A Novel Panel of Serum Biomarkers for MPM Diagnosis.

Exposure to asbestos is the main cause of malignant pleural mesothelioma (MPM), a highly aggressive cancer of the pleura. Since the only tools for early detection are based on radiological tests, some authors focused on serum markers (i.e., mesothelin). The aim of this study was the evaluation of new serum biomarkers to be used individually or in combination, in order to improve the outcome of patients whose disease would be diagnosed at an earlier stage. Serum and plasma were available from 43 subjects previously exposed to asbestos and 27 MPM patients, all being epithelioid type. All the new markers found differentially expressed in MPM and healthy subjects, by proteomic and genomic approaches, have been validated in the serum by the use of specific ELISA. The combined approach, using tools of genomics and proteomics, is found to be highly innovative for this type of disease and led to the identification of new serum markers in the diagnosis of MPM. These results, if confirmed in a larger series, may have a strong impact in this area, because early detection of this cancer in people at high risk could significantly improve the course of the disease and the clinical approach to an individualized therapy.

Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n=45 CFS; n=45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments.

The activation of mitochondrial BK potassium channels contributes to the protective effects of naringenin against myocardial ischemia/reperfusion injury.

Naringenin (NAR), flavonoid abundant in the genus Citrus, has been reported to interact with the large-conductance calcium-activated potassium channels (BK). Since activators of BK channels expressed in cardiac mitochondria trigger protective effects in several models of myocardial ischemia/reperfusion (I/R), this work aimed to evaluate the potential cardioprotective effects of NAR and the involvement of mitochondrial BK channels. In an in vivo model of acute infarct in rats, NAR (100mg/kg i.p.) significantly reduced the heart injury induced by I/R. This effect was antagonized by the selective BK-blocker paxilline (PAX). The cardioprotective dose of NAR did not cause significant effects on the blood pressure. In Largendorff-perfused rat hearts submitted to ischemia/reperfusion, NAR improved the post-ischemic functional parameters (left ventricle developed pressure and dP/dt) with lower extension of myocardial injury. On isolated rat cardiac mitochondria, NAR caused a concentration-dependent depolarization of mitochondrial membrane and caused a trans-membrane flow of thallium (potassium-mimetic cation). Both these effects were antagonized by selective blockers of BK channels. Furthermore, NAR half-reduced the calcium accumulation into the matrix of cardiac mitochondria exposed to high calcium concentrations. In conclusion, NAR exerts anti-ischemic effects through a "pharmacological preconditioning" that it is likely to be mediated, at least in part, by the activation of mitochondrial BK channels.

Role of translocator protein (18 kDa) in adult separation anxiety and attachment style in patients with depression.

BACKGROUND AND OBJECTIVES: A role for the protein that mediates the rate-limiting step of steroidogenesis, the 18 kDa Translocator Protein (TSPO), has been suggested in the pathophysiology of Adult Separation Anxiety Disorder (ASAD). It has been shown that ASAD patients have 1) low TSPO expression levels and 2) a high frequency of the allele that substitutes Ala with Thr at position 147 of TSPO. The Thr147 ASAD-associated allele has been recently related with a low pregnenolone production. The aim of the present work was to evaluate the relationship between TSPO expression levels and Ala147Thr single nucleotide polymorphism (SNP), which are the two TSPO biological parameters that we have previously examined separately. A further aim was to confirm the genetic association of Ala147Thr SNP with ASAD in an extended case-control sample and to investigate whether this SNP was related to an anxious attachment style that is thought to be connected to ASAD. METHODS: TSPO expression levels were compared among patients with ASAD (n=26), without ASAD (n=26) and control samples (n=10) stratified into the two genotype groups: those with the Ala147 genotype (named "normal pregnenolone production") and those with the Thr147 genotype (named "reduced pregnenolone production"). The case-control genetic study included patients with (n=87) or without (n=101) ASAD and 236 controls. In the patient group, the association between the Ala147Thr SNP and an anxious attachment style was analysed by stepwise logistic regression analysis. RESULTS: The genotype with the lowest TSPO expression levels was the "normal pregnenolone production" genotype in the ASAD group. The genetic Ala147Thr SNP confirmed an excess of the Thr147 allele in ASAD patients. Stepwise logistic regression analysis did not show an association with an anxious attachment style. CONCLUSIONS: ASAD individuals who expressed normal TSPO levels exhibited the "reduced pregnenolone production" genotype. In contrast, the ASAD individuals with the "normal pregnenolone production" genotype expressed low TSPO levels. It is possible that low TSPO expression levels could compromise normal pregnenolone production. Such evidence may have therapeutic implications because it has been documented that drugs targeting TSPO increased pregnenolone production and have anxiolytic effects.

