Erythropoietin (Epo) has been shown to have potent anti-apoptotic activity in central nervous system neurons in animal models of ischaemic injury. Recently, Epo and its receptor (EpoR) have been identified in the peripheral nervous system [Campana & Myers (2001), FASEB J., 15, 1804-1806]. Herein, we demonstrate that in painful neuropathy caused by L5 spinal nerve crush (SNC), therapy with recombinant human Epo (rhEpo) reduced dorsal root ganglion (DRG) apoptosis and pain behaviours. Quantification of both DRG neurons and satellite cells revealed that vehicle-treated, crush-injured DRGs had 35.5 +/- 8.3% apoptotic neurons and 23.5 +/- 2.36% satellite cells compared with 7.5 +/- 6.3% apoptotic neurons and 6.4 +/- 3.94% satellite cells in rhEpo-treated, crush-injured DRGs (P < 0.05). While rhEpo-treated animals were not initially protected from mechanical allodynia associated with L5 SNC, rhEpo did significantly improve recovery rates compared to vehicle-treated animals (P < 0.01). Systemic rhEpo therapy increased JAK2 phosphorylation, a key anti-apoptotic signalling molecule for Epo-induced neuroprotection, in DRGs after crush. Dual immunofluorescence demonstrated Epo-induced JAK2-p was associated with both neuronal and glial cells. JAK2-p was associated with NF200-positive large neurons and with smaller neurons. This population of small neurons did not colocalize with IB4, a marker of nonpeptidergic, glial derived growth factor-responsive neurons. The findings link anti-apoptosis activities of Epo/EpoR/JAK2 in DRG neurons capable of inducing protracted pain states with reductions in pain behaviours, and therefore support a role for Epo therapy in the treatment of neuropathic pain.

Hypoxia-inducible factors are the key elements in the essential process of oxygen homeostasis of vertebrate cells. Stabilisation and subsequent nuclear localisation of HIF-alpha subunits results in the activation of target genes such as vegf, epo and glut1. The passage of transcription factors e.g. HIF-1alpha into the nucleus through the nuclear pore complex is regulated by nuclear transport receptors. Therefore nucleocytoplasmic shuttling can regulate transcriptional activity by facilitating the cellular traffic of transcription factors between both compartments. Here, we report on the identification of specific interactions of hypoxia-inducible factors with nuclear transport receptors importin alpha/beta. HIF-1alpha, -1beta, and HIF-2alpha are binding to importin alpha1, alpha3, alpha5, and alpha7. The direct interaction of HIF-1alpha to alpha importins is dependent on a functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS is not effective. Our findings provide new insight into the mechanism of the regulation of nuclear transport of hypoxia-inducible factors.

This paper is a summary of the 2007 European Position Paper on Rhinosinusitis and Nasal Polyps (EP3OS)1 which was published in Rhinology in 2007. In order to widen dissemination of the EP3OS paper, the editors of Rhinology and the Primary Care Respiratory Journal (PCRJ) have agreed to publish this summary - which is focussed on the needs of general practitioners and community-based non-specialist clinicians - in the PCRJ. In the EP3OS process, an evidence-based methodology was used to identify evidence and to grade recommendations for clinical practice for the management of rhinosinusitis. The EP3OS Taskforce was commissioned by the European Academy of Allergology and Clinical Immunology (EAACI) with the aims of: * Presenting specialist and generalist clinicians with an updated summary of knowledge of rhinosinusitis and nasal polyposis * Providing clinicans with an evidence-based summary of diagnostic methods appropriate for specialist and generalist settings * Providing evidence-based recommendations for management in specialist and generalist settings * Proposing guidance for definitions and outcome measurements in clinical practice and in research in different settings. The current document aims to distil the information presented in the full EP3OS document1 into a shorter and more concise format suitable for use in primary care generalist settings. The summary recommendations for generalists are that clinicians should be aware that rhinitis and sinusitis usually co-exist, and that management strategies should encompass this. Acute rhinosinusitis is an inflammatory condition that may be diagnosed on the basis of acute symptoms of nasal blockage, obstruction, congestion with or without facial pain or reduced smell; many episodes are self-limiting, but where symptoms persist or increase after five days, topical nasal steroids may be considered, with addition of antibiotics in patients with more severe or increasing symptoms. Non-resolution in 14 days, or the presence of atypical symptoms, should prompt consideration of referral to specialist care. Chronic rhinosinusitis occurs when symptoms have been present for >12 weeks, and anterior rhinoscopy or more detailed endoscopy should be performed to identify polyps. Topical nasal corticosteroids, nasal douching, and use of antihistamines in allergic patients, may be used in patients without, or with less symptomatic, polyps; referral to specialist care is needed for patients whose symptoms do not respond or who have large polyps.

