Erythropoietic Activity Research Articles

Soluble Transferrin Receptor and Its Ratio to Erythroblasts in Bone Marrow May Be a New Diagnostic Tool to Distinguish between Aplastic and Refractory Anemia

Myelodysplastic syndromes (MDS), especially refractory anemia (RA) are very heterogeneous diseases regarding their morphological, biological and clinical features. One important clinical problem is the difficulty of diagnosis. Soluble transferrin receptors (sTfRs) reflect the erythropoietic activity in the bone marrow (BM). To establish whether determination of serum sTfR could be useful for the differential diagnosis between RA and aplastic anemia (AA), we measured the serum sTfR concentrations, BM cellularity and BM erythroblast percentages in 14 untreated AA and 7 untreated RA patients. The serum sTfR levels of the RA patients (820.1 ± 402.8 ng/ml) were significantly higher than those of the AA patients (491.1 ± 195.2 ng/ml; p = 0.0207). However, the serum sTfR values of RA and AA patients also overlapped. A new index, the ‘sTfR-E index’ [the ratio of serum sTfR level (ng/ml) to BM cellularity (%) × BM erythroblasts (%)] is proposed, which is expected to reflect the number of transferrin receptors (TfR) on the cell membrane per BM erythroblast. The sTfR-E index values of the 7 RA patients (0.395 ± 0.234) were significantly lower than those of the 14 AA patients (2.669 ± 1.633; p = 0.0003). The sTfR-E index values of AA and RA patients overlapped only marginally. In conclusion, the sTfR-E index may be a useful new diagnostic tool to distinguish between AA and RA patients.

Genetic insights into the clinical diversity of beta thalassaemia.

Genetic insights into the clinical diversity of beta thalassaemia.

Effects of intraperitoneal application of toluene dissolved in propylene glycol on erythropoiesis in Wistar rats

The potential toxic effect of toluene on erythropoiesis was investigated during the enhanced erythropoietic activity provoked by application of propylene glycol (known as a mild hemolytic agent) in adult female Wistar rats. The animals were treated daily for 3, 7 or 11 days, with an intraperitoneal dose of toluene dissolved in propylene glycol (T+PG) or propylene glycol (PG) alone. The effects of T+PG and PG on some hematological parameters in peripheral blood and bone marrow were evaluated at the time of sacrifice. The number of red blood cells (RBC), reticulocytes (Rt), hematocrit values (PCV), and hemoglobin concentration (Hb) were determined in peripheral blood samples using standard laboratory procedures. Total bone marrow nucleated cells (TBMNC) were counted and the myelogram was analysed as well. The number of early bone marrow erythroid progenitor cells - Burst Forming Unit – Erythroid (BFU-E) was assessed using a colony forming assay on methylcellulose. Toluene dissolved in PG induced a decrease in RBC number, PCV and Hb concentration and an increase within the bone marrow BFU-E and precursor erythroid cell compartments, as well as the percentage of peripheral blood Rt, indicating enhanced erythropoietic activity. Very similar changes occurred when PG was administered alone. Therefore, it can be assumed that short-term application of a low dose of toluene (3 mg/kg) dissolved in PG did not have a toxic effect during PG induced enhanced erythropoietic activity.

Sugar coating extends half-lives and improves effectiveness of cytokine hormones

Recombinant human erythropoietin (rhEPO) is an effective and widely used therapeutic agent that is produced by bioengineering. Modification of the rhEPO protein by glycoengineering increased its already abundant N-glycosylation, which enhances its erythropoietic activity in vivo by decreasing its metabolic clearance. Elliott et al. recently reported increased in vivo activities of thrombopoietin (Mpl ligand) and leptin following carbohydrate addition to both, which suggests that such glycoengineering could be applied to a variety of hormones, cytokines and growth factors.

