Red Cell Production Research Articles

Relative Efficiency of Leucocyte Removal Procedures for the Production of Leucocyte-Poor Red Cell Concentrates Assessed by Flow Cytometry

Flow cytometry was used to: (1) determine residual leucocyte numbers in red cell suspensions following the range of leucocyte depletion procedures used in our organisation, and (2) to characterise phenotypically the leucocytes using direct immuofluorescence with monoclonal antibodies to cell surface receptors. Under the conditions used, a lower limit of detection of 2.5 leucocytes per µl (equivalent to 3.43 log(10) or 99.96% removal) could be achieved. Filtration through polyester filters was found to remove up to >99.96% of the initial leucocytes; however, a significant differential efficacy was observed between filters from different manufacturers even when filters with similar costs were compared. The order of filter brands with respect to leucocyte removal found was Pall BPF4 = Erypur Optima G-O > Sepacell R500 > Pall RC50. Pheno- typing revealed that increasing filtration efficacy was associated with a preferential removal of lymphocytes; conversely, a second filtration over one brand of filter allowed proportionately more lymphocytes to pass through compared with the first filtration. A saline wash following filtration removed a further 0.5% of the initial leucocyte content, and was associated with a preferential loss of granulocytes. Freeze-thawing the red cell suspension removed fewer leucocytes (96.3%) than did filtration (98.74% to >99.6%) or filtration followed by washing (99.22%), and also led to preferential loss of granulocytes. Flow cytometry provides a reliable tool for the quality control of leuco-depleted red cells, and allows a qualitative assessment of the residual leucocytes. This information is of value in choosing procedures aimed at decreasing the risk of alloimmunisation and post-transfusion reactions.

Loss of Red Blood Cell Viability Associated with Limited Thermal Inactivation of Extracellular HIV-1

The effects of incubation at mildly elevated temperatures on HIV-1 inactivation and in vitro red blood cell properties were investigated. Red cells (55% Hct) were leukodepleted (3 log(10)) by filtration, maintained at 45 or 47 °C for 4 or 8 h, and then stored at 4°C. Hemolysis was twice that of controls after 42-day storage for samples treated for 4h at 45°C, and five times larger for samples heated at 47°C. There was also a significant increase in the rate of potassium loss, an early decrease in ATP levels, and an initial drop in pH for samples treated at either temperature. Larger differences were observed for samples exposed to these elevated temperatures for 8 h. Osmotic deformability curves obtained by ektacytometry showed dramatic decreases in red cell deformability at both temperatures and for both time periods. HIV-1 inactivation in red cells treated at 45 °C (approximately 0.25 log(10)/h) was considerably less than that obtained in tissue culture medium (1-2 log(10)/h). Since the decrease in red cell deformability is likely to indicate reduced red cell function and survival, and the rate of HIV-1 inactivation is low, mild heat treatment is not an adequate process for viral inactivation of red cell products.

Recombinant Erythropoietin in Anemia of Prematurity: Five Years Later

In 1988, Dr James Stockman discussed the anemia of prematurity in a fascinating editorial that anticipated much of what has unfolded as therapeutic trials using recombinant human erythropoietin (r-HuEPO) were initiated. Dr Stockman began by addressing the implications of then-recent results from my laboratory and from that of Robert Christensen which demonstrated large numbers of committed erythroid progenitors in the blood and bone marrow of small premature babies that responded normally to r-HuEPO in vitro. These data effectively closed the pathophysiologic loop in anemia of prematurity and strongly implicated inadequate production of erythropoietin (EPO) as the underlying reason erythropoiesis is quantitatively insufficient in this clinical setting (see reference 4 for a review). By then, early studies had shown that treatment with r-HuEPO corrects the anemia associated with chronic renal failure, a disorder that resembles anemia of prematurity in that it is characterized by low EPO levels and inadequate red cell production.

