重组人红细胞生成素6000U每周1次治疗非透析患者肾性贫血的临床观察

观察重组人红细胞生成素(rHu EPO)每周1次皮下注射治疗非透析患者肾性贫血的临床疗效及副反应。方法 选择慢性肾功能衰竭并肾性贫血的非透析患者30例，治疗组15例，采用rHu EP06000U每周1次皮下注射；对照组15例，采用相同产品rHu EPO3000U每周2次皮下注射。治疗期间，监测血常规、肝肾功能、电解质、血压及各种不良反应。结果 试验组显效7例、有效5例、无效2例；对照组显效6例、有效4例、无效2例，治疗前后两组治疗观察指标及副反应均无统计学差异。结论 rHu EPO每周1次注射方法与分次注射有同样治疗价值，且减少病人负担，有助于提高患者的生活质量。     

促红细胞生成素及血红蛋白与老年慢性心力衰竭患者的相关性

目的探讨促红细胞生成素(EPO)和血红蛋白(Hb)与慢性心力衰竭(CHF)的相关性。方法选择老年CHF患者1 69例,根据本次住院确诊的心功能(NYHA)Ⅰ、Ⅱ、Ⅲ和Ⅳ级依次分为1组33例、2组34例、3组59例和4组4 3例。对4组患者Hb、EPO及是否死亡进行logistic回归分析。结果与1组比较,2、3和4组lg EPO水平明显上升,Hb水平明显下降(P<0.01);4组患者lg EPO和Hb水平两两比较,差异均有统计学意义(P<0.01)。Hb、EPO及肌酐(OR=0.978,35.820,1.013,P<0.01)均是影响老年CHF患者住院死亡的危险因素。结论 EPO随患者CHF程度的加重而升高,Hb值则降低。Hb、EPO及肌酐均为影响CHF患者住院死亡的危险因素。     

高渗盐液经骨髓腔通路复苏失血性休克犬后EPO、IL-6的变化

目的探讨经骨髓腔通路输注不同复苏液复苏失血性休克犬后促红细胞生成素（EPO）、白细胞介素6（IL-6）的变化。方法健康杂种犬随机分为3组：7.5%氯化钠羟乙基淀粉40溶液（HSH）组、0.9%生理盐水（NS）组、假手术（Sham）组。建立犬失血性休克模型,分别从胫骨骨髓腔输注HSH、NS两种复苏液,观察复苏后EPO、IL-6的变化。结果复苏后48 h,NS组、HSH组EPO均较Sham组升高（P〈0.05）;复苏后1周HSH组EPO水平仍高于Sham组（P〈0.05）。休克终点,NS组、HSH组IL-6水平均较Sham组升高（P〈0.05）;复苏后2 h HSH组IL-6水平低于NS组（P〈0.05）;复苏后48 h HSH组IL-6水平降至正常。结论HSH经骨髓腔通路复苏失血性休克犬,能够减轻机体的炎症反应,是一种有效的复苏方案。     

Combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) anf erythropoietin (EPO) for the treatment of advanced non-responsive chronic lymphocytic leukemia

We report the use of a colony-stimulating granulocyte-macrophage factor (GM/CSF) and erythropoietin (EPO) combination as salvage treatment in four heavily-pretreated patients with refractory/ recurrent B-CLL. Induction therapy was subcutaneous GM-CSF 2.5 microg/ kg, and EPO, 150 units/kg both daily for the first 14 d. Maintenance therapy was GM-CSF on days 1, 3 and 5 and Epo on days 2, 4 and 6 at the same doses with weekly recycling. All the patients responded favourably. A significant reduction of lymphocytosis, lymphoadenomegaly, and organomegaly was obtained within one month of therapy. The number of infections and transfusional requirement decreased dramatically. The hemoglobin increased to over 11 g/dl in 3 out of 4 patients. With a median follow-up of 11 months (range 5-13) we observed 4 partial responses (NCI/IWCLL) and only one progression after a 10-month partial response. This combination regimen seems very active, safe and easy to administer. It may represent a promising therapeutical option in heavily pretreated patients. Further clinical and biological studies on a larger cohort of patients are needed to confirm these preliminary data, to clarify the hypothetical interactions between these cytokines and B-CLL cell proliferation pathways, and to establish if this therapy may have an impact on survival.     

