Plasmodium Falciparum-infected Red Cells Research Articles

The Severity of Malarial-Anemia in Pregnant Women in Biyem-Assi, Yaounde

Malaria is a parasitic disease endemic in Sub-Saharan Africa especially in Cameroon. Pregnancy is associated with increased susceptibility to malaria. Closely associated to malaria is anaemia. It is generally accepted that pregnancy ends with childbirth. The outcome of the pregnancy is highly affected by the presence of malaria and anaemia. The aim of ths study was to determine the prevalence and severity of malaria and anemia among pregnant women and the influence of socio-demographic factors on malaria and anaemia prevalences in pregnant women in Biyam- Assi Yaounde. Information on socio-demographic factors was collected from 302 pregnant women attending prenatal clinics in health institutions within the Biyem-Assi Health District of Yaounde VI subdivision using a pretested questionnaire. Peripheral blood was collected and thick smears were prepared for screening Plasmodium parasites. The level of hemoglobin was measured using a haemoglobinometer URIT- 12. Analysis was done using SPSS version 20.0. Out of the 302 women, 146 (51.7%) had Plasmodium falciparum infected red cells. Among the 146 infected women, 36% had mild parasitaemia, 45% had moderate parasitaemia and 19% had severe parasitaemia. The prevalence of anemia in the study population was 53%. There was no significant association between the severity of malaria and the severity of anaemia (p > 0, 05). Parity had a significant influence on the severity of malaria. The level of school education significantly (p < 0, 05) influenced the severity of anaemia. Multiparous women were more affected by malaria than primiparous women. However, in relation to disease severity, primiparous women parasitized by Plasmodium presented a significantly higher risk of severe malaria compared to multiparous women. Malaria and anaemia prevalences in the study population were high (>50%), anaemia severity was directly associated to level of education. We recommend that more sensitization of women should be done to reduce ignorance, prevent malaria and in turn reduce anaemia and improve on the health of mother and baby.

The role of heme-oxygenase-1 in pathogenesis of cerebral malaria in the co-culture model of human brain microvascular endothelial cell and ITG Plasmodium falciparum-infected red blood cells.

To investigate the role of human host heme-oxygenase-1 (HO-1) in pathogenesis of cerebral malaria in the invitro model. The effect of human host HO-1 [human brain microvascular endothelial cell (HBMEC)] on hemoglobin degradation in the co-culture model of HBMEC and ITG Plasmodium falciparum-infected red cells (iRBC) through measurement of the enzymatic products iron and bilirubin. Following exposure to the HO-1 inducer CoPPIX at all concentrations, the HBMEC cells apoptosis occurred, which could be prominently observed at 15μM of 3h exposure. In contrast, there was no significant change in the morphology in the non-exposed iRBC at all concentrations and exposure time. This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin, of which the highest levels (106.03 and 1753.54% of baseline level, respectively) were observed at 15μM vs. 20μM at 3h vs. 24h exposure. For the effect of the HO-1 inhibitor ZnPPIX, HBMEC cell morphology was mostly unchanged, but significant inhibitory effect on cell apoptosis was seen at 10μM for the exposure period of 3h (37.17% of baseline level). The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed (highest effect at 10μM and 3h exposure). Results provide at least in part, insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model.

High density of ‘spiky’ excrescences covering the surface of an erythrocyte infected with Plasmodium malariae

This Atomic Force Microscope image shows the surface of a Plasmodium malariae trophozoite-infected red blood cell isolated from a Burmese patient suffering from quartan malaria (top). Of note, the P. malariae-infected cell (bottom left) is covered with dense ‘spike-like’ excrescences (mean height: 7·59 nm; mean diameter: 52·95 nm), which are morphologically distinct from the larger, more rounded ‘knob’ structures found on a Plasmodium falciparum-infected red cell (mean height: 19·65 nm; mean diameter: 96·64 nm) (bottom right). The ‘knobs’ on red cells containing mature asexual forms of P. falciparum assist the infected cells to bind/sequester to the vascular endothelium under shear flow conditions and thus avoid splenic clearance. The function of P. malariae spikes (which we have observed on every sexual and asexual stage examined in isolates from Indonesia and Thailand) is not known. The sample from which the P. malariae pictured was isolated (Mae Sod, Tak, Thailand) had the diagnosis confirmed by polymerase chain reaction. This picture is artificially coloured. The horizontal black scale bars represent 1 μm. The two lower images are identical in scale and orientation.

FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells.

