Autoantibodies Profile in the Sera of Patients with Sjogren]s Syndrome: The ANA Evaluation—A Homogeneous, Multiplexed System

Background: Flow-based, multiplex bead arrays (MBA) have been developed for a variety of applications including the detection of antibodies to extractable nuclear antigens (ENA). It offers a rapid and sensitive method to assess multiple analyses in a single tube/well

Functional Improvement of Regulatory T Cells From Rheumatoid Arthritis Subjects Induced by Capsular Polysaccharide Glucuronoxylomannogalactan

Objective: Regulatory T cells (Treg) play a critical role in the prevention of autoimmunity, and the suppressive activity of these cells is impaired in rheumatoid arthritis (RA). The aim of the present study was to investigate function and properties of Treg of RA patients in response to purified polysaccharide glucuronoxylomannogalactan (GXMGal). Methods: Flow cytometry and western blot analysis were used to investigate the frequency, function and properties of Treg cells. Results: GXMGal was able to: i) induce strong increase of FOXP3 on CD4+ T cells without affecting the number of CD4+CD25+FOXP3+ Treg cells with parallel increase in the percentage of non-conventional CD4+CD25-FOXP3+ Treg cells; ii) increase intracellular levels of TGF-beta1 in CD4+CD25-FOXP3+ Treg cells and of IL-10 in both CD4+CD25+FOXP3+ and CD4+CD25-FOXP3+ Treg cells; iii) enhance the suppressive activity of CD4+CD25+FOXP3+ and CD4+CD25-FOXP3+ Treg cells in terms of inhibition of effector T cell activity and increased secretion of IL-10; iv) decrease Th1 response as demonstrated by inhibition of T-bet activation and down-regulation of IFN-gamma and IL-12p70 production; v) decrease Th17 differentiation by down-regulating pSTAT3 activation and IL-17A, IL-23, IL-21, IL-22 and IL-6 production. Conclusion: These data show that GXMGal improves Treg functions and increases the number and function of CD4+CD25-FOXP3+ Treg cells of RA patients. It is suggested that GXMGal may be potentially useful for restoring impaired Treg functions in autoimmune disorders and for developing Treg cell-based strategies for the treatment of these diseases

One year in review 2017: primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is a complex and heterogeneous disease. Last year, a great deal of basic and clinical research was carried out in pSS. Following the previous reviews of this publishing series, we will herewith provide a critical digest of the most recent literature on pSS pathogenesis, clinical manifestations and treatment. More specifically, we will focus on the heterogeneity of the disease, on the underlying pathogenetic pathways and on the possible new targeted treatments on the horizon

Functional improvement of regulatory T cells from rheumatoid arthritis subjects induced by capsular polysaccharide glucuronoxylomannogalactan.

Regulatory T cells (Treg) play a critical role in the prevention of autoimmunity, and the suppressive activity of these cells is impaired in rheumatoid arthritis (RA). The aim of the present study was to investigate function and properties of Treg of RA patients in response to purified polysaccharide glucuronoxylomannogalactan (GXMGal).Flow cytometry and western blot analysis were used to investigate the frequency, function and properties of Treg cells.GXMGal was able to: i) induce strong increase of FOXP3 on CD4+ T cells without affecting the number of CD4+CD25+FOXP3+ Treg cells with parallel increase in the percentage of non-conventional CD4+CD25-FOXP3+ Treg cells; ii) increase intracellular levels of TGF-β1 in CD4+CD25-FOXP3+ Treg cells and of IL-10 in both CD4+CD25+FOXP3+ and CD4+CD25-FOXP3+ Treg cells; iii) enhance the suppressive activity of CD4+CD25+FOXP3+ and CD4+CD25-FOXP3+ Treg cells in terms of inhibition of effector T cell activity and increased secretion of IL-10; iv) decrease Th1 response as demonstrated by inhibition of T-bet activation and down-regulation of IFN-γ and IL-12p70 production; v) decrease Th17 differentiation by down-regulating pSTAT3 activation and IL-17A, IL-23, IL-21, IL-22 and IL-6 production.These data show that GXMGal improves Treg functions and increases the number and function of CD4+CD25-FOXP3+ Treg cells of RA patients. It is suggested that GXMGal may be potentially useful for restoring impaired Treg functions in autoimmune disorders and for developing Treg cell-based strategies for the treatment of these diseases

