Eosinophils (EOs) participate in a variety of inflammatory states characterized by endothelial cell damage, such as vasculitis, pneumonitis, and endocarditis. We find that 100 U/ml TNF-alpha/cachectin (TNF), a concentration attainable in the blood of humans with parasitic infestations, stimulates highly purified populations of EOs to damage human umbilical vein endothelial cells (HUVEC), a model of human endothelium. This TNF-dependent EO cytotoxicity is strongly inhibited by heparin and methyprednisolone but unaffected by the platelet-activating factor antagonist BN52012 or scavengers of superoxide anion and H2O2, superoxide dismutase and catalase. However, addition of a physiologically relevant concentration of Br- (100 microM) enhances EO/TNF damage to HUVEC, implicating the possible participation of EO peroxidase (EPO) in the killing mechanism. EOs adherent to FCS-coated plastic wells more than double their production of superoxide anion and the cytotoxic EPO-derived oxidant HOBr when exposed to TNF, showing that TNF activates the respiratory burst of EOs attached to a "physiologic" surface. Unlike PMNs, EOs were not irreversibly activated to kill unopsonized endothelium by previous exposure to TNF, and did not degranulate or upregulate CR3 expression as detected by Mo1 in the presence of 100 U/ml TNF. HUVEC exposed 18 h to TNF were considerably more susceptible to lysis by PMA-activated EOs and reagent H2O2, demonstrating a direct effect of TNF upon endothelium, perhaps through inhibition of antioxidant defenses. These findings suggest that abnormally elevated serum levels of TNF may provoke EOs to damage endothelial cells and thereby play a role in the pathogenesis of tissue damage in hypereosinophilic states.

AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoietin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Down-regulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation.

Erythropoietin (Epo) controls the proliferation, differentiation and survival of the erythroid progenitors. This cytokine was cloned in 1985 and rapidly became used for treatment of anemia of renal failure, opening the way to the first clinical trials of a hematopoietic growth factor. The clonage of one chain of the Epo receptor followed in 1989, thereby opening the research on intracellular signal transduction induced by Epo. Epo is synthesized mainly by the kidney and the liver and sequences required for tissue-specific expression have been localized in the Epo gene. A 3'enhancer is responsible for hypoxia-inducible Epo gene expression. HIF-1 alpha and beta proteins bind to this enhancer. Gene regulation by hypoxia is widespread in many cells and involves numerous genes in addition to the Epo gene. The Epo receptor belongs to the cytokine receptor family and includes a p66 chain which is dimerized upon Epo activation; two accessory proteins defined by cross-linking remain to be characterized. Epo binding induces the stimulation of Jak2 tyrosine kinase. Jak2 activation leads to the tyrosine phosphorylation of several proteins including the Epo receptor itself. As a result, different intracellular pathways are activated: Ras/MAP kinase, phosphatidylinositol 3-kinase and STAT transcription factors. However, the exact mechanisms by which the proliferation and/or the differentiation of erythroid cells are regulated after Epo stimulation are not known. Furthermore, target disruption of both Epo and Epo receptor showed that Epo was not involved in the commitment of the erythroid lineage and seemed to act mainly as a survival factor.

This study aimed to determine whether brief hypoxic stimuli in a hypobaric chamber are able to elicit erythropoietin (EPO) secretion, and to effectively stimulate erythropoiesis in the short term. In two different experiments, a set of haematological, biochemical, haemorheological, aerobic performance, and medical tests were performed in two groups of healthy subjects. In the first experiment, the mean plasma concentration of EPO ([EPO]) increased from 8.7 to 13.5 mU.ml-1 (55.2%; P < 0.01) after 90 min of acute exposure at 540 hPa, and continued to rise until a peak was attained 3 h after the termination of hypoxia. In the second experiment, in which subjects were exposed to a simulated altitude of up to 5500 m (504 hPa) for 90 min, three times a week for 3 weeks, all haematological indicators of red cell mass increased significantly, reaching the highest mean values at the end of the programme or during the subsequent 2 weeks, including packed cell volume (from 42.5 to 45.1%; P < 0.01), red blood cell count (from 4.55 x 10(6) to 4.86 x 10(6).l-1; P < 0.01), reticulocytes (from 0.5 to 1.4%; P < 0.01), and haemoglobin concentration (from 14.3 to 16.2 g.dl-1; P < 0.01), without an increase in blood viscosity. Arterial blood oxygen saturation during hypoxia was improved (from 60% to 78%; P < 0.05). Our most relevant finding is the ability to effectively stimulate erythropoiesis through brief intermittent hypoxic stimuli (90 min), in a short period of time (3 weeks), leading to a lower arterial blood desaturation in hypoxia. The proposed mechanism for these haematological and functional adaptations is the repeated triggering effect of EPO production caused by the intermittent hypoxic stimuli.

