Erythropoietin enhances iron bioavailability in HepG2 cells by downregulating hepcidin through mTOR, C/EBPα and HIF-1α.

The regulation of iron (Fe) levels is essential to maintain an adequate supply for erythropoiesis, among other processes, and to avoid possible toxicity. The liver-produced peptide hepcidin is regarded as the main regulator of Fe absorption in enterocytes and release from hepatocytes and macrophages, as it impairs Fe export through ferroportin. The glycoprotein erythropoietin (Epo) drives erythroid progenitor survival and differentiation in the bone marrow, and has been linked to the mobilization of Fe reserves necessary for hemoglobin production. Herein we show that Epo inhibits hepcidin expression directly in the HepG2 hepatic cell line, thus leading to a decrease in intracellular Fe levels. Such inhibition was dependent on the Epo receptor-associated kinase JAK2, as well as on the PI3K/AKT/mTOR pathway, which regulates nutrient homeostasis. Epo was also found to decrease binding of the C/EBP-α transcription factor to the hepcidin promoter, which could be attributed to an increased expression of its inhibitor CHOP. Epo did not only hinder the stimulating effect of C/EBP-α on hepcidin transcription, but also favored hepcidin inhibition by HIF-1α, by increasing is nuclear translocation as well as its protein levels. Moreover, in assays with the inhibitor genistein, this transcription factor was found necessary for Epo-induced hepcidin suppression. Our findings support the involvement of the PI3K/AKT/mTOR pathway in the regulation of Fe levels by Epo, and highlight the contrasting roles of the C/EBP-α and HIF-1α transcription factors as downstream effectors of the cytokine in this process.

Erythropoietin and derivatives: Potential beneficial effects on the brain.

Erythropoietin (Epo), the main erythropoiesis-stimulating factor widely prescribed to overcome anemia, is also known nowadays for its cytoprotective action on non-hematopoietic tissues. In this context, Epo showed not only its ability to cross the blood-brain barrier, but also its expression in the brain of mammals. In clinical trials, recombinant Epo treatment has been shown to stimulate neurogenesis; improve cognition; and activate antiapoptotic, antioxidant, and anti-inflammatory signaling pathways. These mechanisms, proposed to characterize a neuroprotective property, opened new perspectives on the Epo pharmacological potencies. However, many questions arise about a possible physiological role of Epo in the central nervous system (CNS) and the factors or environmental conditions that induce its expression. Although Epo may be considered a strong candidate to be used against neuronal damage, long-term treatments, particularly when high Epo doses are needed, may induce thromboembolic complications associated with increases in hematocrit and blood viscosity. To avoid these adverse effects, different Epo analogs without erythropoietic activity but maintaining neuroprotection ability are currently being investigated. Carbamylated erythropoietin, as well as alternative molecules like Epo fusion proteins and partial peptides of Epo, seems to match this profile. This review will focus on the discussion of experimental evidence reported in recent years linking erythropoietin and CNS function through investigations aimed at finding benefits in the treatment of neurodegenerative diseases. In addition, it will review the proposed mechanisms for novel derivatives which may clarify and, eventually, improve the neuroprotective action of Epo.

Role of protein tyrosine phosphatase 1B (PTP1B) in the increased sensitivity of endothelial cells to a promigratory effect of erythropoietin in an inflammatory environment.

The proliferation and migration of endothelial cells are vascular events of inflammation, a process which can also potentiate the effects of promigratory factors. With the aim of investigating possible modifications in the activity of erythropoietin (Epo) in an inflammatory environment, we found that Epo at a non-promigratory concentration was capable of stimulating EA.hy926 endothelial cell migration when TNF-α was present. VCAM-1 and ICAM-1 expression, as well as adhesion of monocytic THP-1 cells to endothelial layers were also increased. Structurally modified Epo (carbamylation or N-homocysteinylation) did not exhibit these effects. The sensitizing effect of TNF-α on Epo activity was mediated by the Epo receptor. Inhibition assays targeting the PI3K/mTOR/NF-κB pathway, shared by Epo and TNF-α, show a cross-talk between both cytokines. As observed in assays using antioxidants, cell migration elicited by TNF-α + Epo depended on TNF-α-generated reactive oxygen species (ROS). ROS-mediated inactivation of protein tyrosine phosphatase 1B (PTP1B), involved in Epo signaling termination, could explain the synergistic effect of these cytokines. Our results suggest that ROS generated by inflammation inactivate PTP1B, causing the Epo signal to last longer. This mechanism, along with the cross-talk between both cytokines, could explain the sensitizing action of TNF-α on the migratory effect of Epo.

