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Immunoglobulin G and subclass responses to Plasmodium falciparum antigens: a study in highly exposed Cameroonians.

We conducted a cross-sectional study in Bolifamba village in the South West Province of Cameroon to determine antibody responses to crude Plasmodium falciparum antigens. A total of 347 subjects were examined. Parasite counts were obtained on thick blood films stained with Field's stain. Total immunoglobulin (Ig)G and IgG subclass levels were determined in serum samples from four groups comprising children 1 to 5 years old and adults > or = 18 years with or without falciparum malaria parasites, using enzyme-linked immunosorbent assay with crude blood-stage antigens of Plasmodium falciparum strain F32 as target. Depending on the age group, malaria prevalence varied between 10% and 65% with a mean of 30.8%. Prevalence rate and parasite density declined with increasing age. Total IgG and IgG1-3 levels were significantly higher in adults than in children (p < 0.05). Parasite-bearing individuals in both age groups had higher IgG titres than their non-infected counterparts, while subtype levels were not significantly different (p = 0.05). These findings indicate that Bolifamba village could be a convenient site to study further the protective immunity to malaria.

Adolescent↗

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus.

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.

Animals↗

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria.

In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weight ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection, in parallel to the parasitaemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced) stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced) triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that the polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

Animals↗

The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages.

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strains so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich in glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that the Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.

Amino Acid Sequence↗

Infected erythrocyte choline carrier inhibitors: exploring some potentialities inside Plasmodium phospholipid metabolism for eventual resistance acquisition.

We have developed a model for designing antimalarial drugs based on interference with an essential metabolism developed by Plasmodium during its intraerythrocytic cycle, phospholipid (PL) metabolism. The most promising drug interference is choline transporter blockage, which provides Plasmodium with a supply of precursor for synthesis of phosphatidylcholine (PC), the major PL of infected erythrocytes. Choline entry is a limiting step in this metabolic pathway and occurs by a facilitated-diffusion system involving an asymmetric carrier operating according to a cyclic model. Choline transport in the erythrocytes is not sodium dependent nor stereospecific as demonstrated using stereoisomers of alpha and beta methylcholine. These last two characteristics along with distinct effects of nitrogen substitution on transport rate demonstrate that choline transport in the infected erythrocyte possesses characteristics quite distinct from that of the nervous system. This indicates a possible discrimination between the antimalarial activity (inhibition of choline transport in the infected erythrocyte) and a possible toxic effect through inhibition of choline entry in synaptosomes. Apart from the de novo pathway of choline, PC can be synthesized by N-methylation from phosphatidylethanolamine (PE). There is a de novo pathway for PE biosynthesis from ethanolamine in infected cells but phosphatidylserine (PS) decarboxylation also occurs. In addition, PE can be directly and abundantly synthesized from serine decarboxylation into ethanolamine, a pathway which is absent from the host. The variety of the pathways that exist for the biosynthesis of one given PL led us to investigate whether an equilibrium can occur between all PL metabolic pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Bacteria in midguts of field-collected Anopheles albimanus block Plasmodium vivax sporogonic development.

Bacterial infections were investigated in midguts of insectary and field-collected Anopheles albimanus Weidemann from southern Mexico. Serratia marcescens, Enterobacter cloacae and Enterobacter amnigenus 2, Enterobacter sp., and Serratia sp. were isolated in field samples obtained in 1998, but only Enterobacter sp. was recovered in field samples of 1997 and no bacteria were isolated from insectary specimens. These bacteria were offered along with Plasmodium vivax infected blood to aseptic insectary An. albimanus, and the number of infected mosquitoes as well as the oocyst densities assessed after 7d. Plasmodium vivax infections in mosquitoes co-infected with En. amnigenus 2, En. cloacae, and S. marcensces were 53, 17, and 210 times, respectively, lower than in control mosquitoes, and the mean oocyst density in mosquitoes co-infected with En. cloacae was 2.5 times lower than in controls. Mortality was 13 times higher in S. marcensces-infected mosquitoes compared with controls. The overall midgut bacterial infection in mosquito field populations may influence P. vivax transmission, and could contribute to explain the annual variations in malaria incidence observed in the area.

Animals↗

Dentification of four vectors of human Plasmodium spp. by multiplex PCR: Anopheles rangeli, An. strodei, An. triannulatus, and An. trinkae (Diptera: Culicidae: Nyssorhynchus).

