RESUMEN
BACKGROUND AND PURPOSE: Pulpitis is associated with tooth hypersensitivity and results in pulpal damage. Thermosensitive transient receptor potential (TRP) ion channels expressed in the dental pulp may be key transducers of inflammation and nociception. We aimed at investigating the expression and role of thermo-TRPs in primary human dental pulp cells (hDPCs) in normal and inflammatory conditions. EXPERIMENTAL APPROACH: Inflammatory conditions were induced in hDPC cultures by applying polyinosinic:polycytidylic acid (poly(I:C)). Gene expression and pro-inflammatory cytokine release were measured by RT-qPCR and ELISA. Functions of TRPA1 channels were investigated by monitoring changes in intracellular Ca2+ concentration. Mitochondrial superoxide production was measured using a fluorescent substrate. Cellular viability was assessed by measuring the activity of mitochondrial dehydrogenases and cytoplasmic esterases. TRPA1 activity was modified by agonists, antagonists, and gene silencing. KEY RESULTS: Transcripts of TRPV1, TRPV2, TRPV4, TRPC5, and TRPA1 were highly expressed in control hDPCs, whereas TRPV3, TRPM2, and TRPM3 expressions were much lower, and TRPM8 was not detected. Poly(I:C) markedly up-regulated TRPA1 but not other thermo-TRPs. TRPA1 agonist-induced Ca2+ signals were highly potentiated in inflammatory conditions. Poly(I:C)-treated cells displayed increased Ca2+ responses to H2O2, which was abolished by TRPA1 antagonists. Inflammatory conditions induced oxidative stress, stimulated mitochondrial superoxide production, resulted in mitochondrial damage, and decreased cellular viability of hDPCs. This inflammatory cellular damage was partly prevented by the co-application of TRPA1 antagonist or TRPA1 silencing. CONCLUSION AND IMPLICATIONS: Pharmacological blockade of TRPA1 channels may be a promising therapeutic approach to alleviate pulpitis and inflammation-associated pulpal damage.
Asunto(s)
Pulpa Dental , Estrés Oxidativo , Pulpitis , Canal Catiónico TRPA1 , Regulación hacia Arriba , Humanos , Estrés Oxidativo/efectos de los fármacos , Canal Catiónico TRPA1/metabolismo , Canal Catiónico TRPA1/antagonistas & inhibidores , Pulpitis/metabolismo , Pulpitis/patología , Pulpa Dental/citología , Pulpa Dental/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Células Cultivadas , Canales de Potencial de Receptor Transitorio/metabolismo , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Poli I-C/farmacología , Supervivencia Celular/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Calcio/metabolismo , Superóxidos/metabolismoRESUMEN
BACKGROUND: There is numerous empirical evidence supporting that college students studying in fields with rigorous curriculum and high requirements, such as medical training, are characterized by a higher risk of attrition than their peers. Since Hungarian medical training attracts more and more international students every year, the issue of dropout can have a global impact. Our study aimed to examine attrition risks of local and international students in Hungarian medical training. METHODS: In our study, we examined the dropout behaviour of all medical students who started their studies in 2010 in Hungary (N = 2391) by analysing longitudinal administrative data of those who studied between 2010 and 2017. Doing this, we conducted descriptive statistics and uncovered the risks of dropout using binary logistic regression. RESULTS: Our results indicate that the danger is primarily increased by factors directly linked to or indicating poor academic performance (slow pace of credit accumulation, tuition-based forms of finance). Individual characteristics, namely gender, and citizenship, also have a moderate but significant effect on the latter. CONCLUSIONS: Thus a policy proposal can be formulated consisting of making the training network less rigid, devoting more educational attention to and providing targeted mentoring for students with learning difficulties and academic hardships. Foreign medical students studying in Hungary comprise a large group that has a high attrition rate, making it a prime target for dropout-reducing programs.