Aquaporin-1 plays a key role in erythropoietin-induced endothelial cell migration.

Water influx through aquaporin-1 (AQP-1) has been linked to the ability of different cell types to migrate, and therefore plays an important part in processes like metastasis and angiogenesis. Since the erythroid growth factor erythropoietin (Epo) is now recognized as an angiogenesis promoter, we investigated the participation of AQP-1 as a downstream effector of this cytokine in the migration of endothelial cells. Inhibition of AQP-1 with either mercury ions (Hg2+) or a specific siRNA led to an impaired migration of EA.hy926 endothelial cells exposed to Epo (wound-healing assays). Epo also induced the expression of AQP-1 at mRNA and protein levels, an effect which was dependent on the influx of extracellular calcium through L-type calcium channels as well as TRPC3 channels. The relationship between Epo and AQP-1 was further confirmed at shorter exposure times, as the cytokine was unable to trigger calcium influxes in cells where AQP-1 had previously been knocked down. Moreover, Epo promoted changes in the subcellular localization of AQP-1 as well as rearrangements in the actin cytoskeleton, which are consistent with a migratory phenotype. Worthy of note, carbamylated erythropoietin (cEpo), the non-erythropoietic and non-promigratory derivative of Epo, was incapable of AQP-1 modulation. The therapeutical implications of aquaporin targeting in angiogenesis-related diseases highlight the importance of the present results in the context of the relationship between AQP-1 and Epo.

Esferocitosis hereditaria: experiencia clínica y diagnóstica en Argentina / Hereditary spherocytosis: clinical and diagnostic experience in Argentina / Esferocitose hereditária: experiência clínica e diagnóstica na Argentina

Los objetivos del presente estudio fueron: a) Analizar las características demográficas y clínicas de nuestra población al diagnóstico; b) Evaluar si las pruebas más recientes presentan ventajas sobre las tradicionales; c) Confirmar la frecuencia de las distintas deficiencias de proteínas de membrana; d) Establecer la relación entre severidad y resultado de las pruebas o tipo de deficiencia. Se analizaron 359 individuos estudiados desde 2007, cuando se incorporaron criohemólisis hipertónica (CH), citometría de flujo con eosina-5'- maleimida (5'EMA-CF), FOE por citometría de flujo (FOE-CF) y electroforesis de proteínas de membrana (SDS-PAGE) al estudio de laboratorio clásico, fragilidad osmótica eritrocitaria (FOE) y autohemólisis (AH). Criterios diagnósticos para Esferocitosis Hereditaria (ESH): esferocitos en frotis y dos pruebas positivas. Se identificaron 174 pacientes con ESH y 22 portadores sanos. El 74,9% eran menores de 12 años. La transmisión fue dominante en el 83,1% de los casos. Tuvieron manifestaciones neonatales 89,1%. Las pruebas con mayor sensibilidad fueron CH (92,0%), FOE diferida (91,1%) y 5'EMA-CF (88,5%). En los 125 pacientes en quienes se realizaron CH, 5'EMA-CF y FOE-CF se observó que todos tenían al menos una prueba positiva; 122 (97,6%) tuvieron dos o tres positivas. Las deficiencias más frecuentes fueron ankirina y espectrina. No hubo diferencia en el resultado de las pruebas entre los subgrupos de severidad. Se concluye que las deficiencias más frecuentes en Argentina son ankirina y espectrina, coincidiendo con otras poblaciones latinoamericanas. El uso simultáneo de CH, 5'EMA-CF y FOE-CF permite diagnosticar más del 97% de los casos. La incidencia de manifestaciones neonatales es elevada.

