Rocaglates as dual-targeting agents for experimental cerebral malaria.

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.

Synthetic oleanane triterpenoids enhance blood brain barrier integrity and improve survival in experimental cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection associated with high mortality and neurocognitive impairment in survivors. New anti-malarials and host-based adjunctive therapy may improve clinical outcome in CM. Synthetic oleanane triterpenoid (SO) compounds have shown efficacy in the treatment of diseases where inflammation and oxidative stress contribute to pathogenesis. METHODS: A derivative of the SO 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), CDDO-ethyl amide (CDDO-EA) was investigated for the treatment of severe malaria in a pre-clinical model. CDDO-EA was evaluated in vivo as a monotherapy as well as adjunctive therapy with parenteral artesunate in the Plasmodium berghei strain ANKA experimental cerebral malaria (ECM) model. RESULTS: CDDO-EA alone improved outcome in ECM and, given as adjunctive therapy in combination with artesunate, it significantly improved outcome over artesunate alone (p = 0.009). Improved survival was associated with reduced inflammation, enhanced endothelial stability and blood-brain barrier integrity. Survival was improved even when administered late in the disease course after the onset of neurological symptoms. CONCLUSIONS: These results indicate that SO are a new class of immunomodulatory drugs and support further studies investigating this class of agents as potential adjunctive therapy for severe malaria.

CD47-SIRPα Interactions Regulate Macrophage Uptake of Plasmodium falciparum-Infected Erythrocytes and Clearance of Malaria In Vivo.

CD47 engagement by the macrophage signal regulatory protein alpha (SIRPα) inhibits phagocytic activity and protects red blood cells (RBCs) from erythrophagocytosis. The role of CD47-SIRPα in the innate immune response to Plasmodium falciparum infection is unknown. We hypothesized that disruption of SIRPα signaling may enhance macrophage uptake of malaria parasite-infected RBCs. To test this hypothesis, we examined in vivo clearance in CD47-deficient mice infected with Plasmodium berghei ANKA and in vitro phagocytosis of P. falciparum-infected RBCs by macrophages from SHP-1-deficient (Shp-1(-/-)) mice and NOD.NOR-Idd13.Prkdc(scid) (NS-Idd13) mice, as well as human macrophages, following disruption of CD47-SIRPα interactions with anti-SIRPα antibodies or recombinant SIRPα-Fc fusion protein. Compared to their wild-type counterparts, Cd47(-/-) mice displayed significantly lower parasitemia, decreased endothelial activation, and enhanced survival. Using macrophages from SHP-1-deficient mice or from NS-Idd13 mice, which express a SIRPα variant that does not bind human CD47, we showed that altered SIRPα signaling resulted in enhanced phagocytosis of P. falciparum-infected RBCs. Moreover, disrupting CD47-SIRPα engagement using anti-SIRPα antibodies or SIRPα-Fc fusion protein also increased phagocytosis of P. falciparum-infected RBCs. These results indicate an important role for CD47-SIRPα interactions in innate control of malaria and suggest novel targets for intervention.

Chronic HIV infection impairs nonopsonic phagocytosis of malaria parasites.

Malaria-specific immune responses are altered in HIV/malaria-coinfected individuals and are associated with higher parasite burdens and more severe clinical disease. Monocyte/macrophage phagocytosis is a major mechanism of malaria parasite clearance. We hypothesized that phagocytosis of malaria-parasitized erythrocytes is impaired in coinfected individuals and could contribute to the increased parasite burdens observed. We show that nonopsonic phagocytosis of Plasmodium falciparum parasitized erythrocytes is impaired in monocytes isolated from HIV-infected individuals. The observed defects in phagocytic capacity were rescued after 6 months of antiretroviral therapy, demonstrating the importance of HIV treatment and immune reconstitution in the context of coinfection.

HIV infection deregulates Tim-3 expression on innate cells: combination antiretroviral therapy results in partial restoration.

