Neutralizing anti-IL-1 receptor antagonist autoantibodies induce inflammatory and fibrotic mediators in IgG4-related disease.

BACKGROUND: IgG4-related disease (IgG4-RD) is a fibroinflammatory condition involving loss of B-cell tolerance and production of autoantibodies. However, the relevant targets and role of these aberrant humoral immune responses are not defined. OBJECTIVE: Our aim was to identify novel autoantibodies and autoantigen targets that promote pathogenic responses in IgG4-RD. METHODS: We sequenced plasmablast antibody repertoires in patients with IgG4-RD. Representative mAbs were expressed and their specificities characterized by using cytokine microarrays. The role of anti-IL-1 receptor antagonist (IL-1RA) autoantibodies was investigated by using in vitro assays. RESULTS: We identified strong reactivity against human IL-1RA by using a clonally expanded plasmablast-derived mAb from a patient with IgG4-RD. Plasma from patients with IgG4-RD exhibited elevated levels of reactivity against IL-1RA compared with plasma from the controls and neutralized IL-1RA activity, resulting in inflammatory and fibrotic mediator production in vitro. IL-1RA was detected in lesional tissues from patients with IgG4-RD. Patients with anti-IL-1RA autoantibodies of the IgG4 subclass had greater numbers of organs affected than did those without anti-IL-1RA autoantibodies. Peptide analyses identified IL-1RA epitopes targeted by anti-IL-1RA antibodies at sites near the IL-1RA/IL-1R interface. Serum from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) also had elevated levels of anti-IL-1RA autoantibodies compared with those of the controls. CONCLUSION: A subset of patients with IgG4-RD have anti-IL-1RA autoantibodies, which promote proinflammatory and profibrotic meditator production via IL-1RA neutralization. These findings support a novel immunologic mechanism underlying the pathogenesis of IgG4-RD. Anti-IL-1RA autoantibodies are also present in a subset of patients with SLE and RA, suggesting a potential common pathway in multiple autoimmune diseases.

P2Y6 Receptor Antagonist MRS2578 Inhibits Neutrophil Activation and Aggregated Neutrophil Extracellular Trap Formation Induced by Gout-Associated Monosodium Urate Crystals.

Human neutrophils (polymorphonuclear leukocytes [PMNs]) generate inflammatory responses within the joints of gout patients upon encountering monosodium urate (MSU) crystals. Neutrophil extracellular traps (NETs) are found abundantly in the synovial fluid of gout patients. The detailed mechanism of MSU crystal-induced NET formation remains unknown. Our goal was to shed light on possible roles of purinergic signaling and neutrophil migration in mediating NET formation induced by MSU crystals. Interaction of human neutrophils with MSU crystals was evaluated by high-throughput live imaging using confocal microscopy. We quantitated NET levels in gout synovial fluid supernatants and detected enzymatically active neutrophil primary granule enzymes, myeloperoxidase, and human neutrophil elastase. Suramin and PPADS, general P2Y receptor blockers, and MRS2578, an inhibitor of the purinergic P2Y6 receptor, blocked NET formation triggered by MSU crystals. AR-C25118925XX (P2Y2 antagonist) did not inhibit MSU crystal-stimulated NET release. Live imaging of PMNs showed that MRS2578 represses neutrophil migration and blocked characteristic formation of MSU crystal-NET aggregates called aggregated NETs. Interestingly, the store-operated calcium entry channel inhibitor (SK&F96365) also reduced MSU crystal-induced NET release. Our results indicate that the P2Y6/store-operated calcium entry/IL-8 axis is involved in MSU crystal-induced aggregated NET formation, but MRS2578 could have additional effects affecting PMN migration. The work presented in the present study could lead to a better understanding of gouty joint inflammation and help improve the treatment and care of gout patients.

Combination of anti-citrullinated protein antibodies and rheumatoid factor is associated with increased systemic inflammatory mediators and more rapid progression from preclinical to clinical rheumatoid arthritis.

