Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria

Malaria remains one of the greatest public health challenges worldwide, particularly in sub-Saharan Africa. The clinical outcome of individuals infected with Plasmodium falciparum parasites depends on many factors including host systemic inflammatory responses, parasite sequestration in tissues and vascular dysfunction. Production of pro-inflammatory cytokines and chemokines promotes endothelial activation as well as recruitment and infiltration of inflammatory cells, which in turn triggers further endothelial cell activation and parasite sequestration. Inflammatory responses are triggered in part by bioactive parasite products such as hemozoin and infected red blood cell-derived extracellular vesicles (iRBC-derived EVs). Here we demonstrate that such EVs contain functional miRNA-Argonaute 2 complexes that are derived from the host RBC. Moreover, we show that EVs are efficiently internalized by endothelial cells, where the miRNA-Argonaute 2 complexes modulate target gene expression and barrier properties. Altogether, these findings provide a mechanistic link between EVs and vascular dysfunction during malaria infection

Circulating microparticles: square the circle

Background: The present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance. Results: MPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform. Conclusions: Improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics, but also for the evaluation of treatment regimens and predicting disease outcomes

Red cell and platelet-derived microparticles are increased in G6PD-deficient subjects.

In response to oxidative stress and during apoptosis, cells often shed microparticles (MPs), submicron elements carrying phosphatidylserine and protein antigens. Glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are extremely sensitive to oxidative damage that may lead to the formation of MPs. To determine whether G6PD deficiency alters membrane phospholipid asymmetry and increases MPs production, we determined the concentrations and cellular origins of MPs in G6PD-deficient individuals using flow cytometry. G6PD-deficient individuals showed an increase in circulating MPs concentrations as compared with G6PD-normal individuals [1051/μL (865-2532/μL) vs. 258/μL (235-575/μL), P < 0.01]. MPs concentrations were significantly increased with the severity of G6PD deficiency. Median MPs concentrations from individuals with severe G6PD deficiency, and individuals with moderate G6PD deficiency were 2567/μL (1216-2532/μL) and 984/μL (685-2107/μL), respectively (P < 0.01). Importantly, G6PD enzymatic activity was significantly correlated with MPs concentrations with r(2) = 0.731. MPs found in G6PD deficiency individuals were largely derived from red blood cells (RBCs) (45%) and platelets (30%). Additionally, Atomic Force Microscopy was used to study the morphology and measures the diameter of MPs found in G6PD-deficient individuals. The mean (SD) width and height of RMPs were 0. 41 (0.18) and 2.04 (0.14) μm, respectively. Together, these results indicate that MP concentration is significantly correlated with G6PD enzymatic activity and is increased in G6PD-deficient as compared with G6PD-normal individuals. Our data also provide an evidence for an alteration in cell membrane associated with a decreased in G6PD activity. However, the significance of MPs in G6PD deficiency needs further clarification

The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay.

BACKGROUND: Activation of red blood cell is associated with the formation of red cell-derived microparticles (RMPs). Analysis of circulating RMPs is becoming more refined and clinically useful. A quantitative Trucount tube method is the conventional method uses for quantitating RMPs. In this study, we validated a quantitative method called "flow rate based assay using red cell bead (FCB)" to measure circulating RMPs in the peripheral blood of healthy subjects. METHODS: Citrated blood samples collected from 30 cases of healthy subjects were determined the RMPs count by using double labeling of annexin V-FITC and anti-glycophorin A-PE. The absolute RMPs numbers were measured by FCB, and the results were compared with the Trucount or with flow rate based calibration (FR). Statistical correlation and agreement were analyzed using linear regression and Bland-Altman analysis. RESULTS: There was no significant difference in the absolute number of RMPs quantitated by FCB when compared with those two reference methods including the Trucount tube and FR method. The absolute RMPs count obtained from FCB method was highly correlated with those obtained from Trucount tube (r(2) = 0.98, mean bias 4 cell/microl, limit of agreement [LOA] -20.3 to 28.3 cell/microl), and FR method (r(2) = 1, mean bias 10.3 cell/microl, and LOA -5.5 to 26.2 cell/microl). CONCLUSION: This study demonstrates that FCB is suitable and more affordable for RMPs quantitation in the clinical samples. This method is a low cost and interchangeable to latex bead-based method for generating the absolute counts in the resource-limited areas