Anti-tumor necrosis factor-alpha response in rheumatoid arthritis is associated with an increase in serum soluble CD30

OBJECTIVE: Patients with rheumatoid arthritis (RA) display high serum concentrations of soluble CD30 (sCD30), which correlate with counter-regulatory activity of CD30+ T cells in the inflamed joint. To verify the contribution of this T cell subset to disease remission, sCD30 levels were analyzed longitudinally in patients with active RA following infliximab therapy. METHODS: Infliximab plus methotrexate were started in 39 patients with active RA, while 20 patients with inactive disease, controlled by stable doses of methotrexate, acted as controls. Serial evaluations of sCD30 concentrations and disease activity indexes were performed throughout 38 weeks. RESULTS: sCD30 levels were higher in patients than in healthy controls. Rapid infliximab-induced decrease in disease activity was associated with an overall increase of sCD30 levels. In contrast, levels remained stable in controls. An inverse correlation between sCD30 levels and Disease Activity Score 28 was observed from the 22nd week of infliximab treatment. Analysis of sCD30 levels according to American College of Rheumatology response showed, after an initial general enhancement of sCD30 concentrations, a persistent increase of sCD30 in responders, but not in nonresponders. CONCLUSION: sCD30 serum levels are enhanced by tumor necrosis factor-a (TNF-a) blockade in patients with active RA and inversely correlated with disease activity, but only after some weeks of treatment. Of interest, a sustained increase of sCD30 is present only in subjects with evidence of persistent clinical response to anti-TNF-alpha. As sCD30 serum levels mirror antiinflammatory activity of joint T cells, the present data may suggest a role of synovial counter-regulatory CD30+ T cells in the induction of infliximab-mediated remission in RA

Effects of cytotoxic T-lymphocyte-associated protein 4 compared to TNF inhibitors on lipid profile:Results from an observational multicentre rheumatoid arthritis cohort

AIM: To evaluate the impact of selective cytotoxic T-lymphocyte-associated protein 4 (CTLA-4Ig) compared to tumor necrosis factor inhibitors (TNFi) on cardiovascular (CV) clinical and laboratory outcomes in patients with rheumatoid arthritis (RA).METHODS: We performed a prospective observational multicenter study of RA patients included in the "Cardiovascular Obesity and Rheumatic DISease (CORDIS)" Study Group database, collecting demographic, clinical, and laboratory data of those starting a CTLA-4Ig or TNFi at baseline, 6-month, and 12-month follow-up.RESULTS: Of the 206 RA patients without previous CV events enrolled in the study, 64 received a CTLA-4Ig and 142 a TNFi. The two groups did not differ in age, gender, or smoking habits, and the prevalence of hypertension, diabetes, and metabolic syndrome was similar. Over a follow-up period of 12 months, although no significant differences were found in the disease activity course, we observed that LDL cholesterol levels slightly decreased only in the CTLA-4Ig-treated patients.CONCLUSIONS: Patients treated with both CTLA-4Ig and TNFi did not differ in disease activity response and changes in traditional CV risk factors after 12 months of treatment. However, CTL-A-4Ig treatment is associated with a favorable change in lipid profile at 12-month follow-up.</p

GXMGal effect on Th17 response.

