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Gasdermin D (GSDMD) and mixed lineage kinase domain-like protein (MLKL) are the pore-forming effectors of pyroptosis and necroptosis, respectively, with the capacity to disturb plasma membrane selective permeability and induce regulated cell death. The budding yeast Saccharomyces cerevisiae has long been used as a simple eukaryotic model for the study of proteins associated with human diseases by heterologous expression. In this work, we expressed in yeast both GSDMD and its N-terminal domain (GSDMD(NT)) to characterize their cellular effects and compare them to those of MLKL. GSDMD(NT) and MLKL inhibited yeast growth, formed cytoplasmic aggregates and fragmented mitochondria. Loss-of-function point mutants of GSDMD(NT) showed affinity for this organelle. Besides, GSDMD(NT) and MLKL caused an irreversible cell cycle arrest through TORC1 inhibition and disrupted endosomal and autophagic vesicular traffic. Our results provide a basis for a humanized yeast platform to study GSDMD and MLKL, a useful tool for structure-function assays and drug discovery.
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Endocitosis , Gasderminas , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas Quinasas , Saccharomyces cerevisiae , Humanos , Mitocondrias , Factores de TranscripciónRESUMEN
PURPOSE: Prognostic tools to estimate the risk of relapse for patients with liver-limited metastatic colorectal cancer (LL-mCRC) undergoing resection with curative intent are needed. Circulating tumor DNA (ctDNA) as a surrogate of postsurgical minimal residual disease is a promising marker in localized CRC. We explored the role of postoperative ctDNA as a marker of minimal residual disease in patients with radically resected LL-mCRC. MATERIALS AND METHODS: Seventy-six patients with LL-mCRC were retrospectively included. DNA from tumor tissue was sequenced, and one somatic mutation was then assessed by digital droplet polymerase chain reaction in plasma samples collected after surgery to identify the persistence of ctDNA. Relapse-free survival and postresection overall survival were compared between patients with positive vs negative postoperative ctDNA. RESULTS: ctDNA was found in 39 (51%) of 76 patients with LL-mCRC. At a median follow-up of 77 months, 33 of 39 ctDNA-positive patients and 20 of 37 ctDNA-negative patients experienced disease relapse (P = .008). ctDNA-positive patients reported significantly shorter RFS than ctDNA-negative ones (median RFS 12.7 v 27.4 months hazard ratio, 2.09, P = .008). In the multivariable model including other prognostic covariates, this association was still significant (P = .046) and a trend toward shorter overall survival among ctDNA-positive patients was reported (hazard ratio, 1.65, P = .183). CONCLUSION: The detection of postsurgical ctDNA is an independent negative prognostic marker and identifies patients at high risk of relapse after liver metastases resection.
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ADN Tumoral Circulante , Neoplasias del Colon , Neoplasias Hepáticas , Humanos , ADN Tumoral Circulante/genética , Neoplasia Residual , Estudios Retrospectivos , Biomarcadores de Tumor/genética , Recurrencia Local de Neoplasia/diagnóstico , Estudios Prospectivos , Neoplasias Hepáticas/diagnóstico , Neoplasias del Colon/patologíaRESUMEN
Background: Previous data, mostly from clinical trials, reported that HER2-low status is associated with low pathological complete response (pCR), and favourable prognosis. Since these findings suggest the existence of an additional breast cancer subtype, we questioned if the predictive/prognostic value of HER2-low was also relevant in the real world. Methods: Data from non-metastatic breast cancer patients treated with neoadjuvant chemotherapy and surgery (2009-2020) were retrieved from our institutional prospectively-maintained registry. Univariable and multivariable logistic models were implemented to study the association between pCR and baseline HER2 status. Univariable analysis of disease-free survival (DFS) was performed through Kaplan-Meier survival curves and log-rank tests. Results: Starting from a total of 790 consecutive cases, we identified 444 newly-diagnosed breast cancer patients featuring HER2 immunohistochemistry (IHC) 0 (HER2-0, n = 109), and 1 + or IHC 2+/in situ hybridization negative (HER2-low, n = 335) receiving anthracycline and taxane-based regimens in 88.9% of cases. Most of the patients were diagnosed with stage II (67.3%) and there was no difference of disease presentation according to HER2-status. pCR was attained by 71 (16.0%) patients and was significantly associated with increased DFS (p = 0.031). Compared to HER2-0, HER2-low cases were more likely hormone receptor-positive (81.2% vs. 43.1%, p < 0.001), well-differentiated (47.5% vs. 26.6%, p = 0.001), less proliferative (21.5% vs. 8.3%, p = 0.001) and less responsive to treatment (pCR 11.6% vs. 29.4%, p < 0.0001). There was no difference in DFS according to HER2 status, though hormone-receptor (HR) negative/HER2-low cases tended to have a worse prognosis compared to HR-negative/HER2-0. By pCR achievement, 3-years DFS was 87.5.% (75.1-100%) vs. 71.6% (65.9-77.8%) (p = 0.161) in HER2-low and 89.1% (75.8-100%) vs. 72.1% (59.7-87.0%) (p = 0.092) in HER2-0. Conclusion: Our real-world data show that HER2-low breast cancer patients represent roughly a half of the cases treated with neoadjuvant therapy, and have poor treatment response. In absence of pCR, HER2-low breast cancer patients have a dismal prognosis, especially when primary tumor hormone receptor status is negative. Studies are therefore needed to define the biology of these tumors for new therapeutic targets and to incorporate HER2-targeting agents in early-stage treatment.