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Análisis de Datos , Estudiantes de Medicina , Curriculum , Escolaridad , Humanos , HungríaRESUMEN
RasGRP3 mediates the activation of the Ras signaling pathway that is present in many human cancers. Here, we explored the involvement of RasGRP3 in the formation and maintenance of the prostate cancer phenotype. RasGRP3 expression was elevated in multiple human prostate tumor tissue samples and in the human androgen-independent prostate cancer cell lines PC-3 and DU 145 compared with the androgen-dependent prostate cancer cell line LNCaP. Downregulation of endogenous RasGRP3 in PC-3 and DU 145 cells reduced Ras-GTP formation, inhibited cell proliferation, impeded cell migration, and induced apoptosis. Anchorage-independent growth of the PC-3 cells and tumor formation in mouse xenografts of both cell lines were likewise inhibited. Inhibition of RasGRP3 expression reduced AKT and extracellular signal-regulated kinase 1/2 phosphorylation and sensitized the cells to killing by carboplatin. Conversely, exogenous RasGRP3 elevated Ras-GTP, stimulated proliferation, and provided resistance to phorbol 12-myristate 13-acetate-induced apoptosis in LNCaP cells. RasGRP3-overexpressing LNCaP cells displayed a markedly enhanced rate of xenograft tumor formation in both male and female mice compared with the parental line. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses, caused the cells to resume the LNCaP morphology, and suppressed growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. We conclude that RasGRP3 contributes to the malignant phenotype of the prostate cancer cells and may constitute a novel therapeutic target for human prostate cancer.
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Factores de Intercambio de Guanina Nucleótido/genética , Neoplasias de la Próstata/genética , División Celular , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Masculino , Fenotipo , Neoplasias de la Próstata/patología , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
The muscle Lim protein knock-out (MLP-KO) mouse model is extensively used for studying the pathophysiology of dilated cardiomyopathy. However, explanation is lacking for the observed long survival of the diseased mice which develop until adulthood despite the gene defect, which theoretically predestines them to early death due to heart failure. We hypothesized that adaptive changes of cardiac intracellular calcium (Ca(i)(2+)) handling might explain the phenomenon. In order to study the progression of changes in cardiac function and Ca(i)(2+) cycling, myocardial Ca(i)(2+)-transients recorded by Indo-1 surface fluorometry were assessed with concomitant measurement of hemodynamic performance in isolated Langendorff-perfused hearts of 3- and 9-month old MLP-KO animals. Hearts were challenged with beta-agonist isoproterenol and the sarcoplasmic reticular Ca(2+)-ATPase (SERCA2a) inhibitor cyclopiazonic acid (CPA). Cardiac mRNA content and levels of key Ca(2+) handling proteins were also measured. A decline in lusitropic function was observed in 3-month old, but not in 9-month old MLP-KO mice under unchallenged conditions. beta-adrenergic responses to isoproterenol were similar in all the studied groups. The CPA induced an increase in end-diastolic Ca(i)(2+)-level and a decrease in Ca(2+)-sequestration capacity in 3-month old MLP-KO mice compared to age-matched controls. This unfavorable condition was absent at 9 months of age. SERCA2a expression was lower in 3-month old MLP-KO than in the corresponding controls and in 9-month old MLP-KO hearts. Our results show time-related recovery of hemodynamic function and an age-dependent compensatory upregulation of Ca(i)(2+) handling in hearts of MLP-KO mice, which most likely involve the normalization of the expression of SERCA2a in the affected hearts.
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Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/mortalidad , Corazón/fisiopatología , Hemodinámica , Proteínas Musculares/fisiología , Factores de Edad , Animales , Western Blotting , Índice de Masa Corporal , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Insuficiencia Cardíaca/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Indoles/farmacología , Isoproterenol/farmacología , Proteínas con Dominio LIM , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Miocardio/patología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tasa de SupervivenciaRESUMEN
OBJECTIVES: Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa. Its etiology is still unclear. Neurogenic components might contribute to the inflammatory process. The oral mucosa is richly innervated by sensory fibers. Mediators secreted by inflammatory cells activate sensory nerves via transient receptor potential vanilloid receptor 1 (TRPV1) and lead to the release of neuropeptides. So far, TRPV1 receptor expression was detected on neurons. Only recently, TRPV1 receptors were identified in nonneuronal tissues. The aim of the present study was to detect the presence of TRPV1 receptors and peripheral expression of receptor mRNA in normal oral mucosa and mucous membranes from OLP patients. METHODS: Presence of TRPV1 receptor proteins in the mucosal tissue was assessed by immunohistochemistry. Expression of TRPV1 receptor mRNA was determined by quantitative RT-PCR. RESULTS: We provided qualitative and quantitative immunohistochemical evidence that TRPV1 receptors are present in normal human oral mucosa and that their expression is increased in OLP. The number of immunopositive cells was elevated in the epithelium, and vascular endothelial cells, lymphocytes and fibroblasts of the subepithelium were also labeled in samples obtained from OLP patients. The local expression of nonneuronal TRPV1 receptors was proven at mRNA level using quantitative real-time RT-PCR. CONCLUSIONS: Since the number of TRPV1 receptor-positive nonneural cells is increased in inflammatory conditions, we hypothesize that TRPV1-receptor-mediated processes might play role in the pathogenesis of OLP.
