Search

Background: Although estimates of suboptimal adherence to oral corticosteroids in asthma range from 30% to 50%, no ideal method for measurement exists; the impact of poor adherence in severe asthma is likely to be particularly high. Research Questions: What is the prevalence of suboptimal adherence detected by self-reporting and direct measures? Is suboptimal adherence associated with disease activity? Study Design and Methods: Data were included from individuals with severe asthma taking part in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study and prescribed daily oral corticosteroids. Participants completed the Medication Adherence Report Scale, a five-item questionnaire used to grade adherence on a scale from 1 to 5, and provided a urine sample for analysis of prednisolone and metabolites by liquid chromatography-mass spectrometry. Results: Data from 166 participants were included in this study: mean (SD) age, 54.2 (± 11.9) years; FEV 1, 65.1% (± 20.5%) predicted; female, 58%; 37% completing the Medication Adherence Report Scale reported suboptimal adherence; and 43% with urinary corticosteroid data did not have detectable prednisolone or metabolites in their urine. Good adherence by both methods was detected in 49 of the 142 (35%) of participants in whom both methods were performed; adherence detection did not match between methods in 53%. Self-reported high adherers had better asthma control and quality of life, whereas directly measured high adherers had lower blood eosinophil levels. Interpretation: Low adherence is a common problem in severe asthma, whether measured directly or self-reported. We report poor agreement between the two methods, suggesting some disassociation between self-assessment of medication adherence and regular oral corticosteroid use, which suggests that each approach may provide complementary information in clinical practice.

Background Anticardiolipin (aCL) and anti-β2 glycoprotein I (aβ2GPI) immunoglobulin A (IgA) antiphospholipid antibodies (aPL) have shown to associate with thrombosis and pregnancy morbidity. However, inclusion of IgA aPL in the classification criteria of the antiphospholipid syndrome (APS) has been debated. We investigated the value of aCL and aβ2GPI IgA aPL in the detection of thrombosis and pregnancy morbidity in addition to the current aPL panel for APS. Methods We included 1,068 patients from eight European medical centers: 259 thrombotic APS patients, 122 obstetric APS patients, 204 non-APS thrombosis patients, 33 non-APS obstetric patients, 60 APS patients with unspecified clinical manifestations, 196 patients with autoimmune diseases, and 194 controls. aCL and aβ2GPI IgG/M/A were detected with four commercial assays and lupus anticoagulant was determined by the local center. Results Positivity for IgA aPL was found in 17 to 26% of the patients with clinical manifestations of APS and in 6 to 13% of the control population. Both aCL and aβ2GPI IgA were significantly associated with thrombosis and pregnancy morbidity. Isolated IgA positivity was rare in patients with clinical manifestations of APS (0.3–5%) and not associated with thrombosis and/or pregnancy morbidity. Addition of IgA to the current criterion panel did not increase odds ratios for thrombosis nor pregnancy morbidity. Conclusion aCL and aβ2GPI IgA are associated with clinical manifestations of APS. However, isolated IgA positivity was rare and not associated with thrombosis or pregnancy morbidity. These data do not support testing for aCL and aβ2GPI IgA subsequent to conventional aPL assays in identifying patients with thrombosis or pregnancy morbidity.

Background Classification of the antiphospholipid syndrome (APS) relies predominantly on detecting antiphospholipid antibodies (aPLs). Antibodies against a domain I (DI) epitope of anti-beta 2glycoprotein I (beta 2GPI) proved to be pathogenic, but are not included in the current classification criteria. Objectives Investigate the clinical value of detecting anti-DI IgG in APS. Patients/Methods From eight European centers 1005 patients were enrolled. Anti-cardiolipin (CL) and anti-beta 2GPI were detected by four commercially available solid phase assays; anti-DI IgG by the QUANTA Flash (R) beta 2GPI domain I assay. Results Odds ratios (ORs) of anti-DI IgG for thrombosis and pregnancy morbidity proved to be higher than those of the conventional assays. Upon restriction to patients positive for anti-beta 2GPI IgG, anti-DI IgG positivity still resulted in significant ORs. When anti-DI IgG was added to the criteria aPLs or used as a substitute for anti-beta 2GPI IgG/anti-CL IgG, ORs for clinical symptoms hardly improved. Upon removing anti-DI positive patients, lupus anticoagulant remained significantly correlated with clinical complications. Anti-DI IgG are mainly present in high-risk triple positive patients, showing higher levels. Combined anti-DI and triple positivity confers a higher risk for clinical symptoms compared to only triple positivity. Conclusions Detection of anti-DI IgG resulted in higher ORs for clinical manifestations than the current APS classification criteria. Regardless of the platform used to detect anti-beta 2GPI/anti-CL, addition of anti-DI IgG measured by QUANTA Flash (R) did not improve the clinical associations, possibly due to reduced exposure of the pathogenic epitope of DI. Our results demonstrate that anti-DI IgG potentially helps in identifying high-risk patients.

Background The anti-phospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with persistent presence of anti-phospholipid antibodies (aPL). Laboratory criteria include aPL detection by coagulation tests for lupus anticoagulant (LAC) or solid phase assays measuring anti-β2 glycoprotein I (aβ2GPI) or anti-cardiolipin (aCL) immunoglobulin (Ig) G/IgM antibodies. External quality control programs illustrate that commercially available aPL assays produce variable results. Objective We aimed to investigate the agreement and diagnostic accuracy of solid phase assays. Materials and Methods In this multi-centre study, 1,168 patient samples were tested on one site for aCL and aβ2GPI IgG/IgM antibodies by four solid phase test systems. Samples included APS patients, controls and monoclonal antibodies (MoAB) against different epitopes of β2GPI. LAC was determined by the local centre. Results aCL IgM assays resulted in the most discrepancies (60%), while aCL IgG and aβ2GPI IgM assays resulted in lower discrepancies (36%), suggesting better agreement. Discrepant samples displayed lower median aPL titers. Dependent on the solid phase test system, odds ratios (ORs) for thrombosis and pregnancy morbidity ranged from 1.98 to 2.56 and 3.42 to 4.78, respectively. Three platforms showed lower sensitivity for MoAB directed against the glycine (Gly) 40-arginine (Arg) 43 epitope of domain I of β2GPI. Conclusion Poor agreement was observed between different commercially available aCL and aβ2GPI IgG/IgM assays, hampering uniformity in the identification of aPL-positive patients. Clinical association was globally concordant between solid phase test systems considering results of the four aPL together. An assay sensitive in detecting the MoAB against Gly40-Arg43 of domain I of β2GPI reached the highest OR for thrombosis.

INTRODUCTION ANCA-associated vasculitides (AAV) is a group of rare disorders where inflammation and damage of the small blood vessels lead to dysfunction of the supplied organs. In severe flares of the disease patients may require intensive care unit (ICU) admission and treatment. The study aims to characterize Polish patients with AAV who were admitted to the ICU and compare them to the others. MATERIAL AND METHODS An observational, retrospective study based on the POLVAS - registry of Polish adult patients with AAV was carried out. Patients admitted to the ICU (ICU group) were identified and compared with the patients who did not require ICU admission (non-ICU group). Characteristics and comparison between groups were made using standard statistic descriptive methods. RESULTS 30 patients admitted to the ICU were identified among 573 cases included in the registry. All patients in the ICU group with available data were ANCA positive. The clinical manifestations related to the ICU admission were respiratory, renal and central nervous system involvement. The treatment regimen for remission induction was similar in both groups. Almost half of the patients in the ICU-group (48.3%) required dialysis, whereas in the non-ICU group it was 21.8% (P = 0.01). Infections were also more frequent in the ICU group (72.4% vs. 36.9% P < 0.001). The mortality rate among patients who needed ICU treatment was significantly higher when compared to the rest of the patients (53.6% vs. 7.8%; P < 0.001). CONCLUSIONS In the Polish AAV cohort one in twenty patients required ICU admission. This group was characterized by multiple organ involvement and high mortality.

Background Asthma prevalence is 339 million globally. ‘Severe asthma’ (SA) comprises subjects with uncontrolled asthma despite proper management. Objectives To compare asthma from diverse ethnicities and environments. Methods A cross-sectional analysis of two adult cohorts, a Brazilian (ProAR) and a European (U-BIOPRED). U-BIOPRED comprised of 311 non-smoking with Severe Asthma (SAn), 110 smokers or ex-smokers with SA (SAs) and 88 mild to moderate asthmatics (MMA) while ProAR included 544 SA and 452 MMA. Although these projects were independent, there were similarities in objectives and methodology, with ProAR adopting operating procedures of U-BIOPRED. Results Among SA subjects, age, weight, proportion of former smokers and FEV1 pre-bronchodilator were similar. The proportion of SA with a positive skin prick tests (SPT) to aeroallergens, the scores of sino-nasal symptoms and quality of life were comparable. In addition, blood eosinophil counts (EOS) and the % of subjects with EOS > 300 cells/μl were not different. The Europeans with SA however, were more severe with a greater proportion of continuous oral corticosteroids (OCS), worse symptoms and more frequent exacerbations. FEV1/FVC pre- and post-bronchodilator were lower among the Europeans. The MMA cohorts were less comparable in control and treatment, but similar in the proportion of allergic rhinitis, gastroesophageal reflux disease and EOS >3%. Conclusions ProAR and U-BIOPRED cohorts, with varying severity, ethnicity and environment have similarities, which provide the basis for global external validation of asthma phenotypes. This should stimulate collaboration between asthma consortia with the aim of understanding SA, which will lead to better management.

