Enhanced Eryptosis in Glucose-6-Phosphate Dehydrogenase Deficiency

Background/Aims: Defects in the Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme enhance cellular oxidative damage, thus impairing erythrocytes and radically shortening their lifespan. We aimed to study programmed erythrocyte cell death in G6PD-deficient patients, describe the molecular genetics basis of G6PD and investigate phenotype-genotype correlations. Methods: We explored eryptosis using the annexin V-binding assay, taken as an indicator of PS exposure at the erythrocyte surface. We assessed reactive oxygen species (ROS) production, intracellular calcium concentrations and ceramide formation at the cell surface. Prior to and following treatments, cells were analyzed by flow cytometry. Finally, we explored G6PD gene mutations through PCR-Sanger sequencing. Results: Before stimulation, PS-exposing erythrocytes were significantly higher in G6PD-deficient patients than in healthy volunteers. This was paralleled by a significant increase in reactive oxygen species production, suggesting that oxidative stress is the main trigger of PS exposure in G6PDdeficient erythrocytes. Five previously described mutations were detected in our patients. Two genotypes correlated with a significantly higher percentage of PS-exposing cells. Conclusion: Our study uncovers a novel effect detected in G6PD-deficient erythrocytes which is cell membrane scrambling with PS translocation to the erythrocyte surface. Our findings shed a light on the mechanisms of premature erythrocyte clearance in G6PD deficiency.

Systematic analysis of direct antiglobulin test results in post-artesunate delayed haemolysis

Background Post-artesunate delayed haemolysis (PADH) is common after severe malaria episodes. PADH is related to the “pitting” phenomenon and the synchronous delayed clearance of once-infected erythrocytes, initially spared during treatment. However, direct antiglobulin test (DAT) positivity has been reported in several PADH cases, suggesting a contribution of immune-mediated erythrocyte clearance. The aim of the present study was to compare clinical features of cases presenting a positive or negative DAT. Methods Articles reporting clinical data of patients diagnosed with PADH, for whom DAT had been performed, were collected from PubMed database. Data retrieved from single patients were extracted and univariate analysis was performed in order to identify features potentially related to DAT results and steroids use. Results Twenty-two studies reporting 39 PADH cases were included: median baseline parasitaemia was 20.8% (IQR: 11.2–30) and DAT was positive in 17 cases (45.5%). Compared to DAT-negative individuals, DAT-positive patients were older (49.5 vs 31; p = 0.01), had a higher baseline parasitaemia (27% vs 17%; p = 0.03) and were more commonly treated with systemic steroids (11 vs 3 patients, p = 0.002). Depth and kinetics of delayed anaemia were not associated with DAT positivity. Conclusions In this case series, almost half of the patients affected by PADH had a positive DAT. An obvious difference between the clinical courses of patients presenting with a positive or negative DAT was lacking. This observation suggests that DAT result may not be indicative of a pathogenic role of anti-erythrocytes antibodies in patients affected by PADH, but it may be rather a marker of immune activation.

Cellular mechanisms of erythrodieresis

Normally erythrodieresis is in a dynamic equilibrium with the process of erythropoiesis, and is therefore one of the factors to providing a relatively constant number of red blood cells in the bloodstream. The physiologically old, damaged and non-viable erythrocytes, as well as the erythrocytes which are produced during stress erythropoiesis, are destroyed. Erythrocyte clearance is a selective process. First of all, the cells that have lost their ability to deform are removed from the bloodstream. The deformability of red blood cells depends on the shape of the cells, the viscosity of the cytoplasm and the mechanical properties of the membrane. Old and altered erythrocytes are quite rigid, and are therefore delayed in the narrow capillaries and venous sinuses of the liver and spleen. In addition, macrophages of the liver and spleen phagocytize erythrocytes, which expose “eat me” signaling molecules on their surface. Exposure of phosphatidylserine on the outer cell surface of erythrocytes and vesicles results in their elimination from the bloodstream by Kupffer cells and other mononuclear phagocytes. During the initiation of erythrophagocytosis, the phosphatidylserine of the outer lipid layer of the erythrocyte plasma membrane directly interacts with the receptors Stabilin-2, TIM-1, TIM-4 or CD300 of macrophages. The macrophage’s integrins avb3 and avb5, as well as the Mer receptor tyrosine kinase indirectly interact with the cell surface-exposed phosphatidylserine through the soluble proteins MFG-E8, Gas 6 and protein S. Clustering of the erythrocyte membrane protein band 3 cau­ses the binding of natural antibodies, and opsonization of erythrocytes with C3b enhances this process and facilitates the recognition of such cells by red pulp macrophages in the spleen. In senescent erythrocytes, the formation of the CD47-SIRPα complex («do not eat me» signal), is suppressed, and this is an additional stimulus for erythrocytes removing by splenic and liver macrophages. The purpose of the review is to describe the mechanisms of erythrophagocytosis and the molecular determinants of erythrocyte senescence and death, including eryptosis and neocytolysis, and to illustrate the substantiated facts and contradictions that exist at the present time of the study of this scientific problem.