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of proliferation and differentiation of neutrophilic granulocyte precursor cells. G-CSF activates multiple signaling molecules, including the JAK1 and JAK2 kinases and the STAT transcription factors. To investigate G-CSF signaling events regulated by the JAK-STAT pathway, we have generated UT7-epo cells stably expressing either wild-type (wt) G-CSF receptor or a series of C-terminal deletion mutants. Gel mobility shift and immunoprecipitation/Western analysis showed that STAT5 is rapidly activated by G-CSF in cells expressing the wt G-CSF receptor, in addition to the previously reported STAT3 and STAT1. Mutants lacking any tyrosine residues in the cytoplasmic domain maintain their ability to activate STAT5 and STAT1 but cannot activate STAT3, implying that STAT5 and STAT1 activation does not require receptor tyrosine phosphorylation. We also observed significant changes in the ratio of STAT1:STAT3:STAT5 activated by various G-CSF receptor C-terminal deletion mutants. These mutant receptors were further used to investigate the role of JAKs and STATs in G-CSF-mediated responses in these cells. We found that JAK activation correlates with G-CSF-induced cell proliferation, whereas STAT activation is not required. We have also identified three classes of G-CSF immediate early genes, whose activation correlates with the activation of distinct JAK-STAT pathways. Our data show that, whereas c-fos is regulated through a pathway independent of STAT activation, oncostatin M, IRF-1, and egr-1 are regulated by an STAT5-dependent pathway and fibrinogen is regulated by an STAT3-dependent pathway. In conclusion, our results suggest that G-CSF regulates its complex biologic activities by selectively activating distinct early response genes through different JAK-STAT signaling molecules.

Fluoroquinolone-resistance in Pseudomonas aeruginosa may be due to efflux pump overexpression (EPO) and/or target mutations. EPO can result in multidrug resistance (MDR) due to broad substrate specificity of the pumps. MC-04,124, an efflux pump inhibitor (EPI) shown to significantly potentiate activity of levofloxacin in P. aeruginosa, was used to examine the prevalence of EPO in clinical isolates. MICs were determined for ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin with or without EPI and for other antipseudomonal agents by using broth microdilution against P. aeruginosa isolates from adults (n = 119) and children (n = 24). The prevalence of the EPO phenotype >>/=8-fold MIC decrease when tested with EPI) was compared among subgroups with different resistance profiles. The EPO phenotype was more prevalent among levofloxacin-resistant than levofloxacin-sensitive strains (61%, 48/79 versus 9%, 6/64). EPO was present in 60% of fluoroquinolone-resistant strains without cross-resistance, while it was present at variable frequencies among strains with cross-resistance to other agents: piperacillin-tazobactam (86%), ceftazidime (76%), cefepime (65%), imipenem (56%), gentamicin (55%), tobramycin (48%), and amikacin (27%). The magnitude of MIC decrease with an EPI paralleled the frequency of which the EPO phenotype was observed in different subgroups. EPI reduced the levofloxacin MIC by as much as 16-fold in eight strains for which MICs were 128 microg/ml. Efflux-mediated resistance appears to contribute significantly to fluoroquinolone resistance and MDR in P. aeruginosa. Our data support the fact that increased fluoroquinolone usage can negatively impact susceptibility of P. aeruginosa to multiple classes of antipseudomonal agents.

OBJECTIVE

Individually, the cytokines erythropoietin (EPO) and insulin-like growth factor-I (IGF-I) have both been shown to reduce neuronal damage significantly in rodent models of cerebral ischemia. The authors have previously shown that EPO and IGF-I, when administered together, provide acute and prolonged neuroprotection in cerebrocortical cultures against N-methyl-D-aspartate-induced apoptosis. The aim of this study was to determine whether intranasally applied EPO plus IGF-I can provide acute neuroprotection in an animal stroke model and to show that intranasal administration is more efficient at delivering EPO plus IGF-I to the brain when compared with intravenous, subcutaneous, or intraperitoneal administration.