548 CERA (Continuous erythropolesis receptor activator) is an innovative erythropoietic agent with an extended serum half-life: studies of mode of action, pharmacokinetics and erythropoietic activity

Interferon α-2a plays an essential role in the treatment of chronic hepatitis C, but it is limited in its efficacy by the short in vivo half-life. To improve the half-life and efficacy, interferon α-2a is conjugated with a 40-kDa branched polyethylene glycol moiety (PEG-IFN, PEGASYS). From this preparation the positional PEG-IFN isomers were isolated and characterized by different analytical methods and antiviral assay. Two chromatographic steps were used to separate and purify nine isomers. The analytical methods IE-HPLC, RP-HPLC, SE-HPLC, SDS–PAGE, and MALDI-TOF MS indicated that each of these nine isomers is conjugated to the branched polyethylene glycol chain at a specific lysine. No isomer with a modification at the amino terminus was observed. All positional isomers induced viral protection of MDBK cells in the antiviral assay. When comparing the quantitative potency of the individual isomers with the whole mixture of PEG-IFN, significant differences in the specific activities were observed: PEG-Lys(31) and PEG-Lys(134) showed higher activities than the mixture, PEG-Lys(164) was equal to the mixture, whereas the activities of PEG-Lys(49), PEG-Lys(70), PEG-Lys(83), PEG-Lys(112), PEG-Lys(121), and PEG-Lys(131) were lower.

Anaemia of Chronic Renal Failure

Chronic anaemia is an almost invariable consequence of renal failure. As renal function deteriorates, there is a slow, progressive decrease in haemoglobin concentration that becomes particularly evident once the glomerular filtration rate (GFR) declines below 30 ml/minute (approximately corresponding to serum creatinine > 300 μmol/litre). This anaemia is important because it causes many debilitating symptoms (e.g. tiredness and lethargy, muscle fatigue, intolerance to cold, breathlessness on exertion, poor exercise capacity). It is also a major factor in the high prevalence of cardiovascular disease in renal patients, and their consequent increased morbidity and mortality; cardiovascular disease accounts for more than 50% of deaths in these patients. The anaemia of chronic renal failure (CRF) is normochromic and normocytic (unless superimposed iron deficiency is present), and hypoproliferative (because of reduced erythropoietic activity in the marrow). The reticulocyte count is therefore inappropriately low for the degree of anaemia, but the peripheral blood film often looks normal except for occasional fragmented RBCs ('burr cells', Figure 1). Blood volume studies show a reduced RBC mass, but normal total blood volume.

Role of humoral factors in the regulation of hemopoiesis during cytostatic-induced myelosuppression.

The content of hemopoietic precursors in the bone marrow, its morphological composition, production of hemopoietic growth factors by cells of the hemopoietic microenvironment, erythropoietic activity, and plasma content of endogenous erythropoietin were studied in CBA/CaLac mice with hypoplasia of hemopoiesis induced by adriamycin in a maximum permissible dose. Cytostatic treatment affected production of substances determining erythropoietic activity by adherent and nonadherent cells of the bone marrow. Erythropoietic activity of peripheral blood plasma considerably increased. Recovery of erythroid hemopoiesis was determined by the effects of endogenous erythropoietin and other substances secreted by cells of the hemopoietic microenvironment and present in the peripheral blood.

Erythropoietic activity of preparations containing receptors and antibodies to erythropoietin in ultralow doses.

We compared the erythropoiesis-stimulating effects of ultralow doses of erythropoietin receptors and antibodies to erythropoietin in intact mice. Antibodies to erythropoietin, but not erythropoietin receptors, possessed considerable erythropoiesis-activating properties.

Marked increase in serum transferrin receptor among Thai children with Hb-E-beta-thalassaemia.

