RESUMEN
The dental implant surface plays a crucial role in osseointegration. The topography and physicochemical properties will affect the cellular functions. In this research, four distinct titanium surfaces have been studied: machined acting (MACH), acid etched (AE), grit blasting (GBLAST), and a combination of grit blasting and subsequent acid etching (GBLAST + AE). Human amniotic mesenchymal (hAMSCs) and epithelial stem cells (hAECs) isolated from the amniotic membrane have attractive stem-cell properties. They were cultured on titanium surfaces to analyze their impact on biological behavior. The surface roughness, microhardness, wettability, and surface energy were analyzed using interferometric microscopy, Vickers indentation, and drop-sessile techniques. The GBLAST and GBLAST + AE surfaces showed higher roughness, reduced hydrophilicity, and lower surface energy with significant differences. Increased microhardness values for GBLAST and GBLAST + AE implants were attributed to surface compression. Cell viability was higher for hAMSCs, particularly on GBLAST and GBLAST + AE surfaces. Alkaline phosphatase activity enhanced in hAMSCs cultured on GBLAST and GBLAST + AE surfaces, while hAECs showed no mineralization signals. Osteogenic gene expression was upregulated in hAMSCs on GBLAST surfaces. Moreover, α2 and ß1 integrin expression enhanced in hAMSCs, suggesting a surface-integrin interaction. Consequently, hAMSCs would tend toward osteoblastic differentiation on grit-blasted surfaces conducive to osseointegration, a phenomenon not observed in hAECs.
Asunto(s)
Amnios , Implantes Dentales , Propiedades de Superficie , Titanio , Humanos , Titanio/química , Amnios/citología , Amnios/metabolismo , Osteogénesis , Diferenciación Celular , Células Cultivadas , Oseointegración , Células Madre/citología , Células Madre/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Supervivencia Celular , Fosfatasa Alcalina/metabolismoRESUMEN
During pregnancy, apoptosis is a physiological event critical in the remodeling and aging of the placenta. Increasing evidence has pointed toward the relevance of hypoxia as modulator of trophoblast cell death. Previous reports have shown that leptin, a placental cytokine, promotes cell survival in both cell culture and placental explant models. The aim of this work is to establish the role of leptin in apoptosis under hypoxic condition in trophoblast cells. In this study, we evaluated the effect of cobalt chloride, a hypoxia mimicking agent that stabilizes the expression of hypoxia-inducible factor-1 alpha, on Swan-71 and human placental explants. Hypoxia chamber was also used to generate 2% oxygen. Apoptosis was determined by the presence of apoptotic nucleus, fragmentation of DNA and Caspase-3 and PARP-1 cleavage. The pro-apoptotic proteins BAX, BID, BAD, and BAK and the anti-apoptotic effectors BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1 were also analyzed. We found that hypoxia-inducible factor-1 alpha stabilization increased the appearance of apoptotic nucleus, fragmentation of DNA, and Caspase-3 and PARP-1 cleavage. Hypoxia mimicking conditions enhanced the expression of pro-apoptotic effectors BAX, BID, BAD, and BAK. Hypoxia-inducible factor-1 alpha stabilization also downregulated the level of BCL-2, B-cell lymphoma-extra-large, and myeloid cell leukemia-1. All these apoptotic parameters changes were reversed with leptin treatment. Moreover, we showed that leptin action on apoptosis modulation involves PI3K and MAPK signaling pathways. Obtained data demonstrate that hypoxia-inducible factor-1 alpha stabilization induces apoptosis in human placenta and leptin counteracts this effect, reinforcing its role as a survival cytokine.
Asunto(s)
Apoptosis , Leptina , Placenta , Humanos , Femenino , Placenta/metabolismo , Placenta/efectos de los fármacos , Embarazo , Leptina/metabolismo , Leptina/farmacología , Apoptosis/efectos de los fármacos , Trofoblastos/metabolismo , Trofoblastos/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Cobalto/farmacología , Hipoxia de la Célula/fisiologíaRESUMEN
The placenta and the extraembryonic tissues represent a valuable source of cells for regenerative medicine. In particular, the amniotic membrane possesses cells with stem cells characteristics that have attracted research attention. Human amniotic epithelial cells (hAECs) have unique and desirable features that position them over other stem cells, not only because of the unlimited potential supplied of, the easy access to placental tissues, and the minimal ethical and legal barriers associated, but also due to the embryonic stem cells markers expression and their ability to differentiate into the three germ layers. In addition, they are non-tumorigenic and have immunomodulatory and anti-inflammatory properties. Hepatic failure is one of the major causes of morbidity and mortality worldwide. Organ transplantation is the best way to treat acute and chronic liver failure, but there are several associated obstacles. Stem cells have been highlighted as alternative hepatocytes source because of their potential for hepatogenic differentiation. HAECs, in particular, have some properties that make them suitable for hepatocyte differentiation. In this work, we review the general characteristics of the epithelial stem cells isolated from human amniotic membrane as well as their ability to differentiate to hepatic cells. We also revise their regenerative properties, with the focus on their potential application in the liver disease treatment.