One of the major obstacles for studies of the biology, ecology, and behavior of Neotropical vectors of human Plasmodium has been the lack of reliable and efficient means of identifying many species. Although the subgenus Nyssorhynchus includes most species responsible for human transmission in South America, there are no polymerase chain reaction (PCR)-based techniques for identifying members of this taxon. We describe the first multiplex PCR for identifying four species in the subgenus Nyssorhynchus that are vectors of human Plasmodium spp. Four species specific primers, together with a universal primer that anneals to the 5.8S rDNA region, produce amplicons of the internal transcribed spacer two with base pair sizes of 131,308,371, and 441 for An. triannulatus, An. trinkae, An. strodei, and An. rangeli, respectively.

Animals↗

Detection of Plasmodium yoelii stage mRNA in BALB/c mice.

Relatively little information is available concerning the expression of parasite genes during the liver stage of Plasmodium infection, mostly because of low-level infection of host hepatocytes and the lack of purification techniques for the liver stage parasites. We have determined the optimal dosage of Plasmodium yoelii sporozoite inoculum and routes of inoculation, which are intravenous tail vein and the intrahepatic portal circulation. To determine which route was optimal, BALB/c mice were inoculated via 1 of these routes, and parasitemia was detected by reverse transcription polymerase chain reaction (RT-PCR) detecting both murine beta-actin and P. yoelii-specific 28S ribosomal RNA in the liver samples. Murine beta-actin was detected after 15 cycles of PCR, and its expression levels did not differ between treatment groups. However, P. yoelii-specific 28S ribosomal RNA gene product was detected after 15 cycles of PCR in animals inoculated via the tail vein but was not detected until 25 cycles in animals inoculated via the intrahepatic portal circulation. Experiments were then performed to identify the smallest inoculum required to initiate a liver stage infection that would yield sufficient parasite RNA for analysis. Inoculation with different doses of sporozoite inocula was followed by RT-PCR on the livers of the inoculated animals. The P. yoelii-specific 28S ribosomal RNA gene product was first detected in both treatment groups after 15 cycles, suggesting that both doses of sporozoite inocula provided relatively the same level of liver infection rate. We also have analyzed infected mouse liver for parasite-specific mRNA, which was detectable as early as 24 hr postinfection.

Animals↗

Decreased hemoglobin concentrations, hyperparasitemia, and severe malaria are associated with increased Plasmodium falciparum gametocyte carriage.

To determine factors influencing gametocyte carriage, a cross-sectional study was conducted among 512 patients admitted for Plasmodium falciparum malaria. After adjustments for potential confounders, hemoglobin concentrations were lower in gametocyte carriers 10.5 (+/-2.5) than in patients without gametocytes 12.5 (+/-2.3) (P < 0.0001). Hemoglobin concentrations were negatively correlated with peak gametocyte counts (Spearman's p = -0.37, P < 0.0001) and gametocyte carriage durations (Spearman's p = -(0.30, P < 0.0001). Adjustments for the duration of the malaria episode and other potential confounders did not alter the association (P < 0.0001). After adjustment for potential confounders, the median asexual parasitemia was higher in patients with gametocytes than in patients without gametocytes (P = 0.003). Severe malaria cases were more likely to have gametocytes (65%) than malaria with hyperparasitemia (38%) or mild malaria (31%) (P = 0.0001). These findings suggest that events surrounding anemia and tissue hypoxia stimulate Plasmodium falciparum gametocytogenesis.

Adolescent↗

Plasmodium yoelii: identification and partial characterization of an MDR1 gene in an artemisinin-resistant line.

The molecular mechanisms by which the malarial parasite has managed to develop resistance to many antimalarial drugs remain to be completely elucidated. Mutations in the pfmdr1 gene of Plasmodium falciparum, as well as an increase in pfmdr1 copy number, have been associated with resistance to the quinoline-containing antimalarial drugs. We investigated the mechanisms of drug resistance in Plasmodium using a collection of P. yoelii lines with different drug resistance profiles. The mdr1 gene of P. yoelii (pymdr1) was identified and characterized. A 2- to 3-fold increase in the pymdr1 gene copy number was observed in the P. yoelii ART line (artemisinin resistant) when compared with the NS parental line. The pymdr1 gene was mapped to a chromosome of 2.1 Mb in all lines analyzed. Reverse transcriptase-polymerase chain reaction and Western blot experiments confirmed the expression of the gene at the RNA and protein levels.

Amino Acid Sequence↗

Two Plasmodium species of the crocodile skink Tribolonotus gracilis from Irian Jaya, Indonesia.

