neptune, a Kruppel-like transcription factor that participates in primitive erythropoiesis in Xenopus

The specification of the erythroid lineage from hematopoietic stem cells requires the expression and activity of lineage-specific transcription factors. One transcription factor family that has several members involved in hematopoiesis is the Kruppel-like factor (KLF) family [1]. For example, erythroid KLF (EKLF) regulates beta -globin expression during erythroid differentiation [2-6]. KLFs share a highly conserved zinc finger-based DNA binding domain (DBD) that mediates binding to CACCC-box and GC-rich sites, both of which are frequently found in the promoters of hematopoietic genes. Here, we identified a novel Xenopus KLF gene, neptune, which is highly expressed in the ventral blood island (VBI), cranial ganglia, and hatching and cement glands. neptune expression is induced in response to components of the BMP-4 signaling pathway in injected animal cap explants. Similar to its family member, EKLF, Neptune can bind CACCC-box and GC-rich DNA elements. We show that Neptune cooperates with the hematopoietic transcription factor XGATA-1 to enhance globin induction in animal cap explants. A fusion protein comprised of Neptune's DBD and the Drosophila engrailed repressor domain suppresses the induction of globin in ventral marginal zones and in animal caps. These studies demonstrate that Neptune is a positive regulator of primitive erythropoiesis in Xenopus.

Less frequent dosing of erythropoiesis stimulating agents in patients undergoing dialysis: a European multicentre cost study.

OBJECTIVE: To calculate the variable costs involved with the process of delivering erythropoiesis stimulating agents (ESA) in European dialysis practices. METHODS: A conceptual model was developed to classify the processes and sub-processes followed in the pharmacy (ordering from supplier, receiving/storing/delivering ESA to the dialysis unit), dialysis unit (dose determination, ordering, receipt, registration, storage, administration, registration) and waste disposal unit. Time and material costs were recorded. Labour costs were derived from actual local wages while material costs came from the facilities' accounting records. Activities associated with ESA administration were listed and each activity evaluated to determine if dosing frequency affected the amount of resources required. RESULTS: A total of 21 centres in 8 European countries supplied data for 142 patients (mean) per hospital (range 42-648). Patients received various ESA regimens (thrice-weekly, twice-weekly, once-weekly, once every 2 weeks and once-monthly). Administering ESA every 2 weeks, the mean costs per patient per year for each process and the estimates of the percentage reduction in costs obtainable, respectively, were: pharmacy labour (10.1 euro, 39%); dialysis unit labour (66.0 euro, 65%); dialysis unit materials (4.11 euro, 61%) and waste unit materials (0.43 euro, 49%). LIMITATION: Impact on financial costs was not measured. CONCLUSION: ESA administration has quantifiable labour and material costs which are affected by dosing frequency.

Circulating microRNAs as long-term biomarkers for the detection of erythropoiesis-stimulating agent abuse.

MicroRNAs (miRNAs) are small, non-protein coding transcripts involved in many cellular and physiological mechanisms. Recently, a new class of miRNA called 'circulating miRNAs' was found in cell-free body fluids such as plasma and urine. Circulating miRNAs have been shown to be very stable, specific, and sensitive biomarkers. In this paper, we investigate whether circulating miRNAs can serve as biomarkers for erythropoiesis-stimulating agent abuse. To this end, we analyzed miRNA levels in plasma by miRNA microarrays and quantitative real-time polymerase chain reaction (PCR). Plasma samples are derived from a clinical study with healthy subjects injected with erythropoiesis-stimulating agent (C.E.R.A.). Based on microarray results, we observed a significant difference in the levels of miRNAs in plasma after C.E.R.A. injection. We demonstrated that a specific miRNA, miR-144, exhibit a high increase that lasts 27 days after C.E.R.A. stimulation. Considering the fact that miR-144 is an essential erythropoiesis agent in different organisms, these findings suggest the possibility of using miR-144 as a sensitive and informative biomarker to detect C.E.R.A. abuse. Copyright © 2011 John Wiley & Sons, Ltd.

