Ildiko Fanny Horvath

Cells with regulatory function of the innate and adaptive immune system in primary Sjögren's syndrome

The aim of the present study was to describe subsets of cells with regulatory properties in primary Sjögrens syndrome (pSS), and to correlate these cell populations with clinical symptoms. Among the 32 investigated patients, 23 had extraglandular manifestations (EGMs), while nine had only glandular symptoms. Twenty healthy individuals served as controls. The percentages of natural killer (NK), natural killer T cells (NK T), interleukin (IL)‐10 producing T regulatory type 1 (Tr1) cells and CD4+CD25+ regulatory T cells (Treg) cells were determined by flow cytometry and serum cytokine levels of IL‐4, IL‐6, IL‐10, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were evaluated by enzyme‐linked immunosorbent assay (ELISA). Functional tests were carried out to assess the suppressor properties of Treg cells in patients and controls. Peripheral NK, NK T and Tr1 cell percentages were elevated in pSS, while CD4+CD25+ Treg cells showed reduced frequencies in patients compared to controls. In pSS, elevated percentages of NK T, Tr1 and CD4+CD25+ Treg cells were observed in patients with EGMs, when compared to patients with sicca symptoms only. CD4+CD25+ Treg cell percentages showed a negative correlation with sialometry values. The in vitro functional assay demonstrated lower suppression activity of CD4+CD25+ Treg cells in patients compared to controls. Serum IL‐6 and TNF‐α levels were elevated, while IL‐10 was decreased in patients compared to controls. Negative correlation was found between IL‐10 levels and the percentages of Tr1 cells. Changes in the investigated subsets of regulatory cells in pSS may contribute to the development and progression of the disease.

Altered T-cell and regulatory cell repertoire in patients with diffuse cutaneous systemic sclerosis

Objectives: The aim of this study was to evaluate a wide spectrum of peripheral immune-competent cell types, reflecting overall disturbances in immune homeostasis, characteristic of systemic sclerosis (SSc). We also assessed visceral organ involvement and evaluated the relationship between cell proportions and clinical symptoms of the disease. Methods: Twenty-one patients with diffuse cutaneous SSc (dcSSc) and 15 healthy individuals participated in the study. Peripheral blood lymphocyte subgroups were quantified by flow cytometry, soluble cytokines were assessed by enzyme-linked immunosorbent assay (ELISA), serum complement levels were measured by nephelometry, and autoantibodies were determined by indirect immunofluorescence staining and ELISA technique. Functional tests of regulatory T (Treg) cells were also carried out. Results: Patients with SSc had higher percentages of activated CD3+/HLA-DR+ T cells. Comparing naive vs. memory subsets of CD4+ and CD8+ T cells, a shift towards central memory phenotype was observed in SSc. Natural killer (NK) and T-helper (Th)17 cell percentages were increased, while NKT, Th1, Treg type 1 (Tr1), and CD4+CD25+ Treg cell percentages were decreased in patients. Moreover, the suppressor activity of CD4+CD25+ Treg cells was lower in SSc. Negative correlations occurred between modified Rodnan skin score (MRSS) and Tr1 cell percentages and between complement levels and CD4+CD25+ Treg cells. We also found decreased interleukin (IL)-10 levels in SSc. Conclusions: Our data suggest that the increased Th17/CD4+CD25+ Treg ratio and the altered regulatory function of CD4+CD25+ Treg cells play an important role in the development of SSc. Moreover, our study reveals the potential role of the decreased profile of IL-10-producing Tr1 cells in the progression of disproportionate immune responses in SSc.

Immunological alterations in newly diagnosed primary Sjögren's syndrome characterized by skewed peripheral T-cell subsets and inflammatory cytokines.

