The fusion protein MEN 11303 (granulocyte-macrophage colony stimulating factor/erythropoietin) acts as a potent inducer of erythropoiesis

OBJECTIVE: A fusion protein made of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), referred to as MEN 11303, has been tested for biologic activity using mobilized CD34(+) cells. METHODS AND RESULTS: MEN 11303 and a combination of GM-CSF/EPO produced the same amount of colony-forming unit granulocyte-macrophage (CFU-GM), of burst-forming unit erythroid (BFU-E), and of multipotent CFU-mixed. After 15 days, liquid cultures of CD34(+) cells exposed to MEN 11303 yielded a total cell number larger than that obtained with an equimolar mixture of GM-CSF and EPO, with a clear prevalence of cells exhibiting an erythroid phenotype. A colony-forming cell assay established from CD34(+) cells precultured with MEN 11303 for 7 days yielded a greater amount of BFU-E than GM-CSF/EPO combination. Exposing CD34(+) cells to MEN 11303 for 7 days in liquid culture resulted in higher recoveries of cells expressing a comparatively less differentiated hematopoietic phenotype and of long-term culture initiating cells. A cell-based binding-competition assay using the human EPO-receptor (EPO-R) transfected murine Ba/F3EPOR cell line showed that MEN 11303 bound to EPO-R with a sixfold lower affinity but induced a more sustained receptor phosphorylation. MEN 11303 supported the growth of Ba/F3EPOR cells more efficiently than EPO and remained detectable in the spent culture medium for a longer time. CONCLUSIONS: MEN 11303 and the combination of GM-CSF/EPO are equally potent in recruiting hematopoietic progenitors into cycle, but the fusion protein is superior in promoting the expansion of committed erythroid percursors. Primitive hematopoiesis is less affected by MEN110303 than GM-CSF/EPO combination. Part of these effects may reflect the peculiar interaction of the EPO moiety of MEN 11303 with the EPO-R.     

Heme metabolism and erythropoiesis in abnormal iron states: role of delta-aminolevulinic acid synthase and heme oxygenase

Heme metabolism was examined in bone marrow and hepatic cells from iron-deficient and chronically iron-overloaded rats. Results indicate that the rate limiting enzymes delta-aminolevulinic acid synthase (ALAS) and heme oxygenase were significantly elevated in the iron-overloaded hepatic and bone marrow cells and near normal levels in cells from iron-deficient rats. Conversely, delta-aminolevulinic acid dehydrase (ALAD) was depressed in iron-overloaded cells and elevated in iron-deficient cells. Erythroid colony (CFU-E) cultures demonstrated that iron-overloaded bone marrow cells were poor hemin and CFU-E responders in vitro, whereas iron-deficient marrows grew exuberant numbers of CFU-E and responded to hemin. Succinylacetone (1 mM) inhibited ALAD activity and normal CFU-E growth, and CFU-E inhibition by succinylacetone was completely overcome by the addition of hemin. Results are discussed with respect to the regulation of hepatic and bone marrow heme metabolism in abnormal iron states and the possible role of iron in the induction of heme oxygenase in chronic iron overload.     

Role of c-Kit and erythropoietin receptor in erythropoiesis

Erythropoiesis is regulated by a number of growth factors, among which stem cell factor (SCF) and erythropoietin (Epo) play a non-redundant function. Viable mice with mutations in the SCF gene (encoded by the Steel (Sl) locus), or its receptor gene c-Kit (encoded by the White spotting (W) locus) develop a hypoplastic macrocytic anemia. Mutants of W or Sl that are completely devoid of c-Kit or SCF expression die in utero of anemia between days 14 and 16 of gestation and contain reduced numbers of erythroid progenitors in the fetal liver. Likewise, Epo and Epo receptor (Epo-R)-deficient mice die in utero due to a marked reduction in the number of committed fetal liver derived erythroid progenitors. Thus, committed erythroid progenitors require both c-Kit and Epo-R signal transduction pathways for their survival, proliferation and differentiation. In vitro, Epo alone is capable of generating mature erythroid progenitors; however, a combined treatment of Epo and SCF results in synergistic proliferation and expansion of developing erythroid progenitors. This review summarizes recent advances made towards understanding the signaling mechanisms by which Epo-R and c-Kit regulate growth, survival, and differentiation of erythroid progenitors alone and cooperatively.     