Improving anemia management with less frequently dosed erythropoiesis stimulating agents

Introduction and Aims: The process of delivering erythropoiesis stimulating agents (ESAs) to hemodialysis patients (HD) is complex. Many European countries are requiring centers to document this process. To date, there has not been any comprehensive description of the operational aspects of ESA delivery in Europe. The objective of the Mercurius study was to describe the entire process of ESA delivery in dialysis centers. In addition, we explored the benefits of less frequent dosing. Methods: A conceptual model was developed to classify the sub-processes in the pharmacy, dialysis unit, waste unit, and back office. Within each dialysis unit activities associated with dose determination, ordering procedures, receipt and storage of ESAs, and ESA administration were measured. Within the pharmacy, ordering from supplier, receiving and storing, and delivering ESA to the dialysis unit were measured. The amount of time and materials associated with waste disposal and back office activities were also observed. We also evaluated the impact of less frequent dosing on the resources required to perform anemia management for HD patients. Structured interviews with staff were used to develop a comprehensive list of processes, sub-processes, and activities that are routinely followed to order, register, administer, and dispose of waste associated with ESAs. Each activity was evaluated to determine if less frequent dosing influenced the amount of resources required. A model was developed to estimate the change in resources consumed using less frequent dosing regimens. Results: Eight centers from 5 European countries (Belgium, France, Italy, Sweden, and Switzerland) participated in the study. The number of HD patients in each center ranged from 42 to 707 (mean=175). Across all of the centers, patients received a variety of dosing regimens (eg, TIW, BIW, QW and Q2W). The mean (±SD) time spent for the pharmacy to order an ESA from the supplier was 6.1 (±8.7) minutes; time spent in the dialysis unit and pharmacy for receiving and storing ESPs was 5.3 (±5.3) and 10.0 (±10.9) minutes, respectively; and time spent administering each injection was 6.4 (±6.5) minutes. Switching from current dosing practices to Q2W could decrease the mean number of syringes used from 12,420 to 5,085 per year. We estimate a reduction in the number of disinfective tissues and liquids of 58% and 71%, respectively by switching from current practice to dosing ESAs Q2W. Conclusions: There was significant variation in the time that it takes to perform routine ESA activities. We estimate that a reduction in resources required to manage anemia can be obtained by reducing the frequency of administration from the current mix of ESAs. These resources could be redeployed for patient care.

Detection of erythropoiesis-stimulating agents in human anti-doping control: past, present and future.

Stimulation of erythropoiesis is one of the most efficient ways of doping. This type of doping is advantageous for aerobic physical exercise and of particular interest to endurance athletes. Erythropoiesis, which takes place in bone marrow, is under the control of EPO, a hormone secreted primarily by the kidneys when the arterial oxygen tension decreases. In certain pathological disorders, such as chronic renal failure, the production of EPO is insufficient and results in anemia. The pharmaceutical industry has, thus, been very interested in developing drugs that stimulate erythropoiesis. With this aim, various strategies have been, and continue to be, envisaged, giving rise to an expanding range of drugs that are good candidates for doping. Anti-doping control has had to deal with this situation by developing appropriate methods for their detection. This article presents an overview of both the drugs and the corresponding methods of detection, and thus follows a roughly chronological order.