[MALDI-TOF and SELDI-TOF analysis: "tandem" techniques to identify potential biomarker in fibromyalgia]. / MALDI-TOF e SELDI-TOF: due tecniche "tandem" per l'individuazione di potenziali biomarker nella fibromialgia.

OBJECTIVE: Fibromyalgia (FM) is characterized by the presence of chronic widespread pain throughout the musculoskeletal system and diffuse tenderness. Unfortunately, no laboratory tests have been appropriately validated for FM and correlated with the subsets and activity. The aim of this study was to apply a proteomic technique in saliva of FM patients: the Surface Enhance Laser Desorption/Ionization Time-of-Flight (SELDI-TOF). METHODS: For this study, 57 FM patients and 35 HC patients were enrolled. The proteomic analysis of saliva was carried out using SELDI-TOF. The analysis was performed using different chip arrays with different characteristics of binding. The statistical analysis was performed using cluster analysis and the difference between two groups was underlined using Student's t-test. RESULTS: Spectra analysis highlighted the presence of several peaks differently expressed in FM patients compared with controls. The preliminary results obtained by SELDI-TOF analysis were compared with those obtained in our previous study performed on whole saliva of FM patients by using electrophoresis. The m/z of two peaks, increased in FM patients, seem to overlap well with the molecular weight of calgranulin A and C and Rho GDP-dissociation inhibitor 2, which we had found up-regulated in our previous study. CONCLUSION: These preliminary results showed the possibility of identifying potential salivary biomarker through salivary proteomic analysis with MALDI-TOF and SELDI-TOF in FM patients. The peaks observed allow us to focus on some of the particular pathogenic aspects of FM, the oxidative stress which contradistinguishes this condition, the involvement of proteins related to the cytoskeletal arrangements, and central sensibilization.

Adenylate-cyclase activity in platelets of patients with obsessive-compulsive disorder.

Although the main biological hypothesis on the pathophysiology of obsessive-compulsive disorder (OCD) is centered on the serotonin system, indications are available that other neurotransmitters, and even second messengers, particularly the cyclic adenosine monophosphate (cAMP) signaling, may be involved, though effective data are few. Therefore, the aim of the present study was to evaluate and compare the basal and isoprenaline (ISO)-stimulated velocity of adenylate-cyclase (AC) in human platelet membranes of patients with OCD and healthy control subjects. The results showed that the basal and ISO-stimulated AC activity, as well as the dose-response curves of ISO by using agonist concentrations ranging between 0.1 nM and 10 muM, were not different in the two groups. However, OCD patients showed lower EC(50) and higher E(max) values than healthy subjects. These findings suggest the presence of supersensitive beta-adrenergic receptors in platelets of OCD patients.

Norepinephrine-mediated regulation of 5HT1 receptor functioning in human platelets.

Adaptive changes in serotonergic 5HT1 receptor signalling are believed to underlie the therapeutic effectiveness of antidepressant drugs. Since cells are continuously exposed to neurotransmitters/neuromodulators, spatially and temporally integrated, the responsiveness of a receptor system is dependent upon the physio-pathological state of the cell and the interaction between different neurotransmitters. In the present work, we investigated heterologous regulation of 5HT1 receptors induced by norepinephrine (NE) in human platelets. NE platelet treatment induced a time and concentration dependent 5HT1 receptor desensitisation mediated by both alpha and beta receptors through activation of intracellular protein kinases. In particular NE, through PKC activation, regulated 5HT1 receptor phosphorylation on threonine residues, causing in turn serotonin receptor-G protein uncoupling and functional responsiveness drop. These results suggest that high NE levels (released i.e. during stress disorders) may play an important role in regulating the 5HT1 responsiveness and in controlling effectiveness of drugs acting on these neurotransmitter systems.

