A recent study in dogs suggested that erythropoietin (EPO) not only promotes the synthesis of increased numbers of reticulated platelets but that these newly produced platelets are hyperreactive compared with controls. Because of the increasing use of EPO in the perioperative setting, we characterized the effects of EPO on platelet reactivity in healthy human volunteers. In a randomized, controlled trial, we studied the effects of EPO on platelet reactivity, thrombopoiesis, and endothelial activation in circumstances similar to those of autologous blood donation. Thirty healthy male volunteers received placebo or EPO (100 or 500 U/kg of body weight given intravenously) three times a week for 2 weeks and underwent phlebotomy on days 8 and 15. Thrombin receptor-activating peptide induced expression of P-selectin, and CD63 increased 2- to 3-fold during EPO treatment. The enhanced platelet reactivity was also reflected by a 50% increase in soluble P-selectin in plasma. Plasma E-selectin levels increased in a dose-dependent fashion by more than 100% during EPO treatment, indicating substantial activation of endothelial cells. A 10% to 20% increase in platelet counts was observed in both EPO groups on day 5. In the placebo group, platelets increased only several days after the first phlebotomy. The increase in platelet counts was not reflected by changes in the amounts of reticulated platelets or circulating progenitor cells. In summary, we found that EPO markedly enhances endothelial activation and platelet reactivity, which may adversely affect patients at cardiovascular risk. However, the increased platelet reactivity could be exploited in patients with platelet dysfunction. (Blood. 2000;95:2983-2989)

We have generated 2 zebrafish lines carrying inactivating germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene ortholog vhl. Mutant embryos display a general systemic hypoxic response, including the up-regulation of hypoxia-induced genes by 1 day after fertilization and a severe hyperventilation and cardiophysiologic response. The vhl mutants develop polycythemia with concomitantly increased epo/epor mRNA levels and erythropoietin signaling. In situ hybridizations reveal global up-regulation of both red and white hematopoietic lineages. Hematopoietic tissues are highly proliferative, with enlarged populations of c-myb(+) hematopoietic stem cells and circulating erythroid precursors. Chemical activation of hypoxia-inducible factor signaling recapitulated aspects of the vhl(-/-) phenotype. Furthermore, microarray expression analysis confirms the hypoxic response and hematopoietic phenotype observed in vhl(-/-) embryos. We conclude that VHL participates in regulating hematopoiesis and erythroid differentiation. Injections with human VHLp30 and R200W mutant mRNA demonstrate functional conservation of VHL between mammals and zebrafish at the amino acid level, indicating that vhl mutants are a powerful new tool to study genotype-phenotype correlations in human disease. Zebrafish vhl mutants are the first congenital embryonic viable systemic vertebrate animal model for VHL, representing the most accurate model for VHL-associated polycythemia to date. They will contribute to our understanding of hypoxic signaling, hematopoiesis, and VHL-associated disease progression.

The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15-0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

Recombinant human erythropoietin (rhEPO) induces neurogenesis and angiogenesis. Using a coculture system of mouse brain endothelial cells (MBECs) and neural progenitor cells derived from the subventricular zone of adult mouse, we investigated the hypothesis that neural progenitor cells treated with rhEPO promote angiogenesis. Treatment of neural progenitor cells with rhEPO significantly increased their expression and secretion of vascular endothelial growth factor (VEGF) and activated phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and extracellular signal-regulated kinase (ERK1/2). Selective inhibition of the Akt and ERK1/2 signaling pathways significantly attenuated the rhEPO-induced VEGF expression in neural progenitor cells. The supernatant harvested from neural progenitor cells treated with rhEPO significantly increased the capillary-like tube formation of MBECs. SU1498, a specific VEGF type-2 receptor (VEGFR2) antagonist, abolished the supernatant-enhanced angiogenesis. In addition, coculture of MBECs with neural progenitor cells treated with rhEPO substantially increased VEGFR2 mRNA and protein levels in MBECs. These in vitro results suggest that EPO enhances VEGF secretion in neural progenitor cells through activation of the PI3K/Akt and ERK1/2 signaling pathways and that neural progenitor cells treated with rhEPO upregulate VEGFR2 expression in cerebral endothelial cells, which along with VEGF secreted by neural progenitor cells promotes angiogenesis.