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Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of caspase-8. Overexpression of both caspases in the heterologous model led to their activation and caused mitochondrial hyperpolarization, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and caspase-8 was more aggressive than caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of caspase-1 enhanced its activity, whereas the deletion of the death effector domain (DED) of caspase-8 diminished it. We show that caspase-1 is able to efficiently process its target gasdermin D (GSDMD) when co-expressed in yeast. In sum, we propose that S. cerevisiae provides a manageable tool to explore caspase-1 activity and structure-function relationships.
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Caspasa 1/biosíntesis , Caspasa 8/biosíntesis , Mitocondrias/enzimología , Saccharomyces cerevisiae/enzimología , Citoesqueleto de Actina/enzimología , Citoesqueleto de Actina/genética , Caspasa 1/genética , Caspasa 8/genética , Activación Enzimática , Inducción Enzimática , Galactoquinasa/genética , Galactoquinasa/metabolismo , Regulación Fúngica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Viabilidad Microbiana , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Mitocondrias/genética , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
FENIB (familial encephalopathy with neuroserpin inclusion bodies) is a human monogenic disease caused by point mutations in the SERPINI1 gene, characterized by the intracellular deposition of polymers of neuroserpin (NS), which leads to proteotoxicity and cell death. Despite the different cell and animal models developed thus far, the exact mechanism of cell toxicity elicited by NS polymers remains unclear. Here, we report that human wild-type NS and the polymerogenic variant G392E NS form protein aggregates mainly localized within the endoplasmic reticulum (ER) when expressed in the yeast S. cerevisiae. The expression of NS in yeast delayed the exit from the lag phase, suggesting that NS inclusions cause cellular stress. The cells also showed a higher resistance following mild oxidative stress treatments when compared to control cells. Furthermore, the expression of NS in a pro-apoptotic mutant strain-induced cell death during aging. Overall, these data recapitulate phenotypes observed in mammalian cells, thereby validating S. cerevisiae as a model for FENIB.
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The yeast Saccharomyces cerevisiae is a model organism that has been thoroughly exploited to understand the universal mechanisms that govern signaling pathways. Due to its ease of manipulation, humanized yeast models that successfully reproduce the function of human genes permit the development of highly efficient genetic approaches for molecular studies. Of special interest are those pathways related to human disease that are conserved from yeast to mammals. However, it is also possible to engineer yeast cells to implement functions that are naturally absent in fungi. Along the years, we have reconstructed several aspects of the mammalian phosphatidylinositol 3-kinase (PI3K) pathway in S. cerevisiae. Here, we briefly review the use of S. cerevisiae as a tool to study human oncogenes and tumor suppressors, and we present an overview of the models applied to the study of the PI3K oncoproteins, the tumor suppressor PTEN, and the Akt protein kinase. We discuss the application of these models to study the basic functional properties of these signaling proteins, the functional assessment of their clinically relevant variants, and the design of feasible platforms for drug discovery.