Monitoring of erythropoiesis by the serum transferrin receptor and erythropoietin

Virtually all cells have transferrin receptors (a transmembrane glycoprotein) on their surface but in a normal adult, 80% of them are in the erythroid marrow. Some of them are lost into the circulation where they can be measured by immuno-assays. A direct and highly significant correlation exists between serum transferrin receptor level and erythron transferrin uptake in humans. The measurement of serum transferrin receptor has wide clinical applications for the quantitation of erythropoiesis. It can be used to study erythropoiesis in situations in which ferrokinetics is not acceptable such as pregnancy. It is particularly useful for serial studies, e.i., for monitoring the recovery of erythropoiesis after stem cell transplantation or after treatment with erythropoietin. Combined with the determination of serum erythropoietin, both evaluated in relation to the degree of anemia, they provide a physiological approach to the diagnosis of anemia. Thus, the simultaneous determination of hematocrit, reticulocytes, serum transferrin receptor and serum erythropoietin has high discriminatory value in distinguishing between a defect in erythroid proliferation, maturation or red cell survival. It is also particularly useful for detecting the presence of multiple mechanisms of anemia in the same patient.     

Endotoxin-induced hypercoagulability: a possible aggravating factor of alcoholic liver disease

The present experiments were designed to study the effect of chronic ethanol consumption on endotoxin toxicity. The intravenous injection of endotoxin produced a more pronounced increase of serum AST and ALT activities in chronic ethanol-fed rats, when compared to controls. The activities of hepatic mitochondrial enzymes, succinate dehydrogenase and cytochrome oxidase, were also distinctly decreased by endotoxin treatment in chronic ethanol-fed rats. Consistent with these biochemical alterations, light and electron microscopic examinations revealed severe liver injury after endotoxin injection in chronic ethanol-fed rats. Furthermore, the increase of blood BUN and creatinine levels accompanied by the degeneration of the renal tubulus and slight infiltration of neutrophils into the glomerule were produced by endotoxin treatment and were more conspicuous in chronic ethanol-fed rats than controls. Therefore, the biochemical and histological evidence indicates that endotoxin markedly potentiates organ injury after chronic ethanol consumption. In addition, a more pronounced decrease in blood antithrombin III activity accompanied by an increase in fibrin degradation product level in blood was recognized in chronic ethanol-fed rats receiving endotoxin, when compared to controls receiving endotoxin. This increase of blood fibrin degradation product level correlated well with the decrease of antithrombin III activity (r = -0.6116; p less than 0.005). These findings of blood antithrombin III activity and fibrin degradation product level indicate that the coagulation-fibrinolysis system is more activated by endotoxin treatment after chronic ethanol consumption. Furthermore, the activation of the coagulation-fibrinolysis system was well correlated with biochemical and histological alterations representing hepatorenal involvement.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo suppression of erythropoiesis by tumor necrosis factor-alpha (TNF-alpha): reversal with exogenous erythropoietin (EPO)

Tumor necrosis factor-alpha (TNF-alpha) selectively kills tumor cells in vitro and in vivo and is being tested as a cancer therapeutic agent. We have shown that TNF-alpha significantly suppresses late-stage erythropoiesis, leading to anemia in chronically treated mice. These erythropoietic effects could limit the clinical use of TNF-alpha. Therefore, we have examined whether erythropoietin (EPO) could be used to prevent TNF-alpha-induced erythroid suppression. Normal mice were treated with a single dose of recombinant murine TNF-alpha (10(5) U/mouse, i.p.) with and without various concentrations of recombinant human EPO. After 3 days, effects on late-stage erythropoiesis were measured by determining the number of mature erythroid colony-forming cells (CFU-E) in the spleen and bone marrow. Simultaneous treatment with EPO abrogated the suppressive effect of TNF-alpha in a dose-dependent manner. EPO treatment also prevented the decrease in peripheral blood-hematocrit that was observed with chronic (5 x 10(4) U/mouse/day for 5 days) administration of TNF-alpha. TNF-alpha-induced hemorrhagic necrosis of tumors and stimulation of macrophage (CFU-M) progenitors were unaffected by EPO treatment. These results demonstrate that simultaneous injection of EPO can abrogate the TNF-alpha-induced suppressive effects on erythropoiesis.     

Developments in the therapeutic use of erythropoiesis stimulating agents

Recombinant human erythropoietin (rHuEPO) has become the standard therapy for treatment of the anaemia of chronic kidney disease (CKD) during the past two decades. In addition, rHuEPO can be indicated for the treatment of cancer patients on chemotherapy and surgical patients to avoid allogeneic red blood cell transfusion. This review first describes recent attempts in developing rHuEPO congeners (mutated and pegylated rHuEPOs) and mimetics with prolonged half-lives and improved application requirements. Secondly, the pathophysiological background of the regulatory guideline, that blood haemoglobin levels in anaemic CKD or cancer patients on chemotherapy should not be raised above the target value of 120 g/l, is discussed. Finally, potential novel indications are considered for the use of rHuEPO and its analogues as pleiotropic cytoprotectant agents for cardio-, nephro-, hepato- and neuroprotection.     

