Characteristics of IEF, SDS and SAR-PAGE of Cuba EPO biosimilar

EPO is a glycoprotein produced in the kidney, which stimulates the division and differentiation of red cells in the bone marrow. Erythropoietin is available as a therapeutic agent produced by recombinant DNA technology in mammalian cell culture into which the human EPO gene has been transfected. Biosimilar Epoetins are mostly erythropoietins of the Epoetin alfa, beta or omega type, which are being produced at much lower cost due to expired patents. Recombinant human erythropoietin (rh-EPO) contains the identical amino acid sequence of natural EPO: 165 amino acids, with a molecular weight of 30,400 Da. Since glycosylation is not only dependent on the cell-line used for the expression of Epoetins but also on the entire biotechnological process the glycosylation patterns of biosimilars do not necessarily reflect the patterns of the originator compounds. Today biosimilar Epoetins are manufactured and distributed worldwide and under many different names. The use of recombinant EPOs for doping is prohibited because of its performance enhancing effect. The aim of the present study was to investigated whether biosimilar alpha r-HuEPO – ior®-EPOCIM, produced in Cuba and also available in other countries in all continents, could be differentiated from endogenous one by iso-electro-focusing plus double blotting, SDS-PAGE and SAR-PAGE for antidoping analysis.

Detection of EPO doping and blood doping: the haematological module of the Athlete Biological Passport.

The increase of the body's capacity to transport oxygen is a prime target for doping athletes in all endurance sports. For this pupose, blood transfusions or erythropoiesis stimulating agents (ESA), such as erythropoietin, NESP, and CERA are used. As direct detection of such manipulations is difficult, biomarkers that are connected to the haematopoietic system (haemoglobin concentration, reticulocytes) are monitored over time (Athlete Biological Passport (ABP)) and analyzed using mathematical models to identify patterns suspicious of doping. With this information, athletes can either be sanctioned directly based on their profile or targeted with conventional doping tests. Key issues for the appropriate use of the ABP are correct targeting and use of all available information (e.g. whereabouts, cross sectional population data) in a forensic manner. Future developments of the passport include the correction of all concentration-based variables for shifts in plasma volume, which might considerably increase sensitivity. New passport markers from the genomic, proteomic, and metabolomic level might add further information, but need to be validated before integration into the passport procedure. A first assessment of blood data of federations that have implemented the passport show encouraging signs of a decreased blood-doping prevalence in their athletes, which adds scientific credibility to this innovative concept in the fight against ESA- and blood doping. Copyright © 2012 John Wiley & Sons, Ltd.