The aims of this study were (a) to assess demographic and clinical aspects of our population at diagnosis; (b) to evaluate diagnostic accuracy of hypertonic cryohemolysis (HC), eosin-5'-maleimide flow cytometry (EMA-FC) and flow cytometric osmotic fragility (OF-FC) in relation to standard screening tests osmotic fragility (OF) and autohemolysis (AH); (c) to confirm the previously reported prevalence of membrane proteins defects; and (d) to assess the relationship between severity of anemia and results of confirmatory tests. Since 2007, the following tests were available in our laboratory: OF, AH, HC, EMA-FC, OF-FC and SDS-PAGE of membrane proteins. Diagnostic criteria for hereditary spherocytosis were spherocytes in blood smear plus ≥2 positive tests. Data from 359 individuals were analyzed: 174 HS patients and 22 silent carriers were detected; 74.9% of patients were less than 12 years old; 83.1% of them showed a dominant inheritance pattern; antecedent of neonatal jaundice/anemia was registered in 89.1%. Tests with higher sensitivity were: HC (92.0%), incubated OF (91.1%), and EMA-FC (88.5%). HC, EMA-FC and OF-FC were simultaneously performed on 125 patients: each of them had at least 1 positive test; 122 (97.6%) had 2 or 3 positive tests. Ankyrin and spectrin were the most frequently found protein deficiencies. Comparison of test results in relation to severity of anemia showed no difference between groups. It can be concluded that compared toother Latin American countries, ankyrin and spectrin were the most frequent protein deficiencies. Simultaneous performing of HC, EMA-FC and OF-FC enabled diagnosing HS in more than 97% of patients. A high incidence of neonatal jaundice/anemia was observed.

Os objetivos do presente estudo foram: a) analisar as características demográficas e clínicas de nossa população ao diagnóstico; b) Avaliar se as provas mais recentes apresentam vantagens sobre as tradicionais; c) Confirmar a frequência das diversas deficiências de proteínas de membrana; d) Establecer a relação entre severidade e resultado das provas ou tipo de deficiência. Foram analisados 359 indivíduos estudados desde 2007, quando se incorporaram crio-hemólise hipertônica (CH), citometria de fluxo com eosina-5'-maleimida (5'EMA-CF), FOE por citometria de fluxo (FOE-CF) e eletroforese de proteínas de membrana (SDS-PAGE) ao estudo de laboratório clássico - fragilidade osmótica eritrocitária (FOE) e auto-hemólise (AH). Critérios diagnósticos para ESH: esferócitos em esfregaço e duas provas positivas. Foram identificados 174 pacientes com ESH e 22 portadores sadios. 74,9% eram menores de 12 anos. A transmissão foi dominante em 83,1%. Tiveram manifestações neonatais 89,1%. As provas com maior sensibilidade foram CH (92,0%), FOE diferida (91,1%) e 5'EMA-CF (88,5%). Nos 125 pacientes aos quais lhes realizaram CH, 5'EMA-CF e FOE-CF se observou que todos tinham no mínimo uma prova positiva; 122 (97,6%) tiveram duas ou três positivas. As deficiências mais frequentes foram anquirina e espectrina. Não houve diferença no resultado das provas entre os subgrupos de severidade. Conclui-se que as deficiências mais frequentes na Argentina são anquirina e espectrina, as quais coincidem com outras populações latinoamericanas. O uso simultâneo de CH, 5'EMA-CF e FOE-CF permite diagnosticar mais de 97% dos casos. A incidência de manifestações neonatais é elevada.

Eritropoyetina como agente eritropoyético y no eritropoyético: consideraciones terapéuticas / Erythropoietic and non-erythropoietic functions of erythropoietin: therapeutic considerations / Eritropoietina como agente eritropoiético e não eritropoiético: considerações terapêuticas / Erythropoietic and non-erythropoietic functions of erythropoietin: therapeutic considerations

La producción de glóbulos rojos es controlada continuamente para suplir la desaparición de las células envejecidas y garantizar un aporte de oxígeno adecuado a todo el organismo. La citoquina pleitrópica eritropoyetina (Epo), originalmente definida por su rol en la eritropoyesis para prevenir la muerte programada de progenitores eritroides en la médula ósea, ha demostrado un rol antiapoptótico protector sobre diversos tejidos no hematopoyéticos. A la reconocida eficacia del tratamiento con eritropoyetina recombinante humana (rhuEpo) para contrarrestar la anemia que acompaña a patologías muy diversas, se agregan algunos aspectos que impiden lograr los resultados terapéuticos esperados, ya sea por resistencia al tratamiento o por el desarrollo de efectos adversos. Con el fin de prevenir estos efectos, así como reducir las dosis de rhuEpo en tratamientos crónicos se han desarrollado nuevos agentes que presentan modificaciones estructurales de la Epo, o bien alteraciones en las propiedades/actividad de la Epo nativa. Dado que, actualmente, los resultados sobre los efectos de la Epo sobre morbilidad/ mortalidad en diversas patologías no están suficientemente claros, nuevas investigaciones serán útiles para resolver dudas sobre la efectividad de la eritropoyetina y sus derivados o agentes alternativos con el fin de proveer bases sólidas para el desarrollo de ensayos clínicos concluyentes.

