OBJECTIVE

Delayed ischemic deficits (DIDs), a major source of disability following aneurysmal subarachnoid hemorrhage (aSAH), are usually associated with severe cerebral vasospasm and impaired autoregulation. Systemic erythropoietin (EPO) therapy has been demonstrated to have neuroprotective properties acting via EPO receptors on cerebrovascular endothelia and ischemic neurons. In this trial, the authors explored the potential neuroprotective effects of acute EPO therapy following aSAH.

METHODS

Within 72 hours of aSAH, 80 patients (age range 24-82 years) were randomized to receive intravenous EPO (30,000 U) or placebo every 48 hours for a total of 90,000 U. Primary end points were the incidence, duration, and severity of vasospasm and impaired autoregulation on transcranial Doppler ultrasonography. Secondary end points were incidence of DIDs and outcome at discharge and at 6 months.

RESULTS

Randomization characteristics were balanced except for age, with the EPO group being older (mean age 59.6 vs 53.3 years, p=0.034). No differences were demonstrated in the incidence of vasospasm and adverse events; however, patients receiving EPO had a decreased incidence of severe vasospasm from 27.5 to 7.5% (p=0.037), reduced DIDs with new cerebral infarcts from 40.0 to 7.5% (p=0.001), a shortened duration of impaired autoregulation (ipsilateral side, p<0.001), and more favorable outcome at discharge (favorable Glasgow Outcome Scale score, p=0.039). Among the 71 survivors, the EPO group had fewer deficits measured with National Institutes of Health Stroke Scale (median Score 2 vs 6, p=0.008).

CONCLUSIONS

This preliminary study showed that EPO seemed to reduce delayed cerebral ischemia following aSAH via decreasing severity of vasospasm and shortening impaired autoregulation.

Erythropoiesis stimulating agents (ESAs) have been reported to activate erythropoietin receptors (EpoR) on cell types, including endothelial, neuronal, renal tubule, and cardiac cells. ESAs have also been reported to promote angiogenesis. However, those findings are controversial and confounded by methodologic issues. We show that EpoR mRNA was detected in essentially all cell types examined, including primary human endothelial, renal, cardiac, and neuronal cells but 10- to 100-fold lower than Epo-responsive cells using quantitative reverse-transcribed polymerase chain reaction. Total endothelial EpoR protein examined using a new monoclonal antibody was low to undetectable. Surface EpoR on endothelial cells was not detected using [(125)I]-rHuEpo surface-binding studies. There was no evidence of ESA-induced intracellular signaling in endothelial cells. There was a similar lack of EpoR expression and signaling in other cell types examined. Experiments were performed examining ESA function on these cells. An in vivo rat corneal angiogenesis assay demonstrated neo-vessel formation in response to recombinant human vascular endothelial growth factor (rHuVEGF). However, recombinant mouse Epo did not induce vessel formation. Similarly, ESAs did not reproducibly provide cytoprotection to neuronal, renal, or cardiac cells. Taken together, our data challenge the notion of presence or function of EpoR on nonhematopoietic cells, and call into question the preclinical basis for clinical studies exploring direct, "pleiotropic" actions of ESAs.

BACKGROUND

Erythropoietin (EPO), secreted by fibroblast-like cells in the renal interstitium, controls erythropoiesis by regulating the survival, proliferation, and differentiation of erythroid progenitor cells. We examined whether renal cells that are exposed to EPO express EPO receptors (EPO-R) through which analogous cytokine responses might be elicited.

METHODS

Normal human and rat kidney tissue and defined cell lines of human, rat, and mouse kidney were screened, using reverse transcription-polymerase chain reaction, nucleotide sequencing, ligand binding, and Western blotting, for the expression of EPO-R. EPO's effects on DNA synthesis and cell proliferation were also examined.

RESULTS

EPO-R transcripts were readily detected in cortex, medulla, and papilla of human and rat kidney, in mesangial (human, rat), proximal tubular (human, mouse), and medullary collecting duct cells (human). Nucleotide sequences of EPO-R cDNAs from renal cells were identical to those of erythroid precursor cells. Specific 125I-EPO binding revealed a single class of high- to intermediate-affinity EPO-Rs in each tested cell line (kD 96 pm to 1. 4 nm; Bmax 0.3 to 7.0 fmol/mg protein). Western blots of murine proximal tubular cell membranes revealed an EPO-R protein of approximately 68 kDa. EPO stimulated DNA synthesis and cell proliferation dose dependently.

