A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent 'hypnozoite' forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

The presence of leukocytes in ex vivo assays significantly increases the 50-percent inhibitory concentrations of artesunate and chloroquine against Plasmodium vivax and Plasmodium falciparum.

Plasmodium species ex vivo sensitivity assay protocols differ in the requirement for leukocyte removal before culturing. This study shows that the presence of leukocytes significantly increases the 50% inhibitory concentration (IC50) of P. vivax and P. falciparum to artesunate and chloroquine relative to results with the paired leukocyte-free treatment. Although leukocyte removal is not an essential requirement for the conduct of ex vivo assays, its use has important implications for the interpretation of temporal and spatial antimalarial sensitivity data.

Reversal of chloroquine resistance in malaria parasite Plasmodium falciparum by desipramine.

Desipramine and several other tricyclic antidepressant drugs reverse chloroquine resistance in Plasmodium falciparum in vitro at concentrations observed in the plasma of human patients treated for depression. Reversal of resistance is associated with increased chloroquine accumulation in the parasite, probably because of inhibition of a putative chloroquine efflux pump. When owl monkeys (Aotus lemurinus lemurinus) infected with chloroquine-resistant Plasmodium falciparum were treated with chloroquine plus desipramine, their parasitemias were rapidly suppressed. Desipramine was found to be one of the most effective compounds yet described for the reversal of chloroquine resistance both in vitro and in vivo.

Efflux of chloroquine from Plasmodium falciparum: mechanism of chloroquine resistance.

Chloroquine-resistant Plasmodium falciparum accumulate significantly less chloroquine than susceptible parasites, and this is thought to be the basis of their resistance. However, the reason for the lower accumulation of chloroquine was unknown. The resistant parasite has now been found to release chloroquine 40 to 50 times more rapidly than the susceptible parasite, although their initial rates of chloroquine accumulation are the same. Verapamil and two other calcium channel blockers, as well as vinblastine and daunomycin, each slowed the release and increased the accumulation of chloroquine by resistant (but not susceptible) Plasmodium falciparum. These results suggest that a higher rate of chloroquine release explains the lower chloroquine accumulation, and thus the resistance observed in resistant Plasmodium falciparum.

Association between mutations in Plasmodium falciparum chloroquine resistance transporter and P. falciparum multidrug resistance 1 genes and in vivo amodiaquine resistance in P. falciparum malaria-infected children in Nigeria.

This study investigated the association between Plasmodium falciparum chloroquine resistance transporter (pfcrt) T76 and P. falciparum multidrug resistance gene 1 (pfmdr1) Y86 alleles and in vivo amodiaquine (AQ) resistance, as well as the clearance of parasites harboring these two alleles in children treated with AQ in southwest Nigeria. One hundred one children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of AQ and followed-up for 28 days. Blood samples were collected on filter paper samples at enrollment and during follow-up for identification of parasite genotypes and pfcrt and pfmdr1 mutations using polymerase chain reaction and restriction fragment length polymorphism approaches. Parasitologic assessment of response to treatment showed that 87% and 13% (RI) of patients were cured and failed treatment, respectively. Although infections in patients were polyclonal (as determined by merozoite surface protein 2 genotyping), the presence of both mutants pfcrtT76 and pfmdr1Y86 alleles in parasites is associated with in vivo AQ resistance (odds ratio = 7.58, 95% confidence interval = 1.58-36.25, P = 0.006) and is selected by the drug in children who failed AQ treatment. Treatment failure with the combination of mutant pfcrtT76 and pfmdr1Y86 alleles as well as the ability of patients to clear these resistant parasites is dependent on age, suggesting a critical role of host immunity in clearing AQ-resistant P. falciparum. The combination of mutant pfcrtT76 and pfmdr1Y86 alleles may be useful markers for monitoring the development and spread of AQ resistance, when combining this drug with other antimalarials for treatment of malaria in Africa.

Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine-pyrimethamine resistance in malaria-infected patients from Nigeria.

Mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes have been used as means to predict treatment failure to sulfadoxine-pyrimethamine (SP) and for monitoring/surveillance of resistance to the drug in many areas where malaria is endemic. However, patients responses to treatment are significantly dependent on factors like host immunity profile of treated patients. In order to investigate the relationship between molecular markers of SP resistance, host immunity and clinical outcome, the association between pre-treatment dhfr and dhps genotypes, age and treatment outcomes was evaluated in 109 children treated with SP for acute uncomplicated malaria in Ibadan, Nigeria. Seventy-three percent of the children were cured with the drug, while 27% failed treatment after 28 days of follow-up. All children infected with parasites harboring less than two dhfr/dhps mutations were cured with SP. The dhfr triple (Asn-108/Ile-51/Arg-59) mutants or the dhps double mutants (Gly-437/Glu-540) were independently associated with SP treatment failure in children aged less than 5 years, but not in older children. The dhfr and dhps quintuple mutant (dhfr triple mutant+dhps double mutant) was the genotype most strongly associated with SP treatment failure (OR=24.72, 95%CI=8.24-74.15) in both younger and older children.

Serial analysis of gene expression (SAGE) in Plasmodium falciparum: application of the technique to A-T rich genomes.

The advent of high-throughput methods for the analysis of global gene expression, together with the Malaria Genome Project open up new opportunities for furthering our understanding of the fundamental biology and virulence of the malaria parasite. Serial analysis of gene expression (SAGE) is particularly well suited for malarial systems, as the genomes of Plasmodium species remain to be fully annotated. By simultaneously and quantitatively analyzing mRNA transcript profiles from a given cell population, SAGE allows for the discovery of new genes. In this study, one reports the successful application of SAGE in Plasmodium falciparum, 3D7 strain parasites, from which a preliminary library of 6880 tags corresponding to 4146 different genes was generated. It was demonstrated that P. falciparum is amenable to this technique, despite the remarkably high A-T content of its genome. SAGE tags as short as 10 nucleotides were sufficient to uniquely identify parasite transcripts from both nuclear and mitochondrial genomes. Moreover, the skewed A-T content of parasite sequence did not preclude the use of enzymes that are crucial for generating representative SAGE libraries. Finally, a few modifications to DNA extraction and cloning steps of the SAGE protocol proved useful for circumventing specific problems presented by A-T rich genomes.

Amplification of pfmdr 1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand.

Drug resistance in Plasmodium falciparum is an expanding problem in most endemic areas. Recent studies have suggested the potential involvement of genes in the MDR gene family in resistance to quinoline-containing compounds in P. falciparum. In this study a molecular analysis of pfmdr 1 in recent isolates from Thailand was done (1) to further examine the role of pfmdr 1 in drug-resistant isolates and (2) to examine the reported association of pfmdr 1 intragenic alleles and chloroquine resistance. Most of the isolates (10 of 11) were resistant to all compounds tested. Analysis of pfmdr 1 revealed an apparent association between increased gene copy number and increased level of expression of pfmdr 1 and decreased susceptibility to mefloquine and halofantrine. Sequence analysis of pfmdr 1 in these isolates revealed no association of intragenic alleles with chloroquine resistance.

Syntheses and antimalarial activities of 10-substituted deoxoartemisinins.

Two series of 10-substituted deoxoartemisinin derivatives have been synthesized. The first employed the reaction of dihydroartemisinin acetate with several silyl enol ethers in the presence of titanium tetrachloride. The second utilized the reaction of 10-(2-oxoethyl)deoxoartemisinin with several Grignard reagents. The in vitro antimalarial activities of both series were determined against two drug-resistant clones of P. falciparum. The activities of 13 beta and 15 beta were 5-7 times greater than that of artemisinin.

Antimalarial activity of new dihydroartemisinin derivatives. 7. 4-(p-substituted phenyl)-4(R or S)-[10(alpha or beta)-dihydroartemisininoxy]butyric acids.