Erythropoietin (Epo), the cytokine required for promoting erythropoiesis through the proliferation and differentiation of erythroid cells, has been reported to act as a pleiotropic cytokine beyond the hematopoietic system. In contrast with the potentially beneficial effects attributed to recombinant human erythropoietin (rhuEpo), research has advanced to indicate that mortality and morbidity rates are increased in some patient groups when treated with rhuEpo. Some cardiac and systemic conditions may predispose to adverse events, and other factors, such as proinflammatory agents, may lead to resistance to erythropoietin treatment. Many compounds are currently under investigation in order to avoid these unwanted effects and to reduce the rhuEpo dose during chronic therapies. They are either erythropoiesis-stimulating agents different from erythropoietin or structurally modified erythropoietins with altered properties and activities. In recent reports, contrasting data have raised several concerns regarding the effectiveness of erythropoietin treatment to prevent adverse events. Therefore, much investigation is needed to provide a solid basis for the development of conclusive clinical trials.

A produção de glóbulos vermelhos é controlada continuamente para suprir o desaparecimento das células envelhecidas e garantir uma contribuição de oxigênio adequado a todo o organismo. A citocina pleiotrópica eritropoietina (Epo), originalmente definida por seu papel na eritropoiese para prevenir a morte programada de progenitores eritroides na medula óssea, tem demonstrado um papel anti-apoptótico protetor sobre diversos tecidos não hematopoiéticos. Adicionam-se à reconhecida eficácia do tratamento com eritropoietina recombinante humana (rhuEpo), para contra-arrestar a anemia que acompanha patologias muito diversas, alguns aspectos que impedem alcançar os resultados terapêuticos esperados, quer seja por resistência ao tratamento ou pelo desenvolvimento de efeitos adversos. Com o fim de prevenir estes efeitos, bem como reduzir as doses de rhuEpo em tratamentos crônicos foram desenvolvidos novos agentes que apresentam modificações estruturais da Epo, ou então alterações nas propriedades/atividade da Epo nativa. Devido a que, atualmente, os resultados sobre os efeitos da Epo sobre morbidade/mortalidade em diversas patologias não estão suficientemente claros, novas pesquisas serão úteis para resolver dúvidas sobre a efetividade da eritropoietina e seus derivados ou agentes alternativos visando a fornecer bases sólidas para o desenvolvimento de ensaios clínicos concludentes.

Red blood cell aquaporin-1 expression is decreased in hereditary spherocytosis.

Aquaporin-1 (AQP1) is the membrane water channel responsible for changes in erythrocyte volume in response to the tonicity of the medium. As the aberrant distribution of proteins in hereditary spherocytosis (HS) generates deficiencies of proteins other than those codified by the mutated gene, we postulated that AQP1 expression might be impaired in spherocytes. AQP1 expression was evaluated through flow cytometry in 5 normal controls, 1 autoimmune hemolytic anemia, 10 HS (2 mild, 3 moderate, 2 severe, and 3 splenectomized), and 3 silent carriers. The effect of AQP1 inhibitors was evaluated through water flow-based tests: osmotic fragility and hypertonic cryohemolysis. Serum osmolality was measured in 20 normal controls and 13 HS. The effect of erythropoietin (Epo) on AQP1 expression was determined in cultures of erythroleukemia UT-7 cells, dependent on Epo to survive. Independent of erythrocyte size, HS patients showed a lower content of AQP1 in erythrocyte membranes which correlated with the severity of the disease. Accordingly, red blood cells from HS subjects were less sensitive to cryohemolysis than normal erythrocytes after inhibition of the AQP1 water channel. A lower serum osmolality in HS with respect to normal controls suggests alterations during reticulocyte remodeling. The decreased AQP1 expression could contribute to explain variable degrees of anemia in hereditary spherocytosis. The finding of AQP1 expression induced by Epo in a model of erythroid cells may be interpreted as a mechanism to restore the balance of red cell water fluxes.

Social and environmental health determinants and their relationship with parasitic diseases in asymptomatic children from a shantytown in Buenos Aires, Argentina.