Objectives: To describe how certain peripheral immune parameters reflect the inflammatory alterations in patients with primary Sjögrens syndrome (pSS). We determined lymphocyte subpopulations and their state of activation from peripheral blood, evaluating both soluble serum T‐helper (Th)1/Th2‐type cytokines and intracytoplasmic cytokines. Methods: Forty‐nine patients with newly diagnosed pSS and 40 healthy individuals, all free from immunomodulant or immunosuppressive medication, were studied. Peripheral blood lymphocyte subgroups were quantified by flow cytometry, soluble cytokines were assessed using enzyme‐linked immunosorbent assay (ELISA), and intracellular cytokine levels were measured after phorbol myristate acetate (PMA) stimulation by flow cytometry after staining of intracellular cytokines. Results: Patients with primary SS had higher percentages of activated CD3+/CD69+ T cells than controls. When comparing naïve vs. memory subsets of CD4+ and CD8+ T cells, a shift towards the memory phenotype was observed for both. Natural killer (NK) cell and NK T‐cell (NKT) percentages and Th0 and Th1 cell numbers were increased in patients compared to controls. Among circulating cytokines, interferon (IFN)‐γ was high, whereas interleukin (IL)‐10 was decreased in SS when compared to controls. Conclusions: SS, considered as a systemic autoimmune disease, is characterized by a complex interplay of various cytokines and immune cells. The skewed T‐cell subsets and cytokine imbalance play important roles in an orchestrated proinflammatory cascade.

The immunoregulatory role of vitamins A, D and E in patients with primary Sjögren's syndrome

OBJECTIVE The aim of the present study was to investigate the immunomodulating role of fat-soluble vitamins in 25 patients with primary SS (pSS) and 15 healthy individuals. METHODS Plasma levels of vitamins A, D and E were determined by HPLC. Peripheral NK, NK T cells, T-cell subsets, B cells, IL-10 producing Tr1 cells, CD4(+)CD25(+) Treg cells and Th17 were determined by flow cytometry. Various Th1- and Th2-soluble cytokines were assessed by ELISA, whereas intracytoplasmic cytokines (IFN-gamma, IL-4, -10 and -17) were measured by flow cytometry. Correlation was assessed between vitamin levels and immunological and clinical parameters. RESULTS Vitamin A levels did not differ between patients and controls, yet in patients with extraglandular manifestations (EGMs) a significant decrease in vitamin A levels was apparent compared with pSS patients without EGMs (P = 0.005). Vitamin E levels were increased in patients compared with controls (P = 0.004), whereas vitamin D levels were similar in pSS and control subjects. In patients, vitamin A showed a positive correlation with both NK cell (P = 0.038) and Th17 cell (P = 0.025), and a negative correlation with Schirmers test values (P = 0.035). Positive correlation was found between vitamin E and NK cells (P = 0.043), Th1 cells (P = 0.049) and the Th1/Th2 ratio (P = 0.043). In the control group, we found correlation between vitamin E and serum IL-10 levels (P = 0.003). CONCLUSIONS Our data suggest that fat-soluble vitamins may be important in immunoregulatory processes in patients with pSS.

Clinical Course, Prognosis, and Cause of Death in Primary Sjögren’s Syndrome

The aim of this retrospective, single-centre study was to investigate the clinical and laboratory features and disease outcomes of 547 patients diagnosed with primary Sjögrens syndrome (pSS) between 1975 and 2010. The patients were followed up for 11.4 ± 6.2 years. We evaluated the clinical and laboratory features, and assessed their influence on the time of diagnosis, survival, and mortality ratios, and compared them within subgroups defined by gender, glandular and extraglandular manifestations (EGMs), associated diseases, and immunoserological abnormalities. The most frequent EGMs were polyarthritis, Raynauds phenomenon, and vasculitis among our patients; the most common associated disease was thyroiditis. During the follow-up period, 51 patients died; the median survival time was 33.71 years. Our results revealed a negative effect of cryoglobulinemia on survival ratios; additionally, the presence of vasculitis and lymphoproliferative diseases at the time of diagnosis increased the risk of mortality. The development of vasculitis was the most powerful predictor of mortality. Mortality in the group of patients with extraglandular symptoms was two- to threefold higher than in the glandular group. Attention is drawn to the importance of close monitoring and targeted diagnostic approaches in those pSS subgroups with obviously increased mortality risk.