Asunto(s)
Células Epiteliales , Hepatopatías , Humanos , Femenino , Embarazo , Placenta , Hepatopatías/terapia , Diferenciación Celular , Células Madre EmbrionariasRESUMEN
It is widely accepted that the immune system includes molecular and cellular components that play a role in regulating and suppressing the effector immune response in almost any process in which the immune system is involved. Myeloid-derived suppressor cells (MDSCs) are described as a heterogeneous population of myeloid origin, immature state, with a strong capacity to suppress T cells and other immune populations. Although the initial characterization of these cells was strongly associated with pathological conditions such as cancer and then with chronic and acute infections, extensive evidence supports that MDSCs are also involved in physiological/non-pathological settings, including pregnancy, neonatal period, aging, and vaccination. Vaccination is one of the greatest public health achievements and has reduced mortality and morbidity caused by many pathogens. The primary goal of prophylactic vaccination is to induce protection against a potential pathogen by mimicking, at least in a part, the events that take place during its natural interaction with the host. This strategy allows the immune system to prepare humoral and cellular effector components to cope with the real infection. This approach has been successful in developing vaccines against many pathogens. However, when the infectious agents can evade and subvert the host immune system, inducing cells with regulatory/suppressive capacity, the development of vaccines may not be straightforward. Notably, there is a long list of complex pathogens that can expand MDSCs, for which a vaccine is still not available. Moreover, vaccination against numerous bacteria, viruses, parasites, and fungi has also been shown to cause MDSC expansion. Increases are not due to a particular adjuvant or immunization route; indeed, numerous adjuvants and immunization routes have been reported to cause an accumulation of this immunosuppressive population. Most of the reports describe that, according to their suppressive nature, MDSCs may limit vaccine efficacy. Taking into account the accumulated evidence supporting the involvement of MDSCs in vaccination, this review aims to compile the studies that highlight the role of MDSCs during the assessment of vaccines against pathogens.
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Células Supresoras de Origen Mieloide , Inmunidad , Inmunización , Linfocitos T , VacunaciónRESUMEN
Resumen Introducción: Las enfermedades pulmonares intersticiales difusas son un grupo heterogéneo de enfermedades respiratorias con difícil diagnóstico. El estudio del lavado broncoalvolar mediante citometría de flujo puede definir patrones celulares típicos en diferentes en fermedades, proporcionando algo de ayuda en el diagnóstico diferencial. El objetivo de este estudio ha sido analizar retrospectivamente la utilidad clínica de las subpoblaciones celulares y linfocitarias detectadas en el lavado broncoalveolar por citometría de flujo, con la finalidad de definir patrones celulares típicos que permitan el diagnóstico diferencial de enfermedades granulomatosas pulmonares. Materiales y métodos: En el estudio se han incluido 44 pacientes retrospectivamente. Los sujetos fueron diagnosticados de sar coidosis o neumonitis por hipersensibilidad durante un periodo de 3 años. Se realizó el análisis celular de lavado broncoalveolar por citometría de flujo, pruebas histológicas y de imagen (TACAR), como parte del diagnóstico. Los porcentajes de células T, células B, células NK, CD4, CD8 y CD4 / CD8 se analizaron por citometría de flujo, a través de los marcadores CD3 +, CD19 + CD4 +, CD8 +, CD3 + CD4-CD8- y CD3 + CD16-CD56-. Resultados: Concluimos que los parámetros de mayor utilidad fueron la linfocitosis y sobre todo, el cociente CD4/CD8. Este cociente se presentó alto en patologías como la sarcoidosis y se invirtió en la neumonitis por hipersensibilidad, con respecto a los valores hallados en sangre periférica. Conclusiones: El estudio de BAL es útil para discriminar entre enfermedades pulmonares intersticiales granulomatosas y otras EPID.