CONCLUSIONS

This is the first direct demonstration, to our knowledge, that renal cells possess EPO-Rs through which EPO stimulates mitogenesis. This suggests currently unrecognized cytokine functions for EPO in the kidney, which may prove beneficial in the repair of an injured kidney while being potentially detrimental in renal malignancies.

OBJECTIVE

Anaemia is often observed in patients with chronic heart failure (CHF), and it may be associated with a worse prognosis. Aim of this study was to identify the individual mechanisms of anaemia in CHF patients.

RESULTS

One hundred and forty-eight consecutive patients with haemoglobin concentration <13 g/dL (if males) or <12 g/dL (if females) were enrolled. Factors responsible for anaemia were investigated by evaluating endogenous erythropoietin (Epo) production, serum cytokines levels, body iron status, and iron supply for erythropoiesis. Most patients (57%) presented anaemia of chronic disease and among them, 92% showed evidence of a defective endogenous Epo production. This was indicated by an observed/predicted log(serum Epo) ratio less than 0.8 and/or a defective iron supply for erythropoiesis diagnosed by low transferrin saturation and/or increased value of soluble transferrin receptor. According to regression analysis sex, renal failure, and serum Epo were correlated with anaemia.

CONCLUSIONS

According to our study, about half of anaemic CHF patients showed anaemia of chronic disease with blunted endogenous Epo production and/or a defective iron supply for erythropoiesis. Determination of the individual mechanisms of anaemia in CHF could justify a rational therapeutic approach to anaemia.

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors, namely insulin receptor substrate-2 (IRS2), Src homology 2 domain-containing inositol 5'-phosphatase (SHIP), Grb2-associated binder-1 (Gab1), and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors, Akt, FKHRL1, and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR, or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors, but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2), which, in turn, down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation.

OBJECTIVE

Depending on the specific definition, acute kidney injury (AKI) occurs in 7-40% of patients undergoing cardiac surgery. Even small changes in serum creatinine (SCr) levels are associated with increased mortality after cardiac surgery. However, there are no current methods for preventing AKI after cardiac surgery. Erythropoietin (EPO) has been shown to elicit tissue-protective effects in various experimental models. In this pilot trial, we evaluated the effectiveness of EPO in the prevention of AKI after coronary artery bypass grafting (CABG).

METHODS

71 patients scheduled for elective CABG randomly received either 300 U/kg of EPO or saline intravenously before surgery. AKI was defined as a 50% increase in SCr levels over baseline within the first 5 postoperative days. Estimated glomerular filtration rate (eGFR) was calculated from the Cockcroft-Gault equation.

RESULTS

Of 71 patients, 13 developed postoperative AKI: 3 of the 36 patients in the EPO group (8%) and 10 of the 35 patients in the placebo group (29%; p = 0.035). The increase in postoperative SCr concentration and the decline in postoperative eGFR were significantly lower in the EPO group than in the placebo group.

CONCLUSIONS

In our small, pilot trial, prophylactic administration of EPO prevents AKI and improves postoperative renal function. These data are preliminary and require confirmation in a larger clinical trial.

Erythropoietin can be over-expressed in skeletal muscles by gene electrotransfer, resulting in 100-fold increase in serum EPO and significant increases in haemoglobin levels. Earlier studies have suggested that EPO improves several metabolic parameters when administered to chronically ill kidney patients. Thus we applied the EPO over-expression model to investigate the metabolic effect of EPO in vivo.At 12 weeks, EPO expression resulted in a 23% weight reduction (P<0.01) in EPO transfected obese mice; thus the mice weighed 21.9+/-0.8 g (control, normal diet,) 21.9+/-1.4 g (EPO, normal diet), 35.3+/-3.3 g (control, high-fat diet) and 28.8+/-2.6 g (EPO, high-fat diet). Correspondingly, DXA scanning revealed that this was due to a 28% reduction in adipose tissue mass.The decrease in adipose tissue mass was accompanied by a complete normalisation of fasting insulin levels and glucose tolerance in the high-fat fed mice. EPO expression also induced a 14% increase in muscle volume and a 25% increase in vascularisation of the EPO transfected muscle. Muscle force and stamina were not affected by EPO expression. PCR array analysis revealed that genes involved in lipid metabolism, thermogenesis and inflammation were increased in muscles in response to EPO expression, while genes involved in glucose metabolism were down-regulated. In addition, muscular fat oxidation was increased 1.8-fold in both the EPO transfected and contralateral muscles.In conclusion, we have shown that EPO when expressed in supra-physiological levels has substantial metabolic effects including protection against diet-induced obesity and normalisation of glucose sensitivity associated with a shift to increased fat metabolism in the muscles.