Anaemia in cancer patients is multifactorial and may occur as a either a direct effect of the cancer, as a result of the cancer treatment itself, or due to chemical factors produced by the cancer. The clinical symptoms of anaemia vary according to the individual's capacity to respond to blood loss or reduced red cell production. The haematological features in anaemic patients depend on the different types of malignant disease. Clinical and laboratory evaluation, and examination of the bone marrow can provide important diagnostic clues in many cases. Decisions are commonly made based on subjective consideration rather than on objective data. Blood transfusion involves many hazards, some of which may be reduced or avoided. Erythropoietin (EPO) treatment has been found to be effective in preventing anaemia and in reducing the need for blood transfusions, although it would be useful to identify high-risk patient subgroups who would benefit most from this expensive treatment. In advanced cancer patients the use of blood transfusion should be evaluated on an individual basis, according to the presence of distressing symptoms and life expectancy. These measures are unlikely to have an effect in irreversible and progressive bleeding states.

BACKGROUND

There is some epidemiological and clinical evidence that the anemia seen in chronic kidney disease (CKD) in patients not on dialysis could be due to a significant extent to iron deficiency, and that adequate iron replacement could cause a marked improvement in the anemia even without the use of erythropoietin (EPO). The purpose of this work was to study the effects of intravenous (i.v.) iron administration (ferric gluconate - Ferrlecit) on hemoglobin (Hb) of patients with CKD.

METHODS

Forty-seven consecutive patients with CKD with Hb <12 g/dL in whom no underlying cause for the anemia could be found underwent sternal bone marrow biopsy and had their red cell and blood iron parameters measured. They then received 250 mg of ferric gluconate (Ferrlecit) intravenously twice monthly for 3 months, and had their blood parameters measured 1 month later. No patient received erythropoietin (EPO).

RESULTS

Forty-six patients had no evidence of any iron deposits in the bone marrow - consistent with the presence of severe iron deficiency. The mean serum ferritin and %transferrin saturation prior to treatment were 235.9 +/- 54.3 ug/L and 13.5 +/- 4.1%, respectively, and both increased significantly with the iron treatment. Mean Hb increased from 10.16 +/- 1.32 to 11.96 +/- 1.52 g/dL, an increase of 1.80 +/- 1.72 g/dL (p<0.01). Twenty-six patients (55.3%) reached the target Hb of 12 g/dL. Ten patients (21.3%) had an increase of 0.1-0.9 g/dL, nine patients (19.1%) had an increase of 1-1.9 g/dL and 23 patients (48.9%) had an increase of>>or= 2 g/dL.

CONCLUSIONS

Iron deficiency is frequently seen in anemic CKD patients not on dialysis and its correction with i.v. iron will often cause a marked increase in the Hb level, and the achievement of the target Hb of 12 g/dL even without EPO.

Erythropoietin (EPO) has been shown to exert cytoprotective effects on erythroid progenitor cells as well as various non-erythroid cells. Experimental studies have demonstrated the renoprotective effects of EPO in various acute and chronic renal injury models. These protective effects have been largely attributed to antiapoptotic signalings of EPO. However, injured cells undergoing apoptosis are generally too severely damaged to function properly. Therefore, simply corrupting apoptotic pathway is unlikely to be an effective strategy, because the remaining damaged cells may not function appropriately, or they may eventually undergo necrotic cell death. Recent evidences suggest that EPO also provides cytoprotection by ameliorating oxidative stress, the principal cellular insult. EPO may exert its antioxidative effects directly by exploiting intracellular antioxidative mechanisms such as heme oxygenase-1 and glutathione peroxidase. In addition, EPO may act indirectly by inducing iron depletion and thereby inhibiting iron-dependent oxidative injury. Increasing red blood cells by EPO may also indirectly reduce cellular oxidative stress, as red blood cells are loaded with a substantial amount of antioxidative enzymes. Further investigation regarding the mechanisms of cellular antioxidative responses to EPO would provide a better insight to cytoprotective action of EPO, and would support the development of better cytoprotective drugs in the near future.

Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations.

Anemia, defined as a hemoglobin level of less than 12 g/dL, often is seen in congestive heart failure (CHF). It is associated with an increased mortality and morbidity and increased hospitalizations. Compared with nonanemic patients the presence of anemia also is associated with worse cardiac clinical status, more severe systolic and diastolic dysfunction, a higher beta natriuretic peptide level, increased extracellular and plasma volume, a more rapid deterioration of renal function, a lower quality of life, and increased medical costs. The only way to determine if anemia is merely a marker for more severe CHF or actually is contributing to the worsening of the CHF is to correct the anemia and see if this favorably influences the CHF. In several controlled and uncontrolled studies, correction of the anemia with subcutaneous erythropoietin (EPO) or darbepoetin in conjunction with oral and intravenous iron has been associated with an improvement in clinical status, number of hospitalizations, cardiac and renal function, and quality of life. However, larger, randomized, double-blind, controlled studies still are needed to verify these initial observations. The effect of EPO may be related partly to its nonhematologic functions including neovascularization; prevention of apoptosis of endothelial, myocardial, cerebral, and renal cells; increase in endothelial progenitor cells; and anti-inflammatory and antioxidant effects. Anemia also may play a role in increasing cardiovascular morbidity in chronic kidney insufficiency, diabetes, renal transplantation, asymptomatic left ventricular dysfunction, left ventricular hypertrophy, acute coronary syndromes including myocardial infarction and chronic coronary heart disease, and in cardiac surgery. Again, controlled studies of correction of anemia are needed to assess its importance in these conditions. The anemia in CHF mainly is caused by a combination of renal failure and CHF-induced increased cytokine production, and these can both lead to reduced production of EPO, resistance of the bone marrow to EPO stimulation, and to cytokine-induced iron-deficiency anemia caused by reduced intestinal absorption of iron and reduced release of iron from iron stores. The use of angiotensin-converting enzyme inhibitor and angiotensin receptor blockers also may inhibit the bone marrow response to EPO. Hemodilution caused by CHF also may cause a low hemoglobin level. Renal failure, cardiac failure, and anemia therefore all interact to cause or worsen each other--the so-called cardio-renal-anemia syndrome. Adequate treatment of all 3 conditions will slow down the progression of both the CHF and the chronic kidney insufficiency.

Hypoxia regulates expression of erythropoietin (EPO), a glycoprotein that stimulates erythrocytosis, at the level of transcription and also possibly at the level of messenger RNA (mRNA) stability. A pyrimidine-rich region within the EPO mRNA 3' untranslated region was implicated in regulation of EPO mRNA stability element and shown to bind protein factors. In the present study we wished to identify the protein factor binding to the pyrimidine-rich sequence in the EPO mRNA stability element. Using mobility shift assays, ultraviolet light cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroelution of protein factors from the gel slices corresponding to the ribonucleoprotein complexes, we found that two isoforms of a 40 kD poly(C) binding protein (PCBP, also known as alphaCP or hnRNPE), PCBP1, and PCBP2 are present in that complex. In Hep3B or HepG2 cells hypoxia induces neither expression of PCBP nor formation of the ribonucleoprotein complex associated with EPO mRNA that involves PCBP.

Vertebral fractures are common but usually remain unrecognized in primary care. Data from 2908 women and 2653 men in the EPOS study were used to derive algorithms to indicate the need for a spine X-ray to identify a fracture using easily elicited determinants. At a sensitivity of 50% for identifying cases, the specificity was increased from 50% to 78% in women and from 50% to 72% in men compared with a random allocation of X-rays. Use of X-rays can be optimized by selecting patients at high risk using a short screening procedure.

BACKGROUND

Previous osteoporotic fracture is an independent risk factor for further fractures and an indication for treatment. Vertebral fractures are the most common osteoporotic fractures before age 75, accounting for 48% of all fractures in men and 39% in women over 50. They usually remain unrecognized, so many patients requiring treatment are denied it, doubling their risk of a further fracture. Our objective was to develop an efficient algorithm indicating the need for an X-ray.