Atypical EPO profiles in anti-doping analyses

Résumé : Erythropoietin (EPO) is a glycoprotein hormone endogenously produced by the kidney, whose main physiological role is the stimulation of erythropoiesis. Since the beginning of the nineties, recombinant human EPO (rhEPO), a potent anti-anaemia treatment drug, has been manufactured by pharmaceutical industries. However, the erythropoiesis stimulating power of rhEPO was rapidly misused by unscrupulous athletes in order to improve their performances in endurance sports. Endogenous EPO has the same amino-acid backbone as most of recombinant forms; the molecules however differ through their respective glycosylation patterns. This difference constitutes the basis of the usual EPO screening test (IEF) developed in 2000 and still currently used in all anti-doping laboratories of the world. Nowadays, 3 EPO generations have been commercialized. The fight against EPO abuse is a continuous challenge for anti-doping laboratories. The diversity of recombinant EPO forms and the continuous development of new ones considerably confuse the identification of EPO doping. Several facets of this fight were investigated in this work. One of the limiting aspects of doping agents screening is the availability of positive samples. Therefore, 2nd and 3rd generation EPOS, namely NESP and C.E.R.A., were injected to healthy subjects in the frame of pilot clinical studies. These latter allowed to review the current EPO identification criteria defined by the World Anti-Doping Agency (WADA) in the case of NESP and to validate and implement a new assay targeting C.E.R.A. in human serum. Both studies resulted in the determination of the respective detection windows of NESP and C.E.R.A. in biological fluids. Following that, Dynepo, a 1st generation EPO presenting similarities with the endogenous form, was also in the centre of a similar clinical study. Our work aimed to overcome the actual identification criteria, which are not adapted to Dynpeo, and to propose an alternative pattern classification method based on the discriminant analysis of IEF EPO profiles. This method might be validated for other EPO forms in the future. The detection window of this molecule was also determined. Under particular conditions, confounding effects can complicate the identification of EPO in biological matrices. For example, athletes having performed a strenuous physical effort can excrete modified isoforms of endogenous EPO, making it very similar to some recombinant forms. Such phenomena, called effort urines, were reproduced under controlled conditions and, after characterization of effort EPO, an urinary biochemical marker was proposed to unequivocally identify effort urines. It also happens that EPO analyses fail to detect endogenous levels of EPO. Such profiles were thoroughly investigated and potential causes identified. Natural reasons relying on urine properties and test specificity were underlined, but the possible addition of adulterant agents in urine samples was also considered. Therefore, a simple biochemical assay targeting the suspected substances was set up. Our work was based on the characterization of atypical EPO profiles from different origins. Therefore, 3 EPO molecules representing the 3 generations of the drug and 2 confounding effects confusing the results interpretation were studied. These studies resulted in tangible applications for the laboratory, the best example of which being the C.E.R.A. assay, but also in scientific findings allowing to improve our comprehension of EPO doping in sport.

Sagopilone (ZK-EPO, ZK 219477) for recurrent glioblastoma. A phase II multicenter trial by the European Organisation for Research and Treatment of Cancer (EORTC) Brain Tumor Group.

Background: Sagopilone (ZK 219477), a lipophylic and synthetic analog of epothilone B, that crosses the blood-brain barrier has demonstrated preclinical activity in glioma models.Patients and methods: Patients with first recurrence/progression of glioblastoma were eligible for this early phase II and pharmacokinetic study exploring single-agent sagopilone (16 mg/m(2) over 3 h every 21 days). Primary end point was a composite of either tumor response or being alive and progression free at 6 months. Overall survival, toxicity and safety and pharmacokinetics were secondary end points.Results: Thirty-eight (evaluable 37) patients were included. Treatment was well tolerated, and neuropathy occurred in 46% patients [mild (grade 1) : 32%]. No objective responses were seen. The progression-free survival (PFS) rate at 6 months was 6.7% [95% confidence interval (CI) 1.3-18.7], the median PFS was just over 6 weeks, and the median overall survival was 7.6 months (95% CI 5.3-12.3), with a 1-year survival rate of 31.6% (95% CI 17.7-46.4). Maximum plasma concentrations were reached at the end of the 3-h infusion, with rapid declines within 30 min after termination.Conclusions: No evidence of relevant clinical antitumor activity against recurrent glioblastoma could be detected. Sagopilone was well tolerated, and moderate-to-severe peripheral neuropathy was observed in despite prolonged administration.

Erythropoietin (EPO) immunoaffinity columns--a powerful tool for purifying EPO and its recombinant analogues.

The sample preparation method preceding the urinary erythropoietin (EPO) doping test is based on several concentration and ultrafiltration steps. In order to improve the quality of isoelectric focusing (IEF) gel results and therefore, the sensitivity of the EPO test, new sample preparation methods relying on affinity purification were recently proposed. This article focuses on the evaluation and validation of disposable immunoaffinity columns targeting both endogenous and recombinant EPO molecules in two World Anti-Doping Agency (WADA) accredited anti-doping laboratories. The use of the columns improved the resolution of the IEF profiles considerably when compared with the classical ultrafiltration method, and the columns' ability to ensure the isoform integrity of the endogenous and exogenous EPO molecules was confirmed. Immunoaffinity columns constitute therefore a potent and reliable tool for the preparation of urine samples and their use will significantly improve the sensitivity and specificity of the actual urinary EPO test.