Two undescribed Plasmodium species were present in 1 of 8 New Guinea skinks, Tribolonotus gracilis. Plasmodium tribolonoti n. sp. is characterized by rounded or oblong schizonts 6.1 x 5.3 microm (5-7 x 4-7) that produce on average 14.3 merozoites (10-21), in no particular arrangement of nuclei. All parasitized proerythrocytes and had no effect upon dimensions of host cells or their nuclei. Gametocytes, mostly erythrocytic, averaged 7.2 x 6.3 (6.5-9.0 x 5.5-7.5), with length x width (LW) 45.5 microm2 (38-63) and L/W 1.15 (1.0-1.5). Gametocytes are not sexually dimorphic in size or shape and had no effect upon dimensions of host erythrocytes or their nuclei. Schizonts of P. gracilis n. sp. averaged 4.3 x 3.5 (3-6 x 3-5), with 4.9 merozoites (3-8) usually arranged as a fan, and had no effect upon dimensions of host erythrocytes or their nuclei. Gametocytes averaged 5.9 x 5.5 (5.0-6.6 x 5.0-6.0), with LW 31.9 microm2 (25-40) and L/W 1.06 (1.0-1.2). Gametocytes are not sexually dimorphic in size or shape and had no effect upon dimensions of host erythrocytes or their nuclei.

Analysis of Variance↗

Functional characterization of an LCCL-lectin domain containing protein family in Plasmodium berghei.

Using bioinformatic, proteomic, immunofluorescence, and genetic cross methods, we have functionally characterized a family of putative parasite ligands as potential mediators of cell-cell interactions. We name these proteins the Limulus clotting factor C, Coch-5b2, and Lgl1 (LCCL)-lectin adhesive-like protein (LAP) family. We demonstrate that this family is conserved amongst Plasmodium spp. It possesses a unique arrangement of adhesive protein domains normally associated with extracellular proteins. The proteins are expressed predominantly, though not exclusively, in the mosquito stages of the life cycle. We test the hypothesis that these proteins are surface proteins with 1 member of this gene family, lap1, and provide evidence that it is expressed on the surface of Plasmodium berghei sporozoites. Finally, through genetic crosses of wild-type Pblap1+ and transgenic Pblap1- parasites, we show that the null phenotype previously reported for sporozoite development in a Pblap1- mutant can be rescued within a heterokaryotic oocyst and that infectious Pblap1 sporozoites can be formed. The mutant is not rescued by coparasitization of mosquitoes with a mixture Pblap1+ and Pblap1- homokaryotic oocysts.

Amino Acid Sequence↗

Observations on the Vietnam Palo Alto strain of Plasmodium vivax in two species of Aotus monkeys.

Thirty-three splenectomized Aotus lemurinus griseimembra monkeys with no previous experience with malaria were infected with the Vietnam Palo Alto strain of Plasmodium vivax. The median maximum parasite count was 280,000/microl. Nine splenectomized monkeys with previous infection with Plasmodium falciparum had median maximum parasite counts of 120,000/microl. Splenectomized Aotus nancymai monkeys supported infections at a lower level. Transmission via the bites of Anopheles dirus mosquitoes was obtained in a splenectomized A. lemurinus griseimembra, with a prepatent period of 31 days. It is estimated that between 1.5 x 10(8) and 1.6 x 10(9) parasites can be removed from an infected animal for molecular or diagnostic antigenic studies.

Animals↗

A new species of Plasmodium from Malagasy vangas.

During a recent examination of blood smears from Malagasy birds, a species of avian Plasmodium unlike those currently known was observed. All infected birds were members of the Vangidae, which is endemic to Madagascar and the Comoro Islands. Plasmodium parvulum n. sp. is described, and classified as a member of the subgenus Haemamoeba because of gametocyte and schizont shape, displacement of the host cell nucleus, as well as distortion of the host cell. Round, rosettelike schizonts with 6-8 merozoites, clumped refractile granules, and little cytoplasm were observed. Both schizonts and mature, round gametocytes rotated and displaced the erythrocyte nucleus. A brief comparison to P. relictum is included.

Animals↗

Detection of a malaria parasite (Plasmodium mexicanum) in ectoparasites (mites and ticks), and possible significance for transmission.

Two species of sandflies (Lutzomyia) are competent vectors of Plasmodium mexicanum, a malaria parasite of lizards. The very patchy distribution of sites with high P. mexicanum prevalence in the lizards, and often low or even nil sandfly density at such sites, provoked an evaluation of 2 common lizard ectoparasites, the tick Ixodes pacificus and the mite Geckobiella occidentalis, as potential passive vectors. Plasmodium sp.-specific polymerase chain primers were used to amplify a long segment of the mitochondrial cytochrome b gene that is unlikely to survive intact if the parasite cells are killed within a blood-feeding arthropod. The segment was strongly amplified from sandflies (the positive control for the method) from 1 to 96 hr postfeeding on an infected lizard. For ticks, the gene fragment was poorly amplified at 0 hr postfeed, and not amplified after 2 hr. In contrast, strong amplification of the parasite DNA was observed from mites from 0 to 20 hr postfeed, and weak amplification even at 96 hr.