The cDNA for human stem cell factor (hSCF) has been cloned and expressed in mammalian and bacterial hosts and recombinant protein purified. We have examined the stimulatory effect of recombinant human SCF (rhSCF) on human bone marrow cells alone and in combination with recombinant human colony stimulating factors (CSFs) and erythropoietin (rhEpo). RhSCF alone resulted in no significant colony formation, however, in the presence of rhGM-CSF, rhG-CSF or rhIL-3, rhSCF stimulated a synergistic increase in colony numbers. In addition, increased colony size was stimulated by all combinations. The morphology of cells in the colonies obtained with the CSFs plus rhSCF was identical to the morphology obtained with rhGM-CSF, rhG-CSF or rhIL-3 alone. RhEpo also synergised with rhSCF to stimulate the formation of large compact hemoglobinized colonies which stained positive for spectrin and transferrin receptor and had a morphological appearance consistent with normoblasts. RhSCF stimulation of low density non-adherent, antibody depleted, CD34+ cells suggests that rhSCF directly stimulates progenitor cells capable of myeloid and erythroid differentiation.

This study was carried out to investigate the early changes in erythropoietin (EPO) formation in humans in response to hypoxia. Six volunteers were exposed to simulated altitudes of 3,000 and 4,000 m in a decompression chamber for 5.5 h. EPO was measured by radioimmunoassay in serum samples withdrawn every 30 min during altitude exposure and also in two subjects after termination of hypoxia (4,000 m). EPO levels during hypoxia were significantly elevated after 114 and 84 min (3,000 and 4,000 m), rising thereafter continuously for the period investigated. Mean values increased from 16.0 to 22.5 mU/ml (3,000 m) and from 16.7 to 28.0 mU/ml (4,000 m). This rise in EPO levels corresponds to 1.8-fold (3,000 m) and 3.0-fold (4,000 m) increases in the calculated production rate of the hormone. After termination of hypoxia, EPO levels continued to rise for approximately 1.5 h and after 3 h declined exponentially with an average half-life time of 5.2 h.

The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.

OBJECTIVE

Besides stimulating hematopoiesis, erythropoietin (EPO) protects against experimental ischemic injury in the heart. The present study evaluated the safety and tolerability of EPO treatment in non-anemic patients with acute myocardial infarction (MI).

RESULTS

In this single-center, investigator-initiated, prospective study, patients with a first acute MI were randomized to one bolus of 300 microg darbepoetin alfa or no additional medication before primary coronary intervention. Twenty-two patients (mean age 59 +/- 2 years) were included. In the darbepoetin group, serum EPO-levels increased to 130-270 times that of controls, within the first 24 h. After darbepoetin administration, only small and non-significant changes in hematocrit levels were observed, while endothelial progenitor cells (EPCs, CD34+/CD45-) were increased at 72 h (2.8 vs. 1.0 cells/microl in control group, p < 0.01). No adverse events were recorded during the 30-day follow-up. After 4 months, left ventricular ejection fraction was similar in the two groups (52 +/- 3% in darbepoetin vs. 48 +/- 5% in control group, p = NS).

CONCLUSIONS

Intravenous single high-dose darbepoetin alfa in acute MI is both safe and well tolerated. Darbepoetin treatment after MI stimulates EPCs mobilization. The results of this first pilot study support a larger scale clinical trial to establish efficacy of EPO administration in patients after acute MI.

