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Reversal of ineffective erythropoiesis in pernicious anaemia following vitamin B12 therapy.

Ineffective erythropoiesis was quantitated in a series of patients with pernicious anaemia at different times in relation to vitamin B12 therapy by measuring the incorporation of [15N]delta aminolaevulinic acid and [15N]glycine into early labelled bilirubin. Prior to therapy ineffective erythropoiesis was grossly increased but this was reversed within 24 h of giving vitamin B12, suggesting that most of the existing megaloblasts are enabled to mature into circulating red cells.

Aged↗

Pathophysiological changes in serum erythropoiesis stimulating activity.

The mouse fetal liver cell bioassay has been used to study serum levels of erythropoiesis stimulating activity (ESA) in patients with a variety of haematological disturbances. The results indicate that this assay system provides a quantitative measurement of ESA, the concentration of which varies predictably in a manner which is consistent with it having a function as an in vivo regulator of erythropoiesis. Dose-response curves for serum ESA show highly significant parallelism with those obtained from sheep plasma erythropoietin and the International Reference Preparation of human urinary erythropoietin.

Anemia↗

Thyroid hormones stimulate erythropoiesis in vitro.

Thyroid hormones have important effects on erythropoiesis in man and animals. We performed in vitro culture studies with murine and human bone marrow in order to define the interaction of these hormones with erythroid and granulocyte-monocyte progenitor cells. The methylcellulose clonogenic assay was used with the appropriate addition of erythropoietin or colony-stimulating activity. L-thyroxine, D-thyroxine and L-triiodothyronine potentiated erythropoientin-stimulated erythroid colony formation in concentrations of 50-100 ng/ml. These hormones had no effect on granulocyte-monocyte colony formation at concentrations up to 500 ng/ml. Testing of various thyroid analogues showed no clear correlation between potentiation of erythropoiesis and known calorigenic potency. Reverse triiodothyronine also had potentiating activity in this system. The active thyroid hormones stimulated erythroid colony formation at several concentrations of erythropoietin but could not substitute for erythropoietin. These data suggest that thyroid hormones have a direct effect on erythroid precursor proliferative capacity, a finding which may have relevance to the mechanism of erythropoietic dysfunction in human thyroid disease.

Animals↗

Mechanism of the decreased erythropoiesis in the water deprived rat.

Radioiron uptake by erythrocytes, metabolic rate, erythropoietin formation during hypoxia and erythroid responsiveness to exogenous erythropoietin were determined in both starved and water deprived rats. The feed intake showed a marked and progressive reduction during water deprivation. The metabolic rates of rats deprived of either food of water declined progressively showing a 40% reduction 5d after water deprivation or starvation began. At this time, the 24 h red blood cells 59Fe incorporation was 85% lower in both starved and dehydrated rats than in normal rats. Plasma erythropoietin levels in response to hypoxia were approximately 50% decreased in both starved and dehydrated rats. Both polycythaemic starved and polycythaemic water deprived rats injected with human urinary erythropoietin showed a 75% decrease in 59Fe incorporation into erythrocytes when compared to control rats. It is suggested that depression of erythropoiesis during water deprivation in the rat depends on a reduced sensitivity to erythropoietin, possibly associated with decreased production of the hormone. Since water deprived rats drastically reduce feed intake it is suggested that secondary starvation is the principal cause of the decreased erythropoiesis induced in the rat by water deprivation.

Animals↗

Canine cyclic haematopoiesis: the effect of endotoxin on erythropoiesis.

We have examined the effects of chronic endotoxin treatment on erythropoiesis in six grey collies with cyclic haematopoiesis. Blood reticulocytes, bone marrow erythroid colony (EC) forming cells, serum iron and erythropoietin (ESF) values showed regular periodic fluctuations in untreated grey collies. Daily endotoxin injections eliminated the cyclic fluctuations of reticulocytes and EC. The mean serum iron values were increased and recurrent hypoferraemia eliminated, while the mean serum ESF values were reduced. The cyclic fluctuations of serum ESF values were no longer apparent in the endotoxin treated grey collies. Tritiated thymidine suicide of the marrow EC forming cells failed to show cyclic changes either in untreated or endotoxin treated dogs. The ESF sensitivity of EC in the grey collie was unchanged during endotoxin treatment and was not different from normal dogs. Endotoxin appears to alter periodic erythropoiesis by stabilizing the flux of cells into the committed erythroid precursor cell pool from a more primitive stem cell compartment.

