Acute and chronic Plasmodium falciparum malaria are accompanied by severe immunodepression possibly related to subversion of dendritic cells (DC) functionality. Phagocytosed hemozoin (malarial pigment) was shown to inhibit monocyte functions related to immunity. Hemozoin-loaded monocytes, frequently found in circulation and adherent to endothelia in malaria, may interfere with DC development and play a role in immunodepression. Hemozoin-loaded and unloaded human monocytes were differentiated in vitro to immature DC (iDC) by treatment with GM-CSF and IL-4, and to mature DC (mDC) by LPS challenge. In a second setting, hemozoin was fed to iDC further cultured to give mDC. In both settings, cells ingested large amounts of hemozoin undegraded during DC maturation. Hemozoin-fed monocytes did not apoptose but their differentiation and maturation to DC was severely impaired as shown by blunted expression of MHC class II and costimulatory molecules CD83, CD80, CD54, CD40, CD1a, and lower levels of CD83-specific mRNA in hemozoin-loaded iDC and mDC compared with unfed or latex-loaded DC. Further studies indicated activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) in hemozoin-loaded iDC and mDC, associated with increased expression of PPAR-γ mRNA, without apparent involvement of NF-κB. Moreover, expression of PPAR-γ was induced and up-regulation of CD83 was inhibited by supplementing iDC and mDC with plausible concentrations of 15(S)-hydroxyeicosatetraenoic acid, a PPAR-γ ligand abundantly produced by hemozoin via heme-catalyzed lipoperoxidation.

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Natural hemozoin (nHZ), prepared after schizogony, consists of crystalline ferriprotoporphyrin-IX dimers from undigested heme bound to host and parasite proteins and lipids. Phagocytosed nHZ alters important functions of host phagocytes. Most alterations are long-term effects. We show that host fibrinogen (FG) was constantly present (at ϳ 1 FG per 25 000 HZ-heme molecules) and stably bound to nHZ from plasma-cultured parasites. FG was responsible for the rapid 100-fold stimulation of reactive oxygen species production and 50-fold increase of TNF and monocyte chemotactic protein 1 by human monocytes. Those effects, starting within minutes after nHZ cell contact, were because of interaction of FG with FG-receptors TLR4 and integrin CD11b/CD18. Receptor blockage by specific mAbs or removal of FG from nHZ abrogated the effects. nHZ-opsonizing IgGs contribute to the stimulatory response but are not essential for FG effects. Immediate increase in reactive oxygen species and TNF may switch on previously described long-term effects of nHZ, largely because of HZ-generated lipo-peroxidation products 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid and 4-hydroxynonenal. The FG/HZ effects mediated by TLR4/integrins represent a novel paradigm of nHZ activity and allow expansion of nHZ effects to nonphagocytic cells, such as endothelia and airway epithelia, and lead to a better understanding of organ pathology in malaria. (Blood. 2011;117(21):5674-5682) IntroductionNatural hemozoin (nHZ), as present in the food vacuole of Plasmodium falciparum and largely coincident with residual bodies (RBs) shed during schizogony, consists of a scaffold of crystalline ferriprotoporphyrin IX dimers (␤-hematin, BH) from undigested host hemoglobin-heme 1 bound to a vast array of host and parasite molecules. [2][3][4] nHZ is phagocytosed in vivo and in vitro by host phagocytes and alters important functions in those cells. Most functional alterations were long-term postphagocytic effects. 3,[5][6][7][8] Some of those long-term effects were also reported in human and murine phagocytes fed with BH or variously manipulated HZ. 9,10 By contrast, a powerful, short-term stimulation of oxidative burst by human monocytes was also shown to occur during phagocytosis of nHZ. 8 Here, we show that host fibrinogen (FG) was constantly present and stably bound to nHZ and RBs prepared from parasites cultured in presence of plasma. FG in nHZ was responsible for a prephagocytic rapid and powerful stimulation of oxidative burst, accompanied by release of TNF and monocyte chemotactic protein 1 (MCP-1) by human monocytes. Those prephagocytic events were because of the interaction of FG with TLR4 and integrin CD11b/CD18 (CR3, Mac-1), known receptors for FG as an immune-active molecule. [11][12][13] Present data are the first indication that nHZ-bound FG may act as a powerful self-signal molecule for early immune responses and a trigger for the persistent long-term effects that play multiple roles in malaria pathogenesis. Methods ReagentsUnless otherwise stated, reagen...