To search for water soluble dihydroartemisinin derivatives with higher efficacy and longer plasma half-life than artesunic or artelinic acid, a series of new stereoisomers of 4-(p-substituted phenyl)-4(R or S)-[10(alpha or beta)-dihydroartemisininoxy]butyric acids were synthesized as new potential antimalarial agents. Two approaches were taken in the design of these new molecules in an attempt to (a) increase the lipophilicity of the molecule and (b) decrease the rate of oxidative dealkylation of the target compounds. The new compounds showed a 2-10-fold increase in in vitro antimalarial activity against D-6 and W-2 clones of Plasmodium falciparum than artemisinin or artelinic acid. R-diastereomers are, in general, more potent than the corresponding S-diastereomers. p-Chlorophenyl and p-bromophenyl derivatives showed in vivo oral antimalarial activity against P. berghei (with 3/8 cured) superior to that of artelinic acid (1/8 cured), whereas p-fluorophenyl and p-methoxyphenyl analogs demonstrated activity only comparable (1/8 cured) to that of artelinic acid at the same dosage level (64 mg/kg twice a day). The in vivo antimalarial activity of these new compounds correlates with their SD50 (50% parasitemia suppression dose). The biological results suggested that an electronic effect, besides the lipophylicity, may play a role in determining the efficacy of this class of compounds.

Methyl-substituted dispiro-1,2,4,5-tetraoxanes: correlations of structural studies with antimalarial activity.

Two tetramethyl-substituted dispiro-1,2,4,5-tetraoxanes (7,8,15, 16-tetraoxadispiro[5.2.5.2]hexadecanes) 3 and 4 were designed as metabolically stable analogues of the dimethyl-substituted dispiro-1, 2,4,5-tetraoxane prototype WR 148999 (2). For a positive control we selected the sterically unhindered tetraoxane 5 (7,8,15, 16-tetraoxadispiro[5.2.5.2]hexadecane), devoid of any substituents. Tetraoxanes 3 and 4 were completely inactive in contrast to tetraoxanes 2 and 5. We hypothesize that the two inactive tetraoxanes possess sufficient steric hindrance about the tetraoxane ring due to the two additional axial methyl groups to prevent their activation to presumed parasiticidal carbon radicals by inhibiting electron transfer from heme or other iron(II) species. For each of the tetraoxanes 2-4, the tetraoxane and both spirocyclohexyl rings are in a chair conformation and the bond lengths and angles are all quite normal except for the C1-C2 bond which is slightly lengthened. Comparison of the modeled and X-ray structures for tetraoxanes 2-5 reveals that molecular mechanics (MMX and MM3) and 3-21G calculations each gave accurate structural parameters such as bond lengths, bond angles, and dihedral angles. In contrast, semiempirical methods such as AM1 gave poor results.

Cholic acid derivatives as 1,2,4,5-tetraoxane carriers: structure and antimalarial and antiproliferative activity.

Cholic acid-derived 1,2,4,5-tetraoxanes were synthesized in order to explore the influence of steroid carrier on its antimalarial and antiproliferative activity in vitro. Starting with chiral ketones, cis and trans series of diastereomeric tetraoxanes were obtained, and the cis series was found to be approximately 2 times as active as the trans against Plasmodium falciparum D6 and W2 clones. The same tendency was observed against human melanoma (Fem-X) and human cervix carcinoma (HeLa) cell lines. The amide C(24) termini, for the first time introduced into the carrier molecule of a tetraoxane pharmacophore, significantly enhanced both antimalarial and antiproliferative activity, as compared to the corresponding methyl esters, with cis-bis(N-propylamide) being most efficient against the chloroquine-susceptible D6 clone (IC(50) = 9.29 nM). cis- and trans-bis(N-propylamides) were also screened against PBMC, and PHA-stimulated PBMC, showing a cytotoxicity/antimalarial potency ratio of 1/10 000.

Synthesis and antimalarial activity of sixteen dispiro-1,2,4, 5-tetraoxanes: alkyl-substituted 7,8,15,16-tetraoxadispiro[5.2.5. 2]hexadecanes.

