Objective
To develop and validate an international set of classification criteria for primary Sjögren's syndrome (SS) using guidelines from the American College of Rheumatology (ACR) and the European ...League Against Rheumatism (EULAR). These criteria were developed for use in individuals with signs and/or symptoms suggestive of SS.
Methods
We assigned preliminary importance weights to a consensus list of candidate criteria items, using multi‐criteria decision analysis. We tested and adapted the resulting draft criteria using existing cohort data on primary SS cases and non‐SS controls, with case/non‐case status derived from expert clinical judgment. We then validated the performance of the classification criteria in a separate cohort of patients.
Results
The final classification criteria are based on the weighted sum of 5 items: anti‐SSA/Ro antibody positivity and focal lymphocytic sialadenitis with a focus score of ≥1 foci/4 mm2, each scoring 3; an abnormal ocular staining score of ≥5 (or van Bijsterveld score of ≥4), a Schirmer's test result of ≤5 mm/5 minutes, and an unstimulated salivary flow rate of ≤0.1 ml/minute, each scoring 1. Individuals with signs and/or symptoms suggestive of SS who have a total score of ≥4 for the above items meet the criteria for primary SS. Sensitivity and specificity against clinician‐expert–derived case/non‐case status in the final validation cohort were high, i.e., 96% (95% confidence interval 95% CI 92–98%) and 95% (95% CI 92–97%), respectively.
Conclusion
Using methodology consistent with other recent ACR/EULAR‐approved classification criteria, we developed a single set of data‐driven consensus classification criteria for primary SS, which performed well in validation analyses and are well‐suited as criteria for enrollment in clinical trials.
In this study, we presented clear evidence of the increased expression of citrullinated inter‐alpha‐trypsin inhibitor heavy chain 4 (cit‐ITIH4) both in joints from peptide glucose‐6‐phosphate ...isomerase‐induced arthritis (pGIA) mice and rheumatoid arthritis patients. Neutrophils with PAD4 in joints might contribute to this citrullination, which was shown by decreases in ITIH4 citrullination upon depletion of neutrophils by anti‐Ly6G antibodies in pGIA mice. In addition, we demonstrated that the citrullination of ITIH4 enhanced the migration of human neutrophils, supporting an association between cit‐ITIH4 and the generation of arthritis.
Summary
The citrullinated inter‐alpha‐trypsin inhibitor heavy chain 4 (cit‐ITIH4) was identified as its blood level was associated with the arthritis score in peptide glucose‐6‐phosphate‐isomerase‐induced arthritis (pGIA) mice and the disease activity in patients with rheumatoid arthritis (RA). This study aimed to clarify its citrullination pathway and function as related to neutrophils. In pGIA‐afflicted joints, ITIH4 and cit‐ITIH4 levels were examined by immunohistochemistry (IHC), immunoprecipitation (IP) and Western blotting (WB), while peptidylarginine deiminase (PAD) expression was measured by reverse transcription–quantitative polymerase chain reaction (RT–qPCR), IHC and immunofluorescent methods. The pGIA mice received anti‐lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies to deplete neutrophils and the expression of cit‐ITIH4 was investigated by WB. The amounts of ITIH4 and cit‐ITIH4 in synovial fluid (SF) from RA and osteoarthritis (OA) patients were examined by I.P. and W.B. Recombinant ITIH4 and cit‐ITIH4 were incubated with sera from healthy volunteers before its chemotactic ability and C5a level were evaluated using Boyden’s chamber assay and enzyme‐linked immunosorbent assay (ELISA). During peak arthritic phase, ITIH4 and cit‐ITIH4 were increased in joints while PAD4 was over‐expressed, especially in the infiltrating neutrophils of pGIA mice. Levels of cit‐ITIH4 in plasma and joints significantly decreased upon neutrophil depletion. ITIH4 was specifically citrullinated in SF from RA patients compared with OA patients. Native ITIH4 inhibited neutrophilic migration and decreased C5a levels, while cit‐ITIH4 increased its migration and C5a levels significantly. Cit‐ITIH4 is generated mainly in inflamed joints by neutrophils via PAD4. Citrullination of ITIH4 may change its function to up‐regulate neutrophilic migration by activating the complement cascade, exacerbating arthritis.
We search for the dark photon dark matter (DPDM) using a cryogenic millimeter-wave receiver. DPDM has a kinetic coupling with electromagnetic fields with a coupling constant of χ and is converted ...into ordinary photons at the surface of a metal plate. We search for signal of this conversion in the frequency range 18-26.5 GHz, which corresponds to the mass range 74-110 μeV/c^{2}. We observed no significant signal excess, allowing us to set an upper bound of χ<(0.3-2.0)×10^{-10} at 95% confidence level. This is the most stringent constraint to date and tighter than cosmological constraints. Improvements from previous studies are obtained by employing a cryogenic optical path and a fast spectrometer.
