Patients with Primary Sjögren's Syndrome Have Alterations in Absolute Quantities of Specific Peripheral Leucocyte Populations.

An accurate dissection of peripheral blood enumeration is lacking in primary Sjögren's syndrome (pSS). The purpose of this study was to quantify different leucocyte populations in peripheral blood of patients with pSS. Numbers of specific leucocyte subsets were determined in 86 pSS patients and 74 healthy donors quantifying 21 distinct subtypes by flow cytometry. Subgroups of pSS patients were stratified based on presence of extraglandular manifestations (EGMs) and SSA/SSB autoantibodies. Overall, pSS patients manifested decreased lymphocyte subpopulations compared to healthy donors. Such decreases were more pronounced in SSA/SSB positive patients and patients with EGM. Granulocyte and monocyte subpopulations were increased in pSS patients compared to healthy donors, with the greatest increases in SSA/SSB positive patients. Unsupervised hierarchal clustering based on cell quantities was used to further subgroup the pSS patients into four clusters. One of the clusters characterized by higher concentrations of NKT cells, CD56hi NK cells, CD20+ CD38- B cells and CD8+ CD38- T cells was associated with weaker clinical symptoms than the other clusters, possibly marking a milder disease phenotype. In conclusion, our analyses indicate significant alterations in the cellular profiles of peripheral blood leucocytes in patients with pSS and may help to stratify the patients according to disease severity.

Calprotectin (S100A8/A9) and S100A12 are associated with measures of disease activity in a longitudinal study of patients with rheumatoid arthritis treated with infliximab.

OBJECTIVES: The pro-inflammatory proteins calprotectin (a heterocomplex of S100A8/A9) and S100A12 have been associated with disease activity in rheumatoid arthritis (RA). The aim of this study was to compare their potential as biomarkers in a prospective study of RA patients starting with infliximab as their first biological disease-modifying anti-rheumatic drug (DMARD). METHOD: Thirty-nine RA patients were examined and serum samples collected when starting with infliximab and after 3, 6, and 12 months. Calprotectin and S100A12 were analysed by enzyme-linked immunosorbent assays (ELISAs) and, together with C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), measured at all time points. A disease activity score of 28 joints (DAS28) was calculated. Radiographs of the hands, wrists, and feet were taken at baseline and after 3 years, and assessed according to the modified Sharp/van der Heijde (SvH) score. Responsiveness was evaluated according to the European League of Associations for Rheumatology (EULAR) response criteria based on 28 joints. RESULTS: Both S100 proteins were significantly higher in seropositive than in seronegative patients (p = 0.01). Calprotectin correlated significantly with CRP (ρ = 0.51-0.75), ESR (ρ = 0.32-0.52), and DAS28 (ρ = 0.32-0.62). S100A12 correlated with calprotectin (ρ = 0.62-0.77) and CRP (ρ = 0.32-0.63). The S100 proteins, and especially calprotectin (ρ = 0.23-0.39), showed weak associations with radiographic progression, unlike CRP/ESR. None of the S100 proteins could predict responsiveness. CONCLUSIONS: Calprotectin showed the strongest correlation with measures of disease activity and may be better than S100A12 when evaluating disease activity in RA patients. More extensive studies are needed to further compare the predictive value of the S100 proteins relative to radiographic progression.

TLR-7 and -9 Stimulation of Peripheral Blood B Cells Indicate Altered TLR Signalling in Primary Sjögren's Syndrome Patients by Increased Secretion of Cytokines.

