trust (4 volte all&#39;anno borsa di studio di dottorato di AC e Senior Fellowship in Basic Scienze Biomediche alle JAR, concedere numero 084226).

Raccomandazioni generali Umano microvascolari cerebrali delle cellule endoteliali (HBEC-5i) la cultura è stato precedentemente descritto in 6, 8. P. parassiti falciparum sono stati coltivati ​​come in 9. Entrambi HBEC-5i e P. falciparum culture parassita deve essere conservato in condizioni sterili in ogni momento. Tutti i reagenti devono essere pre-riscaldato a 37 ° C. Si consiglia di controllare regolarmente la contaminazione da micoplasma 10 mediante PCR (Minevera Biolabs, le istruzioni del produttore successivo). Il protocollo è riassunto nella Figura 1. 1. Cellule endoteliali cultura di routine <ol><li> Preparare i reagenti necessari. </li></ol><table border="1"><tbody><tr><td> Medio per preparare </td><td> Reagenti </td><td> Quantità </td></tr><tr><td> &quot;Incompleto DMEM&quot; </td><td> DMEM-F12 Ham </td><td> 500ml </td></tr><tr><td> &amp;nbsp; </td><td> L-glutammina 200mm </td><td> 5ml </td></tr><tr><td></td><td> Penicillina / streptomicina 100X </td><td> 5 ml </td></tr><tr><td></td><td> NaOH 1M </td><td> 1,3 ml (aggiustare il pH a 7,4) </td></tr><tr><td> &quot;Completo DMEM&quot; </td><td> &quot;Incompleto DMEM&quot; </td><td> 450ml </td></tr><tr><td></td><td> Siero fetale bovino inattivato al calore </td><td> 50ml </td></tr><tr><td></td><td> Supplemento di crescita delle cellule endoteliali </td><td> 5 ml </td></tr></tbody></table> <ol start="2"><li> Cultura HBEC-5i in una reflex 25 centimetri 2 pallone con media 10ml DMEM completo in un incubatore a 37 ° C con 5% di CO 2. </li><li> Celle di passaggio quando diventano confluenti. Rimuovere il supporto di vecchia aspirazione e lavare due volte con medium DMEM incompleti o tessuto della cultura di grado PBS (Ca 2 + e Mg 2 + libero) pre-riscaldato a 37 ° C. </li><li> Aggiungere 1 ml di pre-riscaldato Provapsin-EDTA (0,025% tripsina, 0.5MM EDTA), agitare per coprire la totalità del pallone e incubare per circa 2 min a 37 ° C. </li><li> Controllare al microscopio invertito che almeno il 90% delle cellule sono stati staccati. In caso di necessità, bussare delicatamente la parte inferiore del pallone per staccare le cellule aderenti. Aggiungere 10 ml di medium DMEM completo di bloccare le cellule tripsina e trasferire in una provetta da 15 ml conica. Centrifugare per 4 min a 300 gr a temperatura ambiente (RT). </li><li> Eliminare il surnatante e risospendere il pellet con 10 ml di medium DMEM completo. Pipettare la soluzione su e giù per risospendere completamente le cellule. </li><li> Aggiungere 1 o 2 ml di sospensione cellulare in un pallone nuova cultura e aggiungere 8 ml di terreno fresco per mantenere la cultura. Valutare la crescita della cultura ogni giorno sotto un microscopio rovesciato e il cambiamento di media ogni 2 o 3 giorni prima che diventa gialla. </li></ol> 2. Preparazione cellule endoteliali per una selezione <ol><li> Due giorni prima del giorno del Selectio<html> n, aggiungere fibronectina diluito in PBS sterile (2 mg / cm 2) in uno (o più) 60 millimetri piatto di Petri. Incubare il piatto per 5 a 20 min a 37 ° C, quindi rimuovere la soluzione di fibronectina, che possono essere conservati a 4 ° C per un mese e riutilizzati una volta. </li><li> Celle di passaggio, come descritto nei paragrafi 1.3. a 1,5. Assumendo che le cellule del 100% confluenti sono state staccate e risospesi in media 10ml DMEM completo (sezione 1.6., Risospendere con un volume equivalente di media se la confluenza è inferiore, ad esempio, risospendere con 8ml se la confluenza è stata 80%), aggiungere 1,5 ml di sospensione cellule alla fibronectina piatto rivestito di Petri e un altro 1,5 ml di medium DMEM completo. </li><li> Porre la capsula Petri seminati in incubatrice. Nota: Idealmente, la confluenza sarà di circa il 90% al momento della selezione di due giorni dopo. </li><li> Opzionale. Per attivare HBEC-5i, aggiungere la (fattore di necrosi tumorale) citochina TNF ad una concentrazione finale di 50μg/ml 24 ore precedenti la selezione. </li></ol>e_title &quot;&gt; 3. Plasmodium falciparum routine cultura <ol><li> Preparare i reagenti necessari (vedi tabella qui sotto). Preparare umano globuli rossi (RBC) separando sangue intero (gruppo O +) dal passaggio attraverso un filtro a esaurimento dei