Asunto(s)
Enfermedades Pulmonares Intersticiales , Enfermedades Pulmonares Intersticiales/diagnóstico , Lavado Broncoalveolar , Alveolitis Alérgica Extrínseca , Citometría de FlujoRESUMEN
Abstract Introduction: Diffuse interstitial lung diseases are a hard-to-diagnose heterogeneous group of respiratory diseases. The study of bronchoalveolar lavage through flow cytometry may define typical cell patterns in different diseases and so help confirm the differential diagnosis. The purpose of this study was to retrospectively analyze the clinical utility of cell and lymphocyte subpopulations detected in the bronchoalveolar lavage by flow cytometry in order to define typical cell patterns that allow for making a differential diagnosis of granulomatous lung diseases. Materials and methods: The retrospective study included 44 patients. The subjects were diagnosed with sarcoidosis or hypersen sitivity pneumonitis during a period of 3 years. We performed the cellular analysis of bronchoalveolar lavage through flow cytometry and histological and imaging testing (HRCAT, High Resolution Computed Axial Tomography) as part of the diagnosis. The percentages of T cells, B cells, NK cells, CD4, CD8 and CD4/CD8 were analyzed by flow cytometry for the following markers: CD3 +, CD19 + CD4 +, CD8 +, CD3 + CD4-CD8- and CD3 + CD16-CD56-. Results: We conclude that the most important parameters were lymphocytosis and especially the CD4/CD8 quotient. This quotient was high for diseases such as sarcoidosis and low for hypersensitivity pneumonitis, in comparison with the values found in the peripheral blood. Conclusions: The BAL (Bronchoalveolar Lavage) study is useful for differentiating between granulomatous interstitial lung diseases and other DILDs (diffuse interstitial lung diseases).
Asunto(s)
Enfermedades Pulmonares Intersticiales , Enfermedades Pulmonares Intersticiales/diagnóstico , Lavado Broncoalveolar , Alveolitis Alérgica Extrínseca , Citometría de FlujoRESUMEN
A new coronavirus respiratory disease (COVID-19) caused by the SARS-CoV-2 virus, surprised the entire world, producing social, economic, and health problems. The COVID-19 triggers a lung infection with a multiple proinflammatory cytokine storm in severe patients. Without effective and safe treatments, COVID-19 has killed thousands of people, becoming a pandemic. Stem cells have been suggested as a therapy for lung-related diseases. In particular, mesenchymal stem cells (MSCs) have been successfully tested in some clinical trials in patients with COVID-19. The encouraging results positioned MSCs as a possible cell therapy for COVID-19. The amniotic membrane from the human placenta at term is a valuable stem cell source, including human amniotic epithelial cells (hAECs) and human mesenchymal stromal cells (hAMSCs). Interestingly, amnion cells have immunoregulatory, regenerative, and anti-inflammatory properties. Moreover, hAECs and hAMSCs have been used both in preclinical studies and in clinical trials against respiratory diseases. They have reduced the inflammatory response and restored the pulmonary tissue architecture in lung injury in vivo models. Here, we review the existing data about the stem cells use for COVID-19 treatment, including the ongoing clinical trials. We also consider the non-cellular therapies that are being applied. Finally, we discuss the human amniotic membrane cells use in patients who suffer from immune/inflammatory lung diseases and hypothesize their possible use as a successful treatment against COVID-19.
Asunto(s)
Amnios/citología , COVID-19/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre/citología , Ensayos Clínicos como Asunto , Femenino , Humanos , Inflamación , Células Madre Mesenquimatosas/citología , Placenta/citología , Embarazo , RiesgoRESUMEN
Pregnancy success requires a proper fetal maternal interaction at the establishment of implantation. Leptin has been described as a multitasking cytokine in pregnancy, particularly in the placenta, where it acts as an autocrine hormone. The expression of leptin in normal trophoblastic cells is regulated by different endogenous signals. We have previously reported that 17ß-estradiol upregulates placental leptin expression through genomic and non-genomic mechanisms. To improve the knowledge of estrogen receptor mechanisms in regulating leptin gene expression, we examined transcription nuclear factor kappa B (NFκB) effect on estradiol leptin induction in human BeWo cell line and human term placental explants. We demonstrated that estradiol induction effect on leptin expression is blocked by the inhibition of NFκB signaling. We also found that the overexpression of p65 subunit, the active form of NFκB, induces leptin expression. Moreover, downregulation of estrogen receptor alpha (ERα), through a specific siRNA, abolished NFκB effect on leptin expression. We also demonstrated that ERα enhanced NFκB signaling pathway activation in trophoblastic cells. Estradiol treatment significantly increased p65 expression and phosphorylation of the inhibitory protein κB alpha (IκBα). A reporter plasmid containing NFκB elements was also induced in response to estradiol stimulation. Localization experiments revealed that estradiol treatment induced nuclear localization of overexpressed p65. Moreover, the overexpression of ERα produced a complete displacement of p65 protein to the nucleus. Finally, immunoprecipitation experiments showed the presence of a complex containing ERα and NFκB. All these evidences suggest a cooperative behavior between ERα and NFκB transcription factors to induce leptin transcription.
