Thymosin β4 and β10 in Sjögren's syndrome: Saliva proteomics and minor salivary glands expression

Background: In the present study, we investigated whether thymosin β (Tβ) in saliva and in minor salivary glands is differentially expressed in patients with primary Sjögren's syndrome (pSS) and patients with autoimmune diseases (systemic sclerosis [SSc], systemic lupus erythematosus [SLE], and rheumatoid arthritis [RA], with and without sicca syndrome [ss]). Methods: Saliva specimens of nine patients with pSS, seven with ss/SSc, seven with ss/SLE, seven with ss/RA, seven with SSc, seven with SLE, and seven with RA, as well as ten healthy subjects, were analyzed using a high-performance liquid chromatograph coupled with a mass spectrometer equipped with an electrospray ionization source to investigate the presence and levels of Tβ4, Tβ4 sulfoxide, and Tβ10. Immunostaining for Tβ4 and Tβ10 was performed on minor salivary glands of patients with pSS and ss. Results: Tβ4 levels were statistically higher in patients with pSS with respect to the other subgroups. Tβ10 was detectable in 66.7 % of patients with pSS and in 42.8 % of those with ss/SSc, while Tβ4 sulfoxide was detectable in 44.4 % of patients with pSS and in 42.9 % of those with ss/SSc. Tβ10 and Tβ4 sulfoxide were not detectable in patients without associated ss and in healthy control subjects. Regarding thymosin immunostaining, all patients had immunoreactivity for Tβ10, and a comparable distribution pattern in the four different subgroups of patients was observed. Tβ4 immunoreactivity was present in patients with ss/SSc and those with ss/SLE, while it was completely absent in patients with pSS and those with ss/RA. Conclusions: Our data show that higher salivary Tβ expression characterizes patients with pSS, while Tβ4 sulfoxide and Tβ10 salivary expression was selectively present in patients with sicca symptoms. Moreover, at the immunohistochemical level in patients with pSS, minor salivary glands showed a peculiar pattern characterized by immunostaining for Tβ10 in acinar cells in the absence of any reactivity for Tβ4. These findings, taken together, suggest a different role for Tβ4 and Tβ10 in patients with pSS who have ss and other autoimmune disease

Citrullination: the loss of tolerance and development of autoimmunity in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and pannus formation, which can lead to severe destruction of cartilage and bone. Several self proteins have been suggested to be disease-driving autoantigens. Moreover the presence of autoantibodies to citrullinated proteins in sera of patients with RA enhances the strength of this hypothesis. Proteins are encoded by a limited number of genes in our genome. Post-translational modifications such as phosphorylation, glycosylation and citrullination can increase the morphological and the functional diversity of the proteome

JAK inhibition by methotrexate (and csDMARDs) may explain clinical efficacy as monotherapy and combination therapy

Methotrexate (MTX) is recognized as the anchor drug in the algorithm treating chronic arthritis (RA, psoriatic arthritis), as well as a steroid sparing agent in other inflammatory conditions (polymyalgia rheumatica, vasculitis, scleroderma). Its main mechanism of action has been related to the increase in extracellular adenosine, which leads to the effects of A2A receptor in M1 macrophages that dampens TNFα and IL12 production and increases IL1Ra and TNFRp75. By acting on A2B receptor on M2 macrophages it enhances IL10 synthesis and inhibits NF‐kB signaling. MTX has also been shown to exert JAK inhibition of JAK2 and JAK1 when tested in Drosophila melanogaster as a model of kinase activity and in human cell lines (nodular sclerosis Hodgkin's lymphoma and acute myeloid leukemia cell lines). These effects may explain why MTX leads to clinical effects similar to anti‐TNFα biologics in monotherapy, but is less effective when compared to anti‐IL6R in monotherapy, which acting upstream exerts major effects downstream on the JAK1‐STAT3 pathway. The MTX effects on JAK1/JAK2 inhibition also allows to understand why the combination of MTX with Leflunomide, or JAK1/JAK3 inhibitor leads to better clinical outcomes than monotherapy, while the combination with JAK1/JAK2 or JAK1 specific inhibitors does not seem to exert additive clinical benefit

Citrullination: the loss of tolerance and development of autoimmunity in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and pannus formation, which can lead to severe destruction of cartilage and bone. Several self proteins have been suggested to be disease-driving autoantigens. Moreover the presence of autoantibodies to citrullinated proteins in sera of patients with RA enhances the strength of this hypothesis. Proteins are encoded by a limited number of genes in our genome. Post-translational modifications such as phosphorylation, glycosylation and citrullination can increase the morphological and the functional diversity of the proteome