Background Granulomatosis with polyangiitis (GPA) is a rare granulomatous vasculitis affecting small- and medium-sized blood vessels. In optimally treated patients with long-standing disease, the common cause of death is atherosclerosis even in the absence of typical risk factors. Objective To evaluate endothelial dysfunction in GPA patients. Methods 44 patients (21 men and 23 women) diagnosed with GPA and 53 controls matched for age, sex, BMI and typical risk factors for cardiovascular diseases (22 men and 31 women) were enrolled in the study. We measured each participant’s serum levels of vascular cell adhesion molecule-1 (VCAM-1), interleukin 6 (IL-6), and thrombomodulin. We also studied flow-mediated dilatation (FMD) of the brachial artery, intima-media thickness (IMT) of the common carotid artery and aortic stiffness using echocardiography. Results Patients with GPA showed a 15.9% increase in serum levels of VCAM-1 (p = 0.01), 66% of IL-6 (p

Objectives: Neutrophil apoptosis is mandatory for resolving inflammation and is regulated by expression of pro- and anti-apoptotic genes. We studied neutrophils isolated from patients with granulomatosis with polyangiitis (GPA) to investigate apoptosis alterations and to identify transcriptional and circulating factors affecting this process. Method: We enrolled 36 patients (18 in active stage, 18 in remission) and 18 healthy controls. Circulating levels of tumour necrosis factor-α (TNF-α), granulocyte–macrophage colony-stimulating factor (GM-CSF), macrophage migration inhibitory factor, plasminogen activator inhibitor-1, interferon-γ, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, soluble Fas (sFas), sFas ligand, survivin, and pentraxin-3 (PTX3) were evaluated by enzyme-linked immunosorbent assay/Luminex; circulating apoptotic neutrophils by flow cytometry; and apoptosis-related gene transcripts by real-time polymerase chain reaction. Results: Patients had decreased fractions of circulating apoptotic neutrophils and delayed neutrophil apoptosis was present in vitro. Circulating levels of TNF-α, GM-CSF, sFas, and PTX3 were higher in GPA. Delayed neutrophil apoptosis was accompanied by decreased mRNA of pro-apoptotic genes and transcription factors (DIABLO, PMAIP1, BAX, CASP3, CASP7, RUNX3, E2F1, TP53) and increased anti-apoptotic CFLAR and BCL2A1 mRNA. TNF-α and sFas levels correlated with circulating apoptotic neutrophils and expression of apoptosis genes. Stimulation with TNF-α of neutrophils from controls significantly down-regulated E2F1 and CASP3 expression. Conclusions: Circulating neutrophils in GPA have anti-apoptotic phenotype involving both intrinsic and extrinsic pathways of apoptosis. This is accompanied by increased levels of circulating pro-survival factors (GM-CSF, TNF-α, sFas), independent of disease activity. Anti-apoptotic phenotype of neutrophils in GPA is reproduced by exposure to low concentrations of TNF-α.

Bleeding phenotype in patients with congenital factor VII (FVII) deficiency is highly variable. No direct correlation between FVII activity and bleeding tendency is noted. The aim of this study was to analyse clinical and laboratory phenotype of patients with FVII deficiency treated in one haemophilia treatment centre in Little Poland. Clinical and laboratory data of 106 patients were collected retrospectively. Bleeding symptoms were evaluated according to the Bleeding Assessment Tool. The mean FVII activity was 19.5% (range: 1.0-49.2) and the mean prothrombin time (PT) was 29.7 seconds (range: 13.2-64.8), comparable in both sexes. Activity was lower than 1% in 6.6% of individuals. The average age at diagnosis (31.2 years; range: 1-76) did not correlate with FVII activity. Half of the patients were diagnosed incidentally, mostly due to routine PT measurement. The most frequently reported symptoms were gum, nose and tooth extraction bleeds. A total of 22.6% of patients remained asymptomatic and 60.4% never required replacement therapy. Thrombotic episodes were diagnosed in five women (4.7%). In conclusion, the clinical picture of the analysed group is similar to the previously described. It should be stressed that a significant risk of bleeds, including joint haemorrhages, can be expected in patients with mildest FVII deficiency (26%) as well. The rate of thrombosis (4.7%) was similar to other reports, with all affected patients having strong prothrombotic risk factors or being intensively treated with FVII concentrate.Die klinische Ausprägung des angeborenen Faktor-VII-Mangels ist variabel. Die Blutungsneigung korreliert nur wenig mit der Faktor-VII-Aktivität. Ziel dieser Arbeit ist die Darstellung der klinischen und diagnostischen Charakteristika von Patienten mit Hypoprokonvertinämie, betreut im Hämophiliezentrum (Kleinpolen). Die retrospektive Klinik- und Labordaten von 106 Patienten waren analysiert. Blutungsneigung wurde evaluiert anhand des standardisierten Fragebogens (Bleeding Assessment Tool). Durchschnittliche Faktor-VII-Aktivität betrug 19.5% (1.0–49.2) und durchschnittliche Prothrombinzeit 29.7 second (13.2–64.8), vergleichbar bei beiden Geschlechtern. Faktor-VII-Aktivität war niedriger als 1% bei 6.6% der Patienten. Durchschnittlicher Alter zum Zeitpunkt der Diagnose (31.2 Jahre; 1–76) korrelierte nicht mit der Faktor-VII-Aktivität. Bei der Hälfte der Patienten wurde die Krankheit zufällig entdeckt, meistens infolge routinemäßiger Prothrombinzeit Messung. Die am häufigsten gemeldeten Symptome waren Zahnfleischbluten, Nasenbluten und Blutungen nach der Zahnextraktion. Bei 22.6% der Patienten trafen keine Symptome auf und 60.4% der Patienten erhalten niemals Ersatztherapie. Thrombose tritt auf in 5 Frauen (4.7%). Das klinische Bild dieser Blutungsneigung in unserer Patientengruppe ist vereinbar mit anderen Literaturberichten. Zu unterstreichen ist, dass ein signifikantes Risiko der Blutungen (auch Gelenkblutungen) besteht auch bei Patienten mit dem höchsten Faktor-VII-Spiegel (26%). Häufigkeit der Thrombose (4.7%) war ähnlich wie in früheren Studien. Alle Patienten erwiesen dabei starke Risikofaktoren für die Thrombose oder waren intensiv mit Faktor-VII-Konzentrat behandelt.

BACKGROUND Antiphosphatidylserine/prothrombin complex (aPS/PT) antibodies are recognized as a marker for antiphospholipid syndrome (APS). Dense and poorly lysable fibrin clots occur in thrombotic APS. Compact clots predict thromboembolism, but determinants of the unfavorable clot phenotype remain unknown in APS. We hypothesized that elevated aPS/PT antibodies determine unfavorable clot features. METHODS In a cohort study involving 124 consecutive patients with thrombotic APS, we measured at baseline plasma fibrin clot permeability (Ks ), efficiency of fibrinolysis (clot lysis time, CLT), and turbidity (off anticoagulation) along with immunoglobulin (Ig)G/IgM aPS/PT. During follow-up, symptomatic thromboembolic events were recorded. RESULTS Elevated IgG and IgM aPS/PT antibodies >30 international enzyme units (UI) were detected in 54.8% and 42.7% of APS patients, including 76.2% and 54% of lupus anticoagulant- (LA, n = 63) positive patients, respectively. Elevated IgG and IgM aPS/PT antibodies predicted low Ks (lower quartile, 30 UI (hazard ratio [HR] = 3.04, 95% CI 1.20-8.88) and low Ks (HR = 3.00, 95% CI 1.41-6.50). CONCLUSIONS We identified aPS/PT antibodies as a determinant of denser and poorly lysable plasma fibrin clot formation in APS patients. The association of elevated aPS/PT antibodies with thromboembolism in APS could be at least in part mediated by prothrombotic clot properties.

