Objective. The antiphospholipid syndrome (APS) is a disorder of recurrent thrombosis, pregnancy loss, and thrombocytopenia associated with the production of anticardiolipin antibodies (aCL) and lupus anticoagulant (LAC). The mechanisms of thrombus formation remain unknown. Tissue factor (TF), an inducible cell glycoprotein, is a major initiator of coagulation in vivo. The present study was therefore undertaken to investigate TF expression and procoagulant activity on monocytes from patients with primary APS and its correlation with thrombotic events.Methods. Three groups of patients were studied: group 1 comprised 23 primary APS patients with thrombosis, group 2 consisted of 10 primary APS patients without thrombosis, and group 3 contained 20 patients with thrombosis but without antiphospholipid antibodies. Twenty age‐ and sex‐matched healthy blood donors were used as controls (group 4). Anticardiolipin antibodies were measured by enzyme‐linked immunosorbent assay (ELISA) and LAC by standard methodology. Cell surface expression of TF on monocytes was assessed by flow cytometry. The amount of TF in cell lysates (TF_Ag) and soluble TF_Ag plasma levels were analyzed by ELISA, and the TF‐related procoagulant activity (PCA‐TF) on intact cells and cell lysates by a chromogenic assay. Levels of the cytokines that influence TF production, i.e., tumor necrosis factor α (TNFα) and interleukin‐1β (IL‐1β), were determined by ELISA.Results. Cell surface expression of TF was increased in group 1 (mean ± SEM 50.2 ± 4% positive cells) compared with group 2 (14.6 ± 1.6%), group 3 (16.8 ± 3.7%), and group 4 (14.1 ± 1.6%). TF_Ag levels were also elevated in group 1 (215.8 ± 11.2 pg/10⁶) compared with group 2 (150.8 ± 15.2), group 3 (101.4 ± 14.8), and group 4 (80.32 ± 5.5). PCA‐TF on intact cells and cell lysates was significantly increased in group 1 (148.8 ± 16.3 units/10⁵ lysate cells, compared with 54.5 ± 11.5, 38.6 ± 9.7, and 22.5 ± 3.1 in groups 2, 3, and 4, respectively). Among group 1 patients, there was a significant increase in the degree of TF expression in those positive for IgG aCL, but not in those positive for IgM aCL or LAC. TNFα and IL‐1β plasma levels did not differ significantly between any of the groups.Conclusion. These results suggest that monocyte TF expression is directly involved in the pathogenesis of thrombotic complications in patients with the primary APS.

Objective: Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL). In patients with primary APS, expression of tissue factor (TF) on the surface of monocytes is increased, which may contribute to thrombosis in these patients. However, the intracellular mechanisms involved in aPL‐mediated up‐regulation of TF on monocytic cells are not understood. This study was undertaken to investigate the intracellular signals induced by aPL that mediate TF activation in monocytes from APS patients.Methods: We analyzed, both in vivo and in vitro, aPL interactions with proteins that have signaling functions, including mitogen‐activated protein kinases (MAP kinases) and NF‐κB/Rel proteins.Results: In vivo studies demonstrated significantly higher levels of both TF messenger RNA and TF protein in monocytes from APS patients compared with controls. At the molecular level, increased proteolysis of IκBα and activation of NF‐κB were observed. Constitutive activation of both p38 and ERK‐1 MAP kinases was also found. Treatment of normal monocytes with aPL activated ERK‐1 and p38 MAP kinases, as well as the IκB/NF‐κB pathway, in a dose‐dependent manner. NF‐κB activation and IκBα degradation induced by aPL were inhibited by the NF‐κB inhibitor SN50 and the p38 MAP kinase inhibitor SB203580, thus suggesting crosstalk between these pathways. However, the MEK‐1/ERK inhibitor PD98059 did not affect aPL‐induced NF‐κB binding activity. TF expression induced by aPL was significantly inhibited by combined treatment with the 3 inhibitors.Conclusion: Our results suggest that aPL induces TF expression in monocytes from APS patients by activating, simultaneously and independently, the phosphorylation of MEK‐1/ERK proteins, and the p38 MAP kinase–dependent nuclear translocation and activation of NF‐κB/Rel proteins.

