methylcellulose and Anemia--Sickle-Cell

We have examined the possibility that permanent "stress" hemopoiesis in sickle cell anemia (SS) patients leads to the modification of the behavior of circulating 14 day erythroid progenitor cells (BFU-E). In these patients we find that peripheral blood BFU-E are increased in number and have high sensitivity to erythropoietin (Epo). Maximal number of BFU-E are generated from peripheral blood of SS patients at 0.3-0.75 Epo/ml of culture compared to 1.5-2.0 U Epo/ml of culture in normals. Peripheral blood adherent cells depletion leads to the shift of Epo dose response curve, so that the Epo sensitivity of BFU-E significantly decreases. This result suggests that apparent Epo hypersensitivity reflects, in fact, an increased production of a burst promoting activity (BPA) by SS peripheral blood light density adherent (PB-LDA) cells. Experiments with conditioned medium by SS PB-LDA cells confirmed this interpretation. When peripheral blood light density non-adherent (PB-LDNA) cells of SS patients or normal individuals were plated in the presence of various concentrations of SS PB-LD cells conditioned medium and constant amounts of Epo, a dose dependent increase of the number of BFU-E was observed. When the same target cells were plated in the presence of PB-LD cells conditioned medium from normal individuals, such effect does not occur. We conclude that increased BPA production may play a role in the erythropoietic regulation during constant hemopoietic stress in sickle cell anemia and might partially explain the lower than expected Epo levels in these patients.

Topics: Anemia, Sickle Cell; Cells, Cultured; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; Methylcellulose

Topics: Amniocentesis; Anemia, Sickle Cell; Blood Protein Electrophoresis; Carbon Isotopes; Chromatography; Electrophoresis; Female; Fetal Diseases; Fetus; Gestational Age; Globins; Hemoglobins, Abnormal; Humans; Leucine; Methods; Methylcellulose; Placenta; Pregnancy; Tritium; Umbilical Cord

Human hemoglobin messenger RNA was isolated by sucrose gradient centrifugation from reticulocytes of patients having various hemolytic anemias. Using a messenger RNA-dependent cell-free system derived entirely from rabbit reticulocytes, the human hemoglobin messenger RNA has been translated and the products analyzed by carboxymethylcellulose column chromatography. Normal messenger RNA directs synthesis of normal human alpha- and beta-globin chains in nearly equal amounts. Sickle cell anemia messenger RNA directs the synthesis of normal alpha- and sickle beta-chains, beta-thalassemia messenger RNA directs the synthesis of normal alpha- and beta-chains, but the amount of beta-globin synthesized is markedly reduced. Thus the inability of the thalassemia reticulocyte to produce beta-globin is clearly attributable to the beta-globin messenger RNA.

Topics: Anemia, Sickle Cell; Animals; Carbon Isotopes; Cell-Free System; Centrifugation, Density Gradient; Chromatography; Globins; Hemoglobins; Hemoglobins, Abnormal; Humans; Leucine; Methylcellulose; Peptide Biosynthesis; Rabbits; Reticulocytes; Ribosomes; RNA, Messenger; Thalassemia

Human ribosomes obtained from the reticulocytes of patients having either homozygous beta-thalassemia (thalassemia ribosomes) or a hematological disorder unrelated to thalassemia ("normal" ribosomes) have been utilized in a cell-free system highly active in the synthesis of intact human globin chains. This system is dependent on the addition of a ribosomal wash fraction from reticulocytes that contains factors necessary for chain initiation. In response to the ribosomal wash fraction, isolated from either thalassemia, normal human, or rabbit reticulocytes, normal human ribosomes synthesize equal amounts of alpha and beta chains. In contrast, in response to all three types of ribosomal wash fractions, thalassemia ribosomes synthesize 8-times more alpha than beta chains, a ratio similar to that produced in the intact cells of these patients. The molecular defect in beta-thalassemia, therefore, does not appear to be associated with initiation factors.

Topics: Anemia, Hemolytic, Autoimmune; Anemia, Sickle Cell; Animals; Carbon Isotopes; Cell-Free System; Chromatography; Hemoglobins; Humans; In Vitro Techniques; Leucine; Lysine; Methylcellulose; Rabbits; Reticulocytes; Ribosomes; Thalassemia; Tritium