RESUMEN
Adenosine deaminase (ADA) and cytokeratin 19 (CK19) are known pleural biomarkers. Although ADA in humans functions mainly in the immune system, it also appears to be associated with the differentiation of epithelial cells. Keratin filaments are important structural stabilizers of epithelial cells and potent biomarkers in epithelial differentiation. This study aimed to investigate the simultaneous presence of the ADA enzyme and CK19 fragments to assess epithelial differentiation in malignant and benign pleural fluids. Diagnosis of the cause of pleural effusion syndrome was confirmed by means of standard examinations and appropriate surgical procedures. An ADA assay, in which ADA irreversibly catalyzes the conversion of adenosine into inosine, was performed using a commercial kit. The CK19 assay was performed using a CYFRA 21-1 kit, developed to detect quantitative soluble fragments of CK19 using an electrochemiluminescence immunoassay. One hundred nineteen pleural fluid samples were collected from untreated individuals with pleural effusion syndrome due to several causes. ADA levels only correlated with CK19 fragments in adenocarcinomas, with high significance and good correlation (rho = 0.5145, P = 0.0036). However, further studies are required to understand this strong association on epithelial differentiation in metastatic pleural fluids from adenocarcinomas.
Asunto(s)
Adenocarcinoma/metabolismo , Adenosina Desaminasa/metabolismo , Biomarcadores de Tumor/metabolismo , Queratina-19/metabolismo , Derrame Pleural Maligno/metabolismo , Adenocarcinoma/patología , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Derrame Pleural Maligno/patologíaRESUMEN
Although there is a considerable demand for cell culture protocols from invertebrates for both basic and applied research, few attempts have been made to culture neural cells of crustaceans. We describe an in vitro method that permits the proliferation, growth and characterization of neural cells from the visual system of an adult decapod crustacean. We explain the coating of the culture plates with different adhesive substrates, and the adaptation of the medium to maintain viable neural cells for up to 7 days. Scanning electron microscopy allowed us to monitor the conditioned culture medium to assess cell morphology and cell damage. We quantified cells in the different substrates and performed statistical analyses. Of the most commonly used substrates, poly-L-ornithine was found to be the best for maintaining neural cells for 7 days. We characterized glial cells and neurons, and observed cell proliferation using immunocytochemical reactions with specific markers. This protocol was designed to aid in conducting investigations of adult crustacean neural cells in culture. We believe that an advantage of this method is the potential for adaptation to neural cells from other arthropods and even other groups of invertebrates.
RESUMEN
AIM: To evaluate the diagnostic value of pleural adenosine deaminase (P-ADA) as a pleural TB-specific biomarker in lymphocytic pleural effusions. MATERIALS & METHODS: Pleural effusions were classified on the basis of definitive diagnosis. RESULTS: A total of 218 patients (122 tuberculous and 96 nontuberculous) were included in the study. The optimal cut-off value of P-ADA (receiver operating characteristic curve) for the diagnosis of pleural TB was 40.0 U/l (Giusti method). In lymphocytic pleural effusions P-ADA had a sensitivity of 80.3%, a specificity of 96.0% and an accuracy of 86.2%. The positive predictive value was 97.0% and the negative predictive value was 75.0%. The positive likelihood ratio and negative likelihood ratio were 19.8 and 0.2, respectively (p < 0.0001). CONCLUSION: P-ADA activity is recommended for the diagnosis of TB in lymphocytic pleural effusions.
Asunto(s)
Adenosina Desaminasa/análisis , Derrame Pleural/diagnóstico , Tuberculosis Pleural/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Niño , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Adulto JovenRESUMEN
We have previously showed that a phospholipase A2 isolated from Lachesis muta snake venom and named LM-PLA2-I displayed particular biological activities, as hemolysis, inhibition on platelet aggregation, edema induction and myotoxicity. In the present work, we evaluated the effect of LM-PLA2-I on the survival of axotomized rat retinal ganglion cells kept in vitro, as well as its mechanism of action. Our results clearly showed that treatment with LM-PLA2-I increased the survival of ganglion cells (100% when compared to control cultures) and the treatment of LM-PLA2-I with p-bromophenacyl bromide abolished this effect. This result indicates that the effect of LM-PLA2-I on ganglion cell survival is entirely dependent on its enzymatic activity and the generation of lysophosphatidylcholine (LPC) may be a prerequisite to the observed survival. In fact, commercial LPC mimicked the effect of LM-PLA2-I upon ganglion cell survival. To investigate the mechanism of action of LM-PLA2-I, cultures were treated with chelerythrine chloride, BAPTA-AM, rottlerin and also with an inhibitor of c-junc kinase (JNKi). Our results showed that rottlerin and JNK inhibitor abolished the LM-PLA2-I on ganglion cell survival. Taken together, our results showed that LM-PLA2-I and its enzymatic product, LPC promoted survival of retinal ganglion cells through the protein kinase C pathway and strongly suggest a possible role of the PLA2 enzyme and LPC in controlling the survival of axotomized neuronal cells.
