Exploring the Link between Hydrodynamic Size and Immunoglobulins of Circulating Immune Complexes in Rheumatoid Arthritis Patients.

The function of immune complexes in rheumatoid arthritis (RA) is related to their composition and size. Using dynamic light scattering (DLS), we investigated the link between the RA circulating immune complex (CIC) particles' size and the CIC immunoglobulin level. In this study, 30 RA patients and 30 healthy individuals were included. IgA, IgG, and IgM were found in all analyzed CICs, but more IgA and IgG were found in RA than in control CICs. In both control and RA CICs, DLS detected 50 particles that differed in size and clustered around two size groups: with a 7.5-164 nm radius and with a 342-1718 nm radius. An increased level of IgA in RA CICs, compared to control ones, was associated with more than 50% of CIC particles. In RA, compared to the control, a higher number of CICs with 28.2 nm, 531 nm, 712 nm, and 1718 nm particles and a lower number of CICs with 78.8 nm particles were detected. This particle distribution pattern did not reflect the changes in the CIC immunoglobulin level. Thus, RA elevated CIC IgA was linked with all these particles (except the 1718 nm particle), the IgM increase was linked with 43.8 nm and 712 nm particles, and the IgG increase was linked with the 712 nm particle only. This study provides the very first data on the association between CIC particles' size, CIC immunoglobulin level, and RA. It opens the possibility that the size of CICs determined by DLS can be used as a criterion in RA diagnosis or monitoring after a large-scale study confirmation.

Dynamic light scattering analysis of immune complexes in sera of rheumatoid arthritis patients.

The size of circulating immune complexes (CICs) in rheumatoid arthritis (RA) could be an emerging criterion in disease diagnosis. This study analyzed size and electrokinetic potential of CICs from RA patients, healthy young adults, and RA patients age-matched controls aiming to establish their unique CIC features. Pooled CIC of 30 RA patients, 30 young adults, and 30 RA group's age-matched controls (middle-aged and oldеr healthy adults), and in vitro IgG aggregates from pooled sera of 300 healthy volunteers were tested using dynamic light scattering (DLS). Size distribution of CIC in healthy young adults exhibited high polydispersity. RA CIC patients and their age-matched control showed distinctly narrower size distributions compared with young adults. In these groups, particles clustered around two well-defined peaks. Particles of peak 1 were 36.1 ± 6.8 nm in RA age-matched control, and 30.8 ± 4.2 nm in RA patients. Particles of peak 2 of the RA age-matched control's CIC was 251.7 ± 41.2 nm, while RA CIC contained larger particles (359.9 ± 50.5 nm). The lower zeta potential of RA CIC, compared to control, indicated a disease-related decrease in colloidal stability. DLS identified RA-specific, but also age-specific distribution of CIC size and opened possibility of becoming a method for CIC size analysis in IC-mediated diseases.

Impaired Fibrinolysis Is Linked With Digital Vasculopathy and Onset of New Digital Ulcers in Systemic Sclerosis.

OBJECTIVE: To assess thrombin generation, fibrin formation, and structure together with the fibrinolytic status in patients with systemic sclerosis (SSc) in relation to the occurrence of digital ulcers (DUs) during the course of disease. METHODS: We studied variables of endothelial dysfunction, thrombin generation, overall hemostatic potential, and fibrin clot turbidity in plasma from 58 patients with SSc (39 with DU history and 19 DU-naïve) and 46 matched healthy controls (HCs). Fibrin structure was visualized using scanning electron microscopy (SEM). Finally, 39 patients with a history of DUs were followed for 1.5 years and the predictive value of all investigated markers for new DU onset was explored. RESULTS: Significantly enhanced endogenous thrombin potential (ETP) and prolonged clot lysis time (CLT) were found in patients with DUs compared to HCs. CLT was prolonged in patients with DUs compared to those without, showing good validity in identifying DUs with an area under the curve of 0.7 (95% CI 0.6-0.8). The levels of ETP and intercellular adhesion molecule 1 were independently associated with CLT. Over the follow-up period, 20 patients developed new DUs. CLT was prolonged (P < 0.001) in patients with new DU episodes, especially those with recurrent DUs. Regression analysis showed that the Raynaud phenomenon visual analog scale and CLT were predictors of new DUs (OR 1.1, 95% CI 1.0-1.1 and OR 1.2, 95% CI 1.1-1.3, respectively). SEM confirmed denser fibrin clots in patients with new DUs. CONCLUSION: Our results suggest that impaired fibrinolysis might have an emerging role in underlying digital vasculopathy and its progression in SSc.

