A multidisciplinary approach to study the effects of balneotherapy and mud-bath therapy treatments on Fibromyalgia.

Fibromyalgia (FM) is a chronic non-inflammatory musculoskeletal disorder characterized by a variety of symptoms related to pain. A wide range of other unspecific symptoms may characterize the disease including fatigue, sleep disturbances, mood disorders, morning stiffness, anxiety, depression, cognitive dysfunction (e.g., memory problems, concentration difficulties, diminished mental clarity), irritable bowel and bladder syndrome, sexual dysfunction and sicca symptoms. In the last decade, many attempts have been carried out for the research of specific biomarkers in FM, but, at present, there are no specific markers, the diagnosis is basically clinical. The complexity of the disease means that there isn’t a therapeutic treatment standards and this is demonstrated by the fact that pharmacological treatments are often applied in combination with non-pharmacological treatments. Among non-pharmacological interventions the most used there is the spa therapy, which includes hydrotherapy, balneotherapy, physiotherapy, mud-pack therapy and exercise. In the present work, to study the effects of both balneotherapy and mud-bath therapy treatments in patients affected by FM, we used rheumatological, psychiatric, biochemical and proteomic approach. Forty-one FM patients (39 F, 2 M), who fulfilled the American College of Rheumatology criteria received 2-week thermal therapy program comprising therapy once daily for 6 days/week. Twenty-one patients received mud-bath treatment, while the other twenty balneotherapy. Pain, symptoms, and quality of life were assessed. Oxytocin, brain-derived neurotrophic factor (BDNF), ATP and serotonin transporter levels during therapy were assayed by commercial kits. Comparative whole saliva proteomic analysis was performed using a combination of 2-D and nano-LC-ESI-MS/MS spectrometry techniques. The Elisa kit was performed to validate different expression of transaldolase, phosphoglicerate mutase1 and zinc alpha-2-glycoprotein1 proteins. We observed reduction in pain, FIQ values and improvement of SF36 in both groups of patients treated with mud-bath or balneotherapy. The improvement of the outcome measures occurred with different timing and duration in the two spa treatments. A significant decrease of neuropeptide concentrations was observed either after balneo-theraphy or mud-bath therapy when assayed after twelve weeks, while no significant change of oxytocin levels was detected. Significant differences were observed for PGAM1 and zinc-alpha 2 glycoprotein protein expression. Our results showed that the thermal treatment might have a beneficial effect on the specific symptoms of the disease. In particular, while balneotherapy gives results that in most patients occur after the end of the treatment but which are no longer noticeable after 3 months, the mud-bath treatment gives more lasting results

A Direct Aqueous Derivatization GSMS Method for Determining Benzoylecgonine Concentrations in Human Urine

A sensitive and reliable method for extraction and quantification of benzoylecgonine (BZE) and cocaine (COC) in urine is presented. Propyl-chloroformate was used as derivatizing agent, and it was directly added to the urine sample: the propyl derivative and COC were then recovered by liquid-liquid extraction procedure. Gas chromatography-mass spectrometry was used to detect the analytes in selected ion monitoring mode. The method proved to be precise for BZE and COC both in term of intraday and interday analysis, with a coefficient of variation (CV) 2>0.999 and >0.997, respectively) within the range investigated. The method, applied to thirty authentic samples, showed to be very simple, fast, and reliable, so it can be easily applied in routine analysis for the quantification of BZE and COC in urine samples

A multidisciplinary approach to study a couple of monozygotic twins discordant for the chronic fatigue syndrome: a focus on potential salivary biomarkers

BACKGROUND: Chronic Fatigue Syndrome (CFS) is a severe, systemic illness characterized by persistent, debilitating and medically unexplained fatigue. The etiology and pathophysiology of CFS remains obscure, and diagnosis is formulated through the patient’s history and exclusion of other medical causes. Thereby, the availability of biomarkers for CFS could be useful for clinical research. In the present study, we used a proteomic approach to evaluate the global changes in the salivary profile in a couple of monozygotic twins who were discordant for CFS. The aim was to evaluate differences of salivary protein expression in the CFS patient in respect to his healthy twin. METHODS: Saliva samples were submitted to two-dimensional electrophoresis (2DE). The gels were stained with Sypro, and a comparison between CFS subject and the healthy one was performed by the software Progenesis Same Spot including the Analysis of variance (ANOVA test). The proteins spot found with a ≥2-fold spot quantity change and p<0.05 were identified by Nano-liquid chromatography electrospray ionization tandem mass spectrometry. To validate the expression changes found with 2DE of 5 proteins (14-3-3 protein zeta/delta, cyclophilin A, Cystatin-C, Protein S100-A7, and zinc-alpha-2-glycoprotein), we used the western blot analysis. Moreover, proteins differentially expressed were functionally analyzed using the Ingenuity Pathways Analysis software with the aim to determine the predominant canonical pathways and the interaction network involved. RESULTS: The analysis of the protein profiles allowed us to find 13 proteins with a different expression in CFS in respect to control. Nine spots were up-regulated in CFS and 4 down-regulated. These proteins belong to different functional classes, such as inflammatory response, immune system and metabolism. In particular, as shown by the pathway analysis, the network built with our proteins highlights the involvement of inflammatory response in CFS pathogenesis. CONCLUSIONS: This study shows the presence of differentially expressed proteins in the saliva of the couple of monozygotic twins discordant for CFS, probably related to the disease. Consequently, we believe the proteomic approach could be useful both to define a panel of potential diagnostic biomarkers and to shed new light on the comprehension of the pathogenetic pathways of CFS