METHODS

The EPO and IGF-I were administered intranasally to mice that underwent transient middle cerebral artery occlusion (MCAO). Stroke volumes were measured after 1 hour of MCAO and 24 hours of reperfusion. To evaluate the long-term effects of this treatment, behavioral outcomes were assessed at 3, 30, 60, and 90 days following MCAO. Radiography and liquid scintillation were used to visualize and quantify the uptake of radiolabeled 125I-EPO and 125I-IGF-I into the mouse brain after intranasal, intravenous, subcutaneous, or intraperitoneal administration.

RESULTS

Intranasal administration of EPO plus IGF-I reduced stroke volumes within 24 hours and improved neurological function in mice up to 90 days after MCAO. The 125I-EPO and 125I-IGF-I were found in the brain within 20 minutes after intranasal administration and accumulated within the injured areas of the brain. In addition, intranasal administration delivered significantly higher levels of the applied 125I-EPO and 125I-IGF-I to the brain compared with intravenous, subcutaneous, or intraperitoneal administration.

CONCLUSIONS

The data demonstrate that intranasal EPO plus IGF-I penetrates into the brain more efficiently than other drug delivery methods and could potentially provide a fast and efficient treatment to prevent chronic effects of stroke.

To characterize the growth of cord blood progenitor cells, single nonadherent, low-density, T-lymphocyte-depleted CD34 cells were sorted by flow cytometer with an autoclone device into single wells containing culture medium and cytokines. These cells were evaluated for proliferation and for replating ability of their progeny. This latter effect is used as a measure of self-renewal capacity. Colony formation was assessed in 1 degree wells containing various cytokines, alone and in combination, and single colonies deriving after 21 days in semisolid medium were replated into 2 degree wells in the presence of the combination of purified preparations of recombinant human steel factor (SF, a c-kit ligand), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), and erythropoietin (Epo). Replating of single colonies was performed also for 3 degrees, 4 degrees, and 5 degrees cultures. In the presence of serum, colony formation was observed in>> 66% of the wells stimulated with the combination of Epo, SF, GM-CSF, G-CSF, and IL-3, and more than 39% of the colonies formed in these 1 degree wells were very large in size >> 2.5 mm in diameter, dense in the center, and containing>> 10(4) cells/colony). The replating efficiency of these large colonies was up to 93% with generation of subsequent colonies of very large size. Replating could be shown for up to five generations. The cells in these colonies were large, nonspecific esterase positive, and contained large amounts of cytoplasm with one or more nuclei containing several nucleoli per nucleus. Smaller colonies (1 to 2.5 mm in diameter and dense in the center) containing similar cells and making up an additional 14% of the colonies formed in 1 degree wells also showed extensive replating capacity, including generation of larger colonies. These colony-forming cells are likely similar to the murine macrophage high-proliferative potential colony-forming cells. The cells giving rise to these colonies are present in about eightfold higher frequency in cord blood than in adult bone marrow. These cells may at least in part be associated with the successful hematopoietic repopulating capacity of umbilical cord blood cells.

Erythropoietin (Epo) is gaining interest in various neurological insults as a possible neuroprotective agent. We determined the effects of recombinant human Epo (rhEpo, 5000 IU per kg bw) on brain edema induced in rats by traumatic brain injury (TBI; impact-acceleration model; rhEpo administration 30 mins after injury). Magnetic resonance imaging (MRI) and a gravimetric technique were applied. In the MRI experiments, the apparent diffusion coefficient (ADC) and the tissue T(1) relaxation time were measured hourly in the neocortex and caudoputamen, during a 6 h time span after TBI. In the gravimetric experiments, brain water content (BWC) was determined in these two regions, 6 h after TBI. Apparent diffusion coefficient measurements showed that rhEpo decreased brain edema early and durably. Gravimetric measurements showed that rhEpo decreased BWC at H(6) in the neocortex as well as in the caudoputamen. No significant differences in ADC, in T(1), or in BWC were found between rhEpo treated-TBI rats and sham-operated rats. Our findings show that post-traumatic administration of rhEpo can significantly reduce the development of brain edema in a model of diffuse TBI. Further studies should be conducted to identify the biochemical mechanisms involved in these immediate effects and to assess the use of rhEpo as a possible therapy for post-traumatic brain edema.

