In this study, the effects of pH on therapeutically important protein, recombinant human erythropoietin (rhuEPO), production by Pichia pastoris was investigated at pH=4.0, 4.5, 5.0, 5.5 and 6.0. rHuEPO production was started by methanol induction in fed-batch mode. The highest cell concentration was obtained at pH=4.5 as 81.4 g L-1. The co-substrate substrate sorbitol, which was added batch-wise, was consumed at t=15 h of the operations at pH=4.0, 4.5 and 5.0. However as the pH increases above pH=5.0 the sorbitol consumption rate decreases. The highest rHuEPO concentration was achieved at pH=4.5 as 0.158 g L-1 which was 1.43-, 1.24-, 1.95- and 1.23-fold higher than those obtained at pH=4.0, 5.0, 5.5, and 6.0, respectively. Also at pH=4.5 overall cell yield on substrate was 0.51 g g-1 and overall rHuEPO yield on substrate was 1.45 mg g-1. rHuEPO concentration was decreased in the last 3-6 hour of the operation due to proteolysis. Therefore extracellular protease concentrations in the medium were determined. As expected, since the investigated pH range was acidic, the amount of acidic proteases was found to be higher than neutral and basic proteases. Furthermore the total protease concentration increased linearly in the fermentation broth, having close values at different pH values. Thus, pH did not have a significant effect on extracellular protease activity. Alcohol oxidase (AOX) activities showed similar behavior at different pH. The highest specific AOX activity was attained at pH=4.5, at which the highest rHuEPO concentration was achieved, as 110.1 U g-1 CDW. Keywords:

Η αναιμία σχετιζόμενη με τη νόσο στη χρόνια λεμφογενή λευχαιμία (ΧΛΛ) είναι διάγνωση εξ΄ αποκλεισμού και διαπιστώνεται στις περιπτώσεις που δεν ανευρίσκεται άλλο εμφανές αίτιο αναιμίας. Δεδομένου ότι η παθογένεια της αναιμίας αυτής δεν είναι διευκρινισμένη, μελετήσαμε διάφορες παραμέτρους της ερυθροποίησης σε πρωτοδιαγνωσθέντες ασθενείς με ΧΛΛ και αναιμία σχετιζόμενη με τη νόσο και τις συγκρίναμε με τα αντίστοιχα ευρήματα ασθενών με ΧΛΛ χωρίς αναιμία και χωρίς να έχουν λάβει θεραπεία και με αυτά των υγιών μαρτύρων. Η διήθηση του μυελού των οστών από τα κακοήθη Β-λεμφοκύτταρα δεν ήταν αποκλειστικά υπεύθυνη για την πρόκληση αναιμίας. Τα CD34+ κύτταρα του μυελού των οστών στη ΧΛΛ δεν εμφάνισαν ενδογενή διαταραχή στη στροφή και περαιτέρω διαφοροποίησή τους προς την ερυθρά σειρά. Επιπλέον, δε διαπιστώθηκαν ανεπαρκή επίπεδα ερυθροποιητίνης ορού ούτε ελαττωματική απάντηση των ερυθροποιητικών προδρομικών κυττάρων στην ΕΡΟ στον άξονα ΕΡΟ-ΕΡΟ υποδοχέα κατά τη διέγερσή τους με ΕΡΟ ± TNF-α. Από την άλλη μεριά, τα επίπεδα του TNF-α βρέθηκαν αυξημένα στους ασθενείς με ΧΛΛ και αναιμία σχετιζόμενη με τη νόσο και φάνηκε ο TNF-α να ασκεί άμεση κατασταλτική δράση στη διαφοροποίηση των CD34+ κυττάρων προς κύτταρα της ερυθράς σειράς in vitro. Η μελέτη μας έδειξε ότι η αναιμία σχετιζόμενη με τη νόσο στη ΧΛΛ δεν οφείλεται σε ενδογενείς διαταραχές των ερυθροποιητικών προδρομικών κυττάρων, αλλά πιθανότατα να αποδίδεται στην απευθείας κατασταλτική επίδραση του TNF-α . / Disease-related anemia in chronic lymphocytic leukemia (CLL) occurs when the obvious causes are excluded while its pathogenesis is still obscure. To investigate its underlying mechanisms we studied parameters of erythropoiesis at cellular and molecular level in newly diagnosed CLL patients with disease-related anemia in comparison with those of non-anemic CLL patients and normal controls. Bone marrow (BM) infiltration by leukemic B cells was not exclusively responsible for the presence and the severity of disease-related anemia and BM CD34+ cells were intrinsically capable of generating erythroid precursors. No deficiency of serum erythropoietin (EPO) or defective intracellular response of erythroid precursors to EPO ± Tumor Necrosis Factor-α (TNF-α) stimulation was also observed. On the other hand, serum TNF-α levels were found increased in patients with CLL and disease-related anemia and TNF-α appeared to exert a direct inhibitory effect on the differentiation of CD34+ cells towards the erythroid lineage in vitro. Our study showed that disease-related anemia in CLL was not due to intrinsic defects of erythroid precursors, but might result from the direct suppressive effect of TNF-α on the erythroid production.