Summary
Humanized non‐obese diabetic/severe combined immunodeficiency/interleukin‐2 receptor‐γ‐null (NOD/SCID/IL2rγnull) humanized (huNSG) mice engrafted with human hematopoietic cells have been used ...for investigations of the human immune system. However, the epigenetic features of the human regulatory T (Treg) cells of huNSG mice have not been studied. The objective of this study was to clarify the characteristics of human Treg cells in huNSG mice, especially in terms of the epigenetic aspects. We compared the populations, inhibitory molecule expression and suppressive capacity of human Treg cells in spleens harvested from the huNSG mice 120 days after the engraftment of human umbilical cord blood CD34+ cells with human peripheral blood mononuclear cells (PBMCs). Histone modifications and enhancer of zeste homolog 2 (Ezh2), an H3K27 methyltransferase, of human Treg cells were quantified in huNSG mice and human PBMCs. The effect of Ezh2 inhibitor on human Treg cells exposed to interleukin (IL)‐6 was also compared between them. Human Treg cells in the spleens of huNSG mice showed an increased proportion among CD4+ T cells, higher expressions of forkhead box protein 3 (FoxP3), cytotoxic T lymphocyte antigen 4 (CTLA‐4) and glucocorticoid‐induced tumor necrosis factor‐related protein (GITR), a higher production of IL‐10 and enhanced suppressive capacity when compared with those in human PBMCs. H3K27me3 and Ezh2 were specifically up‐regulated in human Treg cells of huNSG mice in comparison with those of human PBMCs. The decrease in Treg cells induced by IL‐6 exposure was attenuated in huNSG mice when compared with human PBMCs, while the difference between them was cancelled by addition of Ezh2 inhibitor. In conclusion, huNSG mice exhibit functionally augmented human Treg cells owing to enzymatic up‐regulation of H3K27me3.
Humanized NOD/SCID/IL2rγnull (huNSG) mice engrafted with human hematopoietic cells exhibited functionally augmented human Treg cells. The higher Treg cell stability in huNSG mice depended on enhanced enhancer of zeste homolog 2 (Ezh2)‐mediated trimethylated histone H3 at lysine 27 (H3K27me3) modification in Treg cells. This is the first report to show the epigenetic features of human Treg cells in huNSG mice.
Summary
To examine genes expressed specifically in labial salivary glands (LSGs) of patients with Sjögren's syndrome (SS) in comparison with those of patients with immunoglobulin (Ig)G4‐related ...disease (IgG4‐RD), and to identify the genes involved in the pathogenesis of SS. Gene expression in LSGs of SS patients, IgG4‐RD patients and healthy controls (HC) was analysed by cDNA microarray. Quantitative polymerase chain reaction (qPCR) was used to validate the up‐regulation of differentially expressed genes (DEGs) in SS. Protein production of the validated gene in LSGs was examined by immunofluorescence (IF) assay. The association of molecular functions of the gene with the pathological conditions in SS was examined using peripheral blood lymphocytes. Among 1320 DEGs up‐regulated in SS, qPCR confirmed the up‐regulation of NR4A2 in LSGs of SS compared with IgG4‐RD. IF staining showed higher production of NR4A2 in nuclei of CD4+ T cells and interleukin (IL)‐17‐producing cells in LSGs of SS, compared with IgG4‐RD. Over‐expression of NR4A2 mRNA was observed in peripheral CD4+ T cells of SS patients, compared with HC. Nuclear NR4A2 expression in T helper type 17 (Th17)‐polarized CD4+ T cells determined by cellular IF was significantly higher in SS than in HC. Importazole, an inhibitor of importin‐β, inhibited nuclear transport of NR4A2 and Th17 polarization along with IL‐21 expression in naive CD4+ T cells under Th17‐polarizing conditions, but did not alter retinoic acid receptor‐related orphan receptor C (RORC) expression. NR4A2 seems to promote Th17 polarization via increased expression and intranuclear localization in CD4+ T cells of SS patients, which could play a critical role in the pathogenesis of SS.
Increased expression and nuclear localization of NR4A2 in CD4+ T cells could be involved in the pathogenesis of Sjögren's syndrome (SS) via enhancing Th17 polarization in patients with SS.