The development of rheumatoid factor (RF) and/or anti-citrullinated protein antibodies (ACPAs) can be observed years prior to clinical diagnosis of rheumatoid arthritis (RA). Nevertheless, the interaction between these two autoantibodies and their combined effect on development of RA is unclear. We measured RF, cytokines, and ACPA subtypes in serial pre-clinical serum samples collected from 83 US veterans who all developed RA. Levels of cytokines and ACPAs were compared between the following groups: anti-cyclic citrullinated peptide (anti-CCP)-/RF- (double negative), anti-CCP+/RF-, anti-CCP-/RF+, or anti-CCP+/RF+ (double-positive). The double-positive subgroup had significantly higher levels of 20 inflammatory cytokines and 29 ACPA reactivities, and the shortest interval, 1.3 years, between the preclinical sample timepoint and diagnosis of RA. Thus, the combined presence of ACPAs and RF is associated with a more rapid progression to RA, suggesting that anti-CCP+/RF+ individuals have a more advanced preclinical disease state and that the onset of RA may be imminent.

Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens.

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.

CCL2/CCR2, but not CCL5/CCR5, mediates monocyte recruitment, inflammation and cartilage destruction in osteoarthritis.

OBJECTIVES: While various monocyte chemokine systems are increased in expression in osteoarthritis (OA), the hierarchy of chemokines and chemokine receptors in mediating monocyte/macrophage recruitment to the OA joint remains poorly defined. Here, we investigated the relative contributions of the CCL2/CCR2 versus CCL5/CCR5 chemokine axes in OA pathogenesis. METHODS: Ccl2-, Ccr2-, Ccl5- and Ccr5-deficient and control mice were subjected to destabilisation of medial meniscus surgery to induce OA. The pharmacological utility of blocking CCL2/CCR2 signalling in mouse OA was investigated using bindarit, a CCL2 synthesis inhibitor, and RS-504393, a CCR2 antagonist. Levels of monocyte chemoattractants in synovial tissues and fluids from patients with joint injuries without OA and those with established OA were investigated using a combination of microarray analyses, multiplexed cytokine assays and immunostains. RESULTS: Mice lacking CCL2 or CCR2, but not CCL5 or CCR5, were protected against OA with a concomitant reduction in local monocyte/macrophage numbers in their joints. In synovial fluids from patients with OA, levels of CCR2 ligands (CCL2, CCL7 and CCL8) but not CCR5 ligands (CCL3, CCL4 and CCL5) were elevated. We found that CCR2+ cells are abundant in human OA synovium and that CCR2+ macrophages line, invade and are associated with the erosion of OA cartilage. Further, blockade of CCL2/CCR2 signalling markedly attenuated macrophage accumulation, synovitis and cartilage damage in mouse OA. CONCLUSIONS: Our findings demonstrate that monocytes recruited via CCL2/CCR2, rather than by CCL5/CCR5, propagate inflammation and tissue damage in OA. Selective targeting of the CCL2/CCR2 system represents a promising therapeutic approach for OA.

Molecular basis for increased susceptibility of Indigenous North Americans to seropositive rheumatoid arthritis.

OBJECTIVE: The pathogenetic mechanisms by which HLA-DRB1 alleles are associated with anticitrullinated peptide antibody (ACPA)-positive rheumatoid arthritis (RA) are incompletely understood. RA high-risk HLA-DRB1 alleles are known to share a common motif, the 'shared susceptibility epitope (SE)'. Here, the electropositive P4 pocket of HLA-DRB1 accommodates self-peptide residues containing citrulline but not arginine. HLA-DRB1 His/Phe13ß stratifies with ACPA-positive RA, while His13ßSer polymorphisms stratify with ACPA-negative RA and RA protection. Indigenous North American (INA) populations have high risk of early-onset ACPA-positive RA, whereby HLA-DRB1*04:04 and HLA-DRB1*14:02 are implicated as risk factors for RA in INA. However, HLA-DRB1*14:02 has a His13ßSer polymorphism. Therefore, we aimed to verify this association and determine its molecular mechanism. METHODS: HLA genotype was compared in 344 INA patients with RA and 352 controls. Structures of HLA-DRB1*1402-class II loaded with vimentin-64Arg59-71, vimentin-64Cit59-71 and fibrinogen ß-74Cit69-81 were solved using X-ray crystallography. Vimentin-64Cit59-71-specific and vimentin59-71-specific CD4+ T cells were characterised by flow cytometry using peptide-histocompatibility leukocyte antigen (pHLA) tetramers. After sorting of antigen-specific T cells, TCRα and ß-chains were analysed using multiplex, nested PCR and sequencing. RESULTS: ACPA+ RA in INA was independently associated with HLA-DRB1*14:02. Consequent to the His13ßSer polymorphism and altered P4 pocket of HLA-DRB1*14:02, both citrulline and arginine were accommodated in opposite orientations. Oligoclonal autoreactive CD4+ effector T cells reactive with both citrulline and arginine forms of vimentin59-71 were observed in patients with HLA-DRB1*14:02+ RA and at-risk ACPA- first-degree relatives. HLA-DRB1*14:02-vimentin59-71-specific and HLA-DRB1*14:02-vimentin-64Cit59-71-specific CD4+ memory T cells were phenotypically distinct populations. CONCLUSION: HLA-DRB1*14:02 broadens the capacity for citrullinated and native self-peptide presentation and T cell expansion, increasing risk of ACPA+ RA.

