Plasmodium falciparum maturation across the intra-erythrocytic cycle shifts the soft glassy viscoelastic properties of red blood cells from a liquid-like towards a solid-like behavior.

The mechanical properties of erythrocytes have been investigated by different techniques. However, there are few reports on how the viscoelasticity of these cells varies during malaria disease. Here, we quantitatively map the viscoelastic properties of Plasmodium falciparum-parasitized human erythrocytes. We apply new methodologies based on optical tweezers to measure the viscoelastic properties and defocusing microscopy to measure the erythrocyte height profile, the overall cell volume, and its form factor, a crucial parameter to convert the complex elastic constant into complex shear modulus. The storage and loss shear moduli are obtained for each stage of parasite maturation inside red blood cells, while the former increase, the latter decrease. Employing a soft glassy rheology model, we obtain the power-law exponent for the storage and loss shear moduli, characterizing the soft glassy features of red blood cells in each parasite maturation stage. Ring forms present a liquid-like behavior, with a slightly lower power-law exponent than healthy erythrocytes, whereas trophozoite and schizont stages exhibit increasingly solid-like behaviors. Finally, the surface elastic shear moduli, low-frequency surface viscosities, and shape recovery relaxation times all increase not only in a stage-dependent manner but also when compared to healthy red blood cells. Overall, the results call attention to the soft glassy characteristics of Plasmodium falciparum-parasitized erythrocyte membrane and may provide a basis for future studies to better understand malaria disease from a mechanobiological perspective.

Resistance to Plasmodium falciparum in sickle cell trait erythrocytes is driven by oxygen-dependent growth inhibition.

Sickle cell trait (AS) confers partial protection against lethal Plasmodium falciparum malaria. Multiple mechanisms for this have been proposed, with a recent focus on aberrant cytoadherence of parasite-infected red blood cells (RBCs). Here we investigate the mechanistic basis of AS protection through detailed temporal mapping. We find that parasites in AS RBCs maintained at low oxygen concentrations stall at a specific stage in the middle of intracellular growth before DNA replication. We demonstrate that polymerization of sickle hemoglobin (HbS) is responsible for this growth arrest of intraerythrocytic P. falciparum parasites, with normal hemoglobin digestion and growth restored in the presence of carbon monoxide, a gaseous antisickling agent. Modeling of growth inhibition and sequestration revealed that HbS polymerization-induced growth inhibition following cytoadherence is the critical driver of the reduced parasite densities observed in malaria infections of individuals with AS. We conclude that the protective effect of AS derives largely from effective sequestration of infected RBCs into the hypoxic microcirculation.

Effect of inherited red cell defects on growth of Plasmodium falciparum: An in vitro study.

BACKGROUND & OBJECTIVES: High prevalence of certain polymorphic alleles of erythrocytes in malaria endemic area has been linked to the resistance provided by these alleles against parasitic infestations. Numerous studies undertaken to demonstrate this correlation have generated conflicting results. This study was undertaken to investigate the abilities of various polymorphic erythrocytes to support in vitro growth of Plasmodium falciparum parasites. METHODS: In this study under in vitro condition the ability of P. falciparum parasites to grow was assessed in the erythrocytes obtained from a total of 40 patients with various haemoglobinopathies, such as ß-thalassaemia (ß-Thal), sickle cell anaemia, erythroenzymopathy-like glucose-6-phosphate dehydrogenase deficiency and membranopathy-like hereditary spherocytosis. RESULTS: Significantly reduced in vitro invasion and growth of parasites was seen in the cultures containing abnormal erythrocytes than in control cultures containing normal erythrocytes (P< 0.05). The mean per cent parasitaemia comparison was also carried out among the three polymorphic erythrocyte groups, i.e. ß-Thal, sickle cell anaemia and enzyme-membranopathies. INTERPRETATION & CONCLUSIONS: Erythroenzymopathies and membranopathies were found to provide a more hostile environment for parasites, as the least parasitaemia was observed in these erythrocytes. The present in vitro study showed that P. falciparum did not grow well and did not invade well in erythrocytes obtained from common inherited red cell disorders.

The triumph of good over evil: protection by the sickle gene against malaria.