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Susceptibilidad a Enfermedades , Modelos Biológicos , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Genes Supresores de Tumor , Ingeniería Genética , Humanos , Oncogenes , Saccharomycetales/metabolismo , Sistemas de Mensajero Secundario , Transducción de Señal/efectos de los fármacosRESUMEN
In orthopedic surgery, precise kinematics assessment helps the diagnosis and the planning of the intervention. The correct placement of the prosthetic component in the case of knee replacement is necessary to ensure a correct load distribution and to avoid revision of the implant. 3D reconstruction of the knee kinematics under weight-bearing conditions becomes fundamental to understand existing in vivo loads and improve the joint motion tracking. Existing methods rely on the semiautomatic positioning of a shape previously segmented from a CT or MRI on a sequence of fluoroscopic images acquired during knee flexion. We propose a method based on statistical shape models (SSM) automatically superimposed on a sequence of fluoroscopic datasets. Our method is based on Gaussian mixture models, and the core of the algorithm is the maximization of the likelihood of the association between the projected silhouette and the extracted contour from the fluoroscopy image. We evaluated the algorithm using digitally reconstructed radiographies of both healthy and diseased subjects, with a CT-extracted shape and a SSM as the 3D model. In vivo tests were done with fluoroscopically acquired images and subject-specific CT shapes. The results obtained are in line with the literature, but the computational time is substantially reduced.
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Huesos/patología , Huesos/cirugía , Imagenología Tridimensional , Modelos Estadísticos , Procedimientos Ortopédicos , Planificación de Atención al Paciente , Anciano , Anciano de 80 o más Años , Algoritmos , Femenino , Fluoroscopía , Humanos , Masculino , Persona de Mediana Edad , Distribución Normal , Rotación , Interfaz Usuario-ComputadorRESUMEN
PURPOSE: Precise knee kinematics assessment helps to diagnose knee pathologies and to improve the design of customized prosthetic components. The first step in identifying knee kinematics is to assess the femoral motion in the anatomical frame. However, no work has been done on pathological femurs, whose shape can be highly different from healthy ones. METHODS: We propose a new femoral tracking technique based on statistical shape models and two calibrated fluoroscopic images, taken at different flexion-extension angles. The cost function optimization is based on genetic algorithms, to avoid local minima. The proposed approach was evaluated on 3 sets of digitally reconstructed radiographic images of osteoarthritic patients. RESULTS: It is found that using the estimated shape, rather than that calculated from CT, significantly reduces the pose accuracy, but still has reasonably good results (angle errors around 2[Formula: see text], translation around 1.5 mm).
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Algoritmos , Fémur/diagnóstico por imagen , Articulación de la Rodilla/diagnóstico por imagen , Modelos Estadísticos , Osteoartritis de la Rodilla/cirugía , Fenómenos Biomecánicos , Femenino , Fémur/fisiopatología , Fluoroscopía/métodos , Humanos , Procesamiento de Imagen Asistido por Computador , Prótesis Articulares , Articulación de la Rodilla/fisiopatología , Diseño de Prótesis , Rango del Movimiento Articular , Tomografía Computarizada por Rayos XRESUMEN
In the present study, we found that CBD inhibited U87-MG and T98G cell proliferation and invasiveness in vitro and caused a decrease in the expression of a set of proteins specifically involved in growth, invasion and angiogenesis. In addition, CBD treatment caused a dose-related down-regulation of ERK and Akt prosurvival signaling pathways in U87-MG and T98G cells and decreased hypoxia inducible factor HIF-1α expression in U87-MG cells. Taken together, these results provide new insights into the antitumor action of CBD, showing that this cannabinoid affects multiple tumoral features and molecular pathways. As CBD is a non-psychoactive phytocannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anti-cancer drug in the management of gliomas.
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Antineoplásicos/farmacología , Cannabidiol/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glioma/tratamiento farmacológico , Glioma/metabolismo , Glioma/patología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Invasividad Neoplásica , Proteoma/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Cannabinoids, the active components of Cannabis sativa, have been shown to exert antiproliferative and proapoptotic effects on a wide spectrum of tumor cells and tissues. Of interest, cannabinoids have displayed great potency in reducing the growth of glioma tumors, one of the most aggressive CNS tumors, either in vitro or in animal experimental models curbing the growth of xenografts generated by subcutaneous or intrathecal injection of glioma cells in immune-deficient mice. Cannabinoids appear to be selective antitumoral agents as they kill glioma cells without affecting the viability of non-transformed cells. This review will summarize the anti-cancer properties that cannabinoids exert on gliomas and discuss their potential action mechanisms that appear complex, involving modulation of multiple key cell signaling pathways and induction of oxidative stress in glioma cells.
