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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.

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.

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.

Objective: To evaluate avidity of IgG anti-β2-glycoprotein I antibodies (anti-β2-GPI) in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE) in relation to thrombosis, and to demonstrate a possible affinity maturation of IgG anti-β2-GPI during the disease course. Methods: 64 sera from 32 patients (18 with primary or secondary APS, 14 with SLE without APS) and their respective IgG fractions or affinity purified anti-β2-GPI were studied by anticardiolipin (aCL) and anti-β2-GPI enzyme linked immunosorbent assay and by chaotropic assay. Results: Six, 12, and 14 patients had high, low, and heterogeneous avidity IgG anti-β2-GPI, respectively. In 12 patients an increase in antibody avidity was observed over a period of between four and 12 years. More patients with APS were in the high avidity than in the low avidity anti-β2-GPI group, while the opposite was observed for SLE alone (both p<0.05). The most common clinical feature among patients with high avidity anti-β2-GPI was thrombosis, mainly venous thrombosis (p<0.05 and p<0.02, respectively, v the low avidity anti-β2-GPI group). Conclusions: Patients with APS with or without SLE may have anti-β2-GPI of high, low, or heterogeneous avidity. High avidity anti-β2-GPI appear to be associated with thrombosis and APS, while in pure SLE low avidity anti-β2-GPI may prevail. Monitoring of avidity may help elucidate the role of anti-β2-GPI affinity maturation in the pathogenesis of APS.

Oxidative stress has been shown to play a role in modifying antibodies in favor of higher auto-immunoreactivity. We studied the immunoreactivity of oxidized IgG (oxIgG) to β2-glycoprotein I (β2GPI), six peptide sequences corresponding to amino acid clusters on its different domains, to determine their effects on human coronary artery endothelial cells (HCAEC). Human IgG was purified from seven donors, electro-oxidized and checked for immunoreactivity and avidity to β2GPI and to peptides by ELISA. Conformational stability and antibody-antigen complex formation of oxIgG was analyzed by fluorescence spectroscopy and dynamic light scattering. Resting and activated sub-confluent HCAEC were stimulated with oxIgG or IgG. Secreted cytokines were measured by ELISA. Immunoreactivity of seven oxIgG samples increased to 7.5-fold against β2GPI and to 3.8-fold against six peptides as compared to IgG. oxIgG showed low avidity “properties.” Conformational changes and exposure of protein hydrophobic regions were confirmed by an elevation in fluorescence (2.4- to 5.0-fold) on bis-ANS dye binding to oxIgG. oxIgG significantly elevated the release of GROα and IL-8 in resting and activated states of HCAEC. Oxidation alters IgG in favor of autoreactivity toward whole β2GPI and corresponding peptides on different domains of β2GPI and could lead to dysfunction of arterial endothelium by upregulation of chemokines.

Self-reproduction and the ability to regulate their composition are two essential properties of terrestrial biotic systems. The identification of non-living systems that possess these properties can therefore contribute not only to our understanding of their functioning but also hint at possible prebiotic processes that led to the emergence of life. Growing lipid vesicles have been previously established as having the capacity to self-reproduce. Here it is demonstrated that vesicle self-reproduction can occur only at selected values of vesicle properties. We treat as an example a simple vesicle with membrane elastic properties defined by a membrane bending modulus kappa and spontaneous curvature C0, whose volume variation depends on the membrane hydraulic permeability Lp and whose membrane area doubles in time Td. Vesicle self-reproduction is described as a process in which a growing vesicle first transforms its shape from a sphere into a budded shape of two spheres connected by a narrow neck, and then splits into two spherical daughter vesicles. We show that budded vesicle shapes can be reached only under the condition that Td Lpkappa C0(4)> or =1.85. Thus, in a growing vesicle population containing vesicles of different composition, only the vesicles for which this condition is fulfilled can increase their number in a self-reproducing manner. The obtained results also suggest that at times much longer than Td the number of vesicles with their properties near the \"edge\" in the system parameter space defined by the minimum value of the product Td Lpkappa C0(4), will greatly exceed the number of any other vesicles.