The mechanisms underlying Plasmodium falciparum resistance in persons with sickle trait have been under active investigation for more than a half century. This Perspective reviews progress in solving this challenging problem, including recent studies that have exploited the genomics and proteomics of the parasite. The formation of Hb S polymer in the parasitized AS RBC leads to impaired parasite growth and development along with enhanced clearance from the circulation and reduced deposition in deep postcapillary vascular beds. Enhanced generation of reactive oxygen species in sickled AS RBCs is a pathogenetic feature shared by parasitized thalassemic and G6PD-deficient RBCs, triggering abnormal topology of the RBC plasma membrane with decreased and disordered display of PfEMP-1, a P falciparum adhesion protein critical for endothelial adherence. A mouse model of Hb S confers host tolerance to P berghei, through inhibition of pathogenic CD8(+) T cells and induction of heme oxygenase-1. An additional and apparently independent mode of protection is provided by the selective expression in AS RBCs of 2 species of microRNA that integrate into P falciparum mRNAs and inhibit translation and parasite growth.

Sickling rates of human AS red cells infected in vitro with Plasmodium falciparum malaria.

The kinetics of sickling of malaria-infected red cells from humans with sickle cell trait were studied in vitro in an attempt to obtain direct experimental evidence for a selective advantage of the hemoglobin S heterozygote in a malarious region. The sickling rates of cells infected with Plasmodium falciparum and of non-infected cells were studied both in the total absence of oxygen (by dithionite addition) and at several different concentrations of oxyhemoglobin which might obtain in vivo. In all cases, red cells containing small plasmodium parasite forms (ring forms) sickled approximately eight times as readily as uninfected cells. Cells containing large parasitic forms (trophozoites and schizonts) appeared to sickle less readily than uninfected cells, by light microscopy criteria, but electron micrographs demonstrated the presence of polymerized deoxyhemoglobin S with a high frequency. It is concluded that enhanced sickling of plasmodium-infected AS cells may be one mechanism whereby the hemoglobin S polymorphism is balanced in favor of the heterozygote.

[Anaemia, iron index status and acute phase proteins in malaria (Abidjan, Côte d'Ivoire)]. / Anémie, métabolisme du fer et protéines de la réaction inflammatoire au cours du paludisme (Abidjan, Côte d'Ivoire).

Clinical signs of malaria are the combined expression of several biological mechanisms. During this parasite infection, anaemia can be the consequence of several different pathogenic mechanisms. It can be an acute haemolytic anaemia due to a mechanical and immune action of the parasite or an inflammation. Besides, in Africa malaria matches with iron deficiency area. So, malarial anaemia in tropical area can be a characteristic of iron deficiency The purpose of this survey was to define the features of malarial anaemia and elucidate the link of all biological processes involved. A black population living in tropical urban areas, with fever and diagnosed Plasmodium-infection was assessed. Parasitaemia, haemoglobin, hematocrit, average corpuscular volume and average corpuscular haemoglobin were determined. For each patient, iron index status and acute phase protein were assessed with the plasmatic iron, ferritin, haptoglobin, transferrin and C-reactive protein. Regardless of gender and age, the characteristics of malarial anaemia are microcythaemia and hypochromia. Anaemia occurs as frequently as parasitaemia is high. When parasitaemia is low anaemia gets a haemolytic feature. When parasitaemia is high, anaemia gets haemolytic and inflammatory features. Anaemia occurs more often with a good iron index status.

Red blood cell defects and malaria.

Malaria is a major cause of childhood death throughout much of the tropical world. As a result, it has exerted a powerful force for the evolutionary selection of genes that confer a survival advantage. Identifying which genes are involved, and how they affect malaria risk, is a potentially useful way of exploring the host-parasite relationship. To date, some of the best-described malaria-protective polymorphisms relate to genes that affect the structure or function of red blood cells (RBC). Recent years have seen significant advances in our understanding of the importance of some of these genes, including glycophorin C (GYPC); complement receptor 1 (CR1); band 3 (SLC4A1); pyruvate kinase (Pklr); and the genes for alpha-(HBA) and beta-globin (HBB). The challenge for the future must be to convert these advances into fresh approaches to the prevention and treatment of malaria.

Basis of unique red cell membrane properties in hereditary ovalocytosis.

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.

Comparison of Plasmodium falciparum growth in sickle cells in low oxygen environment and candle-jar.

