Effects of recombinant human erythropoietin on hematopoietic progenitors of chronic hemodialysis patients in vitro and in vivo

Erythroid progenitors were assayed in 20 chronic hemodialysis patients before and after administration of recombinant human erythropoietin (rhEpo) using a methylcellulose culture method. Hemoglobin concentrations, hematocrit values and the proportion of marrow erythroblasts increased significantly during rhEpo treatment. The numbers of erythroid progenitors (CFU-e and BFU-e) in the patients' marrow also increased following rhEpo administration. Our data suggest that rhEpo is an effective drug for treating anemia caused by chronic renal failure and the administration of rhEpo results in an increase in the numbers of erythroid progenitors.     

Recombinant human erythropoietin corrects anemia of blood loss: a study in the dog

In order to evaluate the possibility of using recombinant human erythropoietin (rhEpo) for the prevention and correction of anemia due to blood loss and as an adjuvant for autologous blood transfusion, its preventive and therapeutic effects were evaluated in beagles in which anemia was induced by repeated phlebotomies. Two hundred U/kg of rhEpo were administered i.v. four or nine times every two weeks for six weeks. Phlebotomies (25 ml/kg of body weight) were conducted three times at two-week intervals. rhEpo was found to successfully prevent and correct anemia caused by the phlebotomies. Concurrent administration of iron increased efficacy. The findings obtained in the present study suggest that rhEpo is useful both for the treatment of anemia caused by blood loss due to surgery and as an adjuvant therapy for pre-deposit autologous blood transfusion.     

Effect of recombinant human erythropoietin on the anemia of chronic renal failure

Phase I and Phase II studies of recombinant human erythropoietin (rhEpo) were conducted in normal volunteers and in anemic patients with chronic renal failure on maintenance hemodialysis. Three hundred U/person of rhEpo was administered intravenously to healthy normal volunteers in the Phase I study, resulting in no subjective or objective changes. In the Phase II study, 66 patients with chronic renal failure on maintenance hemodialysis with less than 20% hematocrit values were treated with rhEpo in doses of 50 U/kg to 200 U/kg two or three times a week. Hematocrit values increased significantly during the 12 weeks, and the patients' conditions improved. Patients previously requiring blood transfusions became transfusion-independent during our study. There were no obvious side effects, thus indicating the safety and efficacy of rhEpo in the anemia of chronic renal failure.     

Clinical use of rHuEPO in bone marrow transplantation

Recipients of bone marrow (BMT) or peripheral blood progenitor stem cells (PBSCT) transplant have in the period following transplantation a frequent need for red blood cell transfusions and therefore an increased risk of blood-transmitted infections. The anaemia is caused mainly by myelosuppression after high-dose chemotherapy, but an impaired erythropoietin (EPO) production and an inappropriate EPO response may also contribute. Since recombinant human erythropoietin (rHuEPO) has been established as a treatment for renal anaemia it has been of interest whether treatment may be of benefit in the transplant setting. This paper gives an overview of the studies conducted to date with rHuEPO treatment in patients receiving bone marrow transplants. Current data donot support any transfusional benefits when rHuEPO is used in patients receiving autologous transplants. However, in patients receiving allogeneic transplants several studies clearly indicate a therapeutic role for rHuEPO with patients showing accelerated erythroid engraftment, increased haemoglobin levels, a reduced requirement for red blood cell transfusions, and a shortened time to transfusion independence. Especially patients with immune haemolysis after transplantation seems to benefit from the treatment. In addition, rHuEPO treatment has been used for lateonset anaemia after BMT and to prevent the need for homologous red blood cell transfusions to the BMT donor. To reduce costs, it is important in future studies to identify not only the optimal dose and route of administration of rHuEPO but also the most effective combination with other haematopoietic growth factors and cytokines that are used before and after transplantation.     

Effect of purified recombinant human erythropoietin on anemia in rats with experimental renal failure induced by five-sixth nephrectomy

