miR-214 aggravates oxidative stress in thalassemic erythroid cells by targeting ATF4.

Oxidative damage to erythroid cells plays a key role in the pathogenesis of thalassemia. The oxidative stress in thalassemia is potentiated by heme, nonheme iron, and free iron produced by the Fenton reaction, due to degradation of the unstable hemoglobin and iron overload. In addition, the levels of antioxidant enzymes and molecules are significantly decreased in erythrocytes in α- and ß-thalassemia. The control of oxidative stress in red blood cells (RBCs) is known to be mediated by microRNAs (miRNAs). In erythroid cells, microR-214 (miR-214) has been reported to respond to external oxidative stress. However, the molecular mechanisms underlying this phenomenon remain unclear, especially during thalassemic erythropoiesis. In the present study, to further understand how miR-214 aggravates oxidative stress in thalassemia erythroid cells, we investigated the molecular mechanism of miR-214 and its regulation of the oxidative status in thalassemia erythrocytes. We have reported a biphasic expression of miR-214 in ß- and α-thalassemia. In the present study the effect of miR-214 expression was investigated by using miR -inhibitor and -mimic transfection in erythroid cell lines induced by hemin. Our study showed a biphasic expression of miR-214 in ß- and α-thalassemia. Subsequently, we examined the effect of miR-214 on erythroid differentiation in thalassemia. Our study reveals the loss-of-function of miR-214 during translational activation of activating transcription factor 4 mRNA, leading to decreased reactive oxygen species levels and increased glutathione levels in thalassemia erythroid cell. Our results suggest that the expression of activating transcription factor 4 regulated by miR-214 is important for oxidative stress modulation in thalassemic erythroid cells. Our findings can help to better understand the molecular mechanism of miRNA and transcription factors in regulation of oxidative status in erythroid cells, particularly in thalassemia, and could be useful for managing and relieving severe anemia symptoms in patients in the future.

Latex C-serum from Hevea brasiliensis induces apoptotic cell death in a leukemic cell line.

BACKGROUND: Hevea brasiliensis latex is generally cultivated for the use of rubber particles. Previous studies have shown that the antiproliferative activity of C-serum in hepatocellular carcinoma is not induced through the classical apoptotic signaling pathway. However, in a leukemic cell line, the anti-proliferation effect of latex C serum remained unclear. METHODS: Leukemic cell lines (K562 and U937) and human peripheral blood mononuclear cells (PBMCs) were examined for cell viability using the MTT assay. Flow cytometry was used for apoptotic cell detection by annexin V/PI staining. The expression levels of proapoptotic and antiapoptotic marker genes were measured by qRT‒PCR. Moreover, the caspase activities of the extrinsic and intrinsic apoptotic pathways were detected by enzymatic activities. RESULTS: Latex C-serum inhibited cell proliferation in the K562 and U937 leukemic cell lines but did not affect human PBMCs. Latex C-serum significantly induced the percentage of early and late apoptotic cells in the leukemic cell line. The expression levels of the pro-apoptotic marker genes BAD, BAX, and CASPASE3 significantly increased in the leukemic cell line after post-latex C-serum leukemic cell treatment. The extrinsic, intrinsic and common apoptotic pathways were also studied through caspase-8, -9, and -3 activities. Latex C-serum treatment significantly induced caspase-8, -9, and -3 activation in the K562 cell line and U937 cell line compared to the untreated cells. CONCLUSIONS: These results indicate that latex C-serum enhanced anti-proliferation in leukemic cell lines by inducing apoptosis and caspase activation.

BACH1 regulates erythrophagocytosis and iron-recycling in ß-thalassemia.