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Inflamación/metabolismo , Liquen Plano Oral/metabolismo , Mucosa Bucal/metabolismo , Canales Catiónicos TRPV/metabolismo , Adulto , Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Inflamación/genética , Inflamación/fisiopatología , Liquen Plano Oral/genética , Liquen Plano Oral/fisiopatología , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Mucosa Bucal/patología , Mucosa Bucal/fisiopatología , Nociceptores/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/genética , Regulación hacia Arriba/fisiologíaRESUMEN
PURPOSE: Recently, functional cannabinoid receptor-1 (CB1) and vanilloid receptor-1 (TRPV1) have been described in human prostate and prostate cancer-derived cell lines where the activation of the receptors resulted in inhibition of cellular growth. We, however, lack the description of the expression of these molecules in human prostate cancer (PCC) and in benign prostate hyperplasia (BPH). METHODS: Therefore, immunohistochemistry, Western blotting, and quantitative "real-time Q-PCR were performed to define the expressions of CB1 and TRPV1 in healthy and diseased prostate tissues. RESULTS: CB1 was identified in epithelial and smooth muscle cells types of the human prostate, whereas TRPV1 was exclusively localized to the mucosal cells. We also found that the expression of CB1 and TRPV1 (both at the protein and mRNA levels) were significantly up-regulated in PCC. However, while the increased expression of TRPV1 showed a proper correlation with increasing PCC tumor grades, such phenomenon was not observed with CB1. In addition, we also measured markedly elevated CB1 levels in BPH tissues whilst the expression of TRPV1 was not altered when compared to healthy control prostate. CONCLUSIONS: Our findings strongly argue for that (1) the CB1 and TRPV1 molecules as well as their ligands may indeed possess a promising future role in the treatment of PCC; (2) TRPV1 may also serve as a prognostic factor in PCC; and (3) CB1 may act as a potential target molecule in the therapeutic management of BPH.
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Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/genética , Receptor Cannabinoide CB1/genética , Canales Catiónicos TRPV/genética , Humanos , Inmunohistoquímica , Masculino , Próstata/citología , Próstata/patología , Prostatectomía , Hiperplasia Prostática/genética , Hiperplasia Prostática/patología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , ARN Mensajero/genética , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
BACKGROUND: Tramadol is an effective analgesic substance widely used in medical practice. Its therapeutic action have been mainly attributed to the activation of mu-opioid receptors as well as to the inhibition of neurotransmitter reuptake mechanisms and various voltage- and ligand-gated ion channels of the nociceptive system. As transient receptor potential vanilloid-1 (TRPV1, "the capsaicin receptor") has been shown to function as a central integrator molecule of pain sensation, our aim in the current study was to define the involvement of TRPV1 in the complex mechanism of action of tramadol. METHODS: To achieve these goals, we used single-cell Ca-imaging as well as fluorescent image plate reader assays on Chinese hamster ovary (CHO) cells heterologously over-expressing TRPV1. RESULTS: We found that (1) tramadol, similar to the well-known TRPV1 agonist, capsaicin, significantly increased [Ca(2+)](i) of TRPV1-CHO cells in a concentration-dependent fashion; (2) its effect was reversibly prevented by the TRPV1 antagonist capsazepine; (3) repeated application of tramadol resulted in marked tachyphylaxis; and (4) tramadol did not modify [Ca(2+)](i) in control (empty vector expressing) CHO cells. CONCLUSIONS: Collectively, these findings strongly support the intriguing and novel concept that tramadol acts as an agonist of TRPV1. Considering that activation of TRPV1 on sensory neurons is followed by a local release of vasoactive neuropeptides and a marked desensitization of the afferent fibers (hence termination of pain sensation), our findings may equally explain both the desired analgesic as well as the often-seen, yet "unexpected," local side effects (e.g., initiation of burning pain and erythema) of tramadol.