Erythropoietin response to anaemia is not altered by surgery or recombinant human erythropoietin therapy

Summary. Recombinant human erythropoietin (EPO) therapy has been shown to increase red blood cell (RBC) production and facilitate autologous blood donation before elective surgery. However, recent reports have suggested that surgery and/or EPO therapy may suppress endogenous erythropoietin secretion in response to anaemia. We therefore analysed the haemoglobin/erythropoietin relationship preoperatively and postoperatively in 71 autologous blood donors subjected to aggressive phlebotomy and six treatments with either EPO (150U/kg, n=16, 300U/kg, n=18, or 600 U/kg, n=19) or placebo (n=18). Using data from the three prepoerative study visit, the linear relationship between log erythropoietin and haemoglobin was determined for each of the 18 placebo patients. We found no significant differences in the slopes of the relationships in this group during aggressive phlebotomy. Furthermore, there was no evidence of a significant difference in the erythropoietin level recorded postoperatively for each patient to that predicted from the patient's postoperative haemoglobin level, based on the haemoglobin/log erythropoietin relationship preoperatively. Similarly, for each of the EPO-treated groups, there was no evidence of a significant difference when comparing the recorded erythropoietin level to that predicted from each patient's postoperative haemoglobin level, based on the haemoglobin/log erythropoietin relationship preoperatively. We conclude that preoperative recombinant human erythropoietin therapy and/or surgery do not adversely affect the postoperative erythropoietin response to anaemia.     

Increased cell surface expression of C-terminal truncated erythropoietin receptors in polycythemia

Primary familial and congenital polycythemia (PFCP) is a disorder characterized by an increased number of erythrocytes despite normal blood oxygen pressure and a normal serum erythropoietin (EPO) level. Recent studies revealed that erythroid progenitor cells from certain individuals with PFCP express various forms of EPO receptor (EPOR) truncated at the terminal carboxyl site (EPOR-TTC(PFCP)). EPOR-TTC(PFCP) can transmit EPO-mediated proliferative signals more efficiently than can full-length EPOR (EPOR-F), at least partly because of defective recruitment of SHP-1 phosphatase to these receptors. In agreement with previous studies, Ba/F3 transfectants expressing EPOR-TTC(PFCP) showed higher proliferative responses to EPO. In those transfectants, we found that EPOR-TTC(PFCP) was expressed more abundantly on the cell surface than was EPOR-F. This tendency was confirmed by a transient-expression experiment using COS7 cells. Since expression levels of EPOR protein were not significantly different among these transfectants, differences in cell surface expression were likely dependent on post-translational mechanism(s). In addition to defective recruitment of SHP-1 to EPOR-TTC(PFCP), more efficient transport and expression on the cell surface appear to serve as mechanisms responsible for increased EPO-responsiveness of erythroid progenitor cells in PFCP.     

Serum Erythropoietin Levels by Radioimmunoassay in Polycythaemia

A radioimmunoassay (RIA) method for erythropoietin (Epo) was developed and validated against the polycythaemic mouse assay. The correlation was good, with a r = 0.94. Several other criteria of specificity were also filled by the RIA, which had a lower detection limit of 5 mU/ml. The mean serum-Epo level in 6 patients with secondary polycythaemia, 50.2 + 26.2 mU/ml, was significantly higher than in a group of 11 normal subjects, 28.7 + 7.2 mU/ml (P < 0.0002). However, the Epo level in 31 polycythaemia vera (PV) patients, M = 21.9 + 6.6 mU/ml, was not significantly different from normal (P = 0.006). Since previous studies with bioassay of heat-treated and concentrated plasma samples have shown a decreased serum-Epo level in PV, Epo levels were measured before and after heat treatment and concentration of samples from normals and polycythaemics. It was found that the levels of immunoreactive material increased after heat treatment and 40 times concentration in samples from normals and patients with secondary polycythaemias, but decreased in PV. We conclude that the Epo levels in serum in the low range measured by our and previous RIA:s probably are not true Epo levels but are partly due to an unspecific serum effect, that was removed by heat treatment.     

促红细胞生成素衍生物研究进展

促红细胞生成素（EPO）除了具有造血活性外,还具有抗凋亡等广泛的组织保护活性。近年来的研究发现促红细胞生成素衍生物———氨甲酰化促红细胞生成素（CEPO）,不表现任何促红细胞生成活性,不能与经典促红细胞生成素受体（EPOR）结合,但可通过新发现的一种受体EPOR-βcR（由EPOR和βcR形成的异源二聚体）起到与EPO类似的组织保护作用。与EPO不同,CEPO无明显的造血活性,CEPO的抗凋亡保护作用与其改善组织重塑及抑制瘢痕形成有关。     

Biological functions and therapeutic use of erythropoiesis-stimulating agents: perplexities and perspectives