Matrix Metalloproteinases-3 Baseline Serum Levels in Early Rheumatoid Arthritis Patients without Initial Radiographic Changes: A Two-Year Ultrasonographic Study.

OBJECTIVE: To investigate the association of high baseline serum levels of metalloproteinases-3 (MMP-3) with structural damage to hand and feet joints, assessed by ultrasonography (US), in patients with early, treatment-naïve rheumatoid arthritis (RA), without initial X-ray-visible erosions, during 24 months follow-up. METHODS: Sixty-three early RA (European League Against Rheumatism/American College of Rheumatology 2010), disease-modifying anti-rheumatic drugs/glucocorticoid naïve patients (mean age 53.4 ± 14.1) with symptom duration ≤12 months, had baseline serum levels of MMP-3 tested. OMERACT US group definition was used to detect the presence, as well as longitudinal diameter of erosions by US at study entry and after 24 months, at the level of wrists, metacarpophalangeal (MCP2/MCP5) joints of both hands, and fifth metatarsophalangeal joints. RESULTS: Complete data were collected from 52 out of 63 patients. High baseline serum levels of MMP-3 (MMP-3-positive) were found in 46/63 patients. 122 bone erosions in total (1.9 bone erosions/patients) were detected by US at baseline visit and 213 erosions (4.3/patients) after 24 months. MMP-3 positive patients had significantly higher total number of erosions than MMP-3-negative (p = 0.039) and higher increase in size of bone erosions in the feet but not in the hand joints after follow-up (OR 4.82 [1.23-18.9], p = 0.024; OR 1.17 [0.320-4.26], p = 0.816 respectively). CONCLUSION: After 2 years of follow-up, US assessment showed a higher number of new bone erosions in MMP-3-positive compared to MMP-3-negative patients with early RA and no visible initial radiographic changes. High baseline levels of MMP-3 predict significantly higher structural damage progression at the level of feet, but not at the level of hand joints.

Influence of Promoter Polymorphisms of the TNF-α (-308G/A) and IL-6 (-174G/C) Genes on Therapeutic Response to Etanercept in Rheumatoid Arthritis.

BACKGROUND: The study was undertaken to assess the influence of functional -308G/A TNF-α (rs 1800629) and -174G/C IL-6 (rs1800795) promoter polymorphisms on the therapeutic response to etanercept, a TNF-α blocker, in patients with rheumatoid arthritis (RA). METHODS: Seventy-three patients suffering from active RA were studied, at baseline and 6 and 12 months after therapy. The therapeutic response was estimated according to the European League Against Rheumatism response criteria. Patients were genotyped for -308G/A TNF-α and -174G/C IL-6 polymorphisms by the PCR-RFLP method, and the influence of genotype on etanercept response was assessed. RESULTS: No difference in the percentage of responders (patients who had DAS28 improvement > 1.2) between patients with the TNF-α-308GG and GA and AA genotype was detected after 6 and 12 months of treatment. After 12 months of treatment the percentage of responders was significantly increased in patients with the IL-6 -174GG genotype compared with those with the GC or CC genotype (p=0.006 by Chi-square test). Evaluation of the patients according to their combined IL-6/TNF-α genotypes showed that patients with the IL-6 -174GG / TNF-α-308GG genotype were more frequent among the responders compared to those with other combined genotypes (p=0.022 by Chi-square test). More precisely, all patients with the combined IL-6 -174GG / TNF-α-308GG genotype were responders after 12 months of etanercept treatment. CONCLUSIONS: The study suggests that patients who are genetically low TNF-α and IL-6 producers are the best responders to etanercept therapy.

Immunoserological parameters in SLE: high-avidity anti-dsDNA detected by ELISA are the most closely associated with the disease activity.

