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Decreased ribosomal RNA content and in vitro RNA synthesis in purified bone marrow erythroblasts of patients with idiopathic ineffective erythropoiesis and DiGuglielmo disease.

Ribsomal RNA content and in vitro 3H-uridine and 3H-thymidine incorporation with examined in purified marrow erythroblasts of patients with idiopathic ineffective erythropoiesis (IIE) and DiGuglielmo disease (DD) and compared with cells from normal marrows. Whereas 3H-thymidine incorporation rates were normal in all patients, 3H-uridine incorporation was 42%--75% of normal in cells from patients examined. Ribosomal RNA content was measured by spectrophotometric scanning of RNA electrophoresed on polyacrylamide gels and corrections made for contamination of the erythroblast preparations with ribosome bearing reticulocytes. In cells from every patient examined RNA content was decreased to 60--76% of normal. Uridine incorporation and rRNA content of patients with beta thalassemia minor and megaloblastic anemia were normal, suggesting that the defects observed in IIE and DD were not due to ineffective erythropoiesis per se. Since erythroblast ribosomes may be rate limiting in protein synthesis, we postulate that even a minor decrease in ribosome content might engender the ubiquitous abnormalities reported in erythroid cells in both IIE and DD.

Bone Marrow Cells↗

Mechanism of arsenic-induced inhibition of erythropoiesis in mice.

Anemia accompanies arsenic intoxication in man. The present studies were undertaken to clarify further the effects of arsenic on erythropoiesis. A dose-related inhibition of red cell 59 Fe incorporation and reticulocyte response was observed in normal mice treated with a single injection of arsenic. Arsenite was approximately two times as inhibitory as arsenate. The effects of arsenic on erythropoietin-induced erythroid differentation revealed a significant inhibitory effect on young, proliferating marrow nucleated erythroid precursor cells. More mature, nonproliferating nucleated erythr oid cells were resistant to the toxic action of arsenic. A dose-related inhibitory effect of arsenic on DNA synthesis was observed in fetal liver nucleated erythroid cells incubated with 3 H thymidine. Ineffective erythropoiesis as well as the megaloblastic morphology accompanying aberrant DNA synthesis -- manifestations of arsenic toxicity in man -- were not evident in the present studies.

Animals↗

Erythropoiesis in primary (idiopathic) osteomyelofibrosis: quantification, PCNA-reactivity, and prognostic impact.

In 64 patients with primary (idiopathic) osteomyelofibrosis (OMF), a morphometric analysis has been performed on bone marrow trephine biopsies following sequential double-immunostaining with monoclonal antibodies against proliferating cell nuclear antigen (PCNA) and erythroid precursor cells (glycophorin C). The purpose of this study was to quantify erythropoiesis and its PCNA-staining capacity and, further, to determine the impact of these parameters for the development of anemia and for prognosis. In comparison with a control group (15 patients), a significant reduction in the number of erythro-normoblasts could be demonstrated, associated with an increase in PCNA-labelling. Moreover, significant correlations between the amount of nucleated erythroid marrow cells and degree of anemia (hemoglobin level, hematocrit, erythrocyte count) and survival could be calculated. Adverse relationships were assessed between number of erythroid cells, thrombocyte count, and spleen size, and also argyrophilic (reticulin/collagen) fiber density. These interactions were thought to reflect the biological behaviour of the disease process, i.e., the progression or extent of myeloid metaplasia. Our findings support ferrokinetic studies suggesting erythroid hypoplasia as one of the major causes of anemia in OMF. The remarkable high PCNA-labelling index of the macrocytic-megaloblastoid appearing erythropoiesis is probably caused by an overexpression of this marker protein. A comparative evaluation of Ki-67 antigen immunostaining in splenic tissue (myeloid metaplasia) and of the PCNA-labelling in pernicious anemia lend support to the assumption of an undue prolongation of the S-phase generated by secondary folate (hematinic) deficiency.

Aged↗

Pure red cell aplasia after chemotherapy for Hodgkin's lymphoma: in vitro evidence for T cell mediated suppression of erythropoiesis and response to sequential cyclosporin and erythropoietin.