Asunto(s)
Coriocarcinoma/patología , Estrógenos/farmacología , Leptina/metabolismo , FN-kappa B/metabolismo , Placenta/metabolismo , Neoplasias Uterinas/patología , Núcleo Celular , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Femenino , Humanos , Leptina/genética , FN-kappa B/genética , Fosforilación , Placenta/efectos de los fármacos , Embarazo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMEN
The placental stem cells have called the focus of attention for their therapeutic potential to treat different diseases, including cancer. There is plenty evidence about the antiproliferative, antiangiogenic and proapoptotic properties of the amniotic membrane. Liver cancer is the fifth cause of cancer in the world, with a poor prognosis and survival. Alternative treatments to radio- or chemotherapy have been searched. In this work we aimed to study the antiproliferative properties of the human amniotic membrane conditioned medium (AM-CM) in hepatocarcinoma cells. In addition, we have analyzed the regulation of pro and antiOncomiRs expression involved in hepatocarcinoma physiology. We have determined by 3H-thymidine incorporation assay that AM-CM inhibits DNA synthesis in HepG2 cells after 72 h of treatment. AM-CM pure or diluted at 50% and 25% also diminished HepG2 and HuH-7 cells viability and cell number. Furthermore, AM-CM induced cell cycle arrest in G2/M. When proliferation mechanisms were analyzed we found that AM-CM reduced the expression of both Cyclin D1 mRNA and protein. Nuclear expression of Ki-67 was also reduced. We observed that this CM was able to promote the expression of p53 and p21 mRNA and proteins, leading to cell growth arrest. Moreover, AM-CM induced an increase in nuclear p21 localization, observed by immunofluorescence. As p53 levels were increased, Mdm-2 expression was downregulated. Interestingly, HepG2 and HuH-7 cells treatment with AM-CM during 24 and 72 h produced an upregulation of antiOncomiRs 15a and 210, and a downregulation of proOncomiRs 206 and 145. We provide new evidence about the promising novel applications of human amniotic membrane in liver cancer.
Asunto(s)
Amnios/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Medios de Cultivo Condicionados/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Amnios/crecimiento & desarrollo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/metabolismo , Ciclina D1/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , MicroARNs/genética , Placenta/metabolismo , Embarazo , Proteínas Proto-Oncogénicas c-mdm2/genética , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Stem cells derived from placental tissues are an attractive source of cells for regenerative medicine. Amniotic epithelial cells isolated from human amnion (hAECs) have desirable and competitive characteristics that make them stand out between other stem cells. They have the ability to differentiate toward all three germ layers, they are not tumorigenic and they have immunosuppressive properties. Although liver transplantation is the best way to treat acute and chronic hepatic failure patients, there are several obstacles. Recently, stem cells have been spotlighted as alternative source of hepatocytes because of their potential for hepatogenic differentiation. In this work, we aimed to study the proliferation and survival of the hAECs during their hepatic differentiation. We have also analyzed the changes in pluripotency and hepatic markers. We differentiated amniotic cells applying a specific hepatic differentiation (HD) protocol. We determined by qRT-PCR that hAECs express significant levels of SOX-2, OCT-4 and NANOG during at least 15 days in culture and these pluripotent markers diminish during HD. SSEA-4 expression was reduced during HD, measured by immunofluorescence. Morphological characteristics became more similar to hepatic ones in differentiated cells and representative hepatic markers significantly augmented their expression, measured by qRT-PCR and Western blot. Cells achieved a differentiation efficiency of 75%. We observed that HD induced proliferation and promoted survival of hAECs, during 30 days in culture, evaluated by 3H-thymidine incorporation and MTT assay. HD also promoted changes in hAECs cell cycle. Cyclin D1 expression increased, while p21 and p53 levels were reduced. Immunofluorescence analysis showed that Ki-67 expression was upregulated during HD. Finally, ERK 1/2 phosphorylation, which is intimately linked to proliferation and cell survival, augmented during all HD process and the inhibition of this signaling pathway affected not only proliferation but also differentiation. Our results suggest that HD promotes proliferation and survival of hAECs, providing important evidence about the mechanisms governing their hepatic differentiation. We bring new knowledge concerning some of the optimal transplantation conditions for these hepatic like cells.
Asunto(s)
Amnios/citología , Proliferación Celular , Supervivencia Celular , Hígado/citología , Biomarcadores/metabolismo , Células Cultivadas , Células Epiteliales/citología , Femenino , Humanos , Hígado/metabolismo , Sistema de Señalización de MAP Quinasas , Fosforilación , Embarazo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Leptin is a homeostatic regulator in the placenta where it promotes proliferation, protein synthesis and the expression of tolerogenic maternal response molecules such as HLA-G. Leptin also exerts an anti-apoptotic action in placenta controlling the expression of p53 master cell cycle regulator under different stress conditions. On the other hand, leptin is an integrative target of different placental stimuli. The expression of leptin in placenta is regulated by hCG, insulin, steroids, hypoxia and many other growth hormones, suggesting that it might have an important endocrine function in the trophoblastic cells. The leptin expression is induced involving the cAMP/PKA or cAMP/Epac pathways which have profound actions upon human trophoblast function. The activation of PI3K and MAPK pathways also participates in the leptin expression. Estrogens play a central role during pregnancy, particularly 17ß-estradiol upregulates the leptin expression in placental cells through genomic and non-genomic actions. The leptin promoter analysis reveals specific elements that are active in placental cells. The transcription factors CREB, AP1, Sp1, NFκB and the coactivator CBP are involved in the placental leptin expression. Moreover, placental leptin promoter is a target of epigenetic marks such as DNA methylation and histone acetylation that regulates not only the leptin expression in placenta during pregnancy but also determines the predisposition of acquiring adult metabolism diseases. Taken together, all these results allow a better understanding of leptin function and regulatory mechanisms of leptin expression in human placental trophoblasts, and support the importance of leptin during pregnancy and in programming adult health.