BACKGROUND

Early referral of patients with chronic kidney disease (CKD) is believed to help with interventions to address risk factors to slow down the rate of progression of kidney failure to end-stage kidney disease (ESKD) and the need for dialysis, hospitalisation and mortality.

OBJECTIVE

We sought to evaluate the benefits (reduced hospitalisation and mortality; increased quality of life) and harms (increased hospitalisations and mortality, decreased quality of life) of early versus late referral to specialist nephrology services in CKD patients who are progressing to ESKD and RRT. In this review, referral is defined as the time period between first nephrology evaluation and initiation of dialysis; early referral is more than one to six months, whereas late referral is less than one to six months prior to starting dialysis. All-cause mortality and hospitalisation and quality of life were measured by the visual analogue scale and SF-36. SF-36 and KDQoL are validated measurement instruments for kidney diseases.

METHODS

We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library, 2012; Issue 1) which contains the Cochrane Renal Group's Specialised Register; MEDLINE (1966 to February 2012), EMBASE (1980 to February 2012). Search terms were approved by the Trial Search Co-ordinator.

METHODS

Randomised controlled trials (RCTs), quasi-RCTs, prospective and retrospective longitudinal cohort studies were eligible for inclusion.

METHODS

Two authors independently assessed study quality and extracted data. Events relating to adverse effects were collected from the studies.

RESULTS

No RCTs or quasi-RCTs were identified. There were 40 longitudinal cohort studies providing data on 63,887 participants; 43,209 (68%) who were referred early and 20,678 (32%) referred late.Comparative mortality was higher in patients referred to specialist services late versus those referred early. Risk ratios (RR) for mortality reductions in patients referred early were evident at three months (RR 0.61, 95% CI 0.55 to 0.67; I² = 84%) and remained at five years (RR 0.66, 95% CI 0.60 to 0.71; I² = 87%). Initial hospitalisation was 9.12 days shorter with early referral (95% CI -10.92 to -7.32 days; I² = 82%) compared to late referral. Pooled analysis showed patients referred early were more likely than late referrals to initiate RRT with peritoneal dialysis (RR 1.74, 95% CI 1.64 to 1.84; I² = 92%).Patients referred early were less likely to receive temporary vascular access (RR 0.47, 95% CL 0.45 to 0.50; I² = 97%) than those referred late. Patients referred early were more likely to receive permanent vascular access (RR 3.22, 95% CI 2.92 to 3.55; I² = 97%). Systolic blood pressure (BP) was significantly lower in early versus late referrals (MD -3.09 mm Hg, 95% CI -5.23 to -0.95; I² = 85%); diastolic BP was significantly lower in early versus late referrals (MD -1.64 mm Hg, 95% CI -2.77 to -0.51; I² = 82%). EPO use was significantly higher in those referred early (RR 2.92, 95% CI 2.42 to 3.52; I² = 0%). eGFR was higher in early referrals (MD 0.42 mL/min/1.73 m², 95% CI 0.28 to 0.56; I² = 95%). Diabetes prevalence was similar in patients referred early and late (RR 1.05, 95% CI 0.96 to 1.15; I² = 87%) as was ischaemic heart disease (RR 1.05, 95% CI 0.97 to 1.13; I² = 74%), peripheral vascular disease (RR 0.99, 95% CI 0.84 to 1.17; I² = 90%), and congestive heart failure (RR 1.00, 95% CI 0.86 to 1.15; I² = 92%). Inability to walk was less prevalent in early referrals (RR 0.66, 95% CI 0.51 to 0.86). Prevalence of chronic obstructive pulmonary disease was similar in those referred early and late (RR 0.89, 95% CI 0.70 to 1.14; I² = 94%) as was cerebrovascular disease (RR 0.90, 95% CI 0.74 to 1.11; I² = 83%).The quality of the included studies was assessed as being low to moderate based on the Newcastle-Ottawa Scale. Slight differences in the definition of early versus late referral infer some risk of bias. Generally, heterogeneity in most of the analyses was high.

CONCLUSIONS

Our analysis showed reduced mortality and mortality and hospitalisation, better uptake of peritoneal dialysis and earlier placement of arteriovenous fistulae for patients with chronic kidney disease who were referred early to a nephrologist. Differences in mortality and hospitalisation data between the two groups were not explained by differences in prevalence of comorbid disease or serum phosphate. However, early referral was associated with better preparation and placement of dialysis access.