BACKGROUND: The Tim-3 receptor has been implicated as a negative regulator of adaptive immune responses and has been linked to T-cell dysfunction in chronic viral infections, such as HIV. Blocking Tim-3 has been proposed as a potential therapeutic intervention in HIV infection. However, a more detailed characterization of Tim-3 expression in the presence of HIV is required before such strategies can be considered. METHODS: In this study, we investigate Tim-3 expression on innate immune cell subsets in chronic HIV-infected individuals pretherapy and posttherapy. RESULTS: We report that, pretherapy, HIV infection is associated with elevated levels of Tim-3 on resting innate lymphocytes (NK, NKT, and γδ T cells), but not resting monocytes. In the absence of HIV infection, stimulation with an inflammatory stimulus resulted in decreased Tim-3 on monocytes and increased Tim-3 on NK, NKT, and γδ T cells. However, innate cells from HIV-infected donors were significantly less responsive to stimulation. Six months of combination antiretroviral therapy (cART) restored Tim-3 levels on resting NK cells but not NKT or γδ T cells. The responses of all subsets to inflammatory stimuli were restored to some extent with cART but only reached HIV-negative control levels in monocytes and NK cells. DISCUSSION: These results demonstrate that, during HIV infection, Tim-3 expression on innate cells is dysregulated and that this dysregulation is only partially restored after 6 months of cART. Our findings suggest that Tim-3 is differentially regulated on innate immune effector cells, and have direct implications for strategies designed to block Tim-3-ligand interactions.

HIV infection deregulates innate immunity to malaria despite combination antiretroviral therapy.

OBJECTIVE: Malaria and HIV-1 adversely interact, with HIV-positive individuals suffering higher parasite burdens and worse clinical outcomes. However, the mechanisms underlying these disease interactions are unclear. We hypothesized that HIV coinfection impairs the innate immune response to malaria, and that combination antiretroviral therapy (cART) may restore this response. Our aim was to examine the innate inflammatory response of natural killer (NK), natural killer T (NKT), and γδ T-cells isolated from the peripheral blood of HIV-infected therapy-naive donors to malaria parasites, and determine the effect of cART on these responses. METHODS: Freshly isolated peripheral blood mononuclear cells from 25 HIV-infected individuals pre-cART (month 0) and post-cART (months 3 and 6), and HIV-negative individuals at matched time-points, were cultured in the presence of Plasmodium falciparum parasitized erythrocytes. Supernatants and cells were collected to assess cytokine production and phenotypic changes. RESULTS: Compared to HIV-negative participants, NKT, NK, and γδ T-cell subsets from participants with chronic HIV infection showed marked differences, including decreased production of interferon γ (IFNγ) and tumor necrosis factor (TNF) in response to malaria parasites. IFNγ production was linked to interleukin-18 receptor (IL-18R) expression in all three cell types studied. Six months of cART provided partial cellular reconstitution but had no effect on IL-18R expression, or IFNγ and TNF production. CONCLUSION: These data suggest that HIV infection impairs the inflammatory response of innate effector cells to malaria, and that the response is not fully restored within 6 months of cART. This may contribute to higher parasite burdens and ineffective immune responses, and have implications for vaccination initiatives in coinfected individuals.

ABO blood groups influence macrophage-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes.

Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A(1), A(2), and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria.

Characterization of a new phosphatase from Plasmodium.

