EPO receptor, Bax and Bcl-x(L) expressions in murine erythropoiesis after cyclophosphamide treatment.

The effect of a single dose of cyclophosphamide (CY, 150 mg/kg i.p.) on the erythropoiesis using an "in vivo" murine model in a time course protocol (0-10 days) was studied through several experimental approaches. Total and differential bone marrow cellularities, apoptosis (TUNEL assays), bone marrow hematopoietic architecture (scanning electronic microscopy), proliferation (DNA assay), BM erythroid progenitors growth (semisolid clonogenic assays) and protein expressions for erythroid commitment and survival: erythropoietin receptor (EPO-R), Bcl-x(L), Bax (immunoblottings) were performed on the scheduled days. Most of the experiences were conducted comparing spontaneous with human recombinant (hr EPO) "ex vivo" stimulated bone marrow (BM) cells. Erythropoiesis was extremely affected by CY. Maximum apoptosis, minimal cellularities and severe disturbances of BM niche were noticed on the second day. During spontaneous recovery post-CY; EPO-R was expressed between 4 and 5 days. Following BM cells "ex vivo" hr EPO stimulation (2U/ml) EPO-R was expressed throughout the study except the period between the first and fourth day. Bax was noticeable all along the experience with and without hr EPO stimulation. Bcl-x(L) was barely detectable without hr EPO, but its expression showed a gradual enhancement from the fifth day onwards in hr EPO stimulated cells. This fact might be related to the end of the erythroid inhibitory stage and to the recovery of BM EPO-dependent proliferation between the fourth and fifth day, and the further recuperation of BFU-E and CFU-E colonies on days 6 and 7 post-CY, respectively. These findings suggest that the proliferation and differentiation of erythroid progenitor cells after the acute early injury inflicted by CY, is associated with changes in EPO-R expression during spontaneous recovery in this particular experimental system.

Human erythroid cells are affected by aluminium. Alteration of membrane band 3 protein.

There is evidence that anaemia is associated with aluminium (Al). We have already reported on the sensitivity to Al, showed by erythroid cell populations of animals chronically exposed to the metal. In order to investigate whether Al could also affect human cells, experiments were carried out both on immature and mature human erythroid cells. Erythroid progenitors (CFU-E, colony-forming units-erythroid) concentrated from human peripheral blood were cultured in an Al-rich medium under erythropoietin stimulation and their development analysed. Human peripheral erythrocytes were aged in the presence of Al. Cells were examined using scanning electron microscopy, and membrane proteins analysed by polyacrylamide gel electrophoresis with sodium dodecyl sulphate and immunoblotting. The development of the Al-treated progenitors was 8750/6600-9200 CFU-E/10(6) cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12300/11200-20700 CFU-E/10(6) cells). Erythrocyte morphological changes were induced by Al during the in vitro ageing. The cells lost their typical biconcave shape, turning into acanthocytes and stomatocytes. Simultaneously, an increased membrane protein breakdown compatible with band 3 degradation was detected. Besides, Al was found within the cells and attached to the membrane. The present in vitro results suggest that Al may disturb human erythropoiesis through combined effects on mature erythrocytes and cellular metabolism in late erythroid progenitors.

Neonatal manifestations of congenital dyserythropoietic anemia type I.

Congenital dyserythropoietic anemia type I is a rare inherited bone marrow disorder characterised by macrocytic anemia with pathognomonic morphological ultrastructural features in erythroid precursors. The disease is usually not diagnosed in the neonatal period. In a retrospective study of 31 patients we found that 17 were first seen in the neonatal age with significant anemia (birth hematocrit 0.34 +/- 0.07); 14 of the 17 infants also had early jaundice. Six infants were small for gestational age and two had syndactyly. Although rare, congenital dyserythropoietic anemia type I should be considered in the differential diagnosis of neonatal anemia.

Comparative study of immature erythroid cells of the diploid Bufo ictericus and the tetraploid Odontophrynus americanus (Amphibia, Anura): ultrastructural cytochemical detection of nucleic acids and polysaccharides, and mapping of the element phosphorus.

In the present study, we used ultrastructural cytochemistry to analyze the distribution of nuclear and cytoplasmic nucleic acids and polysaccharides, and electron spectroscopic imaging to map the element phosphorus in immature erythroid cells taken from two amphibians, the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. In the cytoplasm of cells from the tetraploid species, we detected numerous inclusions containing a material that was similar to the dispersed chromatin seen in the nucleus of these cells. The RNase-gold complex labeled both the dispersed nuclear chromatin and the cytoplasmic inclusions. The Thiéry technique showed that glycoconjugates were present in all the membranous complexes of the erythroid cells of both types of amphibians under study, although they were absent within or around the cytoplasmic RNA inclusions. Electron spectroscopic imaging revealed the presence of phosphorus in these inclusions. These data suggest that an increase in RNA synthesis occurs in tetraploid amphibian cells, probably as a result of an alteration in the mechanisms of gene regulation.