Subject(s)
Humans , Fatty Acids/pharmacology , Globins/genetics , Phenylbutyrates/pharmacology , Cells, Cultured , Erythroid Precursor Cells/drug effects , Erythroid Precursor Cells/metabolism , Fatty Acids/chemistry , Fetal Hemoglobin/biosynthesis , Hemoglobinopathies/blood , Hemoglobinopathies/drug therapy , Hemoglobinopathies/genetics , Leukemia, Erythroblastic, Acute , Phenylacetates/pharmacology , Phenylbutyrates/chemistry , Promoter Regions, Genetic/drug effects , Structure-Activity Relationship , Tumor Cells, Cultured , Up-Regulation/drug effectsABSTRACT
BACKGROUND: Hydroxyurea, which induces Fetal hemoglobin (HbF) synthesis, is the only drug widely used in different hemoglobinopathies; however, the response is very variable. We compared the efficacy of hydroxyurea in-vitro in erythroid cultures and in-vivo in the same patients with different hemoglobinopathies to induce HbF production and enhance γ-messenger RNA expression. MATERIALS AND METHODS: A total of 24-patients with different Hemoglobinopathies were given hydroxyurea and their response was studied in-vivo and in-vitro on mononuclear cells collected from them simultaneously. RESULTS: A total of 57.7% of patients (responders) showed no further crisis or transfusion requirements after hydroxyurea therapy with a mean increase in fetal cells (F-cells) of 63.8 ± 59.1% and γ-mRNA expression of 205.5 ± 120.8%. In-vitro results also showed a mean increase in F-cells of 27.2 ± 24.7% and γ-mRNA expression of 119.6% ± 65.4% among the treated cells. Nearly 19.0% of the partial-responders reduced their transfusion requirements by 50% with a mean increase in F-cells of 61.2 ± 25.0% and 28.4 ± 25.3% and γ-mRNA-expression of 21.0% ± 1.4% and 80.0% ± 14.1% in-vivo and in-vitro respectively. The non-responders (15.3%) showed no change in their clinical status and there was no significant increase in F-cells levels and γ-mRNA expression in-vivo or in-vitro. CONCLUSION: Thus, this method may help to predict the in-vivo response to hydroxyurea therapy; however, a much larger study is required.
Subject(s)
Anemia, Sickle Cell/genetics , Adolescent , Adult , Cells, Cultured , Child , Child, Preschool , Erythroid Precursor Cells/metabolism , Female , Fetal Hemoglobin/analysis , Fetal Hemoglobin/biosynthesis , Fetal Hemoglobin/genetics , Hemoglobinopathies , Hydroxyurea/pharmacology , Humans , India , Male , Middle Aged , RNA, Messenger/genetics , beta-Thalassemia/epidemiology , beta-Thalassemia/genetics , Young AdultABSTRACT
In vitro suspension culture procedures for erythroid progenitor cells make it possible for us to obtain large cultures of erythrocyte populations for the investigation of globin gene switching. In this study we aimed to establish optimized culture systems for neonatal and adult erythroblasts and to explore the globin expression patterns in these culture systems. To culture CD34+ cells purified from human umbilical cord blood (CB) and adult bone marrow (BM), we respectively replaced the fetal bovine serum (FBS) with human cord serum and human adult serum. These CD34+ cells were then induced to erythroid differentiation. All the globin mRNA (including alfa-, xi-, vita-, gama-and epsilón-globin), the hemoglobin (Hb)-producing erythroid cells and the cellular distribution of fetal hemoglobin (Hb F) were identified during the culture process. The results showed that the globin expression pattern during erythroid differentiation in our culture systems closely recapitulated neonatal and adult patterns of globin expression in vivo, suggesting that our specially optimized culture systems not only overcame the higher Hb F levels in the BM-derived CD34+ culture in FBS-containing medium but also eliminated the disadvantages of low cell proliferation rate and low globin mRNA levels in serum-free medium.
Subject(s)
Humans , Adult , Bone Marrow Cells/cytology , Erythroid Precursor Cells/cytology , Fetal Blood/cytology , Gene Expression Regulation, Developmental , Globins/genetics , Cell Culture Techniques , DNA Primers , Erythroid Precursor Cells/metabolism , Flow Cytometry , Globins/metabolism , Microscopy, Fluorescence , Polymerase Chain Reaction , Staining and LabelingABSTRACT
In order to better understand the mechanisms affecting erythropoietin (Epo) synthesis and red cell mass increase under chronic hypoxia, we examined Epo production and erythroid progenitors (CFU-E) in rats exposed to normobaric hypoxia for four weeks. Hypoxia induced the rise of hematocrit (Htc), hemoglobin (Hb) concentration and the red blood cell (RBC) number with a plateau in hematocrit values after two weeks. After 24 h of hypoxia, Epo levels were increased 20 fold, followed by a significant decrease. After the first week of hypoxia, the values were still higher than in the controls, but after two weeks Epo levels did not differ significantly from the normal values. The fall of Epo levels coincided with the plateau values of hematocrit. The changes in the CFU-E number followed the changes in Epo concentration: a two fold increase after 24 h of hypoxia; a further increase during the next two weeks reaching a peak on day 14, and then a progressive decrease at the time when Epo concentration was at a normal level. Although decreased, but still higher than normal, the CFU-E number during the last two weeks of hypoxia could be necessary for the maintenance of an achieved steady state under persistent hypoxic conditions with normal Epo concentration sufficient to maintain the existing rate of erythropoiesis.