BackgroundDuring its intraerythrocytic asexual reproduction cycle Plasmodium falciparum consumes up to 80% of the host cell hemoglobin, in large excess over its metabolic needs. A model of the homeostasis of falciparum-infected red blood cells suggested an explanation based on the need to reduce the colloid-osmotic pressure within the host cell to prevent its premature lysis. Critical for this hypothesis was that the hemoglobin concentration within the host cell be progressively reduced from the trophozoite stage onwards.Methodology/Principal FindingsThe experiments reported here were designed to test this hypothesis by direct measurements of the hemoglobin concentration in live, infected red cells. We developed a novel, non-invasive method to quantify the hemoglobin concentration in single cells, based on Förster resonance energy transfer between hemoglobin molecules and the fluorophore calcein. Fluorescence lifetime imaging allowed the quantitative mapping of the hemoglobin concentration within the cells. The average fluorescence lifetimes of uninfected cohorts was 270±30 ps (mean±SD; N = 45). In the cytoplasm of infected cells the fluorescence lifetime of calcein ranged from 290±20 ps for cells with ring stage parasites to 590±13 ps and 1050±60 ps for cells with young trophozoites and late stage trophozoite/ early schizonts, respectively. This was equivalent to reductions in hemoglobin concentration spanning the range from 7.3 to 2.3 mM, in line with the model predictions. An unexpected ancillary finding was the existence of a microdomain under the host cell membrane with reduced calcein quenching by hemoglobin in cells with mature trophozoite stage parasites.Conclusions/SignificanceThe results support the predictions of the colloid-osmotic hypothesis and provide a better understanding of the homeostasis of malaria-infected red cells. In addition, they revealed the existence of a distinct peripheral microdomain in the host cell with limited access to hemoglobin molecules indicating the concentration of substantial amounts of parasite-exported material.

TGF-β1 Released from Activated Platelets Can Induce TNF-Stimulated Human Brain Endothelium Apoptosis: A New Mechanism for Microvascular Lesion during Cerebral Malaria

Platelets have recently been shown to accumulate in brain microvessels of patients with cerebral malaria and to modulate the binding of Plasmodium falciparum-infected red cells to human brain endothelium in vitro. In the present study we used a platelet-endothelial cell coculture model to investigate the mechanisms by which platelets modify the function of human brain microvascular endothelial cells (HBEC). Platelets were found to have a proapoptotic effect on TNF-activated HBEC, and this was contact-dependent, as inhibiting platelet binding prevented endothelial cell killing. We also showed that the supernatants of thrombin-activated platelets killed TNF-stimulated HBEC and that TGF-beta1 was the main molecule involved in endothelial cell death, because its inhibition completely abrogated the activated-platelet supernatant effect. Our data illustrate another aspect of the duality of TGF-beta1 in malaria and may provide new insights into the pathogenesis of cerebral malaria.

Cytoadherence of Malaria-Infected Red Blood Cells Involves Exposure of Phosphatidylserine

Phosphatidylserine (PS) is a membrane phospholipid which in intact cells is exclusively localized in the inner leaflet of the lipid bilayer. However, once cells undergo apoptosis or oxidative stress, PS molecules are exposed on the external surface of the cells and this contributes to their adherence to macrophages or endothelial cells. PS exposure on Plasmodium falciparum-infected red cells was determined by flow cytometry using fluorescein-labeled annexin V, which specifically binds to PS. Involvement of exposed PS in the adherence of malaria-infected red cells to endothelial cells was examined by in vitro cytoadherence assays. Infected cells exposed PS on their surface as the intracellular parasites matured to trophozoite and schizont stages. Adherence of malaria-infected cells to CD36, CD36-expressing Chinese hamster ovary cells, thrombospondin, and C32 amelanotic melanoma cells was inhibited by annexin V, whereas ICAM-1- and chondroitin sulfate A-mediated binding was not. Further, PS liposomes and glycerophosphorylserine, but not phosphatidylcholine liposomes and glycerophosphorylcholine, inhibited the binding of infected cells to CD36 and thrombospondin. In conclusion, these results demonstrate that PS exposed on the surface of malaria-infected red cells contributes, in part, to the adherence of P. falciparum-parasitized red cells to CD36 and thrombospondin.

Uptake and hydrolysis of sphingomyelin analogues in Plasmodium falciparum-infected red cells

Uptake and hydrolysis of sphingomyelin analogues in Plasmodium falciparum-infected red cells

Identification of complement receptor one (CR1) polymorphisms in west Africa.

Complement receptor one (CR1) is a ligand for the rosetting of Plasmodium falciparum infected red cells with uninfected cells. Since CR1 exhibits three known polymorphisms, we studied European-Americans (n = 112) and African-Americans (n = 330) and Malians (n = 158) to determine if genetic differences existed in an area endemic for malaria that could offer a survival advantage. The frequencies of Knops blood group phenotypes McC(b+) and Sl(a-) were greatly increased in Africans vs Europeans. Although the frequency of McC(b+) was similar between Africans from the USA or Mali, the Sl(a-) phenotype was significantly higher in Mali (39% vs 65%, respectively). There was an increased frequency of the largest size (250 kD) of CR1 in Mali, but this did not differ significantly from the USA (P = 0.09). Both cohorts of Africans had higher expression of red cell CR1 than European-Americans but this showed little difference between the USA and Mali groups. Thus, the most important CR1 polymorphism relevant to rosetting of malaria infected cells appears to be the Knops blood group.

Passive Ca(2+) transport and Ca(2+)-dependent K(+) transport in Plasmodium falciparum-infected red cells.