Recombinant human erythropoietin (rHuEPO) was purified from the conditioned media of Chinese hamster ovary cells with a transfected human erythropoietin gene. We investigated the effects of the rHuEPO in rats with renal anemia induced by partial nephrectomy. Five-sixth nephrectomy resulted in renal failure with anemia. Twenty-five days after the operation plasma urea nitrogen was increased about 2.5 times, and the red blood cell count, hematocrit, and hemoglobin concentration fell to 85% of normal. The reticulocyte count and plasma erythropoietin level did not change such as they do in patients with anemia due to chronic renal failure. Both total red blood cell volume and the plasma iron turnover rate were depressed in five-sixth nephrectomized rats compared with normal rats.The five-sixth nephrectomized rats were injected with rHuEPO (60 IU/kg) intravenously every second day for a total of six injections. After three injections of rHuEPO, circulation volume of total red blood cells was increased from 9.9 ml to 14.6 ml, and the plasma iron turnover rate was increased from 1.03 mg/kg/day to 2.12 mg/kg/day, and the reticulocyte count was also increased. After six injections, a marked increase of the red blood cell count, hematocrit, and hemoglobin concentration were observed. Plasma urea nitrogen and the creatinine levels as indications for renal function did not change after rHuEPO administration in both normal and five-sixth nephrectomized rats.In conclusion rHuEPO has a potent erythropoietic action and it is possible to cure the anemia caused by renal failure.     

Single dose recombinant human erythropoietin reduces transforming growth factor beta in patients on chronic haemodialysis.

Short-term effects of recombinant human erythropoietin on serum levels of transforming growth factor beta-1, interleukin 1-alpha, interleukin 3, interferon gamma, and tumour necrosis factor alpha in patients with chronic renal failure on chronic haemodialysis were investigated. Recombinant human erythropoietin was applied subcutaneously in a dose of 75 IU/kg on 19 patients. Serum levels of transforming growth factor beta-1, interleukin 1-alpha, interleukin 3, interferon gamma, tumour necrosis factor alpha and erythropoietin, red blood cell parameters: red blood cell count, haemoglobin, haematocrit, and erythrocyte indices were determined before and after recombinant human erythropoietin single application. Transforming growth factor beta-1 serum levels were decreased after recombinant human erythropoietin (22.70 +/- 1.51 ng/ml versus 18.77 +/- 1.70 ng/ml (p < 0.01). None of the other investigated parameters was influenced significantly by recombinant human erythropoietin. Recombinant human erythropoietin in patients with chronic renal failure on chronic haemodialysis may influence anaemia not only through its stimulating effect on erythropoiesis, but also by direct oxygen-independent decrease of at least one of the negative regulators of erythropoiesis--the transforming growth factor beta.     

The Erythropoietin/Erythropoietin Receptor Signaling Pathway Promotes Growth and Invasion Abilities in Human Renal Carcinoma Cells

Co-expression of erythropoietin (Epo) and erythropoietin receptor (EpoR) has been found in various non-hematopoietic cancers including hereditary and sporadic renal cell carcinomas (RCC), but the Epo/EpoR autocrine and paracrine mechanisms in tumor progression have not yet been identified. In this study, we used RNA interference method to down-regulate EpoR to investigate the function of Epo/EpoR pathway in human RCC cells. Epo and EpoR co-expressed in primary renal cancer cells and 6 human RCC cell lines. EpoR signaling was constitutionally phosphorylated in primary renal cancer cells, 786-0 and Caki-1 cells, and recombinant human Epo (rhEpo) stimulation had no significant effects on further phosphorylation of EpoR pathway, proliferation, and invasiveness of the cells. Down-regulation of EpoR expression in 786-0 cells by lentivirus-introduced siRNA resulted in inhibition of growth and invasiveness in vitro and in vivo, and promotion of cell apoptosis. In addition, rhEpo stimulation slightly antagonized the anti-tumor effect of Sunitinib on 786-0 cells. Sunitinib could induce more apoptotic cells in 786-0 cells with knockdown EpoR expression. Our results suggested that Epo/EpoR pathway was involved in cell growth, invasion, survival, and sensitivity to the multi-kinases inhibitor Sunitinib in RCC cells.     

The use of recombinant erythropoietin in the reduction of blood transfusion rates in craniosynostosis repair in infants and children