Differences in the composition and in the efficiency of red cell production of normal and CML erythroid progenitor populations are highlighted by response to human c- kit ligand

Previous studies have suggested that erythroid progenitors derived from patients with chronic myelogenous leukemia (CML) in chronic phase may have reduced proliferative capacity. Considering recent evidence that mast cell growth factor (MGF) enhances the proliferative capacity of normal erythroid burst-forming units (BFU-E), we examined whether MGF could increase the proliferative potential of CML erythroid progenitors to normal capacity. To evaluate the total proliferative capacity achieved, the BFU-E were divided into four subpopulations (XL = extra large, L = large, M = medium, S = small) and colonies were aspirated to determine the cellularity of BFU-E from each subpopulation. MGF alone or in combination with MoT cell line conditioned medium (MoCM) or granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-3 (IL-3) significantly increased the proliferative capacity of erythropoietin (EPO) dependent CML and normal BFU-E. Although the total number of BFU-E generated were similar, the number of BFU-E with high proliferative potential were considerably less in CML BFU-E populations. BFU-E designated XL (129,000–431,000 cells) were only found in MGF cultures and only normal BFU-E had this proliferative capacity. BFU-E designated L were increased in both normal and CML BFU-E populations but less CML BFU-E had this proliferative capacity (mean number 25% of normal) and CML L BFU-E from 2 3 CML patients comprised fewer cells than normal L BFU-E. Normal BFU-E populations comprised 16–24% high proliferative BFU-E (XL + L) in contrast to 4–5% high proliferative BFU-E (L only) comprising CML BFU-E populations. This shift resulted in 78–87% of CML erythroid cells being generated by low proliferative BFU-E in contrast to the normal state in which 63–71% of erythroid cells are generated by high proliferative BFU-E. A possible explanation for the difference in the final effect of the Ph 1 translocation on the granulocyte and erythroid lineages is proposed.

A System Approach to Pharmacodynamics. Plasma Iron Mobilization by Endogenous Erythropoietin in the Sheep Fetus; Evidence of Threshold Response in Spontaneous Hypoxemia

At present, nearly all Infants with birth weights of <1kg receive blood transfusions for treatment of clinical signs of tissue hypoxia resulting from anemia of prematurity. In contrast to the successful use of recombinant human erythropoietin (rhEp) in adults, treatment of anemic neonates with rhEp to stimulate red cell production and reduce the need for transfusions that pose serious infectious and immunologic risk has not been effective. The present study investigates the pharmacodynamics (PD) of endogenous erythropoietin (Ep) in sheep fetuses to determine possible causes for the poor rhEp response in early development. The dynamic relationship between plasma Ep and plasma iron resulting from spontaneous hypoxemic episodes is investigated by PD system analysis. The erythropoietic effect of Ep is measured in terms of the mobilization of plasma Iron needed in the production on new erythrocytes. A hysteresis minimization approach is employed to determine the intrinsic PD dose-response relationship (transduction) of Ep. The dose—response relationship shows a well-defined threshold level that has to be exceeded before Ep begins to show a significant effect on plasma iron. It is postulated that the threshold mechanism may serve a useful purpose during early development by reducing the risk of the fetus developing a pathological degree of polycythemia and hyperviscosity in the relatively hypoxemic fetal environment. At the same time, the threshold serves the purpose of providing a needed response to more severe pathologic hypoxemic episodes. The occurrence of anemia during subsequent postnatal life when Pa02 levels increase markedly may be the inevitable, but unfortunate corollary of a continuation of this mechanism. If the poor response to rhEp in early neonatal life is due to a threshold mechanism, then it will be necessary to administer Ep at significantly larger dosing rates to ensure a sufficient and sustained coverage above the threshold level.

Cryopreservation of red blood cells: effect of freezing on red cell quality and residual lymphocyte immunogenicity.