We assessed the relationship between the serum levels of antibodies against double-stranded DNA (dsDNA), C1q, nucleosomes, histones, C3 and C4 complement components with one another, with organ involvement and overall disease activity in patients with systemic lupus erythematosus (SLE). One hundred seventy-five sera from 99 patients with SLE, 31 sera of patients with other connective tissue diseases, and 20 sera from healthy blood donors were tested. SLE disease activity was assessed by modified SLEDAI-2K (M-SLEDAI-2K), not including complement and anti-dsDNA descriptors. Anti-dsDNA antibodies were measured by indirect immunofluorescence on Crithidia luciliae (CLIFT), standard enzyme-linked immunosorbent assay (ELISA) and ELISA for high-avidity antibodies. The most significant risk factor for renal involvement were anti-C1q antibodies (OR = 3.88, p < 0.05), high-avidity anti-dsDNA antibodies for polyserositis (OR = 7.99, p < 0.01), anti-histone antibodies for joint involvement (OR = 2.75, p < 0.05), and low C3 for cytopenia (OR = 11.96, p < 0.001) and mucocutaneous lesions (OR = 3.32, p < 0.01). Multiple linear regression analysis showed that disease activity in SLE could be predicted by the levels of antibodies against dsDNA determined by standard (p < 0.05) and high-avidity (p < 0.001) ELISA, and inversely associated with concentration of C3 (p < 0.001). Using stepwise method, high-avidity anti-dsDNA antibodies were found to be in the closest association to M-SLEDAI-2K. Moreover, positive test for high-avidity anti-dsDNA antibodies appeared as an independent risk factor for moderately to severely active disease (M-SLEDAI-2K>5) (OR = 5.5, p < 0.01). The presence of high-avidity anti-dsDNA antibodies represented a risk for renal, joint, and most importantly for serosal involvement. Our results suggest that simple and reliable ELISA for high-avidity anti-dsDNA antibodies is the test of good clinical utility for the assessment of global SLE activity.

Is there a difference in systemic lupus erythematosus with and without Raynaud's phenomenon?

The aim of this study was to assess the association between Raynaud's phenomenon (RP) and specific capillaroscopic findings in patients with SLE and particular clinical manifestations of the disease. A total of 79 patients with SLE were included in the study: 44 of them (43 women) with RP and 35 (32 women) age-, sex-, and disease-duration-matched patients with SLE without RP. Demographic variables, clinical manifestations, laboratory and nailfold capillaroscopy findings were compared between the two groups. Central nervous systemic involvements (P = 0.0038) and peripheral neuropathy (P = 0.0336) were significantly more common in SLE patients with RP, while secondary Sjögren's syndrome (P = 0.0363) was more common in SLE patients without RP. RP occurred in 52 % of patients before SLE onset while 48 % of patients developed RP after they had been diagnosed with SLE. Arthritis/arthralgia (P = 0.0073) was significantly more common in patients who had been diagnosed with RP before the onset of SLE, while mucosal ulcers were more common in patients who contracted RP after the onset of SLE (P = 0.0258). Enlarged capillaries (P = 0.0482), presence of avascular areas (P = 0.0476), capillary hemorrhages (P = 0.0482), and granular blood flow (P = 0.0482) were more common in patients with SLE who also suffered from RP, than in patients with SLE without RP. The frequency of normal (63.6 vs. 82.9 %, P = 0.100) and nonspecific (25 vs. 17.1 %, P = 0.5696) capillaroscopy findings were similar in either groups. Scleroderma-like pattern of capillaroscopy finding was only found in patients with RP [(11.4 %), P = 0.0482]. RP in our patients with SLE was associated with specific clinical manifestations, indicating that prognostic relevance of RP in SLE should be evaluated.

-174G/C interleukin-6 gene promoter polymorphism predicts therapeutic response to etanercept in rheumatoid arthritis.

