Hereditary erythroblastic anaemia in the laboratory mouse

Hereditary erythroblastic anaemia was found in a homozygous mutant (hea/hea) of an inbred strain CFO, which originated from noninbred CF#1 mice from Carworth Inc. This newly-described anaemia is inherited as an autosomal recessive and is lethal at 15-25 days of age. Erythrocytes of anaemic mice show striking polychromasia, anisocytosis, and poikilocytosis. One of the most marked features of this anaemia is the presence of large numbers of nucleated cells, mainly orthochromatophilic erythroblasts and myeloid cells, in the circulation. They also include immature erythroid and myeloid cells. Many naked nuclei appear on smears from circulating blood of anaemic infants. Erythrocytes, haematocrit percentage, and haemoglobin content of older anaemic infants were only about 50% of those of the normal. Formation of erythroid, myeloid cells, and megakaryocytes in the bone marrow seems to be progressively affected by mutant alleles in the anaemic infants.     

Changes in surface-membrane components during the differentation of rabbit erythroid cells.

The membrane components of rabbit bone-marrow-bound erythroid cells were characterized and compared with those of circulating rabbit erythroid cells. By the criteria of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, radioiodination with lactoperoxidase and binding of radioiodinated lectins, the two circulating forms of erythroid cells (the reticulocyte and erythrocyte) have the same surface components. In contrast, bone-marrow-bound nucleated erythroid cells have a unique set of membrane surface components which are completely different from those found on circulating cells. Of the ten Coomassie-Blue-staining proteins present in nucleated erythroid-cell plasma-membrane preparations, eight are accessible at the extracellular surface, and all of these are lectin-binding glycoproteins. Bone-marrow erythroid cells separated according to age by velocity sedimentation were also studied. The changeover in surface components occurs after the last nucleated stage of the erythroid cells (the orthochromatic normoblast). We discuss the alterations in membrane surface components observed during the differentiation of the erythroid-cell series in relation to the transition from bone-marrow-bound to circulating forms of these cells. We suggest that the change in membrane surface components may be linked to the loss of the nucleus from the normoblast and the entry of the erythroid cell into the circulation.     

Structures and functional roles of the sugar chains of human erythropoietins.

Erythropoietin (EPO) is a haemopoietic hormone specific to cells of erythroid lineage. EPO has recently become available for the treatment of anaemia as the first human recombinant biomedicine produced in heterologous mammalian cells. Human EPO is characterized by its large carbohydrate chains, which occupy close to 40% of its total mass. These sugar moieties were thought to be important for the biological activity of EPO, but detailed studies were not performed until the structures were elucidated. The variety of roles for the sugar chains were then immediately found once the structures were known. EPO is an excellent model for investigating the roles of sugar chains on glycoproteins, since its gene and its multiple glycoforms are available, as well as sensitive bioassays for testing. In this review, we will first summarize the known sugar chain structures of EPO from different host cells, and then discuss the host-cell dependent and peptide structure-dependent glycosylation of glycoproteins. We will then address how one investigates the roles of sugar chains of glycoproteins, show several examples of such investigations, and discuss the functional roles of HuEPO's sugar chains in its biosynthesis and secretion, its in vitro and in vivo biological activities, and its half-life in blood circulation.     

Differentiation studies on separated rabbit early erythroid cells

Haemoglobin-containing cells were removed from cell suspensions of adult rabbit bone marrow by immune lysis, and the remaining cells were layered into BSA density gradients. The top fractions contained early erythroid cells, while fractions near the bottom of the gradient contained granulocytes. Two populations of erythroid cells from anaemic rabbits were resolved by the gradient which differed in their time of maximum stimulation of haem synthesis, in culture with erythropoietin. In addition, a difference in requirement for the presence of erythropoietin in the culture medium was found in separated erythroid cells from rabbits with varying degrees of anaemia.     

