The kidney is a major site of systemic oxygen sensing, regulating blood erythrocyte and hence oxygen content by hypoxia-inducible erythropoietin (Epo) expression. A constant ratio between blood perfusion and oxygen consumption, a stable corticomedullary oxygen gradient, and a relatively low tissue Po(2) are the prerequisites for the function of renal Epo-producing and oxygen-sensing (REPOS) cells, which are located in the juxtamedullary cortex. In kidney disease, renal oxygen consumption is decreased, leading to an increase in Po(2), dysfunction of REPOS cells, and anemia. The molecular principles of cellular oxygen sensing have been elucidated in the last few years, and genetically altered mouse models as well as hereditary diseases causing erythrocytosis have clarified the oxygen-signaling cascade leading to increased Epo expression in REPOS cells. However, the consequences of a number of recently discovered factors for the regulation of oxygen signaling in REPOS cells are unclear, asking for novel cell culture models which might be hampered by the putative neuron-like nature of this enigmatic cell type.

The hematopoietic microenvironment in Steel mutant mice does not support erythropoiesis, megakaryocytopoiesis, or mast cell generation. The question of whether Steel hematopoietic progenitors are present in normal numbers has never been convincingly addressed. In this report, Sl/Sld marrow cells were assessed for long-term competitive repopulation ability in vivo and for short-term growth in vitro. In vivo repopulation assays indicate that the Sl/Sld progenitors are at a distinct disadvantage when they compete against congenic genetically marked +/+ cells in a +/+ host. On the other hand, the Steel erythroid colony-forming cells (CFU-E) respond normally to erythropoietin (Epo) in vitro and are present at normal frequency. Because the Steel marrow is less cellular than normal marrow, the absolute number of CFU-E is decreased. Results suggest that the absence of membrane-bound Steel factor in the mutant donor has a direct effect on Steel hematopoietic progenitors, which is not alleviated during growth for over 6 months in a normal microenvironment. The anomaly does not seem to directly affect the frequency of more mature adult erythroid progenitors.

Using RNAse protection, we have made quantitative measurements of erythropoietin (EPO) mRNA in liver and kidneys of developing rats (days 1-54), to determine the relative contribution of both organs to the total EPO mRNA, to monitor changes which occur with development, and to compare the hypoxia-induced accumulation of EPO mRNA with the changes in serum EPO concentrations. To determine whether developmental and organ-specific responsiveness is related to the type of hypoxic stimulus, normobaric hypoxia was compared with exposure to carbon monoxide (functional anemia). Under both stimuli EPO mRNA concentration in liver was maximal on day 7 and declined during development. In contrast, EPO mRNA concentration in kidney increased during development from day 1 when it was 30-65% the hepatic concentration to day 54 when it was 12-fold higher than in liver. When organ weight was considered the liver was found to contain the majority of EPO mRNA in the first three to four weeks of life, and although, in stimulated animals, the hepatic proportion declined from 85-91% on day 1, it remained approximately 33% at day 54 and was similar for the two types of stimuli. When normalized for body weight the sum of renal and hepatic EPO mRNA in animals of a particular age was related linearly to serum hormone concentrations. However, the slope of this regression increased progressively with development, suggesting age-dependent alterations in translational efficiency or EPO metabolism.

OBJECTIVE

Selective gene expression in response to tumor hypoxia may provide new avenues, not only for radiotherapy and chemotherapy, but also for gene therapy. In this study, we have assessed the extent of hypoxia responsiveness of various DNA constructs by the luciferase assay to help design vectors suitable for cancer therapy.

METHODS

Reporter plasmids were constructed with fragments of the human vascular endothelial growth factor (VEGF) and the erythropoietin (Epo) genes encompassing the putative hypoxia-responsive elements (HRE) and the pGL3 promoter vector. Test plasmids and the control pRL-CMV plasmid were cotransfected into tumor cells by the calcium phosphate method. After 6 h hypoxic treatment, the reporter assay was performed.