To determine the serum transferrin receptor (sTfR) level in patients with Hb-E-beta-thalassaemia attending a paediatric haematology clinic. Sixty-four patients with Hb-E-beta-thalassaemia aged 2.9-10.8 years were attending a paediatric haematology clinic at King Chulalongkorn Memorial Hospital, Bangkok, Thailand. Circulating transferrin receptor was measured, as was the complete blood count and percentage of reticulocytes. A transfusion regimen recommended by the Thalassaemia International Federation was used whenever parents agreed to the treatment. In patients with Hb-E-beta-thalassaemia, the concentration of sTfR varied from 1.88 to 28.44 mg/L (mean +/- SD 13.99 +/- 5.37), while in the control group, it varied from 0.95 to 2.56 mg/L (mean +/- SD 1.58 +/- 0.41; n = 31). The sTfR level correlated inversely to a single measurement of haemoglobin in the same specimen (r = - 0.433; P = 0.00) and was elevated compared to the control group (P = 0.00; alpha < 0.05). In addition, the sTfR level correlated inversely to mean pre-transfusion haemoglobin (r = - 0.36; P = 0.003). The level of sTfR is elevated in children with Hb-E-beta-thalassaemia. The sTfR level can be used as an adjunct real-time assessment of erythropoietic activity, comparable with mean pre-transfusion haemoglobin. Measurement of sTfR is easy using most automated chemical analyzers, and material can be obtained from routine specimen collections. Further studies should be carried out in a larger population to verify the benefits of this test to patients.

Impaired splenic erythropoiesis in phlebotomized mice injected with CL2MDP-liposome: an experimental model for studying the role of stromal macrophages in erythropoiesis

Erythropoiesis occurs in the presence of erythropoietin (EPO) without macrophages in vitro. In hematopoietic tissues, however, erythroid cells associate closely with stromal macrophages, forming erythroblastic islands via interactions with adhesion molecules. To elucidate the role of macrophages in erythropoiesis, we selectively abrogated stromal macrophages of splenic red pulp of phlebotomized mice by injection with dichloromethylene diphosphonate encapsulated in multilamellar liposomes (CL2MDP-liposome). In the spleen, no erythropoietic activity occurred until 5 days after the treatment. Colony assay revealed that the erythropoiesis was suppressed at the level of CFU-E. The splenic erythropoietic activity gradually developed from day 6 after the treatment, when F4/80+ macrophages began to appear in the red pulp. EPO mRNA was expressed in kidney but not in liver or spleen of phlebotomized mice injected with CL2MDP-liposome, and the serum EPO concentration in these mice was higher than that in phlebotomized mice. These findings suggest that abrogation of stromal macrophages by injection with CL2MDP-liposome impairs the splenic microenvironment for erythropoiesis induced by hypoxic stress, and this may be an excellent experimental model for further characterization of the in vivo role of splenic macrophages in erythropoiesis.

Effects of intermittent exposure to high altitude on blood volume and erythropoietic activity.

The purpose of this review is to describe changes in blood volume and erythropoietic activity occurring under different types of intermittent exposure to hypoxia. These hypoxic episodes can vary from a few seconds or minutes to hours, days, or even weeks. Short hypoxic episodes like sleep apnea only lead to a small increase in hemoglobin concentration, which is mainly due to a hormonal-mediated decrease in plasma volume. In most of these cases the cumulative time spent under hypoxia does not exceed the critical threshold of about 90 min. Endurance athletes and mountaineers who voluntarily expose themselves to hypoxia for some hours or during the night while spending the day at normoxia ("sleep high-train low" concept) do improve their physical performance. Despite raising erythropoietic activity, indicated by elevated plasma concentrations of EPO and the transferrin receptor, the postulated increase in red cell volume has not satisfactorily been proved. Frequent changes between low and high altitudes, which are usual in some South American and Asian countries, provoke similar adaptations in red cell mass as occur in high altitude residents. However, the plasma volume decreases at altitude and increases again when staying at sea level. Even after more than 20 yr of regular moving between low and high altitude, the total blood volume, hemoglobin concentration and hematocrit, as well as the plasma EPO concentration, noticeably oscillate during every hypoxic-normoxic cycle. We assume these changes to be an optimal rapid adaptation of the oxygen transport system to the prevailing hypoxic or normoxic environment. However, possible risks for the organism cannot be excluded.