On the kinetics of erythroid cell differentiation in fetal mice. I. Microspectrophotometric determination of the hemoglobin content in erythroid cells during gestation

In a microspectrophotometric study, photographic emulsions and a computer are used for measuring the hemoglobin content of a large number (about 50,000) of erythroid cells in fetal mice. Histograms of the hemoglobin content in erythroid cells illustrate the kinetics of erythropoiesis in yolk sac derived nucleated cells in the fetal peripheral blood, in fetal liver, and in fetal spleen. After the occasional extrusion of their nucleus, yolk sac derived erythrocytes remain as “macrocytes” in fetal circulation two or three days longer than the nucleated yolk sac derived erythrocytes do. Erythrocytes in fetal liver have a constant hemoglobin content of 28 pg ² until day 17 of gestation. During further erythropoiesis in liver and then in the spleen, this amount is gradually adapted to the normal hemoglobin content in red blood cells of 16 pg.     

Blood and Bone Marrow Studies in Cattle Feeding on Brassica Species

Daily administration of 40—60 kg rape for 9 weeks to 8 cows produced no changes in the red blood picture. On the other hand a reduction of the myeloid: erythroid ratio and the maturity ratio of the erythroid cells of the bone marrow was found. This is interpreted as a sign of increased erythropoiesis. The Brassica-induced anaemia found in other investigations is assumed on several grounds to be haemolytic. The present study confirms this assumption, the increased breakdown of erythrocytes being entirely compensated by an increased erythropoiesis. The difference between the present and earlier reports, in which anaemia was found after a shorter time of feeding on roughly the same quantities of rape, is discussed, one possible explanation being that differences exist in rhodanid content between Brassica species.     

Synchrony of bone marrow proliferation and maturation as the origin of cyclic haemopoiesis.

Cyclic haemopoiesis in Grey Collie dogs is characterized by stable oscillations in all haemopoietic lineages. It is proposed that in these animals, in contrast to normal animals, the maturation process of haemopoietic (in particular granuloid) cells from the primitive progenitors to the functional cells is characterized by an abnormally strong synchrony. It is conjectured that the marrow maturation time has a very small variance compared with non-cyclic normal dogs. With a mathematical model of haemopoiesis it is shown that small fluctuations are amplified via regular feedback processes such that stable granuloid oscillations are established. Erythroid oscillations are induced indirectly by granuloid feedback to the stem cell pool. The model calculations further show that the synchrony hypothesis of bone marrow maturation can quantitatively explain the following experimental results: (1) the maintenance of stable cycles of granuloid and erythroid bone marrow and blood cells with a period of approximately 14 d; (2) the disappearance of granuloid and erythroid cycles during the administration of the colony stimulating factor rhG-CSF; (3) the reappearance of oscillations when the administration of CSF is discontinued; (4) the cessation of cycles during endotoxin application; and (5) the persistence of cycles during erythroid manipulations (bleeding anaemia, hypoxia, hypertransfusion). We therefore conclude that cyclic haemopoiesis is not caused by a defect in the regulatory control system but by an unusual maturation process.     

Mathematical study of feedback control roles and relevance in stress erythropoiesis

This work is devoted to mathematical modelling of erythropoiesis. We propose a new multi-scale model, in which we bring together erythroid progenitor dynamics and intracellular regulatory network that determines erythroid cell fate. All erythroid progenitors are divided into several sub-populations according to their maturity. Two intracellular proteins, Erk and Fas, are supposed to be determinant for regulation of self-renewal, differentiation and apoptosis. We consider two growth factors, erythropoietin and glucocorticoids, and describe their dynamics. Several feedback controls are introduced in the model. We carry out computer simulations of anaemia and compare the obtained results with available experimental data on induced anaemia in mice. The main objective of this work is to evaluate the roles of the feedback controls in order to provide more insights into the regulation of erythropoiesis. Feedback by Epo on apoptosis is shown to be determinant in the early stages of the response to anaemia, whereas regulation through intracellular regulatory network, based on Erk and Fas, appears to operate on a long-term scale.     

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.     