RESULTS

The construct pGL3/VEGF containing the 385 bp fragment of the 5' flanking region in human VEGF gene showed significant increases in luciferase activity in response to hypoxia. The hypoxic/aerobic ratios were about 3-4, and 8-12 for murine and human tumor cells, respectively. Despite the very high degree of conservation among the HREs of mammalian VEGF genes, murine cells showed lower responsiveness than human cells. We next tested the construct pGL3/Epo containing the 150 bp fragment of the 3' flanking region in the Epo gene. Luciferase activity of pGL3/Epo was increased with hypoxia only in human cell lines. The insertion of 5 copies of the 35-bp fragments derived from the VEGF HREs and 32 bp of the E1b minimal promoter resulted in maximal enhancement of hypoxia responsiveness.

CONCLUSIONS

The constructs with VEGF or Epo fragments containing HRE may be useful for inducing specific gene expression in hypoxic cells. Especially, the application of multiple copies of the HREs and an E1b minimal promoter appears to have the advantage of great improvement in hypoxia responsiveness.

Oxidative stress significantly impacts multiple cellular pathways that can lead to the initiation and progression of varied disorders throughout the body. It therefore becomes imperative to elucidate the components and function of novel therapeutic strategies against oxidative stress to further clinical diagnosis and care. In particular, both the growth factor and cytokine erythropoietin (EPO), and members of the mammalian forkhead transcription factors of the O class (FoxOs), may offer the greatest promise for new treatment regimens, since these agents and the cellular pathways they oversee cover a range of critical functions that directly influence progenitor cell development, cell survival and degeneration, metabolism, immune function, and cancer cell invasion. Furthermore, both EPO and FoxOs function not only as therapeutic targets, but also as biomarkers of disease onset and progression, since their cellular pathways are closely linked and overlap with several unique signal transduction pathways. Yet, EPO and FoxOs may sometimes have unexpected and undesirable effects that can raise caution for these agents and warrant further investigations. Here we present the exciting as well as the complex role that EPO and FoxOs possess to uncover the benefits as well as the risks of these agents for cell biology and clinical care in processes that range from stem cell development to uncontrolled cellular proliferation.

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl.html) constitutes Europe's primary nucleotide sequence resource. Main sources for DNA and RNA sequences are direct submissions from individual researchers, genome sequencing projects and patent applications. While automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO), the preferred submission tool for individual submitters is Webin (WWW). Through all stages, dataflow is monitored by EBI biologists communicating with the sequencing groups. In collaboration with DDBJ and GenBank the database is produced, maintained and distributed at the European Bioinformatics Institute (EBI). Database releases are produced quarterly and are distributed on CD-ROM. Network services allow access to the most up-to-date data collection via Internet and World Wide Web interface. 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 etc) are available for external users to compare their own sequences against the most currently available data in the EMBL Nucleotide Sequence Database and SWISS-PROT.

