Lack of Association of the HbE Variant with Protection from Cerebral Malaria in Thailand

Hemoglobin E (HbE; beta26Glu --> Lys) is the most common variant of the beta-globin gene in Southeast Asia; it has been suggested that it confers resistance against Plasmodium falciparum malaria. In this study 306 adult patients with P. falciparum malaria (198 mild and 108 cerebral malaria patients) living in northwest Thailand were investigated to examine whether the HbE variant is associated with protection from cerebral malaria. Our results revealed that the sample allele frequency of HbE was not significantly different between mild (7.3%) and cerebral malaria (7.4%) patients. Thus, the HbA/HbE polymorphism would not be a major genetic factor influencing the onset of cerebral malaria in Thailand.     

Fcγ receptor IIA and IIIB polymorphisms are associated with susceptibility to cerebral malaria

Human FcγRIIA and FcγRIIIB exhibit genetic polymorphisms, FcγRIIA-131H/R and FcγRIIIB-NA1/NA2, coding for different capacities for IgG binding and phagocytosis. Recently, FcγRIIA-131R was reported to be associated with protection against high-density Plasmodium falciparum infection in Kenya. Furthermore, FcγRIIIB-NA1/NA2 polymorphism was shown to influence FcγRIIA function in an allele-specific manner. In this study, we examined a possible association of FcγRIIA-131H/R and FcγRIIIB-NA1/NA2 polymorphisms with malaria severity in 107 cerebral malaria patients, 157 non-cerebral severe malaria patients, and 202 mild malaria controls living in northwest Thailand. This study reveals that, with the FcγRIIIB-NA2 allele, the FcγRIIA-131H/H genotype is associated with susceptibility to cerebral malaria (OR 1.85, 95% CI 1.14–3.01; P=0.012), although these polymorphisms are not individually involved in the disease severity. Our results suggest that FcγRIIA-131H/R and FcγRIIIB-NA1/NA2 polymorphisms have an interactive effect on host defense against malaria infection.     

Intestinal helminth infections are associated with increased incidence of Plasmodium falciparum malaria in Thailand

In a prospective study of the total population of 5 hamlets on the western border of Thailand, all subjects were screened for helminth infections; during the following year, the incidence of malaria was recorded. Patients were not treated for helminth infections. Among 731 villagers, helminth-infected subjects were more likely to develop falciparum malaria during the following year (adjusted risk ratio 2.24, range 1.4-3.6; P = 0.001). The risk of developing falciparum malaria increased with the number of helminth species (P =0.036). Whereas in other studies helminths were associated with protection from severe complications of malaria, it seemed here that helminth-infected patients were more likely to develop malaria. It is suggested that a helminth-mediated Th2 shift may have complex consequences on malaria, decreasing antisporozoite immunity, but protecting against severe malaria.     

Management of malaria in Thailand

The purpose of treatment for uncomplicated malaria is to produce a radical cure using the combination of: artesunate (4 mg/kg/day) plus mefloquine (8 mg/kg day) for 3 days: a fixed dose of artemether and lumefantrine (20/120 mg tablet) named Coartem (4 tablets twice a day for three days for adults weighing more than 35 kg): quinine 10 mg/kg 8-hourly plus tetracycline 250 mg 6-hourly for 7 days (or doxycycline 200 mg as an alternative to tetracycline once a day for 7 days) in patients aged 8 years and over: Malarone (in adult 4 tablets daily for 3 days). In treating severe malaria, early diagnosis and treatment with a potent antimalarial drug is recommended to save the patient's life. The antimalarial drugs of choice are: intravenous quinine or a parenteral form of an artemisinin derivative (artesunate i.v./i.m. for 2.4 mg/kg followed by 1.2 mg/kg injection at 12 and 24 hr and then daily for 5 dayss; artemether i.m. 3.2 mg/kg injection followed by 1.6 mg/kg at 12 and 24 hrs and then daily for 5 days; artemether i.m. (Artemotil) with the same dose of artemether or artesunate suppository (5 mg/kg) given rectally 12 hourly for 3 days. Oral artemisinin derivatives (artesunate, artemether, and dihydroartemisinin with 4 mg/kg/day) could replace parenteral forms when patients can tolerate oral medication. Oral mefloquine (25 mg/kg divided into two doses 8 hrs apart) should be given at the end of the artemisinin treatment course to reduce recrudescence.     

Plasmodium falciparum: effect of anti-malarial drugs on the production and secretion characteristics of histidine-rich protein II

Plasmodium falciparum histidine-rich protein II (HRP2) is one of the best documented malaria proteins. However, little is known about the development of HRP2 concentrations under the influence of anti-malarial drugs. HRP2 levels were determined in cell medium mixture, cellular compartment, and in culture supernatant using a double-site sandwich ELISA specific for HRP2. Characteristic increases in the overall HRP2 levels were found during the later ring and the trophozoite stages. Throughout the later schizont development, rupture, and reinvasion, however, the HRP2 levels remained comparatively stable. When the cultures were exposed to serial dilutions of anti-malarial drugs, a distinct inhibition of HRP2 production was seen with increasing concentrations of drugs, resulting in sigmoid dose-response curves, similar to those obtained from conventional drug sensitivity assays. HRP2 therefore allows for a very accurate estimation of parasite development and its inhibition and may therefore be ideally suited for use in drug sensitivity or bioassays.     