Association of anti-citrullinated protein or peptide antibodies with left ventricular structure and function in rheumatoid arthritis.

Objective: High levels of ACPAs in RA are associated with more severe arthritis and worse prognosis. However, the role of ACPAs in mediating the increased risk of heart failure in RA remains undefined. We examined whether specific ACPAs were associated with subclinical left ventricular (LV) phenotypes that presage heart failure. Methods: Sera from RA patients without clinical cardiovascular disease were assayed for specific ACPAs using a custom Bio-Plex bead assay, and their cross-sectional associations with cardiac magnetic resonance-derived LV measures were evaluated. High ACPA level was defined as ⩾ 75th percentile. Findings were assessed in a second independent RA cohort with an expanded panel of ACPAs and LV measures assessed by 3D-echocardiography. Results: In cohort 1 (n = 76), higher levels of anti-citrullinated fibrinogen 41-60 and anti-citrullinated vimentin antibodies were associated with a 10 and 6% higher adjusted mean LV mass index (LVMI), respectively, compared with lower antibody levels (P < 0.05). In contrast, higher levels of anti-citrullinated biglycan 247-266 were associated with a 13% lower adjusted mean LVMI compared with lower levels (P < 0.001). In cohort 2 (n = 74), the association between ACPAs targeting citrullinated fibrinogen and citrullinated vimentin peptides or protein and LVMI was confirmed: higher anti-citrullinated fibrinogen 556-575 and anti-citrullinated vimentin 58-77 antibody levels were associated with a higher adjusted mean LVMI (19 and 15%, respectively; P < 0.05), but no association with biglycan was found. Conclusion: Higher levels of antibodies targeting citrullinated fibrinogen and vimentin peptides or protein were associated with a higher mean LVMI in both RA cohorts, potentially implicating autoimmune targeting of citrullinated proteins in myocardial remodelling in RA.

Nicotine drives neutrophil extracellular traps formation and accelerates collagen-induced arthritis.

Objectives: The aim was to investigate the effects of nicotine on neutrophil extracellular traps (NETs) formation in current and non-smokers and on a murine model of RA. Methods: We compared spontaneous and phorbol 12-myristate 13-acetate-induced NETosis between current and non-smokers by DNA release binding. Nicotine-induced NETosis from non-smokers was assessed by DNA release binding, NET-specific (myeloperoxidase (MPO)-DNA complex) ELISA and real-time fluorescence microscopy. We also used immunofluorescent staining to detect nicotinic acetylcholine receptors (nAChRs) on neutrophils and performed a functional analysis to assess the role of nAChRs in nicotine-induced NETosis. Finally, we investigated the effects of systemic nicotine exposure on arthritis severity and NETosis in the CIA mouse model. Results: Neutrophils derived from current smokers displayed elevated levels of spontaneous and phorbol 12-myristate 13-acetate-induced NETosis. Nicotine induced dose-dependent NETosis in ex vivo neutrophils from healthy non-smokers, and co-incubation with ACPA-immune complexes or TNF-α facilitated a synergistic effect on NETosis. Real-time fluorescence microscopy revealed robust formation of NET-like structures in nicotine-exposed neutrophils. Immunofluorescent staining demonstrated the presence of the α7 subunit of the nAChR on neutrophils. Stimulation of neutrophils with an α7-specific nAChR agonist induced NETosis, whereas pretreatment with an nAChR antagonist attenuated nicotine-induced NETosis. Nicotine administration to mice with CIA exacerbated inflammatory arthritis, with higher plasma levels of NET-associated MPO-DNA complex. Conclusion: We demonstrate that nicotine is a potent inducer of NETosis, which may play an important role in accelerating arthritis in the CIA model. This study generates awareness of and the mechanisms by which nicotine-containing products, including e-cigarettes, may have deleterious effects on patients with RA.