BACKGROUND

Hypoxic-ischemic (HI) injury to the developing brain remains a major cause of morbidity. Hypothermia is effective but does not provide complete neuroprotection, prompting a search for adjunctive therapies. Erythropoietin (Epo) has been shown to be beneficial in several models of neonatal HI. This study examines combination hypothermia and treatment with erythropoietin in neonatal rat HI.

METHODS

Rats at postnatal day 7 were subjected to HI (Vannucci model) and randomized into four groups: no treatment, hypothermia alone, Epo alone, or hypothermia and Epo. Epo (1,000 U/kg) was administered in three doses: immediately following HI, and 24 h and 1 wk later. Hypothermia consisted of whole-body cooling for 8 h. At 2 and 6 wk following HI, sensorimotor function was assessed via cylinder-rearing test and brain damage by injury scoring. Sham-treated animals not subjected to HI were also studied.

RESULTS

Differences between experimental groups, except for Epo treatment on histopathological outcome in males, were not statistically significant, and combined therapy had no adverse effects.

CONCLUSIONS

No significant benefit was observed from treatment with either hypothermia or combination therapy. Future studies may require older animals, a wider range of functional assays, and postinsult assessment of injury severity to identify only moderately damaged animals for targeted therapy.

Thrombopoietin (TPO), originally described as an activity in the serum of thrombocytopenic animals that leads to increased production of platelets, has recently been isolated and cloned. Its closest relative in the cytokine superfamily, erythropoietin (EPO), is transcriptionally regulated during anemia, and it was expected that TPO would similarly be regulated during thrombocytopenia. We induced thrombocytopenia in mice and confirmed that TPO activity was upregulated, as determined by a bioassay. Liver and kidney were found to be the major sources of TPO mRNA. Surprisingly, TPO mRNA in these tissues was not upregulated in thrombocytopenic mice. Using a sensitive RNase protection assay that can distinguish between TPO isoforms, we found no change in the profile of mRNA for these isoforms. A semiquantitative reverse transcription-polymerase chain reaction assay also did not demonstrate upregulation of TPO mRNA in the spleen. Thus, the increase of TPO activity during thrombocytopenia is not caused by regulation at the level of TPO mRNA. Furthermore, isolated mouse platelets absorbed high amounts of bioactive TPO out of TPO-conditioned medium in a dose-dependent fashion. Our results are consistent with TPO protein being regulated at a posttranscriptional level and/or directly through absorption and metabolism by platelets.

Two forms of erythropoietin, EPO-bi and EPO-tetra, with different biological activities were isolated from the culture medium of a recombinant Chinese hamster ovary cell line, B8-300, into which the human erythropoietin gene had been introduced. EPO-bi, an unusual form, showed only one-seventh the in vivo activity and 3 times higher in vitro activity of the previously described recombinant human EPO (standard EPO). In contrast, EPO-tetra showed both in vivo and in vitro activities comparable to those of the standard EPO. EPO-bi, EPO-tetra, and the standard EPO had the same amino acid composition and immunoreactivity. However, structural analyses of their N-linked sugar chains revealed that EPO-bi contains the biantennary complex type as the major sugar chain, while EPO-tetra and the standard EPO contain the tetraantennary complex type as the major sugar chain. From examination of various preparations of recombinant human EPO, we found a positive correlation between the in vivo activity of EPO and the ratio of tetraantennary to biantennary oligosaccharides. These results suggest that higher branching of the N-linked sugar chains is essential for effective expression of in vivo biological activity of EPO.

Disturbed brain water homeostasis with swelling of astroglial cells is a common complication in stroke, trauma, and meningitis and is considered to be a major cause of permanent brain damage. Astroglial cells possess the water channel aquaporin 4 (AQP4). Recent studies from our laboratory have shown that glutamate, acting on group I metabotropic glutamate receptors (mGluRs), increases the permeability of astrocyte AQP4, which, in situations of hypoxia-ischemia, will increase astrocyte water uptake. Here we report that erythropoietin (EPO), which in recent years has emerged as a potent neuro-protective agent, antagonizes the effect of a group I mGluR agonist on astrocyte water permeability. Activation of group I mGluRs triggers fast and highly regular intracellular calcium oscillations and we show that EPO interferes with this signaling event by altering the frequency of the oscillations. These effects of EPO are immediate, in contrast to the neuroprotective effects of EPO that are known to depend upon gene activation. Our findings indicate that EPO may directly reduce the risk of astrocyte swelling in stroke and other brain insults. In support of this conclusion we found that EPO reduced the neurological symptoms in a mouse model of primary brain edema known to depend upon AQP4 water transport.

