Early and extensive CD55 loss from red blood cells supports a causal role in malarial anaemia

BACKGROUND\ud \ud Levels of complement regulatory proteins (CrP) on the surface of red blood cells (RBC) decrease during severe malarial anaemia and as part of cell ageing process. It remains unclear whether CrP changes seen during malaria contribute to the development of anaemia, or result from an altered RBC age distribution due to suppressive effects of malaria on erythropoiesis.\ud \ud METHODS\ud \ud A cross sectional study was conducted in the north-east coast of Tanzania to investigate whether the changes in glycosylphosphatidylinositol (GPI)-anchored complement regulatory proteins (CD55 and CD59) contributes to malaria anaemia. Blood samples were collected from a cohort of children under intensive surveillance for Plasmodium falciparum parasitaemia and illness. Levels of CD55 and CD59 were measured by flow cytometer and compared between anaemic (8.08 g/dl) and non- anaemic children (11.42 g/dl).\ud \ud RESULTS\ud \ud Levels of CD55 and CD59 decreased with increased RBC age. CD55 levels were lower in anaemic children and the difference was seen in RBC of all ages. Levels of CD59 were lower in anaemic children, but these differences were not significant. CD55, but not CD59, levels correlated positively with the level of haemoglobin in anaemic children.\ud \ud CONCLUSION\ud \ud The extent of CD55 loss from RBC of all ages early in the course of malarial anaemia and the correlation of CD55 with haemoglobin levels support the hypothesis that CD55 may play a causal role in this disorder

Dysregulation of L-arginine metabolism and bioavailability associated to free plasma heme.

Severe Plasmodium falciparum malaria is associated with hypoargininemia, which contributes to impaired systemic and pulmonary nitric oxide (NO) production and endothelial dysfunction. Since intravascular hemolysis is an intrinsic feature of severe malaria, we investigated whether and by which mechanisms free heme [Fe(III)-protoporphyrin IX (FP)] might contribute to the dysregulation of L-arginine (L-Arg) metabolism and bioavailability. Carrier systems "y+" [or cationic amino acid transporter (CAT)] and "y+L" transport L-Arg into red blood cells (RBC), where it is hydrolyzed to ornithine and urea by arginase (isoform I) or converted to NO* and citrulline by endothelial nitric oxide synthase (eNOS). Our results show a significant and dose-dependent impairment of L-Arg transport into RBC pretreated with FP, with a strong inhibition of the system carrier y+L. Despite the impaired L-Arg influx, higher amounts of L-Arg-derived urea are produced by RBC preexposed to FP caused by activation of RBC arginase I. This activation appeared not to be mediated by oxidative modifications of the enzyme. We conclude that L-Arg transport across RBC membrane is impaired and arginase-mediated L-Arg consumption enhanced by free heme. This could contribute to reduced NO production in severe malaria

Oxidative stress and rheology in severe malaria.

There is mounting evidence that the release of haemozoin (beta-haematin), which is produced in large amounts during malaria infection and is released into the circulation during schizont rupture, is associated with damage to cell membranes through an oxidative mechanism. The red blood cell membrane is thus oxidised, causing rigidity of the cell. This can contribute to the pathophysiology of severe malaria, since red blood cells will have to deform considerably in order to squeeze through the microcirculation, the patency of which is disturbed by sequestered red blood cells containing the mature forms of the parasite. Rigidity of red blood cells forms a new target for intervention. Since this seems to be caused by oxidative damage to the red blood cell membrane, the anti-oxidant N-acetylcysteine is a promising candidate for adjunctive treatment in severe malaria, which still has a mortality rate as high as 20%

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

Dysregulation of l-arginine metabolism and bioavailability associated to free plasma heme

Severe Plasmodium falciparum malaria is associated with hypoargininemia, which contributes to impaired systemic and pulmonary nitric oxide (NO) production and endothelial dysfunction. Since intravascular hemolysis is an intrinsic feature of severe malaria, we investigated whether and by which mechanisms free heme [Fe(III)-protoporphyrin IX (FP)] might contribute to the dysregulation of l-arginine (l-Arg) metabolism and bioavailability. Carrier systems “y+” [or cationic amino acid transporter (CAT)] and “y+L” transport l-Arg into red blood cells (RBC), where it is hydrolyzed to ornithine and urea by arginase (isoform I) or converted to NO· and citrulline by endothelial nitric oxide synthase (eNOS). Our results show a significant and dose-dependent impairment of l-Arg transport into RBC pretreated with FP, with a strong inhibition of the system carrier y+L. Despite the impaired l-Arg influx, higher amounts of l-Arg-derived urea are produced by RBC preexposed to FP caused by activation of RBC arginase I. This activation appeared not to be mediated by oxidative modifications of the enzyme. We conclude that l-Arg transport across RBC membrane is impaired and arginase-mediated l-Arg consumption enhanced by free heme. This could contribute to reduced NO production in severe malaria

Effects of malaria heme products on red blood cell deformability.

In falciparum malaria, the deformability of the entire erythrocyte population is reduced in proportion to disease severity, and this compromises microcirculatory blood flow through vessels partially obstructed by cytoadherent parasitized erythrocytes. The cause of rigidity of uninfected erythrocytes in not known but could be mediated by malaria heme products. In this study, we show that red blood cell deformability (RBC-D), measured by laser-assisted optical rotational cell analyzer, decreased in a dose-dependent manner after incubation with hemin and hydrogen peroxide but not with hemoglobin or beta-hematin. Hemin also reduced mean red cell volume. Albumin decreased and N-acetylcysteine (NAC) both prevented and reversed rigidity induced by hemin. Hemin-induced oxidative damage of the membrane seems to be a more important contributor to pathology than cell shrinkage because the antioxidant NAC restored RBC-D but not red blood cell volume. The findings suggest novel approaches to the treatment of potentially lethal malaria