IOX1 Fails to Reduce α-Globin and Mediates γ-Globin Silencing in Adult ß0-Thalassemia/Hemoglobin E Erythroid Progenitor Cells.

The accumulation of unbound α-globin chains in red blood cells is a crucial pathophysiology of ß-thalassemia. IOX1 (5-carboxy-8-hydroxyquinoline) is a broad-spectrum 2-oxoglutarate (2OG)-dependent oxygenase inhibitor that can reduce α-globin mRNA expression in human cord blood erythroid progenitor cells. Therefore, IOX1 has been proposed as a potential compound for ß-thalassemia treatment through the decrease in α-globin chain synthesis. However, there is no empirical evidence regarding the consequences of IOX1 in ß-thalassemia. In this study, the therapeutic effects of IOX1 were investigated in ß0-thalassemia/hemoglobin E (HbE) erythroid progenitor cells during in vitro erythropoiesis. The results indicated that IOX1 had no impact on α-globin gene expression, but it led instead to significant decreases in γ-globin and fetal hemoglobin (HbF, α2γ2) production without affecting well-known globin regulators: KLF1, BCL11A, LRF, and GATA1. In addition, differential mRNA expression of several genes in the hypoxia response pathway revealed the induction of EGLN1, the PHD2-encoding gene, as a result of IOX1 treatment. These findings suggested that IOX1 fails to lower α-globin gene expression; on the contrary, it mediates γ-globin and HbF silencing in ß0-thalassemia/HbE erythroid progenitor cells. Because of the negative correlation of EGLN1 and γ-globin gene expression after IOX1 treatment, repurposing IOX1 to study the hypoxia response pathway and γ-globin regulation may provide beneficial information for ß-thalassemia.

Down-regulation of the transcriptional repressor ZNF802 (JAZF1) reactivates fetal hemoglobin in ß0-thalassemia/HbE.

Reactivating of fetal hemoglobin (HbF; α2γ2) can ameliorate the severity of ß-thalassemia disease by compensating for adult hemoglobin deficiency in patients. Previously, microarray analysis revealed that zinc finger protein (ZNF)802 (also known as Juxta-posed with another zinc finger gene-1 (JAZF1)) was upregulated in human erythroblasts derived from adult peripheral blood compared with fetal liver-derived cells, implying a potential role as a HbF repressor. However, deficiency in ZNF802 induced by lentiviral shRNA in ß0-thalassemia/hemoglobinE erythroblasts had no effect on erythroblast proliferation and differentiation. Remarkably, the induction of HBG expression was observed at the transcriptional and translational levels resulting in an increase of HbF to 35.0 ± 3.5%. Interestingly, the embryonic globin transcripts were also upregulated but the translation of embryonic globin was not detected. These results suggest ZNF802 might be a transcriptional repressor of the γ-globin gene in adult erythroid cells.

In vitro production of functional immune cells derived from human hematopoietic stem cells.

Hematopoietic stem cells (HSC) from cord blood are potentially high sources for transplantation due to their low immunogenicity and the presence of the multipotent cells. These cells are capable of differentiating to produce various lineages of blood cells under specific conditions. We have enriched highly purified CD34(+) cells from cord blood, determined in vitro growth of the cells in culture systems in the absence (condition A) or presence of GM-CSF and G-CSF (condition B), and determined the profile of immune cells during the period of cultivation by using flow cytometry. PhytohemagglutininA (PHA) was used as a mitogen to stimulate T lymphocytes derived from hematopoietic stem cells. GM-CSF and G-CSF prolonged the survival of the growing cells and also maintained expansion of cells in blastic stage. By day 12 of cultivation, when cell numbers peaked, various types of immune cells had appeared (CD14(+) cells, CD40(+)HLA-DR(+) cells, CD3(+)CD56(+) cells, CD19(+) cells, CD3(+)CD4(+) cells, CD3(+)CD8(+)cells and CD3-CD56(+)). A significantly higher percentage of monocytes (p = 0.002) were observed under culture with GM-CSF, G-CSF when compared with culture without GM-CSF, G-CSF. In addition, T lymphocytes derived from HSC responded to 50 µg/ml of PHA. This is the first report showing the complete differentiation and proliferation of immune cells derived from CD34(+) HSC under in vitro culture conditions. Lymphocytes, monocytes, dendritic cells and polymorph nuclear cells derived from HSC in vitro are unique, and thus may benefit various studies such as innate immunity and pathophysiology of immune disorders.

Protection against oxidative stress in beta thalassemia/hemoglobin E erythrocytes by inhibitors of glutathione efflux transporters.