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In the last few years the role and significance of the glia in CNS function and pathology have been drastically reassessed. Glial cells physiology appears very different in healthy versus pathological brain and the recent identification of cannabinoid receptors and their endogenous ligands in glia has triggered a number of studies exploring the role of (endo)cannabinoid system in glia functionality and disease. (Endo)cannabinoids exert their effects in these cells directly affecting some important peculiar functions of the glia and actively promoting biochemical signals ending in a pro-survival fate for these cells. By contrast, (endo)cannabinoids induce a selective death in glia-derived tumor cells. Of special physiological and therapeutic relevance is the reported ability of glial cells during neuropathological conditions to release an increased amount of endocannabinoids and to overexpress cannabinoid receptors. This evidence has suggested that the endocannabinoids production by glial cells may constitute an endogenous defense mechanism preventing the propagation of neuroinflammation and cell damage. The present paper will review the evidence supporting the regulatory role of (endo)cannabinoids in glia function, holding in consideration their therapeutic potential as neuroprotective and/or anticancer agents.
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Moduladores de Receptores de Cannabinoides/metabolismo , Enfermedades del Sistema Nervioso Central/fisiopatología , Endocannabinoides , Neuroglía/metabolismo , Animales , Antineoplásicos/farmacología , Expresión Génica , Humanos , Neuroglía/fisiología , Fármacos Neuroprotectores/farmacología , Receptores de Cannabinoides/metabolismo , Transducción de SeñalRESUMEN
N-arachidonoylethanolamine (anandamide, AEA), is a full agonist at both cannabinoid CB(1) receptors and "transient receptor potential vanilloid" type 1 (TRPV1) channels, and N-palmitoylethanolamine (PEA) potentiates these effects. In neurons of the rat dorsal root ganglia (DRG), TRPV1 is activated and/or sensitised by AEA as well as upon activation of protein kinases C (PKC) and A (PKA). We investigated here the effect on AEA levels of PKC and PKA activators in DRG neurons. AEA levels were significantly enhanced by both phorbol-miristoyl-acetate (PMA), a typical PKC activator, and forskolin (FSK), an adenylate cyclase stimulant, as well as by thrombin, which also activates PKC by stimulating protease-activated receptors (PARs). The levels of the other endocannabinoid and TRPV1-inactive compound, 2-arachidonoylglycerol (2-AG), were enhanced only by thrombin and to a lesser extent than AEA, whereas PEA was not affected by any of the treatments. Importantly, FSK- and PMA-induced elevation of AEA levels was not sensitive to intracellular Ca2+ chelation with BAPTA-acetoxymethyl (AM) ester. In human embryonic kidney (HEK-293) cells, which constitutively express PARs, thrombin, PMA and FSK elevated AEA levels, and the effects of the two former compounds were counteracted by the PKC inhibitor, RO318220, whereas the effect of FSK was reduced by the PKA inhibitor RpcAMPs. In conclusion, we report that AEA levels are stimulated by both PKC, either directly or after thrombin receptor activation, and PKA, possibly in a way independent from intracellular calcium. Since AEA activates TRPV1, these findings may suggest the existence of an amplificatory cascades on this receptor in sensory neurons.