Influence of geolocation and ethnicity on the phenotypic expression of primary Sjögren's syndrome at diagnosis in 8310 patients : a cross-sectional study from the Big Data Sjögren Project Consortium

Objectives To analyse the influence of geolocation and ethnicity on the clinical presentation of primary Sjögrens syndrome (SjS) at diagnosis. Methods The Big Data Sjögren Project Consortium is an international, multicentre registry designed in 2014. By January 2016, 20 centres from five continents were participating. Multivariable logistic regression analyses were performed. Results We included 7748 women (93%) and 562 men (7%), with a mean age at diagnosis of primary SjS of 53 years. Ethnicity data were available for 7884 patients (95%): 6174 patients (78%) were white, 1066 patients (14%) were Asian, 393 patients (5%) were Hispanic, 104 patients (1%) were black/African-American and 147 patients (2%) were of other ethnicities. SjS was diagnosed a mean of 7 years earlier in black/African-American compared with white patients; the female-to-male ratio was highest in Asian patients (27:1) and lowest in black/African-American patients (7:1); the prevalence of sicca symptoms was lowest in Asian patients; a higher frequency of positive salivary biopsy was found in Hispanic and white patients. A north-south gradient was found with respect to a lower frequency of ocular involvement in northern countries for dry eyes and abnormal ocular tests in Europe (OR 0.46 and 0.44, respectively) and Asia (OR 0.18 and 0.49, respectively) compared with southern countries. Higher frequencies of antinuclear antibodies (ANAs) were reported in northern countries in America (OR=1.48) and Asia (OR=3.80) while, in Europe, northern countries had lowest frequencies of ANAs (OR=0.67) and Ro/La (OR=0.69). Conclusions This study provides the first evidence of a strong influence of geolocation and ethnicity on the phenotype of primary SjS at diagnosis.

A suitable protocol for measuring alveolar nitric oxide in asthma with differing severity to assess peripheral airways inflammation: A feasible protocol for measuring alveolar nitric oxide in patients with asthma of differing severity

Abstract Objective: Extended nitric oxide (NO) analysis offers the partitioned monitoring of inflammation in central and peripheral airways. Different mathematical models are used to estimate pulmonary NO dynamics in asthma with variable results and limitations. We aimed to establish a protocol for extended NO analysis in patients with differing asthma severity. Methods: Forty patients with stable asthma and 25 matched control subjects were recruited. Exhaled NO was measured at constant flow rates between 10 and 300 mL/s. Twelve controls performed NO measurements weekly for 4 weeks. Results: The proportions of patients with technically acceptable measurements at 10–30–50–100–150–200–250–300 mL/s exhalation flow rates were 8–58–100–98–98–95–90–80%, respectively. Alveolar NO (CANO) and total flux of NO in the conducting airways (JawNO) were calculated with the linear method from NO values measured at 100–150–200–250 mL/s exhalation flows. The mean intrasubject bias for JawNO and CANO in controls was 0.16 nL/s and 0.85 ppb, respectively. Both JawNO (1.31/0.83–2.97/vs. 0.70/0.54–0.87/nL/s, p < 0.001) and CANO (4.08/2.63–7.16/vs. 2.42/1.83–2.89/ppb, p < 0.001) were increased in patients with asthma compared to controls. In patients, CANO correlated with RV/TLC (r = 0.58, p < 0.001), FEF25-75% (p = 0.02, r = –0.36) and DL,CO (r = –0.46, p = 0.004). JawNO was not related to lung function parameters. Conclusions: Calculation of alveolar NO concentration with the linear method from values obtained at medium flow rates (100–250 mL/s) is feasible even in asthmatic patients with severe airflow limitation and may provide information on small airways dysfunction in asthma.

THU0352 Worldwide Heterogeneous Diagnostic Approach To Primary Sjögren Syndrome in 8315 Patients (EULAR-SS Task Force Big Data Sjögren Project)

