Autoantibodies against dsDNA measured with nonradioactive Farr assay-an alternative for routine laboratories.

Autoantibodies against dsDNA are utilized for the diagnosis and prognosis of SLE as they are highly specific and correlate with disease activity/renal involvement. However, different detection methods are used in routine diagnostic laboratories. Farr radioimmunoassay (Farr-RIA) has been designated as the preferred method, since it provides very specific and at the same time quantitative results, enabling follow-up of level variations over time. Using intercalating fluorescent dsDNA dye would enable all the benefits of Farr-RIA without the radioactive material and organic solvents. To develop a modified fluorescent Farr method (Farr-FIA) and compare it to the classical Farr-RIA in regard to laboratory parameters, as well as clinical utility. Assays were tested on sera of 70 SLE patients, 78 other autoimmune patients, and 145 healthy blood donors. DNA for Farr-FIA was isolated from healthy donor, for Farr-RIA, 14C-labeled dsDNA from E. coli was used and mixed with sera in borate-buffered saline, followed by precipitation with saturated ammonium sulfate solution and centrifugation. The supernatant (S) was separated from the precipitate (P), and content of dsDNA was measured with PicoGreen (Invitrogen) in Farr-FIA or radioactive isotope in scintillation solution in Farr-RIA. The results were calculated as a ratio (P-S)/(P+S). Farr-FIA has a diagnostic sensitivity of 53% and diagnostic specificity of 100% (ROC AUC 0.781). Good correlation and agreement were shown between Farr-RIA and Farr-FIA. Also, there is good correlation between Farr-FIA and SLEDAI, comparable to that of Farr-RIA. Farr-FIA differs from Farr-RIA in the changed detection system yielding comparable results and thus could represent a nonradioactive replacement for Farr-RIA.

Insight into inflammatory cell and cytokine profiles in adult IgA vasculitis.

Immunoglobulin A vasculitis (IgAV) is an immune complex, small vessel vasculitis with dominant IgA deposits in vessel walls, predominantly affecting the pediatric population. However, adults frequently have more severe gastrointestinal tract (GIT) and renal involvements as compared to children. Our aim was to study serological and cellular biomarkers to support clinicians in their diagnosis and the course of IgAV in adult patients. This cross-sectional study included 62 adult IgAV patients and 53 healthy blood donors (HBDs). Demographic and clinical data, as well as routine laboratory tests, were meticulously analyzed. Serum levels of IL-1ß, IL-2, IL-6, IL-8, IL-9, IL-10, IL-17A, IL-23, TNF-α and serum amyloid A (SAA) were measured. Percentages of neutrophils, lymphocytes, and monocytes with neutrophil expression of L-selectin and integrin αM were determined by flow cytometry. SAA (12-fold), IL-6 (3-fold), IL-8 (2-fold), and TNF-α (2-fold) were significantly elevated in sera of adult IgAV patients compared to HBDs. There was a 16% elevation in neutrophils in IgAV patients, with IgAV neutrophils showing significantly higher CD62L surface expression. IgAV patients with GIT involvement exhibited elevated numbers of leukocytes, neutrophils, and neutrophil/lymphocyte (NLR), but lower neutrophil CD11b expression, as compared to IgAV patients without GIT. IgAV patients exhibit a low-medium grade inflammatory, neutrophil-driven response. Patients with GIT can be distinguished by their elevated NLR.

Analysis of Drug Effects on Primary Human Coronary Artery Endothelial Cells Activated by Serum Amyloid A.