Evaluation of formalin-fixed paraffin-embedded tissues in the proteomic analysis of parathyroid glands

<p>Abstract</p> <p>Background</p> <p>Proteomic research in the field of parathyroid tissues is limited by the very small dimension of the glands and by the low incidence of cancer lesions (1%). Formalin-fixed paraffin-embedded (FFPE) tissue specimens are a potentially valuable resource for discovering protein cancer biomarkers. In this study we have verified the applicability of a heat induced protein extraction from FFPE parathyroid adenoma tissues followed by a gel-based or gel-free proteomic approach in order to achieve protein separation and identification.</p> <p>Results</p> <p>The best results for high quality MS spectra and parameters, were obtained by using a gel-free approach, and up to 163 unique proteins were identified. Similar results were obtained by applying both SDS-out and SDS-out + TCA/Acetone techniques during the gel-free method. Western blot analysis carried out with specific antibodies suggested that the antigenicity was not always preserved, while specific immunoreactions were detected for calmodulin, B box and SPRY domain-containing protein (BSPRY), peroxiredoxin 6 (PRDX 6) and parvalbumin.</p> <p>Conclusions</p> <p>In spite of some limitations mainly due to the extensive formalin-induced covalent cross-linking, our results essentially suggest the applicability of a proteomic approach to FFPE parathyroid specimens. From our point of view, FFPE extracts might be an alternative source, especially in the validation phase of protein biomarkers when a large cohort of samples is required and the low availability of frozen tissues might be constraining.</p

Proteomic approach used in the diagnosis of Riedel's thyroiditis: a case report

<p>Abstract</p> <p>Introduction</p> <p>Riedel's thyroiditis, a rare thyroid disease, can be difficult to diagnose prior to surgical removal and can be confused with malignancy both clinically and cytologically.</p> <p>Case presentation</p> <p>We report the case of a 72-year-old Caucasian woman who presented with a goiter, which showed a rapid increase in size at ultrasound check, suggesting malignancy. Because of inconclusive cytology, a total thyroidectomy was performed. Fine-needle aspiration of the removed thyroid was processed by two-dimensional electrophoresis, and the proteome was compared with both anaplastic cancer and control samples. Significant differentially expressed protein spots were identified by Western blot analysis by using specific antibodies.</p> <p>Conclusions</p> <p>The protein pattern of Riedel's fine-needle aspiration revealed a superimposition with that of the control samples. The comparison of the protein pattern of Riedel's thyroiditis fine-needle aspiration with that of anaplastic cancer showed evidence of a different expression of ferritin heavy chains, ferritin light chains, and haptoglobins, as previously reported in thyroid cancers. Therefore, we performed Western blot analysis of these proteins and validated that their expression levels were low or absent in Riedel's thyroiditis and control samples despite the high concentrations present in fine-needle aspiration anaplastic samples. The concurrent absent or low expression levels of haptoglobin, ferritin light chain, and ferritin heavy chain in Riedel's thyroiditis fine-needle aspiration samples strongly indicate the benign nature of the thyroid lesion. These results suggest the potential applicability of fine-needle aspiration proteome analysis for Riedel's thyroiditis diagnosis.</p

Platelet proteome and clopidogrel response in patients with stable angina undergoing percutaneous coronary intervention.

OBJECTIVES: We analyzed the platelet proteome of circulating platelets during the onset of clopidogrel therapy in patients with stable angina underwent percutaneous coronary intervention in order to investigate the mechanisms that control platelet reactivity and clopidogrel response in this context. DESIGN & METHODS: Twenty patients were enrolled in this study. Blood samples were collected before coronary angiography (T0), 12h after 600mg of clopidogrel (T1) and 24h after percutaneous coronary intervention (PCI) (T2). Platelet reactivity, Clopidogrel response and proteomic analysis were examined. RESULTS: Clopidogrel loading dose produced a significant inhibition in all markers of platelet activation in both flow cytometry and aggregation tests. Among the proteins found differentially expressed, eighteen were identified by MS/MS analysis and they resulted involved in the cytoskeleton rearrangement (profilin-1, calpain, alpha-soluble NSF attachment protein, thrombospondin), in the energetic metabolism (ubiquitin-like modifier-activating enzyme 1, protein-L-isoaspartate-(D-aspartate) O-methyltransferase and nucleoside diphosphate kinase B) and in the oxidative stress (heat shock 70kDa protein 5 and anti-stress induced phosphoprotein 1. CONCLUSIONS: The present study provides novel information on platelet proteome changes associated with platelet activation and clopidogrel response. This investigation supports the development of further proteomic studies for the identification of novel platelet biomarkers

New insight into benign tumours of major salivary glands by proteomic approach.