Effects of exercise on soluble transferrin receptor and other variables of the iron status

Background: Soluble transferrin receptor (sTfr) is a new marker of iron status and erythropoietic activity. It has been included in multivariable blood testing models for the detection of performance enhancing...

Determinants of circulating soluble transferrin receptor level in chronic haemodialysis patients.

The aim of this study was to identify the factors determining the circulating soluble transferrin receptor (sTfR) concentrations in haemodialysis (HD) patients on maintenance recombinant human erythropoietin (rHuEpo) treatment. In a prospective cross-sectional study, 91 chronic HD patients and 18 anaemic controls with normal renal function were recruited. For each subject, blood samples were measured for complete blood count, reticulocyte count, percentage of hypochromic red cells (% HRC), serum ferritin, serum iron, transferrin saturation (TS), serum erythropoietin (sEpo), C-reactive protein (CRP), and sTfR. HD patients received constant rHuEpo doses and basal sEpo was measured > or = 86 h after the last injection. The age, gender, dialysis vintage, and the above-mentioned parameters were used as independent variables and logarithmic sTfR (log(10)sTfR) as a dependent variable in the forward stepwise multiple regression model. HD patients were similar to controls regarding haematocrit, serum ferritin, TS, and % HRC, but had significantly lower sTfR, sEpo, and reticulocyte index. Univariate analyses showed that the sTfR level strongly correlated with sEpo (r=0.60, P<0.001) and % HRC (r=0.60, P<0.001), and significantly with serum ferritin (r=-0.29, P<0.01), TS (r=-0.27, P<0.05), and dose of rHuEpo administered (r=0.27, P<0.05) in HD patients. sTfR also had a positive correlation with haematocrit (r=0.26, P<0.05), red blood cell (RBC) count (r=0.23, P<0.05), and reticulocyte count (r=0.24, P<0.05), but not with CRP (r=0.16, P>0.05). Multivariate regression analysis disclosed that sEpo, HRC, and serum ferritin were the independent predictors of sTfR level. Overall, the model explained 58.8% of the variability in sTfR (R(2)=0.588, P<0.001). Circulating sTfR is a good index of marrow erythropoietic activity in HD patients during rHuEpo treatment. Its level is also independently up-regulated by functional iron deficiency in the process of enhanced erythropoiesis. Our study showed that sTfR levels quantitatively reflect the integrated effects of iron availability, iron reserves, and erythropoietic stimulation.

Cloning, characterization and chromosome mapping of the human SMAP1 gene

Stromal membrane associated protein (smap-1) is a new murine cell surface molecule on the stromal cells. The murine smap-1 protein is induced in stromal cells by the contact with erythroid cells, which suggests that this protein may be involved in the haematopoietic progenitor cells to stromal cells interactions.Here we report the structure, map location and expression analysis of the human SMAP1 gene, which cover approximately 100 kb on chromosome 6 between D6S455 and D6S1673 markers. This gene is composed of 11 exons and encodes a 468-amino-acid protein, which shows an 86% of homology with the murine smap-1 protein. The expression of smap-1 in erythropoietic organs as well as the correlation with the erythropoietic activity of the haematopoietic organs suggest that smap-1 is induced in stromal cells by the contact with erythroid cells, defining smap-1 as a key molecule that induced an erythropoietic microenvironment in haematopoietic organs. The high sequence conservation between murine and human SMAP1, as well as its expression in bone marrow, strongly suggest conserved functions of this protein in both organisms. Recently, a constitutional translocation t(6;10)(q13;q22) has been described in a patient with severe aplastic anaemia. SMAP1 gene localizes to 6q13 and is probably implicated in erythropoiesis, therefore it remains as an interesting candidate gene.

Optimizing the use of erythropoietic agents-- pharmacokinetic and pharmacodynamic considerations.