Objectives To analyse the diagnostic approach used in the largest international cohort of patients with primary Sjögren syndrome (pSS) Methods The Big Data Sjögren Project is a multicentre registry formed in 2014 by experts participating in the EULAR-SS Task Force. By January 2016, the database included 8315 consecutive patients fulfilling the 2002 criteria. The main features at diagnosis/recruitment (time of criteria fulfilment) were collected and analysed Results The cohort included 7753 (93%) women (mean age at diagnosis 53 years). Sicca symptoms were reported in more than 90% of patients at diagnosis (92% for dry eye and 92.5% for dry mouth). The diagnostic tests used included the determination of anti-Ro/La antibodies in 8250 (99%) patients, Schirmer test in 6205 (75%), salivary gland biopsy in 5988 (72%), salivary flows in 4941 (59%), corneal stainings in 3304 (40%), scintigraphy in 2578 (31%) and sialography in 873 (10%) patients. The mean number of diagnostic tests included in the 2002 AE criteria was 4.85 ± 1.44 (of a maximum of 8 different tests), and was higher in patients with negative ANA (5.18 vs 4.80 in ANA+, p<0.001) or negative RF (5.06 vs 4.81 in RF+, p<0.001), and was also higher in American patients vs European or Asian patients (5.28 vs 4.83 and 4.63, respectively). A correlation was found between the number of tests carried out and the number of 2002 criteria finally fulfilled (R=0.48) Conclusions We found a heterogeneous diagnostic approach of pSS with respect to the number of 2002AE diagnostic tests carried out; the approach varied significantly according to geographic origin and baseline immunological profile Disclosure of Interest None declared

Cells with regulatory function of the innate and adaptive immune system in primary Sjögren's syndrome: Regulatory cells in Sjögren's syndrome

The aim of the present study was to describe subsets of cells with regulatory properties in primary Sjögrens syndrome (pSS), and to correlate these cell populations with clinical symptoms. Among the 32 investigated patients, 23 had extraglandular manifestations (EGMs), while nine had only glandular symptoms. Twenty healthy individuals served as controls. The percentages of natural killer (NK), natural killer T cells (NK T), interleukin (IL)‐10 producing T regulatory type 1 (Tr1) cells and CD4+CD25+ regulatory T cells (Treg) cells were determined by flow cytometry and serum cytokine levels of IL‐4, IL‐6, IL‐10, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were evaluated by enzyme‐linked immunosorbent assay (ELISA). Functional tests were carried out to assess the suppressor properties of Treg cells in patients and controls. Peripheral NK, NK T and Tr1 cell percentages were elevated in pSS, while CD4+CD25+ Treg cells showed reduced frequencies in patients compared to controls. In pSS, elevated percentages of NK T, Tr1 and CD4+CD25+ Treg cells were observed in patients with EGMs, when compared to patients with sicca symptoms only. CD4+CD25+ Treg cell percentages showed a negative correlation with sialometry values. The in vitro functional assay demonstrated lower suppression activity of CD4+CD25+ Treg cells in patients compared to controls. Serum IL‐6 and TNF‐α levels were elevated, while IL‐10 was decreased in patients compared to controls. Negative correlation was found between IL‐10 levels and the percentages of Tr1 cells. Changes in the investigated subsets of regulatory cells in pSS may contribute to the development and progression of the disease.

Cells with regulatory function of the innate and adaptive immune system in primary Sjögren's syndrome: ORIGINAL ARTICLE

The aim of the present study was to describe subsets of cells with regulatory properties in primary Sjögrens syndrome (pSS), and to correlate these cell populations with clinical symptoms. Among the 32 investigated patients, 23 had extraglandular manifestations (EGMs), while nine had only glandular symptoms. Twenty healthy individuals served as controls. The percentages of natural killer (NK), natural killer T cells (NK T), interleukin (IL)‐10 producing T regulatory type 1 (Tr1) cells and CD4+CD25+ regulatory T cells (Treg) cells were determined by flow cytometry and serum cytokine levels of IL‐4, IL‐6, IL‐10, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were evaluated by enzyme‐linked immunosorbent assay (ELISA). Functional tests were carried out to assess the suppressor properties of Treg cells in patients and controls. Peripheral NK, NK T and Tr1 cell percentages were elevated in pSS, while CD4+CD25+ Treg cells showed reduced frequencies in patients compared to controls. In pSS, elevated percentages of NK T, Tr1 and CD4+CD25+ Treg cells were observed in patients with EGMs, when compared to patients with sicca symptoms only. CD4+CD25+ Treg cell percentages showed a negative correlation with sialometry values. The in vitro functional assay demonstrated lower suppression activity of CD4+CD25+ Treg cells in patients compared to controls. Serum IL‐6 and TNF‐α levels were elevated, while IL‐10 was decreased in patients compared to controls. Negative correlation was found between IL‐10 levels and the percentages of Tr1 cells. Changes in the investigated subsets of regulatory cells in pSS may contribute to the development and progression of the disease.