Animals↗

The effects of therapeutic dialysis and renal transplantation on uraemic serum inhibitors of erythropoiesis in vitro.

The methyl cellulose culture system using human bone marrow and erythropoietin (Ep) at different concentrations was used to examine the effects of uraemic sera on erythropoiesis in vitro. Sera from undialysed patients with advanced uraemia (plasma creatinine greater than 900 mumol/l) when added to cultures at 10% with Ep at 2.0 u/ml were consistently inhibitory to the growth of erythroid burst-forming units (BFU-E). No inhibition of erythroid colony-forming units (CFU-E) was observed at this Ep concentration but inhibition was consistently demonstrated with Ep at 0.2 u/ml. Sera from undialysed patients with less severe uraemia (plasma creatinine less than 900 mumol/l) were not inhibitory to BFU-E or CFU-E at Ep 2.0 u/ml. Sera from patients with stable, functioning renal transplants were stimulatory to erythropoiesis in vitro with Ep at 2.0 u/ml. This finding is consistent with the normal or increased haematocrits often found following renal transplantation. Sera from patients on maintenance haemodialysis and continuous ambulatory peritoneal dialysis (CAPD) were not significantly different from normal in their effect on BFU-E growth in vitro and were slightly but significantly stimulatory to the growth of CFU-E. This suggests that these two forms of dialysis are equally effective in reducing the activity of uraemic inhibitors in serum and that inhibition of the marrow response to Ep and/or burst-promoting activity (BPA) is unlikely to be a major factor in the continued anaemia of dialysis patients.

Bone Marrow Cells↗

A model for erythropoiesis in experimental chronic renal failure.

Chronic renal failure (CRF) in rats was induced by various subtotal nephrectomy procedures. Plasma creatinine concentrations ([Creat]p) were consistently elevated in partially nephrectomized (PNX) rats (P less than 0.001) and renal function ([Creat]p-1) was correlated with the amount of intact renal tissue (r = 0.624, P less than 0.001). Red blood cell (RBC) production in uraemic and non-uraemic rats was assessed using the recovery of packed cell volume (PCV) and the production of reticulocytes in response to acute blood loss (5, 10, 15 and 20 ml kg-1 body wt). PCV recovery was significantly delayed and failed to reach pre-bleed values for up to 28 d in PNX rats compared with full recovery within 8 d in controls. Linear relationships between the index of reticulocyte production (RP1) and blood loss induced falls in PCV were found in PNX (r = -0.679, P less than 0.001) and control rats (r = -0.696, P less than 0.001). The index of RBC production was lower in PNX rats compared with controls. This was attributed to a reduced erythropoietin (Ep) mediated reticulocyte response to acute blood loss in PNX rats. PNX rats developed a normocytic, normochromic form of anaemia in which the red blood cell volume (RBCV) and plasma volume (PV) were both appropriate for measured PCVs. The reduced RBC production response in experimental CRF may explain the development of anaemia in PNX rats, even though steady state erythropoiesis may eventually be established at a reduced level of PCV. Furthermore, the quantitative evaluation of RBC production in response to acute blood loss provides a further model for the assessment of agents which may stimulate erythropoiesis in CRF.

Anemia↗

Pure red cell aplasia: lymphocyte inhibition of erythropoiesis.

The pathogenesis of pure red cell aplasia (PRCA) was studied in a patient who had no evidence of malignancy. In marrow culture, no erythroid colonies (from late erythroid progenitors [CFU-E]) but normal numbers of well-haemoglobinized erythroid bursts (from early erythroid progenitors [BFU-E]) were found, indicating that BFU-E existed in the patient but that their subsequent in vivo differentiation was inhibited. Autologous coculture studies suggested that inhibition was mediated by the patient's ER + lymphocytes. After remission was induced with cyclophosphamide, autologous ER + cells no longer suppressed in vitro erythropoiesis. However, cryopreserved ER + cells, obtained with anaemia, suppressed BFU-E growth from remission marrow. An expanded population of large granular lymphocytes (LGL) with ER +, Fc gamma +, T3+, T8+, HNK-1+, Ia-, M1 -- phenotype and no functional natural killer (NK) cell activity was noted during PRCA that reverted to normal with remission. For this patient, both in vivo and in vitro evidence demonstrates a cellular inhibition of erythropoiesis at the level of differentiation between BFU-E and CFU-E.