Mast cells are important effector cells in IgE-mediated acute allergic reactions. Mast cells also produce cytokines such as interleukin (IL)-3, IL-4, IL-5, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) that regulate the function of eosinophils and the development of a late-phase inflammatory response to antigen challenge. To evaluate the role of mast cells on the development of IgE-mediated allergic pulmonary eosinophilia in vivo, we compared the eosinophil infiltration into lungs of mast cell deficient mice (WBB6F1/J-W/Wv) with their congenic normal littermates (W/W+). Mice were sensitized with alum-precipitated ovalbumin and challenged with aerosolized ovalbumin on day 12 after sensitization. Bronchoalveolar lavage (BAL) fluid, lung tissue biopsies, and blood samples were collected after ovalbumin challenge. Eosinophil numbers in the BAL and lung tissue, lung eosinophil peroxidase (EPO) activity and serum levels of IgE and IgG1 were measured. In sensitized W/W+ mice, there were increased numbers of eosinophils in the BAL fluid and lung tissue, and EPO levels were increased after ovalbumin challenge. Ovalbumin challenge of sensitized mast-cell-deficient mice produced fewer numbers of eosinophils in the BAL fluid and lungs, and EPO levels were also reduced compared with their challenged congenic littermates. On the other hand, levels of serum IgE and IgG1 were not different between W/Wv mice and their congenic littermates.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythropoietin (EPO) provides neuroprotection and neurorestoration after traumatic brain injury (TBI). The EPO doses used for treatment of TBI significantly increase hematocrit, which may affect the efficacy of EPO therapy for TBI. The aim of this study was to investigate whether normalization of hematocrit would affect EPO efficacy for treatment of TBI. Young adult male Wistar rats were randomly divided into four groups: (1) Sham group (n=6); (2) TBI+ saline group (n=6); (3) TBI+ EPO group (n=6); and (4) TBI+ EPO+ hemodilution group (n=7). TBI was induced by controlled cortical impact over the left parietal cortex. EPO (5,000 U/kg) or saline was administered intraperitoneally at days 1, 2, and 3 postinjury. Neurological function was assessed using a modified neurological severity score (mNSS), footfault and the Morris water maze (MWM) tests. Animals were sacrificed 35 days after injury, and brain sections were stained for immunohistochemistry. Compared to the saline treatment, EPO treatment significantly reduced hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved sensorimotor functional outcome (lowered mNSS and foot faults) and spatial learning (MWM test). Normovolemic hemodilution effectively normalized the hematocrit and did not significantly affect the histological and functional outcome of EPO therapy for TBI. These data for the first time demonstrate that increased hematocrit does not affect therapeutic effects of EPO on histological and long-term functional outcomes in rats after TBI and also suggest that neuroprotection and neurorestoration of EPO treatment are independent of hematocrit.

We evaluated the quantitative value of a simple model of erythropoiesis, based on the basic assumptions that the red blood cell (RBC) mass determines erythropoietin (Epo) production, which in turn stimulates erythropoietic activity. The RBC mass was quantitated by direct isotopic measurement (RCM), Epo production by serum Epo levels, and erythropoiesis by the ferrokinetic measurement of the erythron transferrin uptake (ETU), the serum transferrin receptor (TfR) level, and the reticulocyte (retic) index, and was completed by an evaluation of overall marrow erythron cellularity. We studied a total of 195 subjects, including 31 normal individuals, 38 patients with polycythemia, and 126 patients with various forms of anemia. Instead of only quantitating Epo and erythropoiesis in absolute terms, we also evaluated them in relation to the degree of anemia or polycythemia, and expressed the results as a ratio of observed values to values predicted from the regression equations between hematocrit (Hct) on the one hand, and Epo, TfR, and ETU on the other, obtained in a carefully selected subpopulation. The slope of the regression of TfR (as well as ETU) versus Hct was very similar to the slope of the regression of Epo versus Hct. Average EPO and TfR (as well as ETU) values predicted from the regression equations were quite comparable to observed values in most groups of subjects, with exceptions predictable from knowledge of the pathophysiology of these hematologic disorders. We identified four major patterns of erythropoiesis, ie, normal, hyperdestruction (with variants of hemolysis or ineffective erythropoiesis), intrinsic marrow hypoproliferation, and defective Epo production. Dissecting out groups of patients showed much greater heterogeneity than when patients were analyzed by group. This was particularly true in the case of a hypoproliferative component being combined with hyperdestruction, giving what we called a "mixed disorder of erythropoiesis." We conclude that the pathophysiology of anemia can be assessed by a simple measurement of Hct, retic index, Epo, and TfR levels, with Epo and TfR being more informative when expressed in relation to the degree of anemia. The model is particularly useful for detecting the presence of multiple mechanisms of anemia in the same patient. However, it has limitations inherent to the relative invalidity of TfR in iron deficiency, the imprecision of a retic count, and the difficulty in distinguishing hemolysis from ineffective erythropoiesis in some patients and in recognizing a component of hyperdestruction in hypoproliferative anemia.