Evaluation of the phenotypic test and genetic analysis in the detection of glucose-6-phosphate dehydrogenase deficiency.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is particularly prevalent in historically malaria-endemic countries. Although most individuals with G6PD deficiency are asymptomatic, deficiency can result in acute haemolytic anaemia after exposure to oxidative agents. A reliable test is necessary for diagnosing the deficiency to prevent an acute haemolytic crisis following, for example, anti-malarial treatment. The aim of this study was to investigate which method was the best predictor of this disorder. METHODS: The present study investigated four G6PD activity detections (fluorescence spot (FS), methaemoglobin reduction (MR), biochemical and cytochemical test). These methods accompanied with mutation analysis of blood samples were taken from 295 apparently healthy individuals with unknown G6PD deficiency status. RESULTS: Molecular characterization of 295 Thai adults revealed an overall prevalence of 14.2%. The G6PD Viangchan (871 G>A) was the most common (83.3%), followed by G6PD Mahidol (487G>A) (11.9%), and G6PD Union (1360 C>T) (4.8%). There were two cases of G6PD deficiency carrying the double mutations of Viangchan (871G > A)-Mahidol (487G > A) and Viangchan (871G > A)-Union (1360C > T). In comparison, the prevalence of G6PD deficiency was 6.1% by FS test and 7.1% by MR test. G6PD activity was 11 ± 2.5 IU/gHb in non-deficient females (mean ± SD), and 10.9 ± 0.6 IU/gHb in non-deficient males. The upper and lower limit cut-off points for partial and severe deficiency in adults were 5.7 IU/gHb (60% of the normal mean) and 0.95 IU/gHb (10% of the normal mean), respectively. All hemizygote, homozygote and double mutations were associated with severe enzyme deficiency (the residual enzyme activity 20% in the cytochemical assay to define G6PD deficiency, the prevalence of G6PD deficiency was closest to the molecular analysis (12.9% G6PD-deficient) compared to the others methods. CONCLUSION: The cytochemical method is a significant predictor of this disease, while FS and MR test are recommended for the detection of severe G6PD deficiency in developing countries

Affordable Technology for Enumeration of the Absolute CD4 T-Lymphocyte Count by Cell Bead Assay

The quantitative BD Trucount (San Jose, CA) tube method is the conventional but expensive method to quantitate CD4+T-lymphocyte (CD4) counts, and this may be beyond the means of countries with limited resources. In this study, we validated a quantitative method known as a cell-bead (CB) assay to quantitate CD4 counts in the peripheral blood of healthy subjects. The absolute CD4 count obtained from the CB method was highly correlated with those obtained from the Trucount tube (r2=0.98, y=26.73+1.01x, P<0.0001 and a mean bias of 34.8 cell/μL, limit of agreement [LOA] -34.8-104.4 cell/μL) and flow rate-based assay method (r2=0.97; y=69.51 + 0.88x, P<0.0001 and a mean bias -53.5 cell/μL, LOA -149.4-42.3 cell/μL). This study demonstrates that the CB method is suitable and more affordable for CD4 quantitation. This method is inexpensive and interchangeable with the latex bead-based methods for generating absolute counts in resource-limited areas

Increase Membrane Vesiculation in Essential Hypertension

Background: A hypertensive condition is known to be an important risk factor for arterial disease and thrombotic events. The detailed understanding of the pathophysiology in thrombotic events is crucial for the development of both preventive measures and the treatment during the early stage. Microparticles are submicron cell membrane vesicle shed from the cell surface in response to cell injury or apoptosis, and they are an essential element in the process leading to the pathogenesis of thrombosis and hemostasis dysfunction in patients. Activation of blood cells can result in the formation of microparticles, which carry a negatively charged, phosphatidylserine (PS), with a diameter of <1.0 micron. The biological and clinical functions of microparticles have been highlighted in coronary artery disease and heart failure. Objective: To elucidate the functional role of microparticles in essential hypertension. Methods: We quantitated the total number of circulating PS+ microparticles and studied the role of PS+ microparticles to see whether they can shorten the plasma recalcification time in patients with essential hypertension. Results: The PS+ microparticles were detectable at a low level in healthy blood and significantly increased in the patients with essential hypertension. With regard to PS+ microparticles affecting prothrombotic state in hypertension, we enriched microparticles and determined their procoagulant activity with a plasma recalcification time. The clotting time was significantly reduced after the addition of enriched microparticles to plasma poor microparticles (PPMP). A significant negative correlation was detected between numbers of enriched-PS+ microparticles and plasma-clotting time (r=-0.43, P=0.01). Conclusion: Taken together, high levels of PS+ microparticles are present in the circulating blood of essential hypertension and may contribute to the generation and perpetuation of a thrombotic state