Macrophages play essential roles in erythrophagocytosis and iron recycling. ß-thalassemia is characterized by a genetic defect in hemoglobin synthesis, which increases the rate of iron recycling. We previously showed that reduced expression of the BTB and CNC homolog 1 (BACH1) gene leads to increased phagocytosis of abnormal RBCs by activated monocytes. However, the mechanisms underlying this abnormal RBC clearance remained unclear. Herein, the spleen and bone marrow cells of ß-thalassemic mice were examined for erythrophagocytosis CD markers and iron-recycling genes. Higher expression levels of CD47 and CD163 on RBCs and macrophages, respectively, were observed in ß-thalassemic mice than in wild-type cells. The decreased expression of BACH1 caused an increase in Nrf2, Spic, Slc40a1, and HMOX1 expression in splenic red pulp macrophages of thalassemic mice. To investigate BACH1 regulation, a macrophage cell line was transfected with BACH1-siRNA. Decreased BACH1 expression caused an increase in CD163 expression; however, the expression levels were lower when the cells were cultured in media supplemented with ß-thalassemia/HbE patient plasma. Additionally, the iron recycling-related genes SPIC, SLC40A1, and HMOX1 were significantly upregulated in BACH1-suppressed macrophages. Our findings provide insights into BACH1 regulation, which plays an important role in erythrophagocytosis and iron recycling in thalassemic macrophages.

Method Verification of the Caretium XC-A30 Automated Erythrocyte Sedimentation Rate Analyser for Erythrocyte Sedimentation Rate.

Background: The erythrocyte sedimentation rate (ESR) analyser is widely used in haematological testing. In addition to the Westergren method, new automatic methods for ESR measurements have been developed. We aimed to study the reliability, precision, accuracy and stability of the Caretium XC-A30 automated ESR analyser. Methods: Ethylenediamine tetraacetic acid (EDTA)-treated blood samples were analysed via the Caretium XC-A30 automated ESR analyser and the Westergren method to compare accuracy. Precision was assessed using control samples and patient samples were classified into three groups-low, medium and high-according to their rates of sedimentation. Moreover, a stability test was performed. Results: The correlation coefficient of the results of the Caretium XC-A30 and Westergren analyses was 0.97. The correlation coefficient of ESR values obtained from the two methods assessed in the low, medium and high groups were r = 0.80, r = 0.68 and r = 0.74, respectively. The coefficient of variation of within-run (%CVw) and between-run (%CVb), with replicates performed with commercial controls samples, were 7.54% and 8.04% for the normal control and 4.68% and 3.50% for abnormal control, respectively. The %CVw obtained with patient samples in the low, medium and high groups were 10.68%, 13.13% and 4.45%, respectively. The Caretium XC-A30 measurements were stable for up to 24 h when samples were stored at 4 °C. Conclusion: The Caretium XC-A30 ESR analyser proved to be a suitable instrument for routine analysis of ESR.

Increased autophagy leads to decreased apoptosis during ß-thalassaemic mouse and patient erythropoiesis.

ß-Thalassaemia results from defects in ß-globin chain production, leading to ineffective erythropoiesis and subsequently to severe anaemia and other complications. Apoptosis and autophagy are the main pathways that regulate the balance between cell survival and cell death in response to diverse cellular stresses. Herein, the death of erythroid lineage cells in the bone marrow from both ßIVS2-654-thalassaemic mice and ß-thalassaemia/HbE patients was investigated. Phosphatidylserine (PS)-bearing basophilic erythroblasts and polychromatophilic erythroblasts were significantly increased in ß-thalassaemia as compared to controls. However, the activation of caspase 8, caspase 9 and caspase 3 was minimal and not different from control in both murine and human thalassaemic erythroblasts. Interestingly, bone marrow erythroblasts from both ß-thalassaemic mice and ß-thalassaemia/HbE patients had significantly increased autophagy as shown by increased autophagosomes and increased co-localization between LC3 and LAMP-1. Inhibition of autophagy by chloroquine caused significantly increased erythroblast apoptosis. We have demonstrated increased autophagy which led to minimal apoptosis in ß-thalassaemic erythroblasts. However, increased PS exposure occurring through other mechanisms in thalassaemic erythroblasts might cause rapid phagocytic removal by macrophages and consequently ineffective erythropoiesis in ß-thalassaemia.