BACKGROUND: RA patients have a higher incidence of cardiovascular diseases compared to the general population. Serum amyloid A (SAA) is an acute-phase protein, upregulated in sera of RA patients. AIM: To determine the effects of medications on SAA-stimulated human coronary artery endothelial cells (HCAEC). METHODS: HCAEC were preincubated for 2 h with medications from sterile ampules (dexamethasone, methotrexate, certolizumab pegol, and etanercept), dissolved in medium (captopril) or DMSO (etoricoxib, rosiglitazone, meloxicam, fluvastatin, and diclofenac). Human recombinant apo-SAA was used to stimulate HCAEC at a final 1000 nM concentration for 24 hours. IL-6, IL-8, sVCAM-1, and PAI-1 were measured by ELISA. The number of viable cells was determined colorimetrically. RESULTS: SAA-stimulated levels of released IL-6, IL-8, and sVCAM-1 from HCAEC were significantly attenuated by methotrexate, fluvastatin, and etoricoxib. Both certolizumab pegol and etanercept significantly decreased PAI-1 by an average of 43%. Rosiglitazone significantly inhibited sVCAM-1 by 58%. CONCLUSION: We observed marked influence of fluvastatin on lowering cytokine production in SAA-activated HCAEC. Methotrexate showed strong beneficial effects for lowering released Il-6, IL-8, and sVCAM-1. Interesting duality was observed for NSAIDs, with meloxicam exhibiting opposite-trend effects from diclofenac and etoricoxib. This represents unique insight into specific responsiveness of inflammatory-driven HCAEC relevant to atherosclerosis.

A concise review of significantly modified serological biomarkers in giant cell arteritis, as detected by different methods.

Giant cell arteritis (GCA) is a primary systemic vasculitis present in subjects older than 50years with involvement of large- and medium-sized arteries. Early diagnosis for GCA is essential to prevent serious complications, such as permanent vision loss and/or cerebrovascular events. Elevated inflammatory cytokines, with acute phase and other proteins dominate large- and medium-sized arteries leading to stenosis or occlusion of arterial lumen. To date, there are no reliable serological markers for monitoring GCA. The review aims to provide concise overview of published GCA studies in order to: a) identify significantly changed serological biomarkers in GCA and compare the influences of techniques for marker evaluation and b) investigate most promising markers in GCA using analyte frequency and meta-analysis.

Evaluation of phosphatidylserine-dependent antiprothrombin antibody testing for the diagnosis of antiphospholipid syndrome: results of an international multicentre study.

Objective A task force of scientists at the International Congress on Antiphospholipid Antibodies recognized that phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT) might contribute to a better identification of antiphospholipid syndrome (APS). Accordingly, initial and replication retrospective, cross-sectional multicentre studies were conducted to ascertain the value of aPS/PT for APS diagnosis. Methods In the initial study (eight centres, seven countries), clinical/laboratory data were retrospectively collected. Serum/plasma samples were tested for IgG aPS/PT at Inova Diagnostics (Inova) using two ELISA kits. A replication study (five centres, five countries) was carried out afterwards. Results In the initial study ( n = 247), a moderate agreement between the IgG aPS/PT Inova and MBL ELISA kits was observed ( k = 0.598). IgG aPS/PT were more prevalent in APS patients (51%) than in those without (9%), OR 10.8, 95% CI (4.0-29.3), p < 0.0001. Sensitivity, specificity, positive (LR+) and negative (LR-) likelihood ratio of IgG aPS/PT for APS diagnosis were 51%, 91%, 5.9 and 0.5, respectively. In the replication study ( n = 214), a moderate/substantial agreement between the IgG aPS/PT results obtained with both ELISA kits was observed ( k = 0.630). IgG aPS/PT were more prevalent in APS patients (47%) than in those without (12%), OR 6.4, 95% CI (2.6-16), p < 0.0001. Sensitivity, specificity, LR + and LR- for APS diagnosis were 47%, 88%, 3.9 and 0.6, respectively. Conclusions IgG aPS/PT detection is an easily performed laboratory parameter that might contribute to a better and more complete identification of patients with APS.

Antibodies to phosphatidylserine/prothrombin complex as an additional diagnostic marker of APS?

