Uric acid increases IL-1ß secretion and Caspase-1 activation in PBMCs of Behçet's disease patients: The in vitro immunomodulatory effect of xanthine oxidase inhibitor Allopurinol.

Behçet's disease (BD) is a multisystem disease, which shares some features with other diseases belonging to the autoinflammatory disorders panel. Recent studies have postulated that IL-1ß/Caspase-1 may play a cardinal role in autoinflammatory diseases. In this study, we aimed to (i) elucidate the mechanism underlying the involvement of xanthine oxidase (XO) and Uric Acid (UA) in BD (ii) study the direct effects of UA and XO inhibitor "Allopurinol" on nitric oxide (NO) and caspase-1-mediated IL-1ß release in peripheral blood mononuclear cells (PBMCs) of BD patients. In this context, plasma of BD patients and healthy controls (HC) were used to measure XO activity, UA, advanced oxidized proteins products (AOPP) and NO levels. In Addition, PBMCs of BD patients and HC were treated or not with either UA or Allopurinol. Then we quantified NO and IL-1ß levels, and Caspase-1 Activity in the supernatants and lysates of PBMCs, respectively. We showed that plasma levels of XO activity, UA, AOPP and NO are significantly increased in BD patients compared to those of HC. Interestingly, a significant positive correlation between XO and UA was observed in BD patients. Additionally, while UA has markedly increased NO, IL-1ß, and Caspase-1 activity levels in PBMCs of BD patients, Allopurinol has exerted an immunomodulatory effect resulting in reduced NO, IL-1ß and Caspase-1 levels in PBMCs of BD patients particularly during the active stages. Collectively, our results indicate a potential clinical use of XO as a tool for assessing BD activity, and suggest that the in-vitro immunomodulatory effect of Allopurinol may have a promising therapeutic value in BD management.

Increased IL-1ß levels are associated with an imbalance of "oxidant/antioxidant" status during Behçet's disease.

Behçet's disease is a multisystem disease. It stands at the crossroad between the autoimmunity and auto-inflammatory disorders. In this study, we sought to address a relationship that might exist between interleukin-1ß (IL-1ß) and the oxidants/antioxidants markers in Behçet's patients. Behçet's disease patients (n = 78: active stage, n = 28; inactive stage, n = 50) and 41 healthy controls have been included in our study. In this context, we investigated the plasma levels of IL-1ß and the nitrosative/oxidative markers: nitric oxide (NO), advanced oxidative protein products (AOPP) and fatty acids peroxidation-malondialdehyde (MDA). The antioxidant system was assessed by measuring the plasma level of superoxide dismutase (SOD) activity. The Mann-Whitney's U and Pearson's correlation tests were used for statistical analyses. Our case-control study showed that patients in active stage displayed higher plasma levels of IL-1ß, NO, AOPP and MDA versus healthy controls and patients in inactive stage. Patients in active stage showed significantly lower SOD levels related to patients in inactive stage and healthy controls respectively, whereas patients in inactive stage showed statistically insignificant SOD level versus healthy controls. Correlation studies showed a significant positive correlation between IL-1ß and AOPP, IL-1ß and NO, and negative correlation between IL-1ß and SOD among Behçet's disease patients. In addition, we showed positive correlation between AOPP and NO, AOPP and MDA and negative correlation between NO and SOD, AOPP and SOD in Behçet's disease patients. Interestingly, our study revealed that IL-1ß levels increased and correlated with an imbalance of oxidants/antioxidants system, especially during active stage of Behçet disease. Collectively, our study indicates a possible link between IL-1ß production and nitrosative/oxidative markers during Behçet's disease. Exploiting this relationship might provide valuable outputs in the follow-up and prognosis of Behçet's disease with a potential therapeutic value.