METHODS

Data from 2908 women and 2653 men>>or=50 years of age in the European Prospective Osteoporosis Study (EPOS) were analyzed. Lateral thoracic and lumbar spine radiographs were taken at baseline and at an average of 3.8 years later. Prevalent fractures were qualitatively diagnosed by an experienced radiologist. Fracture risk was modeled as a function of age, statural height loss since age 25, gender, and fracture history including limb fractures in the last 3 years using negative binomial regression. Receiver operating characteristic (ROC) curves were used to summarize a model's predictive ability, and a prediction algorithm was devised to identify those most likely to have a fracture.

RESULTS

In a multivariate model for women, the risk of prevalent vertebral fracture significantly increased with age (RR, 1.67 [95% CI, 1.46, 1.93] per decade), statural height loss (1.06, [1.03, 1.10] per centimeter decrease), self-reported history of spine fracture (7.52 [5.52, 10.23]), and history of other major fracture (1.83 [1.46, 2.28]). Higher body weight reduced risk (0.86 [0.79, 0.95] per 10-kg increase). In men, the respective RR estimates were as follows: age (1.32 [1.18, 1.49]); height loss (1.06 [1.04, 1.09]); self-reported spine fracture (5.05 [3.69, 6.90]); other major fracture (1.42 [1.12, 1.81]); and weight (0.86 [0.79, 0.94]). Using algorithms based on these easily elicited determinants, specificity was increased from 50% to 78% in women and from 50% to 72% in men at a sensitivity of 50% compared with a random allocation of X-rays. At a sensitivity of 75%, the specificity was 50% in women and 40% in men. Inclusion of hip BMD (femoral neck or trochanter), measured in 1360 women and 1046 men, significantly improved the area under the ROC curves by 4% in women (p < 0.002) but not in men (p>> 0.350). Spine BMD, measured in 982 women and 847 men, produced a significant 5% AUC improvement in women (p = 0.007) but not in men (p = 0.554).

CONCLUSIONS

A woman 65 years of age with one vertebral fracture has a one in four chance of another fracture over 5 years, which can be reduced to one in eight by treatment. Positive treatment decisions are often contingent on identifying a vertebral fracture. Selective use of lateral vertebral X-rays can be optimized using a 2-minute screening procedure administered by a nurse.

Recombinant human erythropoietin (rHuEPO) variants have been constructed to identify amino acid residues important for biological activity. Immunoassays were used to determine the effect of each mutation on rHuEPO folding. With this strategy, we could distinguish between mutations that affected bioactivity directly and those that affected bioactivity because the mutation altered rHuEPO conformation. Four regions were found to be important for bioactivity: amino acids 11 to 15, 44 to 51, 100 to 108, and 147 to 151. EPO variants could be divided into two groups according to the differential effects on EPO receptor binding activity and in vitro biologic activity. This suggests that rHuEPO has two separate receptor binding sites. Mutations in basic residues reduced the biologic activity, whereas mutations in acidic residues did not. This suggests that electrostatic interactions between rHuEPO and the human EPO receptor may involve positive charges on rHuEPO.

Erythropoietin (EPO) promotes neuronal survival after cerebral ischemia in vivo and after hypoxia in vitro. However, the mechanisms underlying the protective effects of EPO on ischemic/hypoxic neurons are not fully understood. The present in vitro experiments showed that EPO attenuated neuronal damage caused by chemical hypoxia at lower extracellular concentrations (10(- 4)-10(-2) U/ml) than were previously considered. Moreover, EPO at a concentration of 10(-3) U/ml up-regulated Bcl-xL mRNA and protein expressions in cultured neurons. Subsequent in vivo study focused on whether EPO rescued hippocampal CA1 neurons from lethal ischemic damage and up-regulated the expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils. EPO was infused into the cerebroventricles of gerbils immediately after 3 min of ischemia for 28 days. Infusion of EPO at a dose of 5 U/day prevented the occurrence of ischemia-induced learning disability. Subsequent light microscopic examinations showed that pyramidal neurons in the hippocampal CA1 field were significantly more numerous in ischemic gerbils infused with EPO (5 U/day) than in those receiving vehicle infusion. The same dose of EPO infusion caused significantly more intense expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils than did vehicle infusion. These findings suggest that EPO prevents delayed neuronal death in the hippocampal CA1 field, possibly through up-regulation of Bcl-xL, which is known to facilitate neuron survival.