Primary Sjögren's syndrome (pSS) is a chronic, inflammatory autoimmune disease characterised by lymphocytic infiltrations in the exocrine glands, resulting in destruction of salivary and lacrimal glands. B cells have an important role in the disease, as detection of autoantibodies against SSA/Ro or SSB/La is one of the diagnostic criteria, being found in a majority of the patients. Toll-like receptors (TLR) are pattern recognition receptors. TLR-7 and -9 are found in endosomes and bind microbial nucleic acids. We have previously shown that pSS patients and healthy controls have similar expression pattern of TLR-7 and -9 in various B-cell populations. In this study we further analysed the responsiveness of B cells upon TLR stimulation. B cells isolated from peripheral blood of 21 pSS patients and 18 healthy controls were stimulated with TLR-7 and -9 ligands for 24 h before being analysed for the expression of certain surface markers and intracellular cytokine levels by flow cytometry. Secreted cytokines were measured by a multiplex cytokine assay. Patients with pSS had more naïve and less preswitched memory B cells compared to controls in unstimulated as well as via TLR-7 stimulated cells. Unstimulated and via TLR-7 stimulated B cells from pSS patients also had fewer IL-10(+) preswitched memory B cells. Moreover, TLR-7 and -9 stimulated B cells of pSS patients secreted increased amounts of several cytokines. B cells of pSS patients show a different responsiveness upon stimulation of TLR-7 and -9 compared to controls.

No association of primary Sjögren's syndrome with Fcγ receptor gene variants.

The genetic background of primary Sjögren's syndrome (pSS) is partly shared with systemic lupus erythematosus (SLE). Immunoglobulin G Fc receptors are important for clearance of immune complexes. Fcγ receptor variants and gene deletion have been found to confer SLE risk. In this study, four Fcγ receptor single-nucleotide polymorphisms (SNPs) and one copy number variation (CNV) were studied. Swedish and Norwegian pSS patients (N=527) and controls (N=528) were genotyped for the Fcγ receptor gene variant FCGR2A H131R (rs1801274) by the Illumina GoldenGate assay. FCGR3A F158V (rs396991) was analysed in 488 patients and 485 controls, FCGR3B rs447536 was analysed in 471 patients and 467 controls, and FCGR3B rs448740 was analysed in 478 cases and 455 controls, using TaqMan SNP genotyping assays. FCGR3B CNV was analysed in 124 patients and 139 controls using a TaqMan copy number assay. None of the SNPs showed any association with pSS. Also, no FCGR3B CNV association was detected. The lack of association of pSS with Fcγ receptor gene variants indicates that defective immune complex clearance may not be as important in pSS pathogenesis as in SLE, and may point to important differences between SLE and pSS.

Autoantigen-specific memory B cells in primary Sjögren's syndrome.

Sjögren's syndrome (SS) is a systemic rheumatic autoimmune disease affecting the exocrine glandular function and is characterized by the presence of autoantibodies against the ribonucleoprotein particles, SS-A/Ro and SS-B/La, and mononuclear cell infiltration of exocrine tissues. Our aim is to characterize memory B cell pattern and function in relation to the progression of the disease, by analysing samples from a well-defined cohort of patients with primary SS. We have measured the number of Ro/La-specific plasma cells in peripheral blood mononuclear cells (PBMC) from 23 patients and 20 healthy controls by direct enzyme-linked immunospot (ELISPOT) assay. Furthermore, we quantified the Ro- and La-specific memory B cells in these individuals by a 6-day in vitro polyclonal stimulation of PBMC followed by an antigen-specific ELISPOT assay for the detection of memory B cells. In addition to this, ELISA profiling of autoantibodies was carried out using patients' plasma and supernatant, collected post-mitogen stimulation of PBMC. The average Ro60-, Ro52- and La48-specific plasma cells in PB was 9, 17 and 13 cells in 10(5) PBMC, respectively. After in vitro stimulation, these numbers increased to 43, 50 and 26 for Ro60, Ro52 and La48, correspondingly. However, the fraction of memory B cells activated into antibody-secreting cells was lower than the overall IgG B cell population. We conclude that these lower Ro/La-specific memory B cell levels may indicate that a greater portion of the Ro- and La-specific B cells are in an activated stage. This is in tune with previous reports.