[Truncated abstract] Erythropoietin (Epo) is a glycoprotein hormone involved in the formation of erythrocytes, by controlling survival, differentiation and proliferation. The technique of cDNA Representational Difference Analysis was utilized to investigate J2E-NR cells that demonstrate a viability response to Epo, but not differentiation or proliferation. The aim of this project was to identify genes that may be upregulated in response to Epo-induced survival; however, no change in gene expression was detected. This was most probably because any changes were below the limit of detectability for the cDNA RDA technique, or the viability effect was mediated post-transcriptionally. Next, it was decided to investigate HLS5, a putative tumour suppressor that was identified in a myeloid variant of the J2E cell line and had been shown to cause apoptosis. A number of HeLa cell lines inducible for Hls5 expression using the tet-off system were produced; despite extremely low expression, Hls5 was shown to produce marked suppression of growth and proliferation, particularly in colony assays colony size and numbers were halved for one induced clone. … A number of haemopoiesis-associated genes were downregulated (viz. globin genes and the Epo receptor gene), which suggested Hls5s role in the myeloid variant of J2E cells, may be to suppress genes expressed in the erythroid lineage. In addition, several interferon-responsive genes were decreased in cells with elevated HLS5, suggesting it may play a role in negatively regulating interferon signaling. Online databases were also searched for information on HLS5, and showed that it is significantly downregulated in liver, lung and uterine cancers, supporting the proposition that HLS5 is a tumour suppressor gene. In summary, a number of approaches were taken to identify the effects of the Hls5 protein. It appears that it strongly suppresses proliferation and that this is likely mediated through an effect on mitosis. This may also result in apoptosis of overexpressing cells. It is possible that this is the mechanism through which HLS5 exerts its potential tumour suppressor function, as a number of tumour suppressors appear to be associated with mitosis/apoptosis control. Hls5 is also likely to have other functions in haemopoietic cells, which includes downregulation of erythroid-specific genes and suppression of interferon responses.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 133-142). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Erythropoietin (EPO) is a glycoprotein which stimulates the erythropoiesis (production of hemaceas) and is clinically used for the treatment of renal anemia. EPO formulations present usually elevated contamination by aluminum. Aluminum is known to participate in the pathogenesis of osteodystrophy and encephalopathy associated with chronic hemodialysis, and has been proposed to be involved in central nervous system degeneration diseases such as Alzheimer s diseases. In the present work, the presence of Al as contaminant in erythropoietin formulations consumed by chronic renal patients, was investigated by developing and optimizing a chromatographic method for separation and determination of proteins, with subsequent collection of fractions and measured of the Al present by graphite furnace atomic absorption spectrometry (GF AAS). As the commercialization of pharmaceutical forms of EPO are in glass containers with rubber cap (bottle, vial or syringe), it was investigated the ability of the constituents of EPO formulations (salts, amino acids, urea, and mannitol among others) to promote the extraction of aluminum present in both glass and rubber. The tests were performed considering the process of sterilization and the contact of the solutions during storage. The results demonstrated that the interaction of EPO with aluminum is higher than that of albumin (the other proteic constituent). Among the other constituents, citric acid and citrate presented the highest interaction. For each constituent there is probably a different mechanism for the release of aluminum from glass and rubber, but all constituent of EPO formulations interacted with glass and rubber packaging, leading to their contamination by aluminum. As the level of contamination by aluminum in commercial formulations of erythropoietin was determined, and it was observed that the lyophilized powder presented the lowest contamination by aluminum, this form must be the choice for renal patients, due to the susceptibility of these patients to aluminum toxicity. / A