Partially purified human burst-forming unit-erythroid (BFU-E) cells from peripheral blood were cultured for 6 to 8 days to obtain colony-forming unit-erythroid (CFU-E) cells. When these BFU-E-derived CFU-E were further purified and recultured in liquid suspension cultures with erythropoietin (EPO), they matured and differentiated into reticulocytes in vitro. A maximum rate of hemoglobin synthesis was observed at day 10 of cumulative culture time by measuring 59Fe incorporation into heme. Withdrawal of EPO from erythroblast cultures at various times during development showed that between day 10 and day 11 (when the majority of the cells are in the polychromatic erythroblast stage), these cells became independent of EPO. The timing of the disappearance of the EPO requirement in these cells coincided with the marked decline in proliferation. Measurement of EPO receptor messenger RNA (mRNA) levels by Northern analysis showed that there is a slight decline during the day 8 to day 10 time period, followed by a rapid decline between days 10 and 14. Binding of 125I-EPO to erythroblasts also showed a steady decline of the cell surface binding during maturation and terminal differentiation. The half-life of the human EPO receptor was 90 minutes in the presence of the transcriptional inhibitor actinomycin D and the half-life measured at two different times during the 8- to 14-day culture period remained constant. These results indicate that human EPO receptor mRNA must be transcribed continuously to maintain the levels seen by Northern analysis. The human cell system described here is well suited for the study of a wide variety of biochemical events during late erythroid differentiation.

Cell death induced by the combined insult of hypoxia-ischemia in neonatal rodents has been extensively investigated. Ischemia-only-induced cell death, however, has been much less characterized. Based on the notion that 1) ischemic stroke is a relatively common disorder in human neonates, and 2) developing cells are more susceptible to apoptosis, the present study examined whether typical apoptosis was induced by cerebral ischemia in a new neonatal rat model. Erythropoietin (EPO; Epoetin) was tested for its protective effect against ischemia-induced cell death. Postnatal day 7 rats were subjected to permanent occlusion of the middle cerebral artery branch supplying the right whisker-barrel cortex. Terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeled-positive cells in the ischemic region were detectable 4 h after ischemia and reached a peak level 16 h later. The cell death was preceded by caspase activation and cytochrome c release. Cell body shrinkage was evident among damaged cells. Agarose gel electrophoresis showed DNA damage with a smear pattern as well as DNA laddering. Electron microscopy demonstrated apoptotic features such as cell shrinkage, chromatin condensation, and fragmentation; meanwhile, necrotic alterations coexisted in the cytoplasm. EPO treatment increased signal transducers and activators of transcription-5 and Bcl-2 levels, markedly attenuated apoptotic cell death, and reduced ischemic infarct in the cortex. It is suggested that focal ischemia in the developing brain causes cell death with prominent apoptotic features coexisting with some characteristics of necrosis. This is consistent with the concept of hybrid death described previously in cultures and adult or developing brain. EPO may be explored as a potential therapy for neonatal ischemic stroke.