Enrichment of malondialdehyde-acetaldehyde antibody in the rheumatoid arthritis joint.

Objective: To characterize the expression of malondialdehdye-acetaldehyde (MAA) adducts and anti-MAA antibody in articular tissues and serum of patients with RA. Methods: Paired sera and SF were examined from 29 RA and 13 OA patients. Anti-MAA antibody, RF, ACPA and total immunoglobulin were quantified. SF-serum measures were compared within and between disease groups. The presence and co-localization of MAA, citrulline and select leukocyte antigens in RA and OA synovial tissues were examined using immunohistochemistry. Results: Circulating and SF anti-MAA antibody concentrations were higher in RA vs OA by 1.5- to 5-fold. IgG (P < 0.001), IgM (P = 0.006) and IgA (P = 0.036) anti-MAA antibodies were higher in paired RA SF than serum, differences not observed for total immunoglobulin, RF or ACPA. In RA synovial tissues, co-localization of MAA with citrulline and CD19+ or CD27+ B cells was demonstrated and was much higher in magnitude than MAA or citrulline co-localization with T cells, monocytes, macrophages or dendritic cells (P < 0.01). Conclusion: Anti-MAA antibodies are present in higher concentrations in the RA joint compared with sera, a finding not observed for other disease-related autoantibodies. Co-localization of MAA and citrulline with mature B cells, coupled with the local enrichment of anti-MAA immune responses, implicates MAA-adduct formation in local autoantibody production.

Association of Antibodies to Citrullinated Protein Antigens with Blood Pressure in First-Degree Relatives of Rheumatoid Arthritis Patients: The Studies of the Etiology of Rheumatoid Arthritis.

BACKGROUND: Hypertension is more common in patients with rheumatoid arthritis (RA) than in the general population. It is unknown whether hypertension is due to RA-related medications or the disease itself. Therefore, we sought to investigate associations between RA-related autoantibodies, specifically antibodies to citrullinated protein antigens (ACPA) and systolic blood pressure (SBP) and diastolic blood pressure (DBP) in first-degree relatives of RA patients, who were free of RA and RA-related medications. We hypothesized that a greater number of detectable ACPA would be associated with high SBP and DBP, independent of other risk factors in these first-degree relatives. METHODS: We evaluated associations between ACPA and SBP and DBP in a cross-sectional study of 72 first-degree relatives (defined as parent, child, or sibling) of RA patients. Fifteen ACPA were measured using a Bio-Plex bead-based assay; each was dichotomized as positive/negative based on pre-specified cut-points. Analysis of covariance was used to evaluate associations between ACPA positivity and SBP and DBP, adjusting for age, sex, race, body mass index (BMI), pack-years of smoking, high sensitivity C-reactive protein (hsCRP), and current use of anti-hypertensive medications. RESULTS: Average age was 51 and 69% were women. Mean SBP was 119 ± 18 and DBP was 74 ± 9 mm Hg. Thirty-three (46%) first-degree relatives were positive for ≥1 ACPA; and were younger, had lower BMI, more pack-years of smoking, and higher hsCRP concentrations compared to ACPA negative first-degree relatives. For each additional positive ACPA, SBP was 0.98 ± 0.5 mm Hg (p = 0.05) higher, and DBP was 0.66 ± 0.3 mm Hg (p = 0.04) higher. Anti-cit-fibrinogen A (211-230) positive and anti-cit-filaggrin positive first-degree relatives had 11.5 and 13.9 mm Hg higher SBP (p = 0.02) respectively. Anti-cit-clusterin, cit-filaggrin, and cit-vimentin positive first-degree relatives had 7-8 mm Hg higher DBP (p = 0.03, 0.05, 0.05 respectively), compared to being negative for these individual ACPA. Consistent with associations between ACPA, SBP, and DBP, anti-cyclic citrullinated peptides (anti-CCP2) positive first-degree relatives had 16.4± (p = 0.03) higher SBP and 12.1± mm Hg (p = 0.01) higher DBP than anti-CCP2 negative first-degree relatives. CONCLUSION: In first-degree relatives without RA, ACPA positivity is associated with higher SBP and DBP. Subclinical autoimmune processes and ACPA may play a role in the vascular changes potentially leading to hypertension prior to RA onset.