Erythropoietic agents have transformed the management of renal anaemia. Two forms of recombinant human erythropoietin (rHuEPO) have been available since the early 1990s, and more recently, a second-generation erythropoietic agent, darbepoetin alfa, has been granted a licence for treating this condition. The endogenous erythropoietin molecule may contain anything from 4 to 14 sialic acid residues. The various isoforms have different biological potencies in vivo owing to their different metabolic clearance rates. However, there is a continuing debate about those organs potentially involved in the metabolism of the erythropoietin molecule; the kidneys, liver, and bone marrow have all been suggested as possible participants. Darbepoetin alfa contains two extra N-linked glycosylation consensus sequences increasing the potential maximum number of sialic acid residues from 14 up to 22. In vitro, the affinity of darbepoetin alfa for the erythropoietin receptor is less than for the natural ligand, but this is more than compensated for by the increased potency in vivo. One of the most important factors determining the biological activity of erythropoiesis-stimulating agents is the length of time that the serum concentration of the protein remains above the threshold necessary for erythropoiesis. The pharmacokinetic profile of darbepoetin alfa is distinct from that of rHuEPO, the major difference being the much longer elimination half-life and slower clearance in vivo of darbepoetin alfa leading to prolonged erythropoietic activity. Stimulation of erythropoiesis depends both on an ambient circulating level of erythropoietin and on the mechanisms governing the interaction of the hormone with its receptor. Our knowledge regarding the latter is limited and it is difficult to predict the optimum frequency of administration for an erythropoietic agent. In general, rHuEPO is given two or three times weekly. A small number of studies have supported the once-weekly use of rHuEPO. All clinical trials so far conducted on darbepoetin alfa have demonstrated success with once-weekly and once every other week dosing.

Iron and erythropoietin in renal disease.

Our knowledge of erythropoiesis and iron in renal disease is limited. The accepted view of the control of erythropoiesis was founded on observations made in a variety of disorders, but the control mechanism in healthy individuals may not be quite the same. Evidence suggests that mechanisms other than erythropoietic stimulation may play a role in increased red blood cell production. Measuring erythropoiesis is complex. The quantitative reticulocyte count is probably the closest practical assessment of erythropoietic activity we can achieve, yet there is very little correlation between circulating erythropoietin level and reticulocyte count in normal and near normal subjects. Oxygen transport in humans depends entirely upon iron. In renal disease, the failure of the erythropoietin positive feedback mechanism can be readily and directly remedied; recombinant human erythropoietin therapy can replace the missing erythropoietin, but this will be negated if iron supply to the erythroid marrow falls short of demand. Measurement of iron stores is also complex. The use of serum ferritin concentration as a direct quantitative estimate of iron in the stores is not advisable, and in practice we have not found the transferrin receptor assay to be useful in identifying patients who require iron therapy. Use of percentage hypochromia as a measure of iron deficiency is complicated by the fact that hypochromic cells are not exclusively a consequence of functional iron deficiency. There are clearly lessons still to be learned in this field and there is much that we do not yet understand about the control of erythropoiesis and iron metabolism in humans.

Evaluation of intestinal iron absorption by indirect methods in patients on hemodialysis receiving oral iron and recombinant human erythropoietin.

Intestinal iron absorption was evaluated in 40 patients on hemodialysis therapy treated over 4 months with 105 mg/d of oral iron and recombinant human erythropoietin (rHuEPO). The effect of iron stores, erythropoietic activity (EA), and route of rHuEPO administration on absorption was evaluated. Iron was administered after basal determinations had been made and was stopped 15 days before obtaining the final determinations. Intestinal iron absorption was calculated by summing the increase in hemoglobin (Hb) iron (iron used for the synthesis of new Hb) and variations in estimated tissue iron reserves (reserves at the end of the study minus basal reserves). Markers of EA included soluble transferrin receptors (sTfRs) and erythron transferrin uptake (ETU). Iron losses caused by dialysis or normal obligatory iron losses were not measured. Hb levels increased from 8.38 +/- 1.4 to 10.75 +/- 1.5 g/dL (P < 0.05). sTfR levels reached their maximum value at 45 days (3.22 +/- 0.84 mg/mL; P < 0.05), and ETU increased from 40 +/- 26 to 61 +/- 39 micromol/L whole blood/d (P = 0.007). Intestinal iron absorption was 238 mg (interquartile range [Q75 to Q25], 255) at 2 months and 348 mg (Q75 to Q25, 475) at 4 months (P =0.02) and correlated positively with hematocrit at the end of the study (r = 0.826; P = 0.0001). No relationship between iron absorption and basal serum ferritin level or EA markers was observed. Intestinal iron absorption was similar regardless of the route of rHuEPO administration. In conclusion, intestinal iron absorption from medicinal iron covers erythropoietic requirements and allows Hb levels to increase significantly. It is proportional to the degree of efficient erythropoiesis reached and independent of tissue iron stores present before treatment, markers of EA, and rHuEPO administration route.