We have previously shown that center- and sex-specific fall rates explained one-third of between-center variation in upper limb fractures across Europe. In this current analysis, our aim was to determine how much of the between-center variation in fractures could be attributed to repeated falling, bone mineral density (BMD), and other risk factors in individuals, and to compare the relative contributions of center-specific BMD vs. center-specific fall rates. A clinical history of fracture was assessed prospectively in 2451 men and 2919 women aged 50-80 from 20 centers participating in the European Prospective Osteoporosis Study (EPOS) using standardized questionnaires (mean follow-up = 3 years). Bone mineral density (BMD, femoral neck, trochanter, and/or spine) was measured in 2103 men and 2565 women at these centers. Cox regression was used to model the risk of incident fracture as a function of the person-specific covariates: age, BMD, personal fracture history (PFH), family hip fracture history (FAMHIP), time spent walking/cycling, number of 'all falls' and falls not causing fracture ('fracture-free') during follow-up, alcohol consumption, and body mass index. Center effects were modeled by inclusion of multiplicative gamma-distributed random effects, termed center-shared frailty (CSF), with mean 1 and finite variance theta (theta) acting on the hazard rate. The relative contributions of center-specific fall risk and center-specific BMD on the incidence of limb fractures were evaluated as components of CSF. In women, the risk of any incident nonspine fracture (n = 190) increased with age, PFH, FAMHIP,>> or =1 h/day walking/cycling, and number of 'all falls' during follow-up (all P < 0.074). 'Fracture-free' falls (P = 0.726) and femoral neck BMD did not have a significant effect at the individual level, but there was a significant center-shared frailty effect (theta = 0.271, P = 0.001) that was reduced by 4% after adjusting for mean center BMD and reduced by 19% when adjusted for mean center fall rate. Femoral trochanter BMD was a significant determinant of lower limb fractures (n = 53, P = 0.014) and the center-shared frailty effect was significant for upper limb fractures (theta = 0.271, P = 0.011). This upper limb fracture center effect was unchanged after adjusting for mean center BMD but was reduced by 36% after adjusting for center mean fall rates. In men, risk of any nonspine fracture (n = 75) increased with PFH, fall during follow-up (P < 0.026), and with a decrease in trochanteric BMD [RR 1.38 (1.08, 1.79) per 1 SD decrease]. There was no center effect evident (theta = 0.081, P = 0.096). We conclude that BMD alone cannot be validly used to discriminate between the risk of upper limb fractures across populations without taking account of population-specific variations in fall risk and other factors. These variations might reflect shared environmental or possibly genetic factors that contribute quite substantially to the risk of upper limb fractures in women.

Erythropoietin receptor (EpoR) is expressed in the central nervous system (CNS), however, no clear consensus has been obtained whether Epo acts as a prosurvival factor in neurons. Because retinal ganglion cell (RGC) death is a common cause of reduced visual function in several ocular diseases, we explored whether Epo might potentially be beneficial in protecting RGCs from glutamate and nitric oxide (NO)-induced cytotoxicity, using isolated RGCs by a two-step panning method. Brain-derived neurotrophic factor (BDNF) was used as a positive control. EpoR mRNA was expressed in isolated RGCs, and EpoR protein was expressed on the RGCs in the normal and ischemic retinas. Epo had less potential to improve the survival of primary RGCs in serum-free medium than BDNF. In these cells, BDNF, but not Epo, downregulated the expression of Bim, a proapoptotic Bcl-2 family member that plays a key role in cytokine-mediated cell survival, suggesting a possible mechanism for this difference. When RGCs were cultured with glutamate or an NO-generating reagent, the survival of RGCs was compromised, and Bcl-2 expression was decreased in these cells. Both Epo and BDNF significantly reduced RGC death induced by glutamate and NO. In agreement with this, these factors reversed the Bcl-2 expression. These findings suggest that Epo may be a potent neuroprotective therapeutic agent for the treatment of ocular diseases that are characterized by RGC death.

The gene for murine erythropoietin (EPO) was isolated from a mouse genomic library with a human EPO cDNA probe. Nucleotide sequence analysis permitted the identification of the murine EPO coding sequence and the prediction of the encoded amino acid sequence based on sequence conservation between the mouse and human EPO genes. Both the coding DNA and the amino acid sequences were 80% conserved between the two species. Transformation of COS-1 cells with a mammalian cell expression vector containing the murine EPO coding region resulted in secretion of murine EPO with biological activity on both murine and human erythroid progenitor cells. The transcription start site for the murine EPO gene in kidneys was determined. This permitted tentative identification of the transcription control region. The region included 140 base pairs upstream of the cap site which was over 90% conserved between the murine and human genes. Surprisingly, the first intron and much of the 5'- and 3'-untranslated sequences were also substantially conserved between the genes of the two species.