Extended linkage disequilibrium surrounding the hemoglobin E variant due to malarial selection

The hemoglobin E variant (HbE; ( beta )26Glu-->Lys) is concentrated in parts of Southeast Asia where malaria is endemic, and HbE carrier status has been shown to confer some protection against Plasmodium falciparum malaria. To examine the effect of natural selection on the pattern of linkage disequilibrium (LD) and to infer the evolutionary history of the HbE variant, we analyzed biallelic markers surrounding the HbE variant in a Thai population. Pairwise LD analysis of HbE and 43 surrounding biallelic markers revealed LD of HbE extending beyond 100 kb, whereas no LD was observed between non-HbE variants and the same markers. The inferred haplotype network suggests a single origin of the HbE variant in the Thai population. Forward-in-time computer simulations under a variety of selection models indicate that the HbE variant arose 1,240-4,440 years ago. These results support the conjecture that the HbE mutation occurred recently, and the allele frequency has increased rapidly. Our study provides another clear demonstration that a high-resolution LD map across the human genome can detect recent variants that have been subjected to positive selection.     

Malaria blood stage parasites activate human plasmacytoid dendritic cells and murine dendritic cells through a Toll-like receptor 9-dependent pathway

A common feature of severe Plasmodium falciparum infection is the increased systemic release of proinflammatory cytokines that contributes to the pathogenesis of malaria. Using human blood, we found that blood stage schizonts or soluble schizont extracts activated plasmacytoid dendritic cells (PDCs) to up-regulate CD86 expression and produce IFN-alpha. IFN-alpha production was also detected in malaria-infected patients, but the levels of circulating PDCs were markedly reduced, possibly because of schizont-stimulated up-regulation of CCR7, which is critical for PDC migration. The schizont-stimulated PDCs elicited a poor T cell response, but promoted gamma delta T cell proliferation and IFN-gamma production. The schizont immune stimulatory effects could be reproduced using murine DCs and required the Toll-like receptor 9 (TLR9)-MyD88 signaling pathway. Although the only known TLR9 ligand is CpG motifs in pathogen DNA, the activity of the soluble schizont extract was far greater than that of schizont DNA, and it was heat labile and precipitable with ammonium sulfate, unlike the activity of bacterial DNA. These results demonstrate that schizont extracts contain a novel and previously unknown ligand for TLR9 and suggest that the stimulatory effects of this ligand on PDCs may play a key role in immunoregulation and immunopathogenesis of human falciparum malaria.     

Contemporaneous and successive mixed Plasmodium falciparum and Plasmodium vivax infections are associated with Ascaris lumbricoides: an immunomodulating effect?

Following an investigation suggesting a protective role for Ascaris against cerebral malaria, possibly through immunomodulation, we examined whether Ascaris had any impact on mixed Plasmodium falciparum and Plasmodium vivax infections. We studied a cross section of 928 patient files between 1991 and 1999. Forty patients had contemporaneous mixed infections and 40 patients had P. falciparum infections, followed by P. vivax infections. There was a significant association between Ascaris infection and risk of having both contemporaneous or successive mixed P. falciparum and P. vivax infections (adjusted odds ratios respectively 6 [2-18] P = 0.001 and 3.6 [1.2-11.1] P = 0.02). There was a positive linear trend between the burden of Ascaris and the risk of mixed infections P < 0.0001. These results suggested the possibility that pre-existing Ascaris infection may increase tolerance of the host to different Plasmodium spp., thus facilitating their coexistence.     

Efficient expression and purification of recombinant glutaminase from Bacillus licheniformis (GlsA) in Escherichia coli

Glutaminase or L-glutamine aminohydrolase (EC 3.5.1.2) is an enzyme that catalyzes the formation of glutamic acid and ammonium ion from glutamine. This enzyme functions in cellular metabolism of every organism by supplying nitrogen required for the biosynthesis of a variety of metabolic intermediates, while glutamic acid plays a role in both sensory and nutritional properties of food. So far there have been only a few reports on cloning, expression and characterization of purified glutaminases. Microbial glutaminases are enzymes with emerging potential in both the food and the pharmaceutical industries. In this research a recombinant glutaminase from Bacillus licheniformis (GlsA) was expressed in Escherichia coli, under the control of a ptac promoter. The recombinant enzyme was tagged with decahistidine tag at its C-terminus and could be conveniently purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The enzyme could be induced for efficient expression with IPTG, yielding approximately 26,000 units from 1-l shake flask cultures. The enzyme was stable at 30°C and pH 7.5 for up to 6h, and could be used efficiently to increase glutamic acid content when protein hydrolysates from soy and anchovy were used as substrates. The study demonstrates an efficient expression system for the production and purification of bacterial glutaminase. In addition, its potential application for bioconversion of glutamine to flavor-enhancing glutamic acid has been demonstrated.