A cohort of 250 Ghanaian schoolchildren aged 5-15 years was followed clinically and parasitologically for 4 months in 1997/98 in order to study the effect of asymptomatic Plasmodium falciparum infections on haematological indices and bone-marrow responses. Of the 250 children 65 met the predefined study criteria. Thus, 14 children were parasite-free throughout (group 1), 44 had P. falciparum in all blood samples collected but no symptoms of malaria (group 2), and 7 had 1 malaria attack during the study period (group 3). At the end of the study the mean haemoglobin (Hb) level in group 1 was 123 g/L, significantly higher than the value of 114 g/L in groups 2 and 3 (P < 0.02, adjusted for age and splenomegaly). The low Hb in group 2 was associated with subnormal plasma iron. Low Hb was associated with elevated erythropoietin (EPO) levels, and there was a positive correlation between EPO and reticulocyte counts. However, the reticulocyte response to EPO was more pronounced in uninfected than in infected children, suggesting a partial interference with erythropoiesis in asymptomatic infections. Children with asymptomatic infections had significantly higher plasma levels of tumour necrosis factor than uninfected children (geometric means 50 ng/L and 27 ng/L, respectively, P < 0.001) and this cytokine may contribute to bone-marrow suppression and disturbed iron metabolism. We suggest that asymptomatic malaria leads to a homeostatic imbalance in which erythrocyte loss due to parasite replication is only partially compensated for by increased erythropoiesis. The consequences of the reduced Hb levels on the development and cognitive abilities of children with asymptomatic infections, and the risk of precipitation of iron deficiency, deserve further study and should be considered in malaria control programmes that aim at reducing morbidity rather than transmission.

The mouse eosinophil peroxidase (mEPO) gene was cloned by screening a random-primed bone marrow cDNA library at reduced criteria using a hEPO cDNA. An mEPO cDNA was subsequently used to isolate the mEPO gene from a lambda-genomic library. The mEPO gene displays a high degree of conservation with its human homologue: the transcription units are approximately the same size, conserve the relative size and position of the 12 exons associated with each gene, and at a nucleotide level the mouse and human EPO genes are 86% identical in the protein coding regions and 66% identical in the 3'-untranslated trailer regions. This strong conservation extends to the encoded proteins which show approximately 90% amino acid identity. Expression of the mEPO gene is restricted to tissues containing eosinophil progenitor cells (e.g., bone marrow and spleen), a pattern similar to the expression of another murine eosinophil granule protein, major basic protein.

Recent data suggest that eosinophils may cause lung injury. To determine if the eosinophil peroxidase (EPO)-hydrogen peroxide (H2O2)-halide system could mediate this injury, we added human EPO, H2O2 (or glucose and glucose oxidase as a continuous source of H2O2), and various halides to monolayers of 51Cr-labeled human A549 and rat type II pneumocytes. Cell lysis was measured as soluble 51Cr release. In initial experiments, EPO in solution did not induce lysis under these conditions. Therefore, in subsequent experiments, pneumocytes were preincubated with EPO for 15 minutes, washed to remove unbound enzyme, and then glucose, glucose oxidase, and the halides were added. EPO alone was not injurious, nor was the addition of glucose and glucose oxidase in the absence of EPO. In contrast, the combined addition of EPO, glucose, glucose oxidase, and chloride produced marked target-cell lysis. This effect was time and EPO dose dependent and was enhanced by the addition of iodide. Catalase and azide substantially inhibited the lysis produced by the EPO-H2O2-halide system, suggesting that EPO-catalyzed products of halide oxidation mediated this form of injury. Finally, the addition of eosinophil major basic protein at 10(-5) mol/L to EPO-coated pneumocytes incubated with glucose, glucose oxidase, and halides failed to enhance or inhibit lysis. We hypothesize that the EPO-H2O2-halide system may injure the lung in asthma and eosinophilic pulmonary syndromes.