To investigate treatment with glycerol/washing as a potential substitute for freeze-thawing in the production of leucocyte depleted red cell concentrates for patients with a history of non-haemolytic reactions following transfusion. The standard procedure of treatment with glycerol/-80 degrees C freezing/thawing/washing was compared with a similar procedure in which freezing was omitted. The quality of the resulting red cell products was assessed in relation to: (1) standard red cell biochemical parameters; (2) leucocyte and lymphocyte subset composition using flow cytometry with fluorescent labelled monoclonal antibodies; and (3) immunogenicity of the residual lymphocytes in mixed lymphocyte culture. Compared with red cells subjected to the standard freeze-thaw technique, red cells undergoing the non-freezing procedure and suspended in additive solutions had significantly better biochemical preservation after 21 days of storage (p < 0.001). Both procedures removed an average 98% of the initial leucocytes at the expense of 18-20% of the red cells. The non-freezing procedure resulted in higher residual concentrations of HLA class II bearing lymphocytes (p < 0.01), but not higher numbers of dendritic cells. Both procedures were equally effective in annulling the residual lymphocytes' ability to act as stimulator cells in one-way mixed lymphocyte culture. The non-freezing procedure produces a superior product for the provision of red cells to patients with granulocyte antibodies. These products may also offer a lower risk of HLA alloimmunisation to previously unexposed patients.

Erythropoietin, protein, and iron supplementation and the prevention of anaemia of prematurity.

The effectiveness of recombinant human erythropoietin (r-HuEpo) in raising haemoglobin concentrations in very low birthweight infants was examined in a randomised multicentre study. Twenty nine 'healthy' appropriate for gestational age infants with birth weights 900-1400 g entered the study at 3 weeks of age. All infants received breast milk supplemented with 9 g/l human breast milk protein from 3 to 8 weeks of age. Eighteen mg iron was given daily from week 3 and was doubled if serum iron concentration fell below 16.0 mumol/l. Fourteen infants were randomised to receive 100 U/kg r-HuEpo subcutaneously three times a week from week 3 to week 7; 15 infants served as controls. After one week reticulocyte and haemoglobin concentrations were significantly higher in the r-HuEpo treated group and the haemoglobin values remained significantly higher throughout r-HuEpo treatment and at the concentrations observed in full term infants. No adverse effects were associated with the treatment. In stable very low birthweight infants with optimal iron and protein intakes, moderate dose r-HuEpo can produce significant gains in red cell production that may be clinically useful.

The effect of methyldopa and procainamide on suppressor cell activity in relation to red cell autoantibody production

Kirtland et al (1980) suggested that methyldopa caused the production of red cell (RBC) autoantibodies by causing a persistent increase in lymphocyte cyclic AMP, which inhibited suppressor T cell function, leading to unregulated autoantibody production in some patients. They showed that significantly higher lymphocyte cyclic AMP concentrations were generated by lymphocytes from healthy donors after adding methyldopa, and by lymphocytes from patients who were receiving methyldopa compared to lymphocytes from healthy donors without methyldopa present. They also showed that methyldopa affected suppressor cell activity. We measured the effect of methyldopa and procainamide on suppressor cell activity, using a similar approach to Kirtland et al (1980). Suppressor cell activity was measured by measuring the amount of IgG, produced in vitro, by B cells following mitogen stimulation preceded by a 24 h incubation period. We found no significant increase in the amount of IgG generated by normal donor lymphocytes, when methyldopa or procainamide was present during the preincubation period. This is in contrast to the findings of Kirtland et al (1980). We also measured the amount of IgG generated in vitro by mitogen-stimulated lymphocytes from patients (with and without positive direct antiglobulin tests) taking methyldopa and compared this to the amount of IgG generated by lymphocytes from normal donors and patients (with and without positive direct antiglobulin tests). The results were similar for each group. This does not agree with the findings of Kirtland et al (1980) who found that lymphocytes from patients taking methyldopa produced more IgG in vitro than lymphocytes from normal donors. Our results do not support the hypothesis that methyldopa and procainamide induce autoantibodies by affecting suppressor cell function.