Association of EBF1, FAM167A(C8orf13)-BLK and TNFSF4 gene variants with primary Sjögren's syndrome.

We performed a candidate gene association study in 540 patients with primary Sjögren's Syndrome (SS) from Sweden (n=344) and Norway (n=196) and 532 controls (n=319 Swedish, n=213 Norwegian). A total of 1139 single-nucleotide polymorphisms (SNPs) in 84 genes were analyzed. In the meta-analysis of the Swedish and Norwegian cohorts, we found high signals for association between primary SS and SNPs in three gene loci, not previously associated with primary SS. These are the early B-cell factor 1 (EBF1) gene, P=9.9 × 10(-5), OR 1.68, the family with sequence similarity 167 member A-B-lymphoid tyrosine kinase (FAM167A-BLK) locus, P=4.7 × 10(-4), OR 1.37 and the tumor necrosis factor superfamily (TNFSF4=Ox40L) gene, P=7.4 × 10(-4), OR 1.34. We also confirmed the association between primary SS and the IRF5/TNPO3 locus and the STAT4 gene. We found no association between the SNPs in these five genes and the presence of anti-SSA/anti-SSB antibodies. EBF1, BLK and TNFSF4 are all involved in B-cell differentiation and activation, and we conclude that polymorphisms in several susceptibility genes in the immune system contribute to the pathogenesis of primary SS.

Phenotypic diversity of peripheral blood plasma cells in primary Sjögren's syndrome.

Production of autoantibodies is one of the main features of primary Sjögren's syndrome (pSS). Long-lived plasma cells (PC) can produce autoantibodies for prolonged period of times without being affected by immunosuppressive therapies. As of today, little is known about the long-lived PC subset and their contribution to autoimmunity. We have characterized the phenotypic and migratory properties of peripheral blood PC isolated from pSS patients (grouped by focus score, FS) and compared them to PC from rheumatoid arthritis (RA) patients and normal non-autoimmune subjects. We observed two populations of PC in all study groups, CD19+ PC and CD19- PC. Interestingly, the CD19- PC subset was most prominent in autoimmune patients (pSS and RA) compared to normal controls. Further investigation of the PC phenotype revealed that a high percentage of both CD19+ and CD19- PC isolated from pSS and RA patients did not express the CD27 marker, which is normally highly expressed on all types of PC. Differences in the expression of markers such as IgM, IgG, CD95 and CXCR3 in the group with high FS compared to FS = 1, underscore the heterogeneity of pSS patient group and demonstrate that phenotypic pattern of circulating PC associates with the severity of inflammation in the salivary glands of these patients. Our migration experiments show that addition of CXCL12 to PC in vitro, do not alter the migration potential of PC in any group tested. However, we observed an overall higher spontaneous migration of PC from pSS compared to both RA and normal controls.

The point prevalence of clinically relevant primary Sjögren's syndrome in two Norwegian counties.

OBJECTIVE: Primary Sjögren's syndrome (PSS) is a chronic autoimmune inflammatory disease characterized by exocrine gland inflammation producing clinical symptoms such as dryness of the mouth and eyes. The reported prevalence of PSS is variable, probably because of different classification criteria used and selection bias. The aim of this study was to determine the prevalence of PSS in a well-defined Norwegian Caucasian population using the revised American-European Consensus Group (AECG) criteria. METHODS: Three hospitals and three private rheumatology practices provide all of the rheumatology services to the local population in Hordaland and Rogaland counties, which included 852 342 Caucasian inhabitants as of 1 January 2009. Patients on file fulfilling the new revised AECG criteria for PSS were included, and patients with incomplete data were invited to a screening visit. RESULTS: A total of 424 PSS patients were identified. Their mean age was 61.6 ± 13.2 years; 28 (7%) were men and 396 (93%) were women. The point estimate for the proportion of PSS was 0.050% [95% confidence interval (CI) 0.048-0.052]. CONCLUSION: The prevalence of PSS in this Norwegian population of Caucasians is lower than previously reported when less stringent criteria for identifying PSS were used, but is in line with more recent studies using the same criteria and methods as in this study.