Erythropoietin (Epo) is a hematopoietic cytokine that is crucial for the differentiation and proliferation of erythroid progenitor cells. Epo acts on its target cells by inducing homodimerization of the erythropoietin receptor (EpoR), thereby triggering intracellular signaling cascades. The EpoR encompasses eight tyrosine motifs on its cytoplasmic tail that have been shown to recruit a number of regulatory proteins. Recently, the feedback inhibitor suppressor of cytokine signaling-3 (SOCS-3), also referred to as cytokine-inducible SH2-containing protein 3 (CIS-3), has been shown to act on Epo signaling by both binding to the EpoR and the EpoR-associated Janus kinase 2 (Jak2) [Sasaki, A., Yasukawa, H., Shouda, T., Kitamura, T., Dikic, I. & Yoshimura, A. (2000) J. Biol. Chem 275, 29338-29347]. In this study tyrosine 401 was identified as a binding site for SOCS-3 on the EpoR. Here we show that human SOCS-3 binds to pY401 with a Kd of 9.5 microm while another EpoR tyrosine motif, pY429pY431, can also interact with SOCS-3 but with a ninefold higher affinity than we found for the previously reported motif pY401. In addition, SOCS-3 binds the double phosphorylated motif pY429pY431 more potently than the respective singly phosphorylated tyrosines indicating a synergistic effect of these two tyrosine residues with respect to SOCS-3 binding. Surface plasmon resonance analysis, together with peptide precipitation assays and model structures of the SH2 domain of SOCS-3 complexed with EpoR peptides, provide evidence for pY429pY431 being a new high affinity binding site for SOCS-3 on the EpoR.

Erythropoietin (EPO) can induce a series of cytoprotective effects in many non-hematopoietic tissues through interaction with the erythropoietin receptor (EPOR), but whether EPO can prevent the overproduction of reactive oxygen species (ROS) and apoptosis in diabetes remains unclear. Here, we report that renal tubular cells possess EPOR and that EPO reduces high glucose-induced oxidative stress in renal tubular cells. Further, we found that EPO inhibited high glucose-induced renal tubular cell apoptosis and that this protective effect was dependent on reduction of Bax/caspase-3 expression as well as elevation of Bcl-2 expression. Our results suggest that EPO can inhibit high glucose-induced renal tubular cell apoptosis through direct effect on anti-oxidative stress and that EPOR may play a key role in this process.

Resistance to erythropoiesis-stimulating agents (ESAs) has been observed in a considerable proportion of patients with chronic kidney disease (CKD) and it is reportedly associated with adverse outcomes, such as increased cardiovascular morbidity, faster progression to end-stage renal disease (ESRD) and all-cause mortality. The major causes of ESA resistance include chronic inflammation producing suppressive cytokines of early erythroid progenitor proliferation. In addition, pro-inflammatory cytokines stimulate hepcidin synthesis thus reducing iron availability for late erythropoiesis. Recent studies showing an association in deficiencies of the vitamin D axis with low haemoglobin (Hb) levels and ESA resistance suggest a new pathophysiological co-factor of renal anaemia. The administration of either native or active vitamin D has been associated with an improvement of anaemia and reduction in ESA requirements. Notably, these effects are not related to parathyroid hormone (PTH) values and seem to be independent on PTH suppression. Another possible explanation may be that calcitriol directly stimulates erythroid progenitors; however, this proliferative effect by extra-renal activation of 1α-hydroxylase enzyme is only a hypothesis. The majority of studies concerning vitamin D deficiency or supplementation, and degree of renal anaemia, point out the prevalent role of inflammation in the mechanism underlying these associations. Immune cells express the vitamin D receptor (VDR) which in turn is involved in the modulation of innate and adaptive immunity. VDR activation inhibits the expression of inflammatory cytokines in stromal and accessory cells and up-regulates the lymphocytic release of interleukin-10 (IL-10) exerting both anti-inflammatory activity and proliferative effects on erythroid progenitors. In CKD patients, vitamin D deficiency may stimulate immune cells within the bone marrow micro-environment to produce cytokines, inducing impaired erythropoiesis. Immune activation involves the reticuloendothelial system, increasing hepcidin synthesis and functional iron deficiency. Consequences of this inflammatory cascade are erythropoietin (EPO) resistance and anaemia. Given the key role of inflammation in the response to EPO, the therapeutic use of agents with anti-cytokines properties, such as vitamin D and paricalcitol, may provide benefit in the prevention/treatment of ESA hyporesponsiveness.

