Combination Of Erythropoietin Research Articles

P-80 Improved predictive factors ofresponse for myelodysplastic syndrome patients treated by the combination of erythropoietin and G-CSF

P-80 Improved predictive factors ofresponse for myelodysplastic syndrome patients treated by the combination of erythropoietin and G-CSF

Combined therapy with methylprednisolone and erythropoietin in a model of multiple sclerosis

Neurodegenerative processes determine the clinical disease course of multiple sclerosis, an inflammatory autoimmune CNS disease that frequently manifests with acute optic neuritis. None of the established multiple sclerosis therapies has been shown to clearly reduce neurodegeneration. In a rat model of experimental autoimmune encephalomyelitis, we recently demonstrated increased neuronal apoptosis under methylprednisolone therapy, although CNS inflammation was effectively controlled. In the present study, we combined steroid treatment with application of erythropoietin to target inflammatory as well as neurodegenerative aspects. After immunization with myelin oligodendrocyte glycoprotein (MOG), animals were randomly assigned to six treatment groups receiving different combinations of erythropoietin and methylprednisolone, or respective monotherapies. After MOG-induced experimental autoimmune encephalomyelitis became clinically manifest, optic neuritis was monitored by recording visual evoked potentials. The function of retinal ganglion cells, the neurons that form the axons of the optic nerve, was measured by electroretinograms. Functional and histo pathological data of retinal ganglion cells and optic nerves revealed that neuron and axon protection was most effective when erythropoietin treatment that was started at immunization was combined with high-dose methylprednisolone therapy given from days 1 to 3 of MOG-induced experimental autoimmune encephalomyelitis. In contrast, isolated neuronal or axonal protection without clinical benefit was achieved under monotherapy with erythropoietin or methylprednisolone, respectively.

An RNA Interference Model of RPS19 Deficiency in Diamond Blackfan Anemia Recapitulates Defective Erythropoiesis and Rescue by Dexamethasone: Identification of Dexamethasone Responsive Genes by Microarray.

Diamond Blackfan Anemia (DBA), a congenital erythroblastopenia, is a model disease for the study of erythroid differentiation, but is poorly understood. RPS19 is the only gene yet to have been associated with DBA, but the relevance of this ubiquitously expressed ribosomal gene to erythroid differentiation is unclear. Glucocorticoids are the primary pharmacological therapy for patients with DBA, but the molecular basis for the activity of glucocorticoids in erythroid differentiation has not been identified. Through retroviral expression of short hairpin RNAs (shRNAs), we demonstrate that targeted degradation of the RPS19 gene in cultured human CD34+ cells blocks the proliferation and differentiation of erythroid progenitor cells. Decreased RPS19 expression limited production of erythroid colony formation on methylcellulose, decreased cell surface expression of glycophorin-A, and decreased cellular proliferation. Treatment of RPS19 deficient cells with dexamethasone restored erythroid differentiation to normal levels. We investigated the molecular basis of pharmacologic therapies for DBA using oligonucleotide microarrays to survey gene expression in CD34+ cells treated with combinations of erythropoietin, dexamethasone, IL-3, and SCF. None of these agents had a direct effect on the expression of RPS19 or on the coordinate expression of other ribosomal genes. Instead, dexamethasone activated a genetic program that includes a set of key hematopoietic regulatory genes, including Flt-3 and PLZF, that increase proliferation of hematopoietic progenitor cells. Genes specific to erythroid progenitor cells were up-regulated by dexamethasone, while genes specific to non-erythroid lineages were powerfully down-regulated. Deficiency of RPS19 therefore blocks proliferation of immature erythroid progenitor cells, and dexamethasone activates proliferation of the same cell population through mechanisms independent of RPS19. Identification of the key regulatory genes affected by dexamethasone may aid in the development of novel therapeutics that de-couple the beneficial hematopoietic effects of dexamethasone from detrimental non-hematopoietic side effects.

The enhanced in vitro hematopoietic activity of leridistim, a chimeric dual G-CSF and IL-3 receptor agonist.