OBJECTIVE

Erythropoietin (EPO), a cytokine best known for its ability to increase red blood cell mass, has recently been shown to protect cardiomyocytes from apoptotic cell death. The objective of the present study was to investigate the role of endothelial nitric oxide synthase (eNOS) in the anti-apoptotic effects of EPO in cardiomyocytes.

RESULTS

Neonatal mouse ventricular cardiomyocytes were isolated and cultured from wild-type and eNOS(-/-) mice. Treatment with EPO significantly reduced apoptosis induced by norepinephrine (NE) in the wild-type cardiomyocytes. The reduction of apoptosis was associated with significant increases in eNOS expression, phosphorylation and NO production. However, the anti-apoptotic effects of EPO were significantly decreased in wild-type cardiomyocytes treated with L-NAME, which inhibits nitric oxide synthase activity. The results were further confirmed using eNOS(-/-) cardiomyocytes. To investigate the in vivo significance of eNOS in mediating the anti-apoptotic effects of EPO, wild-type and eNOS(-/-) mice were subjected to myocardial ischemia and reperfusion. EPO decreased myocardial apoptosis and infarct size in wild-type mice. However, the protective effects of EPO were significantly diminished in eNOS(-/-) mice.

CONCLUSIONS

EPO increases eNOS expression and NO production in cardiomyocytes. The anti-apoptotic effects of EPO in cardiomyocytes are mediated by eNOS-derived NO production.

Erythropoietin (EPO) is the primary regulator of erythropoiesis, stimulating growth, preventing apoptosis, and promoting differentiation of red blood cell progenitors. The EPO receptor belongs to the cytokine receptor superfamily. Although the primary role of EPO is the regulation of red blood cell production, EPO and its receptor have been localized to several nonhematopoietic tissues and cells, including the central nervous system (CNS), endothelial cells, solid tumors, the liver, and the uterus. The presence of EPO receptors and the possibility of EPO signaling in these tissues and cells have led to numerous studies of the effects of EPO at these sites. In particular, expression of EPO and the EPO receptor in cancer cells has generated much interest because of concern that administration of recombinant human erythropoietin (rHuEPO) to patients with breast and other cancer cells expressing the EPO receptor may promote tumor growth via the induction of cell proliferation or angiogenesis. However, evidence supporting a growth-promoting effect has been inconclusive. Moreover, several preclinical studies have shown a beneficial effect of EPO on delaying tumor growth. Further, it is conceivable that increased expression of EPO could reduce tumor hypoxia and ameliorate the deleterious effects of hypoxia on tumor growth, metastasis, and treatment resistance. On the other hand, EPO has also been shown to produce an angiogenic effect in vascular endothelial cells in vitro. However, there is no evidence that these effects occur in vivo to promote tumor growth. EPO and EPO receptors are expressed in neural tissue, and they are upregulated there by hypoxia. Animal studies have shown that administration of epoetin alfa (an rHuEPO) reduces tissue injury due to ischemic stroke, blunt trauma, and experimental autoimmune encephalomyelitis. These findings suggest that epoetin alfa may provide a therapeutic benefit in patients with stroke, trauma, epilepsy, and other CNS-related disorders. Clearly, further study of EPO and the EPO receptor in nonhematopoietic tissue is warranted to determine the potential therapeutic usefulness of rHuEPO as well as to determine the signaling pathway responsible for its effect in vivo.

The JAK2 tyrosine kinase is known to associate with the receptors for growth hormone (GH) and erythropoietin (EPO) and with the interleukin-6 receptor signal transducing protein, gp130. Here we demonstrate that chimeric cytokine receptors which contain the cytoplasmic domain of the receptors for GH and EPO or for gp130 can form complexes with JAK2 when transiently co-expressed in HeLa cells. Mutational analyses of chimeras for the the GH and EPO receptors and gp130 demonstrated that box 1, a motif critical for cytokine receptor signal transduction, was required for the association of JAK2. Although JAK2 was capable of associating with all three of the chimeras, JAK1 co-precipitated only with the gp130 chimera. Association of JAK1 and JAK2 with cytokine receptor proteins, therefore, requires the highly conserved box 1 domain, but other sequences within the receptor proteins may influence the specificity of JAK binding. Mutational analysis of JAK2 revealed that multiple or complex protein sequences within JAK2 are required for association with cytokine receptors.

