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.

Protein trafficking in Plasmodium falciparum-infected red cells and impact of the expansion of exported protein families.

SUMMARY Erythrocytes are extensively remodelled by the malaria parasite following invasion of the cell. Plasmodium falciparum encodes numerous virulence-associated and host-cell remodelling proteins that are trafficked to the cytoplasm, the cell membrane and the surface of the infected erythrocyte. The export of soluble proteins relies on a sequence directing entry into the secretory pathways in addition to an export signal. The export signal consisting of five amino acids is termed the Plasmodium export element (PEXEL) or the vacuole transport signal (VTS). Genome mining studies have revealed that PEXEL/VTS carrying protein families have expanded dramatically in P. falciparum compared with other malaria parasite species, possibly due to lineage-specific expansion linked to the unique requirements of P. falciparum for host-cell remodelling. The functional characterization of such genes and gene families may reveal potential drug targets that could inhibit protein trafficking in infected erythrocytes. This review highlights some of the recent advances and key knowledge gaps in protein trafficking pathways in P. falciparum-infected red cells and speculates on the impact of exported gene families in the trafficking pathway.

Platelet Factor 4 and Duffy Antigen Required for Platelet Killing of Plasmodium falciparum

Platelets restrict the growth of intraerythrocytic malaria parasites by binding to parasitized cells and killing the parasite within. Here, we show that the platelet molecule platelet factor 4 (PF4 or CXCL4) and the erythrocyte Duffy-antigen receptor (Fy) are necessary for platelet-mediated killing of Plasmodium falciparum parasites. PF4 is released by platelets on contact with parasitized red cells, and the protein directly kills intraerythrocytic parasites. This function for PF4 is critically dependent on Fy, which binds PF4. Genetic disruption of Fy expression inhibits binding of PF4 to parasitized cells and concomitantly prevents parasite killing by both human platelets and recombinant human PF4. The protective function afforded by platelets during a malarial infection may therefore be compromised in Duffy-negative individuals, who do not express Fy.

SICKLE CELL ANAEMIA AND MALARIA

Sickle cell anaemia is a major chapter within haemolytic anaemias; at the same time, its epidemiology is a remarkable signature of the past and present world distribution of Plasmodium falciparum malaria. In this brief review, in keeping with the theme of this journal, we focus on the close and complex relationship betweeen this blood disease and this infectious disease. On one hand, heterozygotes for the sickle gene (AS) are relatively protected against the danger of dying of malaria, as now firmly established through a number of clinical field studies from different parts of Africa. In addition, experimental work is consistent with a plausibile mechanism: namely, that in AS heterozygotes P falciparum-infected red cells sickle preferentially and are then removed by macrophages. On the other hand, patients who are homozygous for the sickle gene and therefore suffer from sickle cell anaemia (SCA) are highly susceptible to the lethal effects of malaria. The simplest explanation of this fact is that malaria makes the anaemia of SCA more severe; in addition, in SCA there is often hyposplenism, which reduces clearance of parasites. From the point of view of public health it is important that in malaria-endemic countries patients with SCA, and particularly children, be protected from malaria by appropriate prophylaxis.

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.

Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti-phosphotyrosine and anti-phosphoserine antibodies following 2-DE in conjunction with double channel laser-induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans-membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.

Glutathione S-transferase activity of erythrocyte genotypes HbAA, HbAS and HbSS in male volunteers administered with fansidar and quinine