Purpose The aim of this study is to present the treatment modalities and associated side effects in a Polish nation-wide ANCA-associated vasculitides (AAV) patients’ cohort. Materials and methods Retrospective analysis of patients diagnosed with AAV between 1990 and 2016, included in the POLVAS registry was performed. Standard descriptive statistic methods were used with an emphasis on the treatment modalities. Results There were 625 patients diagnosed with AAV included in this study: 417 cases of granulomatosis with polyangiitis (GPA; 66.7%), 106 cases of microscopic polyangiitis (MPA; 17.0%) and 102 cases of eosinophilic granulomatosis with polyangiitis (EGPA; 16.3%). The mean age at the date of diagnosis was 50.4 (±15.7) years and the median observational period amounted to 4.0 (2.0–8.0) years. Glucocorticosteroids (GCs) were the medicaments most frequently used for remission induction (593/622; 95.3%), followed by cyclophosphamide (487/622; 78.3%), rituximab (44/622; 7.1%), and methotrexate (39/622; 6.3%). GCs were also most frequently administered for maintenance therapy (499/592; 84.3%), followed by azathioprine (224/592; 37.8%), methotrexate (136/592; 23.0%) and mycophenolate mofetil (99/592; 16.7%). The median cumulative doses of cyclophosphamide and rituximab equalled 7.99 g (4.18–14.0) and 2000 mg (1500–2800), respectively. The most commonly observed adverse events included: infections - 214/551 cases (38.8%), which were associated with the time of observation (OR = 1.05; 95% CI 1.01–1.10), the use of GCs intravenous pulses (OR = 2.76; 95% CI 1.68–4.54) and need for haemodialysis (OR = 1.73; 95% CI 1.10–2.71). Conclusions Polish patients with AAV were predominantly treated according to appropriate guidelines. The most frequent adverse events were typical for usually administered immunosuppressive treatment.

Objective: The main goal of our study was to assess the impact of vascular procedures on the activity of hemostatic and fibrinolytic pathways. Methods: We enrolled 38 patients with ≥ 45 years old undergoing surgery for abdominal aortic aneurysm or peripheral artery disease under general or regional anesthesia and who were hospitalized at least one night after the procedure. Patients undergoing carotid artery surgery and those who had acute bypass graft thrombosis, cancer, renal failure defined as estimated glomerular filtration rate < 30 ml/min/1.73m2, venous thromboembolism three months prior to surgery, or acute infection were excluded from the study. We measured levels of markers of hemostasis (factor VIII, von Willebrand factor:ristocetin cofactor [vWF:CoR], antithrombin), fibrinolysis (D-dimer, tissue plasminogen activator [tPA], plasmin-antiplasmin complexes), and soluble cluster of differentiation 40 ligand (sCD40L) before and 6-12h after vascular procedure. Results: Significant differences between preoperative and postoperative levels of factor VIII (158.0 vs. 103.3, P

Acquired haemophilia A (AHA) is a rare autoimmune disease caused by antibodies directed against clotting factor VIII. About half of cases are idiopathic, but AHA may also be secondary to autoimmune, dermatologic, or oncologic diseases. In approximately 10% of non-idiopathic cases, the disease occurs after or with the diagnosis of cancer as an extremely rare paraneoplastic syndrome. We describe the case of a 73-year-old male patient diagnosed with AHA and successfully treated with recombinant human activated factor VIIa and immunosuppression. Two and a half years later, however, the disease relapsed and a routine ultrasound revealed a liver tumour that was then diagnosed as hepatocellular carcinoma. We present this case to increase awareness that this life-threatening condition may develop years prior to the diagnosis of cancer.

Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare form of vasculitis associated with asthma and eosinophilia. Endothelial dysfunction has been well documented in other types of vasculitis but not in EGPA. Thirty patients (10 men and 20 women) diagnosed with EGPA and remaining in a remission, and 58 controls (24 men and 34 women) matched for age, sex, and body mass index, were enrolled in the study. We assessed each participants for typical risk factors of cardiovascular diseases and measured serum levels of vascular cell adhesion molecule-1 (VCAM-1), interleukin 6 (IL-6), and thrombomodulin. We also measured flow-mediated dilatation (FMD) of the brachial artery and intima-media thickness (IMT) of the common carotid artery using ultrasonography. Patients with EGPA had 20% higher serum level of VCAM-1 (p

Objective Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are rare small to medium-size vessel systemic diseases. As their clinical picture, organ involvement, and factors influencing outcome may differ between countries and geographical areas, we decided to describe a large cohort of Polish AAV patients coming from several referral centers-members of the Scientific Consortium of the Polish Vasculitis Registry (POLVAS). Methods We conducted a systematic multicenter retrospective study of adult patients diagnosed with AAV between Jan 1990 and Dec 2016 to analyze their clinical picture, organ involvement, and factors influencing outcome. Patients were enrolled to the study by nine centers (14 clinical wards) from seven Voivodeships populated by 22.3 mln inhabitants (58.2% of the Polish population). Results Participating centers included 625 AAV patients into the registry. Their distribution was as follows: 417 patients (66.7%) with GPA, 106 (17.0%) with MPA, and 102 (16.3%) with EGPA. Male-to-female ratios were almost 1:1 for GPA (210/207) and MPA (54/52), but EGPA was twice more frequent among women (34/68). Clinical manifestations and organ involvement were analyzed by clinical phenotype. Their clinical manifestations seem very similar to other European countries, but interestingly, men with GPA appeared to follow a more severe course than the women. Fifty five patients died. In GPA, two variables were significantly associated with death: permanent renal replacement therapy (PRRT) and respiratory involvement (univariate analysis). In multivariate analysis, PRRT (OR = 5.3; 95% confidence interval (CI) = 2.3–12.2), respiratory involvement (OR = 3.2; 95% CI = 1.06–9.7), and, in addition, age > 65 (OR = 2.6; 95% CI = 1.05–6.6) were independently associated with death. In MPA, also three variables were observed to be independent predictors of death: PRRT (OR = 5.7; 95% CI = 1.3–25.5), skin involvement (OR = 4.4; 95% CI = 1.02– 19.6), and age > 65 (OR = 6.3; 95% CI = 1.18–33.7). Conclusions In this first multicenter retrospective study of the Polish AAV patients, we have shown that their demographic characteristics, disease manifestations, and predictors of fatal outcome follow the same pattern as those from other European countries, with men possibly suffering from more severe course of the disease.

Essentials Antibodies to domain 1 of β2 glycoprotein I (aD1) are a subset of antiphospholipid antibodies. We evaluated the added diagnostic value of an automated aD1 assay in antiphospholipid syndrome. AD1 IgG correctly classifies patients at risk for thrombosis. Agreement between aD1 and aβ2GPI IgG is high, limiting the added value of aD1 in our setting. Click to hear Professor de Groot's perspective on new mechanistic understanding in antiphospholipid syndrome SummaryBackground Laboratory diagnosis of antiphospholipid syndrome (APS) includes lupus anticoagulant (LAC), anticardiolipin (aCL) or anti-β2 glycoprotein I (aβ2GPI) antibodies. Antibodies targeting domain 1 of β2GPI (aD1) constitute a pathogenic subset of autoantibodies. Objectives In this cohort study, we determined the clinical performance characteristics, additional diagnostic value and the contribution to APS risk stratification of an automated aD1 assay. Patients/Methods LAC, aCL, aβ2GPI and aD1 IgG were measured in 101 APS patients, 123 patients with autoimmune disorders, 82 diseased controls and 120 healthy controls. aD1 antibodies were detected by QUANTA Flash® Beta2GPI-Domain 1 chemiluminescence immunoassay. Results With a cut-off value of 20.0 CU, the aD1 IgG assay identifies APS patients in a clinically affected patient cohort with a sensitivity of 53.5% and specificity of 98.8%. It implied a high odds ratio (OR) for clinical events (OR, 17.0; 95% confidence interval [CI], 7.1–40.5). aD1 IgG did not add diagnostic value to the formal aPL panel because aβ2GPI IgG was nearly as specific but more sensitive for APS (sensitivity 56.4%) with a higher OR for clinical events (36.2; 95% CI, 11.1–117.9). High aD1 titers identify triple-positive patients and patients with thrombosis in a β2GPI-dependent LAC-positive population. Agreement between aD1 IgG and aβ2GPI IgG was high (positive and negative agreement 91.7% and 98.4%, respectively). Conclusion Detection of aD1 IgG correctly classifies patients at risk of thrombosis. However, the contribution of aD1 IgG to APS diagnosis and risk stratification depends upon the solid phase assays used for aCL and aβ2GPI detection.

Essentials Triple-positivity is associated with a high risk for a first thrombotic event and recurrence. Identification of triple-positives is dependent on the solid phase assay used. In triple-positivity, IgM only adds value in thrombotic risk stratification together with IgG. Thrombotic risk in triple-positive patients with IgM only, depends on the platform. Abstract Background The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPL). Triple-positivity (i.e. positivity for lupus anticoagulant [LAC], anti-cardiolipin [aCL] and anti-β2glycoprotein I [aβ2GPI] antibodies) is associated with a high thrombotic risk. Objectives We investigated the variability in triple-positivity detection by measuring the same samples with four commercially available solid phase assays. In addition, the added clinical value of aPL in LAC-positive patients was investigated, as well as the association of IgM triple-positivity and thrombosis. Patients/Methods We included 851 patients from seven European medical centers. Anti-CL and aβ2GPI IgG/IgM antibodies were determined by four platforms: BioPlex® 2200, ImmunoCap® EliA, ACL AcuStar® and QUANTA Lite ELISA® . Results Triple-positivity detection by solid phase assays varied, ranging from 89 up to 118 in thrombotic APS patients (n = 258), of which 86 were detected independent of the platform. Lupus anticoagulant positivity resulted in an odds ratio (OR) for thrombosis of 3.4; triple-positivity (irrespective of the isotype) increased the OR from 4.3 up to 5.2, dependent on the platform. Triple-positivity solely for the IgM isotype did not increase the OR for thrombosis compared with LAC positivity. The highest OR for thrombosis was reached for positivity for IgG and IgM aβ2GPI and aCL (8.6 up to 28.9). Conclusions Triple-positivity proved to be highly associated with thrombosis, but identification is assay dependent. Within triple-positivity, IgM antibodies only have an added clinical value in patients positive for IgG antibodies.