Sublethal hypoxic or ischemic events can improve the tolerance of tissues, organs, and even organisms from subsequent lethal injury caused by hypoxia or ischemia. This phenomenon has been termed hypoxic or ischemic preconditioning (HPC or IPC) and is well established in the heart and the brain. This review aims to discuss HPC and IPC with respect to their historical development and advancements in our understanding of the neurochemical basis for their neuroprotective role. Through decades of collaborative research and studies of HPC and IPC in other organ systems, our understanding of HPC and IPC-induced neuroprotection has expanded to include: early- (phosphorylation targets, transporter regulation, interfering RNA) and late- (regulation of genes like EPO, VEGF, and iNOS) phase changes, regulators of programmed cell death, members of metabolic pathways, receptor modulators, and many other novel targets. The rapid acceleration in our understanding of HPC and IPC will help facilitate transition into the clinical setting.

OBJECTIVE

To provide an overview of the current knowledge on neuroprotective properties of Erythropoietin (Epo), mechanisms by which Epo produces neuroprotection, and signaling pathways regulated by Epo in the nervous system.

METHODS

The Medline database was searched for articles on the neuroprotective properties of Epo. Experimental and clinical studies were systematically reviewed.

RESULTS

In addition to promoting the survival, proliferation, and differentiation of immature erythroid cells, Epo and the Epo receptor (EpoR) have recently been shown to exist and function in the nervous system. The Epo/EpoR system plays a critical role in neurodevelopment and neuroprotection. Epo ameliorates or prevents neuronal injury by neuroprotective, anti-apoptotic, anti-inflammatory, anti-oxidant, angiogenic, neurogenic and neurotrophic effects in cell culture and animal models of neurological diseases. The clinical effectiveness of recombinant human Epo in ischemic stroke in human patients has also been reported recently.

CONCLUSIONS

Recent studies suggest that Epo is a potential novel neurotherapeutic agent and further clinical studies are warranted.

Mice immunized with irradiated Onchocerca volvulus third-stage larvae developed protective immunity. Eosinophil levels were elevated in the parasite microenvironment at the time of larval killing, and measurements of total serum antibody levels revealed an increase in the immunoglobulin E (IgE) level in immunized mice. The goal of the present study was to identify the role of granulocytes and antibodies in the protective immune response to the larval stages of O. volvulus in mice immunized with irradiated larvae. Immunity did not develop in mice if granulocytes, including both neutrophils and eosinophils, were eliminated, nor did it develop if only eosinophils were eliminated. Moreover, larvae were killed in naïve interleukin-5 transgenic mice, and the killing coincided with an increase in the number of eosinophils and the eosinophil peroxidase (EPO) level in the animals. To determine if EPO was required for protective immunity, mice that were genetically deficient in EPO were immunized, and there were no differences in the rates of parasite recovery in EPO-deficient mice and wild-type mice. Two mouse strains were used to study B-cell function; micro MT mice lacked all mature B cells, and Xid mice had deficiencies in the B-1 cell population. Immunity did not develop in the micro MT mice but did develop in the Xid mice. Finally, protective immunity was abolished in mice treated to eliminate IgE from the blood. We therefore concluded that IgE and eosinophils are required for adaptive protective immunity to larval O. volvulus in mice.