To examine whether -174G/C interleukin-6 (IL-6) gene polymorphism, previously reported to correlate with IL-6 level, influences response to etanercept therapy in patients with rheumatoid arthritis. Seventy-seven patients with active RA were studied, at baseline and 6- and 12-month follow-up after etanercept therapy. Treatment response was estimated according to the European League Against Rheumatism response criteria. RA patients were genotyped for -174G/C IL-6 gene polymorphism by the PCR-RFLP method, and influence of genotype at this polymorphism to clinical response to etanercept was assessed. After 12 months of treatment, the percentage of responders (patients who had DAS28 improvement >1.2) was significantly increased in patients carrying the IL-6 -174G/G genotype (95.7 %) compared with those with the G/C (75.6 %) or CC (44.4 %) genotype (p = 0.006 by Chi-square test). No significant difference in the mean values of DAS28 improvement was observed between groups with different genotype. RA patients with an IL-6 -174GG genotype respond to etanercept better than patients with GC or CC genotype. This finding, if confirmed in future studies, suggests that the -174G/C IL-6 polymorphism may be a genetic marker of responsiveness to tumor necrosis factor-alpha (TNF-α) blockers in RA.

Serum DNase I activity in systemic lupus erythematosus: correlation with immunoserological markers, the disease activity and organ involvement.

BACKGROUND: Decreased activity of serum desoxyribonuclease I (DNase I) in systemic lupus erythematosus (SLE) has been reported, but its role as a biomarker in SLE is still unelucidated. METHODS: Seventy-seven SLE patients (aged 39.6 ± 13.1 years) were studied for serum DNase I activity, levels of antinuclear (ANA), anti-dsDNA [high-avidity ELISA, conventional ELISA and indirect immunofluorescence (IIF)], anti-nucleosome, anti-histone antibodies, complement components C3 and C4. SLE disease activity was evaluated by disease activity index (SLEDAI-2K). Thirty-five patients were serologically and clinically followed for 3-12 months (mean 5.6 ± 2.8). Thirty-seven healthy blood donors were the control group. RESULTS: DNase I activity in SLE patients was lower than in healthy controls (p<0.01). DNase I activity was in positive correlation with SLEDAI-2K (p<0.01), levels of ANA, anti-dsDNA, anti-nucleosome and anti-histone antibodies (p<0.01) and in negative correlation with C3 concentration (p<0.05). The highest correlation was found between DNase I activity and anti-dsDNA concentrations determined by high-avidity ELISA (r=0.624), followed by IIF (r=0.541) and conventional ELISA (r=0.405). In the follow-up study, DNase I activity also correlated with SLEDAI-2K (p<0.01). SLE patients with low DNase I activity more frequently had SLE-specific cutaneous lesions (p<0.05). CONCLUSIONS: Monitoring of DNase I activity simultaneously with SLEDAI-2K might be a useful tool in the follow-up of SLE. An increase of DNase I activity characterized relapse in most SLE patients, although it did not reach the levels of healthy individuals. A decrease of DNase I activity in SLE flare-ups might be a functional biomarker of a subset of patients with specific dysfunction of apoptotic chromatin degradation.

Effects of TNF inhibitor on innate inflammatory and Th17 cytokines in stimulated whole blood from rheumatoid arthritis patients.

BACKGROUND: Recent studies point to important roles for IL-17 and Th17 cells in sustaining chronic inflammation and articular destruction in rheumatoid arthritis (RA). We investigated the effects of TNF inhibitor on innate inflammatory and Th17 cytokines production by ex vivo lipopolysaccharide (LPS)-stimulated whole blood in patients with RA and the associations of cytokine levels in whole blood cultures with autoantibodies and markers of disease activity. MATERIALS AND METHODS: Whole blood cultures from 18 healthy volunteers and 19 RA patients on etanercept therapy were stimulated with LPS and the production of IL-6, TNF-α, IL-23, IL-17A and IL-21 was measured by ELISA. RESULTS: After stimulation with LPS, the interleukin (IL)-17A (p = 0.020) and IL-21 (p = 0.0001) secretions were significantly higher in patients with RA than in controls, while the TNF-α (p = 0.002) was significantly lower at baseline. Etanercept significantly decreased IL-21 production (p = 0.007), while IL-6 production (p = 0.005) significantly increased after 6 months of therapy. IL-21 significantly correlated with RF (r = 0.917, p < 0.01) and antimutated citrullinated vimentin antibodies (r = 0.770, p < 0.01) at baseline. Logistic regression analysis revealed that baseline IL-21 levels (p = 0.004) were significant predictors of DAS28-ESR at 6 months follow-up. DISCUSSION: Stimulation with LPS increased production of Th17 cytokines in whole blood cultures in patients with RA. Etanercept therapy decreased IL-21 secretion, while the capacity of whole blood cells to produce IL-6 increased. IL-21 production is strongly associated with the levels of autoantibodies. Our findings suggest that IL-21 production in LPS-stimulated whole blood cultures may be predictive of clinical response to etanercept treatment in patients with RA.

