The Role of the Akt Signaling Pathway in Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder with diverse clinical picture and high prevalence of B-cell non-Hodgkin lymphoma (NHL), that possibly raises from the chronic activation of B-cells. The mechanisms underlying the development of neoplasia in pSS remain elusive. Activated Akt/mTOR pathway is a uniform finding in cancer, whereas its significance in haematologic malignancies is highlighted by the plethora of inhibitors with promising therapeutic efficacy. PI3K-Akt activation has been involved in the TLR3-induced apoptosis of cultured salivary gland epithelial cells (SGECs), whereas upregulated expression of the phosphorylated ribosomal S6 protein (pS6), an end-result of PI3K signalling, has been detected in the infiltrating T and B lymphocytes at the MSG lesions of pSS patients; nevertheless, without specifying if this was mediated by the Akt/mTOR or Ras/ERK pathways. To this end, the role of Akt/mTOR pathway in pSS and associated lymphomagenesis, will be investigated by the immunohistochemical detection of the entire and phosphorylated protein forms of Akt kinase and two of its substrates, namely the FoxO1 transcription factor and the proline-rich Akt substrate of 40-kDa (PRAS40) in MSGs of pSS patients with variable histological and clinical phenotype, as well as sicca-complaining controls. Subsequently, the role of this pathway will be evaluated in in-vitro inhibition experiments, studying the effect of specific inhibitors in the phenotype, function, and interaction of SGECs and B cells. The current proposal is expected to promote the understanding of pSS pathogenesis, enlighten the mechanisms underlying related lymphomagenesis and possible therapeutic targets.

Incidence of autoantibodies related to systemic autoimmunity in patients with severe COVID-19 admitted to the intensive care unit.

OBJECTIVES: To assess the prevalence of autoantibodies (AAbs) in mechanically ventilated COVID-19 patients and to investigate whether AAbs influence the clinical outcome. METHODS: Serum samples were drawn within the first 48 hours upon admission to the intensive care unit (ICU) from 217 consecutive patients, from January 1st, 2021, to May 10th, 2021, and investigated for the presence of AAbs using conventional techniques. Serum samples (n=117) of age- and sex-matched healthy individuals collected before COVID-19 pandemic were used as controls. RESULTS: COVID-19 patients in the ICU had more commonly AAbs compared to age- and sex-matched controls (174/217, 80.2% vs. 73/117, 62.4%, p<0.001). Patients expressed more frequently ANAs (48.4% vs. 21.4%, p<0.001), anti-dsDNA (5.1% vs. 0%, p=0.01), anti-CCP (8.3% vs. 1.7%, p=0.014) and anti-CL IgM AAbs (21.7% vs. 9.4%, p=0.005) than controls, respectively. Simultaneous reactivity against at least three autoantigens, occurred in 144 out of 174 (82.8%) patients. The two groups did not differ in terms of clinicoepidemiologic characteristics or the mortality ratio within the ICU. Patients who died compared to convalescents were older, had higher ferritin, D-dimers levels, APACHE II score, lower oxygen saturation, higher prevalence of comorbidities and cognitive dysfunction. However, AAbs were not found to correlate with the clinical outcome. CONCLUSIONS: Patients with severe COVID-19 express AAbs more commonly compared to controls. No correlation was found between AAbs and disease outcome.

Clinical Phenotype and Mechanisms of Leukopenia/Neutropenia in Patients with Primary Sjögren's Syndrome.