Interferon-ß inhibits inflammatory responses mediators via suppression of iNOS signaling pathway in PBMCs from patients with primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) represents a chronic, systemic autoimmune disorder, characterized by lymphocytic infiltration of exocrine glands, inducing compromised secretory function and tissue destruction. Increasing evidence had revealed that inflammatory mediators, such as nitric oxide (NO) and pro-inflammatory cytokines, are critical in the development and perpetuation of pSS systemic manifestations. In our current study, we aimed to investigate the ex vivo immunomodulatory effect of interferon (IFN)-ß on iNOS expression, as well as on pro-inflammatory (tumor necrosis factor (TNF)-α, interleukin (IL)-6) and immunoregulatory (IL-10) cytokine production. Furthermore, we examined potential associations between the influence of IFN-ß treatment on NO production, and pSS clinical and serological manifestations. METHODS: In 41 pSS patients documented for their clinical and serological features, NO and cytokines levels were measured by the Griess method and enzyme-linked immunosorbent assay, respectively. Inducible nitric oxide synthase expression was analyzed by fluorescence immunostaining assay, using peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and pSS patients. RESULTS: Our results revealed a strong down-modulating effect of IFN-ß in the secretion of pro-inflammatory mediators including TNF-α, IL-6, and NO production. Interestingly, IFN-ß exerts an increase in IL-10 levels. The most suppressive effect exerted by IFN-ß on NO production was importantly reported for patients with neurological manifestation. This immunomodulatory effect of IFN-ß on NO production is highly related to the decrease of inducible nitric oxide synthase (iNOS) expression. CONCLUSION: Our findings highlight a consistent ex vivo inhibitory effect of IFN-ß on pro-inflammatory cytokine production and NO pathway in pSS patients. Our data suggest that IFN-ß could represent a potential candidate for targeting inflammation during pSS.

Cardamonin inhibits pro-inflammatory cytokine production and suppresses NO pathway in PBMCs from patients with primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disorder with a complex pathophysiology primarily affecting exocrine glands, leading to compromised secretory function. Recent studies imply that many inflammatory mediators, such as pro-inflammatory cytokines and nitric oxide, are critical in the development and perpetuation of pSS systemic manifestations. In the current study, we aimed to investigate the ex vivo immunomodulatory effect of cardamonin (C16H14O4), on pro-inflammatory cytokines, TNF-α, IL-6 and inducible nitric oxide synthase (iNOS) expression during pSS. For this purpose, peripheral blood mononuclear cells isolated from pSS patients and healthy controls were cultured with different concentrations of cardamonin. Cytokine levels were measured by ELISA and NO production was assessed using the Griess method. Inducible nitric oxide synthase expression and NF-κB activity were analyzed by immunofluorescence staining. Our results suggest that cardamonin inhibits TNF-α, IL-6 and NO production and downregulates iNOS expression and NF-κB activation. Collectively, our results highlight the ex vivo immunomodulatory effects of cardamonin on pro-inflammatory cytokine production and NO pathway in pSS patients. Therefore, cardamonin is a potential candidate for controlling inflammation during pSS.

CD5 expression promotes IL-10 production through activation of the MAPK/Erk pathway and upregulation of TRPC1 channels in B lymphocytes.

CD5 is constitutively expressed on T cells and a subset of mature normal and leukemic B cells in patients with chronic lymphocytic leukemia (CLL). Important functional properties are associated with CD5 expression in B cells, including signal transducer and activator of transcription 3 activation, IL-10 production and the promotion of B-lymphocyte survival and transformation. However, the pathway(s) by which CD5 influences the biology of B cells and its dependence on B-cell receptor (BCR) co-signaling remain unknown. In this study, we show that CD5 expression activates a number of important signaling pathways, including Erk1/2, leading to IL-10 production through a novel pathway independent of BCR engagement. This pathway is dependent on extracellular calcium (Ca2+) entry facilitated by upregulation of the transient receptor potential channel 1 (TRPC1) protein. We also show that Erk1/2 activation in a subgroup of CLL patients is associated with TRPC1 overexpression. In this subgroup of CLL patients, small inhibitory RNA (siRNA) for CD5 reduces TRPC1 expression. Furthermore, siRNAs for CD5 or for TRPC1 inhibit IL-10 production. These findings provide new insights into the role of CD5 in B-cell biology in health and disease and could pave the way for new treatment strategies for patients with B-CLL.

Human regulatory B cells control the TFH cell response.

BACKGROUND: Follicular helper T (TFH) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of TFH cell-dependent humoral immune responses is unknown. OBJECTIVE: We sought to assess the role of Breg cells on TFH cell development and function. METHODS: Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate TFH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. RESULTS: B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing TFH cell maturation. In cocultures they differentiated B cells into CD138+ plasma and IgD-CD27+ memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented TFH cell development. Added to TFH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3+CXCR5+PD-1+ follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on TFH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-ß. CONCLUSION: Human Breg cells control TFH cell maturation, expand follicular regulatory T cells, and inhibit the TFH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the TFH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses.

