Azita Sohrabian

Anti-type II collagen immune complex-induced granulocyte reactivity is associated with joint erosions in RA patients with anti-collagen antibodies

IntroductionRheumatoid arthritis (RA) patients with autoantibodies against collagen type II (CII) are characterized by acute RA onset with elevated inflammatory measures and early joint erosions as well as increased production of tumor necrosis factor-α (ΤΝF-α) by peripheral blood mononuclear cells (PBMC) stimulated by anti-CII immune complexes (IC) in vitro. Polymorphonuclear granulocytes (PMN) are abundant in RA synovial fluids, where they might interact directly with anti-CII IC in the articular cartilage, but no studies have investigated PMN responses towards anti-CII IC. The aim was to investigate whether PMN react towards anti-CII IC, and to what extent such reactivity might relate to the clinical acute onset RA phenotype associated with elevated levels of anti-CII.MethodsPMN and PBMC isolated from healthy donors were stimulated with IC made with a set of 72 baseline patient sera (24 anti-CII positive, 48 anti-CII negative) chosen from a clinically well-characterized RA cohort with two-year radiological follow-up with Larsen scoring. PMN expression of cluster of differentiation (CD)11b, CD66b, CD16 and CD32 was measured by flow cytometry, whereas PMN production of myeloperoxidase (MPO) and interleukin (IL)-17, and PBMC production of ΤΝF-α was measured with enzyme linked immunosorbent assay.ResultsPMN expression of CD11b, CD66b and MPO, and PBMC production of ΤΝF-α were upregulated whereas PMN expression of CD16 and CD32 were downregulated by anti-CII IC. CD16, CD66b, and MPO production correlated to serum anti-CII levels (Spearman’s ρ = 0.315, 0.675 and 0.253, respectively). CD16 was associated with early joint erosions (P = 0.024, 0.034, 0.046 at baseline, one and two years) and CD66b was associated with changes in joint erosions (P = 0.017 and 0.016, at one and two years compared to baseline, respectively). CD66b was associated with baseline C-reactive protein and PBMC production of ΤΝF-α was associated with baseline erythrocyte sedimentation rate, in accordance with our earlier findings. No clinical associations were observed for MPO or IL-17.ConclusionPMN responses against anti-CII IC are more closely associated with early joint erosions than are PBMC cytokine responses. PMN reactivity against anti-CII IC may contribute to joint destruction in newly diagnosed RA patients with high levels of anti-CII.

Smoking and pre-existing organ damage reduce the efficacy of belimumab in systemic lupus erythematosus

OBJECTIVES Belimumab is the first biologic drug approved for Systemic Lupus Erythematosus (SLE). Here, we aimed to investigate the effects of belimumab on clinical and serologic outcomes, and sought to identify predictors of treatment response in three Swedish real-life settings. METHODS Fifty-eight patients were enrolled at initiation of belimumab and followed longitudinally for up to 53months. Surveillance outcomes included the SLE Disease Activity Index 2000 (SLEDAI-2K), 100mm Visual Analogue Scales for Physicians Global Assessment (PGA), fatigue, pain and general health, and the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SDI). Assessment of treatment response included the SLE responder index (SRI). B lymphocyte stimulator (BLyS) levels were determined using ELISA. RESULTS SLEDAI-2K (median baseline score: 8.0; IQR: 4.0-13.8), PGA and corticosteroid use decreased during therapy, and patients reported improvements on fatigue, pain, and general health (p<0.0001 for all). SDI scores remained stable (p=0.08). Patients with baseline SDI scores >1 showed decreased probability and prolonged time to attain SRI response (HR: 0.449; 95% CI: 0.208-0.967), as did current smokers compared with non-smokers (HR: 0.103; 95% CI: 0.025-0.427). In contrast, baseline BLyS levels ≥1.2ng/mL predicted increased probability and shorter time to attain SRI response (HR: 2.566; 95% CI: 1.222-5.387). CONCLUSIONS Disease activity and corticosteroid usage decreased, patient-reported outcomes improved, and no significant organ damage was accrued during follow-up. Smoking and organ damage predicted reduced treatment efficacy. These findings might contribute to a better selection of patients who are likely to benefit from belimumab.