Summary The objective of this study was to investigate the mechanisms of T helper type 17 (Th17) expansion in lupus nephritis (LN) patients, and to determine whether or not it is associated with impaired function of regulatory T cells (Treg). Major effector subsets of peripheral blood CD4+ T cells were assessed by flow cytometry in 33 LN patients with different activity of the disease and 19 healthy controls. The percentage of circulating Th17 cells was increased in LN (median = 1·2% of CD4+ compared to 0·6% in the control group, P

Severe asthma patients with a significant smoking history have airflow obstruction with reported neutrophilia. We hypothesise that multi-omic analysis will enable the definition of smoking and ex-smoking severe asthma molecular phenotypes.The U-BIOPRED cohort of severe asthma patients, containing current-smokers (CSA), ex-smokers (ESA), nonsmokers and healthy nonsmokers was examined. Blood and sputum cell counts, fractional exhaled nitric oxide and spirometry were obtained. Exploratory proteomic analysis of sputum supernatants and transcriptomic analysis of bronchial brushings, biopsies and sputum cells was performed.Colony-stimulating factor (CSF)2 protein levels were increased in CSA sputum supernatants, with azurocidin 1, neutrophil elastase and CXCL8 upregulated in ESA. Phagocytosis and innate immune pathways were associated with neutrophilic inflammation in ESA. Gene set variation analysis of bronchial epithelial cell transcriptome from CSA showed enrichment of xenobiotic metabolism, oxidative stress and endoplasmic reticulum stress compared to other groups. CXCL5 and matrix metallopeptidase 12 genes were upregulated in ESA and the epithelial protective genes, mucin 2 and cystatin SN, were downregulated.Despite little difference in clinical characteristics, CSA were distinguishable from ESA subjects at the sputum proteomic level, with CSA patients having increased CSF2 expression and ESA patients showing sustained loss of epithelial barrier processes.

Plasmapheresis is a therapeutic method based on removal of high molecular weight particles from blood. It is used in a variety of clinical entities in which pathogenic role of such particles has been proven e.g. ANCA-associated vasculitides (granulomatosis with polyangiitis [GPA] and microscopic polyangiitis [MPA]). Efficacy of plasmapheresis in ANCA antibodies removal and its impact on disease activity has not been adequately investigated so far. Influence of antibodies levels on disease activity also remains unknown.Analysis of plasmapheresis effect on serum ANCA levels in patients with ANCA-associated vasculitides.Seven patients with diagnoses of ANCA-associated vasculitides were enrolled in the study between November 2015 and April 2016. All of them underwent plasmapheresis procedures. Serum ANCA levels were measured before and after plasmaphereses using fluoroimmunoenzymatic assay (FEIA). Disease activity was assessed using Birmingham Activity Vasculitis Score; BVAS ver. 3 before first plasma exchange procedure.Patients in disease exacerbation (BVAS 5-10) with positive ANCA antibodies were enrolled in the study: 2 patients with GPA and 5 patients with MPA. Number of performed plasmapheresis procedures ranged from 5 to 7 (median=7). Decrease of serum antibodies concentration was observed in all patients (mean decrease of 88.3% (±10.1%)) with statistically significant difference in mean antibodies concentration before and after plasmapheresis procedures (113.0 vs 15.4; p=0.014).Plasmapheresis is an effective method of ANCA antibodies removal in patients in disease exacerbation.

IntroductionRenal involvement is one of the most serious manifestations of systemic lupus erythematosus, but non-invasive assessment of inflammatory response in kidneys is challenging. In this study we aimed to validate markers of active lupus nephritis (LN) using urine immune profiling.MethodsUrine and serum cytokines (17-plex array) and urine mRNA expression (∼40 immune and glomerular injury genes) were measured in LN patients with active disease ( n = 17) during remission ( n = 16) and in healthy subjects ( n = 18).ResultsUrine and serum levels of CCL2, CCL5 and CXCL10 were elevated in active LN as compared with disease remission (best discrimination for urine CXCL10 and CCL2) and correlated with LN activity. In the active disease, urinary cell transcriptome showed marked upregulation of proinflammatory cytokines (e.g. TNF, CCL2, CCL5, CXCL10), and type-1 immunity-related genes (e.g. CD3G, CD4, TBX21, IFNG). An active pattern of gene expression was also observed in four patients in remission, who had moderately increased urinary leucocyte count. Two patients from this group developed renal exacerbation during the following 3 months. Markers of type-17 immune axis (e.g. IL-17A) were not significantly increased in active LN.ConclusionsActive LN patients were characterized by marked increase of proinflammatory mediators in the urine. Urine cytokines (CCL2 and CXCL10) and type-1 T-cell-related gene markers in the urine sediment had similar diagnostic performance in detection of active LN.

Purpose Systemic autoimmune diseases are a heterogeneous group of disorders associated with dysfunction of multiple organs and unpredictable course. Complicated management and treatment become even more challenging when patients require critical care. This study aims to compare outcomes of small-vessel vasculitides (SVV) and other systemic autoimmune diseases (SAD) patients admitted to the intensive care unit (ICU). Materials and methods Retrospective, observational study conducted in the ICU of Allergy and Immunology Department at the University Hospital in Krakow, Poland, between years 2001–2014, with 5-years follow-up and no lost to follow-up patients. Results 74 patients with autoimmune diseases were enrolled in the study - 23 with SVV and 51 with SAD. Patients in the SVV group achieved higher scores in APACHE II and III SAPS II and SOFA at ICU admission. The SVV patients required renal replacement techniques, blood products transfusion and immunosuppressive treatment more often. SVV patients had higher ICU mortality (60.9% vs. 35.3%, p = .04), however after discharge from ICU, in long term follow-up (1 year and 5 years) mortality was similar in both studied groups. Conclusions Among systemic autoimmune diseases small vessel vasculitides appear to be associated with the highest ICU mortality, higher requirement for advanced procedures and aggressive immunosuppressive therapy.

The aim of this study is to assess the relationship between autoimmunity and endothelial activation/damage (ICAM-1 and vWF serum levels) and the degree of prothrombotic activity (thrombin-antithrombin complexes-TAT serum levels) in SLE. In 60 clinically stable SLE patients, levels of the following parameters were estimated in their serum: lupus anticoagulant (LA), anticardiolipin antibodies in both IgG and IgM classes (aCL-IgG and aCL-IgM, respectively), antiβ2GPI antibodies in both IgG and IgM classes (antiβ2GPI-IgG and antiβ2GPI-IgM, respectively), ICAM, von Willebrand factor (vWF), TAT, CRP, C3c, C4, and IL-6. ICAM-1 values exceeded the upper reference limit in 9 (15%) patients. vWF levels were increased in 21 (35%) patients. In all patients with elevated ICAM-1 values, vWF were also increased. TAT concentrations were elevated in 12 (20%) people. ICAM-1 were significantly higher in patients with elevated aCL-IgM ( 30 MPL vs ≤ 30 MPL; p 0.05). Similarly, ICAM-1 were significantly higher in patients with elevated antiβ2-GPI-IgM ( 20 SMU vs ≤ 20 SMU; p 0.05). There was no significant difference in ICAM-1 levels in relation to LA-positivity. vWF were not significantly different in relation to antiphospholipid antibodies nor the inflammation marker levels. TAT were significantly higher in patients with elevated aCL-IgM ( 30 MPL vs ≤ 30 MPL; p 0.05). In one third of young patients with stable SLE, signs of endothelial activation/damage were found, as shown by elevated plasma ICAM-1 or vWF. Increased prothrombotic tendency manifested by elevated TAT was found in one fifth of the patients. Elevated anticardiolipin (IgM) and anti-β2-glycoprotein I (IgM) antibodies influence endothelial dysfunction and enhance prothrombotic state.