Acquired pure red cell aplasia (PRCA) has been associated with various lymphoproliferative conditions but its occurrence with Hodgkin's disease is rare. We report a case of PRCA occurring immediately following the completion of induction chemotherapy in a patient with Stage IIIB nodular sclerosing Hodgkin's disease. In vitro erythroid colony studies documented evidence for T cell mediated suppression of erythropoiesis and lack of a serum inhibitor. Addition of cyclosporin to the in vitro cultures stimulated erythroid colony growth. Following in vivo treatment with cyclosporin peripheral blood CD4/CD8 ratios returned to normal. However, serum erythropoietin levels were inappropriately low. Subsequent treatment with erythropoietin induced a reticulocytosis and transfusion independence. Since discontinuing the erythropoietin, the patient has been able to maintain a hemoglobin of 100 g/L. This case illustrates that red cell aplasia occurring in the setting of Hodgkin's disease may be due to T cell mediated suppression of erythropoiesis. A response to cyclosporin may be masked by inappropriately low erythropoietin levels.

Adult↗

Erythropoiesis in the yolk sac of the bat Tadarida brasiliensis cynocephala.

The process of erythropoiesis and vasculogenesis in the yolk sac of the bat (Tadarida brasiliensis cynocephala) has been studied through the use of both light and electron microscopy. Stem cells arise from the leading edge of the migrating splanchnic mesoderm and transform into primitive erythroblasts. Differentiation involves either contact or association with the endodermal cells, since all erythropoietic activity occurs on the endodermal side of the expanding vascular bed, and many of the cells are in close apposition to the lateral or basal plasma membranes of the endodermal cells. Endodermal cells also phagocytize developing primitive erythroblasts during the later stage of the process when erythropoiesis is subsiding in the yolk sac. Cells destined to become the endothelium of the expanding vascular bed also arise from the leading edge of the migrating splanchnic mesoderm. Their process of differentiation involves the development of cytoplasmic extensions that may surround a group of differentiating erythroblasts, enclosing them in the newly formed lumen of the blood vessel. The cytoplasmic extensions make contact and develop junctional complexes with similar processes from other cells to complete the lumen of the lengthening vascular bed. Cells of the granulocyte series or megakaryocytes are not observed in the yolk sac of the bat as has been described in certain other species.

Animals↗

Micronucleus test and erythropoiesis: effect of cobalt on the induction of micronuclei by mutagens.

The micronucleus test is used widely as an in vivo short-term assay for potential carcinogens. In the present study, results of the micronucleus test were affected by cobalt dichloride pretreatment. Cobalt dichloride was used to induce erythropoietin, a growth factor for erythropoiesis. The increase in mutagen-induced micronucleus response following cobalt pretreatment, therefore, may have been due to a change in the rate of erythropoiesis. The greatest interaction between cobalt pretreatment and mutagen treatment for the induction of micronucleated polychromatic erythrocytes (MPCE) occurred when mice were injected with 1,1-dimethylhydrazine (DMH) 12-24 hr after pretreatment with cobalt dichloride and killed 30 hr later. Increased sensitivity of the micronucleus test was attributable to the administration of mutagen during the differentiation and multiplication of erythroblast, which is presumed to have been accelerated by pretreatment with cobalt dichloride. An increased induction of MPCE in the bone marrow by two chemicals--benzo(a)pyrene, 2-naphthylamine--was also observed following pretreatment with cobalt dichloride.

2-Naphthylamine↗

The transcription factor KLF11 can induce gamma-globin gene expression in the setting of in vivo adult erythropoiesis.

Previous studies in a fetal erythroid cell line demonstrated that the transcription factor, Krüppel-like factor 11 (KLF11), could specifically induce transcription from a gamma-globin gene promoter, and that this induction was mediated through a specific canonical CACCC cis-DNA binding motif. We report here that ectopic expression of KLF11 can also induce fetal gamma-globin gene expression in the setting of adult erythropoiesis both in vitro and in vivo. Studies in an adult-stage murine erythroleukemia (MEL) cell line demonstrated that retrovirus vector-mediated transduction of KLF11 could increase both the amount of expression from a basally active, but not from a overtly silenced, recombinant gamma-globin transgene, as well as the frequency of cells expressing this transgene. A similar pattern of gamma-globin gene induction was also observed both in vitro and in vivo following KLF11 transduction of bone marrow from mice containing a basally active gamma-globin transgene. These studies provide the first evidence that ectopic expression of a transcription factor can induce gamma-globin gene expression in vivo during adult erythropoiesis.

Animals↗

Erythropoiesis in murine long-term bone-marrow cell cultures: dependence on erythropoietin and endogenous production of an erythropoietic stimulating activity.

Erythropoiesis was obtained in murine long-term bone-marrow cell cultures (LTBMCs) in the presence of erythropoietin (Epo) when the medium was frequently renewed. The level of the erythropoietic differentiation was shown to be a function of the erythropoietin concentration. In response to Epo addition, an activity which stimulates CFU-E proliferation in semisolid cultures of fresh bone marrow cells was detected in the LTBMC supernatants. These results suggest that another factor, whose synthesis may be under Epo control, participates in the stimulation of erythropoiesis in vitro.