eritropoetina (EPO) é uma glicoproteína que estimula a eritropoiese (produção das hemáceas) e é usada clinicamente para o tratamento de anemias associadas à insuficiência renal crônica. Formulações de eritropoetina apresentam geralmente uma elevada contaminação por alumínio, que é um metal extremamente tóxico para pacientes com insuficiência renal. Este metal é relacionado a doenças neurológicas e ao comprometimento da estrutura óssea dos pacientes, podendo causar a morte se administrado cronicamente ou em concentrações elevadas aos pacientes. Alguns autores também relacionam o desenvolvimento de doenças degenerativas como a doença de Alzheimer com intoxicação por alumínio. A presença do alumínio em formulações farmacêuticas destinadas ao tratamento da insuficiência renal e nutrição parenteral é um problema sério devido a toxicidade do alumínio e ao contato direto com o sangue do paciente. Em nosso laboratório, nos dedicamos a estudar a origem do aluminio nestas soluções e de que modo ele interage com os seus constituintes. No presente trabalho, investigou-se a presença de Al como contaminante em formulações farmacêuticas de eritropoetina consumida por pacientes renais crônicos através do desenvolvimento e otimização de um método cromatográfico para separação e determinação de proteínas, com posterior coleta de frações e medidas do Al presente por espectrometria de absorção atômica com forno de grafite (GF AAS). Como as formas farmacêuticas de comercialização da EPO são em recipientes de vidro com tampa de borracha (frasco-ampola ou seringas), investigou-se a capacidade dos constituintes das formulações de EPO (sais, aminoácidos, uréia e manitol entre outros) em promover a extração do alumínio presente tanto no vidro quanto na borracha. Os ensaios foram realizados considerando o processo de esterilização e o contato das soluções durante o período de armazenamento. Os resultados encontrados demonstraram que a EPO apresenta maior interação com o alumínio que a albumina (o outro constituinte proteico das formulações). Entre os outros constituintes, o ácido cítrico e o citrato apresentaram a maior interação. Para cada constituinte há provavelmente um mecanismo diferenciado de liberação de Al do vidro e da borracha, porém todos os constituintes das formulações de EPO interagiram com o vidro e a borracha, levando a contaminação destas por alumínio. Como foi determinado o nível de contaminação por alumínio das formulações de eritropoetina comerciais, observou-se que a forma farmacêutica de pó liofilizado apresentou menor contaminação por alumínio devendo, portanto, ser a forma de escolha para os pacientes renais, tão sucetíveis a toxicidade do alumínio.

A lesão de isquemia e reperfusão (IRI) continua a ser um problema clínico e o estresse do retículo endoplasmático (ERS) parece ser um importante mediador desse processo. A presença da heme oxigenase-1 (HO-1) ou do monóxido de carbono (CO), parece proteger da IRI. O objetivo do nosso trabalho foi avaliar a papel da HO-1 e CO na IRI renal. A indução da HO-1 em camundongos promoveu uma proteção na IRI renal, com melhora da função renal, menos inflamação e atenuação do ERS. Ao avaliarmos o papel do CO, verificamos que há também uma proteção, mediada por p38, vias purinérgicas, estabilização de HIF-1a e eritropoietina. Há ainda uma melhora do metabolismo energético celular após o tratamento com CO. Enfim, podemos concluir que, na presença da HO-1 ou do CO, há uma melhora da lesão isquêmica, através de uma maior ativação de vias citoprotetoras, com atenuação do ERS, redução da inflamação e consequente melhora da função renal. / Ischemia-reperfusion injury (IRI) remains a clinical problem and endoplasmic reticulum stress (ERS) seems to be an important mediator of this process. The presence of heme oxygenase-1 (HO-1) or carbon monoxide (CO) appears to protect from IRI. The aim of our study was to evaluate the role of HO-1 and CO in renal IRI. The induction of HO-1 in mice promoted protection in renal IRI with improved renal function, less inflammation and attenuation of ERS. When evaluating the role of CO, we found that there is also a protection mediated by p38, purinergic signaling, HIF-1a stabilization and erythropoietin. There is still an improvement of cellular energy metabolism after treatment with CO. Finally, we conclude that, in the presence of HO-1 or CO, there is an improvement of the ischemic lesion, through greater activation of cytoprotective pathways, with reduced ERS, reducing inflammation and consequent improvement in renal function.