In vitro studies of erythropoiesis. II. The effect of anoxia on heme synthesis

The effect of various oxygen tensions on the in vitro synthesis of heme by rabbit bone marrow was measured. At levels above 4 per cent oxygen there was noeffect of oxygen tension on heme synthesis. Total anoxia stopped heme synthesiscompletely. No level of oxygen tension was found to stimulate heme synthesis.

Submitted on November 16, 1954 Accepted on January 1, 1955     

The role of erythropoiesis stimulating agents and intravenous (IV) iron in the cardio renal anemia syndrome

Anemia is common in Congestive Heart Failure (CHF) and is associated with an increased mortality, morbidity and progressive renal failure. The most common causes of the anemia in CHF are (1) the associated Chronic Kidney Disease (CKD), which causes depression of erythropoietin (EPO) production in the kidney, and (2) excessive cytokine production in CHF, which can cause both depression of erythropoietin production in the kidney and depression of erythropoietin response in the bone marrow. The cytokines can also induce iron deficiency by increasing hepcidin production from the liver, which both reduces gastrointestinal iron absorption and reduces iron release from iron stores located in the macrophages and hepatocytes. It appears that iron deficiency is very common in CHF and is rarely recognized or treated. The iron deficiency can cause a thrombocytosis that might contribute to cardiovascular complications in both CHF and CKD and is reversible with iron treatment. Thus, attempts to control this anemia in CHF will have to take into consideration both the use of both Erythropoiesis Stimulating Agents (ESA) such as EPO and oral and, probably more importantly, intravenous (IV) iron. Many studies of anemia in CHF with ESA and oral or IV iron and even with IV iron without ESA have shown a positive effect on hospitalization, New York Heart Association functional class, cardiac and renal function, quality of life, exercise capacity and reduced Beta Natriuretic Peptide and have not demonstrated an increase in cardiovascular damage related to the therapy. However, adequately powered long-term placebo-controlled studies of ESA and of IV iron in CHF are still needed and are currently being carried out.     

Mechanisms underlying the suppression of erythropoiesis by hyperoxia

The possible mechanisms underlying the suppression of erythropoiesis in hyperoxic animals were studied. Male Long-Evans rats were injected with cobaltous chloride hexahydrate, a known erythropoietic stimulant. One group was exposed to a hyperoxic environment for ten hours. Both serum levels of erythropoietin (Ep) and renal levels of erythrogenin were significantly lower (p less than 0.005) in the hyperoxic animals compared to those left at room air. In addition, inhibitors to Ep or erythrogenin could not be detected in either the serum or renal tissue of the hyperoxic rats. These results indicate that the primary factor responsible for the erythropoietic suppression observed in a hyperoxic environment is a decreased production of erythrogenin which results in turn, in a lowered level of circulating Ep.     

In vivo effects of interleukin-11 and stem cell factor in combination with erythropoietin in the regulation of erythropoiesis

Summary. In this study we evaluated the in vivo effects of interleukin-11 (IL-11) and stem cell factor (SCF), in combination with erythropoietin (EPO) on murine erythropoiesis. Mice were treated for 7d with IL-11, SCF and EPO, each at three dose levels. In total, 27 different dose combinations were tested. IL-11 as well as SCF could only marginally stimulate erythroid progenitor cell numbers, but IL-11 in combination with SCF was able to increase BFU-E and CFU-E numbers 4-fold, in the absence of exogenous EPO. This resulted in an increased reticulocyte count. In contrast with the stimulatory effect on immature erythroid cell stages, IL-11 treatment induced a mild anaemia, which probably resulted from a plasma volume expansion. The additional treatment with EPO resulted in strong synergistic effects on CFU-E numbers. The combination of high-dose IL-11 and high-dose SCF was able to increase the overall efficiency of EPO-induced erythroid amplification, which was reflected by a left-shift of the in vivo EPO dose-response curve. The stimulating effects of IL-11 and SCF were further demonstrated when the effects on the reticulocyte count of a single high-dose EPO injection were assessed in normal and SCF + IL-11 treated mice. Whereas a single EPO dose increased the reticulocyte count by a factor of 3, IL-11 + SCF pretreatment increased this to a factor of 7. This study shows that in vivo SCF and IL-11 are important modulators of red blood cell production. First, these factors probably increase the input from the stem cell compartment into the erythroid lineage, where subsequently EPO is required for further amplification. Additionally, however, IL-11 and SCF increase the overall efficiency of EPO-induced amplification, probably due to a stimulatory effect on late-stage erythroid cells and to a redistribution of cells from marrow to spleen.     