Bone Marrow↗

Adult 'fetal-like' erythropoiesis characterizes recovery from bone marrow transplantation.

The transient fetal-like erythropoiesis which appears during recovery from bone marrow transplantation has now been examined at the level of erythroid progenitor cells. A 7-year-old boy with beta +-thalassaemia major was studied during engraftment from his beta-thalassaemia trait sister. Hb F and i antigen rose as expected. Macrocytosis never developed, but red cell size distribution became very heterogeneous. Bone marrow CFU-E and BFU-E were detected by 30 d, prior to the appearance of reticulocytes. Marrow erythroid progenitor cell numbers were normal by 146 d, while those in the blood became normal by 360 d. After transplantation globin synthesis ratios in erythroid colonies were diagnostic of thalassaemia trait, indicating engraftment. Individual erythroid colonies derived from both blood and marrow at all times during reconstitution showed no correlation of G gamma and gamma. Thus the fetal-like stress erythropoiesis of marrow expansion following transplantation was derived from adult and not fetal progenitor cells.

Bone Marrow↗

Dyserythropoiesis and ineffective erythropoiesis in Plasmodium vivax malaria.

Nine Thai adults with P. vivax malaria were investigated. Light and electron microscope studies of marrow aspirates revealed morphological evidence of dyserythropoiesis in six of them. Dyserythropoiesis was most marked in the four most anaemic patients. In these four patients the electron microscope also revealed the presence of erythroblasts at various stages of degradation within the cytoplasm of macrophages. Neither the dyserythropoiesis nor the ineffective erythropoiesis could be attributed to a deficiency of vitamin B12, folate or iron. The abnormalities of erythropoiesis seemed to result from the P. vivax infection itself. Other bone marrow reactions seen in this infection included macrophage hyperplasia, plasmacytosis and increased eosinophil granulocytopoiesis. Unlike in severe P. falciparum malaria, the microvasculature of the marrow was not obstructed by parasitized red cells.

Adolescent↗

Effect of subcutaneously administered human recombinant erythropoietin on erythropoiesis in patients with myelodysplasia.

In a phase II study, 12 patients with a myelodysplastic syndrome (MDS) and anaemia (nine transfusion-dependent) were treated with recombinant human erythropoietin (rHuEpo) to assess the therapeutic effect on erythropoiesis and on transfusion requirement. Patients with a low risk of developing acute leukaemia were included, i.e. refractory anaemia (RA), RA with ringed sideroblasts (RARS) and RA with excess blasts (RAEB), providing the percentage of myeloblasts in the bone marrow did not exceed 10%. Recombinant HuEpo treatment was initiated at a dose of 50 units/kg body weight and administered subcutaneously three times weekly. At 3-week intervals the dose was increased with 50 units/kg per injection, until after 15 weeks a maximum dose of 250 units/kg three times weekly was reached. All patients completed the study. Recombinant HuEpo was well tolerated and no serious side effects were seen. There was no evidence of the emergence of a new malignant clone in response to rHuEpo as shown by sequential karyotyping. In none of the patients was an increase in haemoglobin level or a diminished red blood cell transfusion requirement seen. In four out of 10 evaluable sequential bone marrow smears, an increase in erythropoiesis was seen, suggesting stimulation of ineffective red cell production. One of these patients also showed a rise in reticulocyte count. The number of erythroid progenitor cells (BFU-E and CFU-E) in blood and bone marrow was not affected by rHuEpo treatment. Also no change in the number of myeloid progenitor cells (CFU-GM) in blood and bone marrow was noted. In conclusion, subcutaneous treatment with rHuEpo at dosages up to 250 units/kg body weight (three times weekly) fails to increase the haemoglobin level or to diminish the transfusion requirement in patients with MDS and anaemia. It is unclear whether higher doses of rHuEpo are effective or whether patients with less severe anaemia who are transfusion independent, have a higher likelihood of response.