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Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Quinasa C/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Trombina/farmacología , Animales , Animales Recién Nacidos , Ácidos Araquidónicos/metabolismo , Moduladores de Receptores de Cannabinoides , Células Cultivadas , Colforsina/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Endocannabinoides , Ganglios Espinales/citología , Glicéridos/metabolismo , Humanos , Ratas , Ratas Sprague-Dawley , Receptores Proteinasa-Activados/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
OBJECTIVE: Recently, an activation of the endocannabinoid system during obesity has been reported. More particularly, it has been demonstrated that hypothalamic levels of both endocannabinoids, 2-arachidonoylglycerol and anandamide (N-arachidonoylethanolamine), are up-regulated in genetically obese rodents. Circulating levels of both endocannabinoids were also shown to be higher in obese compared with lean women. Yet, the direct production of endocannabinoids by human adipocytes has never been demonstrated. Our aim was to evaluate the ability of human adipocytes to produce endocannabinoids. RESEARCH METHODS AND PROCEDURES: The production of endocannabinoids by human adipocytes was investigated in a model of human white subcutaneous adipocytes in primary culture. The effects of leptin, adiponectin, and peroxisome proliferator-activated receptor (PPAR)-gamma activation on endocannabinoid production by adipocytes were explored. Endocannabinoid levels were determined by high-performance liquid chromatography (HPLC)-atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) analysis, leptin and adiponectin secretion measured by enzyme-linked immunosorbent assay (ELISA), and PPAR-gamma protein expression examined by Western blotting. RESULTS: We show that 2-arachidonoylglycerol, anandamide, and both anandamide analogs, N-palmitoylethanolamine and N-oleylethanolamine, are produced by human white subcutaneous adipocytes in concentrations ranging from 0.042+/-0.004 to 0.531+/-0.048 pM/mg lipid extract. N-palmitoylethanolamine is the most abundant cannabimimetic compound produced by human adipocytes, and its levels are significantly down-regulated by leptin but not affected by adiponectin and PPAR-gamma agonist ciglitazone. N-palmitoylethanolamine itself does not affect either leptin or adiponectin secretion or PPAR-gamma protein expression in adipocytes. DISCUSSION: This study has led to the identification of human adipocytes as a new source of endocannabinoids and related compounds. The biological significance of these adipocyte cannabimimetic compounds and their potential implication in obesity should deserve further investigations.
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Tejido Adiposo/metabolismo , Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Obesidad/metabolismo , Adipocitos/metabolismo , Adiponectina/metabolismo , Adulto , Ácidos Araquidónicos/metabolismo , Regulación hacia Abajo , Femenino , Glicéridos/metabolismo , Humanos , Lípidos/química , Persona de Mediana Edad , PPAR gamma/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Regulación hacia ArribaRESUMEN
The endocannabinoid system is upregulated in both human inflammatory bowel diseases and experimental models of colitis. In this study, we investigated whether this upregulation is a marker also of celiac disease-induced atrophy. The levels of the cannabinoid CB(1) receptor, of the endocannabinoids, anandamide, and 2-arachidonoyl-glycerol (2-AG), and of the anti-inflammatory mediator palmitoylethanolamide (PEA) were analyzed in bioptic samples from the duodenal mucosa of celiac patients at first diagnosis assessed by the determination of antiendomysial antibodies and histological examination. Samples were analyzed during the active phase of atrophy and after remission and compared to control samples from non-celiac patients. The levels of anandamide and PEA were significantly elevated (approx. 2- and 1.8-fold, respectively) in active celiac patients and so were those of CB(1) receptors. Anandamide levels returned to normal after remission with a gluten-free diet. We also analyzed endocannabinoid and PEA levels in the jejunum of rats 2, 3, and 7 days after treatment with methotrexate, which causes inflammatory features (assessed by histopathological analyses and myeloperoxidase activity) similar to those of celiac patients. In both muscle/serosa and mucosa layers, the levels of anandamide, 2-AG, and PEA peaked 3 days after treatment and returned to basal levels at remission, 7 days after treatment. Thus, intestinal endocannabinoid levels peak with atrophy and regress with remission in both celiac patients and methotrexate-treated rats. The latter might be used as a model to study the role of the endocannabinoid system in celiac disease.
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Moduladores de Receptores de Cannabinoides/metabolismo , Enfermedad Celíaca/metabolismo , Duodeno/metabolismo , Endocannabinoides , Yeyuno/metabolismo , Receptor Cannabinoide CB1/metabolismo , Adolescente , Adulto , Amidas , Animales , Ácidos Araquidónicos/metabolismo , Atrofia , Estudios de Casos y Controles , Enfermedad Celíaca/inducido químicamente , Enfermedad Celíaca/dietoterapia , Enfermedad Celíaca/patología , Niño , Dieta con Restricción de Proteínas , Modelos Animales de Enfermedad , Duodeno/patología , Etanolaminas , Femenino , Glicéridos/metabolismo , Humanos , Yeyuno/patología , Masculino , Metotrexato , Persona de Mediana Edad , Ácidos Palmíticos/metabolismo , Peroxidasa/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Ratas , Ratas Wistar , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Dendritic cells (DCs) are the single most central player in all immune responses. To assess whether DC alterations may contribute to the immune dysregulation that affects the elderly, we investigated the effects of ageing on DCs. We analyzed the number, phenotype and function of peripheral blood DCs from 70 healthy subjects aged 20-92 years by using flow cytometric methods that allow cell characterization directly in whole blood samples. We demonstrated that the number of myeloid DCs progressively declines with age. This finding was accompanied by a decrease of CD34+ precursors and increase of circulating monocytes, suggesting that the entire differentiation process of antigen presenting cells is partially dysregulated in the elderly. DCs from aged individuals also appeared to have a more mature phenotype and impaired ability to produce IL-12 upon stimulation. These results may help to clarify the contribution of innate immunity to the development of immunosenescence.