Transcription factor GATA-1 and erythroid development.

In summary, we derived an experimental system that allows us to dissect the function of GATA-1 in red cell development at a genetic level. We have established the essential nature of GATA-1 during both primitive and definitive erythropoiesis. By ablating the expression of the endogenous GATA-1 gene, we are in a position to introduce a variety of constructs that harbor subtle modifications in flanking or protein-coding sequences. We can now study regulatory regions and functional domains of the protein in the context of a true erythroid environment, experiments that have not been possible heretofore. Although the assay involves the dramatic loss of red cell production, it should be possible to define important regulatory domains that can then be assayed using less stringent systems, such as cell-free extracts for in vitro transcription. The ideal situation would be analyses conducted in GATA-1- erythroid cells. However, these cells have been impossible to generate given the requirement of GATA-1 for Epo receptor expression and red cell viability (C. Simon and S. Orkin, unpublished observations). It may be possible to produce such cells by first expressing the Epo receptor under the influence of a constitutive promoter and then targeting the GATA-1 gene. If GATA-1- red cells were available, the analyses would involve the actual transcription of or chromatin structure surrounding the globin genes. Structure-function studies of the GATA-1 protein could be greatly simplified and a larger number of mutants studied. However, the ES cell system can be used as an alternative until targeted erythroleukemia cells become available. Other applications involve the introduction of other GATA-binding protein family members to determine whether they rescue the mutation. If they cannot, chimeric proteins can be tested to identify which amino acids distinguish the different family members. We feel that these experiments are vital to understanding the function of GATA-1 during erythroid ontogeny. How does GATA-1 regulate red cell genes like globin or the Epo receptor? Once we identify the functional domains of the GATA-binding proteins, we hope to learn what proteins GATA-1 binds to in the basic transcription machinery or in chromatin. Is GATA-1 necessary for globin gene switching? GATA-1 may be modified differently during development so that the locus control region can interact with different globin promoters. We may find that one region of the protein is required for embryonic expression and another for adult globin gene expression.

Erythropoietin Does Not Cross the Placenta into the Fetus

Erythropoiesis in the fetus is controlled by erythropoietin (Ep). To determine the role of maternal Ep in this process, we used catheterized preparations of sheep and monkey fetuses to assess the ability of Ep administered to the mother to cross the placental barrier into the fetus. Ep was injected into pregnant sheep (3,600 IU/sheep) or monkeys (800-2,000 IU/animal) as a single intravenous dose, or into sheep in intravenous doses of 2,000 IU once every 12 h for a total of 4 injections. Maternal and fetal blood samples for Ep and reticulocyte determinations were obtained before and at intervals after Ep injections. The administration of Ep resulted in significant increases in maternal circulating Ep levels in sheep and monkeys. Despite the presence of high levels of maternal Ep, however, no increase in fetal plasma Ep levels was detected. The administration of Ep to the mother caused significant increases in reticulocyte production in the mother but not the fetus; injection of Ep directly to the fetus stimulated fetal erythropoiesis. These results demonstrate that Ep does not cross the placenta into the fetus even under conditions of chronically elevated maternal Ep levels, and suggest that red cell production in the fetus is regulated by Ep produced from sites within the fetus.

地方中規模病院におけるＭＳＢＯＳ，Ｔｙｐｅ ａｎｄ Ｓｃｒｅｅｎ導入の試み

To make the most of the blood products, we have introduced maximum blood order schedule (MSBOS) and Type and Screen (T & S) into Nagahama City Hospital. By investigating the transfused blood products according to major surgical procedures of last 2 years, we set up MSBOS and the procedure of blood preparations for elective surgery in our hospital.As a result of one year's experience, the cross-match to transfusion ratio (C/T ratio) in many surgical procedures and that of red cell products in our hospital have decreased, and the effective use of red cell products has elevated.However, because our hospital dose not have the blood products in deposit system and we can not return the products, it was impossible to decrease the out-date.

