Infection of erythroid-lineage cells by human parvovirus B19 is characterized by a gradual cytocidal effect. Accumulating evidence now implicates the nonstructural (NS1) protein of the virus in cytotoxicity, but the mechanism underlying the NS1-induced cell death is not known. Using a stringent regulatory system, we demonstrate that NS1 cytotoxicity is closely related to apoptosis, as evidenced by cell morphology, genomic DNA fragmentation, and cell cycle analysis with the human erythroleukemia cell line K562 and the erythropoietin-dependent megakaryocytic cell line UT-7/Epo. Apoptosis was significantly inhibited by an interleukin-1beta (IL-1beta)-converting enzyme (ICE)/CED-3 family protease inhibitor, Ac-DEVD-CHO (CPP32; caspase 3), whereas a similar inhibitor of ICE (caspase 1), Ac-YVAD-CHO, had no effect. Furthermore, stable expression of the human Bcl-2 proto-oncogene resulted in near-total protection from cell death in response to NS1 induction. Mutations engineered into the nucleoside triphosphate-binding domain of NS1 significantly rescued cells from NS1-induced apoptosis without having any effect on NS1-induced activation of the IL-6 gene expression which is mediated by NF-kappaB. Furthermore, using pentoxifylline, an inhibitor of NF-kappaB activation, we demonstrate that the NF-kappaB-mediated IL-6 activation by NS1 is uncoupled from the apoptotic pathway. This functional dissection indicates a complexity underlying the biochemical function of human parvovirus NS1 in transcriptional activation and induction of apoptosis. Our findings indicate that NS1 of parvovirus B19 induces cell death by apoptosis in at least erythroid-lineage cells by a pathway that involves caspase 3, whose activation may be a key event during NS1-induced cell death.

Hypoxic preconditioning (8% O2, 3 h) produces tolerance 24 h after hypoxic-ischemic brain injury in neonatal rats. To better understand the ischemic tolerance mechanisms induced by hypoxia, we used oligonucleotide microarrays to examine genomic responses in neonatal rat brain following 3 h of hypoxia (8% O2) and either 0, 6, 18, or 24 h of re-oxygenation. The results showed that hypoxia-inducible factor (HIF)-1- but not HIF-2-mediated gene expression may be involved in brain hypoxia-induced tolerance. Among the genes regulated by hypoxia, 12 genes were confirmed by real time reverse transcriptase-PCR as follows: VEGF, EPO, GLUT-1, adrenomedullin, propyl 4-hydroxylase alpha, MT-1, MKP-1, CELF, 12-lipoxygenase, t-PA, CAR-1, and an expressed sequence tag. Some genes, for example GLUT-1, MT-1, CELF, MKP-1, and t-PA did not show any hypoxic regulation in either astrocytes or neurons, suggesting that other cells are responsible for the up-regulation of these genes in the hypoxic brain. These genes were expressed in normal and hypoxic brain, heart, kidney, liver, and lung, with adrenomedullin, MT-1, and VEGF being prominently induced in brain by hypoxia. These results suggest that a number of endogenous molecular mechanisms may explain how hypoxic preconditioning protects against subsequent ischemia, and may provide novel therapeutic targets for treatment of cerebral ischemia.

Advances in the optimization of in vitro-transcribed mRNA are bringing mRNA-mediated therapy closer to reality. In cultured cells, we recently achieved high levels of translation with high-performance liquid chromatography (HPLC)-purified, in vitro-transcribed mRNAs containing the modified nucleoside pseudouridine. Importantly, pseudouridine rendered the mRNA non-immunogenic. Here, using erythropoietin (EPO)-encoding mRNA complexed with TransIT-mRNA, we evaluated this new generation of mRNA in vivo. A single injection of 100 ng (0.005 mg/kg) mRNA elevated serum EPO levels in mice significantly by 6 hours and levels were maintained for 4 days. In comparison, mRNA containing uridine produced 10-100-fold lower levels of EPO lasting only 1 day. EPO translated from pseudouridine-mRNA was functional and caused a significant increase of both reticulocyte counts and hematocrits. As little as 10 ng mRNA doubled reticulocyte numbers. Weekly injection of 100 ng of EPO mRNA was sufficient to increase the hematocrit from 43 to 57%, which was maintained with continued treatment. Even when a large amount of pseudouridine-mRNA was injected, no inflammatory cytokines were detectable in plasma. Using macaques, we could also detect significantly-increased serum EPO levels following intraperitoneal injection of rhesus EPO mRNA. These results demonstrate that HPLC-purified, pseudouridine-containing mRNAs encoding therapeutic proteins have great potential for clinical applications.