Development of anti-citrullinated protein antibody and rheumatoid factor isotypes prior to the onset of rheumatoid arthritis

Development of anti-citrullinated protein antibody and rheumatoid factor isotypes prior to the onset of rheumatoid arthritis

Particle enhanced turbidimetric immunoassay for the determination of urine cystatin C on Cobas c501

OBJECTIVE Urinary cystatin C has been reported to be a good marker for tubular damage and acute kidney injury. The aim of this study was to develop a high throughput assay for the quantification of urine cystatin C. METHODS Antigen-excess, imprecision, interference, linearity, recovery, sample stability and reference values were evaluated on Cobas c501. RESULTS The assay was linear over the dynamic range of the study (R²=0.9994). The total assay imprecision was below 5%. The assay recovery was estimated at 87-100%. No tendency to antigen-excess (up to 35 mg/L), nor interference with haemoglobin (1.25-10 g/L) was observed. Cystatin C was stable for 1 day at ambient temperature (19-23°C) but for 2 days at +4°C. The reference interval for cystatin C in urine was <0.414 mg/L. CONCLUSIONS The urinary cystatin C assay verified to be a reliable assay with convenient performance characteristics, enabling routine testing on clinical chemistry platforms.

Granulocyte-augmented chemokine production induced by type II collagen-containing immune complexes is mediated via TLR4 in rheumatoid arthritis patients

Rheumatoid arthritis (RA) patients with early elevations of antibodies against collagen type II (CII) have a distinct acute onset phenotype, associated with cytokine induction by surface‐bound anti‐CII‐containing immune complexes (ICs) and high C‐reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Polymorphonuclear granulocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) are abundant in the vicinity of CII in RA joints, and both PMN and PBMC reactivity against anti‐CII IC individually relate to early joint destruction and early elevation of CRP and ESR in RA. We searched for CII‐dependent mechanisms that might attract PMNs and PBMCs to RA joints. Human PBMCs and PMNs were stimulated with anti‐CII ICs and control ICs, either individually or in cocultures. Cocultured PMNs and PBMCs stimulated with anti‐CII ICs synergistically augmented production of the chemokines CXCL8, RANTES and MCP‐1, whereas downregulation was seen with control IC. This upregulation was unique to chemokines, as TNF‐α, IL‐1β, and GM‐CSF were downregulated in anti‐CII IC‐stimulated cocultures. The coculture‐associated chemokine upregulation depended on endogenous TLR4 ligand(s) and functionally active PMN enzymes, and was partially mediated by GM‐CSF. As anti‐CII levels peak around the time of RA diagnosis, this mechanism can attract inflammatory cells to joints in early RA and intensify the anti‐CII‐associated acute onset RA phenotype.

Cluster Analysis of an Array of Autoantibodies in Neuropsychiatric Systemic Lupus Erythematosus

Cluster Analysis of an Array of Autoantibodies in Neuropsychiatric Systemic Lupus Erythematosus

Normal serum levels of immune complexes in postpolio patients

Objective The pathophysiology of the postpolio syndrome is not fully understood. Increased cytokine levels in cerebrospinal fluid and peripheral blood indicate a systemic inflammatory process. Decreased cytokine levels and the clinical effect of intravenous immunoglobulin treatment further indicate an inflammatory/immunological pathogenesis. The aim of the present study was to evaluate whether an autoimmune process follows the initial infection, by means of analyzing immune complexes. Patients and methods Circulating immune complexes were analyzed from blood samples of 20 postpolio patients and 95 healthy controls. To compensate for differences in age between patients and controls, a sub-analysis was performed using only the 30 oldest controls. Tumor necrosis factor-inducing properties of polyethylene glycol-precipitated immune complexes were compared between the postpolio patients and 10 healthy controls. Results When comparing levels in postpolio patients to the whole control group, including the 30 oldest investigated, there were no statistically significant differences. No difference was found in tumor necrosis factor levels induced by immune complexes when comparing patients and controls. Conclusions There was no increase in circulating immune complex or in tumor necrosis factor-inducing effects of circulating immune complex between postpolio patients and healthy controls, indicating that the postpolio syndrome is not due to an autoimmune reaction.

High Anti-Dsdna Content in Sle Immune Complexes is associated with Clinical Remission Following Belimumab Treatment