Health inequities are a common problem for all countries and are the result of not only adverse social conditions but also poor public policies. Today chronic diseases represent the most relevant threats and are a current challenge. Parasitic infections, a leading cause of child morbidity affecting low-income populations, can be transmitted because of an unhealthy environment. Notwithstanding, scarce data have been published on the epidemiological profile of intestinal parasitoses in asymptomatic children living in shantytowns. Vulnerable populations settled in slums are growing in Argentina, particularly in Buenos Aires city. Consequently, this work intended to screen healthy carriers of enteric parasites and determine the epidemiologic profile in asymptomatic children residing in one of those communities, to explore risk factors associated with the transmission of parasites, and to initiate a basic health education campaign to promote healthy behavior in the community. Fecal samples (n = 138) were analyzed by conventional parasitological methods and a survey gathered data on symptoms, family composition, and environmental and hygiene-related variables. High prevalence of feco-orally-transmitted parasitoses (83·3%) and polyparasitism were remarkable findings. The main environmental health determinants were those related to excreta disposal and water provision. Health promotion actions were performed through the diffusion of a set of posters with iconic images and brief messages for health education. Results suggest the need for an environmental sanitation policy to complement health promotion actions. It is essential to spread the results of investigations that address inequities and social determinants of health in order to integrate data with local political processes and alert on acceptable actions for developing appropriate interventions.

Signaling pathways of cell proliferation are involved in the differential effect of erythropoietin and its carbamylated derivative.

It is now recognized that in addition to its activity upon erythroid progenitor cells, erythropoietin (Epo) is capable of stimulating survival of different non-erythroid cells. Since stimulation of erythropoiesis is unwanted for neuroprotection, Epo-like compounds with a more selective action are under investigation. Although the carbamylated derivative of erythropoietin (cEpo) has demonstrated non-hematopoietic tissue protection without erythropoietic effect, little is known about differential mechanisms between Epo and cEpo. Therefore, we investigated signaling pathways which play a key role in Epo-induced proliferation. Here we show that cEpo blocked FOXO3a phosphorylation, allowing expression of downstream target p27(kip1) in UT-7 and TF-1 cells capable of erythroid differentiation. This is consistent with the involvement of cEpo in slowing down G1-to-S-phase progression compared with the effect of Epo upon cell cycle. In contrast, similar antiapoptotic actions of cEpo and Epo were observed in neuronal SH-SY5Y cells. Inhibition and competition assays suggest that Epo may act through both, the homodimeric (EpoR/EpoR) and the heterodimeric (EpoR/ßcR) receptors in neuronal SH-SY5Y cells and probably in the TF-1 cell type as well. Results also indicate that cEpo needs both the EpoR and ßcR subunits to prevent apoptosis of neuronal cells. Based on evidence suggesting that cell proliferation pathways were involved in the differential effect of Epo and cEpo, we went forward to studying downstream signals. Here we provide the first evidence that unlike Epo, cEpo failed to induce FOXO3a inactivation and subsequent p27(kip1) downregulation, which is clearly shown in the incapacity of cEpo to induce erythroid cell growth.

Protective action of erythropoietin on neuronal damage induced by activated microglia.

Inflammation is a physiological defense response, but may also represent a potential pathological process in neurological diseases. In this regard, microglia have a crucial role in either progression or amelioration of degenerative neuronal damage. Because of the role of hypoxia in pro-inflammatory mechanisms in the nervous system, and the potential anti-inflammatory protective effect of erythropoietin (Epo), we focused our investigation on the role of this factor on activation of microglia and neuroprotection. Activation of microglial cells (EOC-2) was achieved by chemical hypoxia induced by cobalt chloride (CoCl2 ) and characterized by increased levels of nitrite, tumor necrosis factor-α and reactive oxygen species production, as well as up-regulation of inducible nitric oxide synthase expression. Under these conditions, cell proliferation data and proliferating cell nuclear antigen (PCNA) staining demonstrated a mitogenic effect of chemical hypoxia. Even though pre-treatment with Epo did not prevent nitrite production, inducible nitric oxide synthase protein expression or tumor necrosis factor-α secretion, it prevented the oxidative stress induced by CoCl2 as well as cell proliferation. Neuronal cells (SH-SY5Y) cultured in the presence of conditioned medium from activated EOC-2 cells or macrophages (RAW 264.7) developed significant apoptosis, an effect that was abolished by Epo via Epo/Epo receptor activation. The results show that even though Epo did not exert a direct anti-inflammatory effect on microglia activation, it did increase the resistance of neurons to subsequent damage from pro-inflammatory agents. In addition to its anti-apoptotic ability, the Epo antioxidant effect may have an indirect influence on neuronal survival by modulation of the pro-inflammatory environment.

Oxidative stress due to aluminum exposure induces eryptosis which is prevented by erythropoietin.