Randomized clinical studies, carried out in patients with haematological malignancies and with solid tumours, have consistently demonstrated that treatment with recombinant human erythropoietin (Epo) increases haemoglobin levels, reduces blood transfusion requirements, and improves the quality of life. In addition, identification of erythropoietin receptor (EpoR) expression on many types of non-erythroid and cancer cells has spurred an interest in the extra-haematological activities of Epo itself and other erythropoiesis-stimulating agents (ESAs). Epo and its derivatives have emerged as major tissue-protective cytokines in ischaemic and degenerative damage of cardiovascular, neurological and renal diseases, while their angiogenetic and immunomodulatory properties indicate that their therapeutic potential may extend well beyond erythropoiesis alone. Both preclinical and clinical data, however, have suggested that they may contribute to tumour progression and prejudice survival when administered to anaemic cancer patients, though the results are equivocal and the assumed mechanisms by which tumour growth could be promoted are not fully understood. While these findings offer new perspectives, they nonetheless demand caution in the employment of ESAs. Further, well-designed experimental and clinical studies are warranted.     

Therapeutic effect of recombinant human erythropoietin on anaemia with erythropoietin deficiency in diabetic patients.

AIMS: The aim of this study was to investigate the therapeutic effect of recombinant human erythropoietin (rHuEpo) on anaemia with erythropoietin deficiency in diabetic patients. METHODS: Twenty diabetic patients with anaemia and Epo deficiency were enrolled. All patients were treated with rHuEpo (Epokine; 4000 U/day s.c., three times a week) for 8 weeks. RESULTS: The responder group (n = 14) had significant increments in haemoglobin compared with the non-responder group (n = 6) (P < 0.05). No significant differences were found between the responder and non-responder groups in terms of duration of diabetes mellitus, serum creatinine level, 24-h urine albumin excretion rates, frequency of diabetic microangiopathy, or HbA1c. There was no difference between the two groups in terms of serum iron and total iron-binding capacity (TIBC). Serum ferritin level was significantly higher in the responder group than in the non-responder group (240.3 +/- 108.4, 25.8 +/- 3.0 micro g/l, P < 0.05), as was transferrin saturation (32.7 +/- 7.9%, 21.2 +/- 5.3%, P < 0.05). CONCLUSIONS: rHuEpo could be useful in the treatment of anaemia with erythropoietin deficiency in diabetic patients, and the degree of iron storage and functional iron deficiency might be the main cause of hyporesponsiveness to rHuEpo.     

The in Vitro Production of Erythropoietin and Thrombopoietin

In order to determine if there were any relationships between thrombopoietin, erythropoietin and erythrogenin, supernatant fluids from culture lines of foetal mouse liver, foetal and adult bovine kidney, and adult rabbit kidney cells have been assayed for their content of the presumptive humoral regulators of haematopoiesis. Hormone production varied markedly from one culture line to another confirming other studies that the optimal culture conditions for the generation of haematologically-active hormones have not been delineated. The lack of any discernable relationship between the production of thrombopoietin and erythropoietin, thrombopoietin and erythrogenin or erythropoietin and erythrogenin suggests that these materials represent three distinct entities.     

Serum concentrations of erythropoietin measured by radioimmunoassay in hematologic disorders and chronic renal failure

Bioassays for human erythropoietin are cumbersome, time-consuming, and insensitive. The purification of human erythropoietin (EP) has provided small quantities of highly bioactive EP (～70,000 U/mg) required for the development of an EP radioimmunoassay (RIA). The RIA for EP described in this investigation, can detect 5 mU/ml of EP in the assay tube; the serum concentration of EP in normal individuals ranged from <18 to 81 mU/ml with a mean value of 29 mU/ml. In contrast, nine patients with severe aplastic anemia had markedly elevated serum EP concentrations with a mean value of 3,487 mU/ml, range 984–6,434 mU/ml. In this RIA, patients with Polycythemia vera had consistently undetectable EP concentrations, <18 mU/ml. Eleven patients with chronic renal failure had a higher mean serum EP concentration (40.5 mU/ml) than normal individuals, but the range (<18–115 mU/ml) overlapped that of normals. By immunologic and gel chromatographic criteria, EP measured in serum was similar to standard urinary EP. The EP immunoassay that we have developed has sufficient sensitivity and specificity not only to quantitate the elevated serum EP levels found in aplastic anemia but also to discriminate decreased from normal serum concentrations of EP in most circumstances. This simple, reliable RIA has provided the necessary framework upon which to increase our understanding of the importance of EP in hematopoiesis.     