Recent studies from our lab and others have shown that the hematopoietic cytokine erythropoietin (EPO) can protect the heart from ischemic damage in a red blood cell-independent manner. Here we examined any protective effects of the long-acting EPO analog darbepoetin alfa (DA) in a rat model of ischemia-reperfusion (I/R) injury. Rats were subjected to 30-min ischemia followed by 72-h reperfusion. In a dose-response study, DA (2, 7, 11, and 30 mug/kg) or vehicle was administered as a single bolus at the start of ischemia. To determine the time window of potential cardioprotection, a single high dose of DA (30 mug/kg) was given at either the initiation or the end of ischemia or at 1 or 24 h after reperfusion. After 3 days, cardiac function and infarct size were assessed. Acute myocyte apoptosis was quantified by TUNEL staining on myocardial sections and by caspase-3 activity assays. DA significantly reduced infarct size from 32.8 +/- 3.5% (vehicle) to 11.0 +/- 3.3% in a dose-dependent manner, while there was no difference in ischemic area between groups. Treatment with DA as late as 24 h after the beginning of reperfusion still demonstrated a significant reduction in infarct size (17.0 +/- 1.6%). Consistent with infarction data, DA improved in vivo cardiac reserve compared with vehicle. Finally, DA significantly decreased myocyte apoptosis and caspase-3 activity after I/R. These data indicate that DA protects the heart against I/R injury and improves cardiac function, apparently through a reduction of myocyte apoptosis. Of clinical importance pointing toward a relevant therapeutic utility, we report that even if given 24 h after I/R injury, DA can significantly protect the myocardium.

The tissue protective functions of the hematopoietic growth factor erythropoietin (EPO) are independent of its action on erythropoiesis. EPO and its receptors (EPOR) are expressed in multiple brain cells during brain development and upregulated in the adult brain after injury. Peripherally administered EPO crosses the blood-brain barrier and activates in the brain anti-apoptotic, anti-oxidant and anti-inflammatory signaling in neurons, glial and cerebrovascular endothelial cells and stimulates angiogenesis and neurogenesis. These mechanisms underlie its potent tissue protective effects in experimental models of stroke, cerebral hemorrhage, traumatic brain injury, neuroinflammatory and neurodegenerative disease. The preclinical data in support of the use of EPO in brain disease have already been translated to first clinical pilot studies with encouraging results with the use of EPO as a neuroprotective agent.

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene and characterized by chronic anemia caused by ineffective erythropoiesis, and accompanied by a variety of serious secondary complications such as extramedullary hematopoiesis, splenomegaly, and iron overload. In the past few years, numerous studies have shown that such secondary disease conditions have a genetic basis caused by the abnormal expression of genes with a role in controlling erythropoiesis and iron metabolism. In this article, the most recent discoveries related to the mechanism(s) responsible for anemia/ineffective erythropoiesis and iron overload are discussed in detail. Particular attention is paid to the pathway(s) controlling the expression of hepcidin, which is the main regulator of iron metabolism, and the Epo/EpoR/Jak2/Stat5 signaling pathway, which regulates erythropoiesis. Better understanding of how these pathways function and are altered in β-thalassemia has revealed several possibilities for development of new therapeutic approaches to treat of the complications of this disease.