The first human transfusion was performed by the pioneer Dr Jean-Baptiste Denis in France in 1667 and now, three centuries later, around 50 millions blood units are transfused every year, saving millions of lives. Today, there is a new application for red blood cells (RBCs) in cellular therapy: the effective use of erythrocytes as vehicles for chemical or biological drugs. Using this approach, the therapeutic index of RBC-entrapped molecules can be significantly improved with increased efficacy and reduced side effects. This cell-based medicinal product can be manufactured at an industrial scale and is now used in the clinic for different therapeutic applications.A seminar dedicated to this field of research, debating on this inventive formulation for drugs, was held in Lyon (France) on 28 January 2011. Drs KC Gunter and Y Godfrin co-chaired the meeting and international experts working on the encapsulation of drugs within erythrocytes met to exchange knowledge on the topic 'The Red Blood Cells as Vehicles for Drugs'. The meeting was composed of oral presentations providing the latest knowledge and experience on the preclinical and clinical applications of this technology. This Meeting Highlights article presents the most relevant messages given by the speakers and is a joint effort by international experts who share an interest in studying erythrocyte as a drug delivery vehicle. The aim is to provide an overview of

Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S), which are the most abundant hormones secreted by the adrenal cortex and are present in plasma at approximately 6 M, as well as their analogue, 16␣-bromoepiandrosterone (EPI), exerted antimalarial activities against two chloroquine-sensitive Plasmodium falciparum strains (Palo Alto, 50% inhibitory concentration [IC 50 ] of EPI, 4.8 ؎ 0.68 M; T996/86, IC 50 of EPI, 7.5 ؎ 0.91 M, and IC 50 of DHEA-S, 19 ؎ 2.6 M) and one mildly chloroquine-resistant strain (FCR-3, IC 50 of EPI, 6.5 ؎ 1.01 M). Both EPI and DHEA/DHEA-S are potent inhibitors of glucose-6-phosphate dehydrogenase (G6PD), and G6PD deficiency is known to exert antimalaria protection via enhanced opsonization and phagocytosis of rings, the early forms of the parasite. Plasma-compatible antimalarial EPI concentrations did not inhibit G6PD activity and did not induce ring opsonization by immunoglobulin G and complement fragments, as observed in G6PD deficiency, but nevertheless remarkably stimulated ring phagocytosis. Plasmacompatible, low-micromolar concentrations of EPI induced exposure on the ring surface of phosphatidylserine, a signal for phagocytic removal independent of opsonization. We propose that enhanced ring phagocytosis due to exposure of negatively charged membrane phospholipids may explain the antimalarial activity of EPI.Recently, it has been shown that 16␣-bromoepiandrosterone (EPI), an analogue of the human adrenal steroid hormones dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S), was endowed with antimalarial activity against several strains of Plasmodium falciparum in vitro and against Plasmodium berghei in a mouse model (7). Interestingly, another study (16) has indicated that age-related decreases in the frequency and density of P. falciparum parasitemia were greater during puberty and that the blood level of DHEA-S was a significant predictor of resistance. Both EPI and DHEA/DHEA-S inhibit glucose-6-phosphate dehydrogenase (G6PD) (10) and cell proliferation (13, 24). G6PD deficiency is known to afford antimalaria protection (12), possibly mediated by enhanced phagocytosis of rings, the early parasite stage within the erythrocytes (RBC) (3). Therefore, we explored whether the antimalarial activity of EPI could be related to inhibition of erythrocytic G6PD. The present results confirm the antimalarial activities of EPI and DHEA-S and indicate that sub-and low-micromolar EPI concentrations remarkably stimulated ring phagocytosis. EPI did not inhibit G6PD activity and did not enhance deposition of phagocytic opsonins, but enhanced exposure on the ring surface of phosphatidylserine (PS), a signal for phagocytic removal independent of opsonization (30). We propose that enhanced ring stage phagocytosis due to exposure of negatively charged membrane phospholipids may explain the antimalarial activity of EPI. MATERIALS AND METHODS Materials.Buffers, culture media, substrates, enzymes and coenzymes, adenine, heparin, gentamicin, mannitol, dehydroepiandrosterone sulfate (5-androsten-...