A role for tyrosine phosphorylation in the signal-transducing mechanisms of several hematopoietic growth factors has been hypothesized. To extend these observations, we have examined the effects of erythropoietin (Epo) on tyrosine phosphorylation in an Epo-responsive cell that was obtained by transfecting the murine erythropoietin receptor (EpoR) into an interleukin-3 (IL-3)-dependent cell line. By two-dimensional analysis of phosphotyrosine-containing proteins isolated with a monoclonal antibody (1G2) against phosphotyrosine, Epo and IL-3 were found to rapidly induce tyrosine phosphorylation of comparable substrates of 92, 70, and 56 kDa. In addition, Epo uniquely induced phosphorylation of a 72-kDa substrate while IL-3 uniquely induced phosphorylation of a 140-kDa substrate. Immunoprecipitation and mixing experiments indicated that the 72-kDa substrate may represent a small fraction of the EpoR. To explore the significance of tyrosine phosphorylation, we generated two mutants of the EpoR that lacked 108 or 146 amino acids at their carboxyl termini. In addition we constructed an internally deleted mutant that lacked 20 amino acids in a region of sequence homology with the IL-2 receptor beta chain. Although all mutants were expressed at comparable levels and had comparable binding affinities for Epo, only the mutant lacking 108 amino acids at the carboxyl terminus retained significant mitogenic activity or the ability to induce tyrosine phosphorylation.

Hemopoietin-1 (H-1) is known to act synergistically with CSF-1, a mononuclear phagocyte growth factor, to induce the development of primitive hemopoietic cells. To determine whether purified H-1 also acts on multipotent hemopoietic cells, its ability to act synergistically with interleukin-3 (IL-3) and erythropoietin (Epo) was tested in methyl cellulose cultures of murine bone marrow cells. In the presence of IL-3, H-1 increased the number of colonies formed by primitive, multipotent cells by approximately 30-fold. H-1 alone or H-1 plus Epo produced no colonies. Forty percent of the colonies induced by H-1 plus IL-3 contained cells that could be subcultured at least twice, whereas cells from colonies induced by IL-3 alone could not be similarly subcultured. Thus H-1 permits CSF-1 or IL-3 to act on cells more primitive than those acted on by either growth factor alone. The results indicate that H-1 acts on the most primitive hemopoietic cells yet shown to proliferate and differentiate in culture.

Inflammatory microglia modulate a host of cellular processes in the central nervous system that include neuronal survival, metabolic fluxes, foreign body exclusion, and cellular regeneration. Elucidation of the pathways that oversee microglial survival and integrity may offer new avenues for the treatment of neurodegenerative disorders. Here we demonstrate that erythropoietin (EPO), an emerging strategy for immune system modulation, prevents microglial early and late apoptotic injury during oxidant stress through Wnt1, a cysteine-rich glycosylated protein that modulates cellular development and survival. Loss of Wnt1 through blockade of Wnt1 signaling or through the gene silencing of Wnt1 eliminates the protective capacity of EPO. Furthermore, endogenous Wnt1 in microglia is vital to preserve microglial survival since loss of Wnt1 alone increases microglial injury during oxidative stress. Cellular protection by EPO and Wnt1 intersects at the level of protein kinase B (Akt1), the mammalian target of rapamycin (mTOR), and p70S6K, which are necessary to foster cytoprotection for microglia. Downstream from these pathways, EPO and Wnt1 control "anti-apoptotic" pathways of microglia through the modulation of mitochondrial membrane permeability, the release of cytochrome c, and the expression of apoptotic protease activating factor-1 (Apaf-1) and X-linked inhibitor of apoptosis protein (XIAP). These studies offer new insights for the development of innovative therapeutic strategies for neurodegenerative disorders that focus upon inflammatory microglia and novel signal transduction pathways.