Additive effects of the major risk alleles of IRF5 and STAT4 in primary Sjögren's syndrome.

Primary Sjögren's syndrome (SS) shares many features with systemic lupus erythematosus (SLE). Here we investigated the association of the three major polymorphisms in IRF5 and STAT4 found to be associated with SLE, in patients from Sweden and Norway with primary SS. These polymorphisms are a 5-bp CGGGG indel in the promoter of IRF5, the single nucleotide polymorphism (SNP) rs10488631 downstream of IRF5 and the STAT4 SNP rs7582694, which tags the major risk haplotype of STAT4. We observed strong signals for association between all three polymorphisms and primary SS, with odds ratios (ORs) >1.4 and P-values <0.01. We also found a strong additive effect of the three risk alleles of IRF5 and STAT4 with an overall significance between the number of risk alleles and primary SS of P=2.5 x 10(-9). The OR for primary SS increased in an additive manner, with an average increase in OR of 1.78. For carriers of two risk alleles, the OR for primary SS is 1.43, whereas carriers of five risk alleles have an OR of 6.78. IRF5 and STAT4 are components of the type I IFN system, and our findings emphasize the importance of this system in the etiopathogenesis of primary SS.

Exocrine function in primary Sjögren syndrome: natural course and prognostic factors.

OBJECTIVES: Few studies have addressed the natural course of, or prognostic factors for the salivary and lacrimal function in primary Sjögren syndrome (SS). Except for the early stages, glandular function has been seemingly stable, and SS A antigen (SSA) seropositivity and hypocomplementemia may predict a decline in the van Bijsterveld score. The aim of the present study was to assess the natural course of the exocrine function in a larger cohort based on the American-European consensus criteria for SS, and to address possible predictive factors for a declining exocrine function. METHODS: We performed a retrospective cohort study. A total of 141 patients were investigated with the Schirmer I test and unstimulated whole saliva (UWS). Historical data regarding these tests and focus score were collected from the files of 111 patients. Median time from diagnosis to follow-up investigation was 5.0 years. RESULTS: Median UWS was unchanged during follow-up. Median Schirmer I test improved from 5.0 to 7.0 mm/5 min (p<0.05). Present Schirmer I test was associated with historical high IgG and IgA, positive SSA and SS B antigen (SSB) tests and high focus score, and present UWS with historical low C3/C4. Logistic regression identified high focus scores (odds ratio (OR) = 1.343), and low UWS (OR = 0.692) as factors predicting a 30% or more worsening of the Schirmer I test. High focus scores (OR = 1.488) predicted a 30% or more worsening of the UWS. CONCLUSION: We confirmed previous studies showing a stable or slightly improved exocrine function over time. High focus scores and low UWS were identified as independent predictors of a worsened exocrine function.

The complex role of vitamin D in autoimmune diseases.

Vitamin D, besides having well-known control functions of calcium and phosphorus metabolism, bone formation and mineralization, also has a role in the maintenance of immune-homeostasis. The immune-regulatory role of vitamin D affects both the innate and adaptive immune system contributing to the immune-tolerance of self-structures. Impaired vitamin D supply/regulation, amongst other factors, leads to the development of autoimmune processes in animal models of various autoimmune diseases. The administration of vitamin D in these animals leads to improvement of immune-mediated symptoms. Moreover, in human autoimmune diseases, such as multiple sclerosis, or rheumatoid arthritis the pathogenic role of vitamin D has been described. The review aims at describing the complex immune-regulatory role of vitamin D from the cellular level through autoimmune animal models and depicting the known contribution of vitamin D in the pathogenesis of human autoimmune diseases.

Synovial inflammation in active rheumatoid arthritis and psoriatic arthritis facilitates trapping of a variety of oral bacterial DNAs.