In beta thalassemia/hemoglobin E (Hb E), abnormally high levels of oxidative stress account for accelerated senescence and increased destruction of erythrocytes. The present study aimed to investigate the role of glutathione efflux transporters, namely cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein 1 (MRP1), in the control of glutathione levels and protection against oxidative challenges in beta thalassemia/Hb E erythrocytes. We found that CFTR protein was expressed in the erythrocytes of beta thalassemia/Hb E patients. Treatments with GlyH-101 (50 µM), a small molecule CFTR inhibitor, and MK571 (50 µM), an MRP1 inhibitor, reduced H(2)O(2)-induced free radical generation in the erythrocytes by ∼80% and 50%, respectively. Furthermore, combined treatment with GlyH-101 and MK571 completely abolished the induction of reactive oxygen radicals. Increased oxidative stress in the erythrocytes following H(2)O(2) challenges was accompanied by a decrease in intracellular level of reduced glutathione (GSH), which was prevented by treatments with GlyH-101 and MK571. CMFDA-based assays revealed that GlyH-101 and MK571 reduced H(2)O(2)-induced glutathione efflux from the erythrocytes by 87% and 66%, respectively. Interestingly, H(2)O(2)-induced osmotic tolerance of erythrocytes, a sign of erythrocyte aging, was ameliorated by treatment with GlyH-101. Our study indicates that oxidative stress induces glutathione efflux via CFTR and MRP1 in beta thalassemia/Hb E erythrocytes. Pharmacological inhibition of glutathione efflux represents a potential therapy to delay aging and premature destruction of erythrocytes in beta thalassemia/Hb E.

Suppression of erythroid development in vitro by Plasmodium vivax.

BACKGROUND: Severe anaemia due to dyserythropoiesis has been documented in patients infected with Plasmodium vivax, however the mechanism responsible for anaemia in vivax malaria is poorly understood. In order to better understand the role of P. vivax infection in anaemia the inhibition of erythropoiesis using haematopoietic stem cells was investigated. METHODS: Haematopoietic stem cells/CD34+ cells, isolated from normal human cord blood were used to generate growing erythroid cells. Exposure of CD34+ cells and growing erythroid cells to P. vivax parasites either from intact or lysed infected erythrocytes (IE) was examined for the effect on inhibition of cell development compared with untreated controls. RESULTS: Both lysed and intact infected erythrocytes significantly inhibited erythroid growth. The reduction of erythroid growth did not differ significantly between exposure to intact and lysed IE and the mean growth relative to unexposed controls was 59.4 ± 5.2 for lysed IE and 57 ± 8.5% for intact IE. Interestingly, CD34+ cells/erythroid progenitor cells were susceptible to the inhibitory effect of P. vivax on cell expansion. Exposure to P. vivax also inhibited erythroid development, as determined by the reduced expression of glycophorin A (28.1%) and CD 71 (43.9%). Moreover, vivax parasites perturbed the division of erythroid cells, as measured by the Cytokinesis Block Proliferation Index, which was reduced to 1.35 ± 0.05 (P-value<0.01) from a value of 2.08 ± 0.07 in controls. Neither TNF-a nor IFN-g was detected in the culture medium of erythroid cells treated with P. vivax, indicating that impaired erythropoiesis was independent of these cytokines. CONCLUSIONS: This study shows for the first time that P. vivax parasites inhibit erythroid development leading to ineffective erythropoiesis and highlights the potential of P. vivax to cause severe anaemia.

Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/ß-thalassemia.

BACKGROUND: Hemoglobin E/ß-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of ß-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear. METHODS: The phosphoproteome of bone marrow HSCs/CD34⁺ cells from HbE/ß-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34⁺ cells were compared with HbE/ß-thalassemia and normal HSCs. RESULTS: A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/ß-thalassemia. CONCLUSIONS: Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/ß-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in ß-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/ß-thalassemia.

Use of in vivo gene expression of isolated bone marrow mesenchymal stromal cells to study the pathophysiology of osteoporosis in patients with severe thalassemia.

We compared osteoblast differentiation gene expressions in the isolated CD105 mesenchymal stromal cells from bone marrow in 10 patients with severe thalassemia and 12 normal donor controls. The fold expressions of osteoblast differentiation genes of CD105 cells from patients with thalassemia were lower than those of normal donors but increased after being cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Moreover, the fold expressions of these genes of CD105 cells from normal donors when cultured with 10% pooled serum of patients with thalassemia were lower than when cultured with 10% pooled serum of normal donors. We have also presented the evidence of reversible suppressed expression of these genes in CD105cells from patients with thalassemia when cultured in pooled serum of normal donors. Moreover, healthy donor CD105 cells exhibited lower expression of these genes when cultured in pooled serum of patients with thalassemia compared with pooled serum of normal donors indicating the existence of circulating factors in thalassemic serum impairing the differentiation of mesenchymal stromal cells to osteoblasts.