a-simple-protocol-for-platelet-mediated-clumping-plasmodium

A Simple Protocol for Platelet-mediated Clumping of <em>Plasmodium falciparum</em>-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 ...

New York University School of Medicine

Cerebral malaria (CM), one of the most serious complications of Plasmodium falciparum infection, is characterized by the sequestration of infected erythrocytes (IEs) in cerebral microvascular beds. The precise mechanisms involved in the onset of neuropathology remain unknown, but parasite sequestration in the brain, metabolic disturbances, and host immune responses all play a role. Studies in a murine model of CM showed a potential role for host cells, especially platelets, in the pathogenesis of CM. Indeed, urokinase plasminogen activator receptor (uPAR; CD87) deficiency attenuates the severity of CM, most likely by its important role in platelet kinetics and trapping. These results led us to evaluate whether platelets have a role in the human disease. By immunostaining of brain samples from Malawian patients, we determined that the surface of platelet accumulation and the proportion of vessels filled with platelets were significantly higher in patients who died of CM than in those who died of other causes. We then investigated the role of platelets in IE cytoadhesion in vitro, using CD36-binding IE (IECD36) and CD36-deficient (CD36DEF) brain microvascular endothelial cells (ECs). Coincubation studies indicated that platelets can induce strong IECD36 binding to CD36DEF ECs and, conversely, can hide constitutively expressed falciparum receptors such as chondroitin sulfate A. Thus, platelets may provide an adhesion receptor to microvascular beds originally devoid of it. This novel mechanism of cytoadhesion may reorient the sequestration of different parasite phenotypes and play an important role in the pathogenesis of severe malaria.

Pathophysiology of cerebral malaria: role of host cells in the modulation of cytoadhesion.

Neuropaludisme (CM), une des complications plus graves de l&#39;infection &agrave; Plasmodium falciparum, est caract&eacute;ris&eacute;e par la s&eacute;questration des &eacute;rythrocytes infect&eacute;s (IEs) dans des lits microvasculaires c&eacute;r&eacute;brales. Les m&eacute;canismes pr&eacute;cis impliqu&eacute;s dans l&#39;apparition de la neuropathologie demeurent inconnues, mais la s&eacute;questration du parasite dans le cerveau, des troubles du m&eacute;tabolisme et immunitaire h6te tous jouer un r&ocirc;le. Des &eacute;tudes dans un mod&egrave;le murin de CM ont montr&eacute; un r&ocirc;le potentiel pour les cellules de l&#39;h&ocirc;te, en particulier des plaquettes, dans la pathogen&egrave;se de la CM. En effet, l&#39;urokinase plasminogen activator receptor (uPAR ; Carence CD87) att&eacute;nue la gravit&eacute; de la CM, tr&egrave;s probablement par son r&ocirc;le important sur la cin&eacute;tique de plaquettes et de pi&eacute;geage. Ces r&eacute;sultats nous a conduit &agrave; &eacute;valuer si les plaquettes ont un r&ocirc;le dans la maladie humaine. Par immunomarquage des &eacute;chantillons de cerveau de patients du Malawi, nous avons d&eacute;termin&eacute; que la surface de l&#39;accumulation des plaquettes et la proportion de navires remplis de plaquettes &eacute;taient significativement plus &eacute;lev&eacute;es chez les patients qui sont d&eacute;c&eacute;d&eacute;s de CM que dans ceux qui sont morts d&#39;autres causes. Nous avons ensuite &eacute;tudi&eacute; le r&ocirc;le des plaquettes dans IE cytoadhesion in vitro, en utilisant IE CD36-liaison (IECD36) et CD36-deficient (CD36DEF) du cerveau des cellules endoth&eacute;liales microvasculaires (ECs). Co-incubation &eacute;tudes ont indiqu&eacute; que les plaquettes peuvent induire la liaison forte de IECD36 &agrave; CD36DEF ECs et, inversement, peuvent cacher des r&eacute;cepteurs exprim&eacute;s constitutivement falciparum tels que le sulfate de chondro&iuml;tine A. Ainsi, les plaquettes peuvent fournir un r&eacute;cepteur d&#39;adh&eacute;sion aux lits microvasculaires initialement d&eacute;pourvu il. Ce nouveau m&eacute;canisme de cytoadhesion peut r&eacute;orienter la s&eacute;questration des ph&eacute;notypes diff&eacute;rents parasites et jouent un r&ocirc;le important dans la pathog&eacute;nie du paludisme grave.