leucociti (vedere &quot;Metodi di Ricerca in Malaria&quot; pubblicazione 11 per la coltura generale parassiti della malaria). Lavare RBC due volte per centrifugazione a 400 g per 5 min e risospendere con 10 ml RPMI incompleta. Tenere RBC lavati a 4 ° C in media incompleta al 50% di ematocrito. </li></ol><table border="1"><tbody><tr><td> Medio per preparare </td><td> Reagenti </td><td> Quantità </td></tr><tr><td> &quot;Incompleto RPMI&quot; </td><td> RPMI 1640 (con il bicarbonato) </td><td> 500ml </td></tr><tr><td></td><td> Hepes 1M </td><td> 12.5ml </td></tr><tr><td></td><td> Glucosio 20% </td><td> 5ml </td></tr><tr><td></td&gt; <td> L-glutammina 200mm </td><td> 5ml </td></tr><tr><td></td><td> Gentamicina 50mg/ml </td><td> 250μl </td></tr><tr><td></td><td> NaOH 1M </td><td> 0,7 ml (aggiustare il pH a 7,2) </td></tr><tr><td> &quot;RPMI completo&quot; </td><td> &quot;Incompleto RPMI&quot; </td><td> 450ml </td></tr><tr><td></td><td> Pool umano (non immune) nel siero </td><td> 50ml </td></tr></tbody></table> <ol start="2"><li> Cultura P. falciparum dei globuli rossi infetti con il mezzo RPMI completo al 2% di ematocrito ed incubare a 37 ° C con il 3% di CO 2, 1% O 2, e 96% N 2. Fai striscio Giemsa 11 al giorno per valutare lo stadio di sviluppo di parassiti (Figura 2). </li><li> Regolarmente (circa una volta alla settimana), sincronizzare la cultura da un trattamento sorbitolo 12. Il giorno prima il test di selezione, allo scopo di un anello allo stadio cultura parassitemia al 5% o più (idealmente almeno 10%). </li></ol> 4. Selezione di P. falciparum per cytoadhesion alle cellule endoteliali <ol><li> Il giorno del test, la cultura parassita dovrebbe essere in fase vegetativa pigmentate (Figura 2) (idealmente parassitemia 10%), mentre HBEC-5i cultura dovrebbe essere da 50 a 100% confluenza (idealmente 90%). 30μl di ematocrito di cultura parassita è necessaria per HBEC-5i piastra di Petri. </li><li> Lavare i parassiti due volte per centrifugazione (500 g per 5 min) 1,5 ml di cultura parassita. Gettare il surnatante e risospendere con 10 ml di appena fatto, riscaldato, medio DMEM incompleta. Ripetere il lavaggio una seconda volta. Risospendere il volume di 30μl di ematocrito, con 1,5 ml di incompleto con 1% di BSA DMEM. </li><li> Lavare i HBEC-5i piatto rivestito di Petri due volte aspirante medio e aggiungendo 3 ml di DMEM incompleta. </li><li> Aggiungere la soluzione di parassiti al HBEC-5i piatto e incubare a 37 ° C per 75 min. Risospendere parassiti due volte (dopo 30 e 60 minuti) durante l&#39;incubazionedelicatamente a dondolo il piatto nelle quattro direzioni, così come in senso orario e antiorario. </li><li> Dopo l&#39;incubazione, lavare il piatto 5 volte aspirando mezzo, utilizzando una pipetta Pasteur di plastica per aggiungere 3 ml di medium caldo DMEM incompleti, rock e leggera. Se molti piatti vengono utilizzati, tenerli su di una superficie calda, come un gran fiasco pieno d&#39;acqua a 37 ° C. </li><li> Controllare il piatto sotto un microscopio invertito. Se molti dei globuli rossi infetti sono ancora visibili, fare di più lavaggi come descritto sopra. Maneggiare la piastra Petri con molta attenzione per evitare qualsiasi rischio di contaminazione. </li><li> Rimuovere media dal piatto e aggiungere 3 ml di medium caldo RPMI completo con 40μl di ematocrito di uRBC fresca. </li><li> Porre la capsula in una camera stagna in incubazione, il gas per 3 minuti e il luogo della camera in un incubatore a 37 ° C durante la notte. </li><li> Il giorno dopo, raccogliere i parassiti dal lavaggio con media RPMI incompleta, in modo simile a quanto descritto nel paragrafo 4.4. ma più vigorosamente. Keep tutti mezzo utilizzato (contenente risospeso RBC) in un tubo 15ml conica. Controllare al microscopio invertito che tutte le RBC sono state rimosse dal piatto. </li><li> Pellet i parassiti, scartare il surnatante, risospendere in 5ml media RPMI completo e inserire il composto in un recipiente per la coltura normale. </li></ol> 5. Rappresentante Risultati Parassiti non selezionati mostrano un basso livello di legame HBEC-5i (Figura 3A). Così, dopo il primo turno di selezione, parassiti molto pochi saranno raccolti e può richiedere fino a un mese di coltura per raggiungere una parassitemia sufficiente per il secondo round di selezione. Dopo ogni turno di selezione, parassiti sempre più si legano alle cellule