Asunto(s)
Venenos de Crotálidos/enzimología , Lisofosfatidilcolinas/metabolismo , Fosfolipasas A2/farmacología , Células Ganglionares de la Retina/efectos de los fármacos , Acetonitrilos/farmacología , Acetofenonas/farmacología , Animales , Benzofenantridinas/farmacología , Benzopiranos/farmacología , Benzotiazoles/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Lisofosfatidilcolinas/farmacología , Ratas , Células Ganglionares de la Retina/patologíaRESUMEN
It was already shown that ouabain treatment can stimulate PKC isoenzymes leading to the activation of intracellular pathways involved in cell survival, growth and proliferation. We have previously demonstrated that ouabain or PMA treatment increases retinal ganglion cell survival, an effect mediated by PKC activation. The aim of this work was to investigate the role of EGF receptors in the ouabain effect and also to study which PKC isoform is activated by treatment with ouabain and PMA. Our results show that 2.5 microM tyrphostin, 1.0 microM PP1, 4.0 microM U73122, 1.0 microM JNK inhibitor V and 2.0 microM rottlerin blocked the ouabain effect indicating an involvement of receptors for EGF, Src, PLC, JNK and PKC delta respectively. The effect of PMA was only abolished when cultures were treated with rottlerin or with the JNK inhibitor suggesting the involvement of PKC delta and JNK. These results indicate that PKC delta could be a key regulator of retinal ganglion cell survival.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Receptores ErbB/metabolismo , Ouabaína/farmacología , Proteína Quinasa C-delta/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Células Cultivadas , Activación Enzimática , Isoenzimas/metabolismo , Ratas , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismoRESUMEN
One of the central issues in neuroscience today is the study of the mechanisms of neuronal survival. Since the discovery of nerve growth factor (almost 60 years ago), many groups have clearly demonstrated the central role of neurotrophins on the regulation of neuronal cell survival during developmental stages as well as during adult life. However, neurotrophins are not alone in regulating neuronal survival, and many groups have demonstrated the effect of different cytokines on this phenomenon. In this brief review we will address the effect of interleukins (IL), particularly IL-2, IL-6, and IL-4, on the survival of neuronal cells.
Asunto(s)
Interleucinas/metabolismo , Neuronas/citología , Animales , Supervivencia Celular , Humanos , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Interleucina-6/metabolismoRESUMEN
Protein kinase C (PKC) plays a key role in cellular events including proliferation, survival and differentiation. Our previous study showed the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, inducing a decrease in retinal cells proliferation. This effect was mediated by muscarinic type 1 receptors (M1) activation and brain derived neurotrophic factor (BDNF) treatment also induced a decrease in cell proliferation. Based on these results we analyzed the expression of either M1 receptors or BDNF following PMA treatment of retinal cell cultures. Our data demonstrated that PMA induced a decrease in both protein expressions after 48 h in culture. However, after 45 min, PMA induced a transient increase in BDNF expression and a decrease in M1 receptors expression. Analyzing the expression of M1 receptors and BDNF during the postnatal development in vivo, we observed a decrease in both proteins. Taken together our results suggest the involvement of PKC in the control of M1 expression in retinal cells.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Proteína Quinasa C/fisiología , Receptor Muscarínico M1/biosíntesis , Retina/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática , Regulación de la Expresión Génica , Ratas , Retina/citología , Retina/crecimiento & desarrollo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Since 1973, multiple effects of basic fibroblast growth factor have been described in a large number of cells. These effects include proliferation, survival and differentiation. The aim of this work was to study the intracellular pathways involved in the basic fibroblast growth factor (FGF2) effect on rat retinal cells proliferation in vitro. Our data show that treatment with FGF2 increases proliferation in a concentration- and time-dependent manner. The effect of 25 ng/ml FGF2 was blocked by 10 microM genistein, a tyrosine kinase inhibitor and by 25 microM LY294002, a PI3 kinase inhibitor. The concomitant treatment with 0.3 microM chelerythrine chloride, a protein kinase C inhibitor, and 6.25 microM LY294002 also inhibited the effect of FGF2. Our results suggest that the proliferative effect of FGF2 on retinal cell cultures involves the activation of distinct kinases.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Retina/efectos de los fármacos , Animales , Benzofenantridinas/farmacología , Cromonas/farmacología , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Retina/citología , Retina/enzimologíaRESUMEN
Immune dysfunction has been reported in hypertensive rats, and circulating levels of ouabain are increased in some experimental models of hypertension. Ouabain is an inhibitor of the Na+/K+-ATPase capable of diverse effects on cells of the immune system, but its mode of action on these cells is still unknown. The levels of cytoplasmic calcium ions play an important role in cell signaling, and ouabain may induce an increase in intracellular calcium indirectly through the Na+/Ca2+ exchanger. The current work examined the possibility that this drug could be exerting its effects on thymocytes through calcium mobilization and an increase in the cytosolic calcium concentration. Intracellular calcium was evaluated by using Balb-c mouse thymocytes loaded with FURA-2. Both intracellular and extracellular calcium pools were mobilized by ouabain (3 to 1000 nmol). The influx of extracellular calcium depended on the Na+/Ca2+ exchanger and on voltage-dependent calcium channels, as it was inhibited by amiloride and benzamil, consistent with the inhibition of the Na+/K+ pump. In addition, the increase of calcium from intracellular stores was extremely rapid. Furthermore, an increase in cytosolic calcium levels was obtained with the combination of ouabain and thapsigargin, which was greater than that seen with either drug alone. Our data suggest that low concentrations of ouabain may be acting on thymocytes through a mechanism different from the traditional inhibition of the Na+/K+-ATPase, as the cytosolic calcium rise was partly dependent on the release from intracellular stores.