The widespread use of aluminum (Al) provides easy exposure of humans to the metal and its accumulation remains a potential problem. In vivo and in vitro assays have associated Al overload with anemia. To better understand the mechanisms by which Al affects human erythrocytes, morphological and biochemical changes were analyzed after long-term treatment using an in vitro model. The appearance of erythrocytes with abnormal shapes suggested metal interaction with cell surface, supported by the fact that high amounts of Al attached to cell membrane. Long-term incubation of human erythrocytes with Al induced signs of premature erythrocyte death (eryptosis), such as phosphatidylserine externalization, increased intracellular calcium, and band 3 degradation. Signs of oxidative stress, such as significant increase in reactive oxygen species in parallel with decrease in the amount of reduced glutathione, were also observed. These oxidative effects were completely prevented by the antioxidant N-acetylcysteine. Interestingly, erythrocytes were also protected from the prooxidative action of Al by the presence of erythropoietin (EPO). In conclusion, results provide evidence that chronic Al exposure may lead to biochemical and morphological alterations similar to those shown in eryptosis induced by oxidant compounds in human erythrocytes. The antieryptotic effect of EPO may contribute to enhance the knowledge of its physiological role on erythroid cells. Irrespective of the antioxidant mechanism, this property of EPO, shown in this model of Al exposure, let us suggest potential benefits by EPO treatment of patients with anemia associated to altered redox environment.

c-FLIP is involved in erythropoietin-mediated protection of erythroid-differentiated cells from TNF-alpha-induced apoptosis.

The TNF-alpha (tumour necrosis factor) affects a wide range of biological activities, such as cell proliferation and apoptosis. Cell life or death responses to this cytokine might depend on cell conditions. This study focused on the modulation of factors that would affect the sensitivity of erythroid-differentiated cells to TNF-alpha. Hemin-differentiated K562 cells showed higher sensitivity to TNF-induced apoptosis than undifferentiated cells. At the same time, hemin-induced erythroid differentiation reduced c-FLIP (cellular FLICE-inhibitory protein) expression. However, this negative effect was prevented by prior treatment with Epo (erythropoietin), which allowed the cell line to maintain c-FLIP levels. On the other hand, erythroid-differentiated UT-7 cells - dependent on Epo for survival - showed resistance to TNF-alpha pro-apoptotic action. Only after the inhibition of PI3K (phosphatidylinositol-3 kinase)-mediated pathways, which was accompanied by negative c-FLIP modulation and increased erythroid differentiation, were UT-7 cells sensitive to TNF-alpha-triggered apoptosis. In summary, erythroid differentiation might deregulate the balance between growth promotion and death signals induced by TNF-alpha, depending on cell type and environmental conditions. The role of c-FLIP seemed to be critical in the protection of erythroid-differentiated cells from apoptosis or in the determination of their sensitivity to TNF-mediated programmed cell death. Epo, which for the first time was found to be involved in the prevention of c-FLIP down-regulation, proved to have an anti-apoptotic effect against the pro-inflammatory factor. The identification of signals related to cell life/death switching would have significant implications in the control of proliferative diseases and would contribute to the understanding of mechanisms underlying the anaemia associated with inflammatory processes.

Differential antiapoptotic effect of erythropoietin on undifferentiated and retinoic acid-differentiated SH-SY5Y cells.

Erythropoietin (Epo) is known to have a significant role in tissues outside the hematopoietic system. In this work, we investigated the function of Epo in cells of neuronal origin subjected to differentiation. Treatment of SH-SY5Y cells with all-trans-retinoic acid (atRA) generated differentiated neuron-like cells, observed by increased expression of neuronal markers and morphological changes. Exposure of undifferentiated cells to proapoptotic stimuli such as staurosporine, TNF-alpha, or hypoxia, significantly increased programmed cell death, which was prevented by previous treatment with Epo. In contrast, atRA-differentiated cultures showed cell resistance to apoptosis. No additional effect of Epo was detected in previously differentiated cells. The inhibition of the PI3K/Akt pathway by Ly294002 abrogated the protective effects induced by either Epo or atRA. The effect of atRA was mediated by an increased expression of Bcl-2 whereas the Epo treatment upregulated not only Bcl-2 but also Bcl-xL. This upregulation by Epo was not detected in atRA-differentiated cells, thus confirming the lack of the protective effect of Epo. As expected, assays with AG490, an inhibitor of Jak2, blocked the Epo action only in undifferentiated cells. This reduced neuroprotective function of Epo on SH-SY5Y differentiated cells could be explained at least in part by downregulation of the Epo receptor expression, which was observed in atRA-differentiated cells. This study shows differential cellular protection induced by Epo at two stages of SH-SY5Y differentiation. The results allow us to suggest that this differential cell behavior can be ascribed to the interaction between atRA and the signaling pathways mediated by Epo.