Neuropeptide Y in the brain and retina of the adult teleost gilthead seabream (Sparus aurata L.).

The presence of neuropeptide Y (NPY) in the brain and retina of gilthead seabream (Sparus aurata L.) was investigated for the first time. For this investigation we employed an immunoperoxidase technique and the western immunoblot analysis using an antiserum raised against porcine NPY. The results showed that NPY-immunoreactivity was widely distributed in the brain of S. aurata. In particular, we have found NPY-immunoreactive (ir) neurons in the area ventralis telencephali pars centralis and pars lateralis, in the area dorsali telencephali pars centralis subdivision two and in nucleus intermedius thalami. An intense NPY-ir was detected in the telencephalon, in the optic tectum, in the thalamus, hypothalamus and in the vagal lobes. Scarce positive fibres were seen in the olfactory bulbs. NPY-ir amacrine cells were observed in the retina. The western immunoblot analysis revealed a protein band with a mobility corresponding to that of synthetic NPY. Our findings are, in general, in agreement with those obtained in other teleosts. The extensive distribution of NPY indicates for this peptide a key role in basic physiological actions, including visual and gustatory inputs processing.

Proteomic analysis of human thyroid fine needle aspiration fluid.

The aim of this study was to determine the protein pattern of human thyroid fine needle aspiration fluid (FNA) using a proteomic approach. FNA proteins were separated using 2-dimensional gel electrophoresis (2DE), digested and then analyzed by peptide mass fingerprinting. For the first time, we provided an image of the protein components of the FNA, in which approximately 220 protein spots can be identified. The proteome analysis revealed a specific fingerprint of FNA with proteins appertaining to various functional systems. Our preliminary results of FNA protein pattern could be a starting point in studying the presence of potential markers implicated in thyroid diseases.

Peripheral benzodiazepine receptors on platelets of fibromyalgic patients.

OBJECTIVE: The aim of the present study was to analyze if alterations of peripheral-type benzodiazepine receptor (PBR) characteristics occurred in platelet membranes of patients affected by primary fibromyalgia (FM). DESIGN AND METHODS: Platelets were obtained from 30 patients with FM. Evaluation of kinetic parameters of PBR was performed using [(3)H] PK11195 as specific radioligand compared with 16 healthy volunteers. RESULTS: The results showed a significant increase of PBR binding sites value in platelet membranes from FM patients (B(max) was 5366+/-188 fmol/mg vs. controls, 4193+/-341 fmol/mg, mean+/-SEM) (**p<0.01) but not for affinity value (K(d) was 4.90+/-0.39 nM vs. controls, 4.74+/-0.39 nM, mean+/-SEM) (p>0.05). Symptom severity scores (pain and tiredness) were positively correlated with B(max). CONCLUSIONS: Our results showed an up-regulation of PBR in platelets of FM patients, and this seems to be related to the severity of fibromyalgic symptoms.

Common alterations in the serotonin transporter in platelets and lymphocytes of psychotic patients.

INTRODUCTION: Given the controversial data concerning the role of the serotonin (5-HT) transporter in psychosis, our study was aimed to investigate this structure by means of the measurements of the re-uptake kinetics and of the protein density, in both platelets and lymphocytes of 25 out- and inpatients with different psychotic disorders. METHODS: Diagnoses, according to DSM-IV criteria, were bipolar 1 disorders with mood incongruent psychotic features (14), mixed states (7) and schizophrenia (4). Twenty-five matched healthy subjects were also selected as the control group. Platelet and lymphocyte membranes were prepared according to standardized protocols, as were the [3H]5HT re-uptake and [3H]paroxetine ([3H]Par) binding. RESULTS: The results of this study showed a decreased density of the [3H]Par binding sites coupled with a reduced velocity of [3H]5-HT re-uptake in both platelets and lymphocytes of psychotic patients, as compared with healthy control subjects. CONCLUSION: These findings would suggest a general abnormality of the 5-HT system in psychotic patients, probably not confined only to the brain.

In vitro effects of lead ions on peripheral benzodiazepine receptors and adenylyl cyclase activity in the mantle of Mytilus galloprovincialis.