Objectives APS is associated with arterial and venous thrombosis. The unfavourable fibrin clot phenotype, including formation of dense and poorly lysable clots, has been reported in thrombotic APS. We investigated whether abnormal plasma clot properties are predictive of recurrent thromboembolism in APS. Methods We followed 126 consecutive patients with thrombotic APS and 105 control subjects, without APS, matched for thrombotic events. Plasma fibrin clot permeability (Ks), turbidity measurements and clot lysis time were evaluated ⩾5 months after a thrombotic event. The primary composite end point was symptomatic recurrent venous thromboembolism, ischaemic stroke and/or myocardial infarction. Results During follow-up (median, 62 months; range 46-74 months; 1183.2 patient-years), the primary outcome was observed in 33 (26.2%) APS patients and 16 (15.2%) controls, including 25 (19.8%) and 14 (13.3%) subjects with recurrent venous thromboembolism, respectively. Reduced Ks and prolonged clot lysis time predicted recurrent thromboembolic events in APS patients [per 1 × 10-9 cm2: hazard ratio (HR) = 0.37; 95% CI: 0.24, 0.56; and per 10 min: HR = 1.20; 95% CI: 1.01, 1.40, respectively] and in controls (per 1×10-9 cm2: HR = 0.23; 95% CI: 0.11, 0.42; and per 10 min: HR = 1.51; 95% CI: 1.08, 2.16, respectively). A multivariate analysis showed that positive IgG and IgM anti-β2 glycoprotein I antibodies, withdrawal of anticoagulation, lower platelet count and reduced Ks predicted thromboembolic events in APS patients. Conclusion Formation of denser fibrin networks could be a novel risk factor for recurrent thromboembolism in APS, which highlights the importance of fibrin phenotype in thrombotic disorders.

Leukocytoclastic vasculitis (LCV) is a heterogenous group of disorders that may manifest as a mild disease isolated to the skin or be a part of life-threatening systemic vasculitis. According to the 2012 Chapel Hill Consensus Conference nomenclature, patients presenting symptoms of LCV confined only to the skin should be defined as suffering from a single-organ cutaneous small vessel vasculitis (SoCSVV). SoCSVV is a benign disease with a good clinical outcome but with a significant risk of relapse and skin ulcer formation. The aim of the current study was to characterize SoCSVV and to identify factors that may be associated with the risk of recurrence and skin ulcers. Medical records of patients with LCV hospitalized at the Department of Dermatology at University Hospital in Cracow in the years 2010 to 2015 were analyzed. A total of 24 patients fulfilled criteria of SoCSVV. Drugs and preceding infections were identified as precipitating factors in 40% and 20% of cases, respectively. Skin lesions other than palpable purpura (i.e., macules, urticarial vasculitis, or ulcers) were identified in almost half of the patients. Interestingly, the presence of macules independently increased the risk of skin ulcer formation (odds ratio = 16; 95% confidence interval: 1.5–176.6; P = 0.0075) in the multivariate logistic regression analysis. One-quarter of patients with SoCSVV experienced relapse during the 6-month follow-up. The greater number of affected skin areas was an independent risk factor of recurrence (odds ratio = 5; 95% confidence interval: 2–45; P = 0.02). SoCSVV was usually associated with drugs and preceding infections. The disease relapses in approximately one-quarter of the patients. The more severe the skin involvement in the course of SoCSVV, the higher is the risk of recurrence.

The objective of our study was to evaluate the T-helper (Th) and regulatory T (Treg) cell profile in ANCA-positive granulomatosis with polyangiitis (GPA) and its relation to disease activity. In a prospective study, we studied two groups of GPA patients: (i) disease flare (active-GPA, BVAS>6, n = 19), (ii) sustained remission (≥ 1-year prior enrollment, inactive-GPA, BVAS = 0, n = 18). 24 age-sex matched healthy subjects served as controls. Active-GPA patients were followed for 6 months and reevaluated during remission (early remission; n = 13). We analyzed subsets of Th-cells (flow cytometry), production of signature cytokines by in vitro stimulated lymphocytes, and broad spectrum of serum cytokines (Luminex). In all GPA patients we observed expansion of effector Th17 cells, and increased production of IL-17A by in vitro stimulated T cells, as compared to controls. Disease flare was characterized by marked reduction in Treg cells, whereas in sustained remission we showed expansion of both Treg and Th2 subset. Finally, analyzing the cytokine profile, we identified CCL23 and LIGHT, as potential biomarkers of active disease. We conclude that in GPA, expansion of Treg and Th2 lymphocytes in parallel to increased Th17 response is a characteristic feature of sustained remission. In contrast, Treg cells are markedly decreased in disease flare.

Antiphospholipid syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies (aPL) in patients with thrombosis and/or pregnancy morbidity. In APS patients anti-domain 1 β2-glycoprotein I (anti-D1 β2GPI) IgG antibodies correlate strongly with thrombosis and to the lesser extent, with pregnancy complications. The aim of this study was to assess clinical utility of the anti-D1 β2GPI antibodies in the diagnosis and risk stratification of antiphospholipid syndrome.In this retrospective study 202 autoimmune patients were studied (primary APS - 58, secondary - 45 SLE - 99). Anticardiolipin (aCL) and anti-βThe highest anti-D1 values were observed in triple positive patients as compared to patients with other antiphospholipid antibody profiles. A strong correlation was found between levels of anti-D1 IgG and a β2GPI IgG antibodies for all patients analyzed (Spearman's ρ=0.87; p0.0001). Anti-D1 IgG antibodies increase specificity resulting from classic aPL positivity but at the expense of sensitivity. Anti-D1 test does not add accuracy in predicting APS thrombotic complications on the top of accuracy offered by classic aPL tests and their profiles.Anti-D1 IgG antibodies did not add diagnostic power to the standard laboratory aPL tests as assessed by this retrospective study. A true clinical significance of anti-D1 antibodies in thrombotic risk stratification of aPL positive patients will require a properly designed clinical prospective trials.

INTRODUCTION Inherited deficiencies of natural anticoagulants such as antithrombin (AT; gene: SERPINC1), protein C (PC; PROC), and protein S (PS; PROS1), with the prevalence in the general European population of 0.02% to 0.17%, 0.2% to 0.3%, and 0.5%, respectively, are associated with increased risk of thromboembolic events. Only a few case reports of Polish deficient patients with known causal mutations have been published so far. OBJECTIVES The aim of the study was to characterize the frequency of SERPINC1, PROC, and PROS1 mutations and their thromboembolic manifestations in patients with AT, PC, or PS deficiencies, inhabiting southern Poland. PATIENTS AND METHODS Ninety unrelated patients (mean [SD] age, 40.1 [13.2] years) with AT (n = 35), PC (n = 28), or PS (n = 27) deficiencies, with a history of venous 73 (81%) or arterial 17 (19%) thromboembolism, were screened for mutations using the Sanger sequencing or multiplex ligation‑dependent probe amplification. RESULTS Twenty mutations (29%) described here were new, mostly in the SERPINC1 and PROC genes. Missense mutations accounted for 84% of all mutations in the PROC gene and approximately 50% of those in the SERPINC1 and PROS1 genes. In all 3 genes, the ratio of nonsense and splice-site mutations was 8% to 31% and 8% to 23%, respectively. The mutation detection rate was 90% for AT or PC when anticoagulant activity was below 70%, while for the PROS1 gene, the rate reached 80% at the free PS levels below 40%. CONCLUSIONS To our knowledge, this is the largest cohort of Polish patients deficient in natural anticoagulants and evaluated for the causal genetic background. Several new Polish detrimental mutations were detected, mostly in AT- and PC‑deficient patients.

Myocardial injury after non-cardiac surgery (MINS), a complication with unclear pathogenesis, occurs within the first 30 days after surgery and worsens prognosis. Hypercoagulability induced by surgery might contribute to plaque rupture, with subsequent thrombosis and myocardial injury. This study assessed haemostatic markers before surgery and evaluated their association with MINS.This is a substudy of VISION, a prospective cohort study of perioperative cardiovascular events. Of 475 consecutive vascular surgery patients, 47 (9.9%) developed MINS, defined as postoperative high-sensitivity troponin ≥50 ng litre -1 , with ≥20% elevation from the preoperative concentration. The control group consisted of 84 non-MINS patients matched for patient characteristics and co-morbidities. The following preoperative markers of hypercoagulability and fibrinolysis were measured: antithrombin, factor VIII activity, von Willebrand factor concentration and activity, fibrinogen, D-dimer, plasmin-antiplasmin complex, and tissue plasminogen activator. Moreover, C-reactive protein and CD40L concentrations were measured to assess inflammatory activity.Patients with MINS compared with the non-MINS group had a significantly higher concentration of factor VIII (186 vs 155%, P =0.006), von Willebrand factor activity (223 vs 160%, P0.001), von Willebrand factor concentration (317 vs 237%, P =0.02), concentrations of fibrinogen (5.6 vs 4.2 g litre -1 , P =0.03), D-dimer (1680.0 vs 1090.0 ng ml -1 , P =0.04), plasmin-antiplasmin complex (747 vs 512 ng ml -1 , P =0.002) and C-reactive protein (10 vs 4.5 mg litre -1 , P =0.02) but not antithrombin (95 vs 94%, P =0.89), tissue plasminogen activator (11 vs 9.7 ng ml -1 , P =0.06) and CD40L (8790 vs 8580 pg ml -1 , P =0.73).Preoperative elevation of blood markers of hypercoagulability in patients undergoing vascular surgery is associated with a higher risk of MINS.NCT00512109.

