In humans, studies of the erythroid cell lineage are hampered by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, these progenitors in bone marrow or peripheral blood are scarce and no specific antibodies are available. We describe a new method which allows proliferation in liquid culture of large numbers of pure normal human erythroid progenitors. CD34+ cells were cultured for 7 d in serum-free conditions with the cytokine mixture interleukin (IL)-3/IL-6/stem cell factor (SCF). This resulted in cell expansion and the appearance of a high proportion of CD36+ cells which were purified on day 7. Methylcellulose clones from these cells were composed of 96.6% late BFU-E and 3.4% CFU-GM. These CD36+ cells could be recultured with the same cytokine mixture plus or minus erythropoietin (Epo) for a further 2-7 d. In both conditions further amplification of CD36+ cells was observed, but Epo induced a more dramatic cell expansion. Glycophorin-positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 d of secondary culture. Cells obtained from adult CD34+ progenitors mostly contained adult haemoglobin, whereas cord blood-derived cells contained equal proportions of adult and fetal haemoglobin. Activation of STAT5 and tyrosine phosphorylation of the Epo receptor and JAK2 were observed after Epo stimulation of these cells. This new method represents a straightforward alternative to the procedures previously described for the purification of normal erythroid progenitors and is useful in the study of erythropoietic regulation.

The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.

Eosinophilic granulocytes are found in tissues with an interface with the external environment, such as the gastrointestinal, genitourinary and respiratory tracts. These leucocytes have been associated with tissue damage in a variety of diseases. The aim of this study was to evaluate whether necrotic epithelial cells can activate eosinophils. The danger theory postulates that cells of the innate immune system primarily recognize substances that signal danger to the host. We damaged epithelial cell lines derived from the genital (HeLa cells), respiratory (HEp-2 cells) and intestinal tracts (HT29 cells) and assessed their capacity to cause eosinophilic migration, release of putative tissue-damaging factors, such as eosinophil peroxidase (EPO) and eosinophil cationic protein (ECP), as well as secretion of tissue-healing factors, e.g. fibroblast growth factors (FGF)-1 and -2 and transforming growth factor (TGF)-beta1. We found that necrotic intestinal cells induced chemotaxis in human eosinophils. EPO release was elicited in eosinophils stimulated with necrotic cells derived from all cell lines, as well as from viable HEp-2 and HT29 cells. Release of ECP was only seen in eosinophils incubated with necrotic intestinal or genital cells, not viable ones. Both necrotic intestinal and genital cells elicited FGF-2 secretion from eosinophils. TGF-beta1 was released from eosinophils exposed to viable and necrotic HT29 cells. These findings indicate that eosinophils are able to recognize and be activated by danger signals released from damaged epithelial cells, which may be of importance in understanding the role of eosinophils in the various inflammatory conditions in which they are involved.

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The Epo receptor (EpoR) belongs to the cytokine receptor family and lacks a tyrosine kinase domain. However, Epo induces tyrosine phosphorylation of cellular substrates including the EpoR. To explore the functional significance of receptor tyrosine phosphorylation, we examined the possible interaction of the receptor with the 85-kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3-kinase. After Epo stimulation, p85 was found to associate with the tyrosine-phosphorylated 72-kDa form of EpoR as well as a 92-kDa phosphotyrosyl protein, and PI 3-kinase activity was detectable in anti-EpoR immunoprecipitates. Anti-EpoR blotting of anti-p85 immunoprecipitates revealed that p85 binds specifically to the 72-kDa form of the EpoR and not to unphosphorylated 66- and 64-kDa forms. Association of p85 with the EpoR was Epo dose- and time-dependent and correlated with tyrosine phosphorylation of the receptor. Consistent with a role for tyrosine phosphorylation of the EpoR, PI 3-kinase did not associate with a mitogenically active receptor mutant that lacked tyrosine-phosphorylation sites in the carboxyl-terminal region. A recombinant fusion protein containing the carboxyl-terminal SH-2 domain of p85 was shown to bind to tyrosine-phosphorylated EpoR in vitro. Taken together, these results indicate that, following Epo stimulation, the EpoR recruits PI 3-kinase to the cell membrane by binding between the carboxyl-terminal SH-2 domain of p85 and the tyrosine-phosphorylated carboxyl-terminal region of the receptor. The association with PI 3-kinase is, however, not required for the growth signal transduction from the EpoR.