Animals↗

Splenic plaque-forming cells (PFC) and stem cells (CFU-s) during acute phenylhydrazine-induced enhanced erythropoiesis.

The erythroid status and levels of splenic plaque-forming cells (PFC) to sheep red blood cells (SRBC) were monitored in mice subsequent to acute phenylhydrazine (PHZ)-induced hemolytic anemia. From ferrokinetic measurements, we noted a shift in erythropoiesis from bone marrow to spleen. The levels of splenic PFC were significantly depressed following PHZ-induced erythroid differentiation. Although this immune depression may reflect competition at the stem cell levels, whereby pluripotent stem cells (CFU-s) are preferentially differentiated into the erythroid line at the expense of lymphopoietic pathways, other possibilities cannot be excluded. In this regard, we have shown that loading of the mononuclear phagocyte system (MPS) by PHZ-damaged erythrocytes effected profound depressions in splenic PFC numbers. Lastly, in addition to the well-documented increases in CFU-s migration from marrow to spleen during enhanced erythropoiesis, we noted increased migration of B lymphocytes (as assessed by PFC) in marrow-shielded lethally-irradiated mice given PHZ. We also provide data which show that PHZ-damaged RBC evoke increased migration of CFU-s in normal mice, indicating a possible involvement of the MPS in stem cell migration.

Anemia, Hemolytic↗

Effect of absolute and relative changes of hematocrit on erythropoiesis in mice.

The serial changes of peripheral reticulocytes and marrow erythroid progenitor cells (CFU-e) in mice were monitored under the conditions of absolute or relative changes in red cell mass to study the regulatory mechanism of erythropoiesis. A decreased number of marrow CFU-e and peripheral reticulocytes was observed in the mice with relative polycythemia induced by dehydration as well as in the mice with absolute polycythemia induced by hypertransfusion. On the other hand, a transient increase in the number of marrow CFU-e followed by a gradual increase in the number of peripheral reticulocytes was seen after a considerable amount of exsanguination. Similar stimulatory effects on marrow CFU-e were also observed either by rehydrating the dehydrated mice or by overhydrating the untreated mice to relatively decrease the level of hematocrit. The results suggested that in addition to factors relating to the balance between oxygen supply and requirement, which has been well known, erythropoiesis is greatly affected by hematocrit.

Animals↗

Flow cytometric detection of abnormal fetal erythropoiesis: application to 5-fluorouracil-induced anemia.

We sought to determine whether flow cytometric analysis of circulating fetal blood cells could be used to rapidly detect perturbations of fetal erythropoiesis. In addition, we wanted to determine whether this approach would allow sample collection by exsanguination instead of fetal cardiac puncture, a difficult technique used to prevent contamination of samples with maternal erythrocytes. To monitor fetal erythropoiesis from gestational day (GD) 14-20, we analyzed the cell size, RNA content, and percentage of circulating liver-derived reticulocytes relative to yolk-sac-derived erythroblasts. As a model toxicant, we chose 5-fluorouracil (5-FU), since we previously observed that maternal administration at 20-40 mg/kg on gestational day (GD) 14 produced fetal anemia on GD 16-17, as evidenced by dose-dependent decreases in the cell counts, hematocrit, and hemoglobin content of fetal blood obtained by cardiac puncture. We report herein that 48 hr after maternal 5-FU administration, both cardiac and peripheral blood samples exhibited a dose-dependent decrease in the relative percentage of reticulocytes, indicating a reduced rate of reticulocyte release from the fetal liver. Moreover, at 30 and 40 mg/kg, reticulocytes exhibited increased size and reduced RNA content on GD 16, but elevated RNA content (indicative of premature release) by GD 18. These data suggest that 5-FU inhibits both erythroid cell proliferation and RNA synthesis reversibly, resulting in an anemia that triggers compensatory release of immature reticulocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia↗

Contribution of ventral and dorsal mesoderm to primitive and definitive erythropoiesis in the salamander Hynobius retardatus.