Oncostatin M (OSM) is a member of the interleukin-6 (IL6)-related cytokine subfamily that includes IL6, IL11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor and cardiotrophin-1. While human OSM has been characterized and the bovine OSM gene was recently cloned, the murine counterpart had not been identified. Here we describe molecular cloning of murine OSM as an immediate early gene induced by a subset of cytokines including IL2, IL3 and erythropoietin (EPO) in myeloid and lymphoid cell lines. The induction kinetics of OSM are rapid and transient, reaching a maximal level within 30-60 min and decreasing thereafter. Induction of OSM depends on the signals generated by the membrane-proximal region of the EPO receptor as well as that of the beta chain of the IL3/GM-CSF receptor, which activate JAK2 and STAT5. About 100 bases upstream of the transcription initiation site of the OSM gene contains a possible STAT5 binding site which is essential for IL2, IL3 and EPO-dependent promoter activity of the OSM gene. Expression of STAT5 and the EPO receptor in COS cells conferred EPO-dependent activation of the OSM promoter. Moreover, the mutant IL2 receptor lacking the ability to activate STAT5 induced c-myc but failed to induce OSM. Thus OSM is one of the common targets of a subset of cytokines that activate STAT5. The murine OSM gene is located near to the LIF gene, expressed at high levels in bone marrow and possesses similar biological activity to human OSM. Identification of murine OSM as a cytokine-inducible immediate early gene provides a new insight into the physiological function of this unique cytokine.

Apoptosis is a contributing cause of myocyte loss in ischemic heart disease. Recent work has shown that erythropoietin (EPO) offers protection against apoptosis in a wide variety of tissues. We demonstrate that the erythropoietin receptor (EPOR) is expressed in the neonatal rat ventricular myocyte (NRVM). Exposure of NRVMs to hypoxia, with recombinant human EPO, significantly decreased apoptosis as measured by TUNEL, flow cytometry, and caspase 3/7 like activity when compared to hypoxia treatment alone. EPO administered at the initiation of coronary artery occlusion in the rat significantly decreased apoptosis in the myocardial ischemic region. In the NRVM, EPO increased the activity of Akt. The anti-apoptotic effect of EPO was abrogated by co-treatment with LY294002, a specific blocker of phosphatidylinositol 3-kinase (PI3-K). Our study demonstrates that EPO inhibits apoptosis in the NRVM exposed to hypoxia, through an Akt-dependent pathway. EPO also inhibits apoptosis in the in vivo rat model of myocardial ischemia.

Iron regulatory proteins (Irps) 1 and 2 posttranscriptionally control the expression of transcripts that contain iron-responsive element (IRE) sequences, including ferritin, ferroportin, transferrin receptor, and hypoxia-inducible factor 2α (HIF2α). We report here that mice with targeted deletion of Irp1 developed pulmonary hypertension and polycythemia that was exacerbated by a low-iron diet. Hematocrits increased to 65% in iron-starved mice, and many polycythemic mice died of abdominal hemorrhages. Irp1 deletion enhanced HIF2α protein expression in kidneys of Irp1(-/-) mice, which led to increased erythropoietin (EPO) expression, polycythemia, and concomitant tissue iron deficiency. Increased HIF2α expression in pulmonary endothelial cells induced high expression of endothelin-1, likely contributing to the pulmonary hypertension of Irp1(-/-) mice. Our results reveal why anemia is an early physiological consequence of iron deficiency, highlight the physiological significance of Irp1 in regulating erythropoiesis and iron distribution, and provide important insights into the molecular pathogenesis of pulmonary hypertension.

Red blood cell production is a finely tuned process that requires coordinated oxygen- and iron-dependent regulation of cell differentiation and iron metabolism. Here, we show that translational regulation of hypoxia-inducible factor 2α (HIF-2α) synthesis by iron regulatory protein 1 (IRP1) is critical for controlling erythrocyte number. IRP1-null (Irp1(-/-)) mice display a marked transient polycythemia. HIF-2α messenger RNA (mRNA) is derepressed in kidneys of Irp1(-/-) mice but not in kidneys of Irp2(-/-) mice, leading to increased renal erythropoietin (Epo) mRNA and inappropriately elevated serum Epo levels. Expression of the iron transport genes DCytb, Dmt1, and ferroportin, as well as other HIF-2α targets, is enhanced in Irp1(-/-) duodenum. Analysis of mRNA translation state in the liver revealed IRP1-dependent dysregulation of HIF-2α mRNA translation, whereas IRP2 deficiency derepressed translation of all other known 5' iron response element (IRE)-containing mRNAs expressed in the liver. These results uncover separable physiological roles of each IRP and identify IRP1 as a therapeutic target for manipulating HIF-2α action in hematologic, oncologic, and other disorders.