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Analgésicos Opioides/farmacología , Canales Catiónicos TRPV/agonistas , Tramadol/farmacología , Animales , Células CHO , Calcio/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacología , Cricetinae , Cricetulus , Citofotometría , Relación Dosis-Respuesta a Droga , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Taquifilaxis , Transfección , Regulación hacia ArribaRESUMEN
Itch (pruritus) is a sensory phenomenon characterized by a (usually) negative affective component and the initiation of a special behavioral act, i.e. scratching. Older studies predominantly have interpreted itch as a type of pain. Recent neurophysiological findings, however, have provided compelling evidence that itch (although it indeed has intimate connections to pain) rather needs to be understood as a separate sensory modality. Therefore, a novel pruriceptive system has been proposed, within which itch-inducing peripheral mediators (pruritogens), itch-selective receptors (pruriceptors), sensory afferents and spinal cord neurons, and defined, itch-processing central nervous system regions display complex, layered responses to itch. In this review, we begin with a current overview on the neurophysiology of pruritus, and distinguish it from that of pain. We then focus on the functional characteristics of the large family of transient receptor potential (TRP) channels in skin-coupled sensory mechanisms, including itch and pain. In particular, we argue that - due to their expression patterns, activation mechanisms, regulatory roles, and pharmacological sensitivities - certain thermosensitive TRP channels are key players in pruritus pathogenesis. We close by proposing a novel, TRP-centered concept of pruritus pathogenesis and sketch important future experimental directions towards the therapeutic targeting of TRP channels in the clinical management of itch.
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Prurito/etiología , Prurito/terapia , Canales de Potencial de Receptor Transitorio/fisiología , Animales , Cannabinoides/metabolismo , Capsaicina/metabolismo , Sistema Nervioso Central/fisiología , Histamina/fisiología , Humanos , Mediadores de Inflamación/fisiología , Factores de Crecimiento Nervioso/fisiología , Neuronas/fisiología , Neuronas Aferentes/metabolismo , Neuronas Aferentes/fisiología , Neuropéptidos/fisiología , Dolor/etiología , Dolor/metabolismo , Prurito/clasificación , Prurito/fisiopatología , Médula Espinal/fisiología , Sustancia P/fisiología , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/fisiología , TemperaturaRESUMEN
In this study, we have investigated the effects of insulin-like growth factor-I (IGF-I) on cellular responses of primary human skeletal muscle cells and mouse C2C12 myoblasts. In human muscle, IGF-I stimulated proliferation and fusion of the cells and the expression of the differentiation marker desmin. These effects were completely inhibited by Rottlerin, the inhibitor of the protein kinase C (PKC)delta, but were not affected by the inhibition of the mitogen-activated protein kinase (MAPK) or the phosphatidylinositide 3-kinase (PI-3K) pathways. Furthermore, IGF-I initiated the selective translocation of PKCdelta to the nucleus. In C2C12 myoblasts, the growth-promoting effects of IGF-I were abrogated by inhibition of PKCdelta, but not by the inhibition of the PI-3K system. However, in contrast to the human data, the MAPK inhibitor PD098059 partially (yet significantly) also inhibited the action of IGF-I and, furthermore, IGF-I induced phosphorylation of the MAPK Erk-1/2. In addition, overexpression of constitutively active form of PKCdelta in C2C12 cells fully mimicked, whereas overexpression of kinase inactive mutant of the isoform prevented the action of IGF-I. Finally, the inhibition of PKCdelta suspended the IGF-I-induced phosphorylation of Erk-1/2 and, moreover, the inhibition of the MAPK pathway partially (yet significantly) inhibited the accelerated growth of C2C12 cells overexpressing PKCdelta. Taken together, these results demonstrate a novel, central and exclusive involvement of PKCdelta in mediating the action of IGF-I on human skeletal muscle cells, with an additional yet PKCdelta-dependent contribution of the MAPK pathway on C2C12 myoblasts.
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Factor I del Crecimiento Similar a la Insulina/metabolismo , Mitosis/fisiología , Músculo Esquelético/citología , Mioblastos/metabolismo , Proteína Quinasa C-delta/metabolismo , Transducción de Señal/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Desmina/metabolismo , Humanos , Isoenzimas/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mioblastos/citología , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
The vanilloid receptor (VR1) is a central integrator molecule of nociceptive stimuli. In this study, we have measured the effects of various neurotrophins (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and -4) on recombinant rat VR1-mediated intracellular calcium rise in response to capsaicin in VR1/C6 cells. Our results clearly show that all neurotrophins sensitize the VR1 to capsaicin. Furthermore, using K252a, an inhibitor of tyrosine kinases, we present that actions of neurotrophins are mediated by the trk (A, B, C) receptors expressed in these cells. These data argue for the putative roles of neurotrophins in inducing inflammatory (thermal) hyperalgesia via VR1.