Asunto(s)
Leptina/metabolismo , Placenta/fisiología , Animales , Femenino , Humanos , Placenta/citología , Embarazo , Transducción de SeñalRESUMEN
Leptin, a 16-kDa polypeptide hormone, is produced by the adipocyte and can also be synthesized by placenta. We previously demonstrated that leptin promotes proliferation and survival in placenta, in part mediated by the p53 pathway. In this work, we investigated the mechanisms involved in leptin down-regulation of p53 level. The human first trimester cytotrophoblastic Swan-71 cell line and human placental explants at term were used. In order to study the late phase of apoptosis, triggered by serum deprivation, experiments of DNA fragmentation were carried out. Exogenous leptin added to human placental explants, showed a decrease on DNA ladder formation and MAPK pathway is involved in this leptin effect. We also found that under serum deprivation condition, leptin decreases p53 levels and the inhibitory leptin effect is lost when cells were pretreated with 50 µM PD98059 or 10 µM LY29004; or were transfected with dominant negative mutants of intermediates of these pathways, suggesting that MAPK and PI3K signaling pathways are necessaries for leptin action. Additionally, leptin diminished Ser-46 p53 phosphorylation and this effect in placental explants was mediated by the activation of MAPK and PI3K pathways. Finally, in order to assess leptin effect on p53 half-life experiments with cycloheximide were performed and MDM-2 expression was analyzed. Leptin diminished p53 half-life and up-regulated MDM-2 expression. In summary, we provided evidence suggesting that leptin anti-apoptotic effect is mediated by MAPK and PI3K pathways.
Asunto(s)
Leptina/metabolismo , Trofoblastos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis , Línea Celular , Proliferación Celular , Humanos , Sistema de Señalización de MAP Quinasas , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismoRESUMEN
Leptin, a peripheral signal synthetized by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work, we aimed to study the molecular mechanisms that mediate the survival effect of leptin in placenta. We used the human placenta choriocarcinoma BeWo and first trimester Swan-71 cell lines, as well as human placental explants. We tested the late phase of apoptosis, triggered by serum deprivation, by studying the activation of Caspase-3 and DNA fragmentation. Recombinant human leptin added to BeWo cell line and human placental explants, showed a decrease on Caspase-3 activation. These effects were dose dependent. Maximal effect was achieved at 250 ng leptin/ml. Moreover, inhibition of endogenous leptin expression with 2 µM of an antisense oligonucleotide, reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. We analyzed the presence of low DNA fragments, products from apoptotic DNA cleavage. Placental explants cultivated in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. Taken together these results reinforce the survival effect exerted by leptin on placental cells. To improve the understanding of leptin mechanism in regulating the process of apoptosis we determined the expression of different intermediaries in the apoptosis cascade. We found that under serum deprivation conditions, leptin increased the anti-apoptotic BCL-2 protein expression, while downregulated the pro-apoptotic BAX and BID proteins expression in Swan-71 cells and placental explants. In both models leptin augmented BCL-2/BAX ratio. Moreover we have demonstrated that p53, one of the key cell cycle-signaling proteins, is downregulated in the presence of leptin under serum deprivation. On the other hand, we determined that leptin reduced the phosphorylation of Ser-46 p53 that plays a pivotal role for apoptotic signaling by p53. Our data suggest that the observed anti-apoptotic effect of leptin in placenta is in part mediated by the p53 pathway. In conclusion, we provide evidence that demonstrates that leptin is a trophic factor for trophoblastic cells.