Decreased Synovial Inflammation in Atraumatic Hip Microinstability Compared With Femoroacetabular Impingement.

PURPOSE: To compare the inflammatory profile of hip synovial tissue in those with atraumatic microinstability to patients with femoroacetabular impingement (FAI). METHODS: Patients with cam and mixed-type FAI (FAI group) and patients with hip instability underwent sampling of the anterolateral synovium. Demographic data, intraoperative measurements, and functional outcome scores (International Hip Outcomes Tool and Short Form-12) were recorded. Cryosections were stained and examined under light microscopy as well as confocal fluorescent microscopy for anti-CD45 (common leukocyte antigen), anti-CD31 (endothelial), and anti-CD68 (macrophage) cell surface markers. A grading system was used to quantify synovitis under light microscopy whereas digital image analysis was used to quantify immunofluorescence staining area. Comparison were made with Student t test, Mann-Whitney U, χ2, and regression analysis. RESULTS: There were 12 patients in the FAI group and 5 in the instability group. Mean age was not significantly different (P > .05), but there was a significantly greater proportion of females in the instability group versus the FAI group (P < .001). There was a significant correlation (r = 0.653; P = .005) between number of turns needed for 10 mm of distraction and increased synovitis. Synovitis scores also were increased significantly in patients with cam morphology and articular cartilage damage (P = .024) versus those without. Immunohistochemistry did not reveal differences (P > .082) between the instability and FAI groups, but CD68 staining was significantly greater in those with cam morphology and cartilage damage (P < .045). CD45+/CD68- cells were noted in the perivascular area while CD45+/CD68+ cells were noted within the synovial lining in both groups. CONCLUSIONS: Increased synovial inflammation was associated with an increased number of turns to achieve joint distraction. Both instability and FAI groups demonstrated baseline levels of synovial inflammation. Synovitis scores also were increased in patients with cartilage damage. CLINICAL RELEVANCE: An understanding of the molecular and cellular mechanisms behind both hip instability and FAI may lead to novel therapeutic anti-inflammatory therapy, which may serve as an adjunct to treatment of mechanical abnormalities in this conditions.

Impact of baseline anti-cyclic citrullinated peptide-2 antibody concentration on efficacy outcomes following treatment with subcutaneous abatacept or adalimumab: 2-year results from the AMPLE trial.

OBJECTIVES: To examine whether baseline anti-cyclic citrullinated peptide-2 (CCP2) antibody status and concentration correlated with clinical outcomes in patients treated with abatacept or adalimumab on background methotrexate (MTX) in the 2-year AMPLE (Abatacept versus adaliMumab comParison in bioLogic-naïvE rheumatoid arthritis subjects with background MTX) study. METHODS: In this exploratory analysis, anti-CCP2 antibody concentration was measured at baseline, and antibody-positive patients were divided into equal quartiles, Q1-Q4, representing increasing antibody concentrations. Clinical outcomes analysed by baseline anti-CCP2 status and quartile included change from baseline in disease activity and disability and remission rates. RESULTS: Baseline characteristics were generally comparable across quartiles and treatment groups. In both treatment groups, anti-CCP2 antibody-negative patients responded less well than antibody-positive patients. At year 2, improvements in disease activity and disability and remission rates were similar across Q1-Q3, but were numerically higher in Q4 in the abatacept group; in contrast, treatment effects were similar across all quartiles in the adalimumab group. CONCLUSIONS: In AMPLE, baseline anti-CCP2 positivity was associated with a better response for abatacept and adalimumab. Patients with the highest baseline anti-CCP2 antibody concentrations had better clinical response with abatacept than patients with lower concentrations, an association that was not observed with adalimumab. TRIAL REGISTRATION NUMBER: NCT00929864.

B cell depletion with rituximab in patients with rheumatoid arthritis: Multiplex bead array reveals the kinetics of IgG and IgA antibodies to citrullinated antigens.