Safety concerns surrounding the use of recombinant human erythropoietin (Epo) to treat anemia in cancer patients were raised after 2 recent clinical studies reported a worse survival outcome in patients who received epoetin alpha or epoetin beta compared with patients who received placebo. Although those findings contrasted with previous clinical studies, which demonstrated no difference in survival for cancer patients who received erythropoiesis-stimulating agents (ESAs), some investigators have suggested a potential role for ESAs in promoting tumor growth through 1) stimulation of Epo receptors (EpoR) expressed in tumors, 2) stimulation and formation of tumor vessels, and/or 3) enhanced tumor oxygenation. The first and second hypotheses appeared to be supported by some EpoR expression and ESA in vitro studies. However, these conclusions have been challenged because of poor specificity of EpoR-detection methodologies, conflicting data from different groups, and the lack of correlation between in vitro data and in vivo findings in animal tumor models. For this report, the authors reviewed the biology of EpoR in erythropoiesis and compared and contrasted the reported findings on the role of ESAs and EpoR in tumors.

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The phosphorylation of proteins at tyrosine residues is critical in the growth signaling induced by Epo. This mechanism is regulated by the activities of both protein-tyrosine kinases and protein tyrosine phosphatases. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase, widely expressed in all tissues in mammals became phosphorylated on tyrosine after stimulation with Epo in M07ER cells engineered to express high levels of human EpoR. Syp was complexed with Grb2 in Epo-stimulated M07ER cells. Direct binding between Syp and Grb2 was also observed in vitro. Furthermore, Syp appeared to bind directly to tyrosine-phosphorylated EpoR in M07ER cells. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the tyrosine-phosphorylated EpoR in vitro. These results suggest that Syp may be an important signaling component downstream of the EpoR and may regulate the proliferation and differentiation of hematopoietic cells.

Erythropoietin (EPO) is the main hormone that promotes proliferation and differentiation of erythroid progenitor cells via binding to its surface receptor (EPO-R). Recent studies suggest that this hormone may affect also other cell types, besides the red blood cell lineage. We have previously demonstrated that the immune system is a target of EPO; however, the direct target cells of EPO, as well as the molecular mechanisms underlying its role as an immunomodulator, are unknown. Here we present evidence for functional effects of EPO on dendritic cells (DCs), which are known to initiate the immune response. In-vivo experiments in EPO-injected mice and in transgenic mice over-expressing human EPO showed an increased splenic DC population with a higher cell surface expression of CD80 and CD86. Further analysis based on mouse models, showed that DCs derived in-vitro from bone marrow (BM-DCs) express EPO-R mRNA. In-vitro stimulation of these DCs with recombinant human EPO enhanced viability, upregulated CD80, CD86 and MHC class II and augmented the secretion of IL-12. Biochemical analysis of EPO mediated signaling in the BM-DCs showed activation of the AKT, MAPK and NF-kappaB pathways. EPO stimulation of the BM-DCs led to Tyr-phosphorylation of STAT3. The inability to detect EPO mediated activation of STAT5 in the BM-DCs, suggests that in DCs, STAT3 may play a more important role than STAT5 in EPO-R signaling. Taken together, our data support the premise that DCs are direct targets of EPO, thereby providing an insight to the immunomodulatory functions of EPO.

OBJECTIVE

To test the possible neuroprotective effect of early high-dose erythropoietin-alpha (Epo-alpha) after out-of-hospital cardiac arrest (OHCA).