Studies on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of the molecule and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor-binding affinity. These observations led to the hypothesis that increasing the carbohydrate content, beyond that found naturally, would lead to a molecule with enhanced biological activity. Hyperglycosylated recombinant human EPO (rHuEPO) analogues were developed to test this hypothesis. Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP, ARANESP, Amgen Inc, Thousand Oaks, CA), which was engineered to contain 5 N-linked carbohydrate chains (two more than rHuEPO), has been evaluated in preclinical animal studies. Due to its increased sialic acid-containing carbohydrate content, NESP is biochemically distinct from rHuEPO, having an increased molecular weight and greater negative charge. Compared with rHuEPO, it has an approximate 3-fold longer serum half-life, greater in vivo potency, and can be administered less frequently to obtain the same biological response. NESP is currently being evaluated in human clinical trials for treatment of anaemia and reduction in its incidence.

The neurodegenerative aspects of chronic progressive multiple sclerosis (MS) have received increasing attention in recent years, since anti-inflammatory and immunosuppressive treatment strategies have largely failed. However, successful neuroprotection and/or neuroregeneration in MS have not been demonstrated yet. Encouraged by the multifaceted neuroprotective effects of recombinant human erythropoietin (rhEPO) in experimental models, we performed an investigator-driven, exploratory open label study (phase I/IIa) in patients with chronic progressive MS. Main study objectives were (i) evaluating safety of long-term high-dose intravenous rhEPO treatment in MS, and (ii) collecting first evidence of potential efficacy on clinical outcome parameters. Eight MS patients, five randomly assigned to high-dose (48,000 IU), three to low-dose (8000 IU) rhEPO treatment, and, as disease controls, two drug-naïve Parkinson patients (receiving 48,000 IU) were followed over up to 48 weeks: A 6-week lead-in phase, a 12-week treatment phase with weekly EPO, another 12-week treatment phase with bi-weekly EPO, and a 24-week post-treatment phase. Clinical and electrophysiological improvement of motor function, reflected by a reduction in expanded disability status scale (EDSS), and of cognitive performance was found upon high-dose EPO treatment in MS patients, persisting for three to six months after cessation of EPO application. In contrast, low-dose EPO MS patients and drug-naïve Parkinson patients did not improve in any of the parameters tested. There were no adverse events, no safety concerns and a surprisingly low need of blood-lettings. This first pilot study demonstrates the necessity and feasibility of controlled trials using high-dose rhEPO in chronic progressive MS.

BACKGROUND

Anemia is common in hemodialysis (HD) patients.

METHODS

Data collected from nationally representative samples of HD patients (n = 11,041) in 2002 to 2003 were used to describe current anemia management for long-term HD patients at 309 dialysis units in 12 countries. Analyses of associations and outcomes were adjusted for demographics, 15 comorbid classes, laboratory values, country, and facility clustering.