Sjögren's syndrome (SS) is a chronic, systemic autoimmune disease which afflicts mainly the exocrine salivary and lachrymal glands, leading to mouth and eye dryness. However, any organ can be affected during the disease course, resulting in a variety of clinical manifestations. Sjögren's syndrome clinical manifestations can be classified into glandular (sicca manifestations or parotid swelling), extra-glandular, either nonspecific (arthralgias, arthritis, Raynaud's phenomenon, fatigue) or peri-epithelial (primary biliary cirrhosis, interstitial nephritis, bronchiolitis), and extra-epithelial (palpable glomerulonephritis, peripheral neuropathy, purpura). In addition, SS patients display high risk for B cell lymphomas due to chronic antigenic stimulation. Although disease pathogenesis remains unclear, genetic, environmental, and immunologic factors are implicated. In the context of systemic autoimmune manifestations, SS patients may also present with hematologic abnormalities including anaemia, leukopenia (mainly neutropenia or lymphopenia), and thrombocytopenia. Although leukopenia has been reported as a laboratory finding in many case series or cohorts of SS patients and in very few studies it has been proposed as an independent risk factor for lymphoma, the clinical phenotype of SS patients with leukopenia/neutropenia and the implicated pathogenetic mechanisms have not been elucidated. In the current study, we intend to analyse the clinical phenotype of leukopenic/neutropenic SS patients and explore the possible pathogenetic mechanisms by detecting anti-neutrophil antibodies and investigate the role of apoptotic pathways, especially the contribution of TRAIL pathway and the cFLIP molecule.

Neutrophil extracellular traps in giant cell arteritis biopsies: presentation, localization and co-expression with inflammatory cytokines.

OBJECTIVES: To explore the presence of neutrophil extracellular traps (NETs) in inflamed temporal artery biopsies (TABs) of patients with GCA. METHODS: Ten patients with GCA [five with limited and five with associated generalized vascular involvement, as defined by 18F-fluorodeoxyglucose PET with CT (PET/CT)] and eight with PMR were studied. The presence, location, quantitation and decoration of NETs with IL-6, IL-1ß and IL-17A were assessed in TABs at the time of disease diagnosis by tissue immunofluorescence and confocal microscopy. Paired serum levels of IL-6 and IL-17A were also evaluated in all patients. RESULTS: All temporal artery biopsies from GCA, but not PMR, patients had NETs located mainly in the adventitia, adjacent to the vasa vasorum. NETs decorated with IL-6 were present in 8/10 TABs of GCA patients, of whom 5 were PET/CT(+) and 3 PET/CT(-) patients. IL-17A(+) NETs were observed in all GCA patients. IL-1ß(+) NETs were not detected in any GCA patient. No relation was found between serum IL-6 and IL-17A levels and NETs containing IL-6 and/or IL-17A. CONCLUSIONS: NETs bearing pro-inflammatory cytokines are present in inflamed GCA-TABs. Future studies with a larger number of patients from different centres will show whether the findings regarding neutrophils/NETs in the TAB are consistent and disclose their clinical impact.

Akt Signaling Pathway Is Activated in the Minor Salivary Glands of Patients with Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is an autoimmune exocrinopathy of mainly the salivary and lacrimal glands associated with high prevalence of lymphoma. Akt is a phosphoinositide-dependent serine/threonine kinase, controlling numerous pathological processes, including oncogenesis and autoimmunity. Herein, we sought to examine its implication in pSS pathogenesis and related lymphomagenesis. The expression of the entire and activated forms of Akt (partially and fully activated: phosphorylated at threonine-308 (T308) and serine-473 (S473), respectively), and two of its substrates, the proline-rich Akt-substrate of 40 kDa (PRAS40) and FoxO1 transcription factor has been immunohistochemically examined in minor salivary glands (MSG) of pSS patients (n = 29; including 9 with pSS-associated lymphoma) and sicca-complaining controls (sicca-controls; n = 10). The entire and phosphorylated Akt, PRAS40, and FoxO1 molecules were strongly, uniformly expressed in the MSG epithelia and infiltrating mononuclear cells of pSS patients, but not sicca-controls. Morphometric analysis revealed that the staining intensity of the fully activated phospho-Akt-S473 in pSS patients (with or without lymphoma) was significantly higher than sicca-controls. Akt pathway activation was independent from the extent or proximity of infiltrates, as well as other disease features, including lymphoma. Our findings support that the Akt pathway is specifically activated in MSGs of pSS patients, revealing novel therapeutic targets.

Serum, but Not Saliva, CXCL13 Levels Associate With Infiltrating CXCL13+ Cells in the Minor Salivary Gland Lesions and Other Histologic Parameters in Patients With Sjögren's Syndrome.