Epoetin (recombinant human erythropoietin, EPO) is of proven benefit in the treatment of renal anaemia, and preliminary reports suggest that it may have a role in the management of other anaemic conditions. Pharmacokinetic and therapeutic studies have examined the use of epoetin administered intravenously, intraperitoneally and subcutaneously, and there is accumulating evidence that the last route has several advantages. After intravenous administration, epoetin is distributed in a volume comparable to the plasma volume, and plasma concentrations decay monoexponentially with a half-life of between 4 and 12 hours. Administration of epoetin in peritoneal dialysis fluid results in detectable concentrations in the bloodstream after 1 to 2 hours, and peak concentrations of the order of 2 to 4% of those obtained with the same intravenous dose are found after approximately 12 hours. The bioavailability of epoetin administered intraperitoneally in dialysis fluid is about 3 to 8%, but this may be increased by injecting the drug into a dry peritoneal cavity. Subcutaneous administration results in peak concentrations at about 18 hours which are 5 to 10% of those found after the same intravenous dose. The bioavailability of subcutaneous epoetin is about 20 to 30% and detectable serum concentrations are still present 4 days after administration, in contrast to intravenous administration after which concentrations have returned to baseline within 2 to 3 days. Remarkably little is known about the metabolic fate of either erythropoietin or epoetin. In addition, there is much controversy surrounding the relative roles of the kidney and liver in the catabolism of epoetin. About 3 to 10% of epoetin is excreted unchanged in the urine. In common with other glycoproteins, the carbohydrate residues which constitute 40% of its molecular size are essential for maintaining the stability of epoetin in circulation. Desialated epoetin, although biologically active in vitro, is cleared very rapidly from plasma with resultant loss of activity. Further work is required, however, in identifying the pathways of metabolism and elimination of this glycoprotein hormone.

BACKGROUND

Epothilones are 16-member ring macrolides with antimicrotubule activity that share a similar mechanism of action to the taxanes but have demonstrated potent antiproliferative activity in several different multidrug-resistant and paclitaxel-resistant tumor cell lines in vitro and in vivo.

METHODS

This review summarizes data from preclinical and phase I clinical studies of epothilone B (patupilone; EPOEPO, ABJ-879) derivatives. Data were identified by searches of PubMed and the Proceedings of the American Society of Clinical Oncology annual meetings from 2000 to 2006.

RESULTS

Epothilones demonstrate a linear dose-dependent pharmacokinetic profile, are well tolerated, and exhibit antitumor activity in a variety of tumor types in phase I studies of patients with cancer. Although similar in chemical structure, the epothilones demonstrate a striking difference in toxicity profile in phase I studies. Diarrhea is the dose-limiting toxicity (DLT) associated with patupilone, whereas neurotoxicity and neutropenia are the DLTs most commonly encountered with other epothilones. Consistent with preclinical data, partial responses were observed with patupilone and ixabepilone in patients with breast cancer previously treated with taxanes.

CONCLUSIONS

The epothilones demonstrate promising antitumor activity in a broad spectrum of taxane-sensitive and -refractory tumors at doses and schedules associated with tolerable side-effects.

Human fetal bone fragments implanted in the immunodeficient C.B-17 scid/scid (SCID) mouse were shown to sustain active human hematopoiesis in vivo. Human progenitor cell activity was maintained for as long as 20 weeks after implantation and was associated with multilineage differentiation in the engrafted bone. Thus, the bone implants provided stem cells as well as the microenvironment requisite for their long-term maintenance and multilineage differentiation. Administration of human erythropoietin (Epo) stimulated human erythropoiesis in human bone implants. This animal model may facilitate direct analysis of a wide variety of physiologic and pathologic conditions of human bone marrow (BM) in vivo.