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca(2+) permeability. The magnitude of the increase is greater than that normally required to activate the Ca(2+)-dependent K(+) channel (K(Ca) channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K(Ca) channel and the Ca(2+) permeability properties of pRBC. For pRBC suspended in media containing Ca(2+), K(Ca) channel activation was elicited by treatment with the Ca(2+) ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca(2+) into pRBC in which the Ca(2+) pump was inhibited by vanadate was found to be within the normal range (30-55 micromol (10(13) cells. hr)(-1)), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca(2+) influx increased to over 1 mmol (10(13) cells. hr)(-1), almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca(2+) into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni(2+). Possible roles for this pathway in pRBC are considered.

Phospholipid composition, cholesterol content and cholesterol exchange in Plasmodium falciparum-infected red cells

The membrane lipid composition and [3H]cholesterol exchange rate were studied in both normal human erythrocytes and those infected with the human malaria Plasmodium falciparum. The host cell membrane was separated from parasite membranes using the Affigel (731) bead method. The purity of the membrane preparation was very high, as judged by SDS-PAGE, and in several instances was estimated to be greater than 98% as determined by the activity of the parasite membrane-specific enzyme, choline phosphotransferase. No difference was found in the content of phosphatidylethanolamine and only small changes were observed for phosphatidylcholine and phosphatidylserine. The sphingomyelin content in red cell membranes of both trophozoite- and schizont-infected cells was up to 47% less than that of uninfected cells, and the cholesterol/phospholipid ratio was decreased 55%. Trophozoite- and schizont-infected cells exchanged 29 and 33% less cholesterol, respectively, than uninfected cells. These changes in lipid composition and cholesterol exchange could have a marked effect on the function of the red cell membrane of malaria-infected cells and may be responsible, in part, for the increased fluidity and permeability of P. falciparum-infected erythrocytes.

In vitro cytoadherence of Plasmodium falciparum-infected erythrocytes to melanoma cells: factors affecting adhesion.

A reproducible and standardized assay for measuring the cytoadherence of knobby Plasmodium falciparum-infected red cells to amelanotic melanoma cells was developed. Adhesion was dependent on temperature, haematocrit, and parasitaemia. Addition of EDTA to the binding medium reduced adhesion. Removal of protease-sensitive molecules on the surface of the infected cell abolished cytoadherence, whereas removal of carbohydrate residues by treatment of cells with neuraminidase or galactosidase promoted adhesion. Calcium, magnesium, fibrinogen or fibronectin in the medium had no effect on adhesion nor was there any enhancement of adhesion by pre-loading infected cells with calcium. Serum was essential for good adhesion. Adhesion was species specific for target cells; human endothelial or amelanotic melanoma cells were suitable target cells whereas bovine cells were not. The amelanotic melanoma cell could be formalin-fixed and still retain its adhesion properties. The binding properties of formalin-fixed amelanotic melanoma cells were not identical to those of endothelial or unfixed target cells.

Thrombospondin Mediates the Cytoadherence of Plasmodium falciparum-Infected Red Cells to Vascular Endothelium in Shear Flow Conditions

Cerebral malaria is thought to involve specific attachment of Plasmodium falciparum-infected knobby red cells to venular endothelium. The nature of surface ligands on host endothelial cells that may mediate cytoadherence is poorly understood. We have investigated the effects of soluble thrombospondin, rabbit antiserum raised against thrombospondin, and human immune serum on cytoadherence of parasitized erythrocytes in ex vivo mesocecum vasculature. Preincubation of infected red cells with soluble thrombospondin or human immune serum inhibits binding of infected red cells to rat venular endothelium. Infusion of the microcirculatory preparation with rabbit antithrombospondin antibodies before perfusion of parasitized erythrocytes also resulted in decreased cytoadherence. In addition, incubation of infected cells with human immune sera obtained from malaria patients significantly inhibited the observed cytoadherence. Our results indicate that thrombospondin mediates binding of infected red cells to venular endothelium and may thus be involved in the pathogenesis of cerebral malaria.

Malarial pigment-dependent error in the estimation of hemoglobin content in Plasmodium falciparum-infected red cells: implications for metabolic and biochemical studies of the erythrocytic phases of malaria.

Measurements of mean corpuscular hemoglobin (MCH) in Plasmodium falciparum-infected red cells cultured in vitro revealed that malarial pigment (hemozoin) interferes with a true estimate of the actual hemoglobin content in Drabkin's reagent. When the hemozoin pigment was removed by passage of the lysate over a Biorex 70 column, a lower MCH value was obtained which allowed one to estimate that, under these conditions, the parasite consumes about 25% of the red cell's initial hemoglobin. Because spectrophotometric examinations of infected red cell lysates in Drabkin's reagent detect the unchanging heme content of infected red cells (hemoglobin + hemozoin), it can be used for expressing enzymatic activity or metabolite content. Results agree with simultaneous measurements on a per cell basis. However, it is suggested that instead of per gram hemoglobin, the activity should be stated as per mmole (or mumole) heme pigment. The ability to estimate accurately the consumption of intracellular hemoglobin will be useful in metabolic and pharmacologic studies of the parasite/red cell interaction.