Recent studies suggest that elevated CXCL13 serum levels in patients with primary Sjögren's syndrome (pSS) associate with minor salivary gland (MSG) histologic features, disease severity, as well as high-risk status for non-Hodgkin lymphoma (NHL) development and NHL itself. In contrast, limited discriminative value of CXCL13 saliva levels has been reported. Prompt by these reports, we sought to validate the clinical utility of CXCL13 by investigating potential correlations of serum and saliva levels with MSG histopathologic [including CXCL13+-cell number, severity of infiltrates and germinal center (GC) formation], serologic and clinical parameters, as well as NHL. CXCL13 levels were evaluated in paired serum and saliva specimens of 45 pSS patients (15 with NHL; pSS-associated NHL: SSL), 11 sicca-controls (sicca-complaining individuals with negative MSG biopsy and negative autoantibody profile), 10 healthy individuals (healthy-controls) and 6 non-SS-NHLs. CXCL13+-cells were measured in paired MSG-tissues of 22 of pSS patients studied (including 7 SSLs) and all sicca-controls. CXCL13 serum levels were significantly increased in pSS and SSL patients compared to sicca- and healthy-controls and were positively correlated with the CXCL13+-cell number and biopsy focus-score. Serum CXCL13 was significantly higher in pSS patients with GCs, rheumatoid factor, hypocomplementemia, high disease activity, NHL and in high-risk patients for NHL development. CXCL13 saliva levels were significantly increased in SSL patients (compared to non-SS-NHLs), patients with GCs and in high-risk for NHL patients. Univariate analysis revealed that CXCL13 serum, but not saliva, levels were associated with lymphoma, an association that did not survive multivariate analysis. Conclusively, our findings confirm that serum, but not saliva, levels of CXCL13 are associated with histologic, serologic and clinical features indicative of more severe pSS.

DNA Methylation Studies in Saliva of Patients with Sjögren's Syndrome.

Sjögren's syndrome (SS) is a relatively common systemic autoimmune disease of unknown aetiology, although genetic, hormonal, immunologic, and environmental factors are thought to be involved in disease pathogenesis. It is also termed "autoimmune epithelitis", and afflicts mainly the epithelial structures of salivary and lachrymal glands, through periepithelial lymphocytic infiltration responsible for the occurrence of dryness symptoms. Sjögren's syndrome (SS) is also characterised by B cell hyperactivity as reflected by the presence of hypergammaglobulinemia and the production of autoantibodies, which seems to be associated with the presence of ectopic germinal centres within the inflamed minor salivary glands. Chronic antigenic stimulation may lead to expansion of B cell autoreactive clones with rheumatoid factor activity, and additional molecular events mediate malignant transformation into non-Hodgkin's lymphomas of B cell origin. Therefore, the interaction between the immune cells of the inflammatory infiltrate and the salivary epithelium seems to have an important contribution in disease process. Recent histopathologic and molecular studies have shown that DNA methylation levels of SS patients compared to healthy individuals differ in epithelial cells of salivary glands and peripheral blood mononuclear cells. In the present study, we intend to analyse the epigenetic modifications of DNA in the saliva of SS patients compared to healthy controls. More specifically, salivary DNA methylation levels of selected genetic loci previously found to differ in other tissues, will be compared between SS patients and healthy controls. The study includes saliva collection from SS patients and healthy individuals, extraction of genomic DNA and methylation assessment. The epigenetic profile of each genetic locus will be correlated with SS patients' clinical characteristics and the possibility of genetic loci with differential differences in methylation to be used as potential diagnostic biomarkers will be explored. The current study is anticipated to reveal potential biomarkers for diagnostic and therapeutic purposes, offering the advantage to utilise the easily collected and handled saliva as the main biologic material.

Occurrence and Antigenic Specificity of Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (P-ANCA) in Systemic Autoimmune Diseases.