Sixteen alkyl-substituted dispiro-1,2,4,5-tetraoxanes (7,8,15, 16-tetraoxadispiro[5.2.5.2]hexadecanes) were synthesized to explore dispiro-1,2,4,5-tetraoxane SAR and to identify tetraoxanes with better oral antimalarial activity than prototype tetraoxane 1 (WR 148999). The tetraoxanes were prepared either by peroxidation of the corresponding cyclohexanone derivatives in H(2)SO(4)/CH(3)CN or by ozonolysis of the corresponding cyclohexanone methyl oximes. Those tetraoxanes with alkyl substituents at the 1 and 10 positions were formed as single stereoisomers, whereas the five tetraoxanes formed without the stereochemical control provided by alkyl groups at the 1 and 10 positions were isolated as mixtures of diastereomers. Three of the sixteen tetraoxanes were inactive (IC(50)'s > 1000 nM), but five (2, 6, 10, 11, 12) had IC(50)'s between 10 and 30 nM against the chloroquine-sensitive D6 and chloroquine-resistant W2 clones of Plasmodium falciparum compared to corresponding IC(50)'s of 55 and 32 nM for 1 and 8.4 and 7.3 nM for artemisinin. We suggest that tetraoxanes 13, 16, and 17 were inactive and tetraoxanes 4 and 7 were weakly active due to steric effects preventing or hindering peroxide bond access to parasite heme. Tetraoxanes 1, 10, 11, and 14, along with artemisinin and arteether as controls, were administered po b.i.d. (128 mg/kg/day) to P. berghei-infected mice on days 3, 4, and 5 post-infection. At this dose, tetraoxanes 10, 11, and 14 cured between 40% and 60% of the infected animals. In comparison, artemisinin and tetraoxane 1 produced no cures, whereas arteether cured 100% of the infected animals. There was no apparent relationship between tetraoxane structure and in vitro neurotoxicity, nor was there any correlation between antimalarial activity and neurotoxicity for these seventeen tetraoxanes.

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I.

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

Reversal of Plasmodium falciparum resistance to chloroquine in Panamanian Aotus monkeys.

An Aotus-Plasmodium falciparum model was used to determine if chloroquine resistance could be reversed in vivo. The putative resistance modulators tested all reverse chloroquine resistance in vitro and included verapamil, chlorpromazine, prochlorperazine, cyproheptadine, ketotifen, a tiapamil analog (Ro 11-2933), and a chlorpromazine analog (SKF 2133-A). Combinations of chloroquine plus chlorpromazine or prochlorperazine confirmed reversal of chloroquine resistance as exhibited by cures obtained in six Aotus monkeys infected with chloroquine-resistant P. falciparum (Vietnam Smith/RE strain) and rapid clearance of parasitemia, followed by recrudescence in six additional monkeys. The results indicate the following order of in vivo efficacy for reversing chloroquine resistance in Aotus: chlorpromazine > prochlorperazine >> desipramine >> Ro 11-2933 (tiapamil analog) > ketotifen. Cyproheptadine and verapamil were not effective in reversing chloroquine resistance and probable drug toxicity was observed with these drugs in combination with chloroquine.

Characteristics of multidrug resistance in Plasmodium and Leishmania: detection of P-glycoprotein-like components.