Mitochondrial kinetics during amphibian erythropoiesis related to haeme synthesis

Flow cytometry, light and epifluorescence microscopies and transmission electron microscopy were used to follow the mitochondrial kinetics during amphibian erythropoiesis. A similar behaviour in response to the induction of anaemia was observed in the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. A high cellular activity was observed ten days after haemolytic anaemia induced by phenylhydrazine, based on the higher Rhodamine 123 uptake by the erythroid cells. In addition, the more intense expression of the mitochondrial enzyme cytochrome oxidase, isocitrate and succinic dehydrogenases were cytochemically detected at this stage. This suggests that erythroid cell mitochondria, at this time, could be in a more active functional state than at other stages. In both species, mitochondrial plasticity was observed during cell maturation. A progressive loss of oxidation-reduction enzyme expression seemed to follow changes at the mitochondrial cristae morphology, from transverse to longitudinal form, mainly at the 20th day of recovery from anaemia, possibly related to a natural loss of function. The presence of these mitochondrial enzymes in mitochondrion-like organelles also favours their participation in the haeme synthesis, although with a reduced expression, since this suggests the presence of a complete and active enzymatic complex in these modified organelles. This also supports the idea that all these organelles are mitochondria in distinct metabolic stages, and not mitochondrion-like organelles or haemosomes, as proposed by some authors.     

Red blood cells

Red blood cells are derived from haemopoietic stem cells in bone marrow. Following a series of maturation steps, directed largely by the hormone erythropoietin (Epo), red cells enucleate and enter the circulatory system. In circulation these small, flexible biconcave cells containing haemoglobin transport O₂ from the lungs to the periphery, and CO₂ back from the periphery to the lungs. The most common disorders associated with red blood cells are anaemias. While there are numerous causes of anaemia, the reduced capacity for gaseous exchange is the underlying theme. Over the past 15 years, recombinant Epo has been used extremely successfully in the treatment of several forms of anaemia. The single gene disorders collectively known as haemoglobinopathies represent one of the best opportunities for gene therapy.     

Malarial anaemia: mechanisms and implications of insufficient erythropoiesis during blood-stage malaria

It has been proposed that the basis of severe malarial anaemia, a major cause of morbidity and mortality in endemic areas, is multifactorial. Inappropriately low reticulocytosis is observed in malaria patients suggesting that insufficient erythropoiesis is a major factor. Clinical studies provide conflicting data concerning the production of adequate levels of erythropoietin (EPO) during malaria. Plasmodium chabaudi AS causes non-lethal infection in resistant C57BL/6 mice, and lethal infection in susceptible A/J mice. In P. chabaudi AS infected C57BL/6 and A/J mice, which experience varying degrees of severity of anaemia, kidney EPO production is appropriate to the severity of anaemia and is regulated by haematocrit level. Neutralisation of endogenous EPO during infection leads to lethal anaemia while timely administration of exogenous EPO rescues mice although reticulocytosis is suppressed in proportion to the parasitemia level. Characterisation of alterations in splenic erythroid compartments in naive and P. chabaudi AS infected A/J mice revealed that infection, with or without EPO treatment, leads to sub-optimal increases in TER119+ erythroblasts compared to EPO-treated naive mice. A lower percentage of TER119+ erythroblasts in infected mice undergo terminal differentiation to become mature haemoglobin-producing cells. Furthermore, there is a shift in transferrin receptor (CD71) expression from TER119+ cells to a non-erythroid population. Deficiencies in the number and maturation of TER119+ erythroblasts during infection coincide with blunted proliferation to EPO stimulation in vitro by splenocytes, although a high frequency express EPO receptor (EPOR). Together, these data suggest that during malaria, EPO-induced proliferation of early EPOR+ erythroid progenitors is suppressed, leading to sub-optimal generation of TER119+ erythroblasts. Moreover, a shift in CD71 expression may result in impaired terminal maturation of erythroblasts. Thus, suppressed proliferation, differentiation, and maturation of erythroid precursors in association with inadequate reticulocytosis may be the basis of insufficient erythropoiesis during malaria.     

Formation of Embryo Thromboblasts in Chick Blastoderm: Morphology, Site of Production and Time of Emergence in the Blood

Thrombocytes in the blood of chick embryos (termed embryo thrombocytes by L ucas and J amroz ) have PAS-positive granules in their cytoplasm. Electron microscopic observations reveal that the embryo thrombocytes contain glycogen granules present singly or in clumps. The presence of these inclusions and other morphological characteristics were used as specific markers to distinguish embryo thrombocytes from primitive erythroid cells. These markers also made it possible to determine the time at which the immature thromboblasts first emerge in blood vessels, and the period of their continued presence in the circulation. In this way we found that thromboblasts were detectable in embryos as early as stage 10⁺ of H amburger and H amilton (after 35 hr incubation) and that the thromboblasts were present in the circulation until day 4 of incubation (stage 23). In ovo and in vitro culture of de-embryonated blastoderm demonstrated that thromboblasts were formed in the area opeca vasculosa. The present observations suggest that embryo thromboblasts are formed at the same time and in the same area as the primitive cells of erythroid line.     