The vast majority of infants and children undergoing craniosynostosis correction receive a blood transfusion. The risks of blood transfusion include, but are not limited to, acute hemolytic reactions (approximately 1 of 250,000), human immunodeficiency virus (approximately 1 of 200,000), hepatitis B and C (approximately 1 of 30,000 each), and transfusion-related lung injuries (approximately 1 of 5000). This prospective, single-blinded, randomized study was undertaken to examine the safety and efficacy of preoperative single weekly dosing with erythropoietin (epoetin alfa) in reducing the rate of transfusion in infants and small children undergoing craniosynostosis repair. A total of 29 patients (<8 years) undergoing craniosynostosis repair were randomized into two groups: one received preoperative erythropoietin (600 U/kg) weekly for 3 weeks, and the other served as a control. All caregivers responsible for blood transfusions were blinded, and strict criteria for transfusion were established. A pediatric hematologist monitored both groups, and all patients received supplemental iron (4 mg/kg). Fourteen patients were randomized to receive erythropoietin, and eight of these 14 patients (57 percent) required transfusion (mean age, 17 months; mean weight, 10.1 kg). Of the six patients not requiring transfusion, three were younger than 12 months old (mean, 6 months). Fourteen of 15 patients (93 percent) in the control group (mean age, 13 months; mean weight, 9.3 kg) required a blood transfusion during the study. The only control patient not requiring transfusion was the eldest (5 years old). The difference between the two groups was statistically significant (Fisher's exact test = 0.03). The control group showed no change in hemoglobin levels from baseline to preoperative levels, but the erythropoietin group increased their average hemoglobin levels from 12.1 to 13.1 g/dl. There were no adverse effects noted among children receiving erythropoietin, nor were there any surgical complications. The authors conclude that the preoperative administration of erythropoietin significantly raised hemoglobin levels and reduced the need for a blood transfusion with craniosynostosis correction. More suggestions are made that may further reduce the need for blood transfusions, and a cost-benefit analysis is discussed.     

Erythropoietin for oncology supportive care

Recombinant human erythropoietin (rhEPO), the prototype erythropoiesis-stimulating agent developed in the 1980s, was among the first recombinant human proteins to be marketed for clinical use in the oncology setting. Anemia is a frequent concern in patients with cancer receiving myelosuppressive chemotherapy and the availability of rhEPO as an alternative to red blood cell transfusions to treat symptomatic anemia created excitement among clinicians, particularly during an era of mounting concern for transfusion-transmissible infections. Early studies of rhEPO for chemotherapy-induced anemia in patients with non-myeloid malignancies showed these agents improved hemoglobin levels and reduced transfusion rates. rhEPO therapy was reported to decrease fatigue and improve quality of life, although the magnitude and clinical meaningfulness of these effects have been debated. More recent clinical trials since 2003 linking rhEPO therapy to increased risk of tumor progression, thrombo-vascular events and mortality prompted implementation of use restrictions to minimize potential for harm. Scientific research to understand the basic mechanisms of the biologic effects of erythropoietin at the cellular receptor and signaling level has revealed pleiotropic cytokine effects extending beyond erythropoiesis regulation. The importance of erythropoietin receptor signaling in normal, non-erythroid tissues and in pre-clinical tumor models has been under intense investigation and scrutiny, as potential mechanisms of the adverse outcomes associated with rhEPO therapy have been debated. Further research will be required to clarify the complex interplay between the diverse hematopoietic and non-hematopoietic effects of erythropoietin in normal and malignant tissues and to optimize the clinical use of rhEPO in the supportive care of cancer patients.     

Effect of recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on human BFU-e in serum-free cultures

The effects of recombinant human hemopoietic growth factors on early and late human erythroid progenitors (BFU-e and CFU-e) were investigated in serum-free cultures. Recombinant human erythropoietin (rhEpo) induced the formation of not only human CFU-e-derived colonies but also human BFU-e-derived bursts. Recombinant human interleukin 3 (rhIL-3) alone did not induce the formation of human BFU-e-derived bursts and human CFU-e-derived colonies. In the presence of rhEpo, rhIL-3 dose dependently increased the number of bursts stimulated by rhEpo, although rhIL-3 did not have the augmentative effect on human CFU-e growth. On the other hand, rhIL-3 did not stimulate the formation of murine BFU-e-derived bursts, and murine IL-3 did not stimulate the formation of human BFU-e-derived bursts. The results indicated that the burst-promoting activity of IL-3 was species-specific between human and murine cells. Recombinant human GM-CSF (rhGM-CSF) or recombinant human G-CSF (rhG-CSF) failed to induce human burst formation and did not augment the effect of rhEpo on human burst formation. The results of the present study suggest that in vitro, IL-3 can stimulate BFU-e in collaboration with Epo, but GM-CSF and G-CSF do not stimulate BFU-e growth in the presence or absence of Epo.     

