Immunization of mice with Plasmodium TCTP delays establishment of Plasmodium infection.

Translationally controlled tumour protein (TCTP) may play an important role in the establishment or maintenance of parasitemia in a malarial infection. In this study, the potential of TCTP as a malaria vaccine was investigated in two trials. In the initial vaccine trial, Plasmodium falciparum TCTP (PfTCTP) was expressed in Saccharomyces cerevisiae and used to immunize BALB/c mice. Following challenge with Plasmodium yoelii YM, parasitemia was significantly reduced during the early stages of infection. In the second vaccine trial, the TCTP from P. yoelii and P. berghei was expressed in Escherichia coli and used in several mouse malaria models. A significant reduction in parasitemia in the early stages of infection was observed in BALB/c mice challenged with P. yoelii YM. A significantly reduced parasitemia at each day leading up to a delayed and reduced peak parasitemia was also observed in BALB/c mice challenged with the nonlethal Plasmodium chabaudi (P.c.) chabaudi AS. These results suggest that TCTP has an important role for parasite establishment and may be important for pathogenesis.

Uncertain outcomes: adjusting for misclassification in antimalarial efficacy studies.

Evaluation of antimalarial efficacy is difficult because recurrent parasitaemia can be due to recrudescence or re-infection. PCR is used to differentiate between recrudescences and re-infections by comparing parasite allelic variants before and after treatment. However, PCR-corrected results are susceptible to misclassification: false positives, due to re-infection by the same variant present in the patient before treatment; and false negatives, due to variants that are present but too infrequent to be detected in the pre-treatment PCR, but are then detectable post-treatment. This paper aimed to explore factors affecting the probability of false positives and proposes a Monte Carlo uncertainty analysis to account for both types of misclassification. Higher levels of transmission intensity, increased multiplicity of infection, and limited allelic variation resulted in more false recrudescences. The uncertainty analysis exploits characteristics of study data to minimize bias in the estimate of efficacy and can be applied to areas of different transmission intensity.

An approach to the development of new drugs for African trypanosomiasis.

The inability of the bloodstream form of Trypanosoma brucei brucei to decompose hydrogen peroxide forms the basis of our attempt to develop new pharmacological agents to kill these organisms. Approximately 1-3% of the oxygen consumed by these parasites appears in the form of hydrogen peroxide. Our previous observation that free radical initiators such as heme and hematoporphyrin D proved to be trypanocidal in vitro and in vivo, respectively, prompted this investigation into the mechanism of action of this class of compounds to enhance their therapeutic efficacy. The locus of H2O2 production within the trypanosome was examined using cell-free homogenates. Experiments described herein suggest that H2O2 is formed by the alpha-glycerol phosphate dehydrogenase in an adventitious manner, and that no enzymatic means of disposing of this potentially toxic compound are present with the organisms. Naphthoquinones were found to substantially increase the rate of both oxygen consumption and H2O2 production by trypanosomal mitochondrial preparations. Presumably, the naphthoquinones are acting as coenzyme Q analogues. The addition of sublytic concentrations of both naphthoquinones and heme leads to a synergistic lysis of the organisms in vitro. Another approach to increasing the susceptibility of T. b. brucei to free radical damage involved reduction of the intracellular concentration of glutathione. This was accomplished through the use of trypanocidal arsenicals. Melarsenoxide and heme acted synergistically in vitro, an effect which was further enhanced via addition of a naphthoquinone. Moreover, hematoporphyrin D and tryparsamide were shown to have a synergistic effect in T. b. brucei-infected mice.

Malaria parasites adopt host cell superoxide dismutase.

Aerobic organisms depend on superoxide dismutase to suppress the formation of dangerous species of activated oxygen. Intraerythrocytic stages of the malaria parasite exist within a highly aerobic environment and cause the generation of increased amounts of activated oxygen. Plasmodium berghei in mice was found to derive a substantial amount of superoxide dismutase activity from the host cell cytoplasm. Plasmodia isolated from mouse red cells contained mouse superoxide dismutase, whereas rat-derived parasites contained the rat enzyme. This is believed to be the first example of the acquisition of a host cell enzyme by an intracellular parasite.

Ultrasound-derived fetal size nomogram for a sub-Saharan African population: a longitudinal study.