Adult↗

Erythroid 5-aminolaevulinate synthase activity during normal and iron deficient erythropoiesis.

Reduced erythroblast 5-aminolaevulinate (ALA) synthase activity was observed during iron/haem deficient erythropoiesis. Enzyme activity was reduced approximately threefold to levels similar to those previously detected during sideroblastic erythropoiesis. This response would appear to be erythroblast specific as haem deficiency is known to stimulate hepatic ALA synthase activity. It is, however, unclear as to whether this reduced enzyme activity relates to iron deficiency or to the consequent haem deficiency.

5-Aminolevulinate Synthetase↗

Quantitative assessment of erythropoiesis in haemodialysis patients demonstrates gradual expansion of erythroblasts during constant treatment with recombinant human erythropoietin.

Recombinant human erythropoietin (rHuEpo) has been shown to be effective in correcting the anaemia of chronic renal failure. It has been reported that reticulocytes as well as erythroid progenitors increase within 1-2 weeks, with no further elevation beyond this time interval. However, the erythroblast pool is quantitatively the most important compartment of erythropoiesis, and the rate, extent and duration of the expansion of erythropoietic activity in response to rHuEpo is not known. Treatment with rHuEpo was given to 64 patients i.v. thrice weekly after haemodialysis. The effect of rHuEpo was obvious from the early elevation of reticulocyte counts, but much of this increase was due to a rapid output of shift reticulocytes which levelled off after a few weeks. Serum transferrin receptor (TfR), a quantitative measure of erythropoiesis, increased progressively over 6 weeks to reach a plateau phase at about twice baseline values. The Hct increased progressively and continued to rise steadily after the TfR plateau was reached. The speed and extent of the expansion of erythropoietic activity correlated with the later haematological response to rHuEpo. When rHuEpo was discontinued, erythropoietic activity returned progressively to baseline values, to rise again gradually when treatment was resumed. Part of the Hct increase was also due to haemoconcentration. The results indicate that changes in the various erythroid compartments vary considerably in intensity and speed, and that the erythroblast compartment in particular is slow to respond to modifications in the erythropoietin stimulus.

Adult↗

Defect of erythropoiesis in non-leukaemic AKR mice.

The kinetics of the CFU population and of erythropoiesis were investigated in the AKR strain mouse prior to the onset of thymic leukaemias: haemopoiesis was compared in syngeneic AKR, semi-allogenic C3H and (C3H X AKR) F1 mice injected with AKR stem cells. These experiments demonstrate that the reduction in The number of spleen colonies previously described by Perkins et al. (1971) in syngeneic hosts, as compared to semi-allogenic C3H hosts, is actually related to defective erythropoiesis resulting from a dysfunction of the AKR haemopoietic inductive microenvironment (HIM). Erythropoietin secretion is normal in AKR mice. The early haemopoietic events related to the stem cell: lodgement of the CFU ('f' factor) and doubling time, are not disturbed, but the onset of CFU proliferation is markedly delayed in the AKR strain. The main expression of the AKR HIM dysfunction is a significant reduction in the number of erythroid (E) colonies and an impaired output of red blood cells per E-colony in the syngeneic host as compared to the allogenic one. In addition, data indicate that a weakly histo-incompatible system, such as that in C3H and hybrid hosts, does not interfere with the stages of haemopoiesis except by lengthening the doubling time of the CFU. The results, on the whole, emphasize the prevalent influence of HIM.

Animals↗

Mediastinal extramedullary erythropoiesis in hereditary spherocytosis.

Evidence of thoracic extramedullary erythropoiesis has been noted before but it is considered rare in the course of chronic haemolytic anaemia. We report an adult who was admitted for splenectomy because of hereditary spherocytosis (HS). The evidence of a large mediastinal mass before surgery led us, after isotopic scanning using 111Indium transferrin, to suspect extramedullary erythropoiesis; this was confirmed by cytology during surgery. The utility of 111Indium transferrin as a bone marrow tracer is also noted.