Asunto(s)
Apoptosis , Regulación hacia Abajo , Leptina/metabolismo , Placenta/citología , Placenta/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Fosforilación , Fosfoserina/metabolismo , Embarazo , Trofoblastos/citología , Trofoblastos/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
La esclerosis múltiple (EM) es una enfermedad infamatoria desmielinizante y autoinmune del sistema nervioso central que afecta al cerebro y a la médula espinal. El objetivo del estudio fue la cuantificación de subpoblaciones linfocitarias en líquido cefalorraquídeo y sangre de pacientes diagnosticados de esclerosis múltiple y en pacientes con enfermedades no degenerativas (controles), para encontrar variables o relaciones entre las mismas que permitan diferenciar el estado inmunológico de los pacientes de cada grupo. Este trabajo se ha llevado a cabo conjuntamente con el Hospital Universitario Virgen Macarena de Sevilla entre los años 2008 y 2010. Es un estudio de tipo descriptivo, transversal y de cohortes. La población seleccionada (n=142) estuvo compuesta por sujetos a los que se les realizó una punción lumbar y una citometría de fujo, tanto de LCR como de sangre. El Grupo 1 (n=70) fue el grupo control, Grupo 2: (n=53): pacientes con esclerosis múltiple remitente recidivante (EMRR), Grupo 3: (n=5), pacientes con esclerosis múltiple de tipo primaria progresiva y Grupo 4 (n=14), pacientes que presentaban un síndrome neurológico aislado. Los resultados mostraron un aumento de células B en LCR en pacientes con EM que sugirieron un aumento de la actividad infamatoria focal en el SNC. En cuanto a NKCD8- se observó una disminución de los niveles totales de NK, así como de los NKCD8 con respecto a los controles y un mayor valor del índice de IgG en los pacientes con EMRR.(AU)
Multiple sclerosis is an infammatory demyelinating autoimmune disease that affects the brain and spinal cord. The aim of the study was to quantify lym-phocyte subpopulations in cerebrospinal fuid and blood of patients diagnosed with multiple sclerosis and in patients whit degenerative diseases not (control) in order to fnd some relationships between them that make it possible to differentiate the immune status of patients in each group. This work was jointly carried out with Hospital Universitario Virgen Macarena in Seville during 2008, 2009 and 2010. It is a descriptive, transversal and cohort study. The selected population is composed of 142 subjects who were subjected to lumbar puncture and a blood sample. Group 1 (n=70), control, Group 2 (n=53), patients with relapsing remitting multiple sclerosis, Group 3 (n=5), patients with primary type progressive multiple sclerosis, and Group 4 (n=14) patients with isolated neurological syndrome. The results show an increase in CSF B cells in MS patients suggesting an increase in focal infammatory activity in the CNS. Regarding NKCD8, reduced total levels of NK and NKCD8 regard-ing controls were observed, and it showed an increased IgG index value in patients with RRMS.(AU)
A esclerose múltipla é uma doenþa infamatória desmielinizante autoimune do sistema nervoso central que afeta o cérebro e a medula espinhal. O objetivo do estudo foi quantificar subpopulaþ§es linfocitarias em líquido cefalorraquidiano e sangue de pacientes com diagnóstico de esclerose múltipla e em pacientes com doenþas nÒo degenerativas (controles) para encontrar variáveis ou relaþ§es entre as mesmas que permitam diferenciar o estado imunológico dos pacientes cada grupo. Este trabalho foi realizado em conjunto com o Hospital Uni-versitário Virgen Macarena de Sevilha, entre os anos 2008 e 2010. E um estudo de tipo descritivo, tranversal e de coortes. A populaþÒo selecionada é (n=142) foi constituída por indivíduos submetidos a punþÒo lombar e citometria de fuxo tanto de LCR como de sangue. O Grupo 1 (n=70), foi o grupo controle, o Grupo 2 (n=53): pacientes com esclerose múltipla recidivante-remitente (EMRR), o Grupo 3 (n=5), pacientes com esclerose múltipla de tipo primária progressiva e o Grupo 4 (n=14) pacientes com síndrome neurológica isolada. Os resultados mostraram um aumento de células B no LCR em pacientes com EM, sugerindo um aumento da atividade infamatória focal no SNC. Quanto a NKCD8, observou-se uma diminuiþÒo dos níveis totais de NK bem como dos NKCD8 com relaþÒo a controles e maior valor do índice de IgG nos pacientes com EMRR.(AU)
RESUMEN
La esclerosis múltiple (EM) es una enfermedad infamatoria desmielinizante y autoinmune del sistema nervioso central que afecta al cerebro y a la médula espinal. El objetivo del estudio fue la cuantificación de subpoblaciones linfocitarias en líquido cefalorraquídeo y sangre de pacientes diagnosticados de esclerosis múltiple y en pacientes con enfermedades no degenerativas (controles), para encontrar variables o relaciones entre las mismas que permitan diferenciar el estado inmunológico de los pacientes de cada grupo. Este trabajo se ha llevado a cabo conjuntamente con el Hospital Universitario Virgen Macarena de Sevilla entre los años 2008 y 2010. Es un estudio de tipo descriptivo, transversal y de cohortes. La población seleccionada (n=142) estuvo compuesta por sujetos a los que se les realizó una punción lumbar y una citometría de fujo, tanto de LCR como de sangre. El Grupo 1 (n=70) fue el grupo control, Grupo 2: (n=53): pacientes con esclerosis múltiple remitente recidivante (EMRR), Grupo 3: (n=5), pacientes con esclerosis múltiple de tipo primaria progresiva y Grupo 4 (n=14), pacientes que presentaban un síndrome neurológico aislado. Los resultados mostraron un aumento de células B en LCR en pacientes con EM que sugirieron un aumento de la actividad infamatoria focal en el SNC. En cuanto a NKCD8- se observó una disminución de los niveles totales de NK, así como de los NKCD8 con respecto a los controles y un mayor valor del índice de IgG en los pacientes con EMRR.