OBJECTIVES: To create a fetal size nomogram for use in sub-Saharan Africa and compare the derived centiles with reference intervals from developed countries. METHODS: Fetal biometric measurements were obtained at entry to antenatal care (11-22 weeks' gestation) and thereafter at 4-week intervals from pregnant women enrolled in a longitudinal ultrasound study in Kinshasa, Democratic Republic of Congo. The study population comprised 144 singleton gestations with ultrasound-derived gestational age within 14 days of the menstrual estimate. A total of 755 monthly ultrasound scans were included with a mean +/- SD of 5 +/- 1 (range, 2-8) scans per woman. Estimated fetal weight (EFW) was calculated at each ultrasound examination using the Hadlock algorithm. A general mixed-effects linear regression model that incorporated random effects for both the intercept and slope was fitted to log-transformed EFW to account for both mean growth and within-fetus variability in growth. Reference centiles (5(th), 10(th), 50(th), 90(th) and 95(th) centiles) were derived from this model. RESULTS: Nomograms derived from developed populations consistently overestimated the 50(th) centile EFW value for Congolese fetuses by roughly 5-12%. Differences observed in the 10(th) and 90(th) centiles were inconsistent between nomograms, but generally followed a pattern of overestimation that decreased with advancing gestational age. CONCLUSIONS: In low-resource settings, endemic malaria and maternal nutritional factors, including low prepregnancy weight and pregnancy weight gain, probably lead to lower fetal weight and utilization of nomograms derived from developed populations is not appropriate. This customized nomogram could provide more applicable reference intervals for diagnosis of intrauterine growth restriction in sub-Saharan African populations.

Pneumocystis carinii mutations associated with sulfa and sulfone prophylaxis failures in AIDS patients.

BACKGROUND: Failures of prophylaxis against Pneumocystis carinii pneumonia (PCP) in AIDS patients do occur, but no evidence for drug resistance has yet been presented. OBJECTIVE: To determine whether mutations in the sulfa and sulfone drug target are associated with failure of prophylaxis using a sulfa-containing agent. METHODS: Portions of the gene for P. carinii dihydropteroate synthase (DHPS), the sulfa and sulfone target, from 27 patients (20 of whom had AIDS) diagnosed with PCP between 1976 and 1997 were amplified using polymerase chain reaction and sequenced. Seven of the 27 patients (all of whom had AIDS) were receiving sulfa or sulfone drugs as prophylaxis for PCP. RESULTS: Mutations were found at only two amino-acid positions and were significantly more common in patients who received sulfa/sulfone prophylaxis. Mutations were observed in five (71%) out of seven isolates from AIDS patients receiving sulfa/sulfone as prophylaxis compared with only two (15%) out of 13 specimens from AIDS patients who did not (P = 0.022). No mutations were seen in isolates from seven non-HIV-infected patients, none of whom were on prophylaxis. Mutations were only observed in specimens obtained in 1995-1997. CONCLUSIONS: Mutations in two amino-acid positions were significantly more common in AIDS patients with PCP who failed sulfa/sulfone prophylaxis. These amino acids appeared to be directly involved in both substrate and sulfa binding, based on homology to the Escherichia coli DHPS crystal structure. Thus, the results were consistent with the possibility that mutations in the P. carinii DHPS are responsible for some of the failures of sulfa/sulfone prophylaxis in AIDS patients.

The Plasmodium falciparum translationally controlled tumor protein: subcellular localization and calcium binding.

Artemisinin derivatives are endoperoxide antimalarials widely used to treat falciparum malaria in areas where drug resistance is common. In Plasmodium falciparum-infected erythrocytes, radiolabeled artemisinin derivatives have been shown to react with malarial proteins, one of which is the Translationally Controlled Tumor Protein (TCTP). The P. falciparum TCTP was found by immunofluorescence to be located in both the cytoplasm and food vacuoles. Immunoelectron microscopy shows that it is present in the parasite cytoplasm as well as in its food vacuolar and limiting membranes. Like other TCTPs, the P. falciparum protein binds to calcium. Further studies on the physiological role of TCTP may aid in understanding the mechanism of action of endoperoxide antimalarials.

Prevalence of dihydropteroate synthase mutants in HIV-infected South African children with Pneumocystis jiroveci pneumonia.