Upregulation of miR-214 Mediates Oxidative Stress in Hb H Disease via Targeting of ATF4.

Thalassemia is a genetic disorder, occurring because of an imbalance in the globin chain production. Oxidative stress in erythroid cells of thalassemia is mainly generated from excess globin chains, by Fenton reaction, leading to hemolysis and ineffective erythropoiesis. Previously, data has shown that microRNAs (miRNAs) are involved in oxidative stress regulation in red blood cells (RBCs). microR-214 has been reported to respond with an external oxidative stress in erythroid cells by modulating activating transcription factor 4 (ATF4). In this study, we illustrated the expressions of miR-214 and ATF4 in Hb H (ß4) disease, and Hb E (HBB: c.79G>A)/ß-thalassemia (ß-thal) reticulocyte samples. Our results showed miR-214 expression was increased in Hb H disease, but not significantly different in Hb E/ß-thal reticulocytes. The ATF4 target was decreased in both thalassemic groups. Moreover, miR-214 expression level positively correlated with the reactive oxygen species (ROS) level, while it was negatively correlated with mean corpuscular volume (MCV), mean corpuscular hemoglobin (Hb) (MCH) and mean corpuscular Hb concentration (MCHC). We suggested that the upregulation of miR-214 correlated with the oxidative stress as well as anemia severity of Hb H disease patients, by suppression of ATF4. Understanding the oxidative pathways in erythrocyte could be useful to manage and relieve the clinical manifestation, such as anemia, in thalassemic patients.

The effects of Phorbol 12-myristate 13-acetate concentration on the expression of miR-155 and miR-125b and their macrophage function-related genes in the U937 cell line.

The phorbol 12-myristate 13-acetate (PMA)-induced U937 cell line has been widely used as an in vitro model for studying the functions of human macrophages. However, there are several concentrations of PMA commonly used to drive the differentiation of monocytic cell line to macrophage. Also, the expression of microRNA-155 (miR-155) and miR-125b in PMA-treated human monocytic cell line has not yet been reported. The five usual concentrations of PMA for stimulating macrophage differentiation are 10, 25, 50, 100, and 200 nM. In this study we compared the expression levels of miR-155, miR-125b and their related genes involved in macrophage functions in U937-derived cells after treatment with those five concentrations. The morphological study results showed that the five concentrations of PMA could induce macrophage differentiation in a similar manner. Moreover, cell proliferation and viability were not significantly different among these five conditions excepted the lower cell viability at 200 nM of PMA treatment. The five concentrations of PMA could upregulate the expression of miR-155 and miR-125b and increase the phagocytic activity of U937-derived cells in dose-reversal manner. The upregulation of miR-155 was correlated with increased expression levels of TNFα and decreased expression levels of BACH1 and CEBPß, while the reduction of IRF4 was correlated with increased expression levels of miR-125b. Our study found that PMA could stimulate macrophage differentiation in a broad range of concentrations, however, the lower concentration could upregulate the higher expression of both miR-155 and miR-125b, and that correlated with the phagocytic functional activity of U937-derived macrophages.

Indole signaling decreases biofilm formation and related virulence of Listeria monocytogenes.

Bacterial communication system known as quorum sensing (QS) is a pivotal system for bacterial survival, adaptation and pathogenesis. Members in the multicellular community may synthesize or acquire a signaling molecule in order to elicit downstream cellular processes. Roles of indole and derivatives, a new class of quorum-sensing signal molecules, in various bacterial physiologies and virulence have been reported recently. Indole is normally found in mammal gastrointestinal tract as a metabolite of tryptophan metabolism by microbiota. Therefore, interspecies connection via indole signaling among commensal bacteria and enteric pathogens could be anticipated. Effects of indole exposure on the virulence of Listeria monocytogenes were investigated by phenotypic and molecular approaches. Results demonstrated that synthetic indole and indole-rich conditioned medium significantly diminished biofilm formation and related virulence of L. monocytogenes including motility, cell aggregation and exopolysaccharide production. Transcript levels of virulence-associated (pssE, dltA, flaA, fliI, motB, agrA and hly) and regulatory genes (codY, sigB, prfA and gmaR) were substantially downregulated in indole-treated cells. Only mogR gene encoding for a repressor of motility genes was upregulated after indole exposure. Our findings raise the possibility that L. monocytogenes may acquire indole signaling from gut microbiota for resource-effective adaptation upon transition to new environment.

