F. Sernissi

Salivary gland ultrasonography: a highly specific tool for the early diagnosis of primary Sjögren’s syndrome

IntroductionRecently, a great interest has arisen for salivary gland ultrasonography (SGUS) as a valuable tool for the assessment of major salivary gland involvement in primary Sjögren’s syndrome (pSS. The aims of this study were to test the accuracy of SGUS for the early detection of pSSand to compare the diagnostic performance of SGUS with minor salivary gland biopsy (MSGB) and unstimulated salivary flow (USFR) in this context.MethodPatients with suspected pSS and symptoms duration of ≤5 years were consecutively enrolled in this study. The diagnosis of pSS was made according to the AECG criteria. SGUS was performed by two radiologists blinded to the diagnosis and a previously reported ultrasound scoring system (De Vita et al. 1992, cut-off ≥ 1) was used to grade the echostructure alterations of the salivary glands. Statistical analysis was performed using SPSS v16.ResultsThis study included 50 pSS patients and 57 controls with no-SS sicca symptoms. The mean(SD) age of the pSS group was lower than non-SS group (47(13) vs 53(12)yrs, p = 0.006). No further differences between the two groups were observed. Patients with pSS showed a significantly higher SGUS score in comparison with controls (mean(SD) = 2.1(1.8) vs 0.0(0.4), p = 0.000). The SGUS cut-off ≥ 1 showed a sensitivity (SE) of 66 %, a specificity (SP) of 98 %, a positive predictive value (PPV) of 97 % and a negative predictive value (NPV) of 73 % for pSS diagnosis. The SGUS score correlated also with patients’ MSGB/FS and USFR.ConclusionsThis study confirmed the good performance of SGUS for the early non-invasive diagnosis of pSS. Further research in larger international cohort of patients is mandatory in order to assess the role of SGUS in the diagnostic algorithm of pSS.

Detection of potential markers of primary fibromyalgia syndrome in human saliva

In the last few years, many attempts have been carried out for the research of specific biological biomarkers in fibromyalgia (FM) since, so far, no laboratory tests have been appropriately validated for the diagnosis and the prognostic stratification of the disease. In our study for the first time, we carried out a proteomic analysis of the whole saliva of FM patients in order to evaluate salivary biomarkers. Twenty‐two FM patients with all fulfilling the American College of Rheumathology diagnostic criteria for FM and 26 sex‐and age‐matched healthy subjects were enrolled in the study. Proteomic analysis was performed by combining 2‐DE and MALDI‐TOF‐MS. The most relevant observation which emerged from the data analysis was the exclusive and significant over‐expression of transaldolase and phosphoglycerate mutase I. These findings were validated by Western blot analysis and the total optical density confirmed the significant up‐regulation of transaldolase and phosphoglycerate mutase I in FM samples with respect to healthy subjects. It was noteworthy that seven further salivary proteins resulted differentially expressed, namely: calgranulin A, calgranulin C, cyclophilin A, profilin 1, Rho GDP‐dissociation inhibitor 2, proteasome subunit‐α‐type‐2 and haptoglobin‐related protein precursor. These preliminary results demonstrated the utility of salivary proteomic analysis in the identification of salivary biomarkers in FM patients and in clarifying some of the pathogenetic aspects of the disease.

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

BackgroundChronic 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.MethodsSaliva 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.ResultsThe 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.ConclusionsThis 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.

Ultrasonography of major salivary glands: a highly specific tool for distinguishing primary Sjögren’s syndrome from undifferentiated connective tissue diseases

OBJECTIVES Recently, convincing data have been published on the value of salivary gland ultrasonography (SGUS) in differentiating primary SS from non-immune-mediated sicca syndrome. Limited data are available regarding the diagnostic accuracy of SGUS in distinguishing SS from other rheumatic diseases. The purpose of this study was to assess the usefulness of SGUS in distinguishing patients with SS from those with xerostomia and/or xerophthalmia and a diagnosis of stable UCTD. METHODS This cross-sectional study consecutively enrolled 150 patients either diagnosed with SS (as established by the American-European Consensus Group criteria) or affected by UCTD but not SS. Parotid and submandibular glands on both sides were assessed for size, parenchymal echogenicity and inhomogeneity by means of SGUS, which was performed by a radiologist blinded to the diagnosis. Echostructural alterations of the salivary glands were graded from 0 to 3 (cut-off >2). RESULTS This study included 109 patients: 55 with SS and 54 with UCTD. Patients with SS showed a higher SGUS score in comparison with those with UCTD [mean 2.2 (s.d. 1.8) vs 0.2 (s.d. 0.5), P < 0.0001]. The SGUS cut-off >2 showed a sensitivity of 65%, a specificity of 96%, a positive predictive value of 95% and a negative predictive value of 73% for SS diagnosis. A significant correlation was also found between the SGUS score and the minor salivary gland biopsy/focus score (r = 0.484, P < 0.0001). CONCLUSION This study confirmed the good sensitivity and the high specificity of SGUS in differentiating SS from other CTDs.

[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.

Salivary psoriasin (S100A7) correlates with diffusion capacity of carbon monoxide in a large cohort of systemic sclerosis patients

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by progressive fibrosis of the skin and the internal organs. In a previous work we suggested a correlation between levels of salivary psoriasin (S100A7) and pulmonary involvement in SSc patients. The goals of this study are to determine the distribution characteristics of psoriasin in whole saliva (WS) of SSc and healthy donor populations and define its predictive value on diffusion capacity of carbon monoxide (DLCO), along with others clinical parameters.MethodsSalivary level of psoriasin was determined by ELISA kit in 134 SSc patients, 63 Raynaud syndrome patients, 40 patients affected by other connective diseases (non-case) and 74 healthy control subjects.ResultsA significant increase of salivary psoriasin was observed in SSc patients when compared with other healthy and pathological controls. Moreover, we confirmed the efficacy of salivary psoriasin to correlate with DLCO in a large cohort of SSc patients.ConclusionsOverall our results suggest a rapid, non invasive and low costing method which can help clinicians in the evaluation of SSc pulmonary involvement.

Cystatin S—a candidate biomarker for severity of submandibular gland involvement in Sjögren’s syndrome

Objectives Salivary cystatin S is a defence protein mainly produced by submandibular glands and involved in innate oral immunity. This study aimed to verify whether cystatin S was diversely expressed in different disease subsets of primary Sjogrens syndrome (pSS) patients, defined on the basis of salivary flow [unstimulated salivary flow rate (USFR)], minor salivary gland (MSG) focus score and submandibular gland ultrasonography abnormalities. We also evaluated miR-126 and miR-335-5p expression in MSG biopsies to verify whether an aberrant regulation of cystatin S at the glandular level may influence its salivary expression. Methods Forty pSS patients and 20 sex- and age-matched healthy volunteers were included. Salivary cystatin S levels were assessed by western blot analysis using a stain-free technology. The expression of miR-126, miR-335-5p and cystatin S was assessed by quantitative PCR in 15 MSG biopsies differing for USFR and MSG focus score. Results We found that salivary cystatin S was significantly decreased in pSS patients vs healthy volunteers ( P = 0.000), especially in those with hyposalivation. A positive correlation was observed between cystatin S and USFR ( r = 0.75, P = 0.01). Salivary cystatin S was also significantly reduced in patients with a submandibular gland ultrasonography score ⩾2. The expression levels of miR-126 and miR-335-5P increased in inverse proportion with USFR. The mRNA of cystatin S did not change significantly, suggesting post-transcriptional regulation. Conclusion Cystatin S emerged as a promising biomarker for pSS, strongly correlated with glandular dysfunction. An upregulation of miR-126 and miR-335-5P might be implicated in its expression.

Biomarkers in fibromyalgia: a review

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Current Biomarker Findings 2014:4 35–41 Current Biomarker Findings Dovepress

FRI0394 Diagnostic Accuracy of Salivary Gland Ultrasonography in Patients with Systemic Sclerosis and Associated Sjögren's Syndrome