Previously, we found that the conversion of hemoglobins (Hbs) from the larval to the adult type occurred within a single erythroid cell population in a salamander, Hynobius retardatus ("Hb switching" model), whereas the transition involves replacement of red-blood-cell (RBC) populations ("RBC replacement" model) in many amphibians (M. Yamaguchi, H. Takahashi, and M. Wakahara, 2000, Dev. Gene Evol. 210, 180-189). To further characterize the Hb transition, developmental changes in the erythropoietic sites have been intensively analyzed using larval- and adult-specific globin antibodies and globin and GATA-3 RNA probes. Cells of the ventral blood island (VBI) and the dorsolateral plate (DLP) in embryos differentiate in situ to erythroid cells that contain larval globin mRNA, suggesting that both the VBI and the DLP contribute to "primitive" erythropoiesis. In contrast, the expression pattern of the GATA-3 gene suggests that cells of the DLP may contribute to "definitive" hematopoiesis. In order to determine whether it is possible to define a definitive erythropoiesis in H. retardatus or not, further experiments were done: (1) when metamorphosing larvae were treated with phenylhydrazine to induce anemia and then bled at the postmetamorphic stage after recovery from the anemia, a precocious Hb transition was observed in these animals; (2) an RBC population expressing only adult Hb was confirmed by subtracting the number of RBCs expressing larval Hb from the total number of RBCs during metamorphosis. All these results support the existence of a definitive erythroid cell population that contributes only adult RBCs in this species.

Amino Acid Sequence↗

Effect of erythropoiesis on splenic cadmium-metallothionein level following an injection of CdCl2 in mice.

The effects of erythropoiesis on cadmium-metallothionein (Cd-MT)2 levels in spleen, a major erythropoietic tissue, was examined following sc injection of 109CdCl2 into mice. As the concentration of Cd in plasma decreased following injection of 109CdCl2 into control mice, the levels in spleen and liver increased rapidly. After 48 hr, when the concentration of Cd in the erythrocyte (RBC) was at maximum, the level of Cd in the spleen, but not liver, had decreased. These variations of Cd concentration in spleen and RBC were more notable in mice made anemic by phenylhydrazine (PH). Zinc (Zn)-MT was detected in the PH-induced hemopoietic spleen. A subsequent injection of CdCl2 further induced Cd-MT in the spleen. The protein in spleen contained Cd and Zn but not Cu as in RBCs. Maximum levels of Cd-MT in spleen and RBCs of the PH-treated mice were about four times higher than those of control mice. The concentration of Cd-MT in the spleen of polyemia mice, induced by RBC transfusion, was 50% lower than that of control mice. When erythropoietin was injected into the polyemia mice prior to CdCl2, the concentration of Cd-MT in spleen increased about sixfold. These changes in Cd-MT levels in spleen were not observed in either liver or kidney. The effects of erythropoiesis on the concentration of Cd-MT in the spleen corresponded with effects seen in RBC. These results suggest that splenic Cd-MT can be the source of Cd-MT in mature RBC.

Anemia↗

Differences in erythropoiesis in normal chicken and quail embryos.

Using antibodies against the fetal and adult forms of alpha- and beta-globin, it has been shown that erythropoiesis in the para-aortic foci (PAF) constitutes a major species-specific difference between chicken and quail embryos. In quail embryos, para-aortic foci are rare, small and rather heterogeneous with regard to their erythropoietic and haemopoietic cell composition. In contrast, the PAFs in chicken embryos are abundant and consist of large numbers of erythropoietic cells. In both species a time difference (approximately 1 day) is observed between the first expression of the fetal alpha- and beta-globin and the adult alpha- and beta-globin in erythropoietic cells. Adult erythropoiesis in both species can be detected first in the stalk of the yolk sac; this is similar to the situation in mammalian and amphibian species. From this time onward the number of circulating adult erythrocytes increases steadily. Whereas in chicken, large intraembryonic foci that can serve as sources for these adult cells arise concomitantly, no such foci can be detected in quail embryos, suggesting that the quail yolk sac is a major source for these adult red blood cells.

Animals↗

Erythropoiesis in the thymus of the spotless starling, Sturnus unicolor.

Although previously described in other avian species, intrathymic erythropoiesis is a remarkable feature of the thymus of Sturnus unicolor. In discrete stages of the life cycle of this species, erythroblasts and mature erythrocytes occupy large areas of the thymic cortex and cortico-medullary border. Simultaneously, degenerated thymocytes and epithelial-reticular cells occur in the same areas. The relationship between intrathymic erythropoiesis, degeneration of cortical lymphocytes and epithelial-reticular cells, and macrophage activity is discussed and related to a possible functional role of sex hormones in this phenomenon.

Animals↗

Morphological investigation on phenylhydrazine-induced erythropoiesis in the adult mouse liver.