The serology of patients with Rheumatoid arthritis (RA) is characterized by persistently raised levels of autoantibodies: Rheumatoid Factors (RhF) against Fc of IgG, and to citrullinated (Cit) protein/peptide sequences: ACPA, recognizing multiple Cit-sequences. B cell depletion therapy based on rituximab delivers good clinical responses in RA patients, particularly in the seropositive group, with responses sometimes lasting beyond the phase of B cell reconstitution. In general, ACPA levels fall following rituximab, but fluctuations with respect to predicting relapse have proved disappointing. In order to identify possible immunodominant specificities within either IgG- or IgA-ACPA we used a Multiplex bead-based array consisting of 30 Cit-peptides/proteins and 22 corresponding native sequences. The kinetics of the serum ACPA response to individual specificities was measured at key points (Baseline, B cell depletion phase, Relapse) within an initial cycle of rituximab therapy in 16 consecutive patients with severe, active RA. All had achieved significant decreases in Disease Activity Scores-28 and maintained B cell depletion in the peripheral blood (<5 CD19+cells/µl) for at least 3 months. At Baseline, mean fluorescence intensity shown by individual IgG- and IgA-ACPA were strongly correlated (R(2) = 0.75; p < 0.0001) but IgA-ACPA were approximately 10-fold lower. Data were Z-normalised in order to compare serial results and antibody classes. At Baseline, a total of 68 IgG- and 51 IgA-ACPA had Z-scores ≥ 1 (above population mean) were identified, with at least one Cit-antigen identified in each serum. ACPA to individual specificities subsequently fluctuated with 3 different patterns. Most 51/68 (75%) IgG- and 48/51 IgA-ACPA (94%) fell between Baseline and Depletion, of which 57% IgG- and 65% IgA-ACPA rebounded pre-Relapse. Interestingly, 17/68 IgG-ACPA (25%) and some IgA-ACPA (3/51; 6%) transiently increased from Baseline, subsequently falling pre-Relapse. Individual responses to particular Cit-epitopes were not linked to particular patterns of fluctuation, but IgG- and IgA-ACPA to individual Cit-antigens often followed similar courses. Some new IgG- and IgA-ACPA, generally to different Cit-antigens however, arose at Relapse in 4 patients. The complexities of the ACPA response after rituximab may therefore reflect its ability to deplete or modify the function of parent B cell clones, which varies between patients. Although relapse following rituximab invariably follows naïve B cell exit from the bone marrow, these studies show that interactions between both 'new' and residual autoreactive memory B cells may be key to resumption of symptoms. The lack of identification of any immunodominant specificity suggests that the process of citrullination, rather than any particular Cit-antigen drives the autoimmune response in RA patients.

Increased inflammation and disease activity among current cigarette smokers with rheumatoid arthritis: a cross-sectional analysis of US veterans.

OBJECTIVES: Cigarette smoking is a major risk factor for RA and has been associated with increased disease severity and lower rates of disease remission. We hypothesized that inflammation and disease activity would be associated with smoking status and this would be related to levels of ACPA. METHODS: RA patients from the Veterans Affairs RA registry were studied (n = 1466): 76.9% anti-CCP2 positive, 89% male, median age 63 years (interquartile range 57-72), median disease duration 8.45 years (interquartile range 2.8-18). Baseline serum samples were evaluated for levels of anti-CCP2, RF, 19 distinct ACPAs and 17 cytokines. Smoking status at baseline was recorded as current, former or never. The association of smoking status with cytokines, autoantibodies and disease activity (DAS28) was evaluated. RESULTS: Among anti-CCP-positive RA patients, RA-associated cytokines (false-discovery rates q < 0.1%) and DAS28 (P < 0.01) were higher in current smokers compared with former or never smokers. DAS28 and cytokine levels were similar between former and never smokers. In contrast, ACPA concentrations were higher among both current and former smokers compared with never smokers, and levels of ACPA were not associated with DAS28 or cytokine levels. CONCLUSION: Among anti-CCP2-positive RA patients, current smoking status is associated with elevations in pro-inflammatory cytokines and increased RA disease activity. Similar levels of inflammation and disease activity among former and never smokers suggests that the detrimental effects of smoking could be ameliorated through tobacco cessation. The effect of tobacco cessation on RA disease activity should be evaluated prospectively.

Association of synovial inflammation and inflammatory mediators with glenohumeral rotator cuff pathology.