Bone Marrow↗

Reticulocyte maturity pattern analysis as a predictive marker of erythropoiesis in paediatrics. Part II: Pilot study for clinical application.

Reticulocyte quantification in peripheral blood samples is a commonly used diagnostic indicator of erythropoietic activity. A methodology based on flow cytometry additionally separates reticulocytes into 3 groups by fluorescence staining of the residual RNA. This identifies cells as 'high (HFR), medium (MFR) and low (LFR) fluorescence intensity' reticulocytes. In part II of the study we looked for the clinical applicability in paediatrics. Selected groups of patients with ineffective erythropoiesis, i.e. suffering from renal failure, oncologic patients with suppressed bone marrow activity caused by chemotherapy and anaemic new-born infants have been observed longitudinally for their reticulocyte maturity profiles. Data were compared to the commonly used parameters RBC, Hb and Hct. In all cases in which effective erythropoiesis returned documented by a normalization of standard blood parameters, HFR cells reacted significantly earlier than the traditional markers. These preliminary observations suggest the reticulocyte maturity pattern analysis can be used as an additional aid in diagnosis and as a helpful parameter for the monitoring of any anaemic situation.

Anemia↗

Post-operative erythropoiesis is limited by the inflammatory effect of surgery on iron metabolism.

The decrease in haemoglobin concentration commonly observed after major surgery is usually corrected by red cell transfusions or oral iron medication. The increased awareness of blood-transmissible diseases has led to the restrictive use of homologous blood and to interest in alternatives for correcting anaemia. We investigated the pathophysiology of postoperative anaemia by studying variables of erythropoiesis, iron metabolism, and inflammation in 48 consecutive patients who underwent total hip replacement. Haemoglobin concentration remained low during 14 days after surgery with only a mild increase in erythropoietin concentration and reticulocyte count. No increase in serum transferrin receptor concentration was observed during the first 2 weeks after surgery. Postoperative serum ferritin increased, whereas serum iron, transferrin and transferrin saturation decreased significantly. There was a marked increase in interleukin-6 and C-reactive protein with maximal values on the 1st and 4th post-operative day, respectively. At 6 weeks after surgery, haemoglobin concentration and variables of iron metabolism were almost at the preoperative level and serum transferrin receptor concentration was significantly increased, indicating increased erythropoietic activity. These changes were preceded by the normalization of interleukin-6 and C-reactive protein levels. Haemoglobin, iron, transferrin, and ferritin concentrations were not influenced by iron therapy during the postoperative period and no differences of erythropoietic and iron variables were observed between transfused and non-transfused patients. In conclusion, post-operative erythropoiesis is associated with an inflammatory effect of surgery on iron metabolism, which can explain, despite a slightly increased production of erythropoietin, the persistence of anaemia and the lack of effect of iron supplementation after surgery.

Aged↗

Protection against malaria by anti-erythropoietin antibody due to suppression of erythropoiesis in the liver and at other sites.

We have previously reported that erythropoiesis commences in the liver and spleen after malarial infection, and that newly generated erythrocytes in the liver are essential for infection of malarial parasites as well as continuation of infection. At this time, erythropoietin (EPO) is elevated in the serum. In the present study, we administered EPO or anti-EPO antibody into C57BL/6 (B6) mice to modulate the serum level of EPO. When mice were infected with a non-lethal strain (17NXL) of Plasmodium yoelii (blood-stage infection of 10(4) parasitized erythrocytes per mouse), parasitemia continued for 1 month, showing a peak at day 17. Daily injection of EPO (200 IU/day per mouse) from day five to day 14 prolonged parasitemia, whereas injection of anti-EPO antibody (1.5 mg/day per mouse) every second day from day five to day 28 decreased it. Erythropoiesis was confirmed in the liver, spleen and bone marrow by the appearance of nucleated erythrocytes (TER119+). When anti-EPO antibody was injected by the same protocol into mice infected with a lethal strain (17XL) of P. yoelii, all mice showed decreased parasitemia and recovered from the infection. These results suggest that the use of anti-EPO antibody after malarial infection may be of therapeutic value in severe cases of malaria.

Animals↗