In vivo investigations were carried out to ascertain the comparative levels of erythrocyte GST activity between non-malarious and malarious male subjects of HbAA, HbAS and HbSS erythrocyte genotypes, administered with fansidar and quinine. The incubation of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) resulted in almost quantitative conjugation of glutathione (GSH) to form S-(2,4-dinitrophenyl) glutathione. Determination of GST activity was carried out at 0, 3, 6 and 18 h, after two antimalarial drugs (Fansidar and quinine) were adminis-tered to the volunteers. The control values of non-malarious and malarious individuals ranged between (3.27 to 12.50 iu/gHb) and (2.75 to 12.21 -iu/gHb) respectively. Erythrocyte GST activity was significantly (p < 0.05) lower in P. falciparum infected red cells compared to normal ones, except parasitized male HbSS erythrocytes. Generally, the pattern of in vivo erythrocyte GST activity in the presence of the 2 antimalarial drugs showed 2 phase profile. The first stage showed decreasing levels of relative GST activity within approximate time range of 6 h after the drugs were administered to the volunteers. The second phase showed recovery of erythrocyte GST activity (6 - 18 h approx.) from the inhibitory effects of the drugs. Key words: Glutathione S-transferase (GST) activity, erythrocytes, fansidar, quinine, 1-chloro-2, 4-dinitrobenzene (CDNB).

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.

Selective killing of the human malaria parasite Plasmodium falciparum by a benzylthiazolium dye

Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium. The most virulent form of the disease is caused by Plasmodium falciparum which infects hundreds of millions of people and is responsible for the deaths of 1–2 million individuals each year. An essential part of the parasitic process is the remodeling of the red blood cell membrane and its protein constituents to permit a higher flux of nutrients and waste products into or away from the intracellular parasite. Much of this increased permeability is due to a single type of broad specificity channel variously called the new permeation pathway (NPP), the nutrient channel, and the Plasmodial surface anion channel (PSAC). This channel is permeable to a range of low molecular weight solutes both charged and uncharged, with a strong preference for anions. Drugs such as furosemide that are known to block anion-selective channels inhibit PSAC. In this study, we have investigated a dye known as benzothiocarboxypurine, BCP, which had been studied as a possible diagnostic aid given its selective uptake by P. falciparum infected red cells. We found that the dye enters parasitized red cells via the furosemide-inhibitable PSAC, forms a brightly fluorescent complex with parasite nucleic acids, and is selectively toxic to infected cells. Our study describes an antimalarial agent that exploits the altered permeability of Plasmodium-infected red cells as a means to killing the parasite and highlights a chemical reagent that may prove useful in high throughput screening of compounds for inhibitors of the channel.

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.

Destabilisation and subsequent lysis of human erythrocytes induced by Plasmodium falciparum haem products

In falciparum malaria, both infected and uninfected red cells have structural and functional alterations. To investigate the mechanisms of these modifications, we studied the effects of two Plasmodium falciparum haem products (haematin and malaria pigment in the synthetic form beta-haematin) on isolated human red blood cells (RBCs) and purified RBC ghosts. A dose- and time-dependent incorporation of haematin into RBC ghosts and intact cells was observed, which was in proportion to the extent of haematin- induced haemolysis. RBCs pre-incubated with haematin were more sensitive to haemolysis induced by hypotonic shock, low pH, H2O2 or haematin itself. Haemolysis was not related to membrane lipid peroxidation and only partially to oxidation of protein sulphydryl groups and it could not be prevented by scavengers of lipid peroxidation or hydroperoxide groups. N-acetylcysteine partly protected the oxidation of SH groups and significantly reduced haemolysis. In contrast, beta-haematin was neither haemolytic nor oxidative towards protein sulphydryl groups. Beta-haematin did destabilise the RBC membrane, but to a lesser extent than haematin, inducing increased susceptibility to lysis caused by hypotonic medium, H2O2 or haematin. This study suggests that the destabilising effect of haematin and, to a much less extent, beta-haematin on the RBC membrane does not result from oxidative damage of membrane lipids but from direct binding or incorporation which may affect the reciprocal interactions between the membrane and cytoskeleton proteins. These changes could contribute to the reduced red cell deformability associated with severe malaria.

Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum

BackgroundThe aim of this study was to develop site-specific antibodies as a tool to capture Plasmodium falciparum-dihydrofolate reductase (Pf-DHFR) from blood samples from P. falciparum infected individuals in order to detect, in a sandwich ELISA, structural alterations due to point mutations in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.MethodsA homology model of Pf-DHFR domain was employed to define an epitope for the development of site-specific antibodies against Pf-DHFR. The homology model suggested an exposed loop encompassing amino acid residues 64–100. A synthetic peptide of 37-mers whose sequence corresponded to the sequence of amino acid residues 64–100 of Pf-DHFR was synthesized and used to immunize mice for antibodies. Additionally, polyclonal antibodies recognizing a recombinant DHFR enzyme were produced in rabbits.Results and conclusionsSerum from mice immunized with the 37-mer showed strong reactivity against both the immunizing peptide, recombinant DHFR and a preparation of crude antigen from P. falciparum infected red blood cells. Five monoclonal antibodies were obtained, one of which showed reactivity towards crude antigen prepared from P. falciparum infected red cells. Western blot analysis revealed that both the polyclonal and monoclonal antibodies recognized Pf-DHFR. Our study provides insight into the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.

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.

Plasmodium falciparum:Naturally Occurring Rabbit Immunoglobulins Recognize Human Band 3 Peptide Motifs and Malaria-Infected Red Cells

The reactivity to overlapping decapeptides based on sequences of human band 3 protein was determined for 24 rabbit serum samples. A high percentage of the sera recognized sequences of amino acid 650–670, as well as several other sequences representing putative exofacial regions of the band 3 protein. When sera were used to stain immunofluorescently human erythrocytes, some of which were infected withP. falciparum,those samples that predominantly reacted with amino acids 650–670 stainedP. falciparum-infected red cells, whereas those that reacted with other regions of band 3 stained all erythrocytes. A positive correlation was found between anti-band 3 reactivity and the capacity of a serum to inhibit the cytoadherence ofP. falciparum-infected erythrocytes to C32 amelanotic melanoma cells. Also, some rabbit sera immunoprecipitated a high-molecular-weight protein from extracts of surface iodinatedP. falciparum-infected red cells

Small GTP-binding proteins in Plasmodium falciparum

During its erythrocytic life cycle Plasmodium falciparum exchanges compounds with host cells through phagocytosis and exocytosis. In eucaryotic cells, small GTP-binding proteins of the Ras superfamily appear to be involved in different steps of membrane trafficking and in intracellular signals. In this paper, we investigate the Rab4, Rab6 and Ras-related proteins in P falciparum infected red cells. We report that P falciparum Rab and Ras-related proteins could be distinguished from their counterparts by iso-electrofocusing and immunoblotting. The localization of P falciparum Rab 4 and Rab 6 was studied by immunogold electron microscopy on ultrathin frozen sections of infected red blood cells. Rab4 parasite-relate protein was found associated with the membranes of early endosome-like structures near the parasite plasma membrane. Rab6-related protein was associated with the Golgi/ trans Golgi network, as already suggested by immunofluorescence microscopy studies and Ras-related protein was cytoplasmic and plasma membrane-associated. These results are in accordance with their mammalian counterparts and support the implication of Rab-related proteins in vesicular trafficking in Plasmodium.

Small GTP‐binding proteins in Plasmodium falciparum

Summary— During its erythrocytic life cycle Plasmodium falciparum exchanges compounds with host cells through phagocytosis and exocytosis. In eucaryotic cells, small GTP‐binding proteins of the Ras superfamily appear to be involved in different steps of membrane trafficking and in intracellular signals. In this paper, we investigate the Rab4, Rab6 and Ras‐related proteins in P falciparum infected red cells. We report that P falciparum Rab and Ras‐related proteins could be distinguished from their counterparts by iso‐electrofocusing and immunoblotting. The localization of P falciparum Rab 4 and Rab 6 was studied by immunogold electron microscopy on ultrathin frozen sections of infected red blood cells. Rab4 parasite‐relate protein was found associated with the membranes of early endosome‐like structures near the parasite plasma membrane. Rab6‐related protein was associated with the Golgi/trans Golgi network, as already suggested by immunofluorescence microscopy studies and Ras‐related protein was cytoplasmic and plasma membrane‐associated. These results are in accordance with their mammalian counterparts and support the implication of Rab‐related proteins in vesicular trafficking in Plasmodium.