METHODS

A matched control study. Following resuscitation with mild hypothermia after OHCA, participants received a first dose of Epo-alpha followed by four additional injections within 48 h (40,000 IU intravenously each injection). Plasma Epo-alpha levels were measured at different time points. Outcome and adverse events were assessed up to day 28 and were compared with those of matched-paired controls.

RESULTS

In all 18 participants received Epo-alpha and were compared with 40 matched controls. Pharmacokinetic variables were similar to those previously reported for healthy people or for persons treated with usual dosages of Epo. At day 28, survival rates among the Epo-treated group and the controls (55% versus 47.5%, p=0.17) and rates of full neurological recovery (55% versus 37.5%) did not differ significantly. Incidences of thrombocytosis in the Epo-treated group and controls were 15% and 5%, respectively; an arterial vascular thrombosis was observed in one case (5%) from the Epo-treated cohort.

CONCLUSIONS

Among victims of OHCA treated with Epo-alpha and hypothermia, we observed a high survival rate, with no minor cerebral sequels but potential haematological side effects. Future studies of Epo should pay particular attention to these findings.

The presence and potential physiological role of the erythropoietin receptor (Epo-R) were examined in human skeletal muscle. In this study we demonstrate that Epo-R is present in the endothelium, smooth muscle cells, and in fractions of the sarcolemma of skeletal muscle fibers. To study the potential effects of Epo in human skeletal muscle, two separate studies were conducted: one to study the acute effects of a single Epo injection on skeletal muscle gene expression and plasma hormones and another to study the effects of long-term (14 wk) Epo treatment on skeletal muscle structure. Subjects (n = 11) received a single Epo injection of 15,000 IU (double blinded, cross over, placebo). A single Epo injection reduced myoglobin and increased transferrin receptor and MRF-4 mRNA content within 10 h after injection. Plasma hormones remained unaltered. Capillarization and fiber hypertrophy was studied in subjects (n = 8) who received long-term Epo administration, and muscle biopsies were obtained before and after. Epo treatment did not alter mean fiber area (0.84 +/- 0.2 vs. 0.72 +/- 0.3 mm(2)), capillaries per fiber (4.3 +/- 0.5 vs. 4.4 +/- 1.3), or number of proliferating endothelial cells. In conclusion, the Epo-R is present in the vasculature and myocytes in human skeletal muscle, suggesting a role in both cell types. In accordance, a single injection of Epo regulates myoglobin, MRF-4, and transferrin receptor mRNA levels. However, in contrast to our hypothesis, prolonged Epo administration had no apparent effect on capillarization or muscle fiber hypertrophy.

The proliferation and differentiation of erythroid cells is a highly regulated process that is controlled primarily at the level of interaction of erythropoietin (Epo) with its specific cell surface receptor (EpoR). However, this process is deregulated in mice infected with the Friend spleen focus-forming virus (SFFV). Unlike normal erythroid cells, erythroid cells from SFFV-infected mice are able to proliferate and differentiate in the absence of Epo, resulting in erythroid hyperplasia and leukemia. Over the past 20 years, studies have been carried out to identify the viral genes responsible for the pathogenicity of SFFV and to understand how expression of these genes leads to the deregulation of erythropoiesis in infected animals. The studies have revealed that SFFV encodes a unique envelope glycoprotein which interacts specifically with the EpoR at the cell surface, resulting in activation of the receptor and subsequent activation of erythroid signal transduction pathways. This leads to the proliferation and differentiation of erythroid precursor cells in the absence of Epo. Although the precise mechanism by which the viral protein activates the EpoR is not yet known, it has been proposed that it causes dimerization of the receptor, resulting in constitutive activation of Epo signal transduction pathways. While interaction of the SFFV envelope glycoprotein with the EpoR leads to Epo-independent erythroid hyperplasia, this is not sufficient to transform these cells. Transformation requires the viral activation of the cellular gene Sfpi-1, whose product is thought to block erythroid cell differentiation. By understanding how SFFV can deregulate erythropoiesis, we may gain insights into the causes and treatment of related diseases in man.