Vasoactive intestinal peptide inhibits TNF-alpha-induced apoptotic events in acinar cells from nonobese diabetic mice submandibular glands.

INTRODUCTION: The role of apoptotic secretory epithelium as a pro-inflammatory triggering factor of exocrine dysfunction is currently explored in Sjogren's syndrome patients and in the nonobese diabetic (NOD) mouse model. Vasoactive intestinal peptide (VIP) has anti-inflammatory effects in various models of chronic inflammation. Our goal was to analyse the effect of TNF-alpha on apoptotic mediators in isolated acinar cells from NOD submandibular gland and their modulation by VIP. METHODS: Acinar cells were isolated from submandibular glands of 16-week-old NOD females with salivary flow decline. Age-matched BALB/c females or eight-week-old NOD females were used as controls. Apoptotic mediators and TNF-alpha receptor expression were assessed by immunoblotting and RT-PCR, caspase 3 activity was assessed by optical density at 405 nm with Ac-DEVD-pNA as a substrate and chromatin condensation by Hoechst stain. They were evaluated in resting conditions and after a 3.5 or 6 hours of culture with TNF-alpha. VIP effects in acinar cells were assessed at 100 nM in TNF-alpha-treated cultures and VIP receptor functional assays by radio immunoassay (cAMP) or enzymatic detection (amylase). RESULTS: NOD acinar cells at 16 weeks present an increased expression of TNF-alpha receptor1 together with increased Bax, tumour protein 53-induced nuclear protein1alpha (TP53INP1alpha), caspase 3 activity and chromatin condensation. Acini from NOD mice were more sensitive to TNF-alpha-induced increases of apoptotic mediators than control cells. VIP inhibited TNF-alpha-induced apoptotic events through functional VPAC1 receptors coupled to the protein kinase A (PKA) signalling pathway. CONCLUSIONS: Our results indicate that acinar cells isolated from submandibular glands of NOD mice with salivary dysfunction are more sensitive to apoptosis induced by TNF-alpha which could be prevented by VIP through a PKA-mediated pathway.

TNF-alpha-induced apoptosis is prevented by erythropoietin treatment on SH-SY5Y cells.

The growth factor erythropoietin (Epo) has shown neuronal protective action in addition to its well known proerythroid activity. Furthermore, Epo has dealt with cellular inflammation by inhibiting the expression of several proinflammatory cytokines, such as IL-1 and TNF-alpha. The action of TNF can have both apoptotic and antiapoptotic consequences due to altered balance between different cell signalling pathways. This work has focused on the apoptotic effects of this cytokine and the potential protective action of Epo. The model we used was neuroblastoma SH-SY5Y cells cultured in the presence of 25 ng/ml TNF-alpha or pretreated with 25 U/ml Epo for 12 h before the addition of TNF-alpha. Apoptosis was evaluated by differential cell count after Hoechst staining, analysis of DNA ladder pattern, and measurement of caspase activity. Despite its ability to induce NF-kappaB nuclear translocation, TNF-alpha induced cell death, which was found to be associated to upregulation of TNF Receptor 1 expression. On the other hand, cells activated by Epo became resistant to cell death. Prevention of death receptor upregulation and caspase activation may explain this antiapoptotic effect of Epo, which may be also favoured by the induction of a higher expression of protective factors, such as Bcl-2 and NF-kappaB, through mechanisms involving Jak/STAT and PI3K signalling pathways.

[Diagnosis of familial amyloid polyneuropathy type I in Argentina]. / Diagnóstico de polineuropatía amiloidótica familiar tipo I en la Argentina.

Familial amyloid polyneuropathy (FAP) is an autosomal dominant inherited disease, characterized by systemic deposition of amyloid fibrils in various tissues, especially in peripheral nerves, being a variant of transthyretin (TTR) the principal component of amyloid fibrils. TTR is a normal plasma protein (previously called prealbumin) that functions as a transport protein binding tiroxine and retinol. Among many mutations that have been found in the TTR gene, the variant with a single amino acid substitution of methionine for valine at position 30 (TTR Val30Met) is the responsible of the Portuguese-type Familial Amyloidotic Polyneuropathy (FAP Type I). Interest in this pathology has arisen in Argentina because of the finding of an endemic area where a group of Portuguese immigrant families is localized. Since liver transplantation is a widely accepted treatment because it results in the disappearance of variant transthyretin from plasma, an early detection of the altered gene is essential. Thus, the objective of the present work was to optimize a methodology to detect the Val30Met mutation introducing modifications into techniques that were previously developed. The simple method here described is useful to confirm the diagnosis of the potential disease and, therefore, make it possible for patients to gain access to early liver transplantation.