OBJECTIVE: To investigate the presence of oral bacterial DNAs in serum and synovial fluid (SF) of patients with active rheumatoid arthritis (RA) and psoriatic arthritis (PsA). METHODS: Serum and SF samples from 16 RA patients, 14 PsA patients, and 9 osteoarthritis (controls) patients were extracted for oral bacterial DNA. This was used in a checkerboard DNA-DNA-hybridization set up, to identify 40 different bacteria. RESULTS: Mean number +/- standard deviation (SD) of oral bacterial species in sera were 6.2 (3.2) in the RA group (p = 0.004) and 5.4 (2.7) in the PsA group (p = 0.009) compared to 2.1 (1.7) in the controls. Periodontitis associated species Porphyromonas gingivalis and Prevotella nigrescens were exclusively detected in RA and PsA. Mean number (+/- SD) of oral bacterial species in SF were 14.0 (6.8) in the RA (p = 0.001) and 19.4 (7.1) in the PsA group (p < 0.001) compared to 4.0 (1.7) in controls. P. gingivalis, Tannerella forsythensis and Prevotella intermedia were exclusively identified in RA and PsA SF. Higher means of DNAs were found in RA SF compared to RA serum (p < 0.001), and in PsA SF compared to PsA serum (p < 0.001). Higher concentrations of bacterial DNAs were found in RA and PsA compared to controls. CONCLUSION: Higher variety and concentrations of oral bacterial DNAs were found in SF compared to serum of RA and PsA patients. These findings indicate that synovial inflammation in RA and PsA may favor trapping of oral bacterial DNAs, suggesting a perpetuating effect of oral pathogens in joint disease.

Immunohistochemical characterization of the cellular infiltrates in Sjögren's syndrome, rheumatoid arthritis and osteoarthritis with special reference to calprotectin-producing cells.

The aim of this study was to analyse the nature of infiltrating cells in minor salivary glands of patients with Sjögren's syndrome (SS). Furthermore, we wanted to characterize the tissue distribution of calprotectin-producing cells in inflamed salivary gland tissue of SS and in synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Cryostat sections of labial salivary gland tissue from patients with SS and synovial tissue from RA and OA patients were stained (ABC-immunoperoxidase technique) using monoclonal antibodies (MoAbs) to T cells (CD3), monocytes/macrophages (CD14, CD68), and calprotectin. Monocytes and macrophages were widely distributed in focal infiltrates of salivary gland tissue from SS patients. Calprotectin markers showed a distinct staining of infiltrating macrophages and around blood vessel walls. In synovial tissue samples, calprotectin was expressed in a high percentage of cells in the synovial lining, the subsynovium, and vessel walls. The percentages of cells stained for calprotectin were significantly higher in RA than in OA and SS tissues. Antibodies to the calprotectin complex stained cells with a similar distribution as antibodies against the separate polypeptide chains of calprotectin. The localization and differentiated expression of calprotectin in these chronic inflammatory conditions indicate a role in the inflammatory process and may be an additional marker of macrophages/granulocytes in SS, RA and OA.

Effects of calprotectin in avridine-induced arthritis.

Plasma levels of calprotectin correlate with disease activity and clinical assessments of arthritis in various rheumatic diseases, and high levels have been demonstrated in the synovial fluid of patients with rheumatoid arthritis. However, the role of calprotectin in rheumatic inflammation is unclear. The purpose of the present study was to investigate potential intra-articular effects of calprotectin. Calprotectin was injected into joints of healthy male Lewis rats and into joints of rats in the latency period before onset of avridine-induced arthritis. In addition, a group of animals had IgG antibodies to rat calprotectin injected into joints before onset of avridine-induced arthritis. Injection of 0.2 or 10 micrograms calprotectin into the ankles of healthy male Lewis rats resulted in histologically minor and reversible inflammatory changes, but without any circulating antibodies to calprotectin. Furthermore, animals with 40 micrograms calprotectin injected into ankles before the expected onset of avridine-induced arthritis had lower scores for cellular infiltration than were seen in control joints. This difference did not quite reach statistical significance in the two-sided test used. However, the induced arthritis increased in joints injected with IgG antibodies to calprotectin. These findings may indicate that increased local concentrations of calprotectin are partially protective against avridine-induced arthritis. In contrast, reduced local concentrations appear to exacerbate the severity of arthritis. Calprotectin may thus be involved in the regulation of inflammatory processes in joints.

