Cell-derived microparticles after exercise in individuals with G6PD Viangchan.

Glucose-6-phospate dehydrogenase (G6PD) deficient cells are sensitive to oxidative damage leading to the formation of microparticles (MPs). Therefore, we examined the concentration of MPs and changes in the antioxidant balance after an acute strenuous exercise (SEx) and moderate-intensity exercise (MEx). Eighteen healthy females (18-24 years) with G6PD normal and eighteen age-matched females with G6PD Viangchan (871G>A) were tested by running on a treadmill at their maximal oxygen uptake for SEx and at 75% of their maximal heart rate for MEx. It was found that SEx triggered the release of total microparticles (TTMPs) above baseline levels and remained significantly higher 45 minutes after the exercise in G6PD normal individuals. However, SEx-induced increase in TTMPs was significantly higher in G6PD Viangchan as compared to G6PD normal. In contrast, MEx did not to alter the release of TTMPs in both G6PD normal and Viangchan. Moreover, TTMPs concentrations were inversely correlated with G6PD activity (r =-0.82, P <  0.05) but positively correlated with MDA concentrations (r = 0.74, P <  0.05). Using cell specific antibodies, we determined that MPs were mainly derived from platelets and erythrocytes. Altogether, the present study indicates that G6PD Viangchan may participate in MEx without higher MPs concentration and oxidative stress compared with G6PD normal.

Reduced ADAMTS13 activity is associated with an ADAMTS13 SNP, fever and microparticles in a malaria-like model.

BACKGROUND: Severe falciparum malaria (SM) remains a major cause of death in tropical countries. The reduced activity of ADAMTS13, increasing levels of ultra-large von Willebrand factor (ULVWF) in SM patients, are assumed as factors that intensify disease severity. However, the reason why ADAMTS13 activity is reduced in SM remains unclear. OBJECTIVES: To investigate whether rs4962153, febrile temperature, and microparticles, contribute to reduced ADAMTS13 activity. METHODS: Genotypic association of rs4962153 with ADAMTS13 antigen and activity was examined in 362 healthy Thai participants. The collagen binding assay was used to study the effects of febrile temperature and microparticles on ADAMTS13 activity. RESULTS: ADAMTS13 antigen and activity were decreased in participants with AA genotype, compared to AG and GG (antigen: p-value = 0.014, and < 0.001; activity: p-value = 0.036, and < 0.002, respectively). There was significantly reduced ADAMTS13 antigen in AG compared to GG (p-value = 0.013), but not in ADAMTS13 activity (p-value = 0.082). The number of rs4962153 A alleles correlated with the reduced level of antigen and activity (p-value <0.001 and p-value = 0.001, respectively). MPs showed an inhibitory effect on ADAMTS13 activity (p-value = 0.025). Finally, ADAMTS13 activity was decreased in a temperature and time-dependent manner. The interaction between these two factors was also observed (p-value <0.001). CONCLUSIONS: These findings suggest that the A allele of rs4962153, MPs, and febrile temperature, contribute to reduce ADAMTS13 activity in plasma. These data are useful in malaria or other diseases with reduced ADAMTS13 activity.

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 <10% of the normal mean), whereas only 14.3% of the heterozygote mutations showed severe enzyme deficiency. Based on the cut-off value <5.7 IU/gHb, the quantitative G6PD assay diagnosed 83% of cases as G6PD-deficient. Using a cut-off number of negative cell >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.

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.

Association of ADAMTS13 polymorphism with cerebral malaria.

BACKGROUND: Cerebral malaria is one of the most severe manifestations of Plasmodium falciparum malaria. The sequestration of parasitized red blood cells (PRBCs) to brain microvascular endothelium has been shown to contribute to the pathophysiology of cerebral malaria. Recent studies reported increased levels of von Willebrand factor (VWF) and reduced activity of VWF-cleaving protease, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), in patients with cerebral malaria. METHODS: Association of six single nucleotide polymorphisms (SNPs) of the ADAMTS13 gene with cerebral malaria was examined in 708 Thai patients with P. falciparum malaria. RESULTS: Among six SNPs, the derived allele of a SNP located in intron 28, rs4962153-A, was significantly associated with protection against cerebral malaria when 115 cerebral malaria patients were compared with 367 mild malaria patients (Fisher's exact P-value = 0.0057; OR = 0.27; 95% CI = 0.096-0.76). Significant association was also detected between 115 cerebral malaria and 593 non-cerebral malaria (226 non-cerebral severe malaria and 367 mild malaria) patients (Fisher's exact P-value = 0.012; OR = 0.30; 95% CI = 0.11-0.83). CONCLUSIONS: Excessive adhesion of PRBCs to the platelet-decorated ultra-large VWF (ULVWF) appears to enhance the sequestration of PRBCs to cerebral microvascular endothelium. The genetic association observed in the present study implies that the regulation of platelet-decorated ULVWF strings by ADAMTS13 may play a role in the development of cerebral malaria.