Iron supply for erythropoiesis in the rabbit

Marrow radioiron uptake and marrow blood flow were measured in order to evaluate iron supply for erythropoiesis. Normal, phenylhydrazine-treated and bled animals were studied. The plasma iron turnover of seven normal rabbits was 1.49 +/- 0.22 mg/dl whole blood per d, of 11 rabbits treated 4 d before with phenylhydrazine was 5.16 +/- 1.81, and of four bled animals the plasma iron turnover was 3.75 +/- 1.61. The cardiac output and the percentage of blood flow to the marrow was increased in phenylhydrazine-treated and bled animals. Marrow iron flow in phenylhydrazine-treated animals was 38.3 +/- 32.6 micrograms/min per kg as compared with control values of 7.0 +/- 1.3 (P less than 0.01). This was due to an increase in marrow flow, an increase in plasma iron, and an increase in plasmatocrit. In bled animals, in spite of an increased marrow blood flow, marrow iron flow of 7.3 +/- 2.2 was similar to that of control animals due to a lower plasma iron concentration. The calculated marrow iron extraction of 3.7 +/- 2.4% in phenylhydrazine-treated animals was not different from that of control animals of 4.3 +/- 1.1, whereas extraction was increased in bled animals to 7.9 +/- 1.3 (P less than 0.01). In additional studies of transfused animals, acutely induced anemia was associated with an increased cardiac output, but also with a relative decrease in marrow flow, which left marrow iron supply unaffected. It would appear from these studies that an important mechanism for meeting the increased iron requirement of the hyperplastic erythroid marrow is an increase in marrow blood flow.

Cell-Cell interaction in erythropoiesis: role of human monocytes

Erythroid burst forming units (BFU-E) are proliferative cells present in peripheral blood and bone marrow which may be precursors of the erythroid colony forming cell found in the bone marrow. To examine the possible role of monocyte-macrophages in the modulation of erythropoiesis, the effect of monocytes on peripheral blood BFU-E proliferation in response to erythropoietin was investigated in the plasma clot culture system. Peripheral blood mononuclear cells from normal human donors were separated into four fractions. Fraction-I cells were obtained from the interface of Ficoll-Hypaque gradients (20-30% monocytes; 60-80% lymphocytes); fraction-II cells were fraction-I cells that were nonadherent to plastic (2-10% monocytes; 90-98% lymphocytes); fraction-III cells were obtained by incubation of fraction-II cells with carbonyl iron followed by Ficoll-Hypaque centrifugation (>99% lymphocytes); and fraction-IV cells represented the adherent population of fraction-II cells released from the plastic by lidocaine (>95% monocytes). When cells from these fractions were cultured in the presence of erythropoietin, the number of BFU-E-derived colonies was inversely proportional to the number of monocytes present (r = ¿0.96, P < 0.001). The suppressive effect of monocytes on BFU-E proliferation was confirmed by admixing autologous purified monocytes (fraction-IV cells) with fraction-III cells. Monocyte concentrations of ¿20% completely suppressed BFU-E activity. Reduction in the number of plated BFU-E by monocyte dilution could not account for these findings: a 15% reduction in the number of fraction-III cells plated resulted in only a 15% reduction in colony formation. These results indicate that monocyte-macrophages may play a significant role in the regulation of erythropoiesis and be involved in the pathogenesis of the hypoproliferative anemias associated with infection and certain neoplasia in which increased monocyte activity and monopoiesis also occur.

Interplay of vitamin D, erythropoiesis, and the renin-angiotensin system.

For many years deficiency of vitamin D was merely identified and assimilated to the presence of bone rickets. It is now clear that suboptimal vitamin D status may be correlated with several disorders and that the expression of 1-α-hydroxylase in tissues other than the kidney is widespread and of clinical relevance. Recently, evidence has been collected to suggest that, beyond the traditional involvement in mineral metabolism, vitamin D may interact with other kidney hormones such as renin and erythropoietin. This interaction would be responsible for some of the systemic and renal effects evoked for the therapy with vitamin D. The administration of analogues of vitamin D has been associated with an improvement of anaemia and reduction in ESA requirements. Moreover, vitamin D deficiency could contribute to an inappropriately activated or unsuppressed RAS, as a mechanism for progression of CKD and/or cardiovascular disease. Experimental data on the anti-RAS and anti-inflammatory effects treatment with active vitamin D analogues suggest a therapeutic option particularly in proteinuric CKD patients. This option should be considered for those subjects that are intolerant to anti-RAS agents or, as add-on therapy, in those already treated with anti-RAS but not reaching the safe threshold level of proteinuria.