Multidrug-resistance (MDR) in neoplastic cells is frequently characterized by the overexpression of P-glycoprotein (PGP), a 170 kDa transmembrane glycoprotein that binds multiple cytotoxic drugs as well as calcium channel antagonists. Chloroquine resistance in Plasmodium falciparum appears to be analogous to MDR in neoplastic cells, where the induction of resistance with one drug confers resistance to other structurally and functionally unrelated drugs. To test the hypothesis that chloroquine resistance in P. falciparum and antimony resistance in Leishmania is mediated by a similar mechanism of MDR in mammalian neoplastic cells, a PGP-specific monoclonal antibody (C219) was used to determine the presence of PGP genes in resistant and sensitive Plasmodium and Leishmania parasites by indirect immunofluorescence assays and Western blotting procedures. These PGP-like components were detected in both drug-sensitive and -resistant Plasmodium and Leishmania cells. A 40-42 kDa component was observed to be greater in a chloroquine-resistant P. berghei (C line) than in a chloroquine-susceptible P line. Differences observed between Pentostam-resistant and -sensitive Leishmania promastigote clones and isolates included the increased expression of 96-106 and 23-25 kDa peptides in drug-resistant L. enrietti, and increased amounts of two different peptides in two drug-resistant L. panamensis clones (i.e., 96-106 and 43-45 kDa in WR-746-CL4, and 53 and 23-25 kDa in kDa) in amastigotes as in MDR KB carcinoma cells (KB-V1). Comparative indirect immunofluorescent studies suggested that a correlation existed between the degree of antimony susceptibility and the concentration of the moiety recognized by C219 in two L. panamensis clones. Binding of the C219 monoclonal antibody to the PGP-like component of Leishmania was blocked by Pentostam, while the binding of C219 to multiple-drug resistant KB-V1 PGP was not inhibited by Pentostam, regardless of the PGP concentration. This suggests some degree of specificity in the binding of Pentostam to the Leishmania PGP-like components. In addition, these studies have demonstrated that drug-sensitive Leishmania accumulate two to five times more 125Sb-Pentostam than resistant clones.

Open randomized trial of oral artemether alone and a sequential combination with mefloquine for acute uncomplicated falciparum malaria.

One hundred fifty-one patients with acute uncomplicated falciparum malaria were enrolled in a randomized, open-label study of oral artemether given alone for five or seven days or a sequential treatment of oral artemether followed by mefloquine. Forty patients received oral artemether, 100 mg initially, then 50 mg every 12 hr for a total dose of 500 mg over a five-day period: Group I. Fifty-eight patients received oral artemether, 100 mg initially, then 50 mg every 12 hr for a total dose of 750 mg over a seven-day period: Group II. Fifty-three patients received oral artemether, 200 mg every 8 hr for a total dose of 600 mg, followed 8 hr later with mefloquine (1,250 mg divided into two doses given 6 hr apart: Group III. All patients were admitted to the hospital for 28 days to exclude reinfection and 131 patients remained through the 28-day follow-up. Only two, nine, and nine patients in Groups I, II, and III, respectively, left the hospital prior to study completion for reasons unrelated to their treatment. Cure rates for the three groups were 74% (28 of 38) for Group I, 98% (48 of 49) for Group II, and 98% (43 of 44) for Group III. Mean fever and parasite clearance times were not significantly different (32.8, 27.5, and 31.4 hr for fever clearance times and 40.2, 40.6, and 36.7 hr for parasite clearance times of Groups I, II, and III, respectively) nor were any adverse effects seen. In vitro drug susceptibility testing of admission and recrudescent parasite isolates was conducted for 10 patients. These data showed no decreased response to artemether or dihydroartemisinin in recrudescent isolates when compared with admission isolates. The results of this study suggest that sequential treatment for two days with oral artemether (600 mg) followed by mefloquine (1,250 mg) is effective and well-tolerated in patients with acute uncomplicated falciparum malaria and may be an alternative treatment for multidrug-resistant falciparum malaria, particularly useful for treating patients in rural areas where the period of admission to the hospital should be as short as possible. A seven-day regimen of artemether alone (750 mg) is also very effective, yet requires prolonged administration of drug after malaria symptoms disappear.

Disposition of proguanil in Thai patients with uncomplicated falciparum malaria.