BACKGROUND: Asthma is a heterogeneous disease in which there is a differential response to asthma treatments. This heterogeneity needs to be evaluated so that a personalised management approach can be provided.OBJECTIVES: We stratified patients with moderate-to-severe asthma based on clinico-physiological parameters and performed an -omics analysis of sputum.METHODS: Partition-around-medoid clustering was applied to a training set of 266 asthma participants from the European U-BIOPRED adult cohort using 8 pre-specified clinic-physiological variables. This was repeated in a separate validation set of 152 asthmatics. The clusters were compared based on sputum proteomic and transcriptomic data.RESULTS: Four reproducible and stable clusters of asthmatics were identified. The training set cluster T1 consists of well-controlled moderate-to-severe asthmatics, while cluster T2 is a group of late-onset severe asthmatics with history of smoking and chronic airflow obstruction. Cluster T3 is similar to cluster T2 in terms of chronic airflow obstruction but is composed of non-smokers. Cluster T4 is predominantly composed of obese female uncontrolled severe asthmatics with increased exacerbations, but with normal lung function. The validation set exhibited similar clusters, demonstrating reproducibility of the classification. There were significant differences in sputum proteomics and transcriptomics between the clusters. The severe asthma clusters, T2, T3 and T4, had higher sputum eosinophilia than T1 with no differences in sputum neutrophil counts, exhaled nitric oxide and serum IgE levels.CONCLUSION: Clustering based on clinico-physiological parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.CLINICAL IMPLICATIONS: The definition of four distinct clusters of asthma linked to different pathobiological pathways provides a better template for the phenotyping and personalised treatment of severe asthma, where high unmet needs remain.CAPSULE SUMMARY: Unsupervised clustering of asthma on clinical features alone has led to the definition of four phenotypes. Sputum 'omics' analysis has revealed different biological pathways pointing towards potential new treatments.

Human rhinoviruses (RVs) are a major cause of exacerbations in asthma and other chronic airway diseases. A characteristic feature of asthmatic epithelium is goblet cell metaplasia and mucus hypersecretion. Bronchial epithelium is also an important source of lipid mediators, including pro- and antiinflammatory eicosanoids. By using air-liquid interface cultures of airway epithelium from patients with asthma and nonasthmatic control subjects, we compared RV16 replication-induced changes in mRNA expression of asthma candidate genes and eicosanoid production in the epithelium with or without IL-13-induced mucus metaplasia. Mucus metaplastic epithelium was characterized by a 20-fold less effective replication of RV16 and blunted changes in gene expression; this effect was seen to the same extent in patients with asthma and control subjects. We identified ciliary cells as the main target for RV16 by immunofluorescence imaging and demonstrated that the numbers of ciliary cells decreased in RV16-infected epithelium. RV16 infection of mucociliary epithelium resulted in overexpression of genes associated with bronchial remodeling (e.g., MUC5AC, FGF2, and HBEGF), induction of cyclooxygenase-2, and increased secretion of prostaglandins. These responses were similar in both studied groups. These data indicate that structural changes associated with mucus metaplasia renders airway epithelium less susceptible to RV infection. Thus, exacerbations of the lung disease caused by RV may result from severe impairment in mucociliary clearance or activation of immune defense rather than from preferential infection of mucus metaplastic epithelium. Repeated rhinoviral infections of compromised epithelium may contribute to the remodeling of the airways.

Background: Severe asthma is a heterogeneous condition, as shown by independent cluster analyses based on demographic, clinical, and inflammatory characteristics. A next step is to identify molecularly driven phenotypes using “omics” technologies. Molecular fingerprints of exhaled breath are associated with inflammation and can qualify as noninvasive assessment of severe asthma phenotypes. Objectives: We aimed (1) to identify severe asthma phenotypes using exhaled metabolomic fingerprints obtained from a composite of electronic noses (eNoses) and (2) to assess the stability of eNose-derived phenotypes in relation to within-patient clinical and inflammatory changes. Methods: In this longitudinal multicenter study exhaled breath samples were taken from an unselected subset of adults with severe asthma from the U-BIOPRED cohort. Exhaled metabolites were analyzed centrally by using an assembly of eNoses. Unsupervised Ward clustering enhanced by similarity profile analysis together with K-means clustering was performed. For internal validation, partitioning around medoids and topological data analysis were applied. Samples at 12 to 18 months of prospective follow-up were used to assess longitudinal within-patient stability. Results: Data were available for 78 subjects (age, 55 years [interquartile range, 45-64 years]; 41% male). Three eNose-driven clusters (n = 26/33/19) were revealed, showing differences in circulating eosinophil (P =.045) and neutrophil (P =.017) percentages and ratios of patients using oral corticosteroids (P =.035). Longitudinal within-patient cluster stability was associated with changes in sputum eosinophil percentages (P =.045). Conclusions: We have identified and followed up exhaled molecular phenotypes of severe asthma, which were associated with changing inflammatory profile and oral steroid use. This suggests that breath analysis can contribute to the management of severe asthma.

The aim of this pilot study was to evaluate changes in the concentration of prostaglandin E2 (PGE2) in induced sputum supernatant in 3 groups: sub- jects with NSAID-exacerbated respira- tory disease (NERD), aspirin tolerant asthma (ATA) and healthy controls (HC), before and after oral aspirin chal- lenge test. The study was conducted in the years 2014-2015 at the Clinical Department of the Pulmonology Clinic at the University Hospital in Cracow. 43 patients were enrolled in the study (NERD - n = 15, ATA - n = 15 and HC - n = 13). All of them underwent a placebo-controlled oral aspirin challenge. Sputum was induced 24 hours before the challenge and immediately after the test. Induced sputum was processed in order to obtain cystospin slides to depict inflammatory cell patterns and supernatants, in which PGE2 was measured. The concentration of PGE2 was determined using mass spectrometry coupled with gas chromatography (gas chromatography/mass spectrometry - GC/MS). After aspirin challenge, the concentration of PGE2 in induced sputum supernatant decreased in both asthmatics hypersensitive to aspirin (p = 0.01) and those who tolerated aspirin well (p = 0.17). The change in the healthy control group was not statistically significant. These results support the cyclooxygenase theory of PGE2 inhibition by aspirin. However, the mechanism of bronchoconstriction after aspirin administration alone in patients with NSAID-exacerbated respiratory disease remains unclear.

Introduction We investigated whether primary antiphospholipid syndrome (PAPS) is characterized by a deficiency in immunoregulatory pathways, a phenomenon recently implicated in the pathogenesis of autoimmune diseases. Methods Serum levels of immunoregulatory (e.g. IL-10 and TGF-β1) and proinflammatory (e.g. IL-17A) cytokines were measured in PAPS, systemic lupus erythematosus (SLE) with secondary APS (SAPS), or without APS, and in healthy controls ( n = 40 in each group). In a subgroup of PAPS patients we also compared phenotype and function (flow cytometry) of regulatory T-cells (Treg) and cytokine production by effector T-cells. Results Our major finding was decreased levels of TGF-β1 in PAPS and SAPS as compared to SLE without APS and controls. TGF-β1 was the lowest in PAPS patients showing high levels of aPL IgG with significant negative correlation with the titer. SLE patients were characterized by lower serum levels of IL-2 and increased IL-17A, as compared to the other groups. The numbers of circulating Treg cells and their phenotype (e.g. FoxP3 isoforms) were not disturbed in PAPS. However, surface expression of latency associated peptide (binds TGF-β) in activated FoxP3 + cells and in vitro production of TGF-β1 were decreased in PAPS patients with high titers of aPL IgG. Moreover, frequencies of cytokine producing effector T-helper cells (including Th17) were significantly elevated in this group. Conclusions PAPS patients with high titers of aPL IgG antibodies were characterized by decreased systemic levels of TGF-β1 and its impaired production in vitro, suggesting impaired immunoregulation and enhanced adaptive autoimmune responses leading to the production of aPL antibodies.

Introduction. Renal involvement in systemic lupus erythematosus (SLE) is associated with production of antibodies to double stranded DNA, deposition of immune complexes and organ damage. These processes have been linked with abnormalities in B- and T-cell memory compartments. The aim of the study was to analyze subsets of peripheral memory B-cells and T-cells in lupus nephritis (LN) patients. Material and methods. We used multicolor flow cytometry to analyze major memory subsets of peripheral blood B-cells (defined by CD27, IgD and CD21) and T-cells (CD45RA, CD45RO, CCR7) in 32 patients with active or inactive LN, and 23 control subjects. Results. Lupus nephritis patients were characterized by increased percentage of immature/early-transitional B-cells (CD27-IgD+CD21-), higher frequency of activated switched memory (SM, CD27+IgD-CD21-) and exhausted memory B-cells (CD27-IgD-), and decrease in non-switched memory (NSM, CD27+IgD+) B-cells. CD21 low subsets (immature and activated B-cells) were particularly expanded in patients with active disease. In both groups of LN patients we observed decline in the absolute count of NSM B-cells. It was paralleled by lymphopenia in naive CD4+ T-cell compartment and increase in the frequency of effector memory T-cells, and these changes were more pronounced in active LN. Conclusions. B-cell memory compartment in LN is deficient in NSM cells and during active disease it is further skewed towards SM and exhausted memory phenotypes, most likely as a cause of chronic antigenic stimulation. Parallel changes in T-helper cell subsets suggest a similar mechanism of SLE-related lymphopenia for both B-cell and T-cell compartment.