endoteliali e le selezioni possono essere ripetuti ad intervalli di tempo più brevi. Dopo 4-5 giri di selezione, ad alta vincolante popolazioni parassita sono ottenuti (Figura 3). Nelle nostre mani, il legame di HB3-HBEC a HBEC-5i o TNF attivato HBEC-5i era di simmili livello (dati non riportati). Il protocollo qui descritto è stato testato con 4 P. ceppi di P. falciparum: HB3, IT/FCR3, 3D7 e DD2 (Tabella 1). Solo quest&#39;ultimo non si è dimostrato in grado di legarsi a HBEC-5i, anche dopo 5 turni di selezione. HB3 parassiti sono stati anche selezionati per cytoadherence di cellule microvascolari cutanea e polmonare endoteliale (HDMEC e HPMEC). Dopo 4 turni di selezione, secondo il metodo descritto qui, un alto legame popolazione sono stati ottenuti su entrambi i HDMEC e HPMEC (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/3122/3122fig1.jpg" /> Figura 1. Panoramica del processo di selezione. <img alt="Figura 2" src="/files/ftp_upload/3122/3122fig2.jpg" /> Figura 2. Stadi di sviluppo di P. falciparum globuli rossi infetti. Striscio Giemsa visualizzati al microscopio con ingrandimento 1000X. <img alt="Figura 3" src="/files/ftp_upload/3122/3122fig3.jpg" /> Figura 3. Tipico esempio di parassiti legame HBEC-5i. (A) Lo strato blu è HBEC-5i fissati con glutaraldeide e colorati con Giemsa, visualizzati al microscopio con ingrandimento 1000X. Le foto sono state scattate dopo uRBC e iRBC non legato sono state spazzate via. Il pannello a sinistra mostra un unico P. falciparum HB3 (non selezionato) parassita legato a HBEC-5i. Nel pannello di destra la HB3-HBEC parassiti erano stati selezionati per 5 round. (B) I dati rappresentano la media di due esperimenti indipendenti, ciascuno eseguito in duplice copia. Il numero di parassiti legati per cellule endoteliali è stato contato. <img alt="Tabella 1" src="/files/ftp_upload/3122/3122table1.jpg" /> Tabella 1. Sintesi dei ceppi di Plasmodium falciparum, che si sono selezionati per il legame a HBEC-5i. HB3 Si noti che è stato anche selezionato per il TNF-attivato HBCE-5i. Dopo 5 turni di selezione, il ceppo DD2 non ha mostrato alcun aumento del legame con HBEC-5i rispetto ai non selezionati-DD2. <img alt="Figura 4" src="/files/ftp_upload/3122/3122fig4.jpg" /> Figura 4. Legame di P. falciparum HB3 parassiti cutanea (HDMEC) e polmonare (HPMEC), le cellule endoteliali, prima e dopo 4 turni di selezione. Anche se il terreno di coltura per HDMEC e HPMEC differisce leggermente (vedi istruzioni del fornitore), il protocollo utilizzato per la selezione era identico come HBEC-5i. Foto scattate con ingrandimento 400X. <table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> DMEM-F12 Ham </td><td> Sigma </td><td> D6421 </td><td> Per le medie DMEM completo </td></tr><tr><td> L-glutammina 200mm </td><td>GIBCO </td><td> 25030 </td><td> Per le medie e DMEM RPMI completo </td></tr><tr><td> Penicillina / streptomicina 100X (10000 unità / ml e 10 mg) </td><td> ScienCell </td><td> 0503 </td><td> Per le medie DMEM completo </td></tr><tr><td> Siero fetale bovino inattivato al calore </td><td> ScienCell </td><td> 0025 </td><td> Per le medie DMEM completo </td></tr><tr><td> Tripsina-EDTA (0,025% tripsina, 0.5mm EDTA) </td><td> ScienCell </td><td> 0103 </td><td></td></tr><tr><td> Supplemento di crescita delle cellule endoteliali </td><td> ScienCell </td><td> 1052 </td><td> Per le medie DMEM completo </td></tr><tr><td> Cultura tessuto trattato 60 mm x 15 millimetri Petri </td><td> BD </td><td> 353002 </td><td></td></tr><tr><td> Fibronectina umana </td><td> Millipore </td><td> FC010 </td><td> Utilizzare a 2 mg / cm 2 </td></tr><tr><td> TNF </td><td> R &amp;D Systems </td><td> 210-TA </td><td> opzionale, l&#39;uso a 50 mcg / ml </td></tr><tr><td> RPMI 1640 </td><td> Lonza </td><td> BE12-167F </td><td> Per le medie RPMI completo </td></tr><tr><td> Gentamicina 50mg/ml </td><td> Lonza </td><td> 17-518Z </td><td> Per le medie RPMI completo </td></tr><tr><td> Hepes 1M </td><td> Lonza </td><td> Be17-737E </td><td> Per le medie RPMI completo </td></tr><tr><td> HDMEC </td><td> ScienCell </td><td> 2000 </td><td> Linea di cellule primarie </td></tr><tr><td> HPMEC </td><td> ScienCell </td><td> 3000 </td><td> Linea di cellule primarie </td></tr><tr><td> HBEC-5i </td><td></td><td></td><td> Ottenuto da Francisco Candal (fcandal@cdc.gov) </td></tr></tbody></table> Tabella 2. Materiali