Plasmodium falciparum malaria is the most important parasitic disease worldwide, responsible for an estimated 1 million deaths annually. Two P. falciparum genes code for putative phosphoglycerate mutases (PGMases), a widespread protein group characterized by the involvement of histidine residues in their catalytic mechanism. PGMases are responsible for the interconversion between 2 and 3-phosphoglycerate, an intermediate step in the glycolysis pathway. We have determined the crystal structures of one of the P. falciparum's PGMases (PfPGM2) and a functionally distinct phosphoglycerate mutase from Cryptosporidium parvum, a related apicomplexan parasite. We performed sequence and structural comparisons between the two structures, another P. falciparum enzyme (PfPGM1) and several other PGM family members from other organisms. The comparisons revealed a distinct conformation of the catalytically active residues not seen in previously determined phosphoglycerate mutase structures. Furthermore, characterization of their enzymatic activities revealed contrasting behaviors between the PfPGM2 and the classical cofactor-dependent PGMase from C. parvum, clearly establishing PfPGM2 as a phosphatase with a residual level of mutase activity. Further support for this function attribution was provided by our structural comparison with previously characterized PGM family members. Genetic characterization of PGM2 in the rodent parasite Plasmodium berghei indicated that the protein might be essential to blood stage asexual growth, and a GFP tagged allele is expressed in both blood and zygote ookinete development and located in the cytoplasm. The P. falciparum PGM2 is either an enzyme implicated in the phosphate metabolism of the parasite or a regulator of its life cycle.

Cysteamine, the natural metabolite of pantetheinase, shows specific activity against Plasmodium.

In mice, loss of pantetheinase activity causes susceptibility to infection with Plasmodium chabaudi AS. Treatment of mice with the pantetheinase metabolite cysteamine reduces blood-stage replication of P. chabaudi and significantly increases survival. Similarly, a short exposure of Plasmodium to cysteamine ex vivo is sufficient to suppress parasite infectivity in vivo. This effect of cysteamine is specific and not observed with a related thiol (dimercaptosuccinic acid) or with the pantethine precursor of cysteamine. Also, cysteamine does not protect against infection with the parasite Trypanosoma cruzi or the fungal pathogen Candida albicans, suggesting cysteamine acts directly against the parasite and does not modulate host inflammatory response. Cysteamine exposure also blocks replication of P. falciparum in vitro; moreover, these treated parasites show higher levels of intact hemoglobin. This study highlights the in vivo action of cysteamine against Plasmodium and provides further evidence for the involvement of pantetheinase in host response to this infection.

Adenosine triphosphate depletion of erythrocytes simulates the phenotype associated with pyruvate kinase deficiency and confers protection against Plasmodium falciparum in vitro.

BACKGROUND: Erythrocytes from individuals with pyruvate kinase deficiency (PKD) are resistant to invasion by Plasmodium falciparum parasites, and erythrocytes infected with ring-stage parasites are preferentially cleared by macrophages in vitro. However, the underlying molecular basis of protection is unknown. In the present study, we examined adenosine triphosphate (ATP) levels in PKD erythrocytes (ie, erythrocytes from individuals with PKD) and determined whether depletion of ATP in normal erythrocytes would recapitulate the phenotype observed with PKD. METHODS: We examined ATP levels in homozygous PKLR(-/-) and heterozygous PKLR(+/-) human erythrocytes and used sodium fluoride treatment to inhibit ATP generation in normal human erythrocytes. RESULTS: We demonstrated that ATP levels are reduced in PKLR(-/-) (percentage of control erythrocytes, 26%; interquartile range [IQR], 21%-48%) and PKLR(+/-) erythrocytes (percentage of control erythrocytes, 64%; IQR, 60%-73%) and that there is a correlation between ATP levels in erythrocytes and both inhibition of parasite invasion and enhancement of phagocytosis of erythrocytes infected with ring-stage parasites. Analysis of ATP distribution in parasitized erythrocytes demonstrated that parasites invading PKD erythrocytes respond to low intraerythrocytic ATP levels by means of a parallel increase in parasite-derived ATP via up-regulation of P. falciparum-specific pyruvate kinase. CONCLUSION: These data suggest that reduced erythrocyte ATP levels may contribute to the protection displayed by PKD erythrocytes in vitro and may provide a model system with which to define the molecular basis of protection in inherited PKD.

Rosiglitazone modulates the innate immune response to Plasmodium falciparum infection and improves outcome in experimental cerebral malaria.