Multiple sclerosis is an infammatory demyelinating autoimmune disease that affects the brain and spinal cord. The aim of the study was to quantify lym-phocyte subpopulations in cerebrospinal fuid and blood of patients diagnosed with multiple sclerosis and in patients whit degenerative diseases not (control) in order to fnd some relationships between them that make it possible to differentiate the immune status of patients in each group. This work was jointly carried out with Hospital Universitario Virgen Macarena in Seville during 2008, 2009 and 2010. It is a descriptive, transversal and cohort study. The selected population is composed of 142 subjects who were subjected to lumbar puncture and a blood sample. Group 1 (n=70), control, Group 2 (n=53), patients with relapsing remitting multiple sclerosis, Group 3 (n=5), patients with primary type progressive multiple sclerosis, and Group 4 (n=14) patients with isolated neurological syndrome. The results show an increase in CSF B cells in MS patients suggesting an increase in focal infammatory activity in the CNS. Regarding NKCD8, reduced total levels of NK and NKCD8 regard-ing controls were observed, and it showed an increased IgG index value in patients with RRMS.
A esclerose múltipla é uma doença infamatória desmielinizante autoimune do sistema nervoso central que afeta o cérebro e a medula espinhal. O objetivo do estudo foi quantificar subpopulações linfocitarias em líquido cefalorraquidiano e sangue de pacientes com diagnóstico de esclerose múltipla e em pacientes com doenças não degenerativas (controles) para encontrar variáveis ou relações entre as mesmas que permitam diferenciar o estado imunológico dos pacientes cada grupo. Este trabalho foi realizado em conjunto com o Hospital Uni-versitário Virgen Macarena de Sevilha, entre os anos 2008 e 2010. É um estudo de tipo descritivo, tranversal e de coortes. A população selecionada é (n=142) foi constituída por indivíduos submetidos a punção lombar e citometria de fuxo tanto de LCR como de sangue. O Grupo 1 (n=70), foi o grupo controle, o Grupo 2 (n=53): pacientes com esclerose múltipla recidivante-remitente (EMRR), o Grupo 3 (n=5), pacientes com esclerose múltipla de tipo primária progressiva e o Grupo 4 (n=14) pacientes com síndrome neurológica isolada. Os resultados mostraram um aumento de células B no LCR em pacientes com EM, sugerindo um aumento da atividade infamatória focal no SNC. Quanto a NKCD8, observou-se uma diminuição dos níveis totais de NK bem como dos NKCD8 com relação a controles e maior valor do índice de IgG nos pacientes com EMRR.
Asunto(s)
Citometría de Flujo , Subgrupos Linfocitarios , Esclerosis Múltiple , Bandas Oligoclonales , BioquímicaRESUMEN
Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)(2)cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway.
Asunto(s)
Gonadotropina Coriónica/fisiología , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Leptina/biosíntesis , Sistema de Señalización de MAP Quinasas , Placenta/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cartilla de ADN , Activación Enzimática , Femenino , Humanos , Técnicas In Vitro , Embarazo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The placenta produces a wide number of molecules that play essential roles in the establishment and maintenance of pregnancy. In this context, leptin has emerged as an important player in reproduction. The synthesis of leptin in normal trophoblastic cells is regulated by different endogenous biochemical agents, but the regulation of placental leptin expression is still poorly understood. We have previously reported that 17ß-estradiol (E(2)) up-regulates placental leptin expression. To improve the understanding of estrogen receptor mechanisms in regulating leptin gene expression, in the current study we examined the effect of membrane-constrained E(2) conjugate, E-BSA, on leptin expression in human placental cells. We have found that leptin expression was induced by E-BSA both in BeWo cells and human placental explants, suggesting that E(2) also exerts its effects through membrane receptors. Moreover E-BSA rapidly activated different MAPKs and AKT pathways, and these pathways were involved in E(2) induced placental leptin expression. On the other hand we demonstrated the presence of ERα associated to the plasma membrane of BeWo cells. We showed that E(2) genomic and nongenomic actions could be mediated by ERα. Supporting this idea, the downregulation of ERα level through a specific siRNA, decreased E-BSA effects on leptin expression. Taken together, these results provide new evidence of the mechanisms whereby E(2) regulates leptin expression in placenta and support the importance of leptin in placental physiology.