Erythropoietin (EPO) produced by the fetal liver and adult kidney is an essential stimulator of erythropoiesis. EPO production is regulated through hypoxic activation of gene transcription and possibly hypoxia-induced stabilization of its mRNA. In the liver of early embryos in which EPO production poorly responds to hypoxia, retinoic acid may be an important stimulator. In this decade, new sites of EPO production have been found: central nervous system and reproductive organs. These tissues have a paracrine and/or autocrine system of EPO, which is independent of the endocrine system (kidney/bone marrow) in adult erythropoiesis. In the central nervous system, astrocytes are the main producers of EPO, while EPO receptor is expressed in neurons. EPO protects neurons from a various types of damage. The uterine EPO is likely involved in the estrogen-dependent angiogenesis of the endometrial layer. The possible functions of EPO in other tissues and tissue-characteristic regulation of EPO production are also discussed in this review.

Erythropoietin (Epo) is a hydrophobic sialoglycoproteic hormone produced by the kidney and responsible for the proliferation, maturation, and differentiation of the precursors of the erythroid cell line. Human recombinant erythropoietin (rHuEpo) is used to treat different types of anemia, not only in uremic patients but also in newborns with anemia of prematurity, in patients with cancer-related anemia or myeloproliferative disease, thalassemias, bone marrow transplants, or those with chronic infectious diseases. The pleiotropic functions of Epo are well known. It has been shown that this hormone can modulate the inflammatory and immune response, has direct hemodynamic and vasoactive effects, could be considered a proangiogenic factor because of its interaction with vascular endothelial growth factor, and its ability to stimulate mitosis and motility of endothelial cells. The multifunctional role of Epo has further been confirmed by the discovery in the central nervous system of a specific Epo/Epo receptor (EpoR) system. Both Epo and EpoR are expressed by astrocytes and neurons and Epo is present in the cerebrospinal fluid (CSF). Therefore, novel functions of Epo, tissue-specific regulation, and the mechanisms of action have been investigated. In this review we have tried to summarize the current data on the role of Epo on brain function. We discuss the different sites of cerebral expression and mechanisms of regulation of Epo and its receptor and its role in the development and maturation of the brain. Second, we discuss the neurotrophic and neuroprotective function of Epo in different conditions of neuronal damage, such as hypoxia, cerebral ischemia, and subarachnoid hemorrhage, and the consequent possibility that rHuEpo therapy could soon be used in clinical practice to limit neuronal damage induced by these diseases.

OBJECTIVE

Perinatal asphyxia, intraventricular hemorrhage and stroke are common causes of neonatal brain injury, with hypoxia-ischemia as the final common pathway of injury. Erythropoietin (Epo) has potential to lessen neurologic sequelae due to hypoxia-ischemia. The purpose of this review is to highlight new clinical trials and experimental evidence that expand our understanding of Epo as a potential treatment for perinatal brain injury.

RESULTS

Several trials of Epo treatment are reviewed: two phase I/II trials of high-dose Epo given to preterm infants established pharmacokinetic and safety profiles, and a trial of Epo treatment for term infants with moderate hypoxic-ischemic encephalopathy found reduced disability. Potential risks and benefits of high-dose Epo are discussed. New evidence related to Epo receptor expression, signal transduction pathways, and mechanisms of neuroprotection are reviewed.

CONCLUSIONS

Cautious optimism is warranted regarding the use of high-dose Epo as a treatment option for neonatal brain injury. To date, Epo has been well tolerated to use in neonatal populations and now studies of neuroprotective efficacy are underway.