Perinuclear anti-neutrophilic cytoplasmic antibodies (P-ANCA) recognize heterogeneous antigens, including myeloperoxidase (MPO), lactoferrin, elastase, cathepsin-G and bactericidal/permeability-increasing protein. Although P-ANCA have diagnostic utility in vasculitides, they may also be found in patients with various other systemic autoimmune rheumatic diseases (SARDs). Nevertheless, the clinical significance and the targets recognized by P-ANCA in such patients remain unclear. For this purpose, herein we investigated the occurrence of ANCA-related antigenic specificities in 82 P-ANCA-positive sera by multiplex ELISA, as well as their association with other autoantibodies. The P-ANCA-positive sera corresponded to patients with vasculitides (n = 24), systemic lupus erythematosus (n = 28), antiphospholipid syndrome (n = 5), Sjögren's syndrome (n = 7), rheumatoid arthritis (n = 3), systemic scleroderma (n = 1), sarcoidosis (n = 1) and Hashimoto's thyroiditis (n = 13). In most P-ANCA-positive patients studied (51/82, 62.3%), these autoantibodies occurred in high titers (>1:160). The analysis of P-ANCA-positive sera revealed reactivity to MPO in only 50% of patients with vasculitides, whereas it was infrequent in the other disease groups studied. Reactivity to other P-ANCA-related autoantigens was also rarely detected. Our findings support that high P-ANCA titers occur in SARD. The P-ANCA-positive staining pattern is associated with MPO specificity in vasculitides, while in other autoimmune diseases, it mostly involves unknown autoantigens.

Autophagy and Metabolism in Normal and Malignant Hematopoiesis.

The hematopoietic system relies on regulation of both metabolism and autophagy to maintain its homeostasis, ensuring the self-renewal and multipotent differentiation potential of hematopoietic stem cells (HSCs). HSCs display a distinct metabolic profile from that of their differentiated progeny, while metabolic rewiring from glycolysis to oxidative phosphorylation (OXPHOS) has been shown to be crucial for effective hematopoietic differentiation. Autophagy-mediated regulation of metabolism modulates the distinct characteristics of quiescent and differentiating hematopoietic cells. In particular, mitophagy determines the cellular mitochondrial content, thus modifying the level of OXPHOS at the different differentiation stages of hematopoietic cells, while, at the same time, it ensures the building blocks and energy for differentiation. Aberrations in both the metabolic status and regulation of the autophagic machinery are implicated in the development of hematologic malignancies, especially in leukemogenesis. In this review, we aim to investigate the role of metabolism and autophagy, as well as their interconnections, in normal and malignant hematopoiesis.

A biomarker for lymphoma development in Sjogren's syndrome: Salivary gland focus score.

The aim of this study is to explore the role of labial minor salivary gland (LMSG) focus score (FS) in stratifying Sjögren's Syndrome (SS) patients, lymphoma development prediction and to facilitate early lymphoma diagnosis. Ιn an integrated cohort of 1997 patients, 618 patients with FS ≥ 1 and at least one-year elapsing time interval from SS diagnosis to lymphoma diagnosis or last follow up were identified. Clinical, laboratory and serological features were recorded. A data driven logistic regression model was applied to identify independent lymphoma associated risk factors. Furthermore, a FS threshold maximizing the difference of time interval from SS until lymphoma diagnosis between high and low FS lymphoma subgroups was investigated, to develop a follow up strategy for early lymphoma diagnosis. Of the 618 patients, 560 were non-lymphoma SS patients while the other 58 had SS and lymphoma. FS, cryoglobulinemia and salivary gland enlargement (SGE) were proven to be independent lymphoma associated risk factors. Lymphoma patients with FS ≥ 4 had a statistically significant shorter time interval from SS to lymphoma diagnosis, compared to those with FS < 4 (4 vs 9 years, respectively, p = 0,008). SS patients with FS ≥ 4 had more frequently B cell originated manifestations and lymphoma, while in patients with FS < 4, autoimmune thyroiditis was more prevalent. In the latter group SGE was the only lymphoma independent risk factor. A second LMSG biopsy is patients with a FS ≥ 4, 4 years after SS diagnosis and in those with FS < 4 and a history of SGE, at 9-years, may contribute to an early lymphoma diagnosis. Based on our results we conclude that LMSG FS, evaluated at the time of SS diagnosis, is an independent lymphoma associated risk factor and may serve as a predictive biomarker for the early diagnosis of SS-associated lymphomas.

Validation of thymic stromal lymphopoietin as a biomarker of primary Sjögren's syndrome and related lymphoproliferation: results in independent cohorts.