BACKGROUND: Pneumocystis jiroveci (formerly Pneumocystis carinii) pneumonia (PCP) is a major cause of mortality in human immunodeficiency virus (HIV)-infected infants in Africa, but the prevalence of mutations in the gene encoding dihydropteroate synthase (DHPS) in isolates from Africa has not been reported. METHODS: This study investigated the prevalence of DHPS mutations in P. jiroveci isolates from South African HIV-infected children with PCP by amplifying DNA using 2 different polymerase chain reactions. RESULTS: P. jiroveci DNA from 30 respiratory specimens was amplified; 26 specimens (86.7%) contained wild-type DHPS alleles. Of the 4 samples (13.3%) with DHPS mutations, 2 contained a homogenous population with single DHPS mutations, 1 contained a homogenous population with 2 DHPS mutations, and the fourth contained a heterogenous population of organisms with both wild-type and single-mutant DHPS genotypes. Only 1 child was receiving trimethoprim-sulphamethoxazole (TMP-SMZ) prophylaxis; this patient was infected with wild-type P. jiroveci. The mortality rate (overall, 20 [66.7%] of 30 children) was not significantly different between children infected with wild-type P. jiroveci (17 [65.4%] of 26) and those infected with mutant strains (3 [75%] of 4; P=.8). CONCLUSIONS: DHPS mutations are uncommon in P. jiroveci isolates from South Africa. However, increasing use of TMP-SMZ prophylaxis may result in widespread development of mutations.

Activated oxygen generation by a primaquine metabolite: inhibition by antioxidants derived from Chinese herbal remedies.

Primaquine is an important antimalarial drug which causes hemolytic anemia in patients with glucose-6-phosphate dehydrogenase (G6PDH) deficiency, probably due to oxidant generation by its metabolites. One of primaquine's metabolites, 5,6-dihydroxy-8-aminoquinoline (AQD), was found to cause chemiluminescence (CL) in vitro when incubated in the presence of luminol. This CL is inhibited by catalase and deferoxamine, unaffected by mannitol, and stimulated by superoxide dismutase (SOD), suggesting that it is mediated by H2O2. Three antioxidants (daphnetin, ferulate, and maltol), derived from Chinese herbal remedies, inhibited AQD- and H2O2-mediated CL, whereas a fourth, anisodamine, had no effect. Daphnetin also potently inhibited H2O2-mediated lipid peroxidation as measured by the production of thibarbituric acid reacting substances (TBARS). Thus, the possibility is raised that an antioxidant might be able to mitigate the oxidant hemolytic effects of primaquine.

Scavenger enzymes and resistance to oxygen mediated damage in Trichinella spiralis.

In order to understand why different stages of Trichinella spiralis vary in their susceptibility to killing by leukocytes, the effects of artificially generated oxidants on different stages of this parasite were compared. More than 90% newborn larvae were killed after incubation in acetaldehyde-xanthine oxidase or glucose-glucose oxidase. On the other hand, fewer than 10% of adult worms or muscle larvae were killed when incubated under identical conditions. Thus, only the stages which are resistant to killing by leukocytes are resistant to killing by oxidants. The larvicidal effect of acetaldehyde-xanthine oxidase was blocked by the addition of either superoxide dismutase or catalase and was partially inhibited by radical scavengers and singlet oxygen quenchers. The oxidant resistant adults and muscle larvae contained 3-5 times more superoxide dismutase and at least five times more glutathione peroxidase than the oxidant sensitive newborn larvae. In contrast, all 3 stages lacked detectable amounts of catalase and contained roughly equivalent amounts of reduced glutathione. Accordingly, adults and muscle larvae may be more resistant to killing by leukocytes than newborn larvae because they contain better oxidant defenses.

Binding of chloroquine to DNA.

Three diverse mechanisms have been proposed to explain the antimalarial activity of chloroquine: binding to DNA, binding to hemin, or alkalinizing parasite lysosomes. In order to assess the importance of DNA binding, we have measured the affinity of DNA for chloroquine by equilibrium dialysis using tritiated chloroquine. The dissociation constant of the DNA-chloroquine complex varied from 27 microM to 2.6 mM, depending on the ionic strength of the buffer. Our results suggest that chloroquine binding to DNA is highly dependent on salt concentration. Nevertheless, because of the large number of sites present, binding to parasite DNA may still account for the antimalarial activity of chloroquine.