Animals↗

Studies on sporozoite-induced and chronic infections with Plasmodium fragile in Macaca mulatta and New World monkeys.

Plasmodium fragile continues to be investigated because of its biologic similarities to the human malaria parasite, Plasmodium falciparum. Two strains of P. fragile are available for study; one strain is able to infect mosquitoes, whereas the other strain is transmissible only by blood inoculation. The Sri Lanka strain of P. fragile was transmitted to Macaca mulatta, Macaca fascicularis, Aotus lemurinus griseimembra, Aotus nancymaae, Aotus vociferans, and Saimiri boliviensis monkeys via sporozoites that developed to maturity only in Anopheles dirus mosquitoes. The prepatent periods ranged from 12 to 35 days for macaques and from 15 to 30 days for New World monkeys after intravenous injection of sporozoites. Eight rhesus monkeys were infected with the Nilgiri strain and followed for 482 days. Parasitemia in 6 animals persisted at relatively high density through the period of observation. Erythrocyte, hematocrit, and hemoglobin values reached their lowest levels 3 wk after infection and slowly recovered; however, the values did not approach preinfection levels as long as parasitemia persisted in the monkeys. The mean corpuscular volume and corpuscular hemoglobin concentration reached their peak and lowest values, respectively, at day 38 and then returned to the preinfection level. The mean corpuscular hemoglobin value decreased to its lowest level at day 87 and then returned to preinfection level.

Animals↗

Zinc protoporphyrin IX binds heme crystals to inhibit the process of crystallization in Plasmodium falciparum.

The intraerythrocytic Plasmodium falciparum parasite converts most of host hemoglobin heme into a nontoxic heme crystal. Erythrocyte zinc protoporphyrin IX, normally present at 0.5 microM, which is a ratio of 1:40,000 hemes, can elevate 10-fold in some of the anemias associated with malaria disease protection. This work examines a binding mechanism for zinc protoporphyrin IX inhibition of heme crystallization similar to the antimalarial quinolines. Zinc protoporphyrin IX neither forms crystals alone nor extends on preformed heme crystals. Inhibition of both seed heme crystal formation and crystal extension occurs with an inhibitory concentration (IC)50 of 5 microM. Field emission in-lens scanning electron microscopy depicts the transition and inhibition of heme monomer aggregates to heme crystals with and without seeding of preformed hemozoin templates. In vitro zinc protoporphyrin IX, like the quinolines, binds to heme crystals in a saturable, specific, pH, and time-dependent manner. The ratio at saturation is approximately 1 zinc protoporphyrin IX per 250 hemes of the crystal. Unlike the quinolines, zinc protoporphyrin IX binds measurably in the absence of heme. Isolated ring and trophozoite stage parasites have an elevated zinc protoporphyrin IX to heme ratio 6 to 10 times that in the erythrocyte cytosol, which also corresponds to elevated ratios found in heme crystals purified from Plasmodium parasites. This work implicates protection from malaria by a mechanism where elevated zinc protoporphyrin IX in anemic erythrocytes binds to heme crystals to inhibit further crystallization. In endemic malaria areas, severe iron deficiency anemia should be treated with antimalarials along with iron replenishment.

Animals↗

Plasmodium ARK1 regulates spindle formation during atypical mitosis and forms a divergent chromosomal passenger complex.

Mitosis in Plasmodium spp., the causative agent of malaria, is fundamentally different from model eukaryotes, proceeding via a bipartite microtubule organising centre (MTOC) and lacking canonical regulators such as Polo and Bub1 kinases. During schizogony, asynchronous nuclear replication produces a multinucleate schizont, while rapid male gametogony generates an octaploid nucleus before gamete formation. Here, we identify Aurora-related kinase 1 (ARK1) as a key component of inner MTOC and spindle formation, controlling kinetochore dynamics and driving mitotic progression. Conditional ARK1 depletion disrupts spindle biogenesis, kinetochore segregation, karyokinesis and cytokinesis in both stages, and affects parasite transmission. Interactome analysis reveals ARK1 as the catalytic core of a non-canonical chromosomal passenger complex (CPC) containing two divergent inner centromere proteins (INCENPs) but lacking Survivin and Borealin. Comparative genomics indicates this CPC architecture arose early in Apicomplexa, replacing canonical centromere-targeting modules. These findings uncover a distinct mitotic machinery in Plasmodium and identify the ARK1-INCENP interface as a potential multistage target for malaria therapeutic intervention.

Aurora kinase↗