RESULTS

For patients on dialysis therapy for longer than 180 days, 23% to 77% had a hemoglobin (Hgb) concentration less than 11 g/dL (<110 g/L), depending on country; 83% to 94% were administered erythropoietin (EPO). Mean Hgb levels were 12 g/dL (120 g/L) in Sweden; 11.6 to 11.7 g/dL (116 to 117 g/L) in the United States, Spain, Belgium, and Canada; 11.1 to 11.5 g/dL (111 to 115 g/L) in Australia/New Zealand, Germany, Italy, the United Kingdom, and France; and 10.1 g/dL (101 g/L) in Japan. Hgb levels were substantially lower for new patients with end-stage renal disease, and EPO use before ESRD ranged from 27% (United States) to 65% (Sweden). By patient, EPO use significantly declined with greater Hgb concentration (adjusted odds ratio, 0.61 per 1-g/dL [10-g/L] greater Hgb level; P < 0.0001), as did EPO dosage. Case-mix-adjusted mortality and hospitalization risk declined by 5% and 6% per 1-g/dL greater patient baseline Hgb level (P < or = 0.003 each), respectively. Furthermore, patient mortality and hospitalization risks were 10% to 12% lower for every 1-g/dL greater facility mean Hgb level. Patients were significantly more likely to have Hgb levels of 11 g/dL or greater >> or =110 g/L) if they were older; were men; had polycystic kidney disease; had greater albumin, transferrin saturation, or calcium levels; were not dialyzing with a catheter; or had lower ferritin levels. Facilities with greater intravenous iron use showed significantly greater facility mean Hgb concentrations. Mean EPO dose varied from 5,297 (Japan) to 17,360 U/wk (United States). Greater country mean EPO doses were significantly associated with greater country mean Hgb concentrations. Several patient characteristics were associated with greater EPO doses. Even in some countries with high intravenous iron use, 35% to 40% of patients had a transferrin saturation less than 20% (below guidelines).

CONCLUSIONS

These findings indicate large international variations in anemia management, with significant improvements during the last 5 years, although many patients remain below current anemia guidelines, suggesting large and specific opportunities for improvement.

BACKGROUND

Erythropoietin (EPO) has recently been shown to exert important cytoprotective and anti-apoptotic effects in experimental brain injury and cisplatin-induced nephrotoxicity. The aim of the present study was to determine whether EPO administration is also renoprotective in both in vitro and in vivo models of ischaemic acute renal failure.

METHODS

Primary cultures of human proximal tubule cells (PTCs) were exposed to either vehicle or EPO (6.25-400 IU/ml) in the presence of hypoxia (1% O(2)), normoxia (21% O(2)) or hypoxia followed by normoxia for up to 24 h. The end-points evaluated included cell apoptosis (morphology and in situ end labelling [ISEL], viability [lactate dehydrogenase (LDH release)], cell proliferation [proliferating cell nuclear antigen (PCNA)] and DNA synthesis (thymidine incorporation). The effects of EPO pre-treatment (5000 U/kg) on renal morphology and function were also studied in rat models of unilateral and bilateral ischaemia-reperfusion (IR) injury.

RESULTS

In the in vitro model, hypoxia (1% O(2)) induced a significant degree of PTC apoptosis, which was substantially reduced by co-incubation with EPO at 24 h (vehicle 2.5+/-0.5% vs 25 IU/ml EPO 1.8+/-0.4% vs 200 IU/ml EPO 0.9+/-0.2%, n = 9, P<0.05). At high concentrations (400 IU/ml), EPO also stimulated thymidine incorporation in cells exposed to hypoxia with or without subsequent normoxia. LDH release was not significantly affected. In the unilateral IR model, EPO pre-treatment significantly attenuated outer medullary thick ascending limb (TAL) apoptosis (EPO 2.2+/-1.0% of cells vs vehicle 6.5+/-2.2%, P<0.05, n = 5) and potentiated mitosis (EPO 1.1+/-0.3% vs vehicle 0.5+/-0.3%, respectively, P<0.05) within 24 h. EPO-treated rats exhibited enhanced PCNA staining within the proximal straight tubule (6.9+/-0.7% vs vehicle 2.4+/-0.5% vs sham 0.3+/-0.2%, P<0.05), proximal convoluted tubule (2.3+/-0.6% vs vehicle 1.1+/-0.3% vs sham 1.2+/-0.3%, P<0.05) and TAL (4.7+/-0.9% vs vehicle 0.6+/-0.3% vs sham 0.3+/-0.2%, P<0.05). The frequency of tubular profiles with luminal cast material was also reduced (32.0+/-1.6 vs vehicle 37.0+/-1.3%, P = 0.05). EPO-treated rats subjected to bilateral IR injury exhibited similar histological improvements to the unilateral IR injury model, as well as significantly lower peak plasma creatinine concentrations than their vehicle-treated controls (0.04+/-0.01 vs 0.21+/-0.08 mmol/l, respectively, P<0.05). EPO had no effect on renal function in sham-operated controls.

