TLR7 Signaling Drives the Development of Sjögren's Syndrome.

Sjögren's syndrome (SS) is a chronic systemic autoimmune disease that affects predominately salivary and lacrimal glands. SS can occur alone or in combination with another autoimmune disease like systemic lupus erythematosus (SLE). Here we report that TLR7 signaling drives the development of SS since TLR8-deficient (TLR8ko) mice that develop lupus due to increased TLR7 signaling by dendritic cells, also develop an age-dependent secondary pathology similar to associated SS. The SS phenotype in TLR8ko mice is manifested by sialadenitis, increased anti-SSA and anti-SSB autoantibody production, immune complex deposition and increased cytokine production in salivary glands, as well as lung inflammation. Moreover, ectopic lymphoid structures characterized by B/T aggregates, formation of high endothelial venules and the presence of dendritic cells are formed in the salivary glands of TLR8ko mice. Interestingly, all these phenotypes are abrogated in double TLR7/8-deficient mice, suggesting that the SS phenotype in TLR8-deficient mice is TLR7-dependent. In addition, evaluation of TLR7 and inflammatory markers in the salivary glands of primary SS patients revealed significantly increased TLR7 expression levels compared to healthy individuals, that were positively correlated to TNF, LT-α, CXCL13 and CXCR5 expression. These findings establish an important role of TLR7 signaling for local and systemic SS disease manifestations, and inhibition of such will likely have therapeutic value.

Assessing the practice of LuPOR for poor responders: a prospective study evaluating follicular fluid cfDNA levels during natural IVF cycles.

PURPOSE: The aim of this study is to provide data on the practice of Luteal Phase Oocyte Retrieval (LuPOR). The authors assess cell-free DNA levels in follicular fluid (ff cfDNA) from poor responders undergoing natural cycles, and comparing it to respective data originating from follicular phase oocyte retrievals. METHODS: Forty-seven women were eligible for this prospective study. Participants were classified as poor responders based on Bologna criteria while being detected with a second follicular wave. Follicular fluid was collected and prepared for cfDNA extraction. Levels of cfDNA were quantified via Q-PCR employing the ALU115 and ALU247 primers. These primers are associated with apoptotic and necrotic events. Levels of ff cfDNA resulting from follicular phase oocyte retrieval (FoPOR) and LuPOR-performed in a single menstrual cycle were associated with the number and maturation status of yielded oocytes and the number and fertilization status of resulting zygotes. Survival rate following thawing of cryopreserved zygotes, along with the resulting number of cleavage stage and blastocyst stage embryos are provided. RESULTS: Mean levels of ALU115 were significantly lower during FoPOR when compared to LuPOR (0.79 ± 0.72 vs 1.46 ± 1.59 ng/µl, p = 0.02). Regarding the FoPOR group, a significant positive correlation of serum estradiol and ALU115 concentration (p = 0.04) was revealed. A significant negative correlation between serum estradiol and cfDNA integrity was observed both during FoPOR (p = 0.03) and LuPOR (p = 0.03). A significant lower number of retrieved (1.09 ± 0.28 vs 1.29 ± 0.58, p = 0.02) and MII oocytes (0.77 ± 0.55 vs 1.08 ± 0.61, p = 0.02) was observed when comparing the FoPOR to LuPOR groups respectively. The integrity of cfDNA was observed to be higher in FoPOR originating embryos that arrested either prior to cleavage (0.28 ± 0.13 vs 0.17 ± 0.10, p = 0.006) or prior to blastocyst formation (0.28 ± 0.12 vs 0.13 ± 0.06, p = 0.04). In the case of LuPOR originating embryos, cfDNA integrity was observed to be higher in embryos that arrested only prior to the blastocyst stage (0.27 ± 0.20 vs 0.11 ± 0.07, p = 0.008). Similarly, cfDNA integrity was observed to be lower in top quality blastocysts originating from FoPOR (0.07 ± 0.04 vs 0.17 ± 0.05, p = 0.03) and in top quality cleavage stage embryos (0.09 ± 0.06 vs 0.31 ± 0.22, p = 0.01) and blastocysts (0.06 ± 0.02 vs 0.14 ± 0.06, p = 0.02) originating from LuPOR. CONCLUSIONS: Our results indicate that ff originating from LuPOR presents with higher levels of cfDNA. The higher cfDNA levels are attributed to mainly apoptotic events, as the ALU247 levels and DNA integrity did not differ statistically significantly between FoPOR and LuPOR. The absolute mean level of ALU247 corresponding to necrotic events was higher in LuPOR. Regarding embryological data, cfDNA integrity was correlated with both number and quality of cleavage stage embryos in both FoPOR and LuPOR, along with blastocyst stage embryos in LuPOR. Necrotic events were associated with poorer blastocyst formation rate and blastocyst quality in LuPOR. As the comparison between FoPOR and LuPOR results to similar IVF laboratory data, the practice of LuPOR may stand as a promising approach for poor responders, while it merits further investigation.