Here we investigate the effects of erythropoietin (EPO) on the tissue/organ injury caused by hemorrhagic shock (HS), endotoxic shock, and regional myocardial ischemia and reperfusion in anesthetized rats. Male Wistar rats were anesthetized with thiopental sodium (85 mg/kg i.p.) and subjected to hemorrhagic shock (HS; i.e., mean arterial blood pressure reduced to 45 mmHg for 90 min, followed by resuscitation with shed blood for 4 h), endotoxemia (for 6 h), or left anterior descending coronary artery occlusion (25 min) and reperfusion (2 h). HS and endotoxemia resulted in renal dysfunction and liver injury. Administration of EPO (300 IU/kg i.v., n = 10) before resuscitation abolished the renal dysfunction and liver injury in hemorrhagic, but not endotoxic, shock. HS also resulted in significant increases in the kidney of the activities of caspases 3, 8, and 9. This increase in caspase activity was not seen in HS rats treated with EPO. In cultured human proximal tubule cells, EPO concentration-dependently reduced the cell death and increase in caspase-3 activity caused by either ATP depletion (simulated ischemia) or hydrogen peroxide (oxidative stress). In the heart, administration of EPO (300 IU/kg i.v., n = 10) before reperfusion also caused a significant reduction in infarct size. In cultured rat cardiac myoblasts (H9C2 cells), EPO also reduced the increase in DNA fragmentation caused by either serum deprivation (simulated ischemia) or hydrogen peroxide (oxidative stress). We propose that the acute administration of EPO on reperfusion and/or resuscitation will reduce the tissue injury caused by ischemia-reperfusion of the heart (and other organs) and hemorrhagic shock.

Since its discovery in early 1990s, hypoxia inducible factor 1 (HIF-1) has been increasingly recognized for its key role in transcriptional control of more than a hundred genes that regulate a wide-spectrum of cellular functional events, including angiogenesis, vasomotor control, glucose and energy metabolism, erythropoiesis, iron homeostasis, pH regulation, cell proliferation and viability. Evidence accumulated during the past 7 years suggests a critical role for HIF-1alpha in mediating cardioprotection. The purpose of our present article is to provide an updated overview on this important regulator of gene expression in the cellular stress-responsive and adaptive process. We have particularly emphasized the involvement of HIF-1 in the induction of cardioprotective molecules, such as inducible nitric oxide synthase (iNOS), hemeoxygenase 1 (HO-1), and erythropoietin (EPO), which in turn alleviate myocardial damages caused by harmful events such as ischemia-reperfusion injury. Despite these advances, further in-depth studies are needed to elucidate the possible coordination or interaction between HIF-1alpha and other key transcription factors in regulating protein expression that leads to cardioprotection.

OBJECTIVE

The purpose of this study was to evaluate erythropoietin (Epo) and Epo receptor (EpoR) expression in the retina and in vitreous fluid from diabetic and nondiabetic donors. To gain insight into the mechanisms responsible for the regulation of Epo production in the retina, we also assessed retinal expression of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha).

METHODS

Eighteen postmortem eyes from 9 diabetic patients without clinically detectable retinopathy were compared with 18 eyes from 9 nondiabetic donors. mRNA of Epo, HIF-1alpha, and HIF-2alpha (quantitative RT-PCR) were measured separately in neuroretina and retinal pigment epithelium (RPE). Epo and EpoR were assessed in the retina (immunofluorescence by confocal laser microscopy) and in the vitreous fluid (radioimmunoassay and enzyme-linked immunosorbent assay, respectively).

RESULTS

Epo and EpoR mRNAs were significantly higher in the RPE than in the neuroretina. Higher expression of Epo was detected in the retinas (both in the RPE and in the neuroretina) from diabetic donors. By contrast, EpoR expression was similar in both groups. We did not find any difference in HIF-1alpha and HIF-2alpha mRNA expression between diabetic and nondiabetic donors (both in RPE and neuroretina). Intravitreal Epo concentration was higher in diabetic donors than in nondiabetic control subjects. However, EpoR concentrations were similar in both groups.

CONCLUSIONS

Epo overexpression is an early event in the retina of diabetic patients, and this is not associated with any change in EpoR. At this early stage, other factors apart from hypoxia seem to be more important in accounting for the Epo upregulation that exists in the diabetic retina.

Apart from its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions in brain, retina and heart upon ischaemic injury. However, the physiological non-erythroid functions of Epo remain unclear. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate Epo's impact on ventilation upon hypoxic exposure. Tg21 mice showed improved ventilatory response to severe acute hypoxia and moreover improved ventilatory acclimatization to chronic hypoxic exposure. Furthermore, following bilateral transection of carotid sinus nerves that uncouples the brain from the carotid body, Tg21 mice adapted their ventilation to acute severe hypoxia while chemodenervated wild-type (WT) animals developed a life-threatening apnoea. These results imply that Epo in brain modulates ventilation. Additional analysis revealed that the Epo receptor (EpoR) is expressed in the main brainstem respiratory centres and suggested that Epo stimulates breathing control by alteration of catecholaminergic metabolism in brainstem. The modulation of hypoxic pattern of ventilation after i.v. injection of recombinant human Epo in WT mice and the dense EpoR immunosignal observed in carotid bodies showed that these chemoreceptors are sensitive to plasma levels of Epo. In summary, our results suggest that Epo controls ventilation at the central (brainstem) and peripheral (carotid body) levels. These novel findings are relevant to understanding better respiratory disorders including those occurring at high altitude.