Asunto(s)
Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Ouabaína/farmacología , Timo/metabolismo , Animales , Canales de Calcio/fisiología , Membrana Celular/fisiología , Células Cultivadas , Citosol/metabolismo , Sinergismo Farmacológico , Espacio Extracelular/metabolismo , Transporte Iónico/efectos de los fármacos , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos BALB C , Canales de Sodio/fisiología , Intercambiador de Sodio-Calcio/fisiología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Tapsigargina/farmacología , Timo/efectos de los fármacos , Timo/fisiologíaRESUMEN
Protein kinase C (PKC) is involved in several cell events including proliferation, survival and differentiation. The aim of this work was to investigate the role of PKC activation on retinal cells proliferation. We demonstrated that PKC activation by phorbol 12-myristate 13-acetate (PMA), a tumor promoter phorbol ester, is able to decrease retinal cells proliferation. This effect was mediated by M1 receptors and dependent on intracellular Ca(2+) increase, tyrosine kinase activity, phosphatidylinositol 3-kinase activity, polypeptide secretion and activation of TrkB receptors. The effect of PMA was not via activation of mitogen-activated protein (MAP) kinase. Carbamylcholine and brain derived neurotrophic factor were both able to decrease retinal cells proliferation to the same level as PMA did. Our results suggest that PKC activation leads to a decrease in retinal cells proliferation through the release of acetylcholine and brain derived neurotrophic factor in the culture, and activation of M1 and TrkB receptors, respectively.
Asunto(s)
Acetilcolina/fisiología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Carbacol/farmacología , Proteínas del Ojo/metabolismo , Inhibidores de Crecimiento/farmacología , Pirenzepina/análogos & derivados , Proteína Quinasa C/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Retina/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Alcaloides , Células Amacrinas/efectos de los fármacos , Animales , Atropina/farmacología , Benzofenantridinas , Calcio/farmacología , División Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas del Ojo/efectos de los fármacos , Isoenzimas/efectos de los fármacos , Isoenzimas/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Oxotremorina/farmacología , Fenantridinas/farmacología , Pirenzepina/farmacología , Proteína Quinasa C/efectos de los fármacos , Ratas , Receptor Muscarínico M1 , Receptores Muscarínicos/fisiología , Retina/citología , Transducción de Señal/efectos de los fármacosRESUMEN
Many evidences clearly demonstrate that Schwann cells provide trophic support for neurons. Different cytokines, including neurotrophins (NTs), are produced and released by Schwann cells. These trophic molecules play an important role on neuronal survival either during the development or during adult life. Cytokines have also a pivotal role on neuronal regeneration after lesions occurring during pathological conditions. The aim of this work was to study the effect of sciatic conditioned medium (SCM) on rat retinal cells maintained in culture. Our results show that treatment with SCM obtained after 14 days in vitro (SCM 14 day) induced a three-fold increase in protein content of the culture after 48 h in vitro and this value remained equally high up to 72 h. This effect was totally blocked either by addition of 30 microM BAPTA-AM, an intracellular calcium chelator, 15 microM fluorodeoxyuridine, an inhibitor of cell division, or 10 microM genistein (geni) plus 1.25 microM chelerythrine chloride (CC), the two last ones inhibitors of tyrosine kinases and protein kinase C, respectively. SCM induced an increase in [(3)H]-choline uptake and [(3)H]-thymidine incorporation of retinal cells. SCM also stimulated an increase in cytoplasmic processes outgrowth of retinal cells and survival of retinal ganglion cells. Our results clearly suggest that soluble molecules released by sciatic nerve fragments are able to increase the proliferation and survival of retinal cells in culture.