N-homocysteinylated (Nepsilon-Hcy) proteins and corresponding antibodies have recently been discovered in humans and animals. Increased autoimmune response to Nepsilon-Hcy-proteins has been reported in stroke patients. The aim of the present study was to investigate whether antibodies against N-homocysteinylated albumin are associated with coronary artery disease (CAD). We studied 88 male patients aged 50 years or under with angiographically documented CAD and 100 age-matched apparently healthy men as controls. Serum levels of IgG antibodies against Nepsilon-Hcy-albumin were determined using an enzymelinked immunosorbent assay. Seropositivity to anti-Nepsilon-Hcy-albumin antibodies was 5-fold more frequent in CAD patients than in controls (52.3% vs 10.0%; p<0.0001). Plasma Hcy levels in CAD patients were also significantly higher in the former than in the latter group (medians, 13.0 microM vs 12.1 microM; p=0.026). Importantly, 41.2% of subjects with plasma total Hcy >14.5 mM were seropositive compared with 25.5% of normohomocysteinemic individuals (p=0.048). There was a weak correlation between anti-Nepsilon-Hcy-albumin antibodies and Hcy levels (r=0.16; p=0.03). By multivariate logistic regression analysis, seropositivity to anti-Nepsilon-Hcy-albumin antibodies was an independent predictor of early CAD (OR, 14.82; 95% CI, 4.47 to 49.19; p=0.00002). Interestingly, anti-Nepsilon-Hcy-albumin antibodies were associated with C-reactive protein levels (r=0.24; p=0.002). Seropositivity to anti-Nepsilon-Hcy-albumin antibodies showed no association with the MTHFR C677T polymorphism. Our results suggest that seropositivity to antibodies against Nepsilon-homocysteinylated albumin is associated with early-onset CAD. An autoimmune response to Nepsilon-Hcy-albumin may represent a novel mechanism involved in the early development of CAD.

We tested the hypothesis that plasma fibrin clot structure/function is unfavourably altered in patients with antiphospholipid syndrome (APS). Ex vivo plasma clot permeability, turbidity and susceptibility to lysis were determined in 126 consecutive patients with APS enrolled five months or more since thrombotic event vs 105 controls. Patients with both primary and secondary APS were characterised by 11% lower clot permeability (p<0.001), 4.8% shorter lag phase (p<0.001), 10% longer clot lysis time (p<0.001), and 4.7% higher maximum level of D-dimer released from clots (p=0.02) as compared to the controls. Scanning electron microscopy images confirmed denser fibrin networks composed of thinner fibres in APS. Clots from patients with "triple-antibody positivity" were formed after shorter lag phase (p=0.019) and were lysed at a slower rate (p=0.004) than in the remainder. Clots from APS patients who experienced stroke and/or myocardial infarction were 8% less permeable (p=0.01) and susceptible to lysis (10.4% longer clot lysis time [p=0.006] and 4.5% slower release of D-dimer from clots [p=0.01]) compared with those following venous thromboembolism alone. Multivariate analysis adjusted for potential confounders showed that in APS patients, lupus anticoagulant and "triple-positivity" were the independent predictors of clot permeability, while "triple-positivity" predicted lysis time. We conclude that APS is associated with prothrombotic plasma fibrin clot phenotype, with more pronounced abnormalities in arterial thrombosis. Molecular background for this novel prothrombotic mechanism in APS remains to be established.

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.

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Airway remodeling in asthma is characterized by reticular basement membrane (RBM) thickening, likely related to epithelial structural and functional changes. Gene expression profiling of the airway epithelium might identify genes involved in bronchial structural alterations. We analyzed bronchial wall geometry (computed tomography (CT)), RBM thickness (histology), and the bronchial epithelium transcriptome profile (gene expression array) in moderate to severe persistent (n = 21) vs. no persistent (n = 19) airflow limitation asthmatics. RBM thickness was similar in the two studied subgroups. Among the genes associated with increased RBM thickness, the most essential were those engaged in cell activation, proliferation, and growth (e.g., CDK20, TACC2, ORC5, and NEK5) and inhibiting apoptosis (e.g., higher mRNA expression of RFN34, BIRC3, NAA16, and lower of RNF13, MRPL37, CACNA1G). Additionally, RBM thickness correlated with the expression of genes encoding extracellular matrix (ECM) components (LAMA3, USH2A), involved in ECM remodeling (LTBP1), neovascularization (FGD5, HPRT1), nerve functioning (TPH1, PCDHGC4), oxidative stress adaptation (RIT1, HSP90AB1), epigenetic modifications (OLMALINC, DNMT3A), and the innate immune response (STAP1, OAS2). Cluster analysis revealed that genes linked with RBM thickness were also related to thicker bronchial walls in CT. Our study suggests that the pro-fibrotic profile in the airway epithelial cell transcriptome is associated with a thicker RBM, and thus, may contribute to asthma airway remodeling.