CONCLUSIONS

The results suggest that, in addition to its well-known erythropoietic effects, EPO inhibits apoptotic cell death, enhances tubular epithelial regeneration and promotes renal functional recovery in hypoxic or ischaemic acute renal injury.

Apart from its hematopoietic function, erythropoietin (Epo) exerts neuroprotective functions in brain hypoxia and ischemia. To examine the mechanisms mediating Epo's neuroprotective activity in vivo, we made use of our transgenic mouse line tg21 that constitutively expresses human Epo in brain without inducing excessive erythrocytosis. We show that human Epo is expressed in tg21 brains and that cortical and striatal neurons carry the Epo receptor. After middle cerebral artery occlusion, human Epo potently protected brains of tg21 mice against ischemic injury, both when severe (90 min) and mild (30 min) ischemia was imposed. Histochemical studies revealed that Epo induced an activation of JAK-2, ERK-1/-2, and Akt pathways in the ischemic brain. This activation was associated with elevated Bcl-XL and decreased NO synthase-1 and -2 levels in neurons. Intracerebroventricular injections of selective inhibitors of ERK-1/-2 (PD98059) or Akt (wortmannin) pathways revealed that both ERK-1/-2 and Akt were required for Epo's neuroprotective function, antagonization of either pathway completely abolishing tissue protection. On the other hand, ERK-1/-2 and Akt blockade did not reverse the neuronal NO synthase-1/-2 inhibition, indicating that Epo down-regulates these NO synthases in an ERK-1/-2 and Akt independent manner. On the basis of our data, the dual activation of ERK-1/-2 and Akt is crucial for Epo's neuroprotective activity.

OBJECTIVE

To explore and evaluate the protective effect of erythropoietin (EPO) on retinal cells of chemically induced diabetic rats after EPO was injected intravitreally at the onset of diabetes.

METHODS

Diabetes was induced in Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ). At the onset of diabetes, a single intravitreal injection of EPO (0.05-200 ng/eye) was performed. In the following 6 weeks, the blood retinal barrier (BRB) was evaluated by Evans blue permeation (EBP). Retinal cell death in different layers was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The retinal thickness and cell counts were examined at the light microscopic level. Electron microscopy (EM) was used to scrutinize retinal vascular and neuronal injury. Neurosensory retinas of normal and diabetic rats were used as the sources of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot for the detection of EPO, EPO receptor (EpoR), and products of the extracellular signal-regulated kinase (ERK) and the signal transducers and activators of transcription 5 (STAT5) pathways. The distribution of EpoR in retinal layers was demonstrated by immunohistochemistry (IHC).

RESULTS

In the diabetic rats, BRB breakdown was detected soon after the onset of diabetes, peaked at 2 weeks, and reached a plateau at 2 to 4 weeks. The number of TUNEL-positive cells increased in the neurosensory retina, especially, the outer nuclear layer (ONL) at 1 week after diabetes onset and reached a peak at 4 to 6 weeks. The retinal thickness and the number of cells in the ONL were reduced significantly. EM observations demonstrated vascular and photoreceptor cell death starting soon after the onset of diabetes. All these changes were largely prevented by EPO treatment. Upregulation of EpoR in the neurosensory retina was detected at both the transcriptional and protein levels 4 to 8 weeks after the onset of diabetes, whereas, the endogenous EPO levels of neurosensory retinas were essentially unchanged during the same period observed. In EPO-treated diabetic groups, EpoR expression remained at upregulated levels. Within 2 weeks of the onset of diabetes, activation of the ERK but not the STAT5 pathway was detected in the diabetic retina treated with EPO.

CONCLUSIONS

These data demonstrate that apoptosis is an major contributor to neuronal cell death in the early course of diabetic retinopathy (DR). The upregulation of EpoR may be a compensatory response of retinal cells and tissue to diabetic stresses. The EPO/EpoR system as a maintenance-survival mechanism of retinal neurons responds to the insults of early diabetes other than ischemia. The protective function of EPO/EpoR at the least acts through the EpoR-mediated ERK pathway. Exogenous EPO administration by intravitreal injection in early diabetes may prevent retinal cell death and protect the BRB function. Therefore, this is a novel approach for treatment of early DR.