Recently, erythropoietin was shown to have both hematopoietic as well as tissue-protective properties. Erythropoietin (EPO) had a protective effect in animal models of cerebral ischemia, mechanical trauma of the nervous system, myocardial infarction, and ischemia-reperfusion (I/R) injury of the kidney. It is not known whether EPO protects the liver against I/R injury. Using a rat model of liver I/R injury, we aimed to determine the effect of the administration of human recombinant erythropoietin (rhEPO) on liver injury. Rats were subjected to 30 min of liver ischemia followed by 2 h of reperfusion. When compared with the sham-operated rats, I/R resulted in significant rises in the serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, gamma-glutamyl transferase, tissue lipid peroxidation, caspase-3 activity and altered histology. Administration of rhEPO 5 min before ischemia was able to reduce the biochemical evidence of liver injury; however, this protection was not evident when rhEPO was administered 5 min before reperfusion. Mechanistically, early administration of rhEPO was able to reduce the oxidative stress and caspase-3 activation, suggesting the subsequent reduction of apoptosis. This study provides the first evidence that rhEPO causes a substantial reduction of the liver injury induced by I/R in the rat.

OBJECTIVE

Patients with diabetes commonly have a greater degree of anemia for their level of renal impairment than those presenting with other causes of renal failure. To clarify the contribution and differing roles of diabetes and nephropathy in the development of anemia in diabetic patients, we examined the hematologic and hematinic parameters of diabetic patients without nephropathy.

METHODS

The study group was comprised of 62 patients with type 2 diabetes who had been followed for a median of 7 years. For the study, these patients had additional samples taken during their annual routine blood testing for the measurement of extra parameters, including serum ferritin, serum erythropoietin (Epo) levels, and the percentage of reticulocytes. These measurements were combined with the routine parameters Hb, hematocrit, HbA(1c), and glomerular filtration rate.

RESULTS

In all, 8 of the 45 male patients (17.8%) and 2 of the 17 female patients (11.8%) were classified as anemic (Hb <13g/dl and <11.5g/dl, respectively). Although only a small number of the patients had anemia as defined by normal values, a retrospective analysis of individual patients over time revealed a sustained though small decrease in Hb from initial presentation. A statistically significant difference in Epo levels (P = 0.016 by Kruskal-Wallis test) was observed from the group with the lowest (Hb < or =11.5) to that with the highest (Hb>> or =14.5) Hb values, with a median Epo value of 37 (interquartile range 24-42) vs. 13 (9-15) IU/l, respectively. In contrast, there was no evidence of an increased reticulocyte response to higher levels of Epo (r = 0.134 [Pearsons], P = 0.36). Reticulocyte counts ranged from 44 (38-57) to 76.5 (56-83) in the lowest and highest Hb groups, respectively.

CONCLUSIONS

Although only a small number of subjects in the group were overtly anemic, all subjects had an ongoing, small but significant decrease in Hb since presentation. This study of diabetic patients without nephropathy shows an expected increase in Epo production in response to lowering levels of Hb but without the expected reticulocyte response.

We examined the cardiac effects of chronic erythropoietin (EPO) therapy initiated 7 days after myocardial infarction (MI) in rats. A single high dose of EPO has been shown to reduce infarct size by preventing apoptosis when injected immediately after myocardial ischemia. The proangiogenic potential of EPO has also been reported, but the effects of chronic treatment with standard doses after MI are unknown. In this study, rats underwent coronary occlusion followed by reperfusion or a sham procedure. Infarcted rats were assigned to one of three treatment groups: 1) 0.75 microg/kg darbepoetin (MI+darb 0.75, n = 12); 2) 1.5 microg/kg darbepoetin (MI+darb 1.5, n = 12); 3) vehicle (MI+PBS, n = 16), once a week from day 7 postsurgery. Sham rats received the vehicle alone (n = 10). After 8 wk of treatment, the animals underwent echocardiography, left ventricular pressure-volume measurements, and peripheral blood endothelial progenitor cell (EPC) counting. MI size and capillary density in the border zone and the area at risk (AAR) were measured postmortem. The AAR was similar in the three MI groups. Compared with MI+PBS, the MI+darb 1.5 group showed a reduction in the MI-to-AAR ratio (20.8% vs. 38.7%; P < 0.05), as well as significantly reduced left ventricle dilatation and improved cardiac function. This reduction in post-MI remodeling was accompanied by increased capillary density (P < 0.05) and by a higher number of EPC (P < 0.05). Both darbepoetin doses increased the hematocrit, whereas MI+darb 0.75 did not increase EPC numbers or capillary density and had no functional effect. We found that chronic EPO treatment reduces MI size and improves cardiac function only at a dose that induces EPC mobilization in blood and that increases capillary density in the infarct border zone.