Erythropoietin (EPO) Affords More Potent Cardioprotection by Activation of Distinct Signaling to Mitochondrial Kinases Compared with Carbamylated EPO

Purpose

Erythropoietin (EPO) and its non-erythrogenic derivative, carbarmylated EPO (CEPO), have been reported to activate different receptors (homomeric EPO receptor vs. heteromeric receptor consisting of EPO receptor monomer and common β-subunit). The aim of this study was to examine differences between EPO and CEPO in efficacy of cardioprotection against infarction and in activation of pro-survival kinases.

Methods

In isolated rat hearts, infarction was induced by global ischemia followed by reperfusion. Infarct size was determined 2 h after reperfusion, and ventricular tissues for immunoblotting were sampled at 5 min after reperfusion.

Results

Pretreatment with EPO (10 units/ml) before ischemia reduced infarct size (% of risk area; %IS/AR) from 47.0?±?2.1% of the control after 20-min ischemia to 24.7?±?4.3% and from 62.0?±?3.0% after 25-min ischemia to 45.5?±?4.1%. Desialylated EPO (asialoEPO, 100 ng/ml) mimicked the protection by EPO. However, CEPO (100 ng/ml) failed to reduce infarct size after 20-min ischemia (%IS/AR?=?47.5?±?5.9%) and that after 25-min ischemia (%IS/AR?=?56.1?±?4.2%). The infarct size-limiting effect of CEPO was not shown either by increasing CEPO dose to 500 ng/ml or by shortening ischemia to 15 min. Both EPO and CEPO enhanced phosphorylation of cytosolic GSK-3β upon reperfusion. In contrast, phosphorylation of GSK-3β, Akt, and PKC-ε in mitochondria upon reperfusion was significantly enhanced by EPO but not by CEPO.

Conclusion

EPO affords more potent protection against infarction than does CEPO by distinct activation of signaling leading to phosphorylation of pro-survival protein kinases in mitochondria upon reperfusion.     

Erythropoietin mediates terminal granulocytic differentiation of committed myeloid cells with ectopic erythropoietin receptor expression

OBJECTIVES: The precise role of hematopoietic cytokine/cytokine receptor interactions in lineage-restricted hematopoietic differentiation giving rise to mature blood cells of diverse function is incompletely defined. To study lineage-specific effects of cytokines during terminal hematopoietic differentiation, we examined the ability of erythropoietin (Epo) to mediate terminal granulocytic differentiation and induction of myeloid gene expression in committed myeloid cells, engineered to ectopically express Epo receptor (EpoR). METHODS: A cell culture model for granulocyte-macrophage colony stimulating factor (GM-CSF)-mediated granulocytic differentiation was used. EpoR was introduced by retrovirus-mediated gene transfer into multipotential, hematopoietic murine cell line EML, from which GM-CSF-responsive, promyelocytic EPRO cells were generated. In EPRO cells ectopically expressing EpoR, we examined the ability of Epo to mediate granulocytic differentiation and determined whether Epo-mediated neutrophil differentiation is associated with a pattern of myeloid gene expression comparable to that induced by GM-CSF. RESULTS: Studies of EpoR function in myeloid EPRO cells revealed that Epo/EpoR interaction can mediate terminal granulocytic differentiation of committed myeloid cells. In EPRO cells expressing EpoR, Epo-mediated neutrophil differentiation was associated with surface CD11b/CD18 (Mac-1) expression and induction of mRNA expression of specific myeloid genes including lactoferrin, gelatinase and C/EBPepsilon, in a manner similar to GM-CSF-mediated differentiation. CONCLUSIONS: These results indicate that Epo can deliver differentiative signals along a non-erythroid lineage, providing evidence for interchangeable cytokine receptor signals that mediate terminal differentiation of committed myeloid cells.     