<ol>
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Non abbiamo nulla da rivelare.

Il segno distintivo della malaria cerebrale è il sequestro di P. falciparum iRBC all&#39;interno del cervello microcircolo 2, 3. Tuttavia, in colture in vitro di P. falciparum solo poco cytoadhere a HBEC-5i, un modello per endotelio microvascolare del cervello umano. Qui abbiamo messo a punto un test semplice per arricchire una popolazione per il legame a HBEC-5i, una più &quot;in vivo come&quot; fenotipo. Tre su 4 P. ceppi di P. falciparum sono stati scelti con successo utilizzando questo metodo. Inoltre, HB3 è stato anche selezionato HDMEC e HPMEC, indicando che questo protocollo può essere utilizzato per vari tipi di parassita delle cellule endoteliali. Diverse ipotesi può spiegare la mancanza di legame con il ceppo DD2. La più probabile è il fatto che questa linea parassita è knobless, che ostacola fortemente il cytoadherence 13, 14. Si consiglia di parassiti coltura selezionata insieme al pro selezionecesso per fornire un controllo per il confronto. Ciò consentirà, ad esempio, confrontando il trascrittoma di parassiti vincolanti e non vincolanti, con la speranza di scoprire i candidati ligando parassita. TNF è una citochina trova ad alto livello in pazienti affetti da malaria cerebrale ed è stato spettacolo di indurre l&#39;espressione delle proteine ​​di superficie molte delle HBEC-5i (Claessens et al, in preparazione e 8, 15, 16). In questo caso, la quantità di iRBC legato era simile nei normali HBEC-5i rispetto al attivato HBEC-5i. Questo &quot;modello di sequestro&quot; può essere usato anche per studiare le interazioni molecolari tra il iRBC e le cellule endoteliali, così come l&#39;effetto del presunto anti-cytoadherence farmaci. In questo caso, si consiglia di placcatura HBEC-5i in piccoli pozzi, come ad esempio &quot;8-bene diapositive da camera&quot; (BD 354628) o &quot;CultureWell&quot; (Sigma C7735-20EA).