Thyroxine suppresses thrombocytopoiesis and stimulates erythropoiesis in mice.

Thyroxine has been shown in vitro to stimulate erythropoiesis by two mechanisms: a direct, beta 2-adrenergic receptor-mediated stimulation of red cell precursors, and an indirect, erythropoietin-mediated mechanism. Clinical reports have suggested that excess thyroxine also exerts depressive effects on thrombocytopoiesis, but the most sensitive methods of assessing platelet production, i.e., percentage of 35S incorporation into platelets and determination of megakaryocyte size and number, are not appropriate for analysis of platelet production in human patients. The purpose of this study was to use a mouse model to investigate the effects of the hyperthyroid state on erythropoiesis and thrombocytopoiesis, and to assess in vivo the two mechanisms by which thyroxine has been described to stimulate erythropoiesis in vitro. We found that thyroxine administration significantly depressed platelet production and stimulated erythropoiesis in mice. Both the D- and L-isomers of thyroxine in appropriate doses produced this depression of thrombocytopoiesis, and the effect was dose dependent for both isomers. Daily administration of thyroxine:increased blood volume; decreased the peripheral platelet count, total circulating platelet count and mass, percentage of 35S incorporation into platelets, and megakaryocyte number and size; and concurrently increased indices of red cell production (packed cell volume, red blood cell count, total circulating red blood cell count and mass, and reticulocyte count). Additionally, propranolol, a nonspecific beta-blocker, partially reversed the suppression of platelet production by L-thyroxine, lending credence to the assertion that the direct, beta 2-adrenergic receptor-mediated stimulation of the erythroid cell line by thyroxine reported to exist in vitro may also be important in vivo.

Erythropoiesis in multiple myeloma: defective red cell production due to inappropriate erythropoietin production.

We investigated the pathophysiology of erythropoiesis in 62 patients with multiple myeloma and examined whether it would establish a rational basis for the treatment of their anaemia with recombinant human erythropoietin. Erythropoietin (Epo) production was evaluated by serum levels and erythropoiesis was quantitated by serum transferrin receptor (TfR) levels, both assessed relative to the degree of anaemia. Instead of the expected stimulation of erythropoiesis in response to anaemia, haematocrit correlated positively with marrow erythropoietic activity, indicating that the mechanism of anaemia was primarily defective red cell production. Erythropoiesis decreased and anaemia worsened significantly with advancing clinical stage. 25% of the patients had inadequate Epo production and this proportion increased to 50% in stage 3. Inappropriate Epo production was seen in 60% of patients with renal impairment but was also observed in a number of patients with normal renal function. Erythropoiesis correlated strongly with the adequacy of Epo production, particularly in advanced disease. We conclude that most myeloma patients have defective red cell production even in the absence of massive marrow infiltration and that inappropriate Epo production contributes to their anaemia.

Phosphorylatable and epitope-tagged human erythropoietins: Utility and purification of native baculovirus-derived forms

The hematopoietic glycopeptide erythropoietin (EPO) is a prime regulator of red cell production in mammals, yet the precise nature of its interaction with specific cell surface receptors is poorly understood. Towards defining domains of EPO that are involved in receptor activation, we have developed (i) conditions for the expression of recombinant human EPO (rhEPO) at high levels in SF9 cells using modified 2- and 5-liter stirred reactors, (ii) a two-step procedure for the purification of this EPO without denaturation, and (iii) forms of EPO tagged with either a hemagglutinin influenza virus epitope or a consensus sequence for in vitro phosphorylation. Compared to EPO expressed in mammalian cells, rhEPO from SF9 cells in N-glycosylated with simple, neutral oligosaccharides of limited size, yet as purified presently using nondenaturing procedures, possesses exceptionally high in vitro activity (⩾500,000 U/mg). Thus, this form of EPO should prove advantageous for direct physicochemical analyses. Regarding epitope-tagged and phosphorylatable EPOS, forms modified at the amino terminus (Ala 1) fully retained receptor binding and in vitro biological activities. In contrast, forms modified at the carboxy terminus (Cys 161) were inactive and did not compete for receptor binding, indicating that integrity of this domain is essential for receptor recognition. For active amino-terminal-modified forms, the specific binding of MAb 12CA5 to native HAI-EPO and the utility of 32 32P-labeled PHOS-EPO in receptor binding and internalization studies also were demonstrated. The development of these unique, highly active forms of human EPO should advance studies of essential interactions between this cytokine and its cell surface receptor.