A case for IL-6, IL-17A, and nitric oxide in the pathophysiology of Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune epithelitis characterized by mononuclear cell (MNC) infiltration of the lacrimal and salivary glands (SG), as well as the presence of serum autoantibodies. This condition is a growing public health concern in Algeria. Herein, we sought to determine if the levels of interleukin (IL)-6, IL-17A, and nitric oxide (NO), were correlated with the extent of MNC infiltration. The expression of inducible NO synthase (NOS2) and CD68 was measured in the SG of all patients, but not in those of the normal controls (NCs). We included 44 primary Sjögren's syndrome (pSS) patients and 15 NCs in this study; we found that the expression of NOS2 and CD68 was elevated in all of the SG of SS patients. Additionally, the serum and saliva levels of IL-6, IL-17A, and NO were higher in the pSS patients, compared with the NCs. Furthermore, the NOS2-induced excess NO was associated with the extent of the MNC infiltration, and thereby with tissue injury. It is also important to note that there were correlations between the levels of IL-6, IL-17A, and NO. Such findings indicate that through the effects of NO, IL-17A participates in the pathophysiology of the disease. With the purpose of improving both the diagnosis and prognosis, IL-6, IL-17A, and NO should be assayed in the serum and saliva of patients suspected of SS.

Anti-alpha-actinin antibodies are part of the anti-cell membrane antibody spectrum that characterize patients with lupus nephritis.

Anti-membrane autoantibodies (MbA) have been reported in sera from patients with lupus nephritis (LN) but the targets of the MbA remain to be explored, which is the aim of the current study. Sera were collected from 40 patients with LN determined by renal biopsy, and from 30 systemic lupus erythematosus (SLE) patients without clinical evidence of LN. Thirty autoimmune disease control patients (rheumatoid arthritis, Sjögren's syndrome and systemic sclerosis), and 30 healthy controls were also included. Using flow cytometry, the presence of anti-MbA was explored revealing that IgG anti-MbA positivity was associated with LN (62.5% vs 13.3%) when compared to non-LN SLE patients, autoimmune disease patients (6.7%) and healthy controls (0%). Next, using purified plasma membrane fractions from human embryonic kidney (HEK) cells, the more prominent targets and their occurrence rates were located at 50 kDa, 60/65 kDa, 90 kDa, 110 kDa, 180 kDa and 220 kDa. Alpha-actinin (110 kDa) autoAb was characterized as a major target in LN patients positive for anti-MbA, and anti-MbA binding activity was reduced (36.9 ± 13.7%) in the presence of α-actinin. Laminin (200 kDa) was also characterized as a minor target, which was not the case for annexin A2 (36 kDa). Finally, anti-MbA IgG subclass analysis indicated a predominance of IgG2. In conclusion, IgG anti-MbA were detected at high levels in LN patients supporting a primary pathogenic role for anti-MbA and anti-MbA/α-actinin+ in LN that needs further research.

Cytokines Modulate the "Immune-Metabolism" Interactions during Behçet Disease: Effect on Arginine Metabolism.

Aim and Methods. In this study, we evaluated NOS and arginase activities and their regulation during Behçet disease, a systemic chronic inflammatory disorder with uncertain etiology. The peripheral blood mononuclear cells of 36 patients and 15 control samples (PBMC) were cultured in either RPMI 1640, MEM, or DMEM complemented with 10% of FBS and antibiotics. Cultures were performed with or without the control or patients plasma. Subsequent treatment contained anticytokines (IL-6, TGF-ß), a mitogenic effector (PHA), or NOS modulators (L-NMMA, BH4). Culture supernatants were harvested after 24 h of incubation. NO and urea measurements were, respectively, performed by modified Griess and Berthelot methods. Results. Higher urea levels were found in patients' plasma compared to the control's (P < 0.05). NOS modulators induced inverted production profiles for NO and urea (P < 0.05). Their results differed depending on the clinical findings (P < 0.05). It was also found that cytokine neutralization induced different response profiles in patients as opposed to control cultures (P < 0.05). Conclusion. Our results suggest that arginases can compete with NOS2 for L-arginine during Behçet disease. Both enzymes are regulated by environmental cytokines and substrate availability. Furthermore, it seems that NOS/arginase balance is dependent on clinical expression.

Specific forms of BAFF favor BAFF receptor-mediated epithelial cell survival.