For severe malarial syndromes such as cerebral malaria, adverse clinical outcomes are often mediated by the immune system rather than caused by the parasite directly. However, few therapeutic agents have been developed to modulate the host's immunopathological responses to infection. Here, we report that the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist rosiglitazone modulated the host response to malaria by enhancing phagocytic clearance of malaria-parasitized erythrocytes and by decreasing inflammatory responses to infection via inhibition of Plasmodium falciparum glycosylphosphatidylinositol-induced activation of the mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) signaling pathways. We found that, in the Plasmodium berghei strain ANKA experimental model of cerebral malaria, rosiglitazone modified the inflammatory response to malarial infection and improved the survival rate even when treatment was initiated as late as day 5 after infection. Furthermore, rosiglitazone reduced the parasitemia in a CD36-dependent manner in the Plasmodium chabaudi chabaudi hyperparasitemia model. These data suggest that PPARgamma agonists represent a novel class of host immunomodulatory drugs that may be useful for treatment of severe malaria syndromes.

Pyruvate kinase deficiency and malaria.

Malaria that is caused by Plasmodium falciparum is a significant global health problem. Genetic characteristics of the host influence the severity of disease and the ultimate outcome of infection, and there is evidence of coevolution of the plasmodium parasite with its host. In humans, pyruvate kinase deficiency is the second most common erythrocyte enzyme disorder. Here, we show that pyruvate kinase deficiency provides protection against infection and replication of P. falciparum in human erythrocytes, raising the possibility that mutant pyruvate kinase alleles may confer a protective advantage against malaria in human populations in areas where the disease is endemic.

C5 deficiency and C5a or C5aR blockade protects against cerebral malaria.

Experimental infection of mice with Plasmodium berghei ANKA (PbA) provides a powerful model to define genetic determinants that regulate the development of cerebral malaria (CM). Based on the hypothesis that excessive activation of the complement system may confer susceptibility to CM, we investigated the role of C5/C5a in the development of CM. We show a spectrum of susceptibility to PbA in a panel of inbred mice; all CM-susceptible mice examined were found to be C5 sufficient, whereas all C5-deficient strains were resistant to CM. Transfer of the C5-defective allele from an A/J (CM resistant) onto a C57BL/6 (CM-susceptible) genetic background in a congenic strain conferred increased resistance to CM; conversely, transfer of the C5-sufficient allele from the C57BL/6 onto the A/J background recapitulated the CM-susceptible phenotype. The role of C5 was further explored in B10.D2 mice, which are identical for all loci other than C5. C5-deficient B10.D2 mice were protected from CM, whereas C5-sufficient B10.D2 mice were susceptible. Antibody blockade of C5a or C5a receptor (C5aR) rescued susceptible mice from CM. In vitro studies showed that C5a-potentiated cytokine secretion induced by the malaria product P. falciparum glycosylphosphatidylinositol and C5aR blockade abrogated these amplified responses. These data provide evidence implicating C5/C5a in the pathogenesis of CM.

HIV impairs opsonic phagocytic clearance of pregnancy-associated malaria parasites.

BACKGROUND: Primigravid (PG) women are at risk for pregnancy-associated malaria (PAM). Multigravid (MG) women acquire protection against PAM; however, HIV infection impairs this protective response. Protection against PAM is associated with the production of IgG specific for variant surface antigens (VSA-PAM) expressed by chondroitin sulfate A (CSA)-adhering parasitized erythrocytes (PEs). We hypothesized that VSA-PAM-specific IgG confers protection by promoting opsonic phagocytosis of PAM isolates and that HIV infection impairs this response. METHODS AND FINDINGS: We assessed the ability of VSA-PAM-specific IgG to promote opsonic phagocytosis of CSA-adhering PEs and the impact of HIV infection on this process. Opsonic phagocytosis assays were performed using the CSA-adherent parasite line CS2 and human and murine macrophages. CS2 PEs were opsonized with plasma or purified IgG subclasses from HIV-negative or HIV-infected PG and MG Kenyan women or sympatric men. Levels of IgG subclasses specific for VSA-PAM were compared in HIV-negative and HIV-infected women by flow cytometry. Plasma from HIV-negative MG women, but not PG women or men, promoted the opsonic phagocytosis of CSA-binding PEs (p < 0.001). This function depended on VSA-PAM-specific plasma IgG1 and IgG3. HIV-infected MG women had significantly lower plasma opsonizing activity (median phagocytic index 46 [interquartile range (IQR) 18-195] versus 251 [IQR 93-397], p = 0.006) and levels of VSA-PAM-specific IgG1 (mean fluorescence intensity [MFI] 13 [IQR 11-20] versus 30 [IQR 23-41], p < 0.001) and IgG3 (MFI 17 [IQR 14-23] versus 28 [IQR 23-37], p < 0.001) than their HIV-negative MG counterparts. CONCLUSIONS: Opsonic phagocytosis may represent a novel correlate of protection against PAM. HIV infection may increase the susceptibility of multigravid women to PAM by impairing this clearance mechanism.