Understanding e-government technology transfer: evidence from the transfer of a patent management system from EPO to INPI

Drawing upon Brazilian experience, this research explores some of the key issues to be addressed in using e-government technical cooperation designed to enhance service provision of Patent Offices in developing countries. While the development of software applications is often seen merely as a technical engineering exercise, localization and adaptation are context bounded matters that are characterized by many entanglements of human and non-humans. In this work, technical, legal and policy implications of technical cooperation are also discussed in a complex and dynamic implementation environment characterized by the influence of powerful hidden agendas associated with the arena of intellectual property (IP), which are shaped by recent technological, economic and social developments in our current knowledge-based economy. This research employs two different theoretical lenses to examine the same case, which consists of transfer of a Patent Management System (PMS) from the European Patent Office (EPO) to the Brazilian Patent Office that is locally named ‘Instituto Nacional da Propriedade Industrial’ (INPI). Fundamentally, we have opted for a multi-paper thesis comprising an introduction, three scientific articles and a concluding chapter that discusses and compares the insights obtained from each article. The first article is dedicated to present an extensive literature review on e-government and technology transfer. This review allowed the proposition on an integrative meta-model of e-government technology transfer, which is named E-government Transfer Model (ETM). Subsequently, in the second article, we present Actor-Network Theory (ANT) as a framework for understanding the processes of transferring e-government technologies from Patent Offices in developed countries to Patent Offices in developing countries. Overall, ANT is seen as having a potentially wide area of application and being a promising theoretical vehicle in IS research to carry out a social analysis of messy and heterogeneous processes that drive technical change. Drawing particularly on the works of Bruno Latour, Michel Callon and John Law, this work applies this theory to a longitudinal study of the management information systems supporting the Brazilian Patent Office restructuration plan that involved the implementation of a European Patent Management System in Brazil. Based upon the ANT elements, we follow the actors to identify and understand patterns of group formation associated with the technical cooperation between the Brazilian Patent Office (INPI) and the European Patent Office (EPO). Therefore, this research explores the intricate relationships and interactions between human and non-human actors in their attempts to construct various network alliances, thereby demonstrating that technologies embodies compromise. Finally, the third article applies ETM model as a heuristic frame to examine the same case previously studied from an ANT perspective. We have found evidence that ETM has strong heuristic qualities that can guide practitioners who are engaged in the transfer of e-government systems from developed to developing countries. The successful implementation of e-government projects in developing countries is important to stimulate economic growth and, as a result, we need to understand the processes through which such projects are being implemented and succeed. Here, we attempt to improve understanding on the development and stabilization of a complex social-technical system in the arena of intellectual property. Our preliminary findings suggest that e-government technology transfer is an inherently political process and that successful outcomes require continuous incremental actions and improvisations to address the ongoing issues as they emerge.

Does recombinant human Epo increase exercise capacity by means other than augmenting oxygen transport?

[EN] This study was performed to test the hypothesis that administration of recombinant human erythropoietin (rHuEpo) in humans increases maximal oxygen consumption by augmenting the maximal oxygen carrying capacity of blood. Systemic and leg oxygen delivery and oxygen uptake were studied during exercise in eight subjects before and after 13 wk of rHuEpo treatment and after isovolemic hemodilution to the same hemoglobin concentration observed before the start of rHuEpo administration. At peak exercise, leg oxygen delivery was increased from 1,777.0+/-102.0 ml/min before rHuEpo treatment to 2,079.8+/-120.7 ml/min after treatment. After hemodilution, oxygen delivery was decreased to the pretreatment value (1,710.3+/-138.1 ml/min). Fractional leg arterial oxygen extraction was unaffected at maximal exercise; hence, maximal leg oxygen uptake increased from 1,511.0+/-130.1 ml/min before treatment to 1,793.0+/-148.7 ml/min with rHuEpo and decreased after hemodilution to 1,428.0+/-111.6 ml/min. Pulmonary oxygen uptake at peak exercise increased from 3,950.0+/-160.7 before administration to 4,254.5+/-178.4 ml/min with rHuEpo and decreased to 4,059.0+/-161.1 ml/min with hemodilution (P=0.22, compared with values before rHuEpo treatment). Blood buffer capacity remained unaffected by rHuEpo treatment and hemodilution. The augmented hematocrit did not compromise peak cardiac output. In summary, in healthy humans, rHuEpo increases maximal oxygen consumption due to augmented systemic and muscular peak oxygen delivery.