The in vitro activity of leridistim was characterized for cell proliferation, generation of colony-forming units (CFU) and differentiation of CD34+ cells. In AML-193.1.3 cells, leridistim exhibited a significant increase in potency compared to rhG-CSF, SC-65303 (an IL-3 receptor agonist) or an equimolar combination of rhG-CSF and SC-65303. CFU-GM assays demonstrated that at 50% of the maximum response, the relative potency of leridistim was 12-fold greater than the combination of rhG-CSF and rhIL-3 and 44-fold more potent than rhG-CSF alone. In multi-lineage CFU assays, a combination of erythropoietin (rhEPO) and leridistim resulted in greater numbers of BFU-E, CFU-GEMM and CFU-Mk than rhEPO alone. Ex vivo culture of peripheral blood or bone marrow CD34+ cells with leridistim substantially increased total viable cells over cultures stimulated with rhG-CSF, SC-65303, or a combination of rhG-CSF and SC-65303. Culture with leridistim, resulted in a greater increase in myeloid (CD15+/CD11b+), monocytic (CD41-/CD14+) and megakaryocytic (CD41+/CD14-) precursor cells without depleting the progenitor pool (CD34+/CD15-/CD11b-). These results demonstrate that leridistim is a more potent stimulator of hematopoietic proliferation and differentiation than the single receptor agonists (rhG-CSF and SC-65303) either alone or combined. These unique attributes suggest that leridistim may enhance hematopoietic reconstitution following myelosuppressive chemotherapy.

Recombinant human erythropoietin in the treatment of anemic patients with hematological malignancies.

Patients with malignant diseases frequently develop anemia. An alternative to blood transfusions is the application of recombinant human erythropoietin. Several nonrandomized and prospective, placebo-controlled studies have demonstrated the effect and safety of erythropoietin in patients with hematological malignancies, particularly in patients with multiple myeloma and low- to intermediate-grade non-Hodgkin's lymphoma. However, in patients with myelodysplastic syndromes, the rather low response rate of erythropoietin is overcome by the combination of erythropoietin with granulocyte colony-stimulating factor. A significant acceleration of the reconstitution of erythropoiesis has been reported in allogeneic, but not in autologous bone marrow transplantation. Especially in large open-label, multicenter studies, a statistically and clinically significant improvement in quality of life independent from chemotherapeutic response or tumor type has been demonstrated. A number of simple algorithms have been proposed using the pretreatment serum erythropoietin level, transfusion requirements, and early changes in hematological parameters.

Effects of hydroxurea, stem cell factor, and erythropoietin in combination on fetal hemoglobin in the baboon.

Augmentation of the level of fetal hemoglobin (HbF) is considered therapeutic for patients with sickle cell disease. The objective of this study was to determine the effect of treatment with a combination of erythropoietin (Epo), stem cell factor (SCF), and hydroxyurea (HU) on HbF levels. The effect of treatment with a combination of Epo, SCF, and HU on HbF, F-cell numbers, and globin chain synthesis was evaluated in a baboon model. Treatment with a combination of SCF+Epo resulted in a two-fold increase in HbF, F-cells, and F-reticulocytes compared to Epo alone. The combination of SCF+Epo+HU resulted in an additional two-fold increase in HbF, whereas F-cells and F-reticulocytes increased only 25% compared to the SCF+Epo regimen. Measurement of differential globin chain synthesis indicated that the SCF+Epo+HU treatment also increased the I gamma/V gamma (homologous to human G gamma and A gamma) synthetic ratio toward the fetal ratio. HU can effectively augment growth factor-induced HbF synthesis in vivo. Because I gamma/V gamma ratios are unaffected by erythropoietic stress and similar increases in this ratio have only been observed following administration of 5-azacytidine, we suggest that these two agents may share a common mechanism of action involving the recruitment of a similar target cell population to terminal erythroid differentiation.

Purified unfractionated G-CSF/chemotherapy mobilized CD34+ peripheral blood progenitors and not bone marrow CD34+ progenitors undergo selective erythroid differentiation in liquid culture in the presence of erythropoietin and stem cell factor.

A combination of erythropoietin (EPO) plus stem cell factor (SCF) drove purified unfractionated granulocyte colony stimulating factor (G-CSF)/chemotherapy mobilized peripheral blood CD34+ cells to selective erythroid differentiation in liquid culture with an average 28-fold increase in the total cell number after 21 d. From day 6 of culture cytologic and cytofluorimetric characterization revealed that cultured cells belonged to the erythroid lineage with a gradual wave of maturation along the erythroid pathway to terminal cells. A similar pattern of erythroid differentiation was observed when the same peripheral blood CD34+ cells were culture with EPO plus SCF in serum-free medium. This cytokine combination produced selective erythroid differentiation with the complete exhaustion of the clonogenic potential on day 21. In parallel experiments the same circulating CD34+ cells underwent granulocytic/ monocytic differentiation in liquid culture in response to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and SCF, demonstrating that these CD34+ progenitors had intact pluripotent differentiating potential. Conversely, bone marrow CD34+ cells isolated from bone marrow allografts were unable to selectively differentiate along the erythroid pathway when they were exposed to EPO plus SCF combination. However, these cells maintained a greater number of colony forming cells on day 21 of culture compared to mobilized peripheral blood CD34+ cells. This model is a simple and reliable way to obtain selective erythroid differentiation of peripheral blood G-CSF/ chemotherapy mobilized CD34+ progenitor cells in liquid culture. The absence of cytokines such as GM-CSF and IL-3 in the culture medium permits studies on in vitro erythropoiesis without disturbance of prevalent myelopoiesis.

Inhibitory effects of zidovudine in erythroid progenitor cells: Reversal with a combination of erythropoietin and interleukin-3

To investigate the mechanisms that may be involved in zidovudine (AZT)-induced hematopoietic toxicity, spleen cells isolated from phenylhydrazine-treated anemic mice or murine bone marrow erythroid progenitor cells were treated with AZT (1–10 μM) for 24 hr. A concentration-dependent inhibition of the binding of 125I-labeled erythropoietin (Epo) was observed, suggesting down-regulation of Epo receptors. To determine if this effect is due to modulation of the levels of Epo receptor mRNA and to assess the effect of AZT on the expression of protooncogenes, mRNA levels were monitored by the slot blot hybridization technique. AZT caused a concentration-dependent inhibition in the levels of the mRNA of Epo receptors and c- fos, whereas the level of c- myc mRNA was unaffected. AZT also inhibited protein kinase C (PKC) activity in a concentration- and time-dependent manner, causing 50% inhibition at 10 μM within 3hr. Simultaneous addition of Epo or interleukin-3 (IL-3) partially reversed the inhibitory effects of AZT on the levels of the mRNAs and on PKC activity; however, a combination of Epo and IL-3 was significantly more effective. These studies demonstrate that (i) AZT-induced down-regulation of Epo receptors and c- fos expression coupled with inhibition of Epo receptor-mediated signal transduction through PKC are significant contributory factors to AZT-induced erythroid toxicity, and (ii) these inhibitory effects can be overcome by treatment with a combination of Epo and IL-3.

44 Zidovudine (AZT)-induced down regulation of transcripts of erythropoietin receptor (Epo-R), c-fos and β-protein kinase C (PKC) in human erythroid progenitor cells (EPC): Reversal with a combination of erythropoietin and interleukin-3 : K.C. Agrawal, S.R. Gogu, B.J. Rider and C.A. Leissinger. Depts. of Pharmacology and Medicine, Tulane University School of Medicine, New Orleans, LA. 70112, USA

44 Zidovudine (AZT)-induced down regulation of transcripts of erythropoietin receptor (Epo-R), c-fos and β-protein kinase C (PKC) in human erythroid progenitor cells (EPC): Reversal with a combination of erythropoietin and interleukin-3 : K.C. Agrawal, S.R. Gogu, B.J. Rider and C.A. Leissinger. Depts. of Pharmacology and Medicine, Tulane University School of Medicine, New Orleans, LA. 70112, USA

Isolation and characterization of an erythroid cell line highly inducible to form erythroid burst-like colonies.

The study of induction of Friend erythroleukemic cell lines during the last decade has enriched our understanding of late erythroid differentiation. In comparison, little information is available on early erythroid differentiation. We describe here the isolation and characterization of a highly inducible clone from a murine erythroid cell line, which is capable of forming colonies that possess properties of the early erythroid burst progenitor. We found that a combination of erythropoietin (Epo), spleen conditioned medium (SCM), and plasma from a patient with aplastic anemia (Apa) induces over 95% of cells from this clone (clone 12) to form colonies with the properties of burst or mixed burst blast-like colonies. Examination of the culture conditions of these cells indicated that alpha medium was more efficient for colony induction than Iscove's medium, and that the addition of two-mercaptoethanol did not improve the induction process. These factors (EPo, SCM, and Apa) must be present for 4 days in order for induction to take place. It is hoped that the isolation of this highly inducible cell clone will enrich our understanding of early erythroid differentiation.