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Envejecimiento/inmunología , Células Dendríticas/inmunología , Monocitos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/sangre , Antígenos CD34/biosíntesis , Antígenos CD34/sangre , Antígenos CD34/metabolismo , Recuento de Células Sanguíneas , Células Dendríticas/metabolismo , Femenino , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Células Madre/inmunología , Células Madre/metabolismoRESUMEN
Direct stimulation of cannabinoid CB1 receptors exerts a protective function in animal models of inflammatory bowel diseases (IBDs). However, it is not known whether endocannabinoids are up-regulated during IBDs in animals or humans, nor whether pharmacological elevation of endocannabinoid levels can be exploited therapeutically in these disorders. In this study we addressed these questions. Colon inflammation was induced in mice and rats with 2,4-dinitrobenzene- and 2,4,6-trinitrobenzene sulfonic acids (DNBS and TNBS), respectively. DNBS-treated mice were treated chronically (for 3 or 7 days) with inhibitors of anandamide enzymatic hydrolysis (N-arachidonoyl-serotonin, AA-5-HT) or reuptake (VDM11), 10 or 5 mg/kg, s.c., or with 5-amino-salicilic acid (5-ASA, 1.4 mg/kg, i.r.). Endocannabinoids (anandamide and 2-arachidonoylglycerol, 2-AG) were quantified in mouse colon, or in rat colon mucosa and submucosa, and in bioptic samples from the colon of patients with untreated ulcerative colitis, by liquid chromatography-mass spectrometry. A strong elevation of anandamide, but not 2-AG, levels was found in the colon of DNBS-treated mice, in the colon submucosa of TNBS-treated rats, and in the biopsies of patients with ulcerative colitis. VDM-11 significantly elevated anandamide levels in the colon of DNBS-treated mice and concomitantly abolished inflammation, whereas AA-5-HT did not affect endocannabinoid levels and was significantly less efficacious at attenuating colitis. 5-ASA also increased anandamide levels and abolished colitis. Thus, anandamide is elevated in the inflamed colon of patients with ulcerative colitis, as well as in animal models of IBDs, to control inflammation, and elevation of its levels with inhibitors of its cellular reuptake might be used in the treatment of IBDs.
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Ácidos Araquidónicos/biosíntesis , Ácidos Araquidónicos/uso terapéutico , Colitis/tratamiento farmacológico , Mesalamina/uso terapéutico , Receptor Cannabinoide CB1/fisiología , Serotonina/análogos & derivados , Adulto , Anciano , Amidohidrolasas/antagonistas & inhibidores , Animales , Ácidos Araquidónicos/análisis , Ácidos Araquidónicos/genética , Ácidos Araquidónicos/farmacología , Ácidos Araquidónicos/fisiología , Bencenosulfonatos/toxicidad , Colitis/inducido químicamente , Colitis/patología , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colon/química , Colon/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Endocannabinoides , Femenino , Glicéridos/análisis , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/química , Mucosa Intestinal/patología , Masculino , Mesalamina/farmacología , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Peroxidasa/análisis , Alcamidas Poliinsaturadas , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/agonistas , Serotonina/farmacología , Serotonina/uso terapéutico , Organismos Libres de Patógenos Específicos , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
In the ventrolateral periaqueductal gray (PAG), activation of excitatory output neurons projecting monosynaptically to OFF cells in the rostral ventromedial medulla (RVM) causes antinociceptive responses and is under the control of cannabinoid receptor type-1 (CB1) and vanilloid transient receptor potential vanilloid type 1 (TRPV1) receptors. We studied in healthy rats the effect of elevation of PAG endocannabinoid [anandamide and 2-arachidonoylglycerol (2-AG)] levels produced by intra-PAG injections of the inhibitor of fatty acid amide hydrolase URB597 [cyclohexylcarbamic acid-3'-carbamoyl-biphenyl-3-yl ester] on 1) nociception in the "plantar test" and 2) spontaneous and tail-flick-related activities of RVM neurons. Depending on the dose or time elapsed since administration, URB597 (0.5-2.5 nmol/rat) either suppressed or increased thermal nociception via TRPV1 or CB1 receptors, respectively. TRPV1 or cannabinoid receptor agonists capsaicin (6 nmol) and (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate [WIN55,212-2 (4 nmol)] also suppressed or enhanced nociception, respectively. URB597 dose dependently enhanced PAG anandamide and 2-AG levels, with probable subsequent activation of TRPV1/CB1 receptors and only CB1 receptors, respectively. The TRPV1-mediated antinociception and CB1-mediated nociception caused by URB597 correlated with enhanced or reduced activity of RVM OFF cells, suggesting that these effects occur via stimulation or inhibition of excitatory PAG output neurons, respectively. Accordingly, several ventrolateral PAG neurons were found by immunohistochemistry to coexpress TRPV1 and CB1 receptors. Finally, at the highest doses tested, URB597 (4 nmol/rat) and, as previously reported, WIN55,212-2 (25-100 nmol) also caused CB(1)-mediated analgesia, correlating with stimulation (possibly disinhibition) of RVM OFF cells. Thus, endocannabinoids affect the descending pathways of pain control by acting at either CB1 or TRPV1 receptors in healthy rats.
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Amidohidrolasas/antagonistas & inhibidores , Benzamidas/farmacología , Moduladores de Receptores de Cannabinoides/fisiología , Carbamatos/farmacología , Endocannabinoides , Dolor/fisiopatología , Sustancia Gris Periacueductal/efectos de los fármacos , Receptor Cannabinoide CB1/fisiología , Canales Catiónicos TRPV/fisiología , Analgesia , Animales , Benzoxazinas , Moduladores de Receptores de Cannabinoides/análisis , Inmunohistoquímica , Masculino , Bulbo Raquídeo/efectos de los fármacos , Bulbo Raquídeo/fisiología , Morfolinas/farmacología , Naftalenos/farmacología , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/análisis , Canales Catiónicos TRPV/análisisRESUMEN
Recent studies have addressed the changes in endocannabinoid ligands and receptors that occur in multiple sclerosis, as a way to explain the efficacy of cannabinoid compounds to alleviate spasticity, pain, tremor, and other signs of this autoimmune disease. Using Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, we recently found a decrease in cannabinoid CB1 receptors mainly circumscribed to the basal ganglia, which could be related to the motor disturbances characteristic of these rats. In the present study, using the same model, we explored the potential changes in several neurotransmitters in the basal ganglia that might be associated with the motor disturbances described in these rats, but we only found a small increase in glutamate contents in the globus pallidus. We also examined whether the motor disturbances and the changes of CB1 receptors found in the basal ganglia of EAE rats disappear after the treatment with rolipram, an inhibitor of type IV phosphodiesterase able to supress EAE in different species. Rolipram attenuated clinical decline, reduced motor inhibition, and normalized CB1 receptor gene expression in the basal ganglia. As a third objective, we examined whether EAE rats also exhibited changes in endocannabinoid levels as shown for CB1 receptors. Anandamide and 2-arachidonoylglycerol levels decreased in motor related regions (striatum, midbrain) but also in other brain regions, although the pattern of changes for each endocannabinoid was different. Finally, we hypothesized that the elevation of the endocannabinoid activity, following inhibition of endocannabinoid uptake, might be beneficial in EAE rats. AM404, arvanil, and OMDM2 were effective to reduce the magnitude of the neurological impairment in EAE rats, whereas VDM11 did not produce any effect. The beneficial effects of AM404 were reversed by blocking TRPV1 receptors with capsazepine, but not by blocking CB1 receptors with SR141716, thus indicating the involvement of endovanilloid mechanisms in these effects. However, a role for CB1 receptors is supported by additional data showing that CP55,940 delayed EAE progression. In summary, our data suggest that reduction of endocannabinoid signaling is associated with the development of EAE in rats. We have also proved that the reduction of CB1 receptors observed in these rats is corrected following treatment with a compound used in EAE such as rolipram. In addition, the direct or indirect activation of vanilloid or cannabinoid receptors may reduce the neurological impairment experienced by EAE rats, although the efficacy of the different compounds examined seems to be determined by their particular pharmacodynamic and pharmacokinetic characteristics.