OBJECTIVE

Spinal cord injury (SCI) is a devastating clinical syndrome for which no truly efficacious therapy has yet been identified. In preclinical studies, erythropoietin (EPO) and its nonerythropoietic derivatives asialoEPO and carbamylated EPO have markedly improved functional outcome when administered after compressive SCI. However, an optimum treatment paradigm is currently unknown. Because the uninjured spinal cord expresses a high density of EPO receptor (EPOR) in the basal state, signaling through these existing receptors in advance of injury (pharmacological preconditioning) might confer neuroprotection and therefore be potentially useful in situations of anticipated damage.

METHODS

The authors compared asialoEPO, a molecule that binds to the EPOR with high affinity but with a brief serum half-life (t1/2 < 2 minutes), to EPO to determine whether a single dose (10 microg/kg of body weight) administered by intravenous injection 24 hours before 1 minute of spinal cord compression provides benefit as determined by a 6-week assessment of neurological outcome and by histopathological analysis. Rats pretreated with asialoEPO or EPO and then subjected to a compressive injury exhibited improved motor function over 42 days, compared with animals treated with saline solution. However, pretreatment efficacy was substantially poorer than efficacy of treatment initiated at the time of injury. Serum samples drawn immediately before compression confirmed that no detectable asialoEPO remained within the systemic circulation. Western blot and immunohistochemical analyses performed using uninjured spinal cord 24 hours after a dose of asialoEPO exhibited a marked increase in glial fibrillary acidic protein, suggesting a glial response to EPO administration.

CONCLUSIONS

These results demonstrate that EPO and its analog do not need to be present at the time of injury to provide tissue protection and that tissue protection is markedly effective when either agent is administered immediately after injury. Furthermore, the findings suggest that asialoEPO is a useful reagent with which to study the dynamics of EPO-mediated neuroprotection. In addition, the findings support the concept of using a nonerythropoietic EPO derivative to provide tissue protection without activating the undesirable effects of EPO.

OBJECTIVE

Critically ill patients often develop anaemia which can be related to a number of factors. However, the exact causes of anaemia in many patients remain unexplained. We hypothesized that the relationship between erythropoietin (EPO) and haematocrit may be altered in critically ill patients.

METHODS

Serum concentrations of EPO were serially determined by the ELISA method in 36 critically ill, non-hypoxaemic patients who stayed more than 7 days in the Intensive Care Unit, including 22 patients with sepsis and 14 without. Eighteen ambulatory patients with iron-deficiency anaemia served as a control group.

METHODS

Two University Hospital Intensive Care Departments.

RESULTS

A significant inverse correlation between serum EPO and haematocrit levels was found in the control patients (r = -0.81, p < 0.001), but not in the critically ill patients (r = -0.09, NS), except in a subgroup of non-septic patients without renal failure (r = -0.61, p < 0.01).

CONCLUSIONS

EPO levels can be inappropriately low in critically ill patients, so that EPO deficiency may contribute to the development of anaemia in these patients. This phenomenon is observed not only in the presence of acute renal failure, but also in the presence of sepsis.

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors share a common beta chain (beta(c)), and both cytokines enhance erythropoietin (Epo)-dependent in vitro erythropoiesis by primary hematopoietic progenitors and factor-dependent cells. These data suggest that the Epo receptor and beta(c) may functionally interact. To determine whether such interactions can be documented, we studied a murine factor-dependent cell line (Ba/F3), which endogenously expresses IL-3R. First, Ba/F3 cells were transfected with murine EpoR, making them responsive to both IL-3 and Epo. Next, the EpoR expressing cells were transfected with murine beta(c). This resulted in an enhanced sensitivity of these cells to Epo, which was especially pronounced at low Epo concentrations. Ba/F3-EpoR were then treated with antisense oligodeoxynucleotides to the murine beta. Control sense and nonsense had no effect on Epo-dependent growth, but the antisense markedly and specifically inhibited Epo-dependent growth. In contrast, the antisense did not affect beta-globin message levels (another Epo-responsive effect in these cells) detectable by Northern blot. Finally, Western blot analysis of proteins immunoprecipitated from cells expressing both receptors with antibody against beta and blotted with antibody against EpoR, or immunoprecipitated with antibody against EpoR and blotted with antibody against beta, showed that EpoR and beta coimmunoprecipitate. These data show that the beta chain functionally and physically associates with the EpoR. This suggests that these cytokine receptors exist as a large supercomplex and offers the first molecular explanation for the synergistic effects of IL-3 and GM-CSF with Epo during erythropoiesis.