HYPOTHESIS: We hypothesized that patients with full-thickness rotator cuff tears would have greater synovial inflammation compared with those without rotator cuff tear pathology, with gene expression relating to histologic findings. METHODS: Synovial sampling was performed in 19 patients with full-thickness rotator cuff tears (RTC group) and in 11 patients without rotator cuff pathology (control group). Cryosections were stained and examined under light microscopy and confocal fluorescent microscopy for anti-cluster CD45 (common leukocyte antigen), anti-CD31 (endothelial), and anti-CD68 (macrophage) cell surface markers. A grading system was used to quantitate synovitis under light microscopy, and digital image analysis was used to quantify the immunofluorescence staining area. Quantitative polymerase chain reaction was performed for validated inflammatory markers. Data were analyzed with analysis of covariance, Mann-Whitney U, and Spearman rank order testing, with significance set at α = .05. RESULTS: The synovitis score was significantly increased in the RTC group compared with controls. Immunofluorescence demonstrated significantly increased staining for CD31, CD45, and CD68 in the RTC vs control group. CD45+/68- cells were found perivascularly, with CD45+/68+ cells toward the joint lining edge of the synovium. Levels of matrix metalloproteinase-3 (MMP-3) and interleukin-6 were significantly increased in the RTC group, with a positive correlation between the synovitis score and MMP-3 expression. CONCLUSIONS: Patients with full-thickness rotator cuff tears have greater levels of synovial inflammation, angiogenesis, and MMP-3 upregulation compared with controls. Gene expression of MMP-3 correlates with the degree of synovitis.

TPH2 gene polymorphisms and bipolar disorder: A meta-analysis.

BACKGROUND: Disturbance of the serotonergic system contributes to the etiology of bipolar disorder (BD). Tryptophan hydroxylase-2 (TPH2) is an important rate-limiting enzyme in the synthetic pathway for brain serotonin and has been suggested to play a role in BD. MATERIALS AND METHODS: We performed a systematic review and meta-analysis of all studies to date investigating the association studies between TPH2 and BD published before Aug 2014. All studies were abstracted from PubMed, Embase, HuGNet, and China National Knowledge Infrastructure (CNKI). Manuscripts and the supplementary documents of published genome-wide association studies in the field were also included. Effect sizes of independent loci that have been studied in more than three articles were synthesized using fixed and random effects models. RESULTS: Eight eligible studies addressed association between 63 TPH2 gene single nucleotide polymorphisms (SNPs) with BD, after linkage disequilibrium analysis, 12 independent SNPs were identified. Finally, three SNPs (rs4760820, rs11178998, and rs7954758) were found associated with BD using fixed effects models, and rs4760820 and rs11178998 were still associated with BD even with the more conservative random effects models. CONCLUSIONS: rs4760820 and rs11178998 were identified to have strong genetic association with BD in present study though confirmation will require larger sample sizes and in additional populations.

Identification of anticitrullinated protein antibody reactivities in a subset of anti-CCP-negative rheumatoid arthritis: association with cigarette smoking and HLA-DRB1 'shared epitope' alleles.

INTRODUCTION: A hallmark of rheumatoid arthritis (RA) is the development of autoantibodies targeting proteins that contain citrulline. Anticitrullinated protein antibodies (ACPAs) are currently detected by the commercial cyclic citrullinated peptide (CCP) assay, which uses a mix of cyclised citrullinated peptides as an artificial mimic of the true antigen(s). To increase the sensitivity of ACPA detection and dissect ACPA specificities, we developed a multiplex assay that profiles ACPAs by measuring their reactivity to the citrullinated peptides and proteins derived from RA joint tissue. METHODS: We created a bead-based, citrullinated antigen array to profile ACPAs. This custom array contains 16 citrullinated peptides and proteins detected in RA synovial tissues. We used the array to profile ACPAs in sera from a cohort of patients with RA and other non-inflammatory arthritides, as well as sera from an independent cohort of RA patients for whom data were available on carriage of HLA-DRB1 'shared epitope' (SE) alleles and history of cigarette smoking. RESULTS: Our multiplex assay showed that at least 10% of RA patients who tested negative in the commercial CCP assay possessed ACPAs. Carriage of HLA-DRB1 SE alleles and a history of cigarette smoking were associated with an increase in ACPA reactivity-in anti-CCP(+) RA and in a subset of anti-CCP(-) RA. CONCLUSIONS: Our multiplex assay can identify ACPA-positive RA patients missed by the commercial CCP assay, thus enabling greater diagnostic sensitivity. Further, our findings suggest that cigarette smoking and possession of HLA-DRB1 SE alleles contribute to the development of ACPAs in anti-CCP(-) RA.