Although B cell activating factor (BAFF) and its receptor BR3 are produced and expressed by many cells, their role has been restricted to the lymphocyte lineage. Using various techniques (RT-PCR, indirect immunofluorescence, flow cytometry analysis), we observed the expression of BR3 and the production of BAFF by the human salivary gland cell line, by epithelial cells from biopsies of Sjögren's syndrome patients and their controls, but also by salivary gland epithelial cells in culture. To decipher the role of BAFF and BR3 on epithelial cells, BAFF and BR3 were neutralized by blocking antibodies or RNA specific inhibitor (siBR3) and epithelial cell survival was analyzed. Blocking BR3 promotes epithelial cell apoptosis in vitro. This apoptosis resulted in the nuclear translocation of PKCδ. BAFF neutralization by various anti-BAFF antibodies leads to different effects depending on the antibody used suggesting that only some forms of BAFF are required for epithelial cell survival. Our study demonstrates that BR3 is involved in the survival of cultured epithelial cells due to an autocrine effect of BAFF. It also suggests that epithelial cells produce different forms of BAFF and that only some of them are responsible for this effect.

TLR9 expressed on plasma membrane acts as a negative regulator of human B cell response.

Toll-like receptors (TLRs) are positioned at the interface between innate and adaptive immunity. Unlike others, those such as TLR9, that recognize nucleic acids, are confined to the endosomal compartment and are scarce on the cell surface. Here, we present evidence for TLR9 expression on the plasma membrane of B cells. In contrast to endosomal TLR9, cell surface TLR9 does not bind CpG-B oligodeoxynucleotides. After B cell-receptor (BCR) stimulation, TLR9 was translocated into lipid rafts with the BCR, suggesting that it could serve as a co-receptor for BCR. Nevertheless, stimulation of B cells with anti-TLR9 antibodies did not modify the BCR-induced responses despite up-regulation of tyrosine phosphorylation of proteins. However, CpG-B activation of B cells, acting synergistically with BCR signals, was inhibited by anti-TLR9 stimulation. Induction of CD25 expression and proliferation of B cells were thus down-regulated by the engagement of cell surface TLR9. Overall, our results indicate that TLR9 expressed on the plasma membrane of B cells might be a negative regulator of endosomal TLR9, and could provide a novel control by which activation of autoreactive B cells is restrained.

Diagnostic criteria for autoimmune neutropenia.

Autoimmune neutropenia denotes that the number of circulating polymorphonuclear neutrophils is below 1.5×10(9)/L. This encompasses a wide range of disorders from primary conditions to complications of systemic autoimmune diseases or hematological neoplasms. Antineutrophil autoantibodies are particularly difficult to detect, and their amount does not correlate with the degree of neutropenia. Granulocyte colony-stimulating factor is the first-line therapy, but should be restricted to patients with total absence of neutrophils and/or severe infections.

DNA methylation modulates HRES1/p28 expression in B cells from patients with Lupus.

Systemic lupus erythematosus (SLE) disease is an autoimmune disease of unknown aetiology that affects predominantly women of child bearing age. Since previous studies, including ours, have demonstrated that CD4+ T cells and B cells from SLE patients are defective in their ability to methylate their DNA upon antigen stimulation, the aim of this study was to investigate whether DNA demethylation affects the transcription of HRES-1 in B cells. HRES-1 is the prototype of Human Endogenous Retrovirus (HERV) overexpressed in SLE. We have observed that SLE B cells were characterized by their incapacity to methylate the HRES-1 promoter, both in unstimulated and in anti-IgM stimulated B cells. In turn, HRES-1/p28 expression was increased in SLE B cells after B cell receptor engagement, but not in controls. In SLE B cells the Erk/DNMT1 pathway was defective. In addition, blocking the autocrine-loop of IL-6 in SLE B cells with an anti-IL-6 receptor monoclonal antibody restores DNA methylation and control of HRES-1/p28 expression became effective. As a consequence, a better understanding of HERV dysregulation in SLE reinforces our comprehension of the disease and opens new therapeutic perspectives.

[Regulatory lymphocytes: a new cooperation between T and B cells for a better control of the immune response]. / Les lymphocytes régulateurs : une nouvelle coopération entre cellules T et B pour un contrôle plus efficace de la réponse immunitaire.