Erythropoietin receptor (EpoR) binding mediates neuroprotection by endogenous Epo or by exogenous recombinant human (rh)Epo. The level of EpoR gene expression may determine tissue responsiveness to Epo. Thus, harnessing the neuroprotective power of Epo requires an understanding of the Epo-EpoR system and its regulation. We tested the hypothesis that neuronal expression of EpoR is required to achieve optimal neuroprotection by Epo. The ventral limbic region (VLR) in the rat brain was used because we determined that its neurons express minimal EpoR under basal conditions, and they are highly sensitive to excitotoxic damage, such as occurs with pilocarpine-induced status epilepticus (Pilo-SE). We report that (i) EpoR expression is significantly elevated in nearly all VLR neurons when rats are subjected to 3 moderate hypoxic exposures, with each separated by a 4-day interval; (ii) synergistic induction of EpoR expression is achieved in the dorsal hippocampus and neocortex by the combination of hypoxia and exposure to an enriched environment, with minimal increased expression by either treatment alone; and (iii) rhEpo administered after Pilo-SE cannot rescue neurons in the VLR, unless neuronal induction of EpoR is elicited by hypoxia before Pilo-SE. This study thus demonstrates using environmental manipulations in normal rodents, the strict requirement for induction of EpoR expression in brain neurons to achieve optimal neuroprotection. Our results indicate that regulation of EpoR gene expression may facilitate the neuroprotective potential of rhEpo.

Treatment with erythropoietin (epo) may improve the anemia of myelodysplastic syndromes (MDS) in approximately 20% of patients. Previous studies have suggested that treatment with the combination of granulocyte colony-stimulating factor (G-CSF) and epo may increase this response rate. In the present phase II study, patients with MDS and anemia were randomized to treatment with G-CSF + epo according to one of two alternatives; arm A starting with G-CSF for 4 weeks followed by the combination for 12 weeks, and arm B starting with epo for 8 weeks followed by the combination for 10 weeks. Fifty evaluable patients (10 refractory anemia [RA], 13 refractory anemia with ring sideroblasts [RARS], and 27 refractory anemia with excess blasts [RAEB]) were included in the study, three were evaluable only for epo as monotherapy and 47 for the combined treatment. The overall response rate to G-CSF + epo was 38%, which is identical to that in our previous study. The response rates for patients with RA, RARS, and RAEB were 20%, 46%, and 37%, respectively. Response rates were identical in the two treatment groups indicating that an initial treatment with G-CSF was not neccessary for a response to the combination. Nine patients in arm B showed a response to the combined treatment, but only three of these responded to epo alone. This suggests a synergistic effect in vivo by G-CSF + epo. A long-term follow-up was made on 71 evaluable patients from both the present and the preceding Scandinavian study on G-CSF + epo. Median survival was 26 months, and the overall risk of leukemic transformation during a median follow-up of 43 months was 28%. Twenty patients entered long-term maintenance treatment and showed a median duration of response of 24 months. The international prognostic scoring system (IPSS) was effective to predict survival, leukemic transformation, and to a lesser extent, duration of response, but had no impact on primary response rates.

Neuroinflammation is a normal and healthy response to neuronal damage. However, excessive or chronic neuroinflammation exacerbates neurodegeneration after trauma and in progressive diseases such as Alzheimer's, Parkinson's, age-related macular degeneration, and glaucoma. Therefore, molecules that modulate neuroinflammation are candidates as neuroprotective agents. Erythropoietin (EPO) is a known neuroprotective agent that indirectly attenuates neuroinflammation, in part, by inhibiting neuronal apoptosis. In this review, we provide evidence that EPO also modulates neuroinflammation upstream of apoptosis by acting directly on glia. Further, the signaling induced by EPO may differ depending on cell type and context possibly as a result of activation of different receptors. While significant progress has been made in our understanding of EPO signaling, this review also identifies areas for future study in terms of the role of EPO in modulating neuroinflammation.