Objective. The objective of this study was to extend the findings of the preliminary study by measuring the avidity of IgG anti-β2-glycoprotein I antibodies (anti-β2-GPI) on a larger group of patients with primary or secondary antiphospholipid syndrome (APS) and anti-β2-GPI positive patients without APS in the frame of the European Forum on antiphospholipid antibodies (aPL). Methods. Serum from 137 patients with primary APS, APS associated with autoimmune diseases, and patients with autoimmune diseases other than APS from five EU rheumatology centres were tested for anti-β2-GPI antibodies. The 109 patients who were sera positive for anti-β2-GPI by the in-house anti-β2-GPI enzyme-linked immunosorbent assay (ELISA) at the Immunology Laboratory, UMC Ljubljana were selected for further testing on avidity with chaotropic anti-β2-GPI ELISA. Results. High, low and heterogeneous avidity IgG anti-β2-GPI was found in 32/109, 17/109 and 60/109 patients respectively. Significantly more patients with APS were in the high avidity than in the low avidity anti-β2-GPI group, while the opposite was observed for non-APS (both p < 0.001). The most common clinical feature among patients with high avidity anti-β2-GPI was thrombosis, mainly due to venous thrombosis (p < 0.01 and p < 0.001, versus low avidity anti-β2-GPI group). Conclusion. Patients with or without APS had anti-β2-GPI of high, low or heterogeneous avidity. High avidity anti-β2-GPI was associated with thrombosis and APS, while in the low avidity anti-β2-GPI group non-APS (predominantly SLE) patients prevailed. Determination of anti-β2-GPI avidity should be considered in the analytical strategies for further differentiation of patients with anti-β2-GPI antibodies.

Antibodies against β2-glycoprotein I (anti-β2GPI) are one of the hallmarks of the antiphospholipid syndrome (APS). However, they are heterogenic regarding their epitope specificity, pathogenic mechanisms and their avidity. In the current study we present some outstanding issues about avidity of anti-β2GPI antibodies. Our results confirmed that high avidity anti-β2GPI are associated with thrombosis and APS, while in low avidity anti-β2GPI group non-APS (predominantly systemic lupus erythematosus) patients prevailed.

The pathogenicity of antibodies against β2-glycoprotein I (anti-β2GPI) depends on multiple factors such as subclass type, epitope binding and avidity. Due to their large heterogeneity, their impact on antiphospholipid syndrome (APS) onset is still not fully clarified. We studied the binding characteristics of IgG anti-β2GPI with known avidity from sera of 201 autoimmune patients (87 with APS, 67 with APS associated with systemic lupus erythematosus (SLE), 47 with only SLE) to six β2GPI peptides corresponding to amino acid clusters on domains I–II, II, III and III–IV by indirect ELISA and evaluated their association with clinical features of APS. Peptides A (LKTPRV; domain I–II), B (KDKATF; domain IV) and C (TLRVYK; domain III) were derived from a hexapeptide phage display library previously shown to react with pathogenic monoclonal anti-β2GPI. Peptides D (NGPANSK; domain III), E (YNPLWFV; domain II) and F (KMDGNHP; domain III–IV) represent surface amino acid clusters on β2GPI. The percentage of patients positive for peptides were observed as follows: 30.3 % for peptide D, 28.90 % for B, 25.9 % for C, 24.9 % for E, 24.4 % for F and 10.0 % for A. The anti-peptide antibodies in studied serum samples were predominantly of heterogeneous avidity, followed by law avidity anti-peptide antibodies, whereas only a few were of high avidity. Positive and negative correlations were found between several anti-peptide antibodies and the rate of thrombosis. Our results indicated diverse reactivity of IgG anti-β2GPI to different epitopes on β2GPI. Classification of IgG anti-β2GPI into subgroups regarding epitope specificity and avidity could represent an additional tool in understanding their pathogenicity in APS.

Background: We analysed the outcomes of 726 cases of primary head and neck cancer patients managed between 1996 and 2008, including those managed in the multidisciplinary clinic or team setting (MDT) and those managed outside of an MDT by individual disciplines (non-MDT) in the same institution. Methods: Data were collected from the Hospital Based Cancer Registry and a database within the Head and Neck Cancer Clinic. Univariable comparisons and multivariable analyses were performed using a logistic regression model. Survival by staging was analysed. Comparisons of management and outcomes were made between MDT and non-MDT patients. Results: 395 patients (54%) had been managed in the MDT vs 331 patients (46%) non-MDT. MDT patients were more likely to have advanced disease (likelihood ratio χ 2 =44.7, P <0.001). Stage IV MDT patients had significantly improved 5-year survival compared with non-MDT patients (hazard ratio=0.69, 95% CI=0.51–0.88, P =0.004) and more synchronous chemotherapy and radiotherapy ( P =0.004), and the non-MDT group had more radiotherapy as a single modality ( P =0.002). Conclusions: The improved survival of MDT-managed stage IV patients probably represents both the selection of multimodality treatment and chemotherapeutic advances that these patients received in a multidisciplinary team setting by head and neck cancer specialists as opposed to cancer generalists in a non-MDT setting.