Systematic Analysis of Direct Antiglobulin Test Results in Post-artesunate Delayed Hemolysis

Background: “Post-Artesunate Delayed Hemolysis” (PADH) is common after severe malaria episodes. PADH is related to the “pitting” phenomenon and the synchronous delayed clearance of once-infected erythrocytes, initially spared during treatment. However, direct antiglobulin test (DAT) positivity has been reported in several PADH cases, suggesting a contribution of immune-mediated erythrocyte clearance. The aim of the present study was to compare clinical features of cases presenting a positive or negative DAT.Method: Articles reporting clinical data of patients diagnosed with PADH, for whom DAT had been performed, were collected from PubMed database. Data retrieved from single patients were extracted and univariate analysis was performed in order to identify features potentially related to DAT results and steroids use.Results: Twenty-two studies reporting 39 PADH cases were included: median baseline parasitemia was 20.8% (IQR: 11.2-30) and DAT was positive in 17 cases (45.5%). Compared to DAT-negative individuals, DAT-positive patients were older (49.5 vs 31; p=0.01), had a higher baseline parasitemia (27% vs 17%; p=0.03) and were more commonly treated with systemic steroids (11 vs 3 patients, p=0.002). Depth and kinetics of delayed anemia were not associated with DAT positivity.Conclusions: In this case series, almost half of the patients affected by PADH had a positive DAT. An obvious difference between the clinical courses of patients presenting with a positive or negative DAT was lacking. This observation strongly suggests that DAT result is not indicative of a pathogenic role of anti-erythrocytes antibodies in patients affected by PADH, but is rather a marker of immune activation.

Ankyrin-1 gene exhibits allelic heterogeneity in conferring protection against malaria

Allelic heterogeneity is a common phenomenon where a gene exhibit different phenotype depending on the nature of its genetic mutations. In the context of genes affecting malaria susceptibility, it allowed us to explore and understand the intricate host-parasite interactions during malaria infections. In this study, we described a gene encoding erythrocytic ankyrin-1 (Ank-1) which exhibits allelic-dependent heterogeneous phenotypes during malaria infections. We conducted an ENU mutagenesis screen on mice and identified twoAnk-1mutations, one resulted in an amino acid substitution (MRI95845), and the other a truncatedAnk-1protein (MRI96570). Both mutations caused hereditary spherocytosis-like phenotypes and confer differing protection againstPlasmodium chabaudiinfections. Upon further examination, theAnk-1(MRI96570)mutation was found to inhibit intra-erythrocytic parasite maturation, whereasAnk-1(MW95845)caused increased bystander erythrocyte clearance during infection. This is the first description of allelic heterogeneity in ankyrin-1 from the direct comparison between twoAnk-1mutations. Despite the lack of direct evidence from population studies, this data further supported the protective roles of ankyrin-1 mutations in conferring malaria protection. This study also emphasized the importance of such phenomenon to achieve a better understanding of host-parasite interactions, which could be the basis of future studies.

A novel ENU-induced ankyrin-1 mutation impairs parasite invasion and increases erythrocyte clearance during malaria infection in mice

Genetic defects in various red blood cell (RBC) cytoskeletal proteins have been long associated with changes in susceptibility towards malaria infection. In particular, while ankyrin (Ank-1) mutations account for approximately 50% of hereditary spherocytosis (HS) cases, an association with malaria is not well-established, and conflicting evidence has been reported. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced ankyrin mutation MRI61689 that gives rise to two different ankyrin transcripts: one with an introduced splice acceptor site resulting a frameshift, the other with a skipped exon.Ank-1(MRI61689/+)mice exhibit an HS-like phenotype including reduction in mean corpuscular volume (MCV), increased osmotic fragility and reduced RBC deformability. They were also found to be resistant to rodent malariaPlasmodium chabaudiinfection. Parasites inAnk-1(MRI61689/+)erythrocytes grew normally, but red cells showed resistance to merozoite invasion. UninfectedAnk-1(MRI61689/+)erythrocytes were also more likely to be cleared from circulation during infection; the “bystander effect”. This increased clearance is a novel resistance mechanism which was not observed in previous ankyrin mouse models. We propose that this bystander effect is due to reduced deformability ofAnk-1(MRI61689/+)erythrocytes. This paper highlights the complex roles ankyrin plays in mediating malaria resistance.