Clinical results of recombinant erythropoietin in transfusion-dependent patients with refractory multiple myeloma: role of cytokines and monitoring of erythropoiesis

Abstract: Recombinant erythropoietin (r-EPO) was administered to 37 patients with advanced, transfusion-dependent and chemo-resistant multiple myeloma (MM), at the fixed dose of 10,000/U s.c, 3 times a week, for 2 months. Thirteen patients (35.1%) achieved a significant response in terms of complete abolition of red cell transfusions. Factors significantly predictive of response were: a) inappropriate production of endogenous EPO, as expressed by a reduced observed/predicted ratio; b) presence of a consistant number of circulating erythroid precursors BFU–E; c) low serum levels of tumor necrosis factor (TNF) and interleukin-1 (IL-1), cytokines with inhibitory activity on erythropoiesis; d) a single line of previously received chemotherapy. Renal failure, bone marrow plasma cell infiltration, serum levels of IL-6 and other main clinical and laboratory parameters did not affect significantly the response to r-EPO. High fluorescence reticulocytes (HFR) and soluble transferrin receptor (sTfR) values were useful to detect an early stimulation of erythropoiesis in responders, while a high percentage of circulating hypochromic erythrocytes (HE), as assessed by an automated counter, identified those patients developing functional iron deficiency during r-EPO treatment. We conclude that about one-third of severely anemic patients with advanced MM, unresponsive to chemotherapy, may benefit by r-EPO therapy. The clinical management of these patients can be accomplished using non-invasive parameters, such as sTfR, HFR and HE.     

The effect of stem-cell factor,interleukin-3 and erythropoietin on in vitro erythropoiesis in myelodysplastic syndromes

An inherent defect of erythroid differentiation at the colony-forming unit blast (CFU-blast) compartment and (or) an impaired response of early erythroid progenitors (BFU-E) to growth stimulation are both considered to contribute to anemia in myelodysplastic syndromes (MDS). With the intention of improving survival and growth of early erythroid progenitors we investigated the effect of stem-cell factor (SCF) and interleukin-3 (IL-3) alone and in combination with erythropoietin, on the in vitro erythropoiesis of 13 patients with MDS and of three normal controls. SCF and IL-3 alone did not promote erythroid colony growth in MDS, while 3 cases responded to erythropoietin alone. In each of these, BFU-E colony growth could be increased by SCF, which was also found in all normal bone marrows. Altogether 6 cases showed a significant enhancement of BFU-E colony numbers by the combination of SCF and erythropoietin as compared to erythropoietin alone (P=0.036). Out of the 6 responding cases, 5 belonged to the FAB-classified subgroups refractory anemia (RA) and refractory anemia with ringed sideroblasts (RA/RS) (5/5), while 1 patient was classified as having refractory anemia with excess of blasts (RAEB) (1/4). No patient with refractory anemia with excess of blasts in transformation (RAEB-T) (0/4) responded. In spite of these positive effects, the absolute number of BFU-E colonies remained reduced in all MDS cases when compared to normal controls. IL-3 proved ineffective in increasing the response to erythropoietin in MDS although it increased erythropoietin-induced BFU-E formation in normal controls significantly. We conclude that the striking synergistic effect of SCF and erythropoietin on erythroid colony formation seen with normal bone marrow is conserved in most cases with RA and RA/RS. In RAEB and RAEB-T the intrinsic defect of the erythroid differentiation pathway cannot be overcome by SCF.Abbreviation MDS myelodysplastic syndrome - SCF stem-cell factor - II-3 interleukin-3 - RA refractory anemia - RA/RS RA with ringed sideroblasts - RAEB RA with excess of blasts - RAEB-T RA with excess of blasts in transformation - Epo erythropoietin     

Case report: role of granulocyte colony stimulating factor in radiotherapy.

Two patients who received granulocyte colony stimulating factor (G-CSF) before or during radiotherapy are reviewed. Both patients developed granulocytopenia after multi-agent chemotherapy. One patient had breast cancer with multiple distant metastasis and received palliative radiotherapy to the spine and hip joint. The other patient had locally advanced anal carcinoma and received concurrent 5-fluorouracil, mitomycin, and radiation therapy. Both patients completed the planned course of radiotherapy without interruption, after administration of G-CSF.     

Endotoxin-induced lung injury--the role of leukocytes and oxidants

Endotoxin shock was made in guinea pigs to understand the initial pathogenesis in connection with the oxygen radicals from leukocytes. The intraperitoneal injection of LD50 E. coli endotoxin induced rapid leukopenia, and leukocytosis appeared at 24 hours of injection. The cell differential counts of bronchoalveolar lavage fluid revealed a significant increase in eosinophil at 10 and 30 minutes. There was also a significant increase in neutrophil at 24 hours. The activity of superoxide dismutase in the lung tissue decreased at 10 minutes of injection and remained low for 24 hours. Malonaldehyde, a lipid peroxidation metabolite by oxidants, gradually increased and reached a maximal value at 60 minutes of injection. The lung damage was histologically characterized by a rapid appearance of the alveolar edema and scattered infiltration of granulocytes. These suggested the endotoxin injection induced activation of granulocytes recruited from peripheral blood to cause oxidant reaction in lung tissues, which finally produced acute lung injury.