Cord serum ferritin levels, fetal iron status, and neurodevelopmental outcomes: Correlations and confounding variables

Cord serum ferritin levels, fetal iron status, and neurodevelopmental outcomes: Correlations and confounding variables

Effect of liposomal muramyl tripeptide phosphatidylethanolamine and indomethacin on hematopoietic recovery in irradiated mice

The effects of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE/MLV, radioprotective immunomodulator; 10 mg/kg) and indomethacin (INDO, inhibitor of prostaglandin production; 2 mg/kg) on post-irradiation recovery of hematopoietic functions in mice were investigated. Two agents with distinct radioprotective mechanisms were administered alone or in combination 24 h and 3 h before exposure to 7 Gy 60Co radiation. In the post-irradiation period (3-14 days) combined pre-treatment of mice accelerated recovery of bone marrow cellularity, weight of spleen and myelopoietic and erythropoietic activity in both hematopoietic organs, compared to treatment with MTP-PE/MLV or indomethacin alone. In the peripheral blood, improved radioprotective effects of combined drug administration were found in the recovery of reticulocytes and platelet count. No further significant differences in the recovery of leukocyte count were observed in the examined groups until post-irradiation day 14. Within the first 3-6 post-irradiation days, the bone marrow and peripheral blood smears of mice pre-treated with indomethacin alone or its combination with MTP-PE/MLV more frequently featured blast cells and large cells with abundant cytoplasm which could be considered the hematopoietic stem cells.

Serum transferrin receptor concentration indicates increased erythropoiesis in Kenyan children with asymptomatic malaria

Serum transferrin receptor concentrations indicate both erythropoietic activity and the deficit of functional iron in the erythron. In contrast with serum ferritin concentrations, serum transferrin receptor concentrations are not or are only marginally influenced by the inflammatory response to infection. We assessed iron status and examined the relation between serum transferrin receptor concentrations and malaria in children aged 2-36 mo who were asymptomatic for malaria. This was a community-based cluster survey (n = 318). Prevalences of malaria, anemia (hemoglobin concentration <110 g/L), iron deficiency (serum ferritin concentration <12 microg/L), and iron deficiency anemia were 18%, 69%, 53%, and 46%, respectively. Malaria was associated with lower mean hemoglobin concentrations (92.7 compared with 104.1 g/L; P = 0.0001) and higher geometric mean serum concentrations of transferrin receptor (11.4 compared with 7.8 mg/L; P = 0.005), ferritin (21.6 compared with 11.9 microg/L; P = 0.05), and C-reactive protein (12.5 compared with 6.8 mg/L; P = 0.004). There was no evidence for an association between serum concentrations of C-reactive protein and transferrin receptor. Children with malaria had higher serum transferrin receptor concentrations than expected for the degree of anemia, even after adjustment for inflammation indicated by serum C-reactive protein concentration quartiles (P = 0.02). Our findings are consistent with the notion that malaria-induced hemolysis is accompanied by increased erythropoiesis. Serum transferrin receptor concentration is not useful for detecting iron deficiency in individuals with malaria. Individuals with high concentrations of serum C-reactive protein or similar acute phase reactants should be excluded from analysis if serum ferritin concentrations <12 microg/L are to be used to measure iron deficiency in malaria-endemic areas.