In adults, the kidneys are the major site of production of the glycoprotein hormone erythropoietin (EPO), but the type of renal cell producing EPO has not yet been identified. In the present study we used non-radioactive in situ hybridization with a digoxigenin-labeled cRNA probe to localize cells that produce erythropoietin (EPO) in kidneys of anemic rats. Cryostat sections from both native and perfusion-fixed tissue were used. Cells containing EPO mRNA were found exclusively in the peritubular space of the renal cortex. Using high-resolution interference contrast optics, we found that cells expressing EPO mRNA were not associated with the lumina of peritubular capillaries but rather were located in the angles between adjacent tubules or between tubules and vessels. These spaces are predominantly occupied by resident interstitial fibroblasts and by their cytoplasmic processes. To further identify the type of cell containing EPO mRNA, a double-labeling protocol was established that permitted on the same tissue section both in situ hybridization for EPO mRNA and parallel immunolabeling of ecto-5'-nucleotidase (5'-Nu), a surface marker of peritubular interstitial fibroblasts. The combined labeling technique revealed that a clear majority of cells expressing EPO mRNA also displayed staining for anti-5'-Nu. Staining for EPO mRNA was localized in central perinuclear parts of the interstitial cells, whereas 5'-Nu label was present on the cell surface, including the cytoplasmic processes. These data indicate that peritubular fibroblasts are cellular sites for production of erythropoietin.

Erythropoietin (EPO) modulates primarily the proliferation of immature erythroid precursors, but little is known of the potential protective mechanisms of EPO in the central nervous system. We therefore examined the ability of EPO to modulate a series of death-related cellular pathways during anoxia and free radical induced neuronal degeneration. Neuronal injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidylserine exposure, protein kinase B phosphorylation, cysteine protease activity, mitochondrial membrane potential, and mitogen-activated protein (MAP) kinase phosphorylation. We demonstrate that constitutive neuronal EPO is insufficient to prevent cellular injury, but that signaling through the EPO receptor remains biologically responsive to exogenous EPO administration. Exogenous EPO is both necessary and sufficient to prevent acute genomic DNA destruction and subsequent phagocytosis through membrane PS exposure, because neuronal protection by EPO is completely abolished by co-treatment with an anti-EPO neutralizing antibody. Through pathways that involve the initial activation of protein kinase B, EPO maintains mitochondrial membrane potential. Subsequently, EPO inhibits caspase 8-, caspase 1-, and caspase 3-like activities linked to cytochrome c release through mechanisms that are independent from the MAP kinase systems of p38 and JNK. Elucidating some of the novel neuroprotective pathways employed by EPO may further the development of new therapeutic strategies for neurodegenerative disorders.

OBJECTIVE

Neuronal replacement has recently gained attention as a potential therapeutic target under ischemic conditions. However, the oligodendrogenic infrastructure is equally critical for restoration of brain function and is also sensitive to ischemic injury. Erythropoietin (EPO) is a neuroprotective molecule that stimulates neuronal replacement after neonatal hypoxia/ischemia (H/I) when delivered soon after the onset of reperfusion. Because EPO can improve recovery of neurological function in the absence of tissue protection, we hypothesize that EPO may improve neurological function via enhancement of white matter recovery after H/I. Thus, we sought to determine the effects of delayed administration of EPO on white matter injury and recovery of neurological function after neonatal H/I.

METHODS

EPO (1000 U/kg) was injected intraperitoneally at multiple time points beginning 48 hours after H/I in postnatal day 7 rats. The effects of EPO on oligodendrogenesis, white matter injury, and neurogenesis were evaluated using bromodeoxyuridine incorporation and cell-specific immunohistochemistry. Neurological function was assessed by sensorimotor behavioral tests.

RESULTS

Delayed administration of EPO was incapable of reducing brain volume loss but significantly increased oligodendrogenesis and maturation of oligodendrocytes and attenuated white matter injury after H/I. These effects occurred concurrently with enhanced neurogenesis. Delayed EPO treatment improved behavioral neurological outcomes 14 days after H/I injury.

CONCLUSIONS

Our study demonstrates that delayed administration of EPO promotes oligodendrogenesis and attenuates white matter injury concurrently with increased neurogenesis. These effects likely contribute to the observed improvement in neurological functional outcomes.