The objective of this study was to examine the disposition of proguanil in malaria-infected Thai patients with acute uncomplicated falciparum malaria. Eleven patients were administered 500 mg of proguanil twice a day for three days (total dose = 3,000 mg). Four patients were tentatively classified as extensive metabolizers (EMs) and seven as poor metabolizers (PMs). The mean plasma clearances of proguanil for EMs and PMs were 1.31 and 1.10 L/hr/kg, respectively. The mean elimination half-life of proguanil was statistically longer in PMs than EMs (19.6 hr versus 16.1 hr; P = 0.01). Plasma clearance and elimination half-life of proguanil in the malaria patients were comparable with those reported in the literature for healthy Thai volunteers. In contrast to other ethnic groups. Thai EM patients had relatively low plasma concentrations of cycloguanil, the active metabolite of proguanil. None of the 11 patients treated with proguanil were cured of malaria and their phenotype status did not affect the treatment outcome. Although high levels of parasite resistance to cycloguanil were probably responsible for the poor response to proguanil treatment, the inability of Thai EM and PM patients to produce cycloguanil may have also contributed to the treatment outcome.

Randomized trial of mefloquine-doxycycline, and artesunate-doxycycline for treatment of acute uncomplicated falciparum malaria.

One hundred nine adult patients with acute uncomplicated falciparum malaria were randomly selected to receive combinations of either doxycycline plus mefloquine or doxycycline plus artesunate. Fifty-four patients received mefloquine (1,250 mg divided between two doses of 750 and 500 mg six hours apart) with doxycycline and 55 patients received artesunate (300 mg total for 2.5 days; 100 mg followed by 50 mg every 12 hr for 2.5 days) with doxycycline. Doxycycline was administered in doses of 200 mg once a day for seven days. All patients were admitted to the hospital for 28 days to exclude reinfection. Ninety-seven patients completed the study; 12 patients left prior to completion of follow-up for reasons unrelated to their treatment. Cure rates for the two groups were 96% (46 of 48) for mefloquine plus doxycycline and 80% (39 of 49) for artesunate plus doxycycline. Mean fever and parasite clearance times were significantly shorter in the group that received artesunate plus doxycycline (38.7 and 41.3 hr) than mefloquine plus doxycycline (64.3 and 69.0 hr), respectively. In vitro drug sensitivity testing of selected isolates obtained prior to treatment indicated that eight of nine admission isolates were resistant to mefloquine; all isolates were susceptible to artesunate. Recrudescent isolates failed to show a pattern of decreased sensitivity to the drugs to which the parasites had been exposed during treatment; the studies showed decreased sensitivity to doxycycline in only two of eight isolates tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of leukocyte adhesion molecules CD11b/CD18 and leukocyte adhesion molecule-1 on phagocytic cells activated by malaria pigment.

There is increasing evidence that inappropriate immune activation induced by parasite products occurs in malaria disease. To further elucidate the role of Plasmodium falciparum-derived products on host immune activation, we studied the expression of leukocyte adhesion molecules (CD11b/CD18 and LAM-1) on neutrophils and monocytes in response to malaria pigment using flow cytometry. Exposure of leukocytes to isolated malaria pigment derived from ruptured schizonts resulted in significant up-regulation of CD11b/CD18 expression and down-regulation of LAM-1 on both neutrophils and monocytes. In contrast, culture supernatants (pigment free) from ruptured schizonts did not alter the expression of CD11b/CD18 and LAM-1. The increase of CD11b/CD18 and the loss of LAM-1 expression occurred simultaneously with the earliest response detected at 10 min and a plateau reached by 60 min. The effect of malaria pigment on leukocyte adhesion molecules was inhibited by EDTA in a dose-dependent manner. Phagocytosis of malaria pigment was also suppressed by EDTA. This observation suggests that phagocytosis of malaria pigment may be a prerequisite for the effect of malaria pigment on the regulation of CD11b/CD18 and LAM-1 expression. Regulation of leukocyte adhesion molecules through up-regulation of CD11b/CD18 and down-regulation of LAM-1 by malaria pigment could promote leukocyte adherence to endothelium in vivo. This increased adherence of malaria pigment-activated leukocytes might induce cytokine (tumor necrosis factor alpha and interleukin-1beta)-mediated increases in capillary permeability resulting in local tissue edema, and a cytokine-mediated increase in adhesion molecule expression causing vascular clogging by adherent red blood cells, and in severe disease by adherent leukocytes.