<p>Activated PBMC (<b>A</b> and <b>C</b>) or purified CD4⁺ T cells (<b>B</b>) (both 5×10⁶/ml) from Control and RA were incubated for 2, 18 and 72 h in the presence or absence (NS) of GXMGal (10 µg/ml), MTX (10 ng/ml) or FLLL31 (5 µM). After 18 h of incubation, cell lysates were analyzed by western blotting. Membranes were incubated with Abs to pSTAT3 and STAT3. Actin was used as an internal loading control. Normalization was shown as mean ± SEM of five independent experiments (<b>A</b>) or as one representative experiment of three with similar results (<b>B</b>). *, <i>p</i><0.05 (triplicate samples of 5 different Control and RA; RA treated <i>vs</i> untreated cells). Culture supernatants were collected after 2, 18 and 72 h to test IL-21, IL-22, IL-23, IL-6 and IL-17A levels by specific ELISA assays. *, <i>p</i><0.05 (triplicate samples of 7 different Control and RA; RA GXMGal-treated <i>vs</i> untreated cells); ^†, <i>p</i><0.05 (triplicate samples of 7 different Control and RA; RA MTX-treated <i>vs</i> untreated cells) (<b>C</b>).</p

GXMGal effect on Th1 response.

<p>Activated PBMC (<b>A</b>, <b>B</b> and <b>D</b>) (5×10⁶/ml) from Control and RA were incubated for 20 min, 2, 18 and 72 h in the presence or absence (NS) of GXMGal (10 µg/ml), MTX (10 ng/ml) or DEX (10 nM). After 20 min of incubation, cell lysates were analyzed by western blotting. Membranes were incubated with Ab to T-bet. Actin was used as an internal loading control. Normalization was shown as mean ± SEM of five independent experiments. *, <i>p</i><0.05 (triplicate samples of 5 different Control and RA; treated <i>vs</i> untreated cells) (<b>A</b>). Culture supernatants were collected after 2, 18 and 72 h to test IFN-γ, IL-12p70 (<b>B</b>) and IL-8 (<b>D</b>) levels by specific ELISA assays. *, <i>p</i><0.05 (triplicate samples of 7 different Control and RA; RA GXMGal-treated <i>vs</i> untreated cells); ^†, <i>p</i><0.05 (triplicate samples of 7 different Control and RA; RA MTX-treated <i>vs</i> untreated cells); ‡, <i>p</i><0.05 (triplicate samples of 7 different Control and RA; RA DEX-treated <i>vs</i> untreated cells). Activated purified CD4⁺ T cells (1×10⁶/ml) (<b>C</b>) from RA were stimulated as above described and intracellular stained for T-bet and IFN-γ. The percentage of T-bet⁺/IFN-γ⁺ CD4⁺ T cells from RA after 18 h of GXMGal or MTX treatment was shown as mean ± SEM of five independent experiments. *, <i>p</i><0.05 (triplicate samples of five different RA; treated <i>vs</i> untreated cells).</p

GXMGal effect on different subsets of Treg cells.

<p>Activated PBMC (1×10⁶/ml) from Control and RA were incubated for 2 h in the presence or absence (NS) of GXMGal (10 µg/ml) or MTX (10 ng/ml). After incubation, cells were stained for cell surface expression of CD4, CD25 and CD127 and then intracellular stained for FOXP3. During the acquisition step a population of PBMC enriched of CD4⁺ T cells was obtained. For analysis, the CD4⁺ lymphocytes were gated on PBMC (based on side light scatter and CD4 staining: R1) and analyzed for CD25 and FOXP3 expression (CD25⁺FOXP3⁺: R2 and CD25⁻FOXP3⁺: R3). The expression of CD127 was shown as FACS histograms in R2 and R3 cells. The gating strategy was shown (<b>A</b>). The percentage of CD25⁺FOXP3⁺ and CD25⁻FOXP3⁺ cells are shown as mean ± SEM of ten independent experiments. <i>p</i> = 0.0211 (triplicate samples of 10 different Control and RA; RA GXMGal-treated <i>vs</i> untreated cells); <i>p</i> = 0.0357 (triplicate samples of 10 different Control and RA; RA MTX-treated <i>vs</i> untreated cells (<b>B</b>). The mean of fluorescence intensity (MFI) of FOXP3 in CD25⁺FOXP3⁺ and CD25⁻FOXP3⁺ cells (<b>C</b>) or magnetically purified Treg (<b>D</b>) from RA after 18 h of GXMGal or MTX treatment was shown as mean ± SEM of five independent experiments. *, <i>p</i><0.05 (triplicate samples of 5 different RA; RA GXMGal-treated <i>vs</i> untreated cells).</p