Antiprothrombin antibodies can be measured by ELISA using either a prothrombin/phosphatidylserine complex (aPS/PT) or prothrombin alone (aPT) as antigen. We aimed to compare the clinical features of autoimmune patients with avidity of aPS/PT and determine the diagnostic efficiency of aPS/PT and aPT for assessing antiphospholipid syndrome (APS). aPS/PT were of low (n = 9), heterogeneous (n = 31) and high (n = 8) avidity out of 48 cases. None of the samples with low avidity were positive in aPT ELISA. Among patients with heterogeneous or high avidity aPS/PT, there was a significantly greater number of patients with APS as compared to patients with low avidity (38/39 vs. 7/9; p < 0.05). No SLE patients had high avidity antiprothrombin antibodies.

Autoimmune response following influenza vaccination in patients with autoimmune inflammatory rheumatic disease.

Vaccines have undoubtedly brought overwhelming benefits to mankind and are considered safe and effective. Nevertheless, they can occasionally stimulate autoantibody production or even a recently defined syndrome known as autoimmune/inflammatory syndrome induced by adjuvants (ASIA). There is scarce data regarding autoimmune response after seasonal/influenza A (H1N1) vaccine in patients with autoimmune inflammatory rheumatic disease (AIRD). The objective of our study was therefore to determine autoimmune response in a large group of AIRD patients vaccinated against seasonal and/or H1N1 influenza. We conducted a prospective cohort study with a 6-month follow-up. Two-hundred and eighteen patients with AIRD (50 vaccinated against seasonal influenza, six against H1N1, 104 against both, 58 non-vaccinated controls) and 41 apparently healthy controls (nine vaccinated against seasonal influenza, three against H1N1, 18 against both, 11 non-vaccinated controls) were included. Blood samples were taken and screened for autoantibodies [antinuclear antibody (ANA), anti-extractable nuclear antigen (anti-ENA), anticardiolipin (aCL) IgG/IgM antibodies, anti-beta 2-glycoprotein I (anti-ß2GPI)] at inclusion in the study, before each vaccination, 1 month after the last vaccination and 6 months after inclusion. For non-vaccinated participants (patients and healthy controls) blood samples were taken at the time of inclusion in the study and 6 months later. We report that after the administration of seasonal/H1N1 vaccine there were mostly transient changes in autoantibody production in AIRD patients and in healthy participants. However, a small subset of patients, especially ANA-positive patients, had a tendency towards anti-ENA development. Although no convincing differences between the seasonal and H1N1 vaccines were observed, our results imply that there might be a slight tendency of the H1N1 vaccine towards aCL induction. Although seasonal and H1N1 vaccines are safe and effective, they also have the potential to induce autoantibodies in selected AIRD patients and healthy adults. Follow-up of such individuals is proposed and further research is needed.

Serum amyloid A in autoimmune thrombosis.

The objectives of this study were (1) to determine how levels of serum amyloid A (SAA), high sensitivity C-reactive protein (CRP) and interleukin-6 (IL-6) correlate to autoimmune diseases in patients with or without thrombosis, and (2) to discuss the parameters that influence the relative SAA values. SAA, CRP and IL-6 concentrations were determined by enzyme linked immunosorbent assay (ELISA). 84 patients with secondary antiphospholipid syndrome (SAPS), primary antiphospholipid syndrome (PAPS), systemic lupus erythematosus with antiphospholipid antibodies (SLE+aPL), SLE, venous thrombosis (VT), arterial thrombosis (AT) were compared to healthy donors (n=60). The percentages of patients above cut-off were highest in the SAPS, SLE and SLE+aPL groups. Significant differences were observed between healthy donors and inflammatory groups of patients (SAPS and SLE+aPL) in all three measured parameters. SAA and CRP were shown to be correlated to a greater extent in SAPS patients than SLE+aPL patients. In summary, this cross-sectional, retrospective, small study and accompanying clinical considerations limit the ability to make definite conclusions. SAA would not serve as a useful marker for venous, arterial thrombosis or PAPS (pro-coagulant events). It could however, be a good predictor of progression from a non-inflammatory thrombotic condition to an inflammatory one.