Antonio Lucacchini

[MALDI-TOF and SELDI-TOF analysis: "tandem" techniques to identify potential biomarker in fibromyalgia].

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.

A1 Adenosine Receptors in Human Neutrophils

Adenosine is an ubiquitous nucleoside that mediates several important physiological effects, trough the binding of three pharmacologically distinct receptor subtypes, named A1 A2 and A 3 .1 Adenosine receptor subtypes has been distinguished on the basis of the effect of adenosine on the cellular content of cAMP: occupancy of A2 adenosine receptors induces an increase in cAMP level in several cells types, by contrast stimulation of A 1 receptors causes the opposite effect. These effects are mediated by a coupling of these receptor subtypes with Gs and Gi transduction-proteins, respectively.2

THU0309 Proteomic Differential Expressed Protein in Fibromyalgic Saliva: Comparison with Other Pain Model as Rheumathoid Arthritis and Migraine

Background Fibromyalgia Syndrome (FM) is a chronic non inflammatory musculoskeletal disorder characterized by widespread pain and by the presence of at least 11 out of 18 specific tender points on physical examination and associated with a plethora of dysfunctional disorders. Currently no validated laboratory biomarkers are available for FM and the diagnosis of the disease remains exclusively clinical. Objectives With the present study, we used two-dimensional electrophoresis (2DE) in combination with mass spectrometry (MS) to evaluate the global changes in salivary profile of FM patients. The aim was the search for any eventual diagnostic or prognostic salivary biomarkers which could be useful for the management of FM patients and compare the profile of FM with two different model of chronic pain: migraine patient such as non-inflammatory chronic pain and rheumathoid arthritis (RA) patients as inflammatory chronic pain. Methods Samples were pooled according to their diagnosis, and submitted to 2DE. The gels were stained with Sypro and images analyzed performing a comparison between FM and control classes (healthy, rheumatoid arthritis, migraine). Proteins spots of interest were identified by NanoLC-ESI-MS/MS analysis. Results The analysis of the protein profiles allowed us to find 26 spots with a different expression in FM respect to RA (p-value<0.05), 28 spots from the comparison of FM with migraine, and 32 in FM respect to healthy subjects. In particular, we found 7 spots differentially expressed exclusively in FM. Six spots were identified as serotransferrin and the other as alpha-enolase. Both serotransferrin and alpha-enolase increased in FM respect to all the control classes. Furthermore, the proteins differentially expressed in FM respect to the control classes, were functionally analyzed by using the Ingenuity Pathways Analysis software with the aim to determine the predominant canonical pathways and the interaction network involved. Serotransferrin is an iron binding transport protein, responsible for the transport of iron from sites of absorption to those of storage and utilization, and alpha-enolase is a multifunctional enzyme that, as well as its role in glycolysis, plays a part in various processes such as growth control, hypoxia tolerance and allergic responses. Moreover, serum transferrin plays an important role in inflammation, while alpha enolase stimulates immunoglobulin production. Therefore our results seem to support the inflammatory and a dis-regulation of immunity system hypothesis for FM. In fact, also the network built with our proteins highlights the involvement of inflammatory response in FM and the immune cell trafficking. In addition, proteomic profile of FM patients is more similar to that of RA patients, rather than migraine and healthy subjects. Conclusions In conclusion, this study shows the presence of differentially expressed proteins in the saliva of FM patients, probably related to the disease. Consequently, 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 FM. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.3714

Diagnosis of Fibromyalgia Syndrome: Potential Biomarkers and Proteomic Approach

In 1990 the American College of Rheumatology (ACR) established criteria for the diagnosis of fibromyalgia (Wolfe et al., 1990), and more recently new criteria have been proposed (Wolfe et al., 2010, 2011). However, the absence of anatomic pathological lesions and of biohumoral abnormalities, demonstrated with classical instrumental methods, has led to considerable difficulties in diagnosis. Until now, many attempts have been made to search for biomarkers in fibromyalgia, but at present no specific markers have been found (Bazzichi et al., 2010). The problem lies in the presence of too many data, often controversial, rather than in a lack of data. We will discuss methods that are state of the art in searching for biomarkers in fibromyalgia. Furthermore, we will focus on the contribution that proteomics can give in the diagnosis of the disease on the basis of the study we carried out on human whole saliva of patients affected by fibromyalgia.

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.