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 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.

BACKGROUND Thrombotic events in antiphospholipid syndrome (APS) involve venous and arterial circulation with the possible involvement of coronary or pulmonary microcirculation. OBJECTIVES To evaluate the influence of antiphospholipid antibodies (aPL) and on myocardial ischaemia assessed by single-photon emission computerized tomography (SPECT), coronary atherosclerosis assessed by multidetector computerized tomography (MDCT) and pulmonary pressure assessed by transthoracic echocardiography (TTE) in patients with primary antiphospholipid syndrome (PAPS). MATERIAL AND METHODS TTE, SPECT (Tc 99m sestamibi) and MDCT-based coronary calcium scoring were performed in 26 consecutive PAPS patients (20 females, 6 males, aged 20-61, mean 39.7) without any signs of other autoimmunological disease and without clinical symptoms of heart disease. RESULTS Out of 26 patients, TEE showed normal left and right ventricle function in 25 (96.2%) and elevated (≥ 30 mm Hg) right ventricle systolic pressure in 7 (26.9%) patients. SPECT revealed myocardial perfusion defects in 15 (57.7%) patients: exercise-induced in 6 (23.1%) and persistent in 11 (42.3%). MDCT revealed coronary calcifications in 4 (15.4%) patients. The number of plaques ranged from 1 to 11 (median 2), volume 3-201.7 mm³ (median 7), calcium scores 1.3-202.6 (median 5.7). In the group with perfusion defects or coronary calcifications (n = 15), all the patients showed elevated aCL IgG. CONCLUSIONS In most of the relatively young APS patients, without any symptoms of ischemic heart disease, SPECT showed myocardial perfusion defects, and coronary calcifications in 1/6 of them. Right ventricle systolic pressure was elevated in 1/4 of APS patients. These pathologies, well known as cardiovascular risk markers, were associated with elevated levels of the IgG class of both anti-cardiolipin and antiB2 GPI antibodies. Thus, in a high percentage of APS patients, clinically silent myocardial ischaemia, pulmonary pressure elevation and coronary atherosclerosis are present and related to the presence of antiphospholipid antibodies.

Granulomatosis with polyangiitis (GPA) is an autoimmune disease with still unknown etiology. Recent studies indicate that neutrophils extra-cellular traps participate in the pathophysiology of GPA. This study investigates the levels of circulating NET formation markers and neutrophil-platelet interaction in patients with GPA.We enrolled 40 GPA patients (20 in the active stage of the disease and 20 in remission). Twenty sex- and age-matched healthy subjects served as a control group. Serum/plasma levels of serine proteases, and histone-, myeloperoxidase-, proteinase-3 DNA complexes and sP-selectin were measured using ELISA or Luminex assays. Circulating platelet-neutrophil aggregates and neutrophils activation markers expression was measured by flow cytometry.Patients in active stage of GPA had higher circulating levels of serine proteases, DNA-histone and myeloperoxidase -DNA complexes. In addition, platelet-neutrophil aggregates and sP-selectin were also elevated in this group. Platelet-neutrophil aggregates and myeloperoxidase -DNA complexes correlated positively with the disease activity score (BVAS).NETs production and activation of platelets in GPA is supported by elevated myeloperoxidase-DNA complexes and platelet-neutrophil aggregates correlating positively with the disease activity score. This mechanism justifies laboratory measurements of myeloperoxidase-DNA complexes and plasma sP-selectin as biomarkers for studying GPA activity.

Objectives: Lupus anticoagulant (LA) is clinically the most relevant among all antiphospholipid antibody tests. Recently, new guidelines for LA detection were published. The objective of this retrospective cohort study was to compare tests recommended under these guidelines with other methods used for LA detection. Methods: The study group consisted of 336 subjects suffering from various autoimmune diseases. We used activated partial thromboplastin time (aPTT), diluted Russell viper venom time (dRVVT) and diluted prothrombin time (dPT) tests for LA detection together with a ratio between sensitive and insensitive aPTT reagent. We also tested if LA was dependent on beta(2)glycoprotein I (beta(2)GPI) using one of the recently described methods. Results: All LA tests performed were associated with a history of thrombosis. The highest odds ratio (OR) for thrombosis was found for beta(2)GPI-dependent LA but sensitivity was low (OR = 8.4; specificity/sensitivity = 98%/15%). All LA tests showed a much stronger association with thrombosis than with pregnancy failure. Conclusions: LA tested by aPTT and/or dRVVT (at least one out of two tests positive), as recommended by the guidelines, was associated less strongly with a history of thrombosis (OR = 4.1) than either of these tests separately (OR = 5.0 and 4.3, respectively). With both tests positive ('double LA positivity') the association with thrombosis was stronger (OR = 6.5) compared with only one positive test. In fact, 'double LA positivity', detected by combinations of any of the tests studied, was markedly associated with a history of thrombosis.

Objective Conventional risk factors for coronary artery disease fail to explain the increased frequency or cardiovascular morbidity in systemic lupus erythematosus (SLE) patients. This study was conducted to determine the possible influence of autoimmune and inflammatory phenomena markers on coronary artery calcifications and myocardial perfusion abnormalities in SLE patients. Materials and methods Multi-detector computed tomography (MDCT)-based coronary calcium scoring and single photon emission computerized tomography (SPECT) studies (Tc-99m sestamibi) were performed in 60 SLE patients in stable clinical condition, without a prior history of coronary artery disease. Laboratory evaluation included serum C-reactive protein (CRP), complement C3c and C4 components and antiphospholipid antibodies (aPL). The latter included anticardiolipin (aCL) and anti-β2-glycoprotein I (aβ2GPI) antibodies, of both IgG and IgM classes, and lupus anticoagulant (LA) in plasma. Results SPECT revealed persistent perfusion defects in 22 (36.7%) patients and exercise-induced defects in eight (13.3%), while MDCT revealed coronary calcifications in 15 (25%). Calcium scores ranged from 1 to 843.2 (mean 113.5 ± 259.7). No association was found between conventional coronary artery disease risk factors (obesity, hypertension, tobacco use, hyperlipidaemia, diabetes) nor CRP, C3c or C4 levels and coronary calcifications or myocardial perfusion defects. On the contrary, in patients with these pathologies, augmented autoimmunization was found, reflected by increased aCL IgG and antiβ2GPI IgG levels. In patients with aCL IgG >20 RU/ml or antiβ2GPI IgG >3 RU/ml, the relative risk of coronary calcification formation was 4.1 compared to patients with normal values. Accordingly, in LA-positive patients the relative risk of coronary calcification formation was 4.4 compared to LA-negative patients. Conclusions Conventional risk factors for coronary artery disease as well as markers of an ongoing inflammation did not show any association with perfusion defects and/or coronary artery calcifications in SLE patients. On the contrary, calcified atherosclerotic plaques and myocardial perfusion defects were observed mainly in patients with elevated levels of anticardiolipin and aβ2GPI antibodies of the IgG class. It might be speculated that coronary artery calcifications and perfusion defects are a result of antiphospholipid antibodies-induced coronary artery microthrombosis.

Although peripheral nervous system involvement in patients with Churg-Strauss syndrome (CSS) has been described, little is known about its autonomic part. Autonomic nervous system (ANS) function can be assessed by studying heart rate variability (HRV) and a decrease in the spectrum of HRV correlates with ANS impairment.Out of 24 CSS patients we chose 12 (four males, eight females, aged 40 ± 8.3 years) in disease remission and without cardiac involvement. Twelve age- and sex-matched healthy volunteers served as a control group. All underwent 24-h electrocardiogram (ECG) Holter recordings. HRV was calculated from 1-h segments, including: total power (TP), ultra-low frequency (ULF), very low frequency (VLF), low frequency (LF), and high frequency (HF) powers as well as normalized LF (LF%) and HF (HF%) powers and the LF to HF power ratio (LF/HF).The CSS patients showed decreased HRV parameters in the 1-h domains: TP (2038 vs. 3622 ms(2), p = 0.001), HF (561 vs. 1574 ms(2), p0.001), LF (672 vs. 1050 ms(2), p0.01), and VLF (544 vs. 738 ms(2), p = 0.016). However, LF% and LF/HF ratio were markedly higher in CSS patients than in controls (53.4% vs. 39%, p0.001 and 1.1 vs. 0.64, p0.001), whereas HF% was lower in CSS than in controls (46.6% vs. 61%, p0.001). These results were independent of duration of the disease, eosinophil count, corticosteroids, or peripheral nerve involvement in the past.The CSS patients show impaired HRV parameters, indicating parasympathetic ANS dysfunction in addition to peripheral nervous system involvement.