Mechanims of peripheral tolerance include molecular controls and the presence of regulatory lymphocytes. Regulatory T lymphocytes (Tregs) correspond to different sub-populations of T cells that control immune responses due to the production of cytokines, such as IL-10 and with direct cell-to-cell contacts. Tregs targets are antigen presenting cells, such as dendritic cells, effector CD4(+) and CD8(+) lymphocytes but also effector antibody-producing B lymphocytes. Regulatory B lymphocytes (Bregs) have been more recently described and likely represent different sub-populations of B cells that control the development of autoimmune and inflammatory diseases due to the production of IL-10 and using intercellular contacts. Bregs targets encompass all the cells involved in the immune responses which are thus under a dual control by regulatory lymphocytes. Development and efficient activity of Tregs appear dependent of Bregs for a better regulation of autoimmune reactions, of anti-infectious reactions, but also of anti-tumor reactions.

Intravenous immunoglobulin induces a functional silencing program similar to anergy in human B cells.

BACKGROUND: Chronic inflammatory and autoimmune diseases are largely due to inappropriate response of hyperactive or autoreactive B cells. These autoreactive B cells can evade central tolerance checkpoints and migrate to the periphery, where they would be silenced by anergy. Such anergic cells are characterized by B-cell receptor (BCR) desensitization and altered downstream signaling. OBJECTIVE: We sought to determine whether intravenous immunoglobulin (IVIg) induces a nonresponsive state of B cells and to address the similarities of this mechanism to those described in anergy. METHODS: Human B cells were stimulated with anti-IgM antibody, and effects of IVIg on several parameters, such as calcium release, tyrosine phosphorylation, BCR aggregation, BCR internalization, or transcriptional activity, were studied by using flow cytometry, confocal microscopy, Western blotting, and a quantitative PCR array. RESULTS: IVIg-treated B cells show defects in activating coreceptor expression, calcium signaling, and BCR aggregation on engagement by antigen. IVIg also induces suppression of phosphoinositide 3-kinase signaling, which plays a central role in determining B-cell fate. All these events ultimately lead to profound modifications in gene expression, resulting in long-term functional but reversible silencing of IVIg-treated B cells. CONCLUSION: Our findings provide insights into the effectiveness of IVIg in treating autoimmune or inflammatory pathologies associated with the loss of B-cell tolerance. Furthermore, these data provide a model to explore the complexity of positive versus negative selection in B cells.

Role of Fms-like tyrosine kinase 3 ligand as a potential biologic marker of lymphoma in primary Sjögren's syndrome.

OBJECTIVE: Patients with primary Sjögren's syndrome (SS) are at greater risk of developing lymphoma. This study was undertaken to evaluate whether the Fms-like tyrosine kinase 3 ligand (Flt-3L) might be associated with lymphoma in primary SS. METHODS: Serum levels of Flt-3L were measured in 369 patients with primary SS from the French Assessment of Systemic Signs and Evolution of Sjögren's Syndrome study cohort and in 10 patients with primary SS at the time of lymphoma diagnosis in an Italian cohort. Associations between increased levels of Flt-3L and a history of lymphoma, history of previously diagnosed criteria related to a high risk of lymphoma, and greater extent of disease activity were evaluated. RESULTS: Among patients with primary SS, higher levels of Flt-3L were significantly associated with a history of lymphoma (P = 0.0001). Previous markers for risk of lymphoma development, such as presence of purpura, low levels of C4, presence of lymphocytopenia, low levels of IgM, high levels of ß2 -microglobulin, and a higher primary SS disease activity score, were all associated with higher levels of Flt-3L. The levels of Flt-3L were also increased in serum obtained from patients with primary SS at the time of lymphoma diagnosis. Furthermore, the Flt-3L levels were elevated in the serum of 6 patients up to 94 months (mean 46 months) prior to the diagnosis of lymphoma. Receiver operating characteristic curve analysis showed that an Flt-3L level of 175 pg/ml was the ideal cutoff value for demonstrating an association with lymphoma (specificity 97.5%, sensitivity 44%, negative predictive value 97%). CONCLUSION: Flt-3L is associated with lymphoma in primary SS, and constitutes a good biologic marker. Higher levels of this cytokine are present several years before the diagnosis of lymphoma, and may be useful as a predictive marker of lymphoproliferative disorders in primary SS.

Regulatory B cells play a key role in immune system balance.