Disruption of CD36 impairs cytokine response to Plasmodium falciparum glycosylphosphatidylinositol and confers susceptibility to severe and fatal malaria in vivo.

CD36 is a scavenger receptor that has been implicated in malaria pathogenesis as well as innate defense against blood-stage infection. Inflammatory responses to Plasmodium falciparum GPI (pfGPI) anchors are believed to play an important role in innate immune response to malaria. We investigated the role of CD36 in pfGPI-induced MAPK activation and proinflammatory cytokine secretion. Furthermore, we explored the role of this receptor in an experimental model of acute malaria in vivo. We demonstrate that ERK1/2, JNK, p38, and c-Jun became phosphorylated in pfGPI-stimulated macrophages. In contrast, pfGPI-induced phosphorylation of JNK, ERK1/2, and c-Jun was reduced in Cd36(-/-) macrophages and Cd36(-/-) macrophages secreted significantly less TNF-alpha in response to pfGPI than their wild-type counterparts. In addition, we demonstrate a role for CD36 in innate immune response to malaria in vivo. Compared with wild-type mice, Cd36(-/-) mice experienced more severe and fatal malaria when challenged with Plasmodium chabaudi chabaudi AS. Cd36(-/-) mice displayed a combined defect in cytokine induction and parasite clearance with a dysregulated cytokine response to infection, earlier peak parasitemias, higher parasite densities, and higher mortality rates than wild-type mice. These results provide direct evidence that pfGPI induces TNF-alpha secretion in a CD36-dependent manner and support a role for CD36 in modulating host cytokine response and innate control of acute blood-stage malaria infection in vivo.

Placental chondroitin sulfate A-binding malarial isolates evade innate phagocytic clearance.

Pregnancy-associated malaria is characterized by the accumulation of parasitized erythrocytes (PEs) and monocytes in the placenta, and they are believed to directly contribute to adverse birth outcomes. Although most parasite isolates adhere to CD36, placental isolates express novel variant surface antigens (VSAs) and bind to chondroitin sulfate A (CSA). CSA-binding PEs are rarely observed outside of pregnancy, and most primigravid women lack immunity and must rely on innate immune mechanisms to clear these placental parasite variants. We hypothesized that differences in VSA expression and adhesive phenotype between pregnancy-associated (CSA-binding) and non-pregnancy-associated (CD36-binding) isolates may have direct implications for the failure of primigravid women to control the placental parasite burden through innate phagocytic pathways. We demonstrate here, both in vitro and in vivo, that there is a nonopsonic phagocytic defect for CSA-binding PEs. The ability of CSA-binding PEs to evade innate clearance pathways may contribute to the parasite accumulation and recruitment of monocytes that characterize placental malaria.

Nonopsonic phagocytosis of erythrocytes infected with ring-stage Plasmodium falciparum.

Ring-stage parasitized erythrocytes (RPEs) were demonstrated to interact with effector cells of the innate immune system. With receptor blockade studies and CD36-null macrophages, human and murine macrophages were shown to phagocytose RPEs through the pattern recognition receptor CD36. These in vitro data implicate scavenger receptors in the clearance of RPEs.