Use of a sicca symptoms questionnaire for the identification of patients with Sjögren's syndrome in a heterogeneous hospital population with various rheumatic diseases.

OBJECTIVE: A six-item questionnaire regarding sicca symptoms recently validated for primary Sjögren's syndrome (SS) was tested on 154 in-patients with a wide range of inflammatory rheumatic diseases. Patients with one or more positive responses underwent objective ocular and oral diagnostic procedures. Of 27 patients thus investigated, 19 could be classified as having SS. RESULTS: The positive answers obtained were mainly in response to 4 of the 6 questions: dry eyes, sensation of sand or gravel in the eyes, dry mouth, and drinking of liquids to aid in swallowing dry foods. Among the 19 patients found with SS, most had had earlier diagnoses of various connective tissue diseases (rheumatoid arthritis included) and most were female. CONCLUSION: In conclusion, this study indicates that the sicca symptom questionnaire may be useful when deciding which patients with inflammatory rheumatic diseases should be subjected to special investigations with regard to SS.

[An original method of making a purse for circular mechanical anastomoses]. / Un procédé original de confection de bourse pour la fabrication d'anastomoses mécaniques circulaires.

A simple and reliable procedure applicable to all segments of the digestive tract, except the oesophagus, is described. A series of superficial U-shaped stitches is inserted along the upper edge of a Kocher's forceps that clamps the entire width of the intestine. The intestine is then cut with a scalpel along the lower edge of the forceps, i.e. on the side opposite to the sutures. After the forceps is released, a purse about 2 mm distant from the intestinal opening is obtained. When the purse is tightened the mucosa, crushed by clamping, does not protrude and therefore does not interfere with the mechanical anastomosis.

[Associated compression of the celiac trunk, superior mesenteric artery and aorta by the arcuate ligament of the diaphragm]. / Compression associée du tronc coeliaque, de l'artère mésentérique supérieure et de l'aorte par le ligament arqué du diaphragme.

Simultaneous compression of the coeliac axis, superior mesenteric artery and aorta by the arcuate ligament of the diaphragm is a very rare condition. The pathogenesis of the abdominal pain observed is debatable, but the simplicity and effectiveness of vascular release fully justify surgery in such cases.

[Parathyroid adenoma disclosed by a massive subcapsular hemorrhage]. / Adénome parathyroïdien révélé par une hémorragie sous-capsulaire massive.

In the case reported here a parathyroid gland adenoma was revealed by the sudden occurrence of a large subcapsular haematoma compressing cervical structures and causing recurrent nerve paralysis; spontaneously regressive hypercalcaemia was present. The place of these haemorrhages among other manifestations of a hitherto silent parathyroid gland adenoma is discussed, together with the diagnostic value of thyroid gland scintigraphy and cervical ultrasonography. In view of their potentially somber prognosis, emergency surgery is mandatory in such cases.

[Colectomy by combined resection-anastomosis using linear mechanical sutures]. / Colectomies par résection-anastomose intégrée pièce en place utilisant les sutures mécaniques linéaires.

In the technique described, anastomosis is started with a GIA mechanical stapler then completed with a TA stapler followed by resection of the segment involved. We prefer this technique to other mechanical procedures (triangulation, circular anastomosis) in all colectomies which do not require lower colorectal anastomosis. It is simple, economical and solves problems of incongruence. Finally--and this is the most important point in surgery of the colon--it considerably shortens the septic operative stages.