Introduction: The advent of anti-tumor necrosis factor alpha (anti-TNFα) drugs has considerably improved medical management in rheumatoid arthritis (RA) patients, although it has been reported to be ineffective in a fraction of them. MicroRNAs (miRNAs) are small, non-coding RNAs that act as fine-tuning regulators of gene expression. Targeting miRNAs by gain or loss of function approaches have brought therapeutic effects in various disease models. The aim of this study was to investigate serum miRNA levels as predictive biomarkers of response to anti-TNFα therapy in RA patients. Methods: In total, 95 RA patients undergoing anti-TNFα/disease-modifying antirheumatic drugs (anti-TNFα/DMARDs) combined treatments were enrolled. Serum samples were obtained at 0 and 6 months and therapeutic efficacy was assessed. miRNAs were isolated from the serum of 10 patients before and after anti-TNFα/DMARDs combination therapy, cDNA transcribed and pooled, and human serum miRNA polymerase chain reaction (PCR) arrays were performed. Subsequently, selected miRNAs were analyzed in a validation cohort consisting of 85 RA patients. Correlation studies with clinical and serological variables were also performed. Results: Ninety percent of RA patients responded to anti-TNFα/DMARDs combination therapy according to European League Against Rheumatism (EULAR) criteria. Array analysis showed that 91% of miRNAS were overexpressed and 9% downregulated after therapy. Functional classification revealed a preponderance of target mRNAs involved in reduction of cells maturation - especially on chondrocytes - as well as in immune and inflammatory response, cardiovascular disease, connective tissue and musculoskeletal system. Six out of ten miRNAs selected for validation were found significantly upregulated by anti-TNFα/DMARDs combination therapy (miR-16-5p, miR-23-3p, miR125b-5p, miR-126-3p, miRN-146a-5p, miR-223-3p). Only responder patients showed an increase in those miRNAs after therapy, and paralleled the reduction of TNFα, interleukin (IL)-6, IL-17, rheumatoid factor (RF), and C-reactive protein (CRP). Correlation studies demonstrated associations between validated miRNAs and clinical and inflammatory parameters. Further, we identified a specific plasma miRNA signature (miR-23 and miR-223) that may serve both as predictor and biomarker of response to anti-TNFα/DMARDs combination therapy. Conclusions: miRNA levels in the serum of RA patients before and after anti-TNFα/DMARDs combination therapy are potential novel biomarkers for predicting and monitoring therapy outcome.

A paradoxical but common finding in the obesity clinic is the identification of individuals who can be considered ‘inappropriately’ healthy for their degree of obesity. We think that studying these obese but metabolically healthy individuals and comparing them with equally obese but insulin-resistant individuals could provide important insights into the mechanistic link between adipose tissue expansion and associated metabolic alterations. In the present study, we investigated whether there are differences in inflammatory and insulin signalling pathways in VAT (visceral adipose tissue) that could account for the metabolic differences exhibited by morbidly obese individuals who are either insulin-resistant (IR-MO) or paradoxically insulin-sensitive (NIR-MO). Our results indicate that there are pathways common to obesity and unrelated to insulin resistance and others that are discriminative for insulin resistance for a similar degree of obesity. For instance, all morbidly obese patients, irrespective of their insulin resistance, showed increased expression of TNFα (tumour necrosis factor α) and activation of JNK1/2 (c-Jun N-terminal kinase 1/2). However, the IR-MO group showed significantly elevated expression levels of IL (interleukin)-1β and IL-6 and increased macrophage infiltrates compared with non-obese individuals and NIR-MO. IκBα [inhibitor of NF-κB (nuclear factor κB) α], the activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) and NF-κB were discriminative of the state of insulin resistance and correlated with differential changes in IRS-1 (insulin receptor substrate 1) expression and Akt activation between IR-MO and NIR-MO individuals. Our results support the concept that NIR-MO individuals lack the inflammatory response that characterizes the IR-MO patient and that IL-6, IL-1β, ERK and NF-κB are important effectors that mediate the inflammation effects promoting insulin resistance.

The induction of polyploidy is considered the reproductive end of cells, but there is evidence that polyploid giant cancer cells (PGCCs) contribute to cell repopulation during tumor relapse. However, the role of these cells in the development, progression and response to therapy in colon cancer remains undefined. Therefore, the main objective of this study was to investigate the generation of PGCCs in colon cancer cells and identify mechanisms of formation. Treatment of HCT-116 and Caco-2 colon cancer cells with the hypoxia mimic CoCl₂ induced the formation of cells with larger cell and nuclear size (PGCCs), while the cells with normal morphology were selectively eliminated. Cytometric analysis showed that CoCl₂ treatment induced G2 cell cycle arrest and the generation of a polyploid cell subpopulation with increased cellular DNA content. Polyploidy of hypoxia-induced PGCCs was confirmed by FISH analysis. Furthermore, CoCl₂ treatment effectively induced the stabilization of HIF-1α, the differential expression of a truncated form of p53 (p47) and decreased levels of cyclin D1, indicating molecular mechanisms associated with cell cycle arrest at G2. Generation of PGCCs also contributed to expansion of a cell subpopulation with cancer stem cells (CSCs) characteristics, as indicated by colonosphere formation assays, and enhanced chemoresistance to 5-fluorouracil and oxaliplatin. In conclusion, the pharmacological induction of hypoxia in colon cancer cells causes the formation of PGCCs, the expansion of a cell subpopulation with CSC characteristics and chemoresistance. The molecular mechanisms involved, including the stabilization of HIF-1 α, the involvement of p53/p47 isoform and cell cycle arrest at G2, suggest novel targets to prevent tumor relapse and treatment failure in colon cancer.