Enhanced phagocytosis of ring-parasitized mutant erythrocytes: a common mechanism that may explain protection against falciparum malaria in sickle trait and beta-thalassemia trait.

High frequency of erythrocyte (red blood cell [RBC]) genetic disorders such as sickle cell trait, thalassemia trait, homozygous hemoglobin C (Hb-C), and glucose-6-phosphate dehydrogenase (G6PD) deficiency in regions with high incidence of Plasmodium falciparum malaria and case-control studies support the protective role of those conditions. Protection has been attributed to defective parasite growth or to enhanced removal of the parasitized RBCs. We suggested enhanced phagocytosis of rings, the early intraerythrocytic form of the parasite, as an alternative explanation for protection in G6PD deficiency. We show here that P falciparum developed similarly in normal RBCs and in sickle trait, beta- and alpha-thalassemia trait, and HbH RBCs. We also show that membrane-bound hemichromes, autologous immunoglobulin G (IgG) and complement C3c fragments, aggregated band 3, and phagocytosis by human monocytes were remarkably higher in rings developing in all mutant RBCs considered except alpha-thalassemia trait. Phagocytosis of ring-parasitized mutant RBCs was predominantly complement mediated and very similar to phagocytosis of senescent or damaged normal RBCs. Trophozoite-parasitized normal and mutant RBCs were phagocytosed similarly in all conditions examined. Enhanced phagocytosis of ring-parasitized mutant RBCs may represent the common mechanism for malaria protection in nonimmune individuals affected by widespread RBC mutations, while individuals with alpha-thalassemia trait are likely protected by a different mechanism.

The Haldane malaria hypothesis: facts, artifacts, and a prophecy.

Heterozygous thalassemia and sickle cell disease produce mild hematological symptoms but provide protection against malaria mortality and severe malaria symptoms. Two explanations for resistance are considered in the literature - impaired growth of the parasite or enhanced removal by the host immune cells. A critical overview of studies that connect malaria resistance with impaired intra-erythrocytic growth is presented. All studies are fraught with two kinds of bias. The first one resides in the impossibility of reproducing the in vivo situation in the simplified model in vitro. The second stems from the generalized use of RPMI 1640 culture medium. RPMI 1640 has critically low levels of several amino acids; is devoid of hypoxanthine (essential for parasite growth) and adenine; and is low in reduced glutathione. Analysis of representative studies indicates that impaired parasite growth in heterozygous red blood cells (RBCs) may derive from nutrient limitations and, therefore, possibly be of artefactual origin. This conclusion seems plausible because studies were performed with RPMI 1640 medium at relatively high hematocrit and for prolonged periods of time. Mutations considered are particularly sensitive to nutrient deprivation because they have higher metabolic demands due to permanent oxidant stress related to unpaired globin chains, sickle hemoglobin and high levels of membrane-free iron. In addition, non-parasitized AS- and thalassemic-RBCs are dehydrated and microcytic. Thus, the number of metabolically active elements per unit of blood volume is remarkably larger in mutant RBCs compared to normocytes. The latter point may represent a confirmation of Haldane's prophetic statement: 'The corpuscles of the anaemic heterozygotes are smaller than normal, and more resistant to hypotonic solutions. It is at least conceivable that they are also more resistant to attacks by the sporozoa which cause malaria.'

16alpha-bromoepiandrosterone, an antimalarial analogue of the hormone dehydroepiandrosterone, enhances phagocytosis of ring stage parasitized erythrocytes: a novel mechanism for antimalarial activity.

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 micro M, as well as their analogue, 16alpha-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 micro M; T996/86, IC(50) of EPI, 7.5 +/- 0.91 micro M, and IC(50) of DHEA-S, 19 +/- 2.6 micro M) and one mildly chloroquine-resistant strain (FCR-3, IC(50) of EPI, 6.5 +/- 1.01 micro 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. Plasma-compatible, 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.