Predicting Lymphoma Development by Exploiting Genetic Variants and Clinical Findings in a Machine Learning-Based Methodology With Ensemble Classifiers in a Cohort of Sjögren's Syndrome Patients.

Lymphoma development constitutes one of the most serious clinico-pathological manifestations of patients with Sjögren's Syndrome (SS). Over the last decades the risk for lymphomagenesis in SS patients has been studied aiming to identify novel biomarkers and risk factors predicting lymphoma development in this patient population. Objective: The current study aims to explore whether genetic susceptibility profiles of SS patients along with known clinical, serological and histological risk factors enhance the accuracy of predicting lymphoma development in this patient population. Methods: The potential predicting role of both genetic variants, clinical and laboratory risk factors were investigated through a Machine Learning-based (ML) framework which encapsulates ensemble classifiers. Results: Ensemble methods empower the classification accuracy with approaches which are sensitive to minor perturbations in the training phase. The evaluation of the proposed methodology based on a 10-fold stratified cross validation procedure yielded considerable results in terms of balanced accuracy (GB: 0.7780 ± 0.1514, RF Gini: 0.7626 ± 0.1787, RF Entropy: 0.7590 ± 0.1837). Conclusions: The initial clinical, serological, histological and genetic findings at an early diagnosis have been exploited in an attempt to establish predictive tools in clinical practice and further enhance our understanding towards lymphoma development in SS.

Contribution of MTHFR gene variants in lupus related subclinical atherosclerosis.

OBJECTIVE: Elevated concentrations of homocysteine have been previously identified as an independent risk factor for subclinical atherosclerosis in patients with systemic lupus erythematosus (SLE). Given that heightened homocysteine levels are known to be strongly influenced by genetic factors, in the current study we investigated the contribution of high homocysteine levels as well as of functional polymorphisms of the gene encoding for the enzyme 5, 10- methylenetetrahydrofolate reductase (MTHFR) to atherosclerotic disease characterizing SLE patients. METHODS: Peripheral DNA samples from 150 SLE patients, 214 rheumatoid arthritis (RA) patients and 561 age/sex matched apparently healthy volunteers (HC) were genotyped by PCR-based assays for the detection of the MTHFR gene polymorphisms (c. 677C > T and c. 1298A > C). All SLE patients and 30 age sex matched RA patients underwent assessment for subclinical atherosclerosis [ultrasound measurement of intima-media thickness scores (IMT) and detection of carotid and/or femoral (C/F) plaque] and complete clinical and laboratory evaluation including serum homocysteine levels. Data were analyzed using univariate and multivariate models (SPSS 21.0). RESULTS: Hyperhomocysteinemia was detected in 26.0% of SLE patients compared to 6.7% of age/sex matched RA controls (p = 0.02). Higher serum B12 levels and decreased frequency of the MTHFR 677TT variant in RA patients could potentially account for the observed differences between the groups. In SLE patients, both hyperhomocysteinemia and MTHFR 677TT genotype were identified as independent contributors for plaque formation, following adjustment for traditional cardiovascular risk factors and disease related features, including age, sex, BMI, cholesterol and triglyceride levels, presence of arterial hypertension, smoking (pack/years), disease duration and total steroid dose [OR 95% (CI): 5.8 (1.0-35.8) and 5.2 (1.1-24.0), respectively]. MTHFR 677TT genotype, but not hyperhomocysteinemia was also found to confer increased risk for arterial wall thickening, after the above confounders were taken into account [OR (95%) CI: 4.9 (1.2-20.6)]. CONCLUSIONS: Hyperhomocysteinemia and MTHFR 677TT genetic variant emerged as independent risk factors for subclinical atherosclerosis in SLE patients, implying genetic influences as potential contributors to the increased burden of atherosclerotic disease characterizing SLE.