In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type-specific expression of the Epo gene.

We have published previously a prototype of a decision model for anaemic patients with myelodysplastic syndromes (MDS), in which transfusion need and serum erythropoietin (S-<em>Epo</em>) were used to define three groups with different probabilities of erythroid response to treatment with granulocyte colony-stimulating factor (G-CSF) + <em>Epo</em>. S-<em>Epo</em> </= 500 U/l and a transfusion need of < 2 units/month predicted a high probability of response to treatment, S-<em>Epo</em>>> 500 U/l and>>/= 2 units/month for a poor response, whereas the presence of only one negative prognostic marker predicted an intermediate response. A total of 53 patients from a prospective study were included in our evaluation sample. Patients with good or intermediate probability of response were treated with G-CSF + <em>Epo</em>. The overall response rate was 42% with 28.3% achieving a complete and 13.2% a partial response to treatment. The response rates were 61% and 14% in the good and intermediate predictive groups respectively. The model retained a significant predictive value in the evaluation sample (P < 0.001). Median duration of response was 23 months. Scores for global health and quality of life (QOL) were significantly lower in MDS patients than in a reference population, and fatigue and dyspnoea was significantly more prominent. Global QOL improved in patients responding to treatment (P = 0.01). The validated decision model defined a subgroup of patients with a response rate of 61% (95% confidence interval 48-74%) to treatment with G-CSF + <em>Epo</em>. The majority of these patients have shown complete and durable responses.

Erythropoietin (EPO) is the principal cytokine regulating erythropoiesis through its receptor, EPOR. Interestingly, EPORs are also found on immune cells with incompletely understood functions. Here, we show that EPO inhibits the induction of proinflammatory genes including tumor necrosis factor (TNF)-α and inducible nitric oxide (NO) synthase in activated macrophages, which is mechanistically attributable to blockage of nuclear factor (NF)-κB p65 activation by EPO. Accordingly, in systemic Salmonella infection, treatment of mice with EPO results in reduced survival and impaired pathogen clearance because of diminished formation of anti-microbial effector molecules such as TNF-α and NO. However, neutralization of endogenous EPO or genetic ablation of Epor promotes Salmonella elimination. In contrast, in chemically induced colitis, EPO-EPOR interaction decreases the production of NF-κB-inducible immune mediators, thus limiting tissue damage and ameliorating disease severity. These immune-modulatory effects of EPO may be of therapeutic relevance in infectious and inflammatory diseases.

The aim of this population-based prospective study was to determine the incidence of limb fracture by site and gender in different regions of Europe. Men and women aged 50-79 years were recruited from population registers in 31 European centers. Subjects were invited to attend for an interviewer-administered questionnaire and lateral spinal radiographs. Subjects were subsequently followed up using an annual postal questionnaire which included questions concerning the occurrence of new fractures. Self-reported fractures were confirmed where possible by radiograph, attending physician or subject interview. There were 6451 men and 6936 women followed for a median of 3.0 years. During this time there were 140 incident limb fractures in men and 391 in women. The age-adjusted incidence of any limb fracture was 7.3/1000 person-years [pyrs] in men and 19 per 1000 pyrs in women, equivalent to a 2.5 times excess in women. Among women, the incidence of hip, humerus and distal forearm fracture, though not 'other' limb fracture, increased with age, while in men only the incidence of hip and humerus fracture increased with age. Among women, there was evidence of significant variation in the occurrence of hip, distal forearm and humerus fractures across Europe, with incidence rates higher in Scandinavia than in other European regions, though for distal forearm fracture the incidence in east Europe was similar to that observed in Scandinavia. Among men, there was no evidence of significant geographic variation in the occurrence of these fractures. This is the first large population-based study to characterize the incidence of limb fracture in men and women over 50 years of age across Europe. There are substantial differences in the descriptive epidemiology of limb fracture by region and gender.

BACKGROUND

Elements of malnutrition-inflammation complex syndrome (MICS) may blunt the responsiveness of anemia of end-stage renal disease (ESRD) to recombinant human erythropoietin (EPO).