OBJECTIVES: Thymic stromal lymphopoietin (TSLP) has been implicated in primary Sjögren's syndrome (pSS) and related B-cell lymphoproliferation and lymphoma (NHL) by studies on salivary pathologic tissues and serum. The purpose of this work was to validate serum TSLP as biomarker of pSS and related lymphoproliferation by the study of two additional independent cohorts. METHODS: Serum TSLP was measured by ELISA in the original published Cohort-1 from Udine, Italy, including 91 patients. Two additional cohorts were then studied for validation: Cohort-2, including 4 sub-cohorts comprising 125 patients from the Universities of Roma, L'Aquila, Pisa and Perugia, belonging to the Italian SS Study Group (GRISS), and Cohort-3, including 59 patients from the University of Athens, Greece. Overall, 159 control subjects were enrolled. Active pSS-NHL, as well as pre-lymphomatous conditions, i.e. persistent salivary gland swelling and mixed cryoglobulinaemia, were investigated in detail. In addition, serum samples from pSS-NHL in complete remission were analysed (n=27). RESULTS: TSLP serum levels were confirmed to be significantly higher in pSS compared to controls in both Cohort-2 and Cohort-3, in particular in patients with lymphoproliferation. Serum TSLP was much higher in pSS pre-lymphomatous conditions. Finally, active NHL showed the highest TSLP serum levels, while in NHL in remission TSLP resulted undetectable or significantly lower than in benign pSS. CONCLUSIONS: By the study of independent cohorts, it was again demonstrated that serum TSLP levels are increased in pSS, above all in more advanced B-cell lymphoproliferation and NHL. Serum TSLP can therefore represent a novel biomarker for pSS-related lymphoproliferation.

Interaction of Human Salivary Gland Epithelial Cells with B Lymphocytes: Implications in the Pathogenesis of Sjögren's Syndrome.

Sjögren's syndrome (SS) is characterized by the aberrant activation of B-cells in both the target organs of autoimmune responses, such as the exocrine glands and the periphery. Furthermore, SS is strongly associated with the development of B-cell non-Hodgkin lymphomas, which are considered to result from chronic aberrant activation of B-cells. Disturbances of the minor salivary gland (MSG) infiltrating and peripheral B-cells subpopulations have been described in SS patients; however, the underlying mechanisms have not been uncovered. SG epithelial cells (SGECs) play a key role in the development and organization of MSG lymphocytic infiltrates in SS patients. SGECs are suitably equipped to mediate the recruitment, activation, and differentiation of immune cells in SS, including CD4+-T cells. B-cell activating factor (BAFF) secretion by SGECs suggests that they can also fruitfully interact with B-cells and mediate their activation, differentiation, and disturbed subpopulations in SS. The effect of SGECs in the activation and differentiation of naïve peripheral B-cells, as this attested by phenotypical flow cytometric and cytokine production analyses, is under investigation in the current proposal. This approach is expected to enlighten the mechanisms underlying the aberrant activation and differentiation of B cells in SS and the discovery of novel therapeutic targets for its reversal.

Predictive markers of lymphomagenesis in Sjögren's syndrome: From clinical data to molecular stratification.

Sjögren's syndrome (SS) is a chronic systemic autoimmune disease, affecting predominantly the exocrine glands, a large array of systemic manifestations and high risk of lymphoma development. The latter constitutes the major adverse outcome of SS contributing in the increased morbidity and mortality of the disease. The vast majority of lymphomas in SS are B-cell non-Hodgkin's lymphomas (NHL), primarily indolent mucosa-associated lymphoid tissue (MALT) lymphomas, followed by nodal marginal zone lymphomas (NMZL) and diffuse large B cell lymphomas (DLBCL). In the last 3 decades and due to the adverse impact of NHL in disease outcome, an effort has been undertaken to identify markers and models predicting patients with SS at high risk for lymphoma development. Several epidemiological, clinical, laboratory and histological parameters, some of which are evident at the time of SS diagnosis, were proved to independently predict the development of NHL. These include salivary gland enlargement, skin vasculitis/purpura, glomerulonephritis, peripheral neuropathy, Raynaud's phenomenon, lymphadenopathy, splenomegaly, cytopenias, hypocomplementemia, cryoglobulinemia, rheumatoid factor, anti-Ro/La autoantibodies, hypergammaglobulinemia, serum monoclonal gammopathy, biopsy focus score and organization of lymphocytic infiltrates in the salivary glands into ectopic germinal centers. Prediction models combining some of the afore-mentioned predictors have also been described. However, the identification of specific and sensitive molecular biomarkers, related to the process of lymphomagenesis is still pending. Recently, we described a novel biomarker the miR200b-5p micro-RNA. Low levels of this miRNA in the minor salivary glands, appears to discriminate with high specificity and sensitivity the SS patients who have from those who do not have NHL. miR200b-5p, being expressed years before the clinical onset of NHL, independently predicts NHL development with a predictive value higher than the previously published multifactorial models and has a possible role in the monitoring of therapeutic response. Thus, it is a strong candidate for the identification and follow-up of patients at risk.