The first successful in vitro cultivation of Plasmodium falciparum in sickle cells in a gas mixture containing 3% oxygen, 4% carbon dioxide and 93% nitrogen has been reported recently, contradicting earlier claims that the parasite does not multiply continuously in sickle cell trait (HbAS) and sickle cell anemia (HbSS) erythrocytes at low oxygen tension. The present study extends that report by growing three P. falciparum strains in erythrocytes from four different sickle cell trait and four sickle cell anemia donors. Because P. falciparum is known to grow normally in sickle cells when incubated in a candle-jar estimated to contain 15-18% oxygen, we have also compared the growth at 3% oxygen with that in a candle-jar. For convenience, we also refer to the 3% oxygen and the candle-jar as low and high oxygen environment, respectively. The three P. falciparum strains were first grown continuously in low oxygen environment for at least 1 month in erythrocytes from one HbAS carrier. These stock cultures were then used to infect erythrocytes from additional three HbAS carriers and four HbSS patients. Results of the experiments showed that parasite growth and hemozoin production in HbAS erythrocytes in low oxygen environment were comparable to those obtained in the candle-jar. There was growth retardation in HbSS erythrocytes in low oxygen environment, but some of the parasites survived and eventually produced high parasitemia levels. Continuous cultivation of different P. falciparum strains in HbAS erythrocytes is necessary for investigation of possible molecular differences between malaria parasites in sickle cells and those in HbAA erythrocytes.

The Haldane malaria hypothesis: facts, artifacts, and a prophecy.

Heterozygous thalassemia and sickle cell disease produce mild hematological symptoms but provide protection against malaria mortality and severe malaria symptoms. Two explanations for resistance are considered in the literature - impaired growth of the parasite or enhanced removal by the host immune cells. A critical overview of studies that connect malaria resistance with impaired intra-erythrocytic growth is presented. All studies are fraught with two kinds of bias. The first one resides in the impossibility of reproducing the in vivo situation in the simplified model in vitro. The second stems from the generalized use of RPMI 1640 culture medium. RPMI 1640 has critically low levels of several amino acids; is devoid of hypoxanthine (essential for parasite growth) and adenine; and is low in reduced glutathione. Analysis of representative studies indicates that impaired parasite growth in heterozygous red blood cells (RBCs) may derive from nutrient limitations and, therefore, possibly be of artefactual origin. This conclusion seems plausible because studies were performed with RPMI 1640 medium at relatively high hematocrit and for prolonged periods of time. Mutations considered are particularly sensitive to nutrient deprivation because they have higher metabolic demands due to permanent oxidant stress related to unpaired globin chains, sickle hemoglobin and high levels of membrane-free iron. In addition, non-parasitized AS- and thalassemic-RBCs are dehydrated and microcytic. Thus, the number of metabolically active elements per unit of blood volume is remarkably larger in mutant RBCs compared to normocytes. The latter point may represent a confirmation of Haldane's prophetic statement: 'The corpuscles of the anaemic heterozygotes are smaller than normal, and more resistant to hypotonic solutions. It is at least conceivable that they are also more resistant to attacks by the sporozoa which cause malaria.'

Does the mechanism of protection from falciparum malaria by red cell genetic disorders involve a switch to a balanced TH1/TH2 cytokine production mode?

The mechanism of protection from falciparum malaria by red cell genetic disorders still remains controversial. Decreased survival of parasites in variant red cells has previously been proposed. However, in vitro experiments were not conclusive and do not seem sufficient to explain the substantial degree of in vivo protection afforded to red cell genetic trait carriers. Evidence has recently been accumulating in favour of enhancement of the host immune response by these genetic traits. Malaria-infected variant red cells undergo modifications to their antigenicity which lead to accelerated and selective removal of early blood-stage parasites by splenic macrophages, resulting in fewer parasites reaching schizogony. Consequently there will be alterations in antigen processing, presentation and recognition which could explain the differences observed in T-cell responses between trait carriers and normal individuals. It is suggested that exposure to a lower dose of early parasite-stage antigens rather than the exoantigens of late mature schizonts could lead during primary and subsequent secondary infections to differentiation of T-helper cells into balanced TH1/TH2 subsets that promote protection, reversing the susceptibility to the fatal complications of falciparum malaria.

Laboratory and field comparisons of adenosine influx in Plasmodium falciparum and Plasmodium vivax infected erythrocytes with genetic abnormalities from patients in Myanmar.

Influx of the purine nucleoside, adenosine, was assessed in erythrocytes from both normal subjects and from subjects with a range of genetically determined erythrocyte disorders from Myanmar. The latter included alpha-thalassemia major (Myanmar variant), beta-thalassemia major (Myanmar variant), beta-thalassemia trait, HbEE and HbAE erythrocytes and two variants of glucose-6-phosphate dehydrogenase (G6PDH) deficiency. Significant reductions (p < 0.01) of adenosine influx were observed in erythrocytes from individuals with alpha- and beta-thalassemia major and severe G6PDH deficiency. Abnormal erythrocytes infected with the malarial parasites, Plasmodium falciparum or Plasmodium vivax, demonstrated a reduction in adenosine transport which correlated with the proportion of abnormal erythrocytes present in the samples obtained. The effect of nitrobenzylthioinosine (NBMPR) on adenosine influx was explored in normal and abnormal erythrocytes. In all these cases, NBMPR completely inhibited the transport of adenosine. However, transport of adenosine into P. falciparum and P. vivax-infected normal erythrocytes and abnormal cells was only inhibited 50-60% by NBMPR. The combination of tubercidin and NBMPR completely blocked adenosine transport into both normal and abnormal erythrocytes infected with either P. falciparum or P. vivax.