Circulating red cell-derived microparticles in human malaria.

In patients with falciparum malaria, plasma concentrations of cell-derived microparticles correlate with disease severity. Using flow cytometry, we quantified red blood cell-derived microparticles (RMPs) in patients with malaria and identified the source and the factors associated with production. RMP concentrations were increased in patients with Plasmodium falciparum (n = 29; median, 457 RMPs/µL [range, 13-4,342 RMPs/µL]), Plasmodium vivax (n = 5; median, 409 RMPs/µL [range, 281-503/µL]), and Plasmodium malariae (n = 2; median, 163 RMPs/µL [range, 127-200 RMPs/µL]) compared with those in healthy subjects (n = 11; median, 8 RMPs/µL [range, 3-166 RMPs/µL]; P = .01). RMP concentrations were highest in patients with severe falciparum malaria (P = .01). Parasitized red cells produced >10 times more RMPs than did unparasitized cells, but the overall majority of RMPs still derived from uninfected red blood cells (URBCs). In cultures, RMP production increased as the parasites matured. Hemin and parasite products induced RMP production in URBCs, which was inhibited by N-acetylcysteine, suggesting heme-mediated oxidative stress as a pathway for the generation of RMPs.

Enumeration of the absolute CD4 T-lymphocyte count by cell-bead assay.

BACKGROUND: We have previously developed an alternative approach for undertaking absolute cell counting based upon flow-rate calibration using cell bead (FCB), in which cell bead (CB) can be used as a flow-rate calibration material for generating the absolute microparticle counts. Here, we extended our work of counting CD4+ T-lymphocytes in HIV-infected blood samples with the FCB method. METHODS: CD4+ T-lymphocyte counts in EDTA blood samples from 30 healthy subjects and 80 HIV-1-infected patients were determined using TriTEST reagent. The absolute CD4+ T-lymphocytes were measured by FCB, and the results were compared with the absolute counting by commercial latex bead (CLB) or with flow rate-based calibration method (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 CD4+ T-lymphocyte counts enumerated by FCB when compared with those two reference methods (CLB and FR). The absolute CD4+ T-lymphocyte counts obtained from FCB method was highly correlated with those obtained from CLB [r(2)= 0.99, y = 1.04x - 12.37, P < 0.001, and mean bias 11.96 cell/microl, limit of agreement (LOA) -57.82 - 81.74 cell/microl], FR method (r(2) = 0.98; y = 0.97x - 3.13, P < 0.001, and mean bias -24.15 cell/microl, LOA -114.44 - 66.13 cell/microl). CONCLUSIONS: The use of FCB is comparable with the use of CLB and FR. This approach showed the effective in reducing cost for generating the absolute CD4+ T-lymphocyte counts. Such an approach should facilitate and ensure the success of the ongoing antiretroviral therapy program in resource-limited countries.

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.

Quantitation of cell-derived microparticles in plasma using flow rate based calibration.

Activation of vascular endothelium and blood cells can result in the formation of microparticles (MPs), which are membrane vesicles with a diameter < 1 microm which can play a pathogenetic role in a variety of infectious and other diseases. In this study, we validated a modified quantitative method called "flow rate based calibration", to measure circulating MPs in plasma of healthy subjects and malaria patients using FACSCalibur flow cytometry. MPs counts obtained from "flow rate based calibration" correlated closely with the standard method (R2 = 0.9, p = 0.001). The median (range) number of MPs in healthy subjects was 163/microl (81-375/microl). We demonstrated a flow rate based calibration for the quantitation of MPs in P. falciparum malaria-infected patients. The median (range) number of MPs was 2,051/microl (222-6,432/microl), n = 28 in patients with falciparum malaria. The number of MPs in plasma from patients with severe falciparum malaria was significantly higher than in uncomplicated falciparum malaria (2,567/microl (366-6,432/microl), n = 18 versus [1,947/microl (222-4,107/microl), n = 10, p < 0.01]. Cellular origin of MPs in malaria patients were mainly derived from red blood cells (35%), platelets (10%), and endothelial cells (5%). There was no significant correlation between the total number of MPs and parasitemia. Flow rate based calibration is a simple, reliable, reproducible method and more affordable to quantitate MPs.