Importance of biologic follow-ons: experience with EPO

The importance of recombinant human erythropoietin (epoetin) therapy has been clearly demonstrated in patients with anemia due to chronic kidney disease. The use of biopharmaceuticals to replace endogenous proteins, which may be inadequately low, carries the risk of stimulating the immune system to develop autoantibodies. Although these proteins are designed to closely mimic the endogenous proteins, they may have potential immunogenic properties. Erythropoietin produced by recombinant DNA technology is the most successful and efficacious agent for treating anemia. It was initially used in treating anemia in chronic kidney disease patients. Pure red cell aplasia (PRCA) ensuing from production of neutralizing anti-erythropoietin antibodies occurred very rarely with epoetin treatment. This agent was initially administered intravenously, but the mode of administration was progressively altered to subcutaneous without apparent increase in immune reaction. However, between 1998 and 2001, a sharp increase in the number of PRCA cases was seen. PRCA had been a very rare complication until this time. All of these patients had high affinity neutralizing anti-erythropoietin antibodies. This observation was made primarily in cases where one brand of epoetin, Eprex, was administered subcutaneously to patients with chronic kidney disease treated outside the United States, although a small number of cases among chronic kidney disease patients treated solely with epoetin beta were also identified. The marked increase in the number of Eprex cases was attributed to a change in the stabilizers, storage, and route of administration of Eprex to patients with chronic kidney disease. Since then changes have been made to the route of administration, storage, and handling of Eprex, and more recently to the rubber stoppers used in the prefilled syringes. Eprex was administered intravenously to chronic kidney disease patients and, with improved storage and handling, there was a subsequent dramatic reduction in the number of cases. More recently, the rubber stoppers have been replaced by Teflon-coated ones to prevent interactions with stabilizers and release of chemicals that have adjuvant properties. However, this concern is still relevant in the new generation of epoetin agents and generic formulations of epoetins. Epoetin treatment for anemia requires regular follow-up of hemoglobin levels but also of reticulocyte counts in chronic kidney disease patients.     

The metabolism of erythropoietin in liver cirrhosis patients compared with healthy volunteers.

The purpose of the investigation was to study the metabolism of erythropoietin (EPO) in patients with liver disease. Twelve patients with liver cirrhosis and 10 healthy volunteers were studied. The patients were moderately anemic with a hematocrit of 33 vs 42% (medians) in the volunteers. The pharmacokinetic parameters were calculated after an intravenous (i.v.) injection of 100 U/kg of recombinant human EPO. The serum EPO was measured by radioimmunoassay at regular intervals until 48 h. The median terminal elimination half life in the cirrhosis patients was 5.15 h vs 5.37 h in the control subjects. The clearance was 7.78 vs 7.52 ml/min/1.73 m² (ns). The steady-state volume of distribution was 3.69 vs 3.09 1/1.73 m² (ns). The estimated endogenous EPO production was significantly higher in liver cirrhosis (486 vs 290 U/d/1.73m², p<0.01). The basal serum EPO was significantly higher in the cirrhosis patients (43.5 vs 26.3 U/***l, p<0.01). The hematocrit correlated inversely with the basal serum EPO level in the cirrhosis patients (r= -0.63, p<0.04). The EPO-clearance was not related to the presence of ascites, esophageal varices, or to abnormal blood chemistry. It was concluded that normal metabolism of EPO was maintained in liver cirrhosis and that the cirrhotic patients had a moderate compensatory increase of EPO production in response to anemia     

The metabolism of recombinant erythropoietin in the isolated perfused rat liver

ABSTRACT— Indirect evidence points to extrarenal organs, presumably the liver, as the site of degradation of erythropoietin (EPO). The metabolism of both fully glycosylated and desialated intrinsically labelled ³⁵S-Cysteine recombinant human erythropoietin (rhEPO) was therefore studied in isolated Wistar rat livers perfused in a recirculating mode for 180 min with a hemoglobin-free medium containing rhEPO. Perfusate and bile levels of rhEPO were measured by RIA. Total ³⁵S-radioactivity in liver, bile and perfusate as well as non-acid precipitable radioactivity in perfusate were determined. In addition, detection of ³⁵S-radioactivity was performed after subcellular fractionation of rat livers perfused with desialo-³⁵S-Cysteine rhEPO. While concentrations of fully glycosylated ³⁵S-Cysteine rhEPO did not exhibit any detectable decrease during perfusion, desialo-³⁵S-Cysteine rhEPO was rapidly cleared from the perfusate. After 60 min of perfusion, only 32% of the initial levels of both immunoreactive rhEPO and total radioactivity remained in the perfusate. Quantitative hepatic accumulation of desialated tracer was demonstrated. Subcellular fractionation showed extensive hepatic degradation of the desialated tracer. Furthermore, during perfusion progressively larger amounts of small molecular weight degradation products of the tracer were found in the perfusate. Bile excretion of both fully glycosylated and desialated tracer was negligible. The significance of hepatic metabolism of desialo-³⁵S-Cysteine rhEPO was supported by reduced removal of desialo-³⁵S-Cysteine rhEPO from plasma in hepatectomized rats. It is hypothesized that continuous in vivo desialation is a crucial rate-limiting step in the degradation of circulating EPO.