As an extension of our previous work, where the density of peripheral benzodiazepine receptors (PBR) increased in mantle mitochondria of the marine mollusk Mytilus galloprovincialis Lmk. under chronic exposure to lead, the present study investigates the in vitro effects of an exogenous source of lead ions on PBR and on adenylyl cyclase (AC) complex in mantle membranes of mussels collected from a non-polluted coastal area. PBR binding experiments used the specific isoquinoline carboxamide derivative [3H]PK 11195, and AC activity was measured using a modified procedure adapted to M. galloprovincialis. Lead ions (Pb2+) dose-dependently decreased either the [3H]PK 11195 specific binding in mitochondria or basal AC velocity in plasma membranes of mussel mantle. The IC50 values for lead ions were 10 microM with [3H]PK 11195 binding and 25 microM with AC activity, with maximal inhibition values of 60% and 70%, respectively. Moreover, lead behaved as a non-competitive inhibitor on [3H]PK 11195 binding and as a 'mixed' inhibitor on AC activity. The present results suggest that some of the early effects induced by lead in mussel cell metabolism consist in significant changes of the PBR density and cyclic AMP production in the mantle of M. galloprovincialis.

Peripheral type benzodiazepine receptor in human parathyroid glands: up-regulation in adenoma.

In this study we report the presence of peripheral benzodiazepine receptors (PBRs) in human parathyroid glands and describe the effect of their benzodiazepine type ligands on parathyroid cell function. PBR binding features in normal parathyroid tissue were characterized and compared to parathyroid adenoma, using a specific and selective ligand for PBR, [3H] 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline-carboxamide ([3H]PK11195). Affinity and density of [3H]PK11195 binding sites in homogenate membrane preparations from adenomatous and normal tissues were determined. Parathyroid adenoma showed a statistically significant 2.2 fold increase of [3H]PK11195 binding sites, while the affinity remained unchanged. Our results represent the first evidence of PBRs in parathyroid glands and suggest for them a role in influencing PTH release. A clear trend of PBR up-regulation in parathyroid adenoma was also found.

Impaired glutathione reductase activity and levels of collagenase and elastase in synovial fluid in rheumatoid arthritis.

OBJECTIVE: To test the activity of elastase, collagenase and glutathione reductase in the synovial fluid (SF) of patients with rheumatoid arthritis (RA) and in patients with osteoarthritis (OA); to correlate the elastase and collagenase activity with the glutathione reductase activity, which is important for the inactivation of oxygen free radicals. METHODS: 24 patients affected by osteoarthrosis and 24 patients affected by rheumatoid arthritis took part in the study. We measured elastase activity towards the substrate metoxysuccinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide (MeOSuc-ala-ala-proval-p-NA) which is highly specific for elastase, and insensitive to the other serine proteases, such as cathepsin G; collagenase activity was measured using [14C]-acetylated collagen as the substrate. Glutathione reductase activity was measured following the oxidation of nicotinamide adenine dinucleotide phosphate reduced (NADPH) in the presence of oxidized glutathione (GSSG). RESULTS: The concentrations of elastase, collagenase and glutathione reductase were statistically higher in patients with RA than in patients with OA. Moreover, in the SF of patients with RA we found positive correlation between enzyme activity levels. CONCLUSION: These results confirm a high activity of collagenase and elastase in the SF of patients with RA, which is about 30 times higher than that found in the SF of patients with OA. These data underline the synergic action of these enzymes in the pathogenesis of joint damage. RA patients also exhibit higher levels of glutathione reductase, which is important for the detoxification pathway of oxygen free radicals. However, compared with findings for collagenase and elastase, the increase in glutathione reductase is only three times higher than level found in the SF of OA patients. The limited increase in glutathione reductase activity during the inflammatory process might lead to an insufficient protective effect at the joint level in rheumatoid arthritis.

7-Nitrobenzofurazan (NBD) derivatives of 5'-N-ethylcarboxamidoadenosine (NECA) as new fluorescent probes for human A(3) adenosine receptors.

New fluorescent ligands for adenosine receptors (ARs), obtained by the insertion, in the N(6) position of NECA, of NBD-moieties with linear alkyl spacers of increasing length, proved to possess a high affinity and selectivity for the A(3) subtype expressed in CHO cells. In fluorescence microscopy assays, compound 2d, the most active and selective for human A(3)-AR, permitted visualization and localization of this human receptor subtype, showing its potential suitability for internalization and trafficking studies in living cells.