Asunto(s)
Membrana Celular/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Leptina/genética , Placenta/citología , Placenta/metabolismo , Membrana Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Estradiol/análogos & derivados , Femenino , Fulvestrant , Silenciador del Gen/efectos de los fármacos , Humanos , Leptina/metabolismo , Modelos Biológicos , Placenta/efectos de los fármacos , Embarazo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Albúmina Sérica Bovina , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Leptin, a 16-kDa protein mainly produced by adipose tissue, has been involved in the control of energy balance through its hypothalamic receptor. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it was found to be expressed. In the current study, we examined the effect of cAMP in the regulation of leptin expression in trophoblastic cells. We found that dibutyryl cAMP [(Bu)(2)cAMP], a cAMP analog, showed an inducing effect on endogenous leptin expression in BeWo and JEG-3 cell lines when analyzed by Western blot analysis and quantitative RT-PCR. Maximal effect was achieved at 100 microM. Leptin promoter activity was also stimulated, evaluated by transient transfection with a reporter plasmid construction. Similar results were obtained with human term placental explants, thus indicating physiological relevance. Because cAMP usually exerts its actions through activation of protein kinase A (PKA) signaling, this pathway was analyzed. We found that cAMP response element-binding protein (CREB) phosphorylation was significantly increased with (Bu)(2)cAMP treatment. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor CREB caused a significant stimulation on leptin promoter activity. On the other hand, the cotransfection with a dominant negative mutant of the regulatory subunit of PKA inhibited leptin promoter activity. We determined that cAMP effect could be blocked by pharmacologic inhibition of PKA or adenylyl ciclase in BeWo cells and in human placental explants. Thereafter, we decided to investigate the involvement of the MAPK/ERK signaling pathway in the cAMP effect on leptin induction. We found that 50 microm PD98059, a MAPK kinase inhibitor, partially blocked leptin induction by cAMP, measured both by Western blot analysis and reporter transient transfection assay. Moreover, ERK 1/2 phosphorylation was significantly increased with (Bu)(2)cAMP treatment, and this effect was dose dependent. Finally, we observed that 50 microm PD98059 inhibited cAMP-dependent phosphorylation of CREB in placental explants. In summary, we provide some evidence suggesting that cAMP induces leptin expression in placental cells and that this effect seems to be mediated by a cross talk between PKA and MAPK signaling pathways.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/farmacología , Leptina/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Placenta/efectos de los fármacos , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Leptina/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Fosforilación/efectos de los fármacos , Placenta/metabolismo , Embarazo , Regiones Promotoras Genéticas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiología , TransfecciónRESUMEN
The process of embryo implantation and trophoblast invasion is considered the most limiting factor in the establishment of pregnancy. Leptin was originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, it has been suggested that leptin is involved in other functions during pregnancy, particularly in the placenta, where it was found to be expressed. In the present work, we have found a stimulatory effect of 17beta-estradiol (E(2)) on endogenous leptin expression, as analyzed by Western blot, in both the BeWo choriocarcinoma cell line and normal placental explants. This effect was time and dose dependent. Maximal effect was achieved at 10 nM in BeWo cells and 1 nM in placental explants. The E(2) effects involved the estrogen receptor, as the antagonist ICI 182 780 inhibited E(2)-induced leptin expression. Moreover, E(2) treatment enhanced leptin promoter activity up to 4-fold, as evaluated by transient transfection with a plasmid construction containing the leptin promoter region and the reporter gene luciferase. This effect was dose dependent. Deletion analysis demonstrated that a minimal promoter region between -1951 and -1847 bp is both necessary and sufficient to achieve E(2) effects. Estradiol action involved estrogen receptor 1, previously known as estrogen receptor alpha, as cotransfection with a vector encoding estrogen receptor 1 potentiated the effects of E(2) on leptin expression. Moreover, E(2) action probably involves membrane receptors too, as treatment with an estradiol-bovine serum albumin complex partially enhanced leptin expression. The effects of E(2) could be blocked by pharmacologic inhibition of MAPK and the phosphoinositide-3-kinase (PI3K) pathways with 50 microM PD98059 and 0.1 microM Wortmannin, respectively. Moreover, cotransfection of dominant negative mutants of MAP2K or MAPK blocked E(2) induction of leptin promoter. On the other hand, E(2) treatment promoted MAPK1/MAPK3 and AKT phosphorylation in placental cells. In conclusion, we provide evidence suggesting that E(2) induces leptin expression in trophoblastic cells, probably through genomic and nongenomic actions via crosstalk between estrogen receptor 1 and MAPK and PI3K signal transduction pathways.
Asunto(s)
Estradiol/metabolismo , Leptina/metabolismo , Sistema de Señalización de MAP Quinasas , Placenta/metabolismo , Receptor Cross-Talk , Línea Celular Tumoral , Estradiol/análogos & derivados , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Fulvestrant , Humanos , Técnicas In Vitro , Fosfatidilinositol 3-Quinasas/metabolismo , Embarazo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 microM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 microm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway.