Extracellular Vesicles: Intercellular Communication Mediators in Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombosis, obstetric complications and the presence of antiphospholipid antibodies (aPL) that cause endothelial injury and thrombophilia. Extracellular vesicles are involved in endothelial and thrombotic pathologies and may therefore have an influence on the prothrombotic status of APS patients. Intercellular communication and connectivity are important mechanisms of interaction between healthy and pathologically altered cells. Despite well-characterized in vitro and in vivo models of APS pathology, the field of extracellular vesicles is still largely unexplored and could therefore provide an insight into the APS mechanism and possibly serve as a biomarker to identify patients at increased risk. The analysis of EVs poses a challenge due to the lack of standardized technology for their isolation and characterization. Recent findings in the field of EVs offer promising aspects that may explain their role in the pathogenesis of various diseases, including APS

Comparison and evaluation of different methodologies and tests for detection of anti-dsDNA antibodies on 889 Slovenian patients’ and blood donors’ sera

Aim To evaluate four different commercially available assays for anti-double stranded DNA (dsDNA) detection and compare them with the in-house radioimmunoassay according to Farr (FARR-RIA) in order to select the optimal primary method for use in combination with FARR-RIA. Methods Sera from 583 consecutive patients sent to our laboratory for routine diagnosis, 156 selected patients with autoimmune diseases (76 systemic lupus erythematosus [SLE] patients and 80 patients with other autoimmune diseases), and 150 blood donors were tested for anti-dsDNA antibodies with two enzyme-linked immunoassays (ELISA), two Crithidia luciliae immunoflourescence tests (CLIFT), and FARR-RIA. The specificities and sensitivities of the tests were calculated and compared. Results FARR-RIA and CLIFT 2 showed the highest specificity for SLE (100%), with CLIFT 2 showing higher sensitivity (33% vs 47%). Both ELISAs showed higher sensitivities (>53%) than FARR-RIA but lower specificities (<93%), whereas CLIFT 1 showed the lowest overall agreement with FARR-RIA. Conclusion CLIFT 2 was selected as the primary test for use in combination with FARR-RIA. The use of CLIFT 2 reduced the number of sera that needed to be tested by FARR-RIA, the time needed to report the results, and environmental toxicity, cancerogenicity, and radioactivity

Epidemiologija i dijagnostika primarnoga Sjögrenovog sindroma

This review paper contains selected aspects of Sjögren’s syndrome. It consists of epidemiology, ultrasound of salivary glands and antimuscarinic antibodies. The first part present studies aimed to determine the prevalence and the incidence of the disease with special emphasize on epidemiological studies performed in Slovenia. This is followed by the demonstration of the role of ultrasound of salivary glands in the diagnosis of Sjögren’s syndrome and the value of antimuscarinic antibodies in global assesment of the secretory failure.U preglednom su članku opisani epidemiologija, ultrazvučni nalaz slinovnica i antimuskarinska antitijela u Sjögrenovom syndromu. U prvomu su dijelu rada prikazani prevalencija i incidencija bolesti u odnosu na epidemiološke studije u Sloveniji. Prikazana je uloga ultrazvučne pretrage slinovnica u dijagnozi Sjögrenova sindroma i vrijednost antimuskarinskih antitijela u općoj procjeni sekrecijske poremetnje

Laboratory Methodology Important in the Diagnosis and Prognosis of Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is an autoimmune disease, characterized by thrombosis and pregnancy complications with persistently elevated levels of antiphospholipid antibodies (aPL). Recently, a unique mathematical calculation has been presented to assess the risk of thrombosis in patients with APS called antiphospholipid score or global antiphospholipid syndrome score (GAPSS). This new approach in the diagnosis of APS leads to the assessment of the risk of thrombosis considering the results of different aPL (lupus anticoagulants (LA), anticardiolipin antibodies (aCL), antibodies against β2GPI (anti-β2GPI), and phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT) (isotypes IgG and IgM). This chapter provides an overview of the algorithm strategy for APS diagnosis with the aims of characterizing in detail the laboratory methodology of criteria aPL (LA, aCL, and anti-β2GPI) and noncriteria aPL, such as IgA aCL and IgA anti-β2GPI, anti-domain I β2GPI, and antiprothrombin antibodies. In order to improve APS diagnosis, several new approaches in aPL detection have recently been suggested, such as multiline immunodot assay, detection of aPL by flow cytometry using beads with particular surface properties, and the newly developed automated BioPlex system technology for parallel detection of aCL and anti-β2GPI antibodies of IgG, IgA, and IgM isotypes. A completely different and promising approach in future research lies in the potential of microRNAs as biomarkers for risk of thrombosis and/or obstetric complication

Zgodnji gigantocelični arteritis

Gigantocelični arteritis (GCA) je najpogostejši primarni sistemski vaskulitis pri odraslih po 50. letu starosti v Evropi. Prizadene velike in srednje velike arterije in vnetni proces, ki zožuje ali popolnoma zapre svetlino žile, lahko dovede do hudih/trajnih ishemičnih zapletov kot so oslepitev, možganska kap ali miokardni infarkt. V zadnjem desetletju se je z vključitvijo slikovnih preiskav v diagnostični postopek pomembno skrajšal čas do prepoznave bolezni (t.i. zgodnji GCA). Pospešena obravnava bolnikov (ang. “fast track clinic”) je vodila v zmanjšanje pojavnosti najresnejših ishemičnih zapletov bolezni in znižanje stroškov zdravljenja. Vendar pa bolezen praviloma poteka kronično, s poslabšanji, kar skupaj s kroničnim glukokortikoidnem zdravljenjem vodi v kopičenje okvar organov in tkiv. Prav zato se intenzivno preučuje patogeneza bolezni, z možnostjo implementacije izsledkov kot so sodobne molekularno in celično usmerjene tarčne terapije. Glavni cilji našega preglednega članka so bili: a) analiza raziskav z navedenim časom trajanja od začetka simptomov do postavitve diagnoze, b) raziskava obetavnih molekularnih tarč za zdravljenje GCA in c) prepoznava klinično pomembnih celičnih podtipov. Najbolj obetavne tarčne molekule za tarčno zdravljenje so IL-6, IL-12/IL-23 in citototoksični z limfociti T povezan protein 4, medtem ko terapija z zaviralci TNF-α ni bila uspešna. Kliničnih raziskav z učinkovinami, usmerjenimi proti IL-17, še ni. V prispevku pa smo se dotaknili tudi drugih potencialnih terapevtskih tarč, vključno z molekulami, ki sodelujejujo v signalnih poteh

Olive Leaf Extract Attenuates Inflammatory Activation and DNA Damage in Human Arterial Endothelial Cells

Olive leaf extract (OLE) is used in traditional medicine as a food supplement and as an over-the-counter drug for a variety of its effects, including anti-inflammatory and anti-atherosclerotic ones. Mechanisms through which OLE could modulate these pathways in human vasculature remain largely unknown. Serum amyloid A (SAA) plays a causal role in atherosclerosis and cardiovascular diseases and induces pro-inflammatory and pro-adhesive responses in human coronary artery endothelial cells (HCAEC). Within this study we explored whether OLE can attenuate SAA-driven responses in HCAEC. HCAEC were treated with SAA (1,000 nM) and/or OLE (0.5 and 1 mg/ml). The expression of adhesion molecules VCAM-1 and E-selectin, matrix metalloproteinases (MMP2 and MMP9) and microRNA 146a, let-7e, and let-7g (involved in the regulation of inflammation) was determined by qPCR. The amount of secreted IL-6, IL-8, MIF, and GRO-α in cell culture supernatants was quantified by ELISA. Phosphorylation of NF-κB was assessed by Western blot and DNA damage was measured using the COMET assay. OLE decreased significantly released protein levels of IL-6 and IL-8, as well as mRNA expression of E-selectin in SAA-stimulated HCAEC and reduced MMP2 levels in unstimulated cells. Phosphorylation of NF-κB (p65) was upregulated in the presence of SAA, with OLE significantly attenuating this SAA-induced effect. OLE stabilized SAA-induced upregulation of microRNA-146a and let-7e in HCAEC, suggesting that OLE could fine-tune the SAA-driven activity of NF-κB by changing the microRNA networks in HCAEC. SAA induced DNA damage and worsened the oxidative DNA damage in HCAEC, whereas OLE protected HCAEC from SAA- and H2O2-driven DNA damage. OLE significantly attenuated certain pro-inflammatory and pro-adhesive responses and decreased DNA damage in HCAEC upon stimulation with SAA. The reversal of SAA-driven endothelial activation by OLE might contribute to its anti-inflammatory and anti-atherogenic effects in HCAEC

Verification, implementation and harmonization of automated chemiluminescent immunoassays for MPO- and PR3-ANCA detection

Objectives: Antineutrophil cytoplasmic antibody (ANCA) testing assists clinicians diagnose ANCA-associated vasculitis (AAV). We aimed to verify and harmonize chemiluminescent immunoassays for the detection of myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. Methods: An in-house ELISA, a capture ELISA, and a chemiluminescent assay QUANTA Flash on a BIO-FLASH analyzer were used to detect MPO- and PR3-ANCA in sera from 39 patients with AAV, 55 patients with various non-AAV, and 66 patients with connective tissue diseases. The results of the assays were evaluated, and their clinical performance was assessed. The precision and linearity of the QUANTA Flash assays were determined, and likelihood ratios (LRs) for AAV at diagnosis were calculated. Results: The precision and linearity of the QUANTA Flash assays were confirmed. Overall agreement between 97.5 and 98.8 % and Cohen’s kappa coefficients between 0.861 and 0.947 were observed for the results of the QUANTA Flash assays and ELISAs. The diagnostic sensitivity, specificity, and ROC analysis of the assays for AAV were statistically similar (in-house ELISA 89.7 %, 95.0 %, and 0.937capture ELISA 92.3 %, 98.3 %, and 0.939and QUANTA Flash 89.7 %, 95.9 %, and 0.972). For the QUANTA Flash assay results, the interval-specific LRs for AAV at diagnosis were: 0–8 CU had LR 0.08, 8–29 CU had LR 1.03, 29–121 CU had LR 7.76, 121–191 CU had LR 12.4, and >191 CU had LR ∞. Conclusions: The QUANTA Flash MPO and PR3 assays provide precise and consistent results and have comparable clinical utility for AAV. The calculated LRs were consistent with published LRs, confirming the utility of LRs for harmonization of ANCA results