Precision and accuracy of the Mindray BC-5000Vet hematology analyzer for canine and feline blood.

BACKGROUND: The Mindray BC-5000Vet hematology analyzer is a flow cytometry-based automated hematology analyzer that generates a complete blood count with a five-part white blood cell (WBC) differential count. OBJECTIVES: We aimed to validate reliability results of the Mindray BC-5000Vet for use in dog and cat blood. METHODS: Imprecision was performed using the manufacturer's quality control material at low, normal, and high levels. Blood sample results of healthy and ill dogs and cats were studied for comparability between manual methods and the Mindray BC-5000Vet analyzer. Forty dogs and 40 cats were included in the study. RESULTS: Precision for red blood cell (RBC) parameters was excellent, with a coefficient of variation within-run (%CVw ) and between-run (%CVb ) of <2%. WBC count and differential count showed %CVw and %CVb <8%; however, %CVw and %CVb of low-level control material gave >9% eosinophils. The correlation between the BC-5000 Vet and manual methods in normal and abnormal canine and feline blood samples showed excellent correlations for the RBC counts, hemoglobin concentrations, hematocrits, and WBC counts (r > .93). The differential WBC analysis of canine blood showed good correlation (r = .80-.92). Feline blood samples showed excellent correlations for neutrophils and lymphocytes, with a good correlation for monocytes and eosinophils. CONCLUSIONS: The Mindray BC-5000Vet hematology analyzer proved a suitable instrument for routine analysis in dogs and cats with various hematologic abnormalities.

Anemia Severity in ß-Thalassemia Correlates with Elevated Levels of microRNA-125b in Activated Phagocytic Monocytes.

ß-Thalassemia (ß-thal), is an inherited blood disorder caused by reduced or absent synthesis of ß-globin chains leading to imbalance of globin chain synthesis. The clearance of ß-thalassemic abnormal red blood cells (RBCs) that result from excessive unbound α-globin is mainly achieved by activated monocytes. The phagocytic activity of ß-thal monocytes significantly increases when co-cultured with normal and ß-thal RBC individuals compare to that of normal monocytes co-cultured with normal RBCs. The present study indicates that microRNA (miR) plays a role in monocyte activation. In this study, we identified the higher miR-125b expression in CD14 marker-positive monocytic cells of ß-thal patients. Moreover, miR-125b expression levels positively correlate with the phagocytic activity of monocytes. Remarkably, miR-125b expression levels are negatively correlated with RBC count, hemoglobin (Hb) and hematocrit [or packed cell volume (PCV)], which are the indices for the severity of anemia. From these findings, our future studies will be to prove the hypothesis that miR-125b expression in activated monocytes may be a genetic modifier related to the severity of anemia in ß-thal patients.

miR-144 regulates oxidative stress tolerance of thalassemic erythroid cell via targeting NRF2.