ERYTHROPOIETIN (EPO) TREATMENT OF PROTEIN AND IRON SUPPLEMENTED PREMATURE AGA INFANTS

Preterm AGA infants with birth weights 900-1400 g are studied in this controlled randomized multicentertrial. The infants are followed from 3 to 8 weeks of age with a further assessment at the age of 16 weeks. Only “healthy preterms” are included. Human milk is given throughout the study, and both treatment group and controls receive extra protein and iron to ensure sufficient supply for increased red cell production. Milk protein (0.9 g/100 ml human milk) and oral iron (18 mg Fe++/day increasing to 36 mg/day if serum iron falls below 16 μmol/l) are given. EPO (Eprex, Cilag AG) is given s.c. thrice weekly; 100 IU/kg/dose from week 3 to week 7. Until now 24 children have been included (10 treated & 14 controls). Preliminary data show : BW of the groups are similar (1294 g and 1244 g). There is a significant increase in reticulocyte count after one week of EPO-treatment (8.5% vs. 4.5%, p < 0.01). Mean Hb concentrations after 3 and 4 weeks of EPO-treatment are 11.6 g/dl and 12.1 g/dl respectively. Similar control values are 10.1 g/dl and 9.8 g/dl, which are significantly lower (p<0.05). The calculated total red cell production increased rapidly and significantly (p<0.01) during the first week of EPO treatment, and remained higher than that of the control group during EPO treatment. Three children in the control group needed blood transfusions during the study, none in the treatment group. No adverse effects have been seen, particularly no low white cell counts or increased trombocytes.

Preoperative red cell production in patients undergoing aggressive autologous blood phlebotomy with and without erythropoietin therapy.

Previous studies have demonstrated that autologous blood donors have a suboptimal endogenous erythropoietin response to the mild anemia induced by blood donation. Recent studies in baboons subjected to aggressive phlebotomy have shown an acceleration of erythropoiesis that may be beneficial perioperatively. To address the issue of accelerated erythropoiesis in autologous blood donors, red cell production during an aggressive preoperative autologous blood donation program was analyzed. The volume of red cells increased 568 mL (27% over baseline) and 911 mL (47% over baseline) for 23 placebo and 21 erythropoietin-treated patients, respectively, by hospital admission (9 days after last drug administration and 26 days after beginning therapy). The mean rate of additional red cell production was 22 mL per day in the placebo group and 34 mL per day in the erythropoietin group (p less than 0.001), which represents a twofold and a 2.5-fold increase over basal erythropoiesis, respectively. The major difference in red cell production in the placebo and erythropoietin groups occurred early in the collection period. It can be concluded that an aggressive autologous blood phlebotomy program results in clinically important increased erythropoiesis at the time of surgery. In patients unsuited for aggressive autologous phlebotomy, a more modest autologous blood procurement program, coupled with the administration of recombinant erythropoietin, may be a preferable approach.