We generated red blood cells (RBC) from cord blood (CB) CD34+ cells using a four-phase culture system. We first cultured CB CD34+ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34+ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34+ cells from a different donor using the same coculture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 x 10(12) RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 x 10(13) RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.

BACKGROUND

Cancer anemia causes fatigue and correlates with poor treatment outcome. Erythropoietin has been introduced in an attempt to correct these defects. However, five recent clinical trials reported a negative impact of erythropoietin on survival and/or tumor control, indicating that experimental evaluation of a possible direct effect of erythropoietin on cancer cells is required. Cancer recurrence is thought to rely on the proliferation of cancer initiating cells (CICs). In breast cancer, CICs can be identified by phenotypic markers and their fate is controlled by the Notch pathway.

METHODS

In this study, we investigated the effect of erythropoietin on CICs in breast cancer cell lines. Levels of erythropoietin receptor (EpoR), CD24, CD44, Jagged-1 expression, and activation of Notch-1 were assessed by flow cytometry. Self-renewing capacity of CICs was investigated in sphere formation assays.

RESULTS

EpoR expression was found on the surface of CICs. Recombinant human Epo (rhEpo) increased the numbers of CICs and self-renewing capacity in a Notch-dependent fashion by induction of Jagged-1. Inhibitors of the Notch pathway and PI3-kinase blocked both effects.

CONCLUSIONS

Erythropoietin functionally affects CICs directly. Our observation may explain the negative impact of recombinant Epo on local control and survival of cancer patients with EpoR-positive tumors.

Over the past few decades, understanding of the physiologic function of erythropoietin (EPO) has evolved significantly. EPO binds to erythropoietin receptors (EPOR), initiating signaling that stimulates growth, inhibits apoptosis, and induces the differentiation of erythroid progenitors to increase red blood cell mass. EPO has additionally been shown to exert tissue-protective effects on multiple tissues, suggesting a pleiotropic mechanism of action. Erythropoiesis-stimulating agents (ESA) are used clinically for treating cancer-related anemia [chemotherapy-induced anemia (CIA)]. Recent clinical trials have reported increased adverse events and/or reduced survival in ESA-treated cancer patients receiving chemotherapy, potentially related to EPO-induced cancer progression. Signaling pathways downstream of EPO/EPOR have been shown to influence numerous cellular functions in both normal and tumor cells, including proliferation, apoptosis, and drug resistance. Some studies have reported effects on proliferation, reduced chemotherapy efficacy, reduction of apoptosis, and resistance to selective therapies on cancer cell lines, whereas others have shown null effects. In addition, newer targeted cancer therapies that are directed toward specific signaling pathways may be antagonized by ESAs. This molecular interplay between anticancer agents and potential survival signals triggered by ESAs may have been underestimated and may contribute toward decreased survival seen in certain trials. As more targeted anticancer therapies become available, these types of interactions may mitigate therapeutic efficacy by allowing tumor cells to acquire drug resistance. Therefore, a more complete understanding of the complex pathways involved will allow for the rational use of ESAs for the safe treatment of CIA in oncology patients.

Hematological traits are important clinical parameters. To test the effects of rare and low-frequency coding variants on hematological traits, we analyzed hemoglobin concentration, hematocrit levels, white blood cell (WBC) counts and platelet counts in 31,340 individuals genotyped on an exome array. We identified several missense variants in CXCR2 associated with reduced WBC count (gene-based P = 2.6 × 10(-13)). In a separate family-based resequencing study, we identified a CXCR2 frameshift mutation in a pedigree with congenital neutropenia that abolished ligand-induced CXCR2 signal transduction and chemotaxis. We also identified missense or splice-site variants in key hematopoiesis regulators (EPO, TFR2, HBB, TUBB1 and SH2B3) associated with blood cell traits. Finally, we were able to detect associations between a rare somatic JAK2 mutation (encoding p.Val617Phe) and platelet count (P = 3.9 × 10(-22)) as well as hemoglobin concentration (P = 0.002), hematocrit levels (P = 9.5 × 10(-7)) and WBC count (P = 3.1 × 10(-5)). In conclusion, exome arrays complement genome-wide association studies in identifying new variants that contribute to complex human traits.