Physiopathologie de la malaria c&eacute;r&eacute;brale : r&ocirc;le des cellules de l&#39;h&ocirc;te dans la modulation de cytoadhesion.

La malaria cerebrale (CM), una delle complicanze pi&ugrave; gravi dell&#39;infezione da Plasmodium falciparum, &egrave; caratterizzata dal sequestro degli eritrociti infetti (IEs) in letti Microvascolari cerebrali. I precisi meccanismi coinvolti nell&#39;insorgenza di Neuropatologia rimangono sconosciuti, ma sequestro del parassita nel cervello, disturbi metabolici e risposte immunitarie ospite giocano un ruolo. Gli studi in un modello murino di CM ha mostrato un ruolo potenziale per cellule ospiti, soprattutto le piastrine, nella patogenesi di CM. Infatti, urochinasi plasminogen activator receptor (uPAR; Deficit di CD87) attenua la severit&agrave; di CM, molto probabilmente dal suo ruolo importante nella cinetica piastrinica e abbondanza. Questi risultati ci hanno portato a valutare se le piastrine hanno un ruolo nella malattia umana. Di immunostaining di campioni di cervello da pazienti malawiani, abbiamo determinato che la superficie di accumulo delle piastrine e la proporzione dei vasi riempiti con le piastrine erano significativamente pi&ugrave; alta nei pazienti che morirono di CM rispetto a coloro che sono morti di altre cause. Abbiamo quindi studiato il ruolo delle piastrine in IE cytoadhesion in vitro, utilizzando IE CD36-associazione (IECD36) e deficit di CD36 (CD36DEF) del cervello cellule endoteliali Microvascolari (ECs). Coincubation gli studi indicarono che le piastrine possono indurre forte associazione IECD36 CD36DEF ECS e, al contrario, possono nascondere costitutivamente espresso falciparum recettori come il solfato di condroitina A. Cos&igrave;, le piastrine possono fornire un recettore di adesione a letto microvascolare originariamente privo di esso. Questo nuovo meccanismo di cytoadhesion pu&ograve; riorientare il sequestro dei fenotipi differenti parassita e gioca un ruolo importante nella patogenesi della malaria severa.

Fisiopatologia della malaria cerebrale: ruolo delle cellule ospiti nella modulazione del cytoadhesion.

Malaria is still a major public health problem, partly because the pathogenesis of its major complication, cerebral malaria (CM), remains incompletely understood. However tumor necrosis factor (TNF) is thought to play a key role in the development of this neurological syndrome, as well as lymphotoxin alpha (LT).

Inhibition of endothelial activation: a new way to treat cerebral malaria?

Le paludisme demeure un probl&egrave;me majeur de sant&eacute; publique, en partie parce que la pathog&eacute;nie de sa complication majeure, neuropaludisme (CM), reste incompl&egrave;tement comprise. Toutefois, le facteur de n&eacute;crose tumorale (TNF) est pens&eacute; pour jouer un r&ocirc;le cl&eacute; dans le d&eacute;veloppement de ce syndrome neurologique, ainsi qu&#39;alpha lymphotoxine (LT).

L&#39;inhibition de l&#39;activation endoth&eacute;liale : une nouvelle fa&ccedil;on de traiter le paludisme c&eacute;r&eacute;bral ?

La malaria &egrave; ancora un problema importante di salute pubblica, in parte perch&eacute; la patogenesi della sua grande complicazione, la malaria cerebrale (CM), rimane non completamente capita. Tuttavia il fattore di necrosi tumorale (TNF) &egrave; pensato per giocare un ruolo chiave nello sviluppo di questa sindrome neurologica, cos&igrave; come Alfa lymphotoxin (LT).

Inibizione dell&#39;attivazione endoteliale: un nuovo modo di trattare la malaria cerebrale?

Brain lesions of cerebral malaria (CM) are characterized by a sequestration of Plasmodium falciparum-parasitized red blood cells (PRBC) and platelets within brain microvessels, as well as by blood-brain barrier (BBB) disruption. In the present study, we evaluated the possibility that PRBC and platelets induce functional alterations in brain endothelium. In a human brain endothelial cell line, named HBEC-5i, exhibiting most of the features demanded for a pathophysiological study of BBB, tumor necrosis factor (TNF) or lymphotoxin alpha (LT-alpha) reduced transendothelial electrical resistance (TEER), enhanced the permeability to 70-kDa dextran, and increased the release of microparticles, a recently described indicator of disease severity in CM patients. In vitro cocultures showed that platelets or PRBC can have a direct cytotoxic effect on activated, but not on resting, HBEC-5i cells. Platelet binding was required, as platelet supernatant had no effect. Furthermore, platelets potentiated the cytotoxicity of PRBC for TNF- or LT-alpha-activated HBEC-5i cells when they were added prior to these cells on the endothelial monolayers. This effect was not observed when platelets were added after PRBC. Both permeability and TEER were strongly affected, and the apoptosis rate of HBEC-5i cells was dramatically increased. These findings provide insights into the mechanisms by which platelets can be deleterious to the brain endothelium during CM.