Phosphorylation levels of JAK2 after addition of increasing Epo-concentrations: model approach (Figure 5a)

The present dataset contain source data for Figure 5a from Schilling et al., 2009. Cell fate decisions are regulated by the coordinated activation of signalling pathways such as the extracellular signal-regulated kinase (ERK) cascade, but contributions of individual kinase isoforms are mostly unknown. The authors combined quantitative data from erythropoietin-induced pathway activation in primary erythroid progenitor (colony-forming unit erythroid stage, CFU-E) cells with mathematical modelling, in order to predict and experimentally confirmed a distributive ERK phosphorylation mechanism in CFU-E cells. The authors found evidences that double-phosphorylated ERK1 attenuates proliferation beyond a certain activation level, whereas activated ERK2 enhances proliferation with saturation kinetics. Phosphorylation levels of JAK2 at 7 min after stimulation for Epo concentrations ranging from 0.1 to 1000 U/ml were simulated.

Phosphorylation levels of JAK2 after addition of increasing Epo-concentrations: experimental data (Figure 5a)

The present dataset data contain source data for Figure 5a from Schilling et al., 2009. Cell fate decisions are regulated by the coordinated activation of signalling pathways such as the extracellular signal-regulated kinase (ERK) cascade, but contributions of individual kinase isoforms are mostly unknown. The authors combined quantitative data from erythropoietin-induced pathway activation in primary erythroid progenitor (colony-forming unit erythroid stage, CFU-E) cells with mathematical modelling, in order to predict and experimentally confirmed a distributive ERK phosphorylation mechanism in CFU-E cells. The authors found evidences that double-phosphorylated ERK1 attenuates proliferation beyond a certain activation level, whereas activated ERK2 enhances proliferation with saturation kinetics. CFU-E cells were stimulated with the indicated Epo concentrations for 7 min and phosphorylation levels were determined by quantitative immunoblotting.

Proliferation of retrovirally transduced CFU-E cells after incubation with increasing Epo-concentration (Figure 5c)

Data contain source data for Figure 5c from Schilling et al., 2009. Cell fate decisions are regulated by the coordinated activation of signalling pathways such as the extracellular signal-regulated kinase (ERK) cascade, but contributions of individual kinase isoforms are mostly unknown. The authors combined quantitative data from erythropoietin-induced pathway activation in primary erythroid progenitor (colony-forming unit erythroid stage, CFU-E) cells with mathematical modelling, in order to predict and experimentally confirmed a distributive ERK phosphorylation mechanism in CFU-E cells. The authors found evidences that double-phosphorylated ERK1 attenuates proliferation beyond a certain activation level, whereas activated ERK2 enhances proliferation with saturation kinetics. Retrovirally transduced CFU-E cells were incubated with increasing Epo concentrations for 14 h and proliferation was measured by [3H]-thymidine incorporation.

A model for assembly and activation of the GM-CSF, IL-3 and IL-5 receptors: Insights from activated mutants of the common beta subunit

Granulocyte-macrophage colony stimulating factor (GM-CSF), Interleukin-3 (IL-3) and Interleukin-5 (IL-5) have overlapping, pleiotropic effects on hematopoietic cells, including neutrophils, eosinophils, monocytes and early progenitor cells. The high-affinity receptors for human GM-CSF, IL-3, and IL-5 share a common beta-subunit (h beta(c)), which is essential for signalling and plays a major role in recruiting intracellular signalling molecules. While activation of the cytoplasmic tyrosine kinase JAK2 appears to be the initiating event for signalling, the immediate events that trigger this are still unclear. We have isolated a number of activated mutants of h beta(c), which can be grouped into classes defined by their state of receptor phosphorylation, their requirement for alpha subunit as a cofactor, and their activities in primary cells and cell lines. We discuss these findings with regard to the stoichiometry, activation, and signalling of the normal GM-CSF/IL-3/IL-5 receptor complexes. Specifically, this work has implications for the role of the ligand-specific alpha-subunits in initiating the signalling through the beta-subunit, the role of beta subunit dimerization as a receptor trigger, and the function of receptor tyrosine phosphorylation in generating growth and survival signals. Based on the properties of the activated mutants and the recent structures of erythropoietin receptor (Epo-R) complexes, we propose a model in which (1) activation of h beta(c) can occur via alternative states that differ with respect to stoichiometry and subunit assembly, but which all mediate proliferative responses, and (2) each of the different classes of activated mutants mimics one of these alternative states. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Functional cross-talk between cytokine receptors revealed by activating mutations in the extracellular domain of the beta-subunit of the GM-CSF receptor

Several reports have suggested an interaction between the erythropoietin receptor (EpoR) and the shared signaling subunit (hbeta(c)) of the human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors, although the functional consequences of this interaction are unclear. We previously showed that in vivo expression of constitutively active extracellular (EC) mutants of hbeta(c) induces erythrocytosis and Epo independence of erythroid colony-forming units (CFU-E). This occurs despite an apparent requirement of these mutants for the GM-CSF receptor alpha-subunit (GMRalpha), which is not expressed in CFU-E. Here, we show that coexpression of hbeta(c) EC mutants and EpoR in BaF-B03 cells, which lack GMRalpha, results in factor-independent proliferation and JAK2 activation. Mutant receptors that cannot activate JAK2 fail to produce a functional interaction. As there is no detectable phosphorylation of hbeta(c). on intracellular tyrosine residues, EpoR displays constitutive tyrosine phosphorylation. These observations suggest that JAK2 activation mediates cross-talk between EC mutants of hbeta(c) and EpoR. The implications of these data are discussed as are our findings that activated hbeta(c) mutants can functionally interact with certain other cytokine receptors.

Corpos estranhos de fossas nasais: descrição de tipos e complicações em 420 casos

Corpos estranhos de fossas nasais são acidentes comuns em crianças, podendo, de acordo com a literatura, levar a complicações supurativas e bronco-aspiração do corpo estranho. O diagnóstico é feito quase sempre pela rinoscopia anterior, mas a nasofibroscopia e exames radiológicos podem ser úteis. OBJETIVO: Analisar um total de 420 casos de corpos estranhos de fossas nasais removidos no serviço de ORL-EPO do Hospital Municipal Souza Aguiar quanto a vários parâmetros como sexo, idade, tipo e complicações. MATERIAL E MÉTODO: 420 casos de corpos estranhos de fossas nasais removidos no serviço de Otorrinolaringologia e Endoscopia Per-oral (ORL-EPO) do Hospital Municipal Souza Aguiar, no período de dezembro de 1992 a dezembro de 1998, quanto aos parâmetros acima referidos. RESULTADOS: Foi encontrada uma maior incidência na faixa etária de 0 a 4 anos, sendo os mais comuns, pela ordem fragmentos de espuma, fragmentos de material plástico, grãos de feijão e fragmentos de papel. As complicações ocorreram em 9,05% dos casos, sendo as mais comuns a epistaxe e a vestibulite. CONCLUSÃO: Os corpos estranhos de fossas nasais são acidentes encontrados principalmente na faixa etária de 0 a 4 anos, sendo os mais comuns, em nossa casuística, os fragmentos de espuma e pequenos artefatos de plástico. Complicações não são freqüentes, sendo as mais encontradas a epistaxe e vestibulite nasal.