Background Systemic lupus erythematosus (SLE) is considered driven by immune complexes (IC), and autoantibodies are supposed to participate in IC formation. Thus, the fraction of autoantibodies participating in IC formation merits interest as a diagnostic and/or prognostic marker. We have developed a technique to quantify autoantibody content in IC (Sohrabian et al. Ann Rheum Dis 2015;74(Suppl 1):A74). Objectives To evaluate quantification of autoantibodies in IC as a measure of disease activity and prognosis for response in belimumab-treated SLE patients. Methods Fifty-five SLE cases classified according to the ACR criteria were treated with belimumab and followed for one year. High disease activity was defined as SLE Disease Activity Index 2000 (SLEDAI-2K) ≥10, or as low levels of complement factors C3 and/or C4. Treatment responses were recorded using the SLE Responder Index (SRI), or as a value 0 for modified SLEDAI-2K with suppression of autoantibody and complement data (clinical remission). Low disease activity was also recorded as Lupus Low Disease Activity State (LLDAS). Response data were recorded at 3, 6 and 12 months. IC were purified from sera by binding to C1q-coated beads, and thereafter eluted. Autoantibody levels were determined in unmodified serum and in solubilised IC with addressable laser bead immunoassay (FIDIS Connective, Theradiag, Paris) for autoantibodies against dsDNA, histones, ribosomal P antigen, proliferating cell nuclear antigen (PCNA), SSA/Ro60, SSA/Ro52, SSB, Sm, U1RNP and the Sm/RNP complex. Autoantibody levels in serum and in IC were compared with Mann-Whitneys U test between patients with high and low disease activity at baseline and between patients with and without treatment response during the follow-up period. Results Antibodies against dsDNA, SSA/Ro60 and Sm/RNP were found in 65%, 54% and 43%, other antibodies with lower percentages. Low complement levels were associated with high serum anti-dsDNA (p=0.003) and anti-ribosomal P antigen (p=0.008) levels, whereas high SLEDAI-2K associated with high anti-dsDNA (p=0.02) and anti-Sm/RNP (p=0.047) levels. Serum levels of antibodies against SSA/Ro60, SSB and SSA/Ro52 were lower in patients attaining LLDAS after 6 months (p=0.02, 0.051 [trend] and 0.04, respectively); these associations were stronger for corresponding IC fractions (p=0.01, 0.005, and 0.002, respectively). Baseline levels of anti-dsDNA and anti-histones in IC associated with clinical remission ever during the follow-up period (p=0.003 and p=0.05). Low levels of anti-Sm and anti-Sm/RNP in serum but not in IC associated with clinical remission at month 6 (p=0.02 and 0.04 respectively), and for Sm/RNP also at month 3 (p=0.04). Serum levels of all antibodies except SSA/Ro52 declined during the first 6 months, most prominently for dsDNA, histones ribosomal P, PCNA and Sm/RNP (p<0.0001 for all). Levels of antibodies in IC declined only for dsDNA (p=0.048). Conclusions Autoantibody levels in serum and in IC show different associations to disease activity and to treatment response. High baseline anti-dsDNA levels in IC were most strongly associated with clinical remission, and decreased during belimumab treatment. Belimumab effect might primarily relate to autoantibodies in IC. Disclosure of Interest None declared

Measurement of Urinary Cystatin C with a Particle‐Enhanced Turbidimetric Immunoassay on Architect Ci8200

Cystatin C is a low‐molecular‐weight protein that is freely filtered by the glomerulus and catabolized after reabsorption by the proximal tubular cells in healthy subjects. Urinary cystatin C is a potential biomarker for tubular damage including acute kidney injury (AKI) in the acute phase when patients are submitted to the intensive care unit.

Clinical significance of IgM and IgA class anti-NMDAR antibodies in herpes simplex encephalitis

BACKGROUND Herpes simplex encephalitis (HSE) is a devastating disease, often leaving patients with severe disabilities. It has been shown that IgG anti-N-methyl-d-aspartate receptor (NMDAR) antibodies appear in approximately 25% of HSE patients and could be associated with impaired recovery of cognitive performance. OBJECTIVES To characterize the prevalence of IgM and IgA anti-NMDAR antibodies in HSE patients, in relation to subsequent development of IgG anti-NMDAR and correlation to cognitive performance. STUDY DESIGN A total of 48 subjects were included from a previously described cohort of patients with HSE verified by HSV-1 PCR. Cerebrospinal fluid (CSF) and serum samples drawn close to onset of disease, after 14-21 days of iv aciclovir treatment and after 90 days of follow-up, were analyzed for the presence of IgM and IgA anti-NMDAR, and related to IgG anti-NMDAR. Antibody levels were correlated to the recovery of cognitive performance, as estimated by the Mattis Dementia Rating Scale (MDRS), for a total of 24 months. RESULTS In total, 27 of 48 (56%) study subjects were anti-NMDAR positive, defined as the presence of IgG (12/48, 25%), IgM (14/48, 29%) or IgA (13/48, 27%) antibodies in CSF and/or serum. IgM or IgA anti-NMDAR did not predict subsequent IgG autoimmunization and did not correlate to cognitive outcome. IgG anti-NMDAR serostatus, but not antibody titers, correlated to impaired recovery of cognitive performance. CONCLUSIONS A majority of HSE patients develop IgG, IgM or IgA anti-NMDAR antibodies. However, the predictive value and clinical relevance of non-IgG isotypes remains to be shown in this setting.