Sándor Baráth

Follicular helper T cells may play an important role in the severity of primary Sjögren's syndrome

The aim of this study was to investigate the possible role of follicular helper T (TFH) cells in the pathogenesis of primary Sjögrens syndrome (pSS) by analyzing immune-competent cells and serological markers with special emphasis on clinical symptoms. We enrolled 50 pSS patients and 16 healthy individuals in the study. Patients had elevated ratio of peripheral TFH cells, however, when dividing patients into two groups defined by the presence of extraglandular manifestations (EGMs), only patients with EGMs differed from controls significantly. Moreover, TFH cell percentages correlated positively with both activated T cell and Tr1 cell values. On the contrary, TFH cell percentages showed negative correlation with both IgM and IgG memory B cell proportions. Elevated TFH percent\ages were observed in the anti-SSA/SSB positive patients, and also in patients with higher IL-12, IL-21 levels and focus score values. Increased TFH cell proportions seem to have an important role in disease development.

Measurement of natural (CD4+CD25high) and inducible (CD4+IL-10+) regulatory T cells in patients with systemic lupus erythematosus

Abnormalities of regulatory T cells may play an important role in the loss of self-tolerance, which is a major characteristic of lupus. The objective of this study was to determine the ratio and the number of natural CD4+CD25highFoxp3+ and inducible CD4+IL-10+ regulatory T cells in lupus patients and to search correlation with disease activity. Seventy-two Hungarian lupus patients were enrolled in the study. Fourty-one age- and sex matched healthy donors served as controls. Flow cytometry was used for the quantification of CD4+CD25high Foxp3+ (nTreg) and CD4+IL-10+ (iTreg) cells. The ratio (3.06 ± 1.45%) and the number (0.019 ± 0.012 × 109/L) of nTreg cells decreased in lupus significantly (P < 0.001 in both) as compared to normal controls (4.26 ± 1.01% and 0.039 ± 0.017 × 109/L). The ratio of iTreg cells were significantly higher in patients than in controls (20.92 ± 14.02% versus 15.49 ± 11.65%, P < 0.03), but the number of these cell type did not differ in significant manner (0.314 ± 0.236 × 109/L versus 0.259 ± 0.183 × 109/L). The 19 active patients were characterised by significantly higher disease activity index (SLEDAI 8.63 ± 2.95 versus 1.74 ± 1.68, P < 0.001) and anti-DNA concentration (117.85 ± 145.89 versus 37.36 ± 68.85 IU/mL, P = 0.001) as compered to the 52 inactive patients. Furthermore, active patients required higher dose of methylprednisolon than inactive ones (14.8 ± 10.6 versus 4.8 ± 3.4 mg/day, P < 0.001). However, we did not find statistical significant difference in the number and ratio of the examined cell populations regarding to disease activity. Altered ratio and number of both natural and inducible regulatory T cells may play a role in the pathogenesis of lupus. There are small but appreciable difference in the number of regulatory T cells between inactive patients and healthy controls. It suggests that immunoregulatory deficiencies are present in the inactive stage of the disease also. Lupus (2007) 16, 489—496.

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.

Vitamin D insufficiency in a large MCTD population

OBJECTIVES The aim of the present study was to evaluate the vitamin D status in patients with mixed connective tissue disease (MCTD) and to determine which clinical symptoms, laboratory parameters and endothelial cell markers are associated with low vitamin D levels. METHODS 125 female MCTD patients and 48 age- and sex-matched healthy controls were enrolled in the study. The clinical symptoms, autoantibodies (anti-U1-RNP, anti-cardiolipin - anti-CL and anti-endothelial cell antibody - AECA), serum cytokines (IFN-γ, IL-6, IL-12, IL-23, IL-17 and IL-10), soluble endothelial cell markers (endothelin, thrombomodulin - TM, and von Willebrand factor antigen - vWFAg) and serum lipids (total cholesterol, triglyceride, LDL-C, HDL-C, apolipoprotein A1, and apolipoprotein B) were investigated for an association with vitamin D levels by univariate and multivariate statistical analyses. RESULTS The mean vitamin D levels were significantly lower in MCTD patients, as compared with the control group (26.16±13.50ng/ml vs. 34.92±9.64ng/ml; p<0.001). In laboratory parameters, vitamin D levels were inversely associated with serum IL-6 (p<0.001), IL-23 (p=0.011), IL-10 (p=0.033) cytokine levels, TM (p=0.001) and endothelin (p=0.033) levels. Low vitamin D levels were also significantly associated with carotid artery intima media thickness (p<0.001), fibrinogen (p=0.010), total cholesterol (p=0.042) and ApoA1 (p=0.004) levels. Among the clinical symptoms, the cardiovascular involvement showed an inverse correlation with vitamin D status in MCTD (p<0.001). CONCLUSIONS The prevalence of vitamin D insufficiency is high in patients with MCTD. We assume that vitamin D insufficiency along with inflammatory parameters and lipid abnormalities may provoke cardiovascular events.

Regulatory T cells in atopic dermatitis: epidermal dendritic cell clusters may contribute to their local expansion

Background  Regulatory T cells (Tregs) have an essential role in tolerance and immune regulation. However, few and controversial data have been published to date on the role and number of these cells in atopic dermatitis (AD).