Moderate-altitude living (2,500 m), combined with low-altitude training (1,250 m) (i.e., live high-train low), results in a significantly greater improvement in maximal O2 uptake (V(02)max) and performance over equivalent sea-level training. Although the mean improvement in group response with this "high-low" training model is clear, the individual response displays a wide variability. To determine the factors that contribute to this variability, 39 collegiate runners (27 men, 12 women) were retrospectively divided into responders (n = 17) and nonresponders (n = 15) to altitude training on the basis of the change in sea-level 5,000-m run time determined before and after 28 days of living at moderate altitude and training at either low or moderate altitude. In addition, 22 elite runners were examined prospectively to confirm the significance of these factors in a separate population. In the retrospective analysis, responders displayed a significantly larger increase in erythropoietin (Epo) concentration after 30 h at altitude compared with nonresponders. After 14 days at altitude, Epo was still elevated in responders but was not significantly different from sea-level values in nonresponders. The Epo response led to a significant increase in total red cell volume and V(O2) max in responders; in contrast, nonresponders did not show a difference in total red cell volume or V(O2)max after altitude training. Nonresponders demonstrated a significant slowing of interval-training velocity at altitude and thus achieved a smaller O2 consumption during those intervals, compared with responders. The acute increases in Epo and V(O2)max were significantly higher in the prospective cohort of responders, compared with nonresponders, to altitude training. In conclusion, after a 28-day altitude training camp, a significant improvement in 5,000-m run performance is, in part, dependent on 1) living at a high enough altitude to achieve a large acute increase in Epo, sufficient to increase the total red cell volume and V(O2)max, and 2) training at a low enough altitude to maintain interval training velocity and O2 flux near sea-level values.

Hypoxia-ischemia (H/I) brain injury results in various degrees of damage to the body, and the immature brain is particularly fragile to oxygen deprivation. Hypothermia and erythropoietin (EPO) have long been known to be neuroprotective in ischemic brain injury. Brain-derived neurotrophic factor (BDNF) has recently been recognized as a potent modulator capable of regulating a wide repertoire of neuronal functions. This review was based on studies concerning the involvement of BDNF in the protection of H/I brain injury following a search in PubMed between 1995 and December, 2011. We initially examined the background of BDNF, and then focused on its neuroprotective mechanisms against ischemic brain injury, including its involvement in promoting neural regeneration/cognition/memory rehabilitation, angiogenesis within ischemic penumbra and the inhibition of the inflammatory process, neurotoxicity, epilepsy and apoptosis. We also provided a literature overview of experimental studies, discussing the safety and the potential clinical application of BDNF as a neuroprotective agent in the ischemic brain injury.

Mutations in the KIT transmembrane protein-tyrosine kinase receptor affect erythropoiesis, resulting in fewer committed late progenitors (colony-forming unit erythroid, CFU-E) in the fetal liver. As the survival and proliferation of CFU-Es depend absolutely on erythropoietin (EPO), these results suggest that CFU-Es cannot proliferate or mature further unless both the KIT and EPO receptor signalling pathways are functional. How KIT affects proliferation or differentiation of CFU-Es is not clear. Here we show that the KIT ligand SCF (for stem-cell factor) can replace EPO in supporting the growth and survival of HCD57 cells, an EPO-dependent erythroid-progenitor cell line expressing high levels of KIT. SCF supports the proliferation of 32D cells that express KIT only if they also express the EPO receptor. In HCD57 cells, SCF rapidly induces tyrosine phosphorylation of the EPO receptor, and KIT physically associates with the extended box 2 region in the cytoplasmic domain of the EPO receptor. Our results indicate that KIT may activate the EPO receptor by tyrosine phosphorylation to induce further proliferation and maturation of CFU-Es.