Terminal erythroid differentiation in normal and leukemic mouse cells.

Friend virus-transformed murine erythroleukemic cells (FL cells) have been used as an in vitro model for the study of the expression of the genetic program involved in the final stages of erythroid differentiation. Treatment of the FL cells with chemical inducers such as dimethylsulfoxide results in their differentiation from 'pro-erythroblasts' to orthochromatic normoblasts and the appearance of several erythroid markers including hemoglobin, enzymes of the heme pathway, heme, glycophorin, and spectrin. These markers appear in an ordered sequence, suggesting that two genetic programs are involved in the erythroid differentiation of the cells. Preliminary studies with erythropoietin-stimulated fetal liver cultures in vitro suggest that the same is true for normal erythroid differentiation.     

Erythroid differentiation denucleation factors (EDDFs) function as intrinsic, post-erythropoietin regulators for mammalian erythroid terminal differentiation

Regulatory factors other than erythropoietin (Epo) dependence, that control mammalian erythroid terminal differentiation, are currently uncertain. Here we report the existence of erythroid differentiation factors in erythroid cytoplasm. Purification of these factors from cultured Friend virus anaemia (FVA)-infected mouse splenic erythroblasts was carried out using isoelectrophoresis and high performance of liquid chromatography techniques. We have identified intracellular erythroid differentiation denucleation factors (EDDFs) that were able to mediate the events of post-Epo-dependent erythroblast terminal differentiation. Purified EDDF proteins bound specifically to the enhancer HS2 sequence of the globin gene activated the expression of haemoglobin in mouse erythroleukaemia and K562 erythroleukaemic cells and promoted them to differentiate into mature erythrocytes. EDDF proteins began to emerge at the pro-early erythroblast stages upon exposure to Epo in culture, and increased dramatically in early erythroblast stage. The dynamic of EDDF expression and its action on the key events of erythroblast differentiation and denucleation appeared to be closely consistent with its spatiotemporal distribution. These results suggest that EDDFs are the critical intracellular regulatory factors that may act as the successive regulators to Epo, responsible for the final stages of erythroid terminal differentiation.     

Characteristics of the pathways of erythroid cell differentiation in birds in anemia

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.     

THE NUMBER AND POSSIBLE FUNCTIONS OF DNA-SYNTHESIZING CELLS IN HUMAN BLOOD

The number of DNA-synthesizing cells in the blood of patients with various disorders was studied autoradiographically after incubation of blood in vitro with [³H]thymidine. The DNA-synthesizing cells were cytologically assigned to the following categories: erythroid, myeloid, lymphoplasmacytoid and unidentifiable (monocytoid or blast-like) cells. The following patient categories were studied: mitral valvular disease (samples obtained from peripheral vein, pulmonary artery and left auricle), ‘autoimmune diseases’(systemic lupus erythematosus, schleroderma, Hashimoto's thyroiditis, immunohaemolytic anaemia), patients with depressed haemopoiesis (aplastic anaemia, nitrogen-mustard induced bone-marrow hypoplasia) and with increased haemopoiesis (haemolytic anaemia, pernicious anaemia before and during initial vitamin-B₁₂ therapy, red-cell mass regeneration after haemorrhage or iron deficiency) and patients with bacterial infection. In all conditions studied, the number of labelled monocytoid and blast-like cells varied between 0 and 4/μl. Similarly, the number of labelled lympho-plasmo-cytoid cells was consistently low (0–8/μl) in all cases studied except two, where values of 37 and 63/μl were found. Both these patients had severe bacterial infections. The function(s) and potential(s) of these cells are discussed. The fate of the blast-like and monocytoid cells remains obscure. The lympho-plasmocytoid cells probably serve an immunological function, perhaps by disseminating immune responses. Whether or not some DNA-synthesizing cells in the blood are haemopoietic stem cells cannot be decided from the available evidence.     

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/10⁶ cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12?300/11?200-20?700 CFU-E/10⁶ 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.