Haemotherapy analysis: the Teaching Hospital Olomouc in 2006

Aims: Annual evaluation of blood transfusion preparation administration at the University Hospital Olomouc, Czech Republic as a contribution to the European Union haemovigilance system. Methods: Analysis of blood transfusion preparations released from the Department of Blood Transfusion of the above university hospital and laboratory examination results in receivers of these products for the year 2006. Total hospital consumption and usage in particular medical disciplines in the hospital were assessed. Results: Red cell concentrates: in total 14 347 TU (deleucotised in 20.4 %). Departments according to usage: surgery, haematooncology, anaesthesiology and surgery intensive care department, and internal medicine department. Haemoglobin levels were above 100 g/L before blood transfusion administration in 24 % of cases. Platelets: in total 1 712 TU (100 % manufactured by apheresis, 56 % deleucotised). Platelet counts below 20 x 10(9)/L were found in all cases before platelet concentrate administration. Plasma: in total 5 959 TU to 1 297 of patients. Departments according to usage: surgery, anaesthesiology and surgery intensive care department, internal medicine and haematooncology. Two hundred and forty nine cases without coagulation parameter monitoring and 333 (25.67 %) patients with only 1 TU of administered plasma were found. Conclusions: The incorrect indications for red cell transfusion preparations were found in eleven patients with haemoglobin levels above 130 g/L. Underdosing in 25.67 % of plasma administrations signifies dubious indications in these cases. Implementation of the haemovigilance system in practice is now mandatory for the Czech Republic after joining the European Union. Therefore more precise data and analysis of questionable cases with further education of staff in clinical departments are essential for the haemovigilance principle to be applied on a hospital basis in the Czech Republic.     

The effect of cytokines on the ploidy of megakaryocytes

The effects of recombinant cytokines on the ploidy of human megakaryocytes derived from megakaryocyte progenitors were studied using serum-free agar cultures. Nonadherent and T cell-depleted marrow cells were cultured for 14 days. Megakaryocyte colonies were identified in situ by the alkaline phosphatase anti-alkaline phosphatase technique, using monoclonal antibody against platelet IIb/IIIa. The ploidy of individual megakaryocytes in colonies was determined by microfluorometry with DAPI (4',6-diamidino-2-phenylindole) staining. Recombinant human interleukin 3 (rhIL-3) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) supported megakaryocyte colony formation in a dose-dependent manner. However, both rhIL-3 and rhGM-CSF had no definite ability to increase the ploidy values. Recombinant human erythropoietin (rhEpo) or recombinant human macrophage colony-stimulating factor (rhM-CSF) by itself did not stimulate the growth of megakaryocyte progenitors. rhEpo or rhM-CSF, however, stimulated increases in the number, size and ploidy values of megakaryocyte colonies in the presence of rhIL-3 or rhGM-CSF. Recombinant human interleukin 6 (rhIL-6) showed no capacity to generate or enhance megakaryocyte colony formation when added to the culture alone or in combination with rhIL-3. rhIL-6, however, increased the ploidy values in colonies when added with rhIL-3. These results show that rhEpo, rhM-CSF and rhIL-6 affect endomitosis and that two factors are required for megakaryocyte development.     

重组人红细胞生成素在妇科围手术期贫血治疗中的应用研究现状

抗对于妇科围手术期患者,由于术前贫血、术中失血或术后炎症,可能导致贫血状况愈加严重,而旨在改善患者预后和减少异体输血的妇科围手术期血液管理措施越来越被重视。重组人红细胞生成素能显著升高贫血患者血红蛋白水平并减少输血需求,其应用于围手术期贫血的纠正,已成为临床研究的新热点。综述重组人红细胞生成素治疗妇科围手术期贫血的作用机制、国内外临床研究及安全性研究。     

Iron overload following red blood cell transfusion and its impact on disease severity

Transfusion of red blood cells can be a life-saving therapy both for patients with chronic anemias and for those who are critically ill with acute blood loss. However, transfusion has been associated with significant morbidity. Chronic transfusion results in accumulation of excess iron that surpasses the binding capacity of the major iron transport protein, transferrin. The resulting non-transferrin bound iron (NTBI) can catalyze the production of highly reactive oxygen species (ROS) leading to significant and wide spread injury to the liver, heart, and endocrine organs as well as increases in infection. Acute transfusion of red blood cells in critically ill patients likewise has significant effects including increased mortality, prolonged hospital stays, and elevated risk of nosocomial infection. These effects appear to be more profound with increasing age of stored blood. The progressive release of free iron associated with storage time suggests that morbidity following acute transfusion, like that seen in chronic transfusion, may be due in part to elevated levels of NTBI. It is clear that transfusion is necessary in many instances; however, its risks and benefits must be carefully balanced before proceeding to avoid unnecessary iron toxicity.     

Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells

One challenge in biotechnology industry is to produce recombinant proteins with prolonged serum half-life. One strategy for enhancing the serum half-life of proteins includes increasing the molecular weight of the protein of interest by fusion to the Fc part of an antibody. In this context, we have expressed a homodimer fusion protein in CHO cells which consists of two identical polypeptide chains, in which our target protein, recombinant human erythropoietin (rhEpo), is N-terminally linked with the Fc part of a human IgG₁ molecule. In the present study, culture supernatant of a stable clone was collected and purified by affinity chromatography prior characterization. We emphasized product quality aspects regarding the fusion protein itself and in addition, post-translational characterization of the subunits in comparison to human antibodies and rhEpo. However, overproduction of recombinant proteins in mammalian cells is well established, analysis of product quality of complex products for different purposes, such as product specification, purification issues, batch to batch consistency and therapeutical consequences, is required. Besides product quantification by ELISA, N-acetylneuraminic acid quantification in microtiterplates, quantitative isoform pattern and entire glycan profiling was performed. By using these techniques for the characterization of the recombinant human Epo-Fc (rhEpo-Fc) molecule itself and furthermore, for the separate characterization of both subunits, we could clearly show that no significant differences in the core glycan structures compared to rhEpo and human antibody N-glycans were found. The direct comparison with other rhEpo-Fc fusion proteins failed, because no appropriate data were found in the literature.     

Radioimmunoassay of erythropoietin

A radioimmunoassay for erythropoietin has been developed using 125I labeled pure human erythropoietin and an anti-erythropoietin antiserum produced in a rabbit immunized with human erythropoietin. Two techniques are presented for labeling erythropoietin, both resulting in an immunologically reactive labeled reactant. One method involves the use of lactoperoxidase and the other a reagent known as IODO-GEN. The second International Reference Preparation of human erythropoietin is used as a standard and a double antibody scheme is used for the separation of the free and antibody bound labeled hormone. The radioimmunoassay is sensitive to an absolute amount of erythropoietin equivalent to 0.4 milliunits. Bioassays for erythropoietin require approximately 100 times this amount. The use of pure erythropoietin as the labeled reactant has removed certain discrepancies seen in previous attempts to develop radioimmunoassays for this hormone, e.g., sera from patients without kidneys do not give the high values previously seen. Sera from anemic individuals not only give rise to high radioimmunoassay values but also show a parallel relationship with the erythropoietin standard when halving dilutions are analyzed. Desialated erythropoietin is also reactive with the same parallelism. Bleeding of a normal individual increases the serum erythropoietin level and transfusion decreases it. Erythropoietin from a variety of laboratory animals is also reactive in the radioimmunoassay, with very high values being observed in hypoxic animals.     

Refinement of cytokine use in the in vitro expansion of erythroid cells

Blood transfusion is indispensable for many clinical applications. However, the supply of transfusable material is insufficient in many countries. Human cord blood contains many hematopoietic stem and progenitor cells, providing a promising resource for the production of transfusable material in vitro. In this study, we have refined a protocol to produce abundant red blood cells (RBC) from human cord blood in an in vitro culture system. We found that erythropoietin and interleukin-3 were most effective when they were added to the culture medium sequentially rather than simultaneously. Although insulin-like growth factor-I (IGF-1) has been reported to function as a positive regulator of RBC production in some in vitro culture systems, we found that IGF-1 had a negative effect upon RBC production. However, IGF-II appeared to function as a positive regulator of RBC production. Finally, stem cell factor functioned to both expand and accelerate the differentiation of immature erythroid cells.     

Improvement of anemia by recombinant erythropoietin in patients with myelodysplastic syndromes and aplastic anemia

Eight patients with myelodysplastic syndromes (MDS) and four patients with aplastic anemia (AA) were treated with recombinant erythropoietin (rEpo) to investigate its effect on the anemia of these patients. rEpo was administered by i.v. injection three times a week for at least four weeks. The doses were 3,000, 6,000, or 12,000 U/day. Despite an elevated "endogenous" Epo level, a greater than 1.5 g/dl increase in hemoglobin (Hb) concentration was observed in one patient with refractory anemia (RA), one patient with refractory anemia with excess of blasts (RAEB), and one patient with AA. A greater than 50% decrease in red cell transfusion requirement was observed in one patient with RA and one patient with AA. One RA patient and one AA patient have received rEpo as maintenance therapy for more than 64 and 100 weeks, respectively. They no longer need red cell transfusions and have had a normal Hb concentration and normal ferrokinetics. No side effect was seen. These results indicate that rEpo may benefit some patients with MDS and AA who are dependent on red cell transfusions while further studies will be necessary to elucidate the mechanism by which rEpo stimulates erythropoiesis and improves anemia in patients with these diseases.     

Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3'-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200,000 U/mg), but was of lower Mr (23,000) than human erythropoietin produced in COS cells (30,000) or purified from urine (30,000 to 38,000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosaccharides from this Mr 23,000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18,000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.