METHODS

The authors examined cross-sectional associations between the required dose of EPO within a 13-week interval as prescribed by practicing nephrologists who were blind to the study and several laboratory values known to be related to nutrition and/or inflammation, as well as the malnutrition-inflammation score (MIS), which is a fully quantitative assessment tool based on the subjective global assessment of nutrition.

RESULTS

A total of 339 maintenance hemodialysis (MHD) outpatients, including 181 men, who were aged 54.7 +/- 14.5 years (mean +/- SD), who had undergone dialysis for 36.3 +/- 33.2 months, were selected randomly from 7 DaVita dialysis units in Los Angeles South/East Bay area. The average weekly dose of administered recombinant human EPO within a 13-week interval was 217 +/- 187 U/kg. Patients were receiving intravenous iron supplementation (iron gluconate or dextran) averaging 39.5 +/- 47.5 mg/wk. The MIS and serum concentrations of high-sensitivity C-reactive protein, interleukin 6 (IL-6), tumor necrosis factor-alpha, and lactate dehydrogenase had positive correlation with required EPO dose and EPO responsiveness index (EPO divided by hemoglobin), whereas serum total iron binding capacity (TIBC), prealbumin and total cholesterol, as well as blood lymphocyte count had statistically significant but negative correlations with indices of refractory anemia. Most correlations remained significant even after multivariate adjustment for case-mix and anemia factors and other relevant covariates. Similar associations were noticed across EPO per body weight tertiles via analysis of variance and after estimating odds ratio for higher versus lower tertile via logistic regression after same case-mix adjustment.

CONCLUSIONS

The existence of elements of MICS as indicated by a high MIS and increased levels of proinflammatory cytokines such as IL-6 as well as decreased nutritional values such as low serum concentrations of total cholesterol, prealbumin, and TIBC correlates with EPO hyporesponsiveness in MHD patients.

Investigators using anti-EpoR antibodies for immunoblotting and immunostaining have reported erythropoietin receptor (EpoR) expression in nonhematopoietic tissues including human tumors. However, these antibodies detected proteins of 66 to 78 kDa, significantly larger than the predicted molecular weight of EpoR (56-57 kDa). We investigated the specificity of these antibodies and showed that they all detected non-EpoR proteins. C-20 detected 3 proteins in tumor cell lines (35, 66, and 100 kDa). Sequences obtained from preparative gels had similarity to the C-20-immunizing peptide. The 66-kDa protein was a heat shock protein (HSP70) to which antibody binding was abrogated in peptide competition experiments. Antibody M-20 readily identified a 59-kDa EpoR protein. However, neither M-20 nor C-20 was suitable for detection of EpoR using immunohistochemical methods. We concluded that these antibodies have limited utility for detecting EpoR. Thus, reports of EpoR expression in tumor cells using these antibodies should be viewed with caution.

Skin plays an essential role, mediated in part by its remarkable vascular plasticity, in adaptation to environmental stimuli. Certain vertebrates, such as amphibians, respond to hypoxia in part through the skin; but it is unknown whether this tissue can influence mammalian systemic adaptation to low oxygen levels. We have found that epidermal deletion of the hypoxia-responsive transcription factor HIF-1alpha inhibits renal erythropoietin (EPO) synthesis in response to hypoxia. Conversely, mice with an epidermal deletion of the von Hippel-Lindau (VHL) factor, a negative regulator of HIF, have increased EPO synthesis and polycythemia. We show that nitric oxide release induced by the HIF pathway acts on cutaneous vascular flow to increase systemic erythropoietin expression. These results demonstrate that in mice the skin is a critical mediator of systemic responses to environmental oxygen.

A common feature among all forms of diabetes mellitus is a functional β-cell mass insufficient to maintain euglycemia; therefore, the promotion of β-cell growth and survival is a fundamental goal for diabetes prevention and treatment. Evidence has suggested that erythropoietin (EPO) exerts cytoprotective effects on nonerythroid cells. However, the influence of EPO on pancreatic β cells and diabetes has not been evaluated to date. In this study, we report that recombinant human EPO treatment can protect against diabetes development in streptozotocin-induced and db/db mouse models of type 1 and type 2 diabetes, respectively. EPO exerts antiapoptotic, proliferative, antiinflammatory, and angiogenic effects within the islets. Using β-cell-specific EPO receptor and JAK2 knockout mice, we show that these effects of EPO result from direct biological effects on β cells and that JAK2 is an essential intracellular mediator. Thus, promotion of EPO signaling in β cells may be a novel therapeutic strategy for diabetes prevention and treatment.