Perturbation of transcriptome in non-neoplastic salivary gland epithelial cell lines derived from patients with primary Sjögren's syndrome.

The data presented here are related to the research article titled "Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation" (Vakrakou et al., Journal of Autoimmunity, in press, 2017). In the cited manuscript, using comparative analyses of salivary gland biopsy specimens and ductal salivary gland epithelial cell (SGEC) lines from SS patients and disease controls, we have demonstrated that the ductal epithelia of SS patients display constitutively reduced PPARγ expression, transcriptional activity and anti-inflammatory function that were associated with cell-autonomously activated NF-κB and IL-1ß pathways in these cells. Herein, the comparative transcriptome analysis of SGEC lines is presented. We show that the ductal epithelia of SS patients with severe lymphoepithelial lesions manifest constitutive perturbation in various inflammation- and metabolism related signaling pathways.

Low miR200b-5p levels in minor salivary glands: a novel molecular marker predicting lymphoma development in patients with Sjögren's syndrome.

OBJECTIVES: Development of non-Hodgkin's lymphoma (NHL) is the major adverse outcome of Sjögren's syndrome (SS) affecting both morbidity and mortality. Preliminary evidence suggested that, although not deregulated compared with sicca controls, miR200b-5p levels are decreased in the minor salivary glands (MSGs) of SS patients with NHL. The aim of the current study was to evaluate the MSG expression of miR200b-5p in SS-associated NHLs and its potential predictive value for the identification of patients with SS susceptible to develop NHL. METHODS: miR200b-5p expression was investigated in MSG tissues of patients with SS who were at: (A) low risk and did not develop NHL during follow-up (n=27; median follow-up time on biopsy performance, range: 8.9 years, 1.33-14 years), (B) high-risk and diagnosed with NHL during follow-up (prelymphoma; n=17; median follow-up to until lymphoma diagnosis, range: 3.67 years, 0.42-8.5 years) and (C) had NHL (n=35), as well as non-SS sialadenitis controls (sarcoidosis and hepatitis C virus (HCV) infection, four each). The differential miR200b-5p expression, correlations with disease features and its discriminative/predictive value, was evaluated by appropriate statistical approaches. RESULTS: The MSG levels of miR200b-5p were significantly downregulated in patients with SS who will develop or have NHL and strongly discriminated (p<0.0001) them from those without lymphoma or non-SS sialadenitis. Furthermore, they were reduced long before clinical onset of lymphoma, did not significantly change on transition to lymphoma and, importantly, were proved strong independent predictors of patients who will develop NHL (p<0.0001). CONCLUSIONS: These findings support that miR200b-5p levels in MSGs represent a novel predictive and possibly pathogenetic mechanism-related factor for the development of SS-associated NHL, since its expression is impaired years before lymphoma clinical onset.

Role of miR200b-5p miRNA in lymphomagenesis associated with Sjögren's syndrome (SS).