Effects of Heat Exposure on Body Water Assessed using Single-Frequency Bioelectrical Impedance Analysis and Bioimpedance Spectroscopy

International Journal of Exercise Science 10(7): 1085-1093, 2017. The purpose of this study was to determine if heat exposure alters the measures of total body water (TBW), extracellular water (ECW), and intracellular water (ICW) in both single-frequency bioelectrical impedance analysis (BIA) and bioimpedance spectroscopy (BIS). Additionally, we sought to determine if any differences exist between the BIA and BIS techniques before and after brief exposure to heat. Body water was evaluated for twenty men (age=24±4 years) in a thermoneutral environment (22°C) before (PRE) and immediately after (POST) 15 min of passive heating (35°C) in an environmental chamber. The mean difference and 95% limits of agreement at PRE demonstrated that BIS yielded significantly higher body water values than BIA (all p0.05; 0.2±1.5kg). Additionally, the ES of the mean differences at POST were trivial to small and the r-values were high (r≥0.96). When analyzing the changes in body water before and after heat exposure, POST values for BIS were significantly higher than PRE (all

Allele *2 of the HS1,2A enhancer of the Ig regulatory region associates with rheumatoid arthritis

Objective: To investigate the role of the HS1.2 enhancer polymorphisms as a new candidate marker for rheumatoid arthritis (RA) and to define the possible association with autoantibody positivity and clinical outcome. Methods: Genomic DNA was obtained from two cohorts of patients with RA (100 with early RA (ERA) and 114 with longstanding RA (LSRA)) and from 248 gender-matched controls from the same geographical area. Clinical and immunological characteristics were recorded for all the patients. Results: The percentage of the 2/2 genotype was higher In patients with ERA (27.0%), and In patients with LSRA (34.2%), than In controls (14.9%) (ERA: OR = 2.11 (95% Cl 1.20 to 3.70) vs controls; LSRA: OR = 2.96 (95% Cl 1.76 to 5.00) vs controls). A lower representation of allele *3 was present In patients with ERA (2.0%) than In controls (6.0%; OR = 0.32 (95% Cl 0.11 to 0.91)). No significant associations were found between polymorphisms and autoantibodies positivity. Conclusion: The HS1.2A allele *2 associates with early and longstanding RA

Association between the response to B cell depletion therapy and the allele*2 of the HS1,2A enhancer in seropositive rheumatoid arthritis patients

Objective. Several studies underline the relevance of the genetic background for the response to therapy. We evaluated the relationship between the polymorphism of the HS1,2A enhancer, located in the 3' regulatory region of the heavy immunoglobulin chain (IgH), and the response to B cell depletion therapy (BCDT) with Rituximab (RTX). Methods. Fifty rheumatoid arthritis (RA) patients (42 women; disease duration 13.9±10.6 years) treated with RTX, not responsive to previous DMARDs and/or TNFα inhibitors therapies, and 220 healthy subjects were enrolled in the study. Patients were genotyped for HS1,2A enhancer polymorphism, as previously described. Disease activity was assessed every three months according to the European League Against Rheumatism's (EULAR) criteria. Results. All RA patients were seropositive for at least one of the tested autoantibodies: rheumatoid factor (FR IgA, FR IgM e FR IgG), anti-cyclic citrullinated peptides (anti-CCP IgA, anti-CCP IgM e anti-CCP IgG) and anti-vimentin antibodies. RA patients had an increased frequency of the allele*2 (60.0%) of the HS1,2A enhancer compared to healthy subjects (42.0%; OR(95%ICs): 2.07 (1.33-3.22)). Patients with a good EULAR response at 6 months follow-up visit had an increased frequency of genotype 2/2 (47.1%) compared to poor-responders RA patients (genotype 2/2: 18.2%, OR(95%ICs): 4.00 (1.09-14.68)). All the patients with a good EULAR response had the allele*2, thus showing a possible association with the allele in this population. Conclusions. The presence of allele*2 seems to be related to a good response to BCDT with RTX in seropositive RA patients, thus highlighting the role of the HS1,2A enhancer in B cell maturation and class-switch recombination.Objective. Several studies underline the relevance of the genetic background for the response to therapy. We evaluated the relationship between the polymorphism of the HS1,2A enhancer, located in the 3' regulatory region of the heavy immunoglobulin chain (IgH), and the response to B cell depletion therapy (BCDT) with Rituximab (RTX). Methods. Fifty rheumatoid arthritis (RA) patients (42 women; disease duration 13.9±10.6 years) treated with RTX, not responsive to previous DMARDs and/or TNFα inhibitors therapies, and 220 healthy subjects were enrolled in the study. Patients were genotyped for HS1,2A enhancer polymorphism, as previously described. Disease activity was assessed every three months according to the European League Against Rheumatism's (EULAR) criteria. Results. All RA patients were seropositive for at least one of the tested autoantibodies: rheumatoid factor (FR IgA, FR IgM e FR IgG), anti-cyclic citrullinated peptides (anti-CCP IgA, anti-CCP IgM e anti-CCP IgG) and anti-vimentin antibodies. RA patients had an increased frequency of the allele*2 (60.0%) of the HS1,2A enhancer compared to healthy subjects (42.0%; OR(95%ICs): 2.07 (1.33-3.22)). Patients with a good EULAR response at 6 months follow-up visit had an increased frequency of genotype 2/2 (47.1%) compared to poor-responders RA patients (genotype 2/2: 18.2%, OR(95%ICs): 4.00 (1.09-14.68)). All the patients with a good EULAR response had the allele*2, thus showing a possible association with the allele in this population. Conclusions. The presence of allele*2 seems to be related to a good response to BCDT with RTX in seropositive RA patients, thus highlighting the role of the HS1,2A enhancer in B cell maturation and class-switch recombination