Hemoglobin catabolism and iron utilization by malaria parasites.

Erythrocytic malaria parasites transport large quantities of erythrocyte cytoplasm to an acidic food vacuole, where hemoglobin is degraded. Globin is hydrolysed to free amino acids, which are subsequently incorporated into parasite proteins. Potentially toxic heme moieties are polymerized to hemozoin and also probably provide necessary parasite iron. Our understanding of the precise mechanisms of hemoglobin processing is incomplete. However, it is clear that hemoglobin catabolism and related events in the malarial food vacuole are the likely targets of both important antimalarial drugs and of promising new compounds. Thus, a more precise characterization of the metabolism of hemoglobin and iron by malaria parasites should expedite the development of new modes of antimalarial chemotherapy.

p-Aminobenzoic acid transport by normal and Plasmodium falciparum-infected erythrocytes.

De novo folate biosynthesis is required for the growth of malarial parasites and is inhibited by several important antimalarial agents. We show here that exogenous p-aminobenzoic acid (pABA) can be utilized by malaria parasites to synthesize folates. The transport of pABA into parasite infected red cells was therefore characterized. Normal red cells transport pABA in a saturable and energy-dependent manner, with a dissociation constant of 83 nM. pABA transport in parasite-infected red cells may use the same mechanism, as demonstrated by similarities in time course, concentration-response, and dissociation constant (111 nM). The transport capacity of red cells is temperature-, energy- and pH-dependent. It is inhibited by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP), but not by the sodium ionophores nigericin and monensin. p-Aminosalicylic acid (PAS) inhibits pABA transport competitively, with a inhibition constant of 378 nM. Phloritin, flufanamic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DITS), which are inhibitors of the anion transporter (band 3), and oxalic acid, a substrate of this transporter, partially inhibit pABA transport into both normal and infected red cells. Interestingly, in both normal and infected red cells, the inhibitory effects of PAS and the anion transport inhibitors are additive, suggesting the involvement of 2 independent mechanisms.

Identification of hemoglobin degradation products in Plasmodium falciparum.

Malaria parasites break down human hemoglobin to its constituent amino acids by cysteine and aspartic proteinases. However, no one has previously been able to identify hemoglobin cleavage products in intact parasites. When isolated parasites were subjected to non-denaturing polyacrylamide gels electrophoresis, a unique protein band was found which contains heme and reacts with anti-human hemoglobin antibodies. This protein does not appear to represent oxidized or glycosylated hemoglobin, and is present in isolated parasites but not in the cytosol of infected or uninfected erythrocytes. When this band was eluted and subjected to SDS polyacrylamide gel electrophoresis, three bands were seen on Western blots. The proteins in these bands contain proteins with the N-terminal sequences of alpha- and beta-globin chains but molecular masses of only 13.2-13.4 kDa. These data suggest that hemoglobin alpha- and beta-chains are initially cleaved within the parasite phagolysosome to release peptides of 15-17 and 23-25 amino acids from the C-termini of alpha- and beta-globin chains, respectively. Production of the hemoglobin breakdown products was inhibited by E-64, a cysteine proteinase inhibitor, suggesting the involvement of a cysteine proteinase in an early step of hemoglobin degradation.

The interaction of artemisinin with malarial hemozoin.

[14C]Artemisinin was taken up by Plasmodium falciparum in culture and concentrated in hemozoin. In vitro, hemin and artemisinin were found to undergo a chemical reaction forming two major products which were isolated by high-performance liquid chromatography (HPLC). The m/z values of the two products were 856 and 871. Thin-layer chromatography (TLC) and HPLC of hemozoin isolated from [14C]artemisinin-treated parasites showed that the majority of the hemozoin-associated radioactivity comigrated with the synthetic adducts. When [14C]artemisinin was incubated with isolated hemozoin, [14C]artemisinin disappeared from the solution in a time-dependent manner. Some of the radioactivity present in the treated hemozoin also comigrated with the adducts on TLC. Thus, artemisinin appears to react covalently with heme in malaria hemozoin both in vitro and in situ.

Effects of antimalarials and protease inhibitors on plasmodial hemozoin production.