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

La maggior parte delle morti per malaria umana è causata dal sangue allo stadio parassiti Plasmodium falciparum. Malaria cerebrale, la complicanza più pericolosa per la vita della malattia, è caratterizzata da un accumulo di Plasmodium falciparum cellule infettate globuli rossi (iRBC) nella prima fase vegetativa pigmentate nel microcircolo del cervello 2-4. Questa ostruzione microvascolare (sequestro) porta ad acidosi, ipossia e nocivi citochine infiammatorie (recensione a 5). Sequestro si trova anche nella maggior parte dei tessuti microvascolari del corpo umano, 2, 3. Il meccanismo con cui iRBC attaccare alle pareti dei vasi sanguigni è ancora poco compresa. Il immortalato umane endoteliali microvascolari cerebrali linea cellulare (HBEC-5i) è stato usato come un modello in vitro di barriera emato-encefalica 6. Tuttavia, Plasmodium falciparum iRBC allegare solo male a HBEC-5i in vitro, a differenza del sequestrat densoione che si verifica in casi di malaria cerebrale. Abbiamo quindi sviluppato un test per selezionare panning (arricchire) varie P. ceppi di P. falciparum per l&#39;adesione al HBEC-5i al fine di ottenere popolazioni di alta vincolante parassiti, più rappresentativo di ciò che accade in vivo. Un campione di una cultura parassita (miscela di iRBC e non infetti RBC) in fase di trofozoite pigmentata viene lavata e incubata su uno strato di HBEC-5i coltivate su una piastra di Petri. Dopo l&#39;incubazione, il piatto viene delicatamente lavato e privo di uRBC iRBC legato. Fresco uRBC vengono aggiunti al iRBC pochi allegata al HBEC-5i e incubato per una notte. Come parassiti fase schizonte scoppio, merozoiti inscatoli reinvade RBC e questi parassiti anello fase vengono raccolte il giorno successivo. I parassiti sono coltivati ​​fino abbastanza materiale si ottiene (in genere 2 a 4 settimane) e un nuovo round di selezione può essere eseguita. A seconda del P. falciparum ceppo, da 4 a 7 giri di selezione sono necessari al fine di ottenere una popolazionedove la maggior parte dei parassiti si legano ai HBEC-5i. Il fenotipo legame è progressivamente perso dopo poche settimane, che indica un interruttore di espressione del gene variante antigene di superficie, quindi la selezione regolari HBEC-5i è necessario per mantenere il fenotipo. In sintesi, abbiamo sviluppato un test di rendering selezione P. falciparum parassiti più &quot;cerebrale adesivo malaria&quot; fenotipo. Siamo stati in grado di selezionare 3 su 4 P. ceppi di P. falciparum in HBEC-5i. Questo test è stato utilizzato con successo anche per selezionare i parassiti per il legame di cellule umane endoteliali cutanea e polmonare. È importante sottolineare che questo metodo può essere usato per selezionare tessuto-specifiche popolazioni di parassiti al fine di individuare ligandi parassita candidato per il legame di endotelio cerebrale. Inoltre, questo test può essere usato per individuare presunti anti-sequestro di droga 7.