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Encéfalo/metabolismo , Encéfalo/fisiopatología , Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Receptores de Cannabinoides/metabolismo , Canales Catiónicos TRPV/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Ganglios Basales/efectos de los fármacos , Ganglios Basales/metabolismo , Ganglios Basales/fisiopatología , Encéfalo/efectos de los fármacos , Capsaicina/análogos & derivados , Capsaicina/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/fisiopatología , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Glicéridos/metabolismo , Masculino , Esclerosis Múltiple/fisiopatología , Inhibidores de Fosfodiesterasa/farmacología , Alcamidas Poliinsaturadas , Ratas , Ratas Endogámicas Lew , Receptor Cannabinoide CB1/efectos de los fármacos , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptores de Cannabinoides/efectos de los fármacos , Rolipram/farmacología , Canales Catiónicos TRPV/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis both of the endocannabinoids (which are known to inhibit intestinal motility) and other bioactive amides (palmitoylethanolamide, oleamide, and oleoylethanolamide), which might affect intestinal motility. The physiologic role of FAAH in the gut is largely unexplored. In the present study, we evaluated the possible role of FAAH in regulating intestinal motility in mice in vivo. METHODS: Motility was measured by evaluating the distribution of a fluorescent marker along the small intestine; FAAH messenger RNA (mRNA) levels were analyzed by reverse-transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by isotope-dilution, liquid chromatography, mass spectrometry. RESULTS: Motility was inhibited by N-arachidonoylserotonin (AA-5-HT) and palmitoylisopropylamide, 2 selective FAAH inhibitors, as well as by the FAAH substrates palmitoylethanolamide, oleamide, and oleoylethanolamide. The effect of AA-5-HT was reduced by the CB1 receptor antagonist rimonabant and by CB1 deficiency in mice but not by the vanilloid receptor antagonist 5'-iodoresiniferatoxin. In FAAH-deficient mice, pharmacologic blockade of FAAH did not affect intestinal motility. FAAH mRNA was detected in different regions of the intestinal tract. CONCLUSIONS: We conclude that FAAH is a physiologic regulator of intestinal motility and a potential target for the development of drugs capable of reducing intestinal motility.
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Amidohidrolasas/metabolismo , Motilidad Gastrointestinal/fisiología , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/genética , Animales , Tránsito Gastrointestinal , Intestino Grueso/fisiología , Intestino Delgado/fisiología , Cinética , Masculino , Ratones , Ratones Endogámicos ICR , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Several selective inhibitors of endocannabinoid inactivation via either the fatty acid amide hydrolase (FAAH) or the putative endocannabinoid transporter have been developed so far. Here, we have studied the effect in rats of a subchronic intraperitoneal treatment with three recently developed selective inhibitors of endocannabinoid uptake (VDM-11, UCM-707 and OMDM-2) or with a selective FAAH inhibitor (N-arachidonoyl-serotonin, AA-5-HT), on the brain levels of anandamide and 2-arachidonoylglycerol (2-AG) measured by means of isotope dilution LC-MS 1, 5 and 12 h after the last treatment. OMDM-2 was the most efficacious compound at enhancing the levels of anandamide at all time points, with a maximal effect (1.9-fold enhancement) after 5h. This compound also enhanced 2-AG levels by approximately 1.3-fold, but only 5 and 12h from administration. VDM-11 slightly, albeit significantly, enhanced anandamide levels (1.3-fold) only at 1h from administration and 2-AG levels (1.3-fold) only after 5h. Finally, UCM-707 only affected 2-AG levels (by two-fold) at only 1h from administration. FAAH inhibition by AA-5-HT significantly enhanced the levels of both anandamide (between 1.3- and 1.5-fold, maximal effect after 1 h) and 2-AG (between 1.3- and 1.6-fold, maximal effect after 12 h) at all time points. Brains from rats treated with AA-5-HT did never exhibit enhanced levels of serotonin, thus pointing to the metabolic stability of this FAAH inhibitor. These data indicate that: (1) the pharmacological effects reported so far for the four compounds under study in animal models of diseases may be due to enhancement of both anandamide and 2-AG levels; (2) 2-AG seems to need a longer time after the last administration in order to be augmented; (3) OMDM-2 and AA-5-HT should be regarded as enhancers of endocannabinoid levels suitable for use in vivo.