BACKGROUND

In selected samples, a considerable number of patients at clinical high risk of psychosis (CHR) are found to meet criteria for co-morbid clinical psychiatric disorders. It is not known how clinical diagnoses correspond to or even predict transitions to psychosis (TTP). Our aim was to examine distributions of life-time and current Axis I diagnoses, and their association with TTP in CHR patients.

METHODS

In the EPOS (European Prediction of Psychosis Study) project, six European outpatient centres in four countries examined 245 young help-seeking patients, who fulfilled the inclusion criteria for clinical risk of psychosis according to the Structured Interview for Prodromal Syndromes (SIPS 3.0) or the Bonn Scale for the Assessment of Basic Symptoms - Prediction List basic symptoms (BASBS-P). Patients who had experienced a psychotic episode lasting more than one week were excluded. Baseline and life-time diagnoses were assessed by the Structured Clinical Interview for DSM-IV (SCID-I). TTP was defined by continuation of BLIPS for more than seven days and predicted in Cox-regression analysis.

RESULTS

Altogether, 71% of the CHR patients had one or more life-time and 62% one or more current SCID-I diagnosis; about a half in each category received a diagnosis of life-time depressive and anxiety disorder. Currently, 34% suffered from depressive and 39% from anxiety disorder. Four percent received a current SCID diagnosis of bipolar, and 6.5% of somatoform disorder. During follow-up, 37 (15.1%) patients had developed full-blown psychosis. In bivariate analyses, current non-psychotic bipolar disorder associated significantly with TTP. In multivariate analyses, current bipolar disorder, somatoform and unipolar depressive disorders associated positively, and anxiety disorders negatively, with TTP.

CONCLUSIONS

Both life-time and current mood and anxiety disorders are highly prevalent among clinical help-seeking CHR patients and need to be carefully evaluated. Among CHR patients, occurrence of bipolar, somatoform and depressive disorders seems to predict TTP, while occurrence of anxiety disorder may predict non-transition to psychosis.

Growth factor independence-1B (Gfi-1B) is a transcription factor with a highly conserved transcriptional repressor snail-Gfi-1 (SNAG) domain and 6 zinc-finger domains at the N- and C-terminus, respectively. Disruption of the Gfi-1B gene is lethal in the embryo with failure to produce definitive enucleated erythrocytes. In this study, we analyzed the role of Gfi-1B in human erythropoiesis. We observed an increase of Gfi-1B expression during erythroid maturation of human primary progenitor cells. We studied the consequences of variations in Gfi-1B expression in 2 transformed cell lines (K562 and UT7 cells), as well as in primary CD36(+)/GPA(-) progenitors. A knock-down of Gfi-1B delayed the terminal differentiation of K562 and primary cells. Forced expression of Gfi-1B in UT7 and K562 cells led to an arrest of proliferation and an induction of erythroid differentiation. Enforced expression of Gfi-1B in primary cells at the colony-forming units-erythroid (CFU-E) stage led to a partial glycophorin A (GPA) induction after erythropoietin (EPO) withdrawal but failed to protect cells from apoptosis. Deletion of the SNAG repressor domain abolished Gfi-1B-induced erythroid maturation, strongly suggesting that Gfi-1B acts in the late stage of erythroid differentiation as a transcriptional repressor.

OBJECTIVE

To determine the incidence of catheter-related infection in patients with pulmonary arterial hypertension (PAH) receiving epoprostenol (EPO), and to note an etiologic role for Micrococcus spp, which is rarely reported as a pathogen in the medical literature.

METHODS

Observational study.