Major salivary gland tumours are uncommon neoplasms of the head and neck. The increase of precise pre-operative diagnosis is crucial for their correct management and the identification of molecular markers would surely improve the required accuracy. In this study we performed a comparative proteomic analysis of fine needle aspiration fluids of the most frequent benign neoplasms of major salivary glands, namely pleomorphic adenoma and Warthin's tumour, in order to draw their proteomic profiles and to point out their significant features. Thirty-five patients submitted to parotidectomy were included in the study, 22 were identified to have pleomorphic adenoma and 14 Warthin's tumour. Fine needle aspiration samples were processed using a two-dimensional electrophoresis/mass spectrometry-based approach. A total of 26 differentially expressed proteins were identified. Ingenuity software was used to search the biological processes to which these proteins belong and to construct potential networks. Intriguingly, all Warthin's tumour up-regulated proteins such as Ig gamma-1 chain C region, Ig kappa chain C region and Ig alpha-1 chain C region and S100A9 were correlated to immunological and inflammatory diseases, while pleomorphic adenomas such as annexin A1, annexin A4, macrophage-capping protein, apolipoprotein E and alpha crystalline B chain were associated with cell death, apoptosis and tumorigenesis, showing different features of two benign tumours. Overall, our results shed new light on the potential usefulness of a proteomic approach to study parotid tumours and in particular up regulated proteins are able to discriminate two types of benign parotid lesions

Search for peripheral biomarkers in patients affected by acutely psychotic bipolar disorder: a proteomic approach.

Data on neurobiological mechanisms underlying mood disorders are elusive; the aetiology of such states is multifactorial, including genetic predisposition and environmental factors. Diagnosis is currently being made only on an interview-based methodology. Biological markers, which could improve the current classification, and in perspective, stratify patients on a biological basis into more homogeneous clinically distinct subgroups, are highly needed. We describe here a comparative proteomic analysis of peripheral lymphocytes from patients affected by acute psychotic bipolar disorder (PBD) (n = 15), major depressive episode (MDE) with no personal or family history of psychosis (n = 11), and a group of demographically matched healthy controls (HC) (n = 15). All patients were evaluated by means of Structured Clinical Interview for DSM-IV-Patient version (SCID-I-P), Positive and Negative Symptoms Scale (PANSS), Young Mania Rating Scale (YMRS), Hamilton Anxiety Rating Scale (HAM-A) and Hamilton Depression Rating Scale (HAM-D-17) questionnaires. Blood lymphocytes were obtained by gradient separation, and 2-DE was carried out on protein extracts. Significant differences in protein patterns among the three groups were observed. Thirty-six protein spots were found to be differentially expressed in patients compared to controls, which collapsed into 25 different proteins after mass spectrometry identification. Twenty-one of these proteins failed to discriminate between PBD and MDE, suggesting common signatures for these disorders. Nevertheless, after the western blot validation only two of the remaining proteins, namely LIM and SH3 domain protein1, and short-chain specific acyl-CoA dehydrogenase mitochondrial protein, resulted in being significantly upregulated in PBD samples suggesting additional mechanisms that could be associated with the psychotic features of bipolar disorder

Comparative proteomic analysis of malignant pleural mesothelioma evidences an altered expression of nuclear lamin and filament related proteins

PURPOSE: Malignant mesothelioma is a neoplastic disease linked to asbestos exposure whose diagnosis is limited, so detection methods for an early diagnosis and treatment result essential. Here, we compared proteomic profiles of malignant pleural mesothelioma (MPM) and benign biopsies to search potential biomarkers useful in differential diagnosis. EXPERIMENT DESIGN: Tissue biopsies were obtained from 53 patients who were subjected to a diagnostic thoracoscopy. Two-dimensional electrophoresis/mass spectrometry based approach was used for proteomic analysis and protein validation was carried out by western blot analysis versus benign and lung carcinoma samples RESULTS: Among the proteins identified we confirmed known MPM biomarkers such as calretinin and suggested the new ones as pre-lamin A/C, desmin, vimentin, calretinin, fructose-bisphosphate aldolase A, myosin regulatory light chain 2, ventricular/cardiac muscle isoform, myosin light chain 3 and myosin light chain 6B. Ingenuity software was used to identify the biological processes to which these proteins belong and to construct a potential network. CONCLUSIONS AND CLINICAL RELEVANCE: Overall, our results suggest potential biomarkers that can be useful in occupational medicine for the early identification of the onset of disease in health surveillance of past asbestos-exposed workers, for monitoring the progress of disease and for assessing the response to treatment