Abstract Abnormal placentation is associated with preeclampsia and placental insufficiency, both of which increase the risk for fetal growth restriction. So far the early recognition of the risk population for preeclampsia has been problematic. The first hypothesis of this study was that in preeclampsia, the maternal serum proteomic profile is different from that in uncomplicated pregnancies, and this difference is detectable already in early pregnancy. The findings of this study demonstrate that in clinical preeclampsia the maternal serum proteomic profile is different from that in uncomplicated pregnancies with increased levels of placental proteins and antiangiogenic factors in pregnancies with clinical preeclampsia. Furthermore, the early pregnancy maternal serum proteomic profile in women who later develop preeclampsia revealed a distinct and different pattern compared with the profile in clinical preeclampsia. In early pregnancy, the differentially expressed proteins belong to placental proteins, vascular and/or transport proteins and matrix and/or acute phase proteins, while angiogenic and antiangiogenic proteins were not significantly expressed in early pregnancy. Preeclampsia, placental insufficiency, fetal growth restriction and type-1 diabetes may have an impact on fetal cardiovascular hemodynamics. The second hypothesis in this thesis was that in placental insufficiency, abnormalities in fetal cardiovascular status correlate with biochemical markers of cardiac dysfunction and chronic hypoxia. In placental insufficiency, increases in fetal N-terminal pro-atrial (NT-proANP) and pro-B-type natriuretic peptide (NT-proBNP) and in fetal erythropoietin concentrations were related to increased pulsatility in the fetal umbilical artery and descending aorta. In addition, these fetuses demonstrated increased pulsatility in their systemic venous blood velocity waveforms. Thus, in placental insufficiency, biochemical markers of cardiac dysfunction and chronic hypoxia are associated with signs of increased fetal cardiac afterload and systemic venous pressure. Increased NT-proANP and NT-proBNP levels were also detected in fetuses of type-1 diabetic mothers with normal umbilical artery velocimetry. In these pregnancies, NT-proANP and NT-proBNP levels were related to poor maternal glycemic control during early pregnancy.

Abstract Interruption of cerebral blood flow during hypothermic circulatory arrest (HCA) predisposes neurons to glutamate excitotoxicity. Reperfusion is followed by leukocyte infiltration, which results in an inflammatory reaction in the brain tissue. In the first study, the presynaptic glutamate release inhibitor lamotrigine (L) and the leukocyte-depleting filter (LF) were studied to determine if their combination could mitigate brain injury after HCA (I). The aim of the second study was to evaluate the possible neuroprotective effect of a 14-hour period of mild (32°C) hypothermia after HCA (II). Recent experimental research has demonstrated the neuroprotective properties of erythropoietin (EPO) and fructose-1,6-bisphosphate (FDP), whose effects during and after HCA were evaluated in the third and the fourth studies (III, IV). A chronic porcine model was used. The animals were randomly assigned to the study groups as follows: 8 animals in the L+LF group, 8 in the L group, and 8 in the control group (I); 10 animals in the hypothermia group and 10 in the normothermia group (II); 10 animals in the EPO group and 10 in the control group (III), and 12 animals in the FDP group and 12 in the control group (IV). Monitoring of hemodynamics, metabolism, temperature, electroencephalogram (EEG), brain microdialysis, intracranial pressure (II-IV), and brain tissue oxygen (II-IV) was carried out. A daily behavioral assessment was performed until death or until elective sacrifice on the seventh postoperative day, after which the brain was prepared for a histopathologic examination. The results of these studies indicate that lamotrigine has a neuroprotective effect during HCA. This is observed in terms of EEG burst recovery, behavioral and histopathologic outcome, and brain microdialytic findings. The combined use of lamotrigine and leukocyte filtration may further improve survival. A 14-hour period of mild hypothermia after HCA is associated with a poor outcome. However, it may preserve its efficacy when used for no longer than 4 hours. Administration of EPO before HCA proved ineffective in reducing mortality or brain histopathologic injury. Findings from brain microdialysis, brain tissue oxygen tension, and neuronal apoptosis, however, suggest that the drug has neuroprotective properties. Administration of FDP before and after HCA is associated with better survival, behavioral outcome, and brain histopathologic scores. The metabolic and brain microdialytic findings also suggest that this drug has supportive effects on myocardial and brain metabolism.