New stages in the program of malaria parasite egress imaged in normal and sickle erythrocytes.

The apicomplexan parasite Plasmodium falciparum causes malignant malaria. The mechanism of parasite egress from infected erythrocytes that disseminate parasites in the host at the end of each asexual cycle is unknown. Two new stages of the egress program are revealed: (1) swelling of the parasitophorous vacuole accompanied by shrinkage of the erythrocyte compartment, and (2) poration of the host cell membrane seconds before erythrocyte rupture because of egress. Egress was inhibited in dehydrated cells from patients with sickle cell disease in accord with experimental dehydration of normal cells, suggesting that vacuole swelling involves intake of water from the erythrocyte compartment. Erythrocyte membrane poration occurs in relaxed cells, thus excluding involvement of osmotic pressure in this process. Poration does not depend on cysteine protease activity, because protease inhibition blocks egress but not poration, and poration is required for the parasite cycle because the membrane sealant P1107 interferes with egress. We suggest the following egress program: parasites initiate water influx into the vacuole from the erythrocyte cytosol to expand the vacuole for parasite separation and vacuole rupture upon its critical swelling. Separated parasites leave the erythrocyte by breaching its membrane, weakened by putative digestion of erythrocyte cytoskeleton and membrane poration.

Malaria and red cell genetic defects.

The study of inherited RBC resistance to malaria has increased our knowledge of the biochemistry and physiology of the host-parasite interaction and suggested potential sites for therapeutic intervention. Discovery by Jensen and Trager of the in vitro culture system for P falciparum has facilitated research in this area. Known RBC defects may affect invasion, growth, or merozoite liberation (Fig 1). Significant advances made in understanding mechanisms underlying protection against malaria should not obscure the fact that the data are far from complete. More knowledge is needed about the influence of the erythrocyte cytoskeleton on invasion and growth of parasites as well as the potential role of phospholipids, erythrocyte enzymes other than G6PD, or other metabolic products. Application of DNA analysis and recombinant technology may have an increasing impact on study of the interaction of RBC defects with malarial parasites.

Ultrastructural damage to the malaria parasite in the sickled cell.

The process by which malaria parasites are killed in sickled erythrocytes was studied by electron microscopy. In vitro cultures of Plasmodium falciparum in sickle cell hemoglobin (HbS) homozygous (SS) and heterozygous (SA) red cells were deoxygenated for up to 6 h and fixed under anaerobic conditions. Parasites in SS cells appeared to be disrupted by intrusions of needle-like deoxyHbS aggregates; disintegration of cytoplasm and membranes followed. In SA red cells, the parasites were generally not disrupted. Instead, extensive vacuolization occurred, a sign of metabolic inhibition. The resistance of HbS gene carriers to malaria results partly from these causes of intracellular parasite death.

Diminished red blood cell deformability in uncomplicated human malaria. A preliminary report.

Six patients with active P. falciparum malaria (two resistant to chloroquine therapy), one with treated and apparently cured P. falciparum malaria, one with active P. vivax malaria and two controls from the same geographic area of Amazonian Brazil were studied. All malaria patients had impaired red cell deformability. This was not correlated with drug resistance or number of parasitized cells. The hypothesis is presented that the presence of parasites in red blood cells and related metabolic effects produce decreased red cell deformability and may lead to microvascular perfusion deficiencies including cerebral malaria.

Ovalocytic erythrocytes from Melanesians are resistant to invasion by malaria parasites in culture.

Ovalocytic erythrocytes from Melanesians in Papua New Guinea have been demonstrated to be resistant to infection by malaria parasites (Plasmodium falciparum) in culture by using a double-label fluorescence assay of merozoite invasion. That merozoites do not bind irreversibly to ovalocytes has been demonstrated by an assay that measures competition between ovalocytes and normocytes. Analysis of behavior on thermal deformation has demonstrated that ovalocytes are more more thermostable than normocytes, suggesting that there is a major difference in cytoskeletal structure. These findings with P. falciparum and epidemiological data demonstrating clinical resistance to P. vivax and P. malariae suggest that the membrane alterations(s) in these ovalocytes affect(s) invasion step(s) common to all three species of malaria parasite.