Strong iron demand during hypoxia-induced erythropoiesis is associated with down-regulation of iron-related proteins and myoglobin in human skeletal muscle

[EN] Iron is essential for oxygen transport because it is incorporated in the heme of the oxygen-binding proteins hemoglobin and myoglobin. An interaction between iron homeostasis and oxygen regulation is further suggested during hypoxia, in which hemoglobin and myoglobin syntheses have been reported to increase. This study gives new insights into the changes in iron content and iron-oxygen interactions during enhanced erythropoiesis by simultaneously analyzing blood and muscle samples in humans exposed to 7 to 9 days of high altitude hypoxia (HA). HA up-regulates iron acquisition by erythroid cells, mobilizes body iron, and increases hemoglobin concentration. However, contrary to our hypothesis that muscle iron proteins and myoglobin would also be up-regulated during HA, this study shows that HA lowers myoglobin expression by 35% and down-regulates iron-related proteins in skeletal muscle, as evidenced by decreases in L-ferritin (43%), transferrin receptor (TfR; 50%), and total iron content (37%). This parallel decrease in L-ferritin and TfR in HA occurs independently of increased hypoxia-inducible factor 1 (HIF-1) mRNA levels and unchanged binding activity of iron regulatory proteins, but concurrently with increased ferroportin mRNA levels, suggesting enhanced iron export. Thus, in HA, the elevated iron requirement associated with enhanced erythropoiesis presumably elicits iron mobilization and myoglobin down-modulation, suggesting an altered muscle oxygen homeostasis.

Twist and shout: one decade of meta-analyses of erythropoiesis-stimulating agents in cancer patients

Anemia associated with cancer and cancer therapy is a common and important issue in the treatment of patients with malignant disease. Conventionally, blood transfusions are used to treat severe cancer-related anemia. Short- and long-acting preparations of recombinant human erythropoiesis-stimulating agents (ESAs) offer an alternative treatment option. Multiple studies and subsequent meta-analyses have demonstrated that ESA treatment increases hemoglobin levels and reduces the likelihood of transfusion for a proportion of treated patients. However, studies that attempted to evaluate whether ESAs improve tumor response and survival have generated conflicting evidence. Results of smaller trials reporting improved survival outcomes were contradicted by large randomized controlled trials that reported more deaths in patients receiving ESAs. In addition, there is strong evidence that cancer patients receiving ESAs have an increased risk of thromboembolic and cardiovascular events. We herein review the main meta-analyses published in the field, their strengths and weaknesses, their contribution to patient management and future perspectives for systematic reviews.

Ten years of meta-analyses on erythropoiesis-stimulating agents in cancer patients

Since erythropoiesis-stimulating agents (ESAs) were licensed in 1993, more than 70 randomized controlled trials and more than 20 meta-analyses and systematic reviews on their effectiveness were conducted. Here, we present a systematic review on the meta-analyses of trials evaluating ESAs in cancer patients.

Is intravenous iron supplementation with erythropoiesis-stimulating agents beneficial in cancer patients with anemia?

Chemotherapy-induced anemia is often an important problem for cancer patients, and this complication can be treated with erythropoiesis-stimulating agents (ESAs). This commentary discusses the findings of a study by Bastit et al., in which 396 patients with nonmyeloid malignancies and chemotherapy-induced anemia were treated with darbepoetin alfa with or without intravenous iron. This phase III trial showed that intravenous iron supplementation increases the hematopoietic response rates to ESAs in cancer patients; however, this study provides no information as to whether all cancer patients with anemia should receive intravenous iron as well as treatment with ESAs. Further data are needed to identify those patients who might benefit from intravenous iron supplementation in addition to ESAs, in order to avoid overtreatment of patients who are unlikely to benefit from the additional iron. As both ESAs and intravenous iron have known short-term and long-term risks, identification of reliable predictors of response that can guide these treatments is necessary before this strategy can be implemented into practice.