Characterization of inflammatory cell infiltrate of scleroderma skin: B cells and skin score progression.

Background: The purpose of this study was to investigate the frequency and the distribution of inflammatory cell infiltrate in two sets of cutaneous biopsies derived from clinically involved and uninvolved skin of patients with systemic sclerosis (SSc) and to find correlations between the cell infiltrate and the progression of skin involvement. Methods: Immunohistochemistry was carried out to identify CD68, CD3, CD20 and CD138-positive (+) cells in clinically involved and uninvolved skin of 28 SSc patients. Patients were followed for 6 months and the characteristics of the infiltrate were analyzed according to disease duration, clinical features and skin involvement progression. Results: In all SSc cutaneous specimens, cellular infiltrates were found in a perivascular location predominantly in the mid and deeper portions of the dermis. All the analysed biopsies showed a CD3+ and CD68+ cell infiltrate and the mean number of CD3+, as well as CD68+ cells, was higher in clinically involved skin (CD3+: 71.7\ub134.6 and CD68+: 26.3\ub18.4) than in clinically uninvolved skin (CD3+: 45.7\ub136.0; CD68+: 13.6\ub16.1), (p<0.001 for both comparisons). CD20+ cells were found in 17 (60.7%) patients and in these patients the mean number of CD20+ cells was higher in involved (4.7\ub15.9) than in uninvolved skin (1.9\ub12.9), (p=0.04). Early SSc had a greater number of CD20+ cells compared with patients with long-standing disease. CD138+ cells were found in 100% of involved skin biopsies and in 89.3% of uninvolved skin samples. The mean number of CD138+ cells was higher in clinically involved skin (3.6\ub12.3) than in clinically uninvolved skin (1.9\ub11.7), (p<0.001). Considering the 7 patients that experienced a worsening of skin score higher than 20% after 6-month of follow-up, all of them had presented a CD20+ skin infiltrate in the involved skin biopsy. Conclusions: Our results confirm that mononuclear cells are present in the skin of all SSc patients underlining the role of inflammatory cell infiltrates in skin involvement in SSc. B cells in the skin seem to characterize patients with early diffuse skin disease and to correlate with skin progression

Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis

In healthy joints, synovial fibroblasts (SFs) provide the microenvironment required to mediate homeostasis, but these cells adopt a pathological function in rheumatoid arthritis (RA). Carbohydrates (glycans) on cell surfaces are fundamental regulators of the interactions between stromal and immune cells, but little is known about the role of the SF glycome in joint inflammation. Here we study stromal guided pathophysiology by mapping SFs glycosylation pathways. Combining transcriptomic and glycomic analysis, we show that transformation of fibroblasts into pro-inflammatory cells is associated with glycan remodeling, a process that involves TNF-dependent inhibition of the glycosyltransferase ST6Gal1 and α2-6 sialylation. SF sialylation correlates with distinct functional subsets in murine experimental arthritis and remission stages in human RA. We propose that pro-inflammatory cytokines remodel the SF-glycome, converting the synovium into an under-sialylated and highly pro-inflammatory microenvironment. These results highlight the importance of glycosylation in stromal immunology and joint inflammation