Diagnóstico de polineuropatía amiloidótica familiar tipo I en la Argentina / Diagnosis of familial amyloid polyneuropathy type I in Argentina

La polineuropatía amiloidótica familiar (PAF) es un tipo de amiloidosis hereditaria. Constituye un desorden autosómico dominante caracterizado por el depósito sistémico de material amiloide en tejidos especialmente en nervios periféricos. El principal componente del amiloide es una variante mutada de la transtiretina (TTR), proteína transportadora de tiroxina y retinol. Han sido descriptas numerosas mutaciones en el gen TTR que causan alteración de la secuencia primaria de la proteína. La PAF portuguesa o PAF Tipo I se origina por la variante TTR Val30Met en la cual una valina en posición 30 es reemplazada por una metionina. Es fundamental la identificación temprana de portadores de la mutación porque una vez declarada la enfermedad el único tratamiento efectivo es el trasplante hepático, órgano de síntesis de la TTR. La PAF Tipo I ha sido muy estudiada en la Argentina debido al hallazgo de un área endémica donde habitan familias descendientes de inmigrantes portugueses. El presente trabajo ha sido enfocado a resolver la necesidad diagnóstica de la comunidad, ya que la ausencia de una metodología apropiada en nuestro país ha impedido, hasta ahora, que individuos con antecedentes familiares de PAF puedan tener un diagnóstico precoz y acceder al trasplante hepático temprano. En consecuencia, nuestro objetivo fue optimizar una metodología para detectar la mutación Val30Met adaptando técnicas previamente descriptas. La fiabilidad, sencillez y rapidez en la obtención de los resultados, así como el requerimiento de pequeño volumen de muestra, hacen que la técnica desarrollada en este trabajo sea una herramienta apropiada para procedimientos de screening, permitiendo contar con un marcador preclínico de la enfermedad.

Familial amyloid polyneuro- pathy (FAP) is an autosomal dominant inherited disease, characterized by systemic deposition of amyloid fibrils in various tissues, especially in peripheral nerves, being a variant of transthyretin (TTR) the principal component of amyloid fibrils. TTR is a normal plasma protein (previously called prealbumin) that functions as a transport protein binding tiroxine and retinol. Among many mutations that have been found in the TTR gene, the variant with a single amino acid substitution of methionine for valine at position 30 (TTR Val30Met) is the responsible of the Portuguese-type Familial Amyloidotic Polyneuropathy (FAP Type I). Interest in this pathology has arisen in Argentina because of the finding of an endemic area where a group of Portuguese immigrant families is localized. Since liver transplantation is a widely accepted treatment because it results in the disappearance of variant transthyretin from plasma, an early detection of the altered gene is essential. Thus, the objective of the present work was to optimize a methodology to detect the Val30Met mutation introducing modifications into techniques that were previously developed. The simple method here described is useful to confirm the diagnosis of the potential disease and, therefore, make it possible for patients to gain access to early liver transplantation.

Effect of erythropoietin on staurosporine-induced apoptosis and differentiation of SH-SY5Y neuroblastoma cells.

Since apoptosis appeared to be related to neurodegenerative processes, neuroprotection has been involved in investigation of therapeutic approaches focused upon pharmacological agents to prevent neuronal programmed cell death. In this regard, erythropoietin (Epo) seems to play a critical role. The present work was focused on the study of the Epo protective effect upon human neuroblastoma SH-SY5Y cells subjected to differentiation by staurosporine. Under this condition, profuse neurite outgrowth was accompanied by programmed cell death (35% of apoptotic cells by Hoechst assay, showing characteristic DNA ladder pattern). A previous treatment with recombinant human Epo (rHuEpo) increased the expression of the specific receptor for Epo while prevented apoptosis. Simultaneously, morphological changes in neurite elongation and interconnection induced by staurosporine were blocked by Epo. These Epo effects proved to be associated to the induction of Bcl-xL at the mRNA and protein levels (RT-PCR and Western blot after immunoprecipitation) and were mediated by activation of pathways inhibited by wortmannin. In conclusion, the fact that both events induced by staurosporine, cell apoptosis and differentiation, were prevented in SH-SY5Y cells previously exposed to rHuEpo suggests interrelated signaling pathways triggered by the Epo/EpoR interaction.