METHODS

Two PAH specialty treatment centers, Harbor-UCLA Medical Center (Torrance, CA), and the College of Physicians and Surgeons, Columbia University (New York, NY).

METHODS

A total of 192 patients with PAH receiving continuous therapy with IV EPO.

METHODS

From 1987 to 2000, 192 patients with PAH received infusions of EPO via central venous catheter. Catheter care included regular dressing changes with dry gauze using a sterile procedure, without the use of flushes. Patients were asked to report on known infections and treatments, and symptoms. All infections were verified by a telephone call to the patient, care provider, and microbiology laboratory whenever possible.

RESULTS

There were 335,285 catheter days (mean +/- SD, 1,325 +/- 974 catheter days). There were 88 clinical catheter infections with 51 blood culture-positive infections, necessitating catheter removal in 38 instances. The following pathogens were isolated: Staphylococcus aureus (25); Micrococcus spp (14); mixed flora (3); coagulase-negative Staphylococcus spp (2); Corynebacterium spp (2); Serratia marcessens (1); Enterobacter spp (1); Pseudomonas aeruginosa (1); enterococci (1); and unidentified Gram-positive cocci (1). The catheter infection rate was 0.26 per 1,000 catheter days.

CONCLUSIONS

The use of long-term therapy with continuous EPO appears to be associated with a low incidence of catheter-related infections. Micrococcus spp were the second most common etiologic agent. Caregivers managing patients with PAH must be aware of the risk of catheter infection, as it may contribute to the morbidity and mortality associated with the use of EPO. When isolated, Micrococcus spp should not be viewed as a contaminant, but rather as a true pathogen that may require therapeutic intervention.

BACKGROUND

Hypoxia-inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease.

METHODS

ARPE-19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme-linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real-time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques.

RESULTS

HIF-1 alpha was readily produced by ARPE-19 cells under hypoxia, but HIF-2 alpha and HIF-3 alpha could not be detected even after HIF-1 alpha silencing. HIF-1 alpha protein levels showed an increasing trend for the first 24 h while HIF-1 alpha mRNA levels fluctuated during this time. After 36 h HIF-1 alpha protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF-1 alpha significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level.

CONCLUSIONS

HIF-1 alpha appears to be the main isoform of HIF functioning in ARPE-19 cells. Under hypoxia, HIF-1 alpha levels are likely self-regulated by a feedback loop that involves both transcriptional and post-translational mechanisms. VEGF production by human RPE cells is regulated by HIF-1 alpha. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.

Erythropoietin (EPO) is a cytokine produced by the kidney whose function is to stimulate red blood cell production in the bone marrow. Previously, it was shown that the affinity of EPO for its receptor, EPOR, is inversely related to the sialylation of EPO carbohydrate. To better understand the properties of EPO that modulate its receptor affinity, various glycoforms were analyzed using surface plasmon resonance. The system used has been well characterized and is based on previous reports employing an EPOR-Fc chimera captured on a Protein A surface. Using three variants of EPO containing different levels of sialylation, we determined that sialic acid decreased the association rate constant (k(on)) about 3-fold. Furthermore, glycosylated EPO had a 20-fold slower k(on) than nonglycosylated EPO, indicating that the core carbohydrate also negatively impacted k(on). The effect of electrostatic forces on EPO binding was studied by measuring binding kinetics in varying NaCl concentrations. Increasing NaCl concentration resulted in a slower k(on) while having little impact on k(off), suggesting that long-range electrostatic interactions are primarily important in determining the rate of association between EPO and EPOR. Furthermore, the glycosylation content (i.e., nonglycosylated vs glycosylated, sialylated vs desialylated) affected the overall sensitivities of k(on) to [NaCl], indicating that sialic acid and the glycan itself each impact the overall effect of these electrostatic forces.