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

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

Un<em&gt; In vitro</emModello&gt; per il sequestro malaria cerebrale è descritto<sup&gt; 1</sup&gt;.<em&gt; Plasmodium falciparum</em&gt; Cellule infettate globuli rossi vengono selezionati per legarsi alle cellule del cervello umano immortalato microvascolari endoteliali. I parassiti selezionati mostrano un fenotipo diverso. Il processo di selezione può essere applicato utilizzando vari<em&gt; P. falciparum</em&gt; Ceppi e linee di cellule endoteliali.

Selezione di Plasmodium falciparum Parassiti per Cytoadhesion alle cellule cerebrali umane endoteliali

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

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JoVE Journal

Immunology and Infection

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

18.9K Views.  University of Edinburgh. Un In vitro per il sequestro malaria cerebrale è descritto 1. Plasmodium falciparum Cellule infettate globuli rossi vengono selezionati per legarsi alle cellule del cervello umano immortalato microvascolari endoteliali. I parassiti selezionati mostrano un fenotipo diverso. Il processo di selezione può essere applicato utilizzando vari P. falciparum Ceppi e linee di cellule endoteliali.

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

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

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

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

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

Un<em&gt; In vitro</em&gt; Co-infezione da modello per lo studio<em&gt; Plasmodium falciparum</em&gt;-HIV-1 in Interazioni umane Le cellule immunitarie primarie derivate da monociti

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

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Immunologia e infezioni

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

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

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

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

Un semplice protocollo per Clumping piastrinica mediata da<em&gt; Plasmodium falciparum</emEritrociti&gt; infette in una risorsa Povero Setting

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.

Isolamento e analisi di Brain-sequestrati da leucociti<em&gt; Plasmodium berghei</em&gt; ANKA topi infettati

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

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

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

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

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

Separazione dei<em&gt; Plasmodium falciparum</em&gt; Fine Stage-eritrociti infettati da mezzi magnetici

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

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitative<em&gt; In vitro</em&gt; Assay misurare Adesione neutrofili attivati ​​per primarie umane endoteliali microvascolari in condizioni statiche

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

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

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

Isolamento di cellule dendritiche mieloidi e cellule epiteliali di timo umano

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

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

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

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

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

High Yield Purificazione di<em&gt; Plasmodium falciparum</em&gt; Merozoiti Per l&#39;uso in opsonizzante anticorpo saggi

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.

Metodi per studiare il ruolo di regolamentazione dei piccoli RNA ribosomiale e di occupazione<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 ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

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

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

Infezione da pneumococco delle cellule primarie endoteliali nel flusso costante

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

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Meningitidis di Neisseria Infezione di cellule endoteliali cerebrali derivate da cellule staminali pluripotenti indotte

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...