A BAFF receptor His159Tyr mutation in Sjögren's syndrome-related lymphoproliferation.

OBJECTIVE: To study the prevalence, clinical associations, and functional implications of the His159Tyr mutation of the BAFF receptor (BAFF-R) in patients with Sjögren's syndrome (SS). METHODS: The BAFF-R His159Tyr mutation was evaluated using polymerase chain reaction (PCR)-based assays in 247 patients with SS (of whom 70 had SS complicated by lymphoma [SS-lymphoma]), 145 with systemic lupus erythematosus (SLE), and 101 with rheumatoid arthritis (RA), as well as 180 healthy controls. Real-time PCR and Western blotting were performed for the quantification of both NF-κB1 and NF-κB2 messenger RNA (mRNA) transcript and protein levels in isolated B cells from patients with SS-lymphoma carrying the mutation (SS-lymphoma-BAFF-RHis159Tyr -derived B cells) compared to B cells from patients with SS-lymphoma who were not carriers of the mutation and healthy controls. RESULTS: Both the SS-lymphoma and SS-nonlymphoma patient subgroups exhibited significantly higher frequencies of the His159Tyr BAFF-R mutation compared to healthy controls (8.6% of SS-lymphoma patients and 6.2% of SS-nonlymphoma patients versus 1.7% of healthy controls; P = 0.02 and P = 0.04, respectively). The corresponding frequencies of the His159Tyr BAFF-R mutation in SLE and RA patients were 3.5% and 3%, respectively. Of interest, 71.4% of the SS patients with mucosa-associated lymphoid tissue (MALT) lymphoma who were between the ages of 31 and 40 years at disease onset were mutation carriers. The generalized odds ratio for the development of SS-related MALT lymphoma in the younger age at onset (age <40 years) group in the presence of the BAFF-R mutation was 6.1 (95% confidence interval 2.0-18.7) (P < 0.01). Expression of NF-κB at both the mRNA and protein level was up-regulated in SS-lymphoma-BAFF-RHis159Tyr -derived B cells. CONCLUSION: This study identifies an increased prevalence of the BAFF-R His159Tyr mutation in patients with SS, particularly in those with SS complicated by MALT lymphoma whose disease onset occurred at a younger age. BAFF-RHis159Tyr -mediated activation of the alternate NF-κB pathway might contribute to the pathogenesis of SS-related lymphoproliferative disease.

Subclinical atherosclerosis and impaired bone health in patients with primary Sjogren's syndrome: prevalence, clinical and laboratory associations.