Asthma is one of the key features of Churg-Strauss syndrome (CSS); however its course in the disease is unclear. The aim of this study was to analyze the asthma course in CSS patients.We conducted a retrospective study of 22 CSS patients. Medical documentation was studied and patients were questioned about asthma symptoms during follow-up visits, which took place at four points of the disease. These points, at which lung function tests were performed, were CSS diagnosis; introduction of treatment at hospital discharge; and assessment of the first and last clinical and laboratory CSS remissions. Asthma control and severity were assessed in compliance with current guidelines.In the asthmatic group examined, 21 patients had an adult-onset asthma, with a mean age of onset 35.5 ± 11.8 years, preceded by sinusitis (100%) and nasal polyposis (36%). Asthma at its onset was severe in 68.5%, moderate in 27%, and very poorly controlled in 21 patients. Atopy was present in 64% of patients. Onset of vasculitic symptoms and CSS diagnosis was accompanied by high blood eosinophilia (maximal 7.2 x 10(3)/l [1.2 - 32], asthma exacerbation with airway obstruction in 16 patients (mean values of FEV1 68.8 ± 17.5% and FVC 84.8 ± 19.6%), and lung involvement. After introducing the treatment and achieving stable remission, asthma severity/control and lung function tests (forced expiratory volume in 1 s 92.7 ± 13.3%, and forced vital capacity 101 ± 15.5%; p.001) improved.Asthma in CSS, although severe as its onset improves after achieving CSS remission.

Summary. Background: Antiphospholipid syndrome (APS) is diagnosed by the simultaneous presence of vascular thrombosis and/or pregnancy morbidity and detection of antiphospholipid antibodies in plasma. Objectives: We have shown that prolongation of clotting time by anti-beta2-glycoprotein I (beta2GPI) antibodies correlates better with thrombosis than a positive classic lupus anticoagulant (LAC) assay in a single center study. To confirm or falsify this finding we have conducted a multicenter study. Methods and results: In 325 LAC-positive samples, we found that the beta2GPI-dependent LAC correlated 2.0 times better with thrombosis than the classic LAC assay. Although significant, this was a minimal improvement compared with the ‘classic’ LAC. It was published that calcium influences the behavior of anti-beta2GPI antibodies in coagulation assays. To investigate whether calcium plays a role in the present study, we divided the patient population into two groups: (i) blood was collected in 0.109 m sodium citrate and (ii) blood was drawn in 0.129 m sodium citrate as anticoagulant. We found that a positive result with the beta2GPI-dependent LAC assay correlated better with thrombosis [odds ratio (OR): 3.3, 95% confidence interval (CI) 1.9–5.8] when 0.109 m sodium citrate was used compared with 0.129 m sodium citrate (OR: 0.4, 95% CI 0.1–1.1). Conclusion: We were able to confirm in an international multicenter study that a positive result in a beta2GPI-dependent LAC assay correlates better with thrombosis than the classic LAC assay, but that the assay needs further study as it is sensitive to external factors such as the sodium citrate concentration used as anticoagulant in the test sample.

Venous thromboembolism (VTE) has been considered the dominant major life threatening vascular complication after non-cardiac surgery, but recent studies have shifted the emphasis toward myocardial injury after non-cardiac surgery (MINS) as a common adverse event in the peri-operative setting. The aim of the present study was to compare the incidence and influence on mortality of two dominant venous and arterial events in the peri-operative period by prospectively screening a consecutive cohort of patients undergoing vascular surgery.This was a sub-study of Vascular Events In Non-cardiac Surgery Patients Cohort Evaluation (VISION), the main objective of which was to evaluate major peri-operative complications after non-cardiac surgery. Patients undergoing vascular surgery had their blood collected to measure the Roche fifth generation high sensitivity troponin T (hsTnT) assay before and four times after surgery (6-12 h post-operatively, on the first, second, and third day following the procedure). MINS was defined as an elevated post-operative hsTnT ≥65 ng/L or an hsTnT ≥20 to65 ng/L with an absolute change of ≥5 ng/L that was judged to be due to ischaemia. All patients underwent ultrasound venous compression testing for deep vein thrombosis (DVT) before, 4, and 7 days after surgery and follow-up was performed by telephone 30 days and 1 year after surgery.In total, 164 consecutive patients were included in this sub-study. MINS was diagnosed in 39 patients (23.8%) and DVT in four patients (2.4%). The 1 year mortality was higher in MINS (9/39 [23.1%]) than non-MINS patients (9/125 [7.2%]; p = .006). None of the patients who developed DVT died in the first year after surgery.MINS is a common complication after vascular surgery. It occurs more frequently than DVT and is associated with high 1 year mortality.

International normalized ratio (INR) is used to monitor chronic oral anticoagulant (OA) treatment; however, it is poorly understood how this simple test reflects in vivo hemostatic reactions, culminating in thrombin generation and clot formation. We studied the process of thrombin generation using an ex vivo model, where thrombin—antithrombin (TAT) complexes are measured in blood emerging from standardized skin incisions in 55 patients (35 with venous thromboembolism [VTE] and 20 with sustained atrial fibrillation [AF]) treated with acenocoumarol (INR 2.0-3.0). In addition, in venous blood, we measured the activity of factor VIII (FVIII) and vitamin K-dependent coagulation proteins. Chronic anticoagulation led to significant reductions in maximum TAT concentrations as compared to 35 healthy controls, in maximum TAT generation rates, and in mean amount of thrombin generated. Parameters of thrombin generation did not correlate with INR or any coagulation factor measured. International normalized ratio was significantly and independently affected by the decrease in the activity of all vitamin K-dependent coagulation proteins. The strongest influence was shown for FVII. Factor VIII activity was increased in all patients studied independently of the duration of anticoagulation and did not change over time. In conclusion, in patients with VTE and AF on OA, there is no correlation between INR values and parameters of ex vivo thrombin generation. This may indicate an important role of protein C (PC) system and possibly other endothelium-dependent mechanisms in controlling hemostasis. Increased FVIII activity in patients with VTE and AF does not change significantly during anticoagulation and is probably related to the pretreatment prothrombotic state.

International audience; Connections between inflammation and thrombosis are intriguing, especially in a condition such as an antiphospholipid syndrome (APS), a disease characterized by immune-mediated thrombosis. Tumor necrosis factor alpha (TNF-α) is a cytokine which shares proinflammatory and prothrombotic actions, while a soluble form of interlukin-2 receptor (sIL-2R) is considered a typical marker of (auto)immune inflammation with not known direct links to thrombosis. The differences in the pathogenesis of APS as compared to other autoimmune diseases might be connected with different serum levels of both mediators. To answer this question, we studied 147 patients with systemic lupus erythematosus (SLE), 21 with SLE-like syndrome (SLE-LS), 20 with isolated APS (primary antiphospholipid syndrome, PAPS), and 32 healthy controls. Thirty-six patients from the SLE group fulfilled the updated APS criteria (secondary APS, SAPS). In comparison to healthy subjects, TNF-α concentration was increased in all patients, while sIL-2R rose significantly in the SLE group only. APS (both SAPS and PAPS) was characterized by the highest levels of TNF-α. Moreover, patients with lupus anticoagulant or elevated levels of IgG anticardiolipin or IgG anti-β-glycoprotein I antibodies had higher TNF-α levels than patients without the presence of any type of antiphospholipid antibodies (aPL). In conclusion, the presence of aPL is associated with higher TNF-α level, whereas increased level of sIL-2R is rather connected with definite SLE where inflammatory processes prevail. It might be hypothesized that TNF-α plays a major role in pathogenesis of APS thrombotic phenomena.

Summary. Background: The classification criteria for antiphospholipid syndrome (APS) were updated in 2006. Objective: The aim of the study was to analyze associations between clinical complications and laboratory test abnormalities typical for APS in a group of patients with autoimmune diseases, based on the recently updated criteria. Patients/methods: Three hundred and thirty-six patients were enrolled into the study, with the majority (n = 235) suffering from systemic lupus erythematosus. Laboratory determinations included: lupus anticoagulant (LA), anticardiolipin (aCL) and anti-beta2-glycoprotein I (anti-β2GPI) antibodies (ABs) [of both immunoglobulin G (IgG) and IgM class]. Results: A significant association was found between laboratory and clinical features of APS; odds ratios (ORs) for thrombosis associated with the presence of LA, aCL, and anti-β2GPI Abs were 4.04 [95% CI: 2.44–6.68], 3.71 (95% CI 2.32–5.92) and 2.57 (95% CI 1.60–4.1), respectively. Detailed analysis showed marked differences between the risk of clinical complications associated with the presence of an antibody in the IgG class (OR 4.15, 95% CI 2.42–7.12, and OR 4.77, 95% CI 2.37–9.61 for aCL and anti-β2GPI, respectively) and in the IgM class (OR 2.2, 95% CI 1.31–3.70, and OR 1.9, 95% CI 1.15–3.14 for aCL and anti-β2GPI, respectively). The postulated inclusion of anti-β2GPI antibody positivity into the previous laboratory criteria changed only slightly the number of patients diagnosed with APS (from 112 to 117). Conclusions: The updated APS classification criteria clearly represent a step forward. However, our results argue against the use of overall positivity for aCL or anti-β2GPI, and favor a clear distinction between the IgG and IgM classes of antiphospholipid ABs. Patients with both LA and anti-β2GPI IgG or LA and aCL IgG positivity may represent the subgroups at the highest risk of thrombotic complications.