Erythropoietin (EPO) regulates proliferation and differentiation of erythroid precursor cells into erythrocytes. The last decade has revealed non-renal sites of EPO production and extrahematopoietic expression of the EPO receptor, thus suggesting that EPO has pleiotropic functions. Here, we addressed the interplay between EPO/glucose metabolism/body weight by employing a panel of relevant experimental murine models. The models focused on situations of increased EPO levels, including EPO-injected C57BL/6 and BALB/c mice, as well as transgenic mice (tg6) constitutively overexpressing human EPO, thus exposed to constantly high EPO serum levels. As experimental models for diabetes and obesity, we employed protein Tyr phosphatase 1B (PTP1B) knockout mice associated with resistance to diabetes (PTP1B(-/-)), and ob/ob mice susceptible to diabetes and obesity. The data presented herein demonstrate EPO-mediated decrease in blood glucose levels in all mice models tested. Moreover, in the ob/ob mice, we observed EPO-mediated attenuation of body weight gain and reduction of hemoglobin A1c. Taken together, our data bear significant clinical implications of EPO treatment in the management of a wide range of metabolic diseases, thus adding an important novel therapeutic potential to this pleiotropic hormone.

Hypoxia can induce erythropoiesis through regulated increase of erythropoietin (Epo) production. We investigated the direct influence of oxygen tension (pO(2)) in the physiologic range (2-8%) on erythroid progenitor cell differentiation using cultures of adult human hematopoietic progenitor cells exposed to decreasing (20% to 2%) pO(2) and independent of variation in Epo levels. Decreases in hemoglobin (Hb)-containing cells were observed at the end of the culture period with decreasing pO(2). This is due, in part, to a reduction in cell growth and, at 2% O(2), a marked increase in cell toxicity. Analysis of the kinetics of cell differentiation showed an increase in the proportion of cells with glycophorin-A expression and Hb accumulation at physiologic pO(2). Cells were characterized by an early induction of gamma-globin expression and a delay and reduction in peak levels of beta-globin expression. Overall, fetal Hb and gamma-globin expression were increased at physiologic pO(2), but these increases were reduced at 2% O(2) as cultures become cytotoxic. At reduced pO(2), induction of Epo-receptor (Epo-R) by Epo was decreased and delayed, analogous to the delay in beta-globin induction. The oxygen-dependent reduction of Epo-R can account for the associated cytotoxicity at 2% O(2). Epo induction of erythroid transcription factors, EKLF, GATA-1, and SCL/Tal-1, was also delayed and decreased at reduced pO(2), consistent with lower levels of Epo-R and resultant Epo signaling. These changes in Epo-R and globin gene expression raise the possibility that the early increase of gamma-globin is a consequence of reduced Epo signaling and a delay in induction of erythroid transcription factors.

Parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells. Although previous studies have led to the theory that the basis of this tropism is receptor expression, this has been questioned by more recent observation. In the study reported here, we have investigated the basis of this tropism, and a potential role of erythropoietin (Epo) signaling, in erythroid progenitor cells (EPCs) expanded ex vivo from CD34(+) hematopoietic cells in the absence of Epo (CD36(+)/Epo(-) EPCs). We show, first, that CD36(+)/Epo(-) EPCs do not support B19V replication, in spite of B19V entry, but Epo exposure either prior to infection or after virus entry enabled active B19V replication. Second, when Janus kinase 2 (Jak2) phosphorylation was inhibited using the inhibitor AG490, phosphorylation of the Epo receptor (EpoR) was also inhibited, and B19V replication in ex vivo-expanded erythroid progenitor cells exposed to Epo (CD36(+)/Epo(+) EPCs) was abolished. Third, expression of constitutively active EpoR in CD36(+)/Epo(-) EPCs led to efficient B19V replication. Finally, B19V replication in CD36(+)/Epo(+) EPCs required Epo, and the replication response was dose dependent. Our findings demonstrate that EpoR signaling is absolutely required for B19V replication in ex vivo-expanded erythroid progenitor cells after initial virus entry and at least partly accounts for the remarkable tropism of B19V infection for human erythroid progenitors.