L'érythropoïétine (EPO) est une cytokine ayant un rôle important dans l'homéostasie de l'oxygène (O2). Lors d'une hypoxie chronique, l'EPO stimule la maturation des progéniteurs érythroïdes en globules rouges augmentant ainsi le transport de l'O2 aux tissus. Outre cet effet érythropoïétique, l'EPO module la réponse ventilatoire à l'hypoxie (RVH) par une action directe sur la commande centrale respiratoire (CCR) et les chémorécepteurs périphériques. Cet effet a été principalement caractérisé chez des souris mutantes surexprimant l'EPO. Cependant, plusieurs aspects de l'effet de l'EPO sur l'activité du réseau respiratoire demeurent inconnus. Nos résultats montrent qu'une application aigüe d'EPO diminue la dépression centrale hypoxique mesurée in vitro chez le nouveau-né. En revanche, elle n'affecte pas la RVH mesurée in vivo au cours du développement postnatal mais diminue la fréquence des apnées survenant en hypoxie sévère à 6% d'O2. Aussi, chez la souris adulte, l'administration chronique d'EPO et de C-EPO augmente la sensibilité des chémorécepteurs périphériques à l'O2 et maintient la ventilation durant la phase tardive de la RVH. Enfin, l'EPO diminue la sensibilité ventilatoire à l'hypercapnie grâce à des effets périphériques et centraux. L'ensemble de nos résultats montrent que l'EPO module la respiration et contribue à l'homéostasie de l'O2 et du CO2 grâce à ses effets plasmatiques et centraux. Elle représente un candidat à fort potentiel thérapeutique pour les pathologies respiratoires où la sensibilité chimique à l'O2 et au CO2 sont altérés telles que l'apnée du nouveau-né ou le mal chronique des montagnes. / Erythropoietin (EPO) is a cytokine that plays a major role in O2 homeostasis. Upon chronic hypoxia, EPO stimulates the maturation of erythroid progenitors into red blood cells, contributing to increased O2 carrying to tissues. Besides this well-known erythropoietic effect, EPO also modulates the respiratory response to hypoxia by interacting with the central respiratory network in the brainstem and the peripheral chemoreceptors. This effect was mainly characterized in adult mutant mice that overexpress EPO. Several aspects regarding EPO's effect on breathing regulation remain unknown. Our results show that acute EPO treatment increases the O2 sensitivity of the central respiratory network in newborn mice in vitro. However, EPO does not impact the hypoxic ventilatory response to hypoxia in vivo, but decreases the apneic events during severe hypoxia in mice at postnatal day 7. In WT adults, chronic but not acute EPO and C-EPO treatment increases the O2 sensitivity by stimulating both peripheral chemoreceptor and central respiratory network. Finally, both cerebral and plasmatic EPO blunt the ventilatory response to increased CO2 levels in adult mice. Taken together, these results imply that EPO, by acting on the ventilatory system, plays a key role in the modulation of the chemical sensitivity to O2 and CO2.

Iron is a metal element with crucial roles in human organism. Both iron deficiency and iron overload are important pathologies. Hepcidin, a peptide synthetized in the liver, is a key iron regulatory hormone. Increased amount of iron and inflammation stimulate its expression while iron deficiency and activated erythropoiesis cause hepcidin downregulation. The regulation of hepcidin expression on the molecular level and its hierarchy and interactions are not completely known. The main regulatory pathway is BMP/ SMAD which reacts to the iron amount in the organism. Several molecules, including hemojuvelin and HFE, are involved in this pathway and their mutations are linked to inappropriately low hepcidin production, iron overload and hereditary hemochromatosis. Erythroid regulation with suppressive action on hepcidin expression is known only partially as well as its connection to the BMP/ SMAD pathway. Recently, two new negative regulators of hepcidin expression have been described. Membrane enzyme present in hepatocytes - matriptase-2 (MT-2, TMPRSS6) and soluble factor secreted by erythroblasts - erythroferrone (ERFE). The aim of our work was to investigate how MT-2 is involved in the erythroid regulatory pathway, and whether it can represent the molecule where various regulatory pathways interact....