BACKGROUND: Development of non-Hodgkin's lymphoma (NHL) is the major adverse outcome of primary Sjögren's Syndrome (pSS) affecting both morbidity and mortality. The high frequency of transformation to lymphoid malignancy in pSS among autoimmune rheumatic diseases (6-10% of patients) and the accessibility of the affected organ (minor salivary glands; MSG), render pSS an ideal model for the study of lymphomagenesis associated with autoimmune diseases and inflammation. Although pSS-related lymphoid transformation is generally considered as an antigen-driven, multi-step process owed to the chronic activation of B-cells in MSGs, the underlying mechanisms remain elusive. Our recent results support that miR200b-5p miRNA is significantly down-regulated in the MSGs of pSS patients who have or will develop lymphoma, long before lymphoma clinical onset, indicating that it may be involved in lymphomagenesis. AIM: To investigate the role of miR200b-5p miRNA in pSS-associated lymphomagenesis. METHODS: At first, the miR200b-5p-expression will be examined by in situ hybridization in MSGs of pSS patients who are at low risk and have not developed NHL during follow-up, high risk and developed NHL in the future (pre-lymphoma) or have NHL, and the expressing cellular types, as well as those with reduced expression during lymphomagenesis, will be identified. Then, the miR200b-5p targeted molecular pathways in those cellular types (epithelial, B-cells and/or other lymphocytes, all non-neoplastic) will be studied in in vitro experiments by over-expressing and silencing of miR200b-5p, followed by transcriptome analysis. This approach is expected to find possibly novel pathogenetic mechanisms underlying SS-related lymphomagenesis. The latter is of high significance, not only for the understanding of lymphomagenesis, but also for its reversal and/or treatment. ANTICIPATED BENEFITS: This approach is anticipated to a) reveal the differentially regulated molecules and pathways by miR200b-5p, b) enlighten novel pathogenetic pathways underlying lymphomagenesis and c) identify novel therapeutic targets and possibly evidence-based therapeutic interventions.

Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation.

Sjögren's syndrome (SS) patients manifest inflammation in the salivary glands (SG) and evidence of persistent intrinsic activation of ductal SG epithelial cells (SGEC), demonstrable in non-neoplastic SGEC lines derived from patients (SS-SGEC). The peroxisome-proliferator-activated receptor-γ (PPARγ) mediates important anti-inflammatory activities in epithelial cells. Herein, the comparative analysis of SG biopsies and SGEC lines obtained from SS patients and controls had revealed constitutively reduced PPARγ expression, transcriptional activity and anti-inflammatory function in the ductal epithelia of SS patients that were associated with cell-autonomously activated NF-κB and IL-1ß pathways. Transcriptome profiling analysis revealed several differentially expressed proinflammatory and metabolism-related gene sets in SS-SGEC lines. These aberrations largely correlated with the severity of histopathologic lesions, the disease activity and the occurrence of adverse manifestations in SS patients studied, a fact which corroborates the key role of the persistently-activated epithelia in the pathogenesis of both local and systemic features of this disease. The treatment of control SGEC lines with PPARγ agonists was found to diminish the NF-κB activation and apoptosis induced by proinflammatory agents. In addition, the in-vitro application of PPARγ agonists and pharmacologic inhibitors of IL-1ß and NF-κB had significant beneficial effects on SS-SGEC lines, such as the restoration of PPARγ functions and the reduction of their intrinsic activation, a fact which may advocate the future clinical study of the above agents as therapeutic modalities for SS.

Insight into pathogenesis of Sjögren's syndrome: Dissection on autoimmune infiltrates and epithelial cells.

Sjögren's syndrome (SS) is a chronic autoimmune disease with broad clinical spectrum, extending from benign exocrinopathy to severe systemic disease and lymphoma development. The glandular and extraglandular dysfunction of SS is associated with lymphocytic infiltrates that invade the epithelial structures of affected organs. The in-depth study of autoimmune lesions in the minor salivary glands (MSG), which are the major target-organ of SS responses, revealed that the lymphocytic infiltrates vary in severity and composition among SS-patients, are full-blown at diagnosis and remain unchanged thereafter. Although the pathogenetic pathways underlying SS have not yet elucidated, it is well-established that glandular epithelial cells are central regulators of local autoimmune responses. Moreover, chronic inflammation affects epithelial function and phenotype, which strengthens or weakens their immunoregulatory/secretory function, leading to deterioration of autoimmune phenomena. Herein, the current findings regarding the autoimmune lesions, the role of epithelial cells and their interaction with infiltrating lymphocytic cells are discussed.