In adult mice suffering from a phenylhydrazine (PHZ)-induced hemolytic anemia, erythropoietic islands were observed in the liver. These islands were studied with the light and electron microscope. Within two days after the beginning of four daily injections of PHZ, erythoid elements appeared in the sinusoids and central veins. A maximum number of erythroblasts was found on day 7. Light and electron microscopic observations revealed that the erythropoietic islands consisted of centrally located macrophages(CM) with a Kupffer cell-like morphology, surrounded by erythroblasts, which were often of the same maturation stage. CM in central veins (CM-V) and in sinusoids (CM-S) were found to have a different morphology. The CM-V phagocytized less circulating red blood cells and were in contact with a smaller number of erythroblasts. Furthermore, the contact areas between erythroblasts and CM-S extended for a much longer distance than those between erythroblasts and CM-V. The progenitor cell for the CM-V is most likely a monocyte, since cells which were morphologically determined as monocytes were found to appear on the first day of the PHZ treatment and differentiated into macrophages within about 2 days. The origin of the CM-S population was less clear, but could be monocytic as well. These data are tentatively explained as a migration of a progenitor of a cellular component of the erythroid micro-environment into the liver after appropriate stimuli. In contrast to fetal liver erythropoiesis, erythroblasts in the adult liver occurred only incidentally extrasinusoidally. Furthermore, specialized membrane contacts between erythroblasts and CM or hepatocytes could not be observed in adult liver. Ferritin could not be detected on the erythroid cell membrane or located in coated vesicles. Also, no ferritin could be observed within or attached to the finger-like processes of CM. The observations suggest that the coated vesicles in erythoid elements are partly exocytotic vesicles and are not specific for ferritin transport. The morphological aspects of PHZ-induced extramedullary erythropoiesis is discussed in relation to the hemopoietic microenvironment.

Animals↗

R-h-erythropoietin counteracts the inhibition of in vitro erythropoiesis by tumour necrosis factor alpha in patients with rheumatoid arthritis.

Anaemia of chronic disease (ACD) is a common extra-articular manifestation of rheumatoid arthritis (RA). Tumour necrosis factor alpha (TNF alpha) plays an important role in the development of ACD. The objective of the present study was to assess inhibition of in vitro colony-forming unit erythrocyte (CFUe) and blast-forming unit erythrocyte (BFUe) growth by TNF alpha and to examine whether this suppression could be counteracted by adding increasing concentrations of recombinant human erythropoietin (EPO) (r-h-EPO) to bone marrow cultures of RA patients with ACD and without anaemia (controls). Bone marrow cells of RA patients with ACD and control patients were cultured. The cultures were incubated with increasing concentrations of r-h-EPO (0.25; 0.5; 1; 2 U/ml), each in combination with increasing quantities of TFN alpha (0; 50; 100; 200; 400 U/ml). CFUe and BFUe were assessed after 7 and 14 days, respectively. Dose-dependent inhibition of BFUe and CFUe by increasing concentrations of TNF alpha was observed in ACD and controls. Regarding CFUe (ACD patients) incubated with 0.25 U/ml EPO, 50 U/ml TNF alpha caused 28% suppression compared to cultures without TNF alpha. Increasing the concentration of r-h-EPO from 0.25 U/ml to 2 U/ml completely restored the number of CFUe. A similar pattern was observed in BFUe growth in both groups. These data demonstrated the suppressive effects of TNF alpha on erythropoiesis in vitro and that the suppressed erythropoiesis could be partly corrected by the addition of excess r-h-EPO to the cultures. No significant differences were observed between ACD and control RA patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia↗

Changes in erythropoiesis due to radiation or chemotherapy as studied by flow cytometric determination of peripheral blood reticulocytes.

Flow cytometric determination of time dependent changes of numbers of reticulocytes in peripheral blood were investigated as a parameter for changes in erythropoiesis induced by radiation- or chemotherapy. Rats irradiated or treated with drugs (such as e.g. cyclophosphamide 100 mg/kg, vincristin 0.2 mg/kg, or mitomycin C 1.0 mg/kg) showed clear changes in erythropoietic activity. Reticulocyte numbers decreased rapidly until day 3-4 after treatment; this period was followed by a gradual increase and normal control values were seen at day 8-11. Radiation effects of doses as low 0.5 Gy could be detected in such a way. Similar studies were performed with patients with ovarian tumors treated with cis-platinum, a drug that may cause non-immune haemolysis. During prolonged treatment some patients showed increasing numbers of reticulocytes, measured at the first day of each hospitalization period, whereas leucocyte and platelet counts stayed more or less constant. Increasing numbers of reticulocytes generally indicates stimulation of erythropoietic activity of the bone marrow (due to increased blood loss); in this study increasing numbers often preceeded a decrease in hemoglobin values later on. Flow cytometric analysis of reticulocytes is therefore a potentially useful tool to detect changes in erythropoiesis, and considered more sensitive for the early recognition of patients that develop anemia, than hemoglobin measurements only.

Altretamine↗