Thalassemia has a high prevalence in Thailand. Oxidative damage to erythroid cells is known to be one of the major etiologies in thalassemia pathophysiology. Oxidative stress status of thalassemia is potentiated by the heme, nonheme iron, and free iron resulting from imbalanced globin synthesis. In addition, levels of antioxidant proteins are reduced in α-thalassemia and ß-thalassemia erythrocytes. However, the primary molecular mechanism for this phenotype remains unknown. Our study showed a high expression of miR-144 in ß- and α-thalassemia. An increased miR-144 expression leads to decreased expression of nuclear factor erythroid 2-related factor 2 (NRF2) target, especially in α-thalassemia. In α-thalassemia, miR-144 and NRF2 target are associated with glutathione level and anemia severity. To study the effect of miR-144 expression, the gain-loss of miR-144 expression was performed by miR inhibitor and mimic transfection in the erythroblastic cell line. This study reveals that miR-144 expression was upregulated, whereas NRF2 expression and glutathione levels were decreased in comparison with the untreated condition after miR mimic transfection, while the reduction of miR-144 expression contributed to the increased NRF2 expression and glutathione level compared with the untreated condition after miR inhibitor transfection. Moreover, miR-144 overexpression leads to significantly increased sensitivity to oxidative stress at indicated concentrations of hydrogen peroxide (H2O2) and rescued by miR-144 inhibitor. Taken together, our findings suggest that dysregulation of miR-144 may play a role in the reduced ability of erythrocyte to deal with oxidative stress and increased RBC hemolysis susceptibility especially in thalassemia.

MiR-155 enhances phagocytic activity of ß-thalassemia/HbE monocytes via targeting of BACH1.

Abnormal red blood cell (RBC) clearance in ß-thalassemia is triggered by activated monocytes. Recent reports indicate that miRNA (miR-) plays a role in monocyte activation. To study phagocytic function, we co-cultured monocytes of normal, non-splenectomized and splenectomized ß-thalassemia/HbE individuals with RBCs obtained from normal, non-splenectomized and splenectomized ß-thalassemia/HbE individuals. The phagocytic activity of ß-thalassemia/HbE monocytes co-cultured with ß-thalassemia/HbE RBCs was significantly higher than that of normal monocytes co-cultured with normal RBCs. Upregulation of monocyte miR-155 was observed in ß-thalassemia/HbE patients. Increased miR-155 was associated with reductions in BTB and CNC Homology1 (BACH1) target gene expression and increased phagocytic activity of ß-thalassemia/HbE monocytes. Taken together, these findings suggested that increased miR-155 expression in activated monocytes leads to enhanced phagocytic activity via BACH-1 regulation in ß-thalassemia/HbE. This provides novel insights into the phagocytic clearance of abnormal RBCs in ß-thalassemia/HbE.

Imbalanced globin chain synthesis determines erythroid cell pathology in thalassemic mice.

BACKGROUND: beta-thalassemia occurs from the imbalanced globin chain synthesis due to the absence or inadequate beta-globin chain production. The excessive unbound alpha-globin chains precipitate in erythroid precursors and mature red blood cells leading to ineffective erythropoiesis and hemolysis. DESIGN AND METHODS: In vitro globin chain synthesis in reticulocytes from different types of thalassemic mice was performed. The effect of imbalanced globin chain synthesis was assessed from changes of red blood cell properties including increased numbers of red blood cells vesicles and apoptotic red blood cells, increased reactive oxygen species and decreased red blood cell survival. RESULTS: The alpha/beta-globin chain ratio in beta(IVSII-654)-thalassemic mice, 1.26+/-0.03, was significantly higher than that of wild type mice, 0.96+/-0.05. The thalassemic mice show abnormal hematologic data and defective red blood cell properties. These values were improved significantly in doubly heterozygous thalassemic mice harboring 4 copies of human beta(E)-globin transgene, with a more balanced globin chain synthesis, 0.92+/-0.05. Moreover, transgenic mice harboring 8 extra copies of the human beta(E)-globin transgene showed inversely imbalanced alpha/beta-globin synthesis ratio, 0.83+/-0.01, that resulted in a mild beta-thalassemia phenotype due to the excessive beta-globin chains. The degree of ineffective erythropoiesis also correlated with the degree of imbalanced globin chain synthesis. Bone marrow and splenic erythroid precursor cells of beta(IVSII-654)-thalassemic mice showed increased phosphatidylserine exposure in basophilic and polychromatophilic stages, which was restored to the normal level in doubly heterozygous mice. CONCLUSIONS: Imbalanced alpha/beta-globin chain as a consequence of either reduction or enhancement of beta-globin chain synthesis can cause abnormal red blood cell properties in mouse models.