Vascular calcifications on hand radiographs in rheumatoid arthritis and associations with autoantibodies, cardiovascular risk factors and mortality.

OBJECTIVE: To examine whether vascular calcifications on hand films in RA might aid in determining mortality risk. METHODS: Hand radiographs from 906 RA patients were scored as positive or negative for vascular calcifications. Patient characteristics associated with vascular calcifications were assessed using multivariable logistic regression, and associations with mortality were examined using Cox proportional hazards regression. Cytokines and multiplex ACPA were measured in both groups. RESULTS: A total of 99 patients (11%) demonstrated radiographic vascular calcifications. Factors independently associated with vascular calcifications included diabetes [odds ratio (OR) 2.85; 95% CI 1.43, 5.66], cardiovascular disease at enrolment (OR 2.48; 95% CI 1.01, 6.09), prednisone use (OR 1.90; 95% CI 1.25, 2.91), current smoking (OR 0.06; 95% CI 0.01, 0.23) and former smoking (OR 0.36; 95% CI 0.27, 0.48) vs never smoking. In cytokine and ACPA subtype analysis, IL-4 and anti-citrullinated apolipoprotein E were significantly increased in patients with vascular calcifications in fully adjusted multivariable models. After multivariable adjustment, vascular calcifications were associated with an increase in all-cause mortality (hazard ratio 1.41; 95% CI 1.12, 1.78; P = 0.004). CONCLUSION: Vascular calcifications on hand radiographs were independently associated with increased all-cause mortality in RA. Mechanisms underpinning the associations of IL-4 and select ACPA with vascular calcifications and their utility as biomarkers predictive of cardiovascular disease risk in RA merit further study.

Eculizumab Induces Sustained Remission in a Patient With Refractory Primary Catastrophic Antiphospholipid Syndrome.

Catastrophic antiphospholipid syndrome (CAPS) is fatal in approximately 44% of patients in whom the diagnosis is made, thus demonstrating the inadequacy of current medical therapy. In this report, we discuss a 47-year-old man with a known history of primary antiphospholipid syndrome, who presented with CAPS after undergoing cholecystectomy and a treatment-refractory early relapse after development of colitis. Given the potential therapeutic efficacy of complement inhibition in antiphospholipid syndrome, the patient was administered eculizumab, a terminal complement inhibitor. Progressive clinical improvement and laboratory improvement were observed upon initiation of eculizumab. He has remained in remission for over 16 months of follow-up while on eculizumab. In conclusion, this case represents successful use of eculizumab for the treatment of primary CAPS.

Rheumatoid arthritis in the Women's Health Initiative: methods and baseline evaluation.

Second-generation assays for anti-cyclic citrullinated peptide (anti-CCP), a highly sensitive and specific marker for rheumatoid arthritis (RA), have redefined the epidemiology of RA. In the Women's Health Initiative (WHI) RA study (2009-2011), we evaluated the prevalence of anti-CCP positivity among 15,691 (10.2% of 161,808) WHI participants aged 50-79 years who reported RA. Using stored baseline specimens, we measured serum anti-CCP, rheumatoid factor (RF), and antinuclear antibody in a defined sample of 9,988 of black, white, and Hispanic women. In a subset of women, we measured plasma cytokine levels and number of copies of the human leukocyte antigen (HLA)-DRB1 (HLA-DRB1) shared epitope in DNA by means of Luminex polymerase chain reaction typing (Luminex Corporation, Austin, Texas). We validated classification of probable clinical RA in 2 clinics as anti-CCP positivity or self-reported validated use of disease-modifying antirheumatic drugs (DMARDs). The prevalence of anti-CCP positivity was 8.1%, and the prevalence of RF positivity was approximately 16.0%. DMARD use including prednisone was reported by 1,140 (11.4%) participants (841 excluding prednisone) but by 57.5% of anti-CCP-positive women. The prevalence of 2 shared epitopes was also much higher for anti-CCP-positive women (18.2%, as opposed to only 5.5% for women with anti-CCP-negative DMARD-positive RA and 6.6% for anti-CCP-negative, RF-negative DMARD nonusers). Median cytokine levels were much higher for anti-CCP-positive/RF-positive women. Women with anti-CCP-positive RA and anti-CCP-negative RA had different characteristics with regard to HLA shared epitope, cigarette smoking, and inflammation (cytokines).