The temporal association between airway inflammation and airway hyperresponsiveness (AHR) has been analyzed in BALB/c mice sensitized to, and subsequently exposed to, a single intranasal challenge of ovalbumin (OVA). In OVA-sensitized/challenged animals only a small increase in responsiveness to methacholine (MCh) was seen at 8 h, peaked at 24 to 48 h, and resolved by 96 h. An early bronchoalveolar lavage fluid (BALF) neutrophil infiltrate (peaking at 8 h postchallenge; approximately 72% total cells was observed) that returned to baseline by 48 h. BALF eosinophil numbers did not increase until 48 h (approximately 32% of total cells), peaked at 96 h (approximately 38% total cells), and remained elevated at 8 d (approximately 27% total cells). Airway tissue eosinophilia preceded changes in BALF. Eosinophil peroxidase (EPO) levels in BALF were elevated in OVA-sensitized/challenged mice at 48 h only. BALF TNF-alpha levels peaked at 8 h, whereas IL-5 and IL-4 levels peaked at 24 h. IL-13 levels were increased at both 24 and 48 h. Mucus-positive cells were not observed in the airway epithelium until 48 h. Administration of IL-5 or VLA-4 antibody prior to OVA challenge prevented the development of AHR in sensitized mice as well as BALF and tissue eosinophilia. These data identify a temporal association between Th2 cytokine production, tissue eosinophil infiltration and activation, and, importantly, both the development and resolution kinetics of AHR. Moreover, the antibody studies further support the association of eosinophilia with the pathogenesis of AHR.

The objective of this study was to determine the probabilities of specific morbid events or death among patients with end-stage renal disease (ESRD) treated by hemodialysis. A prospective cohort study was performed between March 1988 and September 1989 in 18 hemodialysis centers in 13 Canadian cities, representing about one third of the hemodialysis population in Canada. The inception cohort consisted of 496 patients entering hemodialysis who had survived 1 month. The few new hemodialysis patients who received erythropoietin (EPO) in the last 3 months of the study were excluded. Survival curves were compared using the Cox proportional hazards regression model. Older age and history of cardiovascular disease were independently associated with a greater probability of death. Age and history of cardiovascular disease were also associated with a greater probability of nonfatal circulatory events (myocardial infarction, angina requiring hospitalization, or stroke), while a serum albumin level less than or equal to 30 g/L (3.0 g dL) was associated with an increased probability of pulmonary edema. The probability of surviving 12 months without receiving a blood transfusion was 47.2% for males and 27.5% for females. The incidence of non-A, non-B hepatitis, as estimated by unexplained elevations in serum aspartate aminotransferase (AST) values, was not different between patients receiving and not receiving blood transfusions. The probability of hospitalization for any cause was greater for patients with grafts for vascular access than for those with fistulae, for those with a history of cardiovascular disease, for those with a serum albumin level less than or equal to 30 g/L, and for those with renal disease due to diabetes or vascular disease. Hospitalization due to circulatory disease was more likely among those with a history of cardiovascular disease and among those with a lower serum albumin level. Hospitalization for infectious disease was more likely among those with a lower serum albumin level and less likely among those with a fistula for vascular access. Among all patients receiving hemodialysis treatment for more than 6 months, there were 14.8 hospital days per year.(ABSTRACT TRUNCATED AT 400 WORDS)

This study evaluates the use of vectors based on adeno-associated viruses (AAVs) to noninvasively deliver genes to airway epithelial cells as a means for achieving systemic administration of therapeutic proteins. We intranasally delivered AAV vectors to mice in which the same AAV2 genome encoding a cellular marker was packaged in capsids from AAV1, 2, or 5 (AAV2/1, AAV2/2, or AAV2/5, respectively). Gene expression levels achieved in both airways and alveoli were higher with AAV2/5 than with AAV2/1 and were undetectable with AAV2/2. The same set of vectors encoding a secreted therapeutic protein, erythropoietin (Epo), under the control of a lung-specific promoter (CC10) was intranasally delivered to mice, resulting in polycythemia with the highest levels of serum Epo obtained with AAV2/5 vectors. After a single intranasal administration of this vector, secretion of Epo was documented for 150 days. Similarly, intranasal administration of an AAV2/5-CC10-factor IX vector resulted in secretion of functional recombinant protein in the bloodstream of hemophiliac, factor IX-deficient mice. In addition, we demonstrate successful readministration of AAV2/5 to the lung 5 months after the first delivery of the same vector. In conclusion, we show that intranasal administration of AAV vectors results in efficient gene transfer to the lung only when the vector contains the AAV5 capsid and that this noninvasive route of administration results in sustained secretion of therapeutic proteins in the bloodstream.