BACKGROUND

Circulating bone marrow-derived endothelial progenitor cells (EPCs) promote vascular reparative processes. In humans, their number correlate with endothelial function and cardiovascular risk. We tested the hypothesis that darbepoetin alfa [i.e., a recombinant analogue of the cytokine erythropoietin (EPO)] stimulates proliferation and differentiation of EPCs.

METHODS

We assessed CD34+ circulating stem cells (cSCs) in whole blood using flow cytometry and, in addition, proliferation/differentiation of EPCs in an in-vitro assay during 6 weeks of a standard darbepoetin therapy in eight patients with renal anemia.

RESULTS

Darbepoetin treatment caused a significant increase in the number of CD34+ cSCs (week 2, 193%+/- 46%; and week 6, 298%+/- 90%; P < 0.05 vs. baseline). In addition, darbepoetin markedly increased the number of functionally active EPCs (week 2, 256%+/- 48%; and week 6, 299%+/- 59%; both P < 0.01 vs. baseline). The effect of darbepoetin on functional activity of EPCs assessed in a tube formation assay was dose dependent. Administration of darbepoietin caused activation of protein kinase B (Akt) in cultured EPCs.

CONCLUSIONS

A standard treatment with darbepoetin markedly enhances EPC proliferation and differentiation in renal patients. The use of recombinant EPO analogues may be a novel and safe therapeutic approach in patients with vascular pathology.

The response to erythropoietin-stimulating agents (ESA) can vary among different patients and according to the different circumstances over time within a given individual. The aim of this study was to analyze the factors that can modify the response to epoetin in patients on hemodialysis (HD) and its influence on early mortality. Prospective and observational study including 1710 patients from 119 HD units in Spain with a follow-up of 12 months. To evaluate the dose-response effect of EPO therapy, we used the erythropoietin resistance index (ERI), calculated as the weekly weight-adjusted dose of EPO divided by the hemoglobin level. Patients were stratified in three groups according to ERI: group A, ERI <5; group B, ERI=5-15; group C, ERI>15 U/kg/week/g per 100 ml. Mean ERI for the entire group was 10.2+/-7.3 U/kg/week/g per 100 ml. ERI was directly related with incident comorbidity (Charlson Index), age, female gender and low body mass index with no relationship with etiology of chronic kidney disease. Patients with antecedents of heart failure, acute infection or malignant neoplasm had significantly higher ERI than those without. Transferrin saturation index, but not serum ferritin, was inversely related with ERI. Serum levels of albumin and cholesterol were related with lower ERI, but no relation was found with normalized protein catabolic rate. Patients with a permanent catheter for HD had significant higher values of ERI than those with native fistula (P=0.012). One year survival in all three groups of patients according to ERI was 0.916 in group A, 0.877 in group B and 0.788 in group C (log-rank=20.7, P<0.001). The resistance to ESA is directly related with incident comorbidity in patients on hemodialysis and it can be interpreted as a useful marker of early mortality.

Carbamylated erythropoietin (CEPO), a well characterized erythropoietin (EPO) derivative, does not bind to the classical EPO receptor and does not stimulate erythropoiesis. Using neural progenitor cells derived from the subventricular zone of the adult mouse, we investigated the effect of CEPO on neurogenesis and the associated signaling pathways in vitro. We found that CEPO significantly increased neural progenitor cell proliferation and promoted neural progenitor cell differentiation into neurons, which was associated with up-regulation of Sonic hedgehog (Shh), its receptor ptc, and mammalian achaete-scute homolog 1 (Mash1), a pro-neuron basic helix-loop-helix protein transcription factor. Blockage of the Shh signaling pathway with a pharmacological inhibitor, cyclopamine, abolished the CEPO-induced neurogenesis. Attenuation of endogenous Mash1 expression by short-interfering RNA blocked CEPO-promoted neuronal differentiation. In addition, recombinant mouse Shh up-regulated Mash1 expression in neural progenitor cells. These results demonstrate that the Shh signaling pathway mediates CEPO-enhanced neurogenesis and Mash1 is a downstream target of the Shh signaling pathway that regulates CEPO-enhanced neuronal differentiation.

The hormone erythropoietin (EPO) is essential for the survival, proliferation and differentiation of the erythrocytic progenitors. The EPO receptor (EPO-R) of erythrocytic cells belongs to the cytokine class I receptor family and signals through various protein kinases and STAT transcription factors. The EPO-R is also expressed in many organs outside the bone marrow, suggesting that EPO is a pleiotropic anti-apoptotic factor. The controversial issue as to whether the EPO-R is functional in tumor tissue is critically reviewed. Importantly, most studies of EPO-R detection in tumor tissue have provided falsely positive results because of the lack of EPO-R specific antibodies. However, endogenous EPO appears to be necessary to maintain the viability of endothelial cells and to promote tumor angiogenesis. Although there is no clinical proof that the administration of erythropoiesis stimulating agents (ESAs) promotes tumor growth and mortality, present recommendations are that (i) ESAs should be administered at the lowest dose sufficient to avoid the need for red blood cell transfusions, (ii) ESAs should not be used in patients with active malignant disease not receiving chemotherapy or radiotherapy, (iii) ESAs should be discontinued following the completion of a chemotherapy course, (iv) the target Hb should be 12 g/dL and not higher and (v) the risks of shortened survival and tumor progression have not been excluded when ESAs are dosed to target Hb <12 g/dL.

Since the isolation and purification of erythropoietin (EPO) in 1977, the essential role of EPO for mature red blood cell production has been well established. The cloning of the EPO gene and production of recombinant human EPO led to the widespread use of EPO in treating patients with anaemia. However, the biological activity of EPO is not restricted to regulation of erythropoiesis. EPO receptor (EPOR) expression is also found in endothelial, brain, cardiovascular and other tissues, although at levels considerably lower than that of erythroid progenitor cells. This review discusses the survival and proliferative activity of EPO that extends beyond erythroid progenitor cells. Loss of EpoR expression in mouse models provides evidence for the role of endogenous EPO signalling in nonhaematopoietic tissue during development or for tissue maintenance and/or repair. Determining the extent and distribution of receptor expression provides insights into the potential protective activity of EPO in brain, heart and other nonhaematopoietic tissues.

Erythropoietin (EPO), originally discovered as hematopoietic growth factor, has direct effects on cells of the nervous system that make it a highly attractive candidate drug for neuroprotection/neuroregeneration. Hardly any other compound has led to so much preclinical work in the field of translational neuroscience than EPO. Almost all of the >180 preclinical studies performed by many independent research groups from all over the world in the last 12 years have yielded positive results on EPO as a neuroprotective drug. The fact that EPO was approved for the treatment of anemia >20 years ago and found to be well tolerated and safe, facilitated the first steps of translation from preclinical findings to the clinic. On the other hand, the same fact, naturally associated with loss of patent protection, hindered to develop EPO as a highly promising therapeutic strategy for application in human brain disease. Therefore, only few clinical neuroprotection studies have been concluded, all with essentially positive and stimulating results, but no further development towards the clinic has occurred thus far. This article reviews the preclinical and clinical work on EPO for the indications neuroprotection/neuroregeneration and cognition, and hopefully will stimulate new endeavours promoting development of EPO for the treatment of human brain diseases.

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.

Erythropoietin (EPO), a humoral regulator of erythropoiesis and replacement therapy for selected red blood cell disorders in EPO-deficient patients, has been implicated in a wide range of activities on diverse cell, tissue, and organ types. EPO signals via two receptors, one comprising EPO receptor (EPOR) homodimers and the other a heterodimer of EPOR and CD131-the common β chain component of the GM-CSF, interleukin (IL)-3, and IL-5 receptors. Ligation of EPORs triggers various signaling pathways, including the JAK2-STAT5 and MAPK-NF-κB pathways, depending both on the receptor and the target cell type. A new study in this issue reveals a novel EPO-triggered pathway involving a Spi2A serpin-lysosome-cathepsin cascade that is initiated through the homodimeric EPOR complex and is required for the survival of erythroid progenitors. A full understanding of EPO's effects on various cell types and their potential clinical relevance requires more work on the signaling events initiated through both EPORs, the effects of other cytokines and growth factors that modulate EPO's actions, and a comparison of the effects of full-length versus truncated forms of EPO.

BACKGROUND

Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.

RESULTS

We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.

CONCLUSIONS

Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

Blind subterranean mole rats (Spalax, Spalacidae) evolved adaptive strategies to cope with hypoxia that climaxes during winter floods in their burrows. By using real-time PCR, we compared gene expression of erythropoietin (Epo), a key regulator of circulating erythrocytes, and hypoxia-inducible factor 1 alpha (HIF-1 alpha), Epo expression inducer, in the kidneys of Spalax and white rats, Rattus norvegicus. Our results show significantly higher, quicker, and longer responses to different O(2) levels in Spalax compared with Rattus. (i) In normoxia, both Spalax and Rattus kidneys produce small amounts of Epo. Maximal expression of Rattus Epo is noticed after a 4-h hypoxia at 6% O(2). Under these conditions, Spalax Epo levels are 3-fold higher than in Rattus. After 24 h of 10% O(2), Spalax Epo reaches its maximal expression, remarkably 6-fold higher than the maximum in Rattus; (ii) the HIF-1 alpha level in normoxia is 2-fold higher in Spalax than in Rattus. Spalax HIF-1 alpha achieves maximal expression after 4-h hypoxia at 3% O(2), a 2-fold increase compared with normoxia, whereas no significant change was detected in Rattus HIF-1 alpha at any of the conditions studied; (iii) at 6% O(2) for 10 h, in which Rattus cannot survive, Epo and HIF-1 alpha levels in Spalax galili, living in heavily flooded soils, are higher than in Spalax judaei, residing in light aerated soil. We suggest that this pattern of Epo and HIF-1 alpha expression is a substantial contribution to the adaptive strategy of hypoxia tolerance in Spalax, evolved during 40 million years of evolution to cope with underground hypoxic stress.

OBJECTIVE

Hypoxic preconditioning is an endogenous protection against subsequent lethal hypoxia, but the mechanism involved is not understood. Hypoxia is followed by reactive oxygen species (ROS) production and induces hypoxia-inducible factor (HIF) and its downstream factor erythropoietin (Epo), which is associated with neuroprotection. We hypothesized that these endogenous processes may contribute to hypoxic preconditioning.

METHODS

We used a mouse neuronal culture model, with 2 hours of hypoxia as preconditioning followed by 15 hours of hypoxic insult, and examined the expression of HIF-1alpha, Epo, and their downstream proteins by Western blotting. Copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) mice were used to detect the effect of ROS. Cell survival and apoptosis were detected by mitogen-activated protein 2 quantification, apoptotic-related DNA fragmentation, and caspase-3 fragmentation. Antisense Epo was used to block endogenously produced Epo.

RESULTS

Hypoxic preconditioning was protective in wild-type (Wt) neurons but not in neurons obtained from SOD1 Tg mice. In Wt neurons, HIF-1alpha and Epo expression showed a greater increase after hypoxia compared with Tg neurons and reached a higher level with preconditioned hypoxia, followed by pJak2, pStat5, and nuclear factor kappaB (NF-kappaB) expression. Antisense Epo decreased these downstream proteins and the neuroprotection of hypoxic preconditioning.

CONCLUSIONS

Hypoxic preconditioning induces ROS, which may downregulate the threshold for production of HIF-1alpha and Epo expression during subsequent lethal hypoxia, thus exerting neuroprotection through the Jak2-Stat5 and NF-kappaB pathways.

OBJECTIVE

The aim of the present study was to determine if outcome in terms of upper limb function at 6 months after stroke can be predicted in hospital stroke units using clinical parameters measured within 72 hours after stroke. In addition, the effect of the timing of assessment after stroke on the accuracy of prediction was investigated by measurements on days 5 and 9.

METHODS

Candidate determinants were measured in 188 stroke patients within 72 hours and at 5 and 9 days after stroke. Logistic regression analysis was used for model development to predict upper limb function at 6 months measured with the action research arm test (ARAT).

RESULTS

Patients with an upper limb motor deficit who exhibit some voluntary extension of the fingers and some abduction of the hemiplegic shoulder on day 2 have a probability of 0.98 to regain some dexterity at 6 months, whereas the probability was 0.25 for those without this voluntary motor activity. Sixty percent of patients with some early finger extension achieved full recovery at 6 months in terms of action research arm test score. Retesting the model on days 5 and 9 resulted in a gradual decline in probability from 0.25 to 0.14 for those without voluntary motor activity of shoulder abduction and finger extension, whereas the probability remained 0.98 for those with this motor activity.

CONCLUSIONS

Based on 2 simple bedside tests, finger extension and shoulder abduction, functional recovery of the hemiplegic arm at 6 months can be predicted early in a hospital stroke unit within 72 hours after stroke onset.

Anemia is currently defined by the World Health Organization (WHO) as a hemoglobin (Hb) level <13 g/dL in men and <12 g/dL in women. While estimates vary widely, nearly one quarter of community-based octagenerians and one half of the chronically ill elderly have Hb levels that satisfy a diagnosis of anemia according to these criteria. A growing body of evidence has linked adverse events with even "mild" anemia or low-normal Hb in the elderly. Recent studies suggest strongly that aging is associated with dysregulation of pro-inflammatory cytokines, most notably interleukin-6 (IL-6), which may negatively impact hematopoiesis, either by inhibition of erythropoietin (EPO) production or interaction with EPO receptors. Anemia in older individuals is associated with a very wide range of complications, including increased risk for mortality, cardiovascular disease, cognitive dysfunction, longer hospitalization for elective procedures and comorbid conditions, reduced bone density, and falls and fractures. Not surprisingly, anemia also has a significant effect on quality of life (QOL) in the elderly. Most anemia in older individuals results from iron deficiency, chronic inflammation, or chronic kidney disease, or it may be unexplained. Future research on anemia in the elderly should focus on the age-related physiologic changes underlying this condition and whether anemia correction can reduce anemia-associated risks, and improve QOL.

Anemia is common in patients who have both heart failure and chronic kidney disease, and there is an association between anemia and progression of both diseases. The main causes of anemia are deficient production of erythropoietin (EPO), iron deficiency, and chronic disease with endogenous EPO resistance. EPO has been successfully used for over a decade to treat anemia in patients with chronic kidney disease. Less obvious are the safety and efficacy of EPO treatment in patients with both heart failure and renal disease. Up to 10% of patients receiving EPO are hyporesponsive to therapy and require large doses of the agent. Several mechanisms could explain resistance to endogenous and exogenous EPO. Proinflammatory cytokines antagonize the action of EPO by exerting an inhibitory effect on erythroid progenitor cells and by disrupting iron metabolism (a process in which hepcidin has a central role). EPO resistance might also be caused by inflammation, which has a negative effect on EPO receptors. Furthermore, neocytolysis could have a role. As resistance to exogenous EPO is associated with an increased risk of death, it is important to understand how cardiorenal failure affects EPO production and function.

Purified recombinant (r) macrophage inflammatory proteins (MIPs) 1 alpha, 1 beta, and 2 were assessed for effects on murine (mu) and human (hu) marrow colony-forming unit-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) colonies. Recombinant MIP-1 alpha, -1 beta, and -2 enhanced muCFU-GM colonies above that stimulated with 10 to 100 U natural mu macrophage-colony-stimulating factor (M-CSF) or rmuGM-CSF, with enhancement seen on huCFU-GM colony formation stimulated with suboptimal rhuM-CSF or rhuGM-CSF; effects were neutralized by respective MIP-specific antibodies. Macrophage inflammatory proteins had no effects on mu or huBFU-E colonies stimulated with erythropoietin (Epo). However, natural MIP-1 and rMIP-1 alpha, but not rMIP-1 beta or -2, suppressed muCFU-GM stimulated with pokeweed mitogen spleen-conditioned medium (PWMSCM), huCFU-GM stimulated with optimal rhuGM-CSF plus rhu interleukin-3 (IL-3), muBFU-E and multipotential progenitors (CFU-GEMM) stimulated with Epo plus PWMSCM, and huBFU-E and CFU-GEMM stimulated with Epo plus rhuIL-3 or rhuGM-CSF. The suppressive effects of natural MIP-1 and rMIP-1 alpha were also apparent on a population of BFU-E, CFU-GEMM, and CFU-GM present in cell-sorted fractions of human bone marrow (CD34 HLA-DR+) highly enriched for progenitors with cloning efficiencies of 42% to 75%. These results, along with our previous studies, suggest that MIP-1 alpha, -1 beta, and -2 may have direct myelopoietic enhancing activity for mature progenitors, while MIP-1 alpha may have direct suppressing activity for more immature progenitors.

Incorporation of a central polypurine tract (cPPT) and a posttranscriptional regulatory element (PRE) into lentivirus vectors provides increased transduction efficiency and transgene expression. We compared the effects of these elements individually and together on transduction efficiency and gene expression, using lentivirus vectors pseudotyped with vesicular stomatitis virus G protein (VSV-G) and encoding enhanced green fluorescent protein (GFP) and rat erythropoietin (EPO). The transduction efficiency was greater than 2-fold higher in the vector containing the PRE element, 3-fold higher in vector encoding the cPPT element, and 5-fold increased in the GFP virus containing both cPPT and PRE elements relative to the parent virus. In comparison with parent vector the mean fluorescence intensity (MFI) of GFP expression was 7-fold higher in cells transduced with virus containing PRE, 6-fold increased in cells transduced with virus containing cPPT, and 42-fold increased in GFP-virus containing both cPPT and PRE elements. EPO-virus containing a PRE element showed a nearly 5-fold increase in EPO secretion over the parent vector, and the vector encoding both PRE and cPPT showed a 65-fold increase. Thus, lentivirus vectors incorporating both PRE and cPPT showed expression levels significantly increased over the sum of the components alone, suggesting a synergistic effect.

Members of the cytokine receptor superfamily have structurally similar extracellular ligand-binding domains yet diverse cytoplasmic regions lacking any obvious catalytic domains. Many of these receptors form ligand-induced oligomers which are likely to participate in transmembrane signaling. A constitutively active (factor-independent) mutant of the erythropoietin receptor (EPO-R), R129C in the exoplasmic domain, forms disulfide-linked homodimers, suggesting that the wild-type EPO-R is activated by ligand-induced homodimerization. Here, we have taken two approaches to probe the role EPO-R dimerization plays in signal transduction. First, on the basis of the crystal structure of the ligand-bound, homodimeric growth hormone receptor (GH-R) and sequence alignment between the GH-R and EPO-R, we identified residues of the EPO-R which may be involved in intersubunit contacts in an EPO-R homodimer. Residue 129 of the EPO-R corresponds to a residue localized to the GH-R dimer interface region. Alanine or cysteine substitutions were introduced at four other residues of the EPO-R predicted to be in the dimer interface region. Substitution of residue E-132 or E-133 with cysteine renders the EPO-R constitutively active. Like the arginine-to-cysteine mutation at position 129 in the exoplasmic domain (R129C), E132C and E133C form disulfide-linked homodimers, suggesting that constitutive activity is due to covalent dimerization. In the second approach, we have coexpressed the wild-type EPO-R with inactive mutants of the receptor missing all or part of the cytosolic domain. These truncated receptors have a dominant inhibitory effect on the proliferative action of the wild-type receptor. Taken together, these results strengthen the hypothesis that an initial step in EPO- and EPO-R-mediated signal transduction is ligand-induced receptor dimerization.