Erythropoietin (Epo) stimulation of its receptor's downstream signaling pathways and optimum function of GATA-1 transcription factor are both essential for normal erythroid cell development. Epo-receptor (EpoR) signaling and GATA-1 regulate proliferation, survival, differentiation, and maturation of erythroid cells. Whether any signal that is generated by EpoR targets GATA-1 or affects GATA-1 transcriptional activity is not known. Here, we demonstrate that stimulation of EpoR results in phosphorylation of GATA-1 at serine 310 (S310) in primary fetal liver erythroid progenitors and in cultured erythroid cells. We show that phosphorylation of GATA-1 is important for Epo-induced maturation of fetal liver erythroid progenitor cells. The PI3-kinase/AKT signaling pathway is identified as a mediator of Epo-induced phosphorylation of GATA-1. AKT serine threonine kinase phosphorylates GATA-1S310 in vitro and in erythroid cells and enhances GATA-1 transcriptional activity. These data demonstrate that EpoR signaling phosphorylates GATA-1 and modulates its activity via the PI3-kinase/AKT signaling pathway.

Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-β (TGF-β) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

We investigated whether the mobilization of endothelial progenitor cells (EPCs) by exogenous erythropoietin (Epo) promotes the repair of injured endothelium. Recombinant human Epo was injected (1000 IU/kg for the initial 3 days) after wire injury of the femoral artery of mice. Neointimal formation was inhibited by Epo to 48% of the control (P<0.05) in an NO-dependent manner. Epo induced a 1.4-fold increase in reendothelialized area of day 14 denuded vessels, 55% of which was derived from bone marrow (BM) cells. Epo increased the circulating Sca-1(+)/Flk-1(+) EPCs (2.0-fold, P<0.05) with endothelial properties NO dependently. BM replacement by GFP- or beta-galactosidase-overexpressing cells showed that Epo stimulated both differentiation of BM-derived EPCs and proliferation of resident ECs. BM-derived ECs increased 2.2- to 2.7-fold (P<0.05) in the Epo-induced neoendothelium, where the expression of Epo receptor was upregulated. Epo induced Akt/eNOS phosphorylation and NO synthesis on EPCs and exerted an antiapoptotic action on wire-injured arteries. In conclusion, Epo treatment inhibits the neointimal hyperplasia after arterial injury in an NO-dependent manner by acting on the injured vessels and mobilizing EPCs to the neo-endothelium.

The molecular components which mediate cytokine signaling from the cell membrane to the nucleus were studied. Upon the interaction of cytokines with their receptors, members of the janus kinase (Jak) family of cytoplasmic protein tyrosine kinases and of the signal transducers and activators of transcription (Stat) family of transcription factors are activated through tyrosine phosphorylation. It has been suggested that the Stat proteins are substrates of the Jak protein tyrosine kinases. MGF-Stat5 is a member of the Stat family which has been found to confer the prolactin response. MGF-Stat5 can be phosphorylated and activated in its DNA binding activity by Jak2. The activation of MGF-Stat5 is not restricted to prolactin. Erythropoietin (EPO) and growth hormone (GH) stimulate the DNA binding activity of MGF-Stat5 in COS cells transfected with vectors encoding EPO receptor and MGF-Stat5 or vectors encoding GH receptor and MGF-Stat5. The activation of DNA binding by prolactin, EPO and GH requires the phosphorylation of tyrosine residue 694 of MGF-Stat5. The transcriptional induction of a beta-casein promoter luciferase construct in transiently transfected COS cells is specific for the prolactin activation of MGF-Stat5; it is not observed in EPO- and GH-treated cells. In the UT7 human hematopoietic cell line, EPO and granulocyte-macrophage colony stimulating factor activate the DNA binding activity of a factor closely related to MGF-Stat5 with respect to its immunological reactivity, DNA binding specificity and molecular weight. These results suggest that MGF-Stat5 regulates physiological processes in mammary epithelial cells, as well as in hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS)