Gross Cystic Disease Fluid Protein-15(GCDFP-15)/Prolactin-Inducible Protein (PIP) as Functional Salivary Biomarker for Primary Sjogren's Syndrome.
ABSTRACT: BACKGROUND:Gross cystic disease fluid protein-15(GCDFP-15)/prolactin-inducible protein (PIP) is a secretory acinar glycoprotein of 14 KDa which we have recently described as significantly lower in salivary samples of patients with primary Sjögren's syndrome (pSS) in comparison to healthy volunteers by proteomic analysis. AIMS OF THE STUDY:(1) to validate our previous data on the decrease of GCDFP-15/PIP protein in a larger number of subjects with pSS (2) to integrate the proteomic results with complementary immunoassays in order better clarify the pathophysiological relevance of GCDFP-15/PIP in pSS exocrinopathy (3) to assess both the glandular expression of the GCDFP-15/PIP and the levels of glandular GCDFP-15/PIP mRNA in the patients' minor salivary gland (MSG) biopsies in order to verify whether the observed reduction of GCDFP-15/PIP in saliva may be related to a decrease in the protein production. PATIENTS AND METHODS:A total of 123 salivary samples from patients affected by pSS, no-SS sicca syndrome and sex- age-matched healthy volunteers were analyzed by different proteomic techniques (SELDI-TOF-MS, 2DE, MALDI-TOF-MS). The expression of GCDFP-15/PIP was then validated by western blot analysis. Real Time PCR and immunohistochemistry for GCDFP-15/PIP in the minor salivary glands (MSG) biopsies were then carried out. RESULTS:By using complementary proteomic analysis we found that a putative peak of 16547 m/z was among the best independent biomarkers for pSS able to discriminate between patients and healthy controls with a sensitivity of 96 % and a specificity of 70%, with a global cross validated error of 29%. We identified the peak as the GCDFP-15/PIP protein and verified that the intensity of GCDFP-15/PIP was significantly lower in pSS patients when compared to both no-SS sicca subjects and healthy controls (p<0.0001). GCDFP-15/PIP expression also correlated with both the salivary flow rate (r=0.312, p=0.023) and MSG biopsies focus score (r=-0.377, p=0.04). Finally, immunohistochemistry confirmed that GCDFP-15/PIP staining was faint in mucus acini and Real Time PCR showed that GCDFP-15/PIP mRNA was significantly lower in pSS patients when compared to both no-SS sicca subjects and healthy controls (p=0.023) thus supporting the hypothesis that the observed reduction of GCDFP-15/PIP in pSS saliva may be related to a decrease in the protein production. CONCLUSION:In this study by different complementary-omic techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for pSS. This finding might also be functionally important as GCDFP-15/PIP has previously been shown to bind to Aquaporin 5 (AQP5), a salivary gland water channel, critical to saliva formation that is known to be downregulated in pSS. It is likely that exploring the GCDFP-15/PIP/AQP5 axis will help better understand the mechanism of salivary gland dysfunction in pSS.
Project description:<h4>Background</h4>Mononuclear cell infiltration of exocrine glands, production of Ro/SSA and La/SSB autoantibodies, along with oral and ocular dryness, are characteristic features of primary Sjögren's syndrome (pSS). Non-SS sicca subjects, an underexplored group in relation to pSS, display similar sicca symptoms, with possible mild signs of inflammation in their salivary glands, yet with no serological detection of autoantibody production. In this study, we investigated inflammatory manifestations in the salivary gland tissue, tear fluid and saliva of non-SS subjects, as compared to pSS patients and healthy individuals.<h4>Methods</h4>Fifteen non-SS, 10 pSS and 10 healthy subjects were included in the analyses. Histological evaluation of salivary gland biopsies was performed. Liquid chromatography-mass spectrometry (LC-MS) was conducted on tear fluid and stimulated whole saliva, and proteomic biomarker profiles were generated. Extracellular vesicle (EVs) isolation and characterisation from both fluids were also combined with LC-MS. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold. Database for Annotation, Visualization and Integrated Discovery (DAVID) and Functional Enrichment Analysis Tool (FunRich) were applied for functional analyses.<h4>Results</h4>Histopathological evaluation of salivary gland biopsies showed implications of milder inflammation in non-SS subjects through mononuclear cell infiltration, fibrosis and fatty replacement, as compared to pSS patients. Although unaffected in the non-SS group, upregulation of proinflammatory pathways and proteins involved in ubiquitination (LMO7 and HUWE1) and B cell differentiation (TPD52) were detected in tear fluid of pSS patients. Moreover, overexpression of proteins STOM, ANXA4 and ANXA1, regulating cellular innate and adaptive immunological pathways, were further identified in EVs from tear fluid of pSS patients. Finally, whole saliva and EVs isolated from whole saliva of pSS patients expressed proteins vital for innate MHC class I cellular regulation (NGAL) and T cell activation (CD44).<h4>Conclusions</h4>Non-SS sicca subjects may show implications of mild inflammation in their glandular tissue, while their protein profile was strikingly more similar to healthy controls than to pSS patients. Hence, the tear and salivary biomarkers identified could be implemented as potential non-invasive diagnostic tools that may aid in increasing diagnostic accuracy when evaluating non-SS subjects and pSS patients and monitoring disease progression.
Project description:INTRODUCTION: A growing interest has arisen in salivary proteomics as a tool for the identification of biomarkers for primary Sjögren's syndrome (pSS). Nonetheless, only a limited number of preclinical validation studies have been performed, limiting the possibility of translating proteomic results into clinical practice. The primary aim of this study was to refine the diagnostic power of a panel of candidate salivary biomarkers described in pSS with respect to both healthy volunteers and pathological controls. We also explored the pathogenetic function of the detected putative biomarkers both in the local exocrinopathy and in the systemic inflammatory processes of SS. METHODS: One hundred and eighty patients were included in the study overall. In the first "exploratory phase", we enrolled 40 females with pSS, 40 sex- and age-matched healthy volunteers, 10 patients with sicca non-SS and 15 secondary SS (sSS) patients. The testing cohort of the second "challenge phase" of the study was represented by 75 unselected, consecutive subjects: 19 pSS, 21 healthy volunteers, 10 sicca non-SS and 25 sSS patients. Salivary proteomic analysis was performed combining two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Western blot (WB) analysis and enzyme-linked immunosorbent assay (ELISA) were employed to validate 2DE results. Ingenuity Pathway Analysis (IPA) Knowledge base was adopted to associate candidate biomarkers in a signalling pathogenetic network. RESULTS: A total of 28, 6, 7 and 12 protein spots were found to be significantly different in pSS samples with respect to healthy volunteers, non-SS sicca syndrome, SSc-sSS and rheumatoid arthritis-sSS, leading to the identification of 15 differently expressed proteins. Among them, ?-amylases precursor, carbonic anhydrase VI, ?-2 microglobulin, glyceraldehydes-3-phosphate dehydrogenase (G3PDH), epidermal fatty acid binding protein (E-FABP) and immunoglobulin k light chain (IGK-light chain) apparently showed the most significant differences in pSS when compared to healthy volunteers and non-SS pathological controls. On the other hand, as expected, pSS and sSS salivary profiles shared a great number of similarities. CONCLUSIONS: This study demonstrated that salivary fluid might represent a novel ideal milieu for the detection of a diagnostic panel of candidate biomarkers for pSS, and to gain an insight into the pathogenetic processes underlying glandular and systemic autoimmune disorders.
Project description:<h4>Objectives</h4>To elucidate the clinical values of anti-M3R in Sjögren's syndrome (SS) in the largest cohort for an anti-M3R study.<h4>Methods</h4>The plasma of 361 subjects (156 primary SS [pSS], 62 non-SS-sicca [SICCA], 40 systemic lupus erythematosus [SLE], 50 rheumatoid arthritis [RA], and 53 healthy controls [HC]) was screened using our modified On-Cell-Western assay. Saliva from pSS (n=37) compared to SICCA (n=26) was also analysed. The sensitivity and specificity of anti-M3R and its association with comprehensive clinical and laboratory features were determined.<h4>Results</h4>Plasma-anti-M3R was higher in pSS compared to other groups, differentiating pSS with good-to-excellent diagnostic power with a specificity of 85% and a sensitivity between 75% and 98%. pSS plasma-anti-M3R was positively correlated with ocular staining scores, anti-Ro/SSA, IgG, β2-microglobulin, ESR, and ESSDAI. It was negatively correlated with WBC, C4, and salivary scintigraphic indices. Saliva-anti-M3R was 3.59 times higher in pSS than in SICCA. Interestingly, the agreement between the 2002 American European Consensus Group criteria and the criteria substituted with plasma-anti-M3R for the lip biopsy reached 92%, with a significant kappa of 0.824.<h4>Conclusions</h4>Anti-M3R enhances sensitivity and specificity for SS diagnosis, correlating with ocular dryness and glandular hypofunction, and the haematological/biological domains of the ESSDAI. Our findings also highlight the clinical significance of anti-M3R in SS diagnosis, especially where clinical assessments, such as lip biopsy, sialometry, or ocular evaluation, by multi-disciplinary specialists are limited.
Project description:While all salivary glands (SGs) can be involved in primary Sjögren’s syndrome (pSS), their respective role in pathogenesis remains unclear. To assess immunopathway activation in paired parotid and labial gland tissue from biopsy-positive and biopsy-negative pSS and non-SS sicca patients, paraffin-embedded, paired parotid and labial salivary gland tissue and peripheral blood mononuclear cells were obtained from 39 pSS and 20 non-SS sicca patients. The patients were subdivided based on fulfillment of ACR-EULAR criteria and histopathology and the samples were analyzed for differentially expressed genes (DEGs). The principal component analysis of SG gene expression could only separate biopsy-positive pSS from non-SS sicca patients. However, when comparing the transcriptome of biopsy-positive pSS and biopsy-negative non-SS sicca patients, 1235 and 624 DEGs (FDR<-1 or >1) were identified for parotid and labial glands, respectively. The number of DEGs between biopsy negative pSS and non-SS sicca patients was scarce. The overall, transcript expression levels correlated strongly between parotid and labial glands (R2=0.86, p‐value<0.0001). Gene signatures present in both glands of biopsy‐positive pSS patients included IFN‐α signaling, IL-12/IL-18 signaling, CD3/CD28 T-cell activation, CD40 signaling in B-cells, DN2 B-cells, and FcRL4+ B-cells. Signature scores varied considerably amongst pSS patients. In conclusion, the transcriptomes of paired major and minor SGs in pSS were overall comparable, although significant inter-individual heterogeneity in immunopathway activation existed. The SG transcriptome of biopsy-negative pSS was indistinguishable from non-SS sicca patients. The different patterns of SG immunopathway activation in pSS argue for personalized treatment approaches. Overall design: Differential expression analysis of a cohort of 39 patients with primary Sjogren's syndrome and 20 non-SS sicca patients. characteristics: ACR EULAR = positive -> patients with primary Sjogren's syndrome characteristics: ACR EULAR = negative -> non-SS sicca patients
Project description:The diagnostic work-up of primary Sjögren's syndrome (pSS) includes quantifying saliva and tear production, evaluation of autoantibodies in serum and histopathological analysis of minor salivary glands. Thus, the potential for further utilizing these fluids and tissues in the quest to find better diagnostic and therapeutic tools should be fully explored. Ten samples of saliva and tears from female patients diagnosed with pSS and ten samples of saliva and tears from healthy females were included for lipidomic analysis of tears and whole saliva using high-performance liquid chromatography coupled to time-of-flight mass spectrometry. In addition, lipidomic analysis was performed on minor salivary gland biopsies from three pSS and three non-SS females. We found significant differences in the lipidomic profiles of saliva and tears in pSS patients compared to healthy controls. Moreover, there were differences in individual lipid species in stimulated saliva that were comparable to those of glandular biopsies, representing an intriguing avenue for further research. We believe a comprehensive elucidation of the changes in lipid composition in saliva, tears and minor salivary glands in pSS patients may be the key to detecting pSS-related dry mouth and dry eyes at an early stage. The identified differences may illuminate the path towards future innovative diagnostic methodologies and treatment modalities for alleviating pSS-related sicca symptoms.
Project description:Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren's syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.
Project description:<h4>Background</h4>While all salivary glands (SGs) can be involved in primary Sjögren's syndrome (pSS), their respective role in pathogenesis remains unclear. Our objective was to assess immunopathway activation in paired parotid and labial gland tissue from biopsy-positive and biopsy-negative pSS and non-SS sicca patients.<h4>Methods</h4>Paraffin-embedded, paired parotid and labial salivary gland tissue and peripheral blood mononuclear cells were obtained from 39 pSS and 20 non-SS sicca patients. RNA was extracted, complementary DNA libraries were prepared and sequenced. For analysis of differentially expressed genes (DEGs), patients were subdivided based on fulfillment of ACR-EULAR criteria and histopathology.<h4>Results</h4>With principal component analysis, only biopsy-positive pSS could be separated from non-SS sicca patients based on SG gene expression. When comparing the transcriptome of biopsy-positive pSS and biopsy-negative non-SS sicca patients, 1235 and 624 DEGs (FDR<0.05, log2FC<-1 or >1) were identified for parotid and labial glands, respectively. The number of DEGs between biopsy-negative pSS and non-SS sicca patients was scarce. Overall, transcript expression levels correlated strongly between parotid and labial glands (R<sup>2</sup> = 0.86, p-value<0.0001). Gene signatures present in both glands of biopsy-positive pSS patients included IFN-α signaling, IL-12/IL-18 signaling, CD3/CD28 T-cell activation, CD40 signaling in B-cells, DN2 B-cells, and FcRL4+ B-cells. Signature scores varied considerably amongst pSS patients.<h4>Conclusion</h4>Transcriptomes of paired major and minor SGs in pSS were overall comparable, although significant inter-individual heterogeneity in immunopathway activation existed. The SG transcriptome of biopsy-negative pSS was indistinguishable from non-SS sicca patients. Different patterns of SG immunopathway activation in pSS argue for personalized treatment approaches.
Project description:Investigating cytokines in tear fluid and saliva may offer valuable information for understanding the pathogenesis of primary Sjögren's syndrome (pSS). Cytokine profiles in both tear fluid and saliva of pSS patients, non-Sjögren's syndrome (non-SS) subjects with sicca symptoms, and healthy controls without sicca complaints were analysed. Furthermore, relationships associating the severity of clinical ocular and oral manifestations with the upregulated cytokines were assessed. In tear fluid, pSS patients showed elevated levels of IL-1ra, IL-2, IL-4, IL-8, IL-12p70, IL-17A, IFN-γ, IP-10, MIP-1b, and Rantes compared to non-SS subjects and healthy controls. The increased cytokine levels (except IP-10) correlated significantly with reduced tear production, less stable tear film, and greater ocular surface damage. In saliva, pSS patients had a higher IP-10 level, which correlated with higher candida score; and an elevated MIP-1a level, which correlated significantly with lower unstimulated and stimulated whole saliva secretion rates. The upregulated cytokines identified in tear fluid and saliva of pSS patients show a clear interplay between innate and adaptive immune responses that may contribute to disease pathogenesis. The increase of IP-10 and MIP in both tears and saliva further emphasises the essential role of macrophages and innate immunity in pSS.
Project description:OBJECTIVE:Determine the presence and assess the extent of fatty infiltration of the minor salivary glands (SG) of primary SS patients (pSS) as compared to those with non-SS sicca (nSS). METHODS:Minor SG biopsy samples from 134 subjects with pSS (n?=?72) or nSS (n?=?62) were imaged. Total area and fatty replacement area for each glandular cross-section (n?=?4-6 cross-sections per subject) were measured using Image J (National Institutes of Health, Bethesda, MD). The observer was blinded to subject classification status. The average area of fatty infiltration calculated per subject was evaluated by logistic regression and general linearized models (GLM) to assess relationships between fatty infiltration and clinical exam results, extent of fibrosis and age. RESULTS:The average area of fatty infiltration for subjects with pSS (median% (range) 4.97 (0.05-30.2)) was not significantly different from that of those with nSS (3.75 (0.087-41.9). Infiltration severity varied widely, and subjects with fatty replacement greater than 6% were equivalently distributed between pSS and nSS participants (?2 p?=?.50). Age accounted for all apparent relationships between fatty infiltration and fibrosis or reduced saliva flow. The all-inclusive GLM for prediction of pSS versus non-SS classification including fibrosis, age, fatty replacement, and focus score was not significantly different from any desaturated model. In no iteration of the model did fatty replacement exert a significant effect on the capacity to predict pSS classification. CONCLUSIONS:Fatty infiltration is an age-associated phenomenon and not a selective feature of Sjögren's syndrome. Sicca patients who do not fulfil pSS criteria have similar rates of fatty infiltration of the minor SG.
Project description:OBJECTIVE:Salivary gland (SG) progenitor cells (SGPCs) maintain SG homeostasis. We have previously shown that in primary Sjögren's syndrome (pSS), SGPCs are likely to be senescent, and may underpin SG dysfunction. This study assessed the extent of senescence of cells in a SGPC niche in pSS patients' SGs, and its correlation with functional and clinical parameters. METHODS:The expression of p16 and p21 as markers of senescence in both total SG epithelium and a SGPC niche (basal striated duct cells, BSD) was examined in SGs of pSS (n?=?35), incomplete pSS (n?=?13) (patients with some signs of pSS, but not fulfilling all classification criteria) and non-SS sicca control (n?=?21) patients. This was correlated with functional and clinical parameters. RESULTS:pSS patient SGs contained significantly more p16+ cells both in the epithelium in general (P <0.01) and in the BSD layer (P <0.001), than non-SS SGs. Significant correlations were found in pSS patients between p16+ BSD cells and secretion of unstimulated whole saliva, stimulated whole saliva, stimulated parotid saliva, CD45+ infiltrate, ultrasound total score and ACR-EULAR classification score, but not with EULAR Sjögren's syndrome disease activity index (ESSDAI) and EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) scores. Correlations with total epithelium p16+ cells were weaker. Incomplete pSS patients also had increased numbers of p16+ epithelial and BSD cells. Based on protein and mRNA expression, p21+ appears not to play a significant role in the SG in pSS. CONCLUSION:These findings suggest SGPC senescence may be an early feature of primary Sjögren's syndrome and may contribute to defective SG function in pSS but not to systemic disease activity.