Increased Cytokeratin 19 Fragment Levels Are Positively Correlated with Adenosine Deaminase Activity in Malignant Pleural Effusions from Adenocarcinomas.

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.

Culture of neural cells of the eyestalk of a mangrove crab is optimized on poly-L-ornithine substrate.

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.

Combined evaluation of adenosine deaminase level and histopathological findings from pleural biopsy with Cope's needle for the diagnosis of tuberculous pleurisy.

INTRODUCTION: Closed needle pleural biopsy (CNPB) has historically been the gold standard procedure for the diagnosis of pleural tuberculosis. Adenosine deaminase (ADA) is an efficient biomarker for tuberculosis that is measurable in pleural fluids. OBJECTIVE: We compared the diagnostic accuracy of the pleural ADA (P-ADA) level and histopathological findings of CNPB specimens in patients with pleural tuberculosis. METHODS: This prospective study consisted of two groups of examinations with a proven diagnosis of pleural effusion. The P-ADA level was measured in 218 patients with pleural effusion due to a number of causes, and 157 CNPB specimens underwent histopathological analysis. RESULTS: CNPBs were performed in patients with tuberculosis (n=122) and other diseases: adenocarcinoma (n=23), lymphoma (n=5), systemic lupus erythematosus (n=4), squamous cell carcinoma (n=2), and small cell lung cancer (n=1). According to the ROC curve, the optimal cut-off value of the P-ADA level (Giusti and Galanti colorimetric method) was equal to or greater than 40.0 U/L. The diagnostic accuracy of the P-ADA test was 83.0%, and that of histopathological examination of the CNPB tissue, was 78.8% (AUC=0.293, P=0.7695). The association between the P-ADA assay and pleural histopathology was 24.41 (P<0.0001). The tetrachoric correlation coefficient was 0.563 (high correlation). CONCLUSION: In Brazil and other countries with a high incidence of tuberculosis, P-ADA activity is an accurate test for the diagnosis of tuberculous pleural effusions, and its use should be encouraged. The high diagnostic performance of the P-ADA test could to aid the diagnosis of pleural tuberculosis and render CNPB unnecessary.

Evaluation of adenosine deaminase activity for the diagnosis of pleural TB in lymphocytic pleural effusions.

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.

A phospholipase A2 isolated from Lachesis muta snake venom increases the survival of retinal ganglion cells in vitro.

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.

The trophic effect of ouabain on retinal ganglion cell is mediated by EGF receptor and PKC delta activation.

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.

Neuronal cell survival: the role of interleukins.

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.

Protein kinase C regulates the expression of M1 receptors and BDNF in rat retinal cells.

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.

The increase in retinal cells proliferation induced by FGF2 is mediated by tyrosine and PI3 kinases.

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.

Ca2+ mobilization induced by ouabain in thymocytes involves intracellular and extracellular Ca2+ pools.

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.

PMA decreases the proliferation of retinal cells in vitro: the involvement of acetylcholine and BDNF.

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.

Sciatic conditioned medium increases survival, proliferation and differentiation of retinal cells in culture.

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.