Systemic lupus erythematosus progressing to non-Hodgkin's lymphoma complicated by fatal hemophagocytic syndrome: case report.

Hemophagocytic syndrome (HPS) may be provoked by infections, malignancies and autoimmune diseases. We report on a 56-year-old woman with long-lasting systemic lupus erythematosus (SLE) who presented with malar rash, inflammatory livedo reticularis, fever, weight loss, pancytopenia and mild splenomegaly with cervical lymphadenopathy. She had criteria for SLE flare-up (malar rash, high antinuclear antibody titer, complement consumption, pathological urinary sediment, and retinal vasculitis). Despite high-dose glucocorticoid therapy, pancytopenia and fever worsened. Important elevations of triglycerides and ferritin were also found. Bone marrow aspirate demonstrated hemophagocytosis, which confirmed the coexistence of HPS and SLE. The treatment with glucocorticoids, immunoglobulins, cyclophosphamide, filgrastim and antimicrobial therapy was unsuccessful. After one month, the patient developed Pneumocystis jirovecii pneumonia with fatal outcome. Bone marrow biopsy, taken 5 days before death, showed high grade diffuse large B-cell (CD20+, Ki-67+) non-Hodgkin's lymphoma (DLBCL). We are the first to report the association of both SLE and non-Hodgkin's lymphoma complicated by HPS. We showed that, based on clinical and laboratory data, it was difficult to distinguish the early phase of HPS from SLE flare-up and new-onset DLBCL. Therapy of such a complex case of HPS has not been standardized, and opportunistic infections remain a difficult issue.

Tumor necrosis factor blockade differentially affects innate inflammatory and Th17 cytokines in rheumatoid arthritis.

OBJECTIVE: To evaluate the effect of a tumor necrosis factor-α (TNF-α) inhibitor (etanercept) on innate inflammatory and Th17 cytokines in patients with rheumatoid arthritis (RA). METHODS: Serum samples were collected from 40 patients with active RA refractory to conventional disease-modifying antirheumatic drugs who initiated therapy with etanercept plus methotrexate (MTX). Treatment response was assessed at Week 24 according to the European League Against Rheumatism response criteria. Serum levels of interleukin 6 (IL-6), TNF-α, IL-32, IL-23, IL-17A, IL-21, and IL-22 were measured in patients with RA and 25 healthy controls. RESULTS: Patients with RA had increased levels of IL-6 (p < 0.001), IL-32 (p < 0.001), IL-23 (p < 0.001), and a trend toward increased IL-21 in the sera compared to controls. At 24 weeks' posttreatment, followup serum samples of etanercept responders had decreased levels of IL-6 (p < 0.001) and increased IL-21 (p < 0.05) and IL-32 (p < 0.001), while there were no differences in cytokine levels in non-responders. Serum IL-6 levels were positively correlated with levels of erythrocyte sedimentation rate (r = 0.458, p < 0.01), C-reactive protein (r = 0.593, p < 0.01), and 28-joint Disease Activity Score (r = 0.432, p < 0.01) at baseline. Serum IL-21 levels were positively correlated with levels of rheumatoid factor (r = 0.513, r = 0.633, both p < 0.01) and antimutated citrullinated vimentin antibodies (r = 0.515, p < 0.01; r = 0.428, p < 0.05) at baseline and after 24 weeks of treatment with etanercept. CONCLUSION: Multiple inflammatory pathways contribute to persistent chronic inflammation in RA. In contrast to nonresponders, etanercept therapy modulated serum cytokine levels and caused a marked decrease of IL-6 levels in responders. IL-21 might be involved in the regulation of autoantibody production in RA.