Anemia of chronic disease

ACD is probably the most common anemia among hospitalized medical patients. It is variably defined by its clinical and, particularly, its laboratory manifestations. The most consistent features are low serum iron and normal or increased serum ferritin levels, reflecting normal or increased iron stores and distinguishing ACD from iron deficiency anemia. ACD often coexists with iron deficiency and the anemia of renal insufficiency. Most patients have an underlying infectious, inflammatory, or neoplastic disease, but as many as one quarter of patients do not. Several mechanisms have been proposed, the most significant of which are a block in reutilization of hemoglobin iron for red cell production and relative deficiency of erythropoietin, but the pathogenesis and mediators involved remain uncertain. The anemia itself seldom requires treatment and is ameliorated by successful treatment of the underlying disease.

3 - Anaemia of Plasmodium falciparum malaria

The pathophysiology of the anaemia of falciparum malaria is both complex and multifactorial, and results in a condition which is a major cause of mortality and morbidity in patients, especially children and pregnant women, living in malarial endemic areas. The importance of anaemia as a cause of death in malaria may well be underestimated because of difficulty in diagnosis, especially where parasitaemia may be low and the clinical picture may be confused with other causes of anaemia. Two clinical presentations predominate: severe acute malaria in which anaemia supervenes, and severe anaemia in patients in whom there have been repeated attacks of malaria. The major mechanisms are those of red cell destruction and decreased red cell production. Potential causes of haemolysis include loss of infected cells by rupture or phagocytosis, removal of uninfected cells due to antibody sensitization or other physicochemical membrane changes, and increased reticuloendothelial activity, particularly in organs such as the spleen. Decreased production results from marrow hypoplasia seen in acute infections, and dyserythropoiesis, a morphological appearance, which in functional terms results in ineffective erythropoiesis. The role of parvovirus B19 as a possible cause of bone marrow aplasia in a few cases is postulated. Finally, there is now evidence which points to genetic factors, HLA associated, which may protect against the development of malarial anaemia and which has become common in areas endemic for malaria.

The human erythropoietin receptor.

Molecular analysis of the human erythropoietin receptor (EpoR) promises to yield a greater mechanistic understanding of erythropoiesis and disease states that affect red cell production. The cloned receptor molecule is a 66 kDa membrane protein that is structurally related to a large superfamily of haemopoietin/growth factor receptors. The 66 kDa EpoR alone is capable of binding to erythropoietin (Epo) with nanomolar affinity. The native EpoR may form dimers before or after binding Epo. EpoR dimers and/or associated molecules are probably necessary for high-affinity Epo binding. The 66 kDa EpoR probably exists as a protein complex with as yet unidentified proteins of 100 and 85 kDa. The molecular mechanism of Epo signal transduction remains largely undefined. The possible role of the EpoR in human diseases has been studied in a variety of clinical conditions. A structurally abnormal EpoR gene has been identified in a human erythroleukemia cell line. In polycythemia vera, red cell progenitors exhibit exaggerated sensitivity to Epo and express only low-affinity EpoR. Some cases of hereditary polycythemia may be due to a mutant EpoR conferring enhanced Epo sensitivity. Other pathologic conditions may also be associated with abnormalities of the EpoR or its associated molecules. Soluble, immunoreactive EpoR is detectable in human serum, but its physiological significance is unknown.

Functional analysis of an oxygen-regulated transcriptional enhancer lying 3' to the mouse erythropoietin gene.

Erythropoietin, the major hormone controlling red-cell production, is regulated in part through oxygen-dependent changes in the rate of transcription of its gene. Using transient transfection in HepG2 cells, we have defined a DNA sequence, located 120 base pairs 3' to the poly(A)-addition site of the mouse erythropoietin gene, that confers oxygen-regulated expression on a variety of heterologous promoters. The sequence has the typical features of a eukaryotic enhancer. Approximately 70 base pairs are necessary for full activity, but reiteration restores activity to shorter inactive sequences. This enhancer operates in HepG2 and Hep3B cells, but not in Chinese hamster ovary cells or mouse erythroleukemia cells, and responds to cobalt but not to cyanide or 2-deoxyglucose, thus reflecting the physiological control of erythropoietin production accurately.