INTRODUCTION: To determine the prevalence and clinical/laboratory associations of subclinical atherosclerosis and impaired bone health in primary Sjogren's syndrome (SS). METHODS: 64 consecutive patients with primary SS, 77 with rheumatoid arthritis (RA) and 60 healthy controls (HC) οf similar age and sex distribution were enrolled. Demographics, clinical/laboratory features, classical risk factors for atherosclerosis and osteoporosis (OP) were recorded. Intima-medial thickness scores (IMT) and carotid/femoral (C/F) plaque formation, as well as bone mineral density (BMD) and fractures were evaluated. Determinants of IMT/BMD levels and the presence of plaque were assessed by univariate and multivariate models. Serum levels of the Wnt signaling mediators Dickkopf-related protein 1(DKK1) and sclerostin were determined in primary SS patients and HC. RESULTS: Increased arterial wall thickening (IMT > 0.90 mm) and impaired bone health (defined as OP or osteopenia), were detected in approximately two-thirds of primary SS and RA patients, with a mean IMT value being significantly increased compared to HC. The presence of primary SS emerged as an independent risk factor for arterial wall thickening when traditional risk factors for cardiovascular disease (CVD) including age, sex, hypertension, smoking (pack/years), LDL and HDL levels were taken into account in a multivariate model [adjusted OR 95% (CI): 2.8 (1.04-7.54)]. In primary SS, age was revealed as independent predictor of increased IMT scores; age and lymphopenia as well as increased urine pH as independent determinants of C/F plaque formation and OP/osteopenia, respectively. An independent association of OP/osteopenia with plaque formation was observed when independent predictors for both variables were considered, with low DKK1 levels being associated with both plaque formation and lower BMD levels. CONCLUSIONS: Comorbidities such as subclinical atherosclerosis and impaired bone health occur frequently in primary SS, in association with disease related features and traditional risk factors. Wnt signaling mediators are potentially involved in the pathogenesis of both entities.

Methods of detection of circulating melanoma cells: a comparative overview.

Disease dissemination is the major cause of melanoma-related death. A crucial step in the metastatic process is the intravascular invasion and circulation of melanoma cells in the bloodstream with subsequent development of distant micrometastases that is initially clinically undetectable and will eventually progress into clinically apparent metastasis. Therefore, the use of molecular methods to detect circulating melanoma cells may be of value in risk stratification and clinical management of such patients. Herein, we review the currently applied techniques for the detection, isolation, enrichment and further characterization of circulating melanoma cells from peripheral blood samples in melanoma patients. Furthermore, we provide a brief overview of the various molecular markers currently being evaluated as prognostic indicators of melanoma progression.

Molecular detection of tyrosinase transcripts in peripheral blood from patients with malignant melanoma: correlation of PCR sensitivity threshold with clinical and pathologic disease characteristics.

BACKGROUND: Positive molecular detection of tyrosinase transcripts (TYR mRNA) in RNA extracts of peripheral blood (PB) samples from patients with malignant melanoma provides evidence of disease dissemination. METHODS: Total RNA extracted from PB was quantified and subjected to RT-PCR under ultra-sensitive and reduced-sensitivity PCR conditions using SSRT-II. Positive TYR mRNA detection in 78 melanoma patients and 40 healthy volunteers was correlated with clinical stage, Breslow's evaluation of tumor thickness, Clark's assessment of tumor invasion, the location of the primary tumor site, and tumor histology. The assay sensitivity was evaluated by spiking PB with the melanoma cell line SK-MEL-28. RESULTS: Using ultra-sensitive PCR conditions, eight out of 40 RNA (20%) samples from healthy volunteers and 50 out of 78 RNA (64.1%) samples from melanoma patients tested positive. Using reduced-sensitivity PCR conditions, we found only two positives in 40 RNA samples from healthy subjects and 20 positives in 78 RNA samples (25.6%) from melanoma patients. Only positive PCR samples for the reduced-sensitivity PCR assay correlated significantly with stage IV (metastatic) disease (p=0.0395). There was no significant correlation between positive TYR mRNA samples for either PCR condition (ultra-sensitive and reduced-sensitivity) with Breslow's classification of tumor thickness, Clark's assessment of tumor invasion, location of the primary tumor site, and type of tumor histology. CONCLUSIONS: We conclude that reduced-sensitivity rather than ultra-sensitive PCR conditions correlate with clinical stage in melanoma patients.