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.

The severity of systemic lupus erythematosus negatively correlates with the increasing number of CD4+CD25highFoxP3+ regulatory T cells during repeated plasmapheresis treatments of patients

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by increased pathologic autoantibody production. A decrease in the number of CD4+CD25highFoxP3+ regulatory T cells can play a key role in the loss of tolerance to self antigens. Our aim was to determine the absolute number of peripheral CD4+CD25highFoxP3+ T cells in 44 patients with SLE, furthermore, to measure the changes in the number of CD4+CD25highFoxP3+ T cells in 5 patients with severe SLE treated with repeated plasmapheresis for 4–6 days in comparison to the changes in the activity of disease (SLEDAI). Percent of CD4+CD25highFoxP3+ T cells were measured by flow cytometry. The absolute number of peripheral CD4+CD25highFoxP3+ T cells was significantly decreased in the 44 patients with SLE compared to the healthy controls n = 32 (0.012 ± 0.006 vs. 0.038 ± 0.017 G/L, p < 0.05). In the 5 patients with severe SLE the repeated plasmapheresis treatments increased the peripheral number of CD4+CD25highFoxP3+ T cells. As the number of CD4+CD25highFoxP3+ T cells increased during the treatment, the activity of disease (the value of SLE activity index) decreased. In the peripheral blood of SLE patients not only the ratio was decreased (as it was published earlier) but also the absolute number of these regulatory T cells. The repeated plasmapheresis treatments of SLE patients induced a significant increase in the number of peripheral CD4+CD25highFoxP3+ T cells in parallel to the decrease in the values of SLEDAI (the activity of disease). This phenomenon is, among others, possibly due to the elimination of interpheron-alpha and lymphocytotoxic antibodies during plasmapheresis.

Alfacalcidol treatment restores derailed immune-regulation in patients with undifferentiated connective tissue disease

Vitamin D deficiency may contribute to pathological changes in the number and function of CD4+ T helper cell subsets (CD4+Th1, CD4+Th17, CD4+CD25(bright)Foxp3-natural regulatory T cells-nTreg) in patients with undifferentiated connective tissue disease (UCTD). The aim of the present study was to evaluate, whether alfacalcidol could restore immune-regulatory changes in patients with UCTD. We assessed the optimal dose of alfacalcidol that could normalize the elevated levels of IFN-γ expressed by the CD4+Th1 cells and the IL-17 expressed by Th17 cells. Furthermore alfacalcidol decreased the Th1 and Th17 related cytokine levels, repaired the nTreg/Th7 balance, and restored the functional activity of nTreg cells. Twenty one UCTD patients with Vitamin D deficiency (<30 ng/ml) were administered with three different daily doses of alfacalcidol. Seven patients were supplemented with 0.5 μg/day, 7 patients with 1.0 μg/day, and 7 patients with 1.5 μg/day alfacalcidol treatment during 5 weeks. Our results indicated that 1.0 μg/day alfacalcidol during 5 weeks was the optimal therapeutic regime to increase the vitamin D levels, repair the nTreg/Th17 balance and raise the capacity of nTreg cells to suppress the proliferation of autologous CD4+CD25- cells. 1.5 μg daily dose alfacalcidol was not more effective than the 1.0 μg/day treatment. In this study we described that vitamin D deficiency can contribute to the complex immune-regulatory abnormalities in patients with UCTD and vitamin D substitution therapy can improve the fine balance of pro- and anti-inflammatory processes in the disease.

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.

Altered Th17 cells and Th17/regulatory T-cell ratios indicate the subsequent conversion from undifferentiated connective tissue disease to definitive systemic autoimmune disorders

A shift in the balance between Th17-cells and regulatory T-cells (Treg) is an important feature of systemic autoimmune diseases (SAID), and may also contribute to their development. Hereby, we assessed the distribution of peripheral Th17 and Treg-cells in patients with undifferentiated connective tissue disease (UCTD), the forerunner of SAIDs and followed these parameters during the development towards definitive SAIDs. Fifty-one UCTD patients were investigated and followed-up for 3 years. Flow cytometry was used to identify and follow three cell-populations: Th17-cells (CD4+IL-17+ T-cells), natural regulatory T-cells (CD4(+)CD25(bright)FoxP3(+); nTregs) and IL-10 producing Type-1 regulatory T-cells (CD4+IL-10+ T-cells; Tr1). Altogether 37.3% of these patients progressed into SAIDs. Th17-cells were increased in UCTD vs. controls, which further increased in those, whom developed SAIDs eventually. The Th17/nTreg ratio gradually increased from controls through UCTD patients, reaching the highest values in SAID-progressed patients. Regarding the Th17/Tr1 ratios, a similar tendency was observed moreover Th17/Tr1 could distinguish between UCTD patients with, or without subsequent SAID progression in a very early UCTD stage. Various immunoserological markers showed association with Th17 and Th17/nTreg at baseline, indicating the consecutive development of a distinct SAID. The derailed Th17/Treg balance may contribute to disease progression therefore could function as a prognostic marker.