Regulatory B cells (Bregs) may act earlier than regulatory T cells (Tregs) and may play as important a role in autoimmune and allergic diseases. Obstacles to the investigation of Bregs are the same as those encountered for Tregs: the regulatory effects are short-lived in some cases, there is no consistent phenotype (C5 expression is neither indispensable nor sufficient), differences exist across species (e.g., between humans and mice), and there are a number of suppression modalities (IL-10, TGF-beta, expression of proapoptotic membrane molecules) that vary across Breg subtypes. The Breg subtypes may be homologous to the Treg subtypes (Br1 cells expressing IL-10, Br3 cells expressing TGF-beta, and B-Foxp3 cells), although the Br1 subtype seems to predominate. Nevertheless, differences with Treg cells may exist: Breg activation may chiefly involve the toll-like receptors rather than the antigen receptor; and Bregs act earlier, facilitating the recruitment of Tregs then disappearing once the Tregs become operational. Bregs make a major contribution to autoimmune disorders associated with several forms of immune deficiency, as well as to the absence of transplant rejection when there is a strong B cell response. Breg deficiencies have been reported in lupus, and the disappointing effects in this disease of treatments designed to inhibit the B cell response may be related to further Breg impairment. In several animal models, Breg stimulation is effective in correcting a variety of autoimmune disorders, most notably those initiated in the mucous membranes. Research into the interactions between the gut microbiota and Bregs holds considerable promise.

Contribution of salivary gland ultrasonography to the diagnosis of Sjögren's syndrome: toward new diagnostic criteria?

OBJECTIVE: To determine the accuracy of salivary gland ultrasonography (SGUS) for diagnosing primary Sjögren's syndrome (SS) and to suggest modifications of the American-European Consensus Group (AECG) classification criteria. METHODS: We conducted a cross-sectional study in a prospective cohort of patients with suspected primary SS that was established between 2006 and 2011. The echostructure of the bilateral parotid and submandibular glands was graded from 0 to 4, and the gland size was measured; blood flow to the parotid gland was assessed using Doppler waveform analysis. The reference standard was a clinical diagnosis of primary SS as determined by a group of experts blinded to the results of SGUS. Receiver operating characteristic (ROC) curve analysis was performed to compare the diagnostic value of the 0-4-point echostructure grade for each of the 4 major salivary glands, the sum of the grades for the 4 glands, and the highest grade among the 4 glands. RESULTS: Of the 158 patients in the study, 78 had a diagnosis of primary SS according to the experts, including 61 patients (78.2%) who met the AECG criteria. Doppler waveform analysis and gland size measurement showed poor diagnostic performance. The results of ROC curve analysis showed that the highest grade among the 4 glands provided the best diagnostic value. The optimal grade cutoff was 2 (62.8% sensitivity and 95.0% specificity). A weighted score was constructed using scores for the 5 variables selected by logistic regression analysis, as follows: (salivary flow×1.5)+(Schirmer's test×1.5)+(salivary gland biopsy×3)+(SSA/SSB×4.5)+(SGUS×2). According to ROC curve analysis, a score of ≥5 of 12.5 had 85.7% sensitivity and 94.9% specificity, compared with 77.9% sensitivity and 98.7% specificity for the AECG criteria. The addition of SGUS to the AECG criteria increased sensitivity to 87.0% but did not change specificity. CONCLUSION: Modifications of the AECG criteria, including the addition of a SGUS score, notably improved diagnostic performance.

Importance of Toll-like receptors for B lymphocyte survival in primary Sjögren's syndrome.

The Sjögren's syndrome is a systemic autoimmune disease characterized by lymphocytic infiltration of the glands responsible for mouth and eyes dryness. A minority of infiltrating B cells is organized as germinal centers while the majority is aggregated into clusters of transitional and marginal zone B cells. The Toll-like receptor 9 (TLR9) recognizes microbial DNA but also, sometimes, the self DNA. It appears to be a key determinant of the survival and differentiation of B lymphocytes. After laser micro-dissection of B cells from salivary glands, analyses by quantitative RT-PCR showed that transitional B cells express high level of TLR9 mRNA unlike B cells from germinal centers. B lymphocytes from healthy donors were sorted by flow cytometry and stimulated in vitro with their TLR9. It induces survival, activation and proliferation associated with phenotypic changes. Transitional B cells exhibited characteristics of the marginal zone, whereas mature B cells expressed follicular germinal center specificities. Finally, IgM and IgG were secreted by both population, but with elevated production of autoantibodies by the transitional B cells. Increased expression of TLR9 by transitional B cells suggests that they may be highly sensitive to differentiate into autoantibody secreting cells through maturation into the marginal zone into the salivary glands. TLR9 might be a target for forthcoming biotherapies.