Malarial hemozoin may play an important role as a target for antimalarial drugs and in disease pathogenesis. A new assay for hemozoin was developed in which the hemozoin was separated from cells by filtration. Trophozoites have substantially more hemozoin than rings, but there are relatively small differences between chloroquine-sensitive and chloroquine-resistant strains. The effects of hemozoin content of chloroquine and artemisinin, two antimalarial drugs, and E64 and Pepstatin A, two protease inhibitors, were measured. At concentrations at which hypoxanthine incorporation was unaffected, the hemozoin content of rings was decreased by E64, but not by the other three compounds. Artemisinin and Pepstatin A also had little effect on the hemozoin content of trophozoites. Chloroquine and E64 inhibited trophozoite hemozoin formation, but inhibited hypoxanthine uptake to a similar or greater extent. When either rings or trophozoites were exposed to several higher concentrations of chloroquine, hemozoin content was diminished, but significantly less than hypoxanthine uptake. Various concentrations of E64, in contrast, inhibited hemozoin production by both rings and trophozoites significantly more than hypoxanthine incorporation, suggesting that hemozoin production may be directly affected by E64.

Oxidant defense enzymes of Plasmodium falciparum.

We have measured and characterized three oxidant defense enzymes in early and late intraerythrocytic stages of the human malarial parasite, Plasmodium falciparum. Isolated early intraerythrocytic stages contain catalase (24.1 mumol min-1 (mg protein)-1) and superoxide dismutase (SOD; 6.3 units (mg protein)-1) but little or no glutathione peroxidase (GPX; less than 2 mumol min-1 (mg protein)-1). Isolated late intraerythrocytic stages of P. falciparum contain slightly less catalase (17.0 mumol min-1 (mg protein)-1) but significantly more GPX (7.7 mumol min-1 (mg protein)-1) and SOD (25.1 units (mg protein)-1). P. falciparum, like P. berghei, probably acquires most of its SOD from its host, since parasite-associated SOD is predominantly cyanide-sensitive, and has the same pI as host SOD. Unlike P. berghei, however, late stages of P. falciparum contain an additional SOD isozyme which is not cyanide-sensitive and may represent an endogenous enzyme. Parasites grown in red cells that have been partially depleted of SOD are more sensitive to exogenously generated superoxide, suggesting some dependence of the parasite on host SOD.

An iron-containing superoxide dismutase in Tritrichomonas foetus.

Dialysed extracts of Tritrichomonas foetus were found to have superoxide dismutase at substantially higher levels than those found in trypanosomatids and mouse red blood cells. The activity was sensitive to inhibition by H2O2 but not by cyanide, suggesting that this organism has iron-containing superoxide dismutase(s). Three isozymes were seen by isoelectric focusing which appeared to be sensitive to inhibition by H2O2.

Artemisinin (qinghaosu): the role of intracellular hemin in its mechanism of antimalarial action.

Artemisinin (qinghaosu), is a promising new antimalarial drug derived from an ancient Chinese herbal remedy. When [13-14C]artemisinin is added to cultures of Plasmodium falciparum, it is converted into a product with different solubility and chromatographic properties than the parent drug. Artemisinin reacts with hemin in aqueous solution to form an adduct with an apparent molecular weight of 914 which has identical chromatographic, solubility, and electrophoretic behavior to the parasite-derived product. The reaction between artemisinin and hemin, when carried out in the presence of red cell membranes, leads to the oxidation of protein thiols. Malarial parasites are rich in hemin; artemisinin's reactivity toward hemin may explain its selective toxicity to malarial parasites.

Structure-activity relationships of lactone ring-opened analogs of the antimalarial 1,2,4-trioxane artemisinin.

1,2,4-Trioxane benzylic ethers 8a-e were prepared as simplified, tricyclic versions of the clinically used tetracyclic antimalarial drug artemisinin (1). Five additional artemisinin analogs (9-11) were prepared. Neither water solubility (analogs 8e and 11b) nor chelating ability (analogs 9 and 10), however, produced trioxanes of especially high in vitro antimalarial activity. Trioxane fluorobenzyl ether 8b is the most active in this series (more active than artemisinin) against Plasmodium falciparum parasites in vitro, with substantial activity also in mice infected with Plasmodium berghei parasites and with 10 times higher activity than artemisinin (1) in killing immature P. falciparum gametocytes.