Regulation of agonist binding to rat ET(B) receptors by cations and GTPgammaS.

Endothelins exert their physiological effects through interaction with cell surface receptors that are members of the G-protein-coupled receptor family. The endothelin receptor subtype B (ET(B) receptor) is abundantly expressed in rat cerebellum. Since agonist binding to G-protein-coupled receptors may be modulated by cations and guanine nucleotides, we investigated the effects of cations and guanosine 5'-O-(2-thiotriphosphate) (GTPgammaS) on 125I-endothelin-1 (125I-ET-1) binding to rat cerebellar membranes. Both Na+ and Mg2+-stimulated 125I-ET-1 binding causing an increase in receptor affinity for the agonist. While the effect of the divalent cation was evident at relatively low concentrations (5-10 mM), the stimulatory activity of the monovalent cation appeared at relatively high concentrations (50 mM). Additive activities of 25-50 mM NaCl and 1 mM MgCl2 suggested that monovalent and divalent cations increased receptor affinity for ET-1 by different mechanisms. In the presence of 5 mM MgCl2, 50 mM NaCl caused an additional modest reduction of the Kd value. Whereas 5 mM MgCl2 affected the displacement curves of both ET-3 and suc-[Glu9, Ala11,15]-endothelin-1 (8-21) (IRL 1620), the influence of 50 mM NaCl on these curves was less substantial. All together, these results suggest that modulation of receptor affinity by NaCl depends on the nature of the displacing agonist. In the presence of 5 mM MgCl2 or 50 mM NaCl, a partial regulation of 125I-ET-1 binding by GTPgammaS was detectable, while in the absence of cations no GTPgammaS-dependent inhibition was evident.

Correlation between platelet alpha(2)-adrenoreceptors and symptom severity in major depression.

BACKGROUND: Abnormalities in different parameters of the norepinephrine system have been widely described in major depression. The presence of alpha(2)-adrenoreceptors in blood platelets, similar to those in the brain, prompted us to evaluate them in depressed patients, as compared with healthy controls. METHODS: Fifteen outpatients affected by major depression, according to DSM IV criteria, and 15 comparable healthy control subjects, were included in the study. The alpha(2)-adrenoreceptors were measured by means of the specific binding of [(3)H]rauwolscine, a highly selective antagonist for this receptor subtype. The severity of depression was assessed by means of the Hamilton Rating Scale for Depression (HRSD). RESULTS: The results did not show any difference in [(3)H]rauwolscine binding parameters (B(max) and K(d)) between patients and controls. However, in the patients, a significant and positive correlation between B(max), which measures the density of the receptors, and HRSD total score was detected. CONCLUSIONS: Therefore, although no change in alpha(2)-adrenoreceptors seems to occur in major depression, the density of these receptors would seem to be related to the severity of depressive symptoms.

Decreased platelet [3H]paroxetine binding sites in suicide attempters.

Research to date would suggest the possible involvement of the serotonin (5-HT) system in the pathophysiology of suicide. With this study, we aimed to investigate the platelet 5-HT transporter, by means of the specific binding of tritiated paroxetine ([3H]Par), in a sample of 20 suicide attempters recruited at a first-aid service, as compared with healthy control subjects and psychiatric patients with no current or previous history of suicide attempt. The results, showing a decreased number of [3H]Par binding sites in suicide attempters, would suggest the involvement of the presynaptic 5-HT transporter in self-aggressive behavior.

Pharmacological characterization of the serotonin transporter in young and elderly subjects.

The potency of some tricyclics (imipramine and clomipramine) and selective 5-HT reuptake inhibitors (fluoxetine, paroxetine, and citalopram) in displacing the [(3)H]paroxetine binding to platelet membranes was measured in young and elderly subjects of both sexes. The results showed that the most potent compound in all subjects was paroxetine, followed by clomipramine, citalopram, fluoxetine, and imipramine, with no differences between male and female subjects. All drugs, except paroxetine and clomipramine, showed significantly lower pKi values in the elderly subjects of both sexes. These findings would suggest that although the pharmacological profile of the 5-HT transporter is not modified qualitatively by age, quantitative changes in its affinity do perhaps occur which would justify more careful studies on this topic in order to get optimal dosages of drugs acting at this level.