Background Systemic sclerosis (SSc) is a multisystem disease in which sicca symptoms and particularly oral manifestations are quite common. The prevalence of an associated Sjögrens syndrome (SSc-SS) in SSc patients ranges from 20 to 40% whereas in the vast majority of the cases sicca symptoms can be attributed to a fibrosis of the exocrine glands. Recently, a growing interest has arisen for salivary gland ultrasonography (SGUS) as an useful tool for the diagnosis of primary SS. Nonetheless, data on the utility of the SGUS in patients with SSc and suspected associated SS are limited. Objectives To assess the diagnostic accuracy of salivary gland ultrasonography (SGUS) in patients with SSc and suspected concomitant SS Methods From Jan 2014 to November 2014 consecutive, unselected patients with SSc were systematically evaluated for sicca symptoms by using the AECG questionnaire. Patients with suspected SS underwent a complete diagnostic work-up. SGUS was carried out by the same radiologist blinded to the diagnosis and the following US parameters were recorded: size, parenchymal echogenicity and inhomogeneity in the parotid and submandibular glands on both sides, number and location of hypo or anechoic area, presence of lymp nodes and calcifications. A modified version of the De Vita score was adopted to grade the echostructure alterations of the salivary glands. Comparison were made using parametric Students t test and non-parametric Mann-Whitney U test as applicable. Dichotomous variables were compared using contingency table analysis and Fishers Exact test. Spearmans rank correlation was applied for detecting correlations between SGUS score and different study parameters. Results Of 145 patients evaluated for sicca symptoms, 47 females were enrolled. The sample had a mean age (S.D) of 68 (14) yrs, and a mean disease duration (S.D) of 4 (4) yrs. Anticentromere antibodies were detected in 42/47 of the patients. A diagnosis of SSc-SS was made in 32/47 (68%) of the cases. No differences between SSc and SSc-SS patients were observed with respect to gender, age and duration of dry-mouth and dry-eye related symptoms. Patients with SSc-SS showed a higher SGUS score (mean (SD)=1.9 (1.9) vs 0.2 (0,4), p<0.0001). Isolated or localized hypoechoic areas were observed also in a minority of patients with SSc alone (2/15). However, diffuse or scattered hypoechoic areas were detected exclusively in patients with SSc-SS. A strong correlation was found between the grade of the echostructure alteration of the parotid and of the submandibular glands (r=0.754, p<0.0001). We also observed that the fibrosis of the submandibular glands was significantly higher in SSc-SS patients (84% vs 53%, p=0.04). No differences between SSc-SS and SSc patients were detected regarding parotid fibrosis, size of the glands, presence of lymph nodes or calcifications. Overall SGUS showed a sensitivity (SE) of 66%, a specificity (SP) of 87%, a positive predictive value (PPV) of 91% and a negative predictive value (NPV) of 54% for SS diagnosis. Conclusions This study demonstrated the utility of SGUS for the diagnosis of SS in the context of SSc, thus encouraging the use of SGUS in clinical practice. Disclosure of Interest None declared

FRI0296 Diagnostic performance of salivary gland ultrasonography in the early stages of pss in a real-life clinical setting

Background Recently, convincing data have been published on the diagnostic value of salivary gland ultrasonography (SGUS) in primary Sjögren’s syndrome (pSS). A limited number of information are available on the performance of SGUS in the early stages of the disease Objectives The purposes of this single-center study were: a. To explore the performance of SGUS in a cohort of unselected patients with suspected pSS and duration of sicca symptoms ≤ 5 years; b. To correlate the SGUS score to other tests included among the AECG criteria for assessing salivary gland involvement in pSS (i.e. minor salivary gland biopsy focus score (MSGB/FS) and salivary flow rate measured by sialometry) Methods The study population consisted of consecutive unselected patients with dry eye and dry mouth suggestive for pSS and symptoms duration ≤ 5 years. A standardized clinical, serological and histological diagnostic algorithm for pSS was performed in all the cases according to the AECG criteria. Unstimulated whole saliva was collected under standard conditions at the study inclusion according to the AECG sialometry protocol. US of salivary glands was performed by the same radiologist blinded to the diagnosis and the following US parameters were recorded: size, parenchymal echogenicity and inhomogeneity in the parotid and submandibular glands on both sides. A previously reported ultrasound scoring system (De Vita et al 1992) was used to grade the echostructure alterations of the salivary glands. SGUS findings were correlated to patients’ clinical and serological features, to the MSGB/FS and to the patients’ unstimulated whole salivary flow rate. Results Thirty-two female patients were enrolled in this study from March 2012 and December 2012 (age=53±15, mean ±SD, years; duration between onset of symptoms and time of diagnosis = 2.1±1.8, mean ± SD, years). Twenty-two patients met the AECG criteria for pSS and 10/32 who did not fulfill the AECG criteria represented the control group. Patients with pSS showed a significantly higher SGUS score in comparison with controls (1.8±1.6 vs 0.4±1.0, p<0.05). The sensitivity of the SGUS score was 55% and the specificity was 90%. An inverse correlation was observed between the SGUS score and the age of the patients (r=-40, p=0.02) with younger patients frequently presenting multiple hypoechogenic areas especially in the parotids. A significant correlation was also found between the SGUS score and the MSGB/FS (r=37, p=0.05). No correlation was detected between the salivary gland echostructure grading and the unstimulated salivary flow rate. Conclusions This study confirmed that, despite the low sensitivity, SGUS might represent a specific non-invasive tool for the diagnosis of pSS in the early stages of the disease. Further research in larger cohort of patients is mandatory in order to better clarify the role of SGUS in the diagnostic algorithm of pSS Disclosure of Interest: None Declared