Summary Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with poor prognosis and high mortality. However, the pathogenesis of IP remains to be elucidated. The ...aim of this study was to clarify the role of pulmonary gammadeltaT cells in IP. In wild-type (WT) mice exposed to bleomycin, pulmonary gammadeltaT cells were expanded and produced large amounts of interferon (IFN)-gamma and interleukin (IL)-17A. Histological and biochemical analyses showed that bleomycin-induced IP was more severe in T cell receptor (TCR-delta-deficient (TCRdelta-/-) mice than WT mice. In TCRdelta-/- mice, pulmonary IL-17A+CD4+ Τ cells expanded at days 7 and 14 after bleomycin exposure. In TCRdelta-/- mice infused with gammadeltaT cells from WT mice, the number of pulmonary IL-17A+ CD4+ T cells was lower than in TCRdelta-/- mice. The examination of IL-17A-/- TCRdelta-/- mice indicated that gammadeltaT cells suppressed pulmonary fibrosis through the suppression of IL-17A+CD4+ T cells. The differentiation of T helper (Th)17 cells was determined in vitro, and CD4+ cells isolated from TCRdelta-/- mice showed normal differentiation of Th17 cells compared with WT mice. Th17 cell differentiation was suppressed in the presence of IFN-gamma producing gammadeltaT cells in vitro. Pulmonary fibrosis was attenuated by IFN-gamma-producing gammadeltaT cells through the suppression of pulmonary IL-17A+CD4+ T cells. These results suggested that pulmonary gammadeltaT cells seem to play a regulatory role in the development of bleomycin-induced IP mouse model via the suppression of IL-17A production.
Summary
Tumour necrosis factor alpha (TNF)‐α‐induced adipose‐related protein (TIARP) is a negative regulator of inflammation in arthritis model mice. In humans, six‐transmembrane epithelial antigen ...of prostate 4 (STEAP4) (human counterpart of TIARP) is also expressed in CD14+ monocytes from patients with rheumatoid arthritis (RA). Recently, highly levels of exon 3‐spliced variant STEAP4 (v‐STEAP4) expression have been observed in porcine lung. The aim of this study is to elucidate the expression and functional role of v‐STEAP4, comparing it with that of STEAP4, in the pathogenesis of arthritis. We identified v‐STEAP4 in CD14+ cells. The expression of STEAP4 and v‐STEAP4 was higher in patients with RA than in healthy participants. We also found that STEAP4 and v‐STEAP4 were correlated positively with C‐reactive protein and that their expression was decreased after treatment with an interleukin (IL)‐6 antagonist in patients with RA. To investigate further the role of STEAP4 and v‐STEAP4, we produced STEAP4 and v‐STEAP4 over‐expressing human monocytic cell lines (THP‐1) for functional analysis. In the v‐STEAP4 over‐expressing cells, the production of IL‐6 was suppressed significantly, but TNF‐α was increased significantly through lipopolysaccharide (LPS) stimulation. Immunoblot analysis revealed that phosphorylated (p‐)nuclear factor kappa B (NF‐κB) was increased after LPS stimulation and degradation of nuclear factor kappa B inhibitor alpha (IκBα) was sustained, whereas p‐signal transducer and activator of transcription 3 (STAT‐3) was decreased with v‐STEAP4. We identified specific up‐regulation of v‐STEAP4 in RA monocytes. V‐STEAP4 might play a crucial role in the production of TNF‐α and IL‐6 through NF‐κB and STAT‐3 pathways, resulting in the generation of RA.
Exon3‐spliced variant STEAP4 might play a crucial role in the production of TNFα and IL‐6 through NF‐κB and STAT3 pathways, resulting in the generation of RA.
M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory ...autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca²⁺ concentrations (Ca²⁺)i were measured. Antibodies to the N-terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca²⁺)i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca²⁺)i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes.
Background and purpose:
Rheumatoid arthritis (RA) is an autoimmune disorder involving subsets of activated T cells, in particular T helper (Th) 1 and Th17 cells, which infiltrate and damage tissues ...and induce inflammation. Prostaglandin E
2
(PGE
2
) enhances the Th17 response, exacerbates collagen‐induced arthritis (CIA) and promotes inflammatory pain. The current study investigated whether selective antagonism of the PGE
2
EP
4
receptor would suppress Th1/Th17 cell development and inflammatory arthritis in animal models of RA.
Experimental approach:
Effects of PGE
2
and a novel EP
4
receptor antagonist ER‐819762 on Th1 differentiation, interleukin‐23 (IL‐23) production by dendritic cells (DCs), and Th17 development were assessed
in vitro
. The effect of ER‐819762 was evaluated in CIA and glucose‐6‐phosphate isomerase (GPI)‐induced arthritis models. In addition, the effects of ER‐819762 on pain were evaluated in a model of chronic inflammatory pain induced by complete Freund's adjuvant (CFA) in the rat.
Key results:
Stimulation of the EP
4
receptor enhanced Th1 differentiation via phosphatidylinositol 3 kinase signalling, selectively promoted Th17 cell expansion, and induced IL‐23 secretion by activated DCs, effects suppressed by ER‐819762 or anti‐PGE
2
antibody. Oral administration of ER‐19762 suppressed Th1 and Th17 cytokine production, suppressed disease in collagen‐ and GPI‐induced arthritis in mice, and suppressed CFA‐induced inflammatory pain in rats.
Conclusion and implications:
PGE
2
stimulates EP
4
receptors to promote Th1 differentiation and Th17 expansion and is critically involved in development of arthritis in two animal models. Selective suppression of EP
4
receptor signalling may have therapeutic value in RA both by modifying inflammatory arthritis and by relieving pain.
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