The European Molecular Biology Laboratory (EMBL) Nucleotide Sequence Database (http://www.ebi.ac. uk/embl/index.html ) is maintained at the European Bioinformatics Institute (EBI) in an international collaboration with the DNA Data Bank of Japan (DDBJ) and GenBank (USA). Data is exchanged amongst the collaborative databases on a daily basis. The major contributors to the EMBL database are individual authors and genome project groups. WEBIN is the preferred web-based submission system for individual submitters, whilst automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO). Database releases are produced quarterly. Network services allow free access to the most up-to-date data collection via Internet and WWW interfaces. EBI's Sequence Retrieval System (SRS) is a network browser for databanks in molecular biology, integrating and linking the main nucleotide and protein databases plus many specialised databases. For sequence similarity searching a variety of tools (e.g., BLITZ, FASTA, BLAST) are available which allow external users to compare their own sequences against the most currently available data in the EMBL Nucleotide Sequence Database and SWISS-PROT.

OBJECTIVE

To determine expression, cellular distribution, and regulation of hemoglobin (Hb) in normal and glaucomatous tissues.

METHODS

Proteomic analysis of Hb expression was conducted on protein samples from ocular hypertensive and control rat eyes and human donor eyes with or without glaucoma. Proteomic findings were validated by quantitative (q)RT-PCR, Western blot analysis, immunohistochemistry, and the analysis of new Hb synthesis in culture. Hypoxic regulation of Hb expression was also studied in primary cultures of rat RGCs and macroglia and after transfer of the glia-conditioned medium to RGCs. The role of erythropoietin (EPO) signaling in Hb induction and cell survival was determined by applying recombinant (r)EPO treatment and performing EPO neutralization experiments by using soluble EPO receptor treatment of hypoxic cultures.

RESULTS

In vivo findings revealed Hb expression in the retina and optic nerve head macroglia and RGCs, suggesting an approximately two-fold upregulation in ocular hypertensive rat eyes and glaucomatous human donor eyes relative to the control eyes. In vitro findings collectively supported that hypoxia boosts glial Hb expression through hypoxia-inducible EPO signaling in an autocrine manner. Based on passive transfer experiments, hypoxia-induced production of glial EPO was also found to upregulate Hb expression in RGCs in a paracrine manner, thereby increasing the hypoxic survival of these neurons.

CONCLUSIONS

Findings of this study provide new insights into tissue oxygen transport in the inner retina and optic nerve head through the regulated expression of Hb in macroglia and RGCs. Upregulation of Hb expression appears to be an intrinsic protective mechanism to facilitate cellular oxygenation and may also provide free radical scavenging.

Erythropoietin (Epo) and its cognate receptor (EpoR) are required for maintaining adequate levels of circulating erythrocytes during embryogenesis and adulthood. Here, we report the functional characterization of the zebrafish epo and epor genes. The expression of epo and epor was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, revealing marked parallels between zebrafish and mammalian gene expression patterns. Examination of the hypochromic mutant, weissherbst, and adult hypoxia-treated hearts indicate that zebrafish epo expression is induced by anemia and hypoxia. Overexpression of epo mRNA resulted in severe polycythemia, characterized by a striking increase in the number of cells expressing scl, c-myb, gata1, ikaros, epor, and betae1-globin, suggesting that both the erythroid progenitor and mature erythrocyte compartments respond to epo. Morpholino-mediated knockdown of the epor caused a slight decrease in primitive and complete block of definitive erythropoiesis. Abrogation of STAT5 blocked the erythropoietic expansion by epo mRNA, consistent with a requirement for STAT5 in epo signaling. Together, the characterization of zebrafish epo and epor demonstrates the conservation of an ancient program that ensures proper red blood cell numbers during normal homeostasis and under hypoxic conditions.