Platelets potentiate brain endothelial alterations induced by Plasmodium falciparum.

Les l&eacute;sions c&eacute;r&eacute;brales de neuropaludisme (CM) sont caract&eacute;ris&eacute;es par une saisie conservatoire de Plasmodium falciparum-parasit&eacute;s culots globulaires (culots Globulaires) et de plaquettes dans les microvaisseaux, ainsi que par la rupture de la barri&egrave;re h&eacute;mato - enc&eacute;phalique (BHE). Dans la pr&eacute;sente &eacute;tude, nous avons &eacute;valu&eacute; la possibilit&eacute; que les culots Globulaires et plaquettes induisent des alt&eacute;rations fonctionnelles dans l&#39;endoth&eacute;lium c&eacute;r&eacute;bral. En ligne en cellules endoth&eacute;liales du cerveau humain, nomm&eacute; HBEC-5i, pr&eacute;sentant la plupart des fonctionnalit&eacute;s exig&eacute;es pour une &eacute;tude physiopathologique de BBB, facteur de n&eacute;crose tumorale (TNF) ou alpha lymphotoxine (LT-alpha) r&eacute;duit la r&eacute;sistance &eacute;lectrique transendoth&eacute;liale (TEER), am&eacute;lior&eacute; la perm&eacute;abilit&eacute; au dextran 70 kDa et augment&eacute; la lib&eacute;ration de microparticules, un indicateur r&eacute;cemment d&eacute;crit de la gravit&eacute; de la maladie chez les patients de CM. Cocultures in vitro ont montr&eacute; que les plaquettes ou les culots Globulaires peuvent avoir un effet cytotoxique direct sur activ&eacute;, mais les cellules au repos, pas sur HBEC-5i. Liaison de plaquettes &eacute;tait n&eacute;cessaire, comme plaquettes surnageant n&#39;eut aucun effet. En outre, plaquettes potentialisent la cytotoxicit&eacute; des culots Globulaires pour TNF - ou LT-alpha-activ&eacute; HBEC-5i cellules lorsqu&#39;elles ont &eacute;t&eacute; ajout&eacute;es avant ces cellules sur les monocouches endoth&eacute;liales. Cet effet n&#39;a pas observ&eacute; lorsque les plaquettes ont &eacute;t&eacute; ajout&eacute;s apr&egrave;s les culots Globulaires. Perm&eacute;abilit&eacute; et TEER ont &eacute;t&eacute; fortement touch&eacute;s et le taux d&#39;apoptose des cellules HBEC-5i a &eacute;t&eacute; consid&eacute;rablement augment&eacute;. Ces d&eacute;couvertes permettent de mieux comprendre les m&eacute;canismes par lesquels plaquettes peuvent &ecirc;tre nocives &agrave; l&#39;endoth&eacute;lium c&eacute;r&eacute;bral pendant CM.

Plaquettes potentialisent l&#39;alt&eacute;ration endoth&eacute;liale de cerveau induite par le Plasmodium falciparum.

Lesioni cerebrali di malaria cerebrale (CM) sono caratterizzate da un sequestro di Plasmodium falciparum-parassiti globuli rossi (PRBC) e di piastrine all&#39;interno del cervello Talengectasia, nonch&eacute; dalla rottura della barriera emato - encefalica (BBB). Nel presente studio, abbiamo valutato la possibilit&agrave; che PRBC e piastrine inducono alterazioni funzionali dell&#39;endotelio cerebrale. In una linea di cellule endoteliali del cervello umano, denominato HBEC-5i, esibendo la maggior parte delle funzionalit&agrave; richieste per un Studio fisiopatologico della BBB, fattore di necrosi tumorale (TNF) o alpha lymphotoxin (LT-alpha) riduzione transendoteliale resistenza elettrica (TEER), migliorata la permeabilit&agrave; di Destrano 70 kDa e aumentato il rilascio di microparticelle, un recente descritto indicatore della gravit&agrave; della malattia in pazienti CM. Cocultures in vitro hanno dimostrato che le piastrine o PRBC pu&ograve; avere un effetto citotossico diretto attivato, ma non a riposo, le cellule HBEC-5i. Associazione delle piastrine &egrave; stato richiesto, come piastrine surnatante ha avuto alcun effetto. Inoltre, le piastrine potenziate la citotossicit&agrave; di PRBC per TNF - o LT-alfa attivato HBEC-5i cellule quando sono stati aggiunti prima di queste cellule sui monostrati endoteliale. Questo effetto non &egrave; stato osservato quando le piastrine sono stati aggiunti dopo PRBC. Permeabilit&agrave; sia TEER sono stati fortemente influenzati, e il tasso di apoptosi delle cellule HBEC-5i &egrave; stata notevolmente aumentato. Questi risultati forniscono informazioni sul meccanismo mediante il quale le piastrine possono essere deleterie per l&#39;endotelio cerebrale durante CM.

Piastrine potenziare alterazioni endoteliali cerebrali indotte da Plasmodium falciparum.

Platelets have recently been shown to accumulate in brain microvessels of patients with cerebral malaria and to modulate the binding of Plasmodium falciparum-infected red cells to human brain endothelium in vitro. In the present study we used a platelet-endothelial cell coculture model to investigate the mechanisms by which platelets modify the function of human brain microvascular endothelial cells (HBEC). Platelets were found to have a proapoptotic effect on TNF-activated HBEC, and this was contact-dependent, as inhibiting platelet binding prevented endothelial cell killing. We also showed that the supernatants of thrombin-activated platelets killed TNF-stimulated HBEC and that TGF-beta1 was the main molecule involved in endothelial cell death, because its inhibition completely abrogated the activated-platelet supernatant effect. Our data illustrate another aspect of the duality of TGF-beta1 in malaria and may provide new insights into the pathogenesis of cerebral malaria.

TGF-beta1 released from activated platelets can induce TNF-stimulated human brain endothelium apoptosis: a new mechanism for microvascular lesion during cerebral malaria.

Les plaquettes ont r&eacute;cemment d&eacute;montr&eacute;s &agrave; s&#39;accumuler dans les microvaisseaux des personnes atteintes de paludisme c&eacute;r&eacute;bral et de moduler la liaison du Plasmodium falciparum infect&eacute;s par des globules rouges &agrave; l&#39;endoth&eacute;lium de cerveau humain in vitro. Dans la pr&eacute;sente &eacute;tude, nous avons utilis&eacute; un mod&egrave;le de co-culture de cellules endoth&eacute;liales-plaquettes d&#39;&eacute;tudier les m&eacute;canismes par lesquels les plaquettes modifient la fonction des cellules endoth&eacute;liales microvasculaires du cerveau humain (HBEC). Plaquettes ont eu un effet pro-apoptotique sur HBEC activ&eacute;s par le TNF, et c&#39;est d&eacute;pendante du contact, comme liaison de plaquettaire inhibant a emp&ecirc;ch&eacute; la mort cellulaire endoth&eacute;liale. Nous avons &eacute;galement montr&eacute; que les fractions surnageantes des plaquettes activ&eacute;e par la thrombine tu&eacute; HBEC stimul&eacute;e par le TNF et que TGF-beta1 est la principale mol&eacute;cule impliqu&eacute;e dans la mort des cellules endoth&eacute;liales, parce que son inhibition abrog&eacute; compl&egrave;tement l&#39;effet de surnageant de plaquettes activ&eacute;es. Nos donn&eacute;es montrent un autre aspect de la dualit&eacute; du TGF-beta1 dans le paludisme et peuvent apporter un nouvel &eacute;clairage dans la pathog&eacute;nie du paludisme c&eacute;r&eacute;bral.

TGF-beta1, lib&eacute;r&eacute; des plaquettes activ&eacute;es peut induire l&#39;apoptose de l&#39;endoth&eacute;lium de cerveau humain stimul&eacute;e par le TNF : un nouveau m&eacute;canisme pour l&eacute;sion microvasculaire au cours du paludisme c&eacute;r&eacute;bral.

Piastrine hanno recentemente dimostrate ad accumularsi nel cervello Talengectasia dei pazienti con malaria cerebrale e di modulare l&#39;associazione del Plasmodium falciparum-rosso cellule infette all&#39;endotelio del cervello umano in vitro. Nel presente studio abbiamo utilizzato un modello di coculture delle cellule endoteliali delle piastrine per indagare i meccanismi con cui le piastrine modificare la funzione delle cellule endoteliali Microvascolari (HBEC) del cervello umano. Le piastrine sono state trovate per avere un effetto Miselli su TNF-attivato HBEC, e questo &egrave; stato il contatto-dipendente, come associazione piastrinica inibendo impedito uccisione delle cellule endoteliali. Abbiamo anche dimostrato che i supernatanti di piastrine attivato da trombina ucciso HBEC TNF-stimolato e che TGF-beta1 era la principale molecola coinvolta nella morte delle cellule endoteliali, perch&eacute; la sua inibizione abrogato completamente l&#39;effetto di surnatante delle piastrine attivate. I nostri dati illustrano un altro aspetto della dualit&agrave; di TGF-beta1 nella malaria e possono fornire nuove conoscenze nella patogenesi della malaria cerebrale.

TGF-beta1 rilasciato dalle piastrine attivate pu&ograve; indurre il apoptosis di endotelio TNF stimola cervello umano: un nuovo meccanismo per lesione microvascolare durante la malaria cerebrale.

[LMP-420, un nuovo approccio terapeutico per la malaria cerebrale?].

[LMP-420, a new therapeutic approach for cerebral malaria?].

[LMP-420, une nouvelle approche th&eacute;rapeutique contre le paludisme c&eacute;r&eacute;bral?].

Microparticles are plasma membrane fragments that are generated and released under physiological conditions. They are also released when tissue and/or systemic homeostasis is disrupted. These microparticles display different physiological features of the cells from which they originate. They are detected in some pathological conditions, but rarely suspected of participating in the disease's pathogenesis. In the present review, we summarise data about the production of the microparticles, their biological significance and potential role during microorganism-driven processes, especially in cerebral malaria.

Cell vesiculation and immunopathology: implications in cerebral malaria.

Microparticules sont des fragments de membrane plasmique qui sont g&eacute;n&eacute;r&eacute;s et rejet&eacute;es dans des conditions physiologiques. Ils sont &eacute;galement distribu&eacute;s lorsque les tissus et/ou hom&eacute;ostasie syst&eacute;mique est perturb&eacute;. Ces microparticules affichent diff&eacute;rentes caract&eacute;ristiques physiologiques des cellules dont ils sont originaires. Ils sont d&eacute;tect&eacute;s dans certaines conditions pathologiques, mais rarement soup&ccedil;onn&eacute;es d&#39;avoir particip&eacute; dans la pathogen&egrave;se de la maladie. Dans cette revue, nous r&eacute;sume les donn&eacute;es sur la production des microparticules, leur signification biologique et leur r&ocirc;le potentiel au cours des processus ax&eacute;s sur le micro-organisme, en particulier dans le paludisme c&eacute;r&eacute;bral.

Cellules v&eacute;siculaires et immunopathologie : incidences sur le neuropaludisme.

Le microparticelle sono frammenti di membrana del plasma che sono generati e rilasciati in condizioni fisiologiche. Vengono inoltre rilasciate quando tessuto e/o l&#39;omeostasi sistemica &egrave; perturbato. Queste microparticelle visualizzare diverse caratteristiche fisiologiche delle cellule da cui essi provengono. Sono rilevati in alcune condizioni patologiche, ma raramente sospettati di partecipanti nella patogenesi della malattia. Nella presente rassegna, abbiamo riassumere i dati relativi alla produzione di microparticelle, loro significato biologico e il ruolo potenziale durante processi basati su microrganismi, specialmente nella malaria cerebrale.

Article	Year
Pathophysiology of cerebral malaria: role of host cells in the modulation of cytoadhesion. Annals of the New York Academy of Sciences\| PubMed ID: 12794044	2003
Inhibition of endothelial activation: a new way to treat cerebral malaria? PLoS medicine\| PubMed ID: 16104828	2005
Platelets potentiate brain endothelial alterations induced by Plasmodium falciparum. Infection and immunity\| PubMed ID: 16369021	2006
TGF-beta1 released from activated platelets can induce TNF-stimulated human brain endothelium apoptosis: a new mechanism for microvascular lesion during cerebral malaria. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 16394007	2006
[LMP-420, a new therapeutic approach for cerebral malaria?]. MÃ©decine sciences : M/S\| PubMed ID: 16597395	2006
Cell vesiculation and immunopathology: implications in cerebral malaria. Microbes and